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A Conformational Restriction Strategy for the Identification of a Highly Selective Pyrimido-pyrrolo-oxazine mTOR Inhibitor

Chiara Borsari
Chiara Borsari
Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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http://orcid.org/0000-0002-4688-8362
,
Denise Rageot
Denise Rageot
Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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http://orcid.org/0000-0002-2833-5481
,
Alix Dall’Asen
Alix Dall’Asen
PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
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Thomas Bohnacker
Thomas Bohnacker
Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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Anna Melone
Anna Melone
Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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Alexander M. Sele
Alexander M. Sele
Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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http://orcid.org/0000-0002-4903-7934
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Eileen Jackson
Eileen Jackson
Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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Jean-Baptiste Langlois
Jean-Baptiste Langlois
Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
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Florent Beaufils
Florent Beaufils
PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
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Paul Hebeisen
Paul Hebeisen
PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
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Doriano Fabbro
Doriano Fabbro
PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
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Petra Hillmann
Petra Hillmann
PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
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Matthias P. Wymann*
Matthias P. Wymann
Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
*E-mail: [email protected]. Phone: +41 61 207 5046. Fax: +41 61 207 3566.
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http://orcid.org/0000-0003-3349-4281

Cite this: J. Med. Chem. 2019, 62, 18, 8609–8630

Publication Date (Web):August 29, 2019

https://doi.org/10.1021/acs.jmedchem.9b00972

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Abstract

The mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have the potential to overcome limitations of rapamycin derivatives in a wide range of malignancies. Herein, we exploit a conformational restriction approach to explore a novel chemical space for the generation of TORKi. Structure–activity relationship (SAR) studies led to the identification of compound 12b with a ∼450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450 reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR inhibitors for cancer treatment.

Introduction

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The mechanistic target of rapamycin (mTOR, also mammalian TOR) is a key signaling node in the phosphoinositide 3-kinase (PI3K)/mTOR signaling pathway and operates downstream of PI3K. mTOR is a serine/threonine protein kinase and is part of two functionally distinct multiprotein complexes, mammalian target of rapamycin complex 1 (mTORC1) and mammalian target of rapamycin complex 2 (mTORC2). (1,2) TORC1 integrates inputs from cell surface receptors, cellular energy, stress levels, availability of oxygen and amino acids. Cell surface receptors activate PI3K to produce PtdIns(3,4,5)P₃, which serves as a docking site for protein kinase B (PKB/Akt) and phosphoinositide-dependent protein kinase 1 (PDK1). This results in phosphorylation of PKB/Akt at two regulatory sites, Thr308 by PDK1 and Ser473 by mTORC2, and other hydrophobic motif kinases. (3−6)

Through the activation of S6 kinase (S6K), TORC1 promotes phosphorylation of ribosomal protein S6, which is a key regulator of protein and lipid synthesis. (7) TORC1 also regulates lysosome function and autophagy, while TORC2 promotes cytoskeletal rearrangement, cell survival, and cell cycle entry. (8) The PI3K/mTOR axis is involved in many cellular processes including cell growth, proliferation, and metabolism, and it has been found to be dysregulated in fatal diseases, such as cancer, metabolic, cardiovascular, and neurological disorders. (5,9−12)

The pivotal role of mTOR in numerous human cancers has made this Ser/Thr-kinase a therapeutic target for cancer treatment.

Rapamycin and its analogs (rapalogs) are allosteric inhibitors of TORC1 (Figure 1) and form a TORC1/rapalog/FK506 binding protein 12 (FKBP12) complex. (13) Rapalogs have been extensively investigated in clinical trials in oncology (14−17) and have been approved for a number of cancer treatments such as lung, gastrointestinal neuroendocrine, noncancerous kidney, and advanced pancreatic tumors, advanced ER+/HER2-negative breast and endometrial cancer, mantle cell lymphoma, tuberous sclerosis complex (TSC), and pediatric subependymal giant cell astrocytoma (SEGA). (16,18,19)

Figure 1. Chemical structures of rapamycin and rapalogs and a selection of ATP-competitive mTOR kinase inhibitor (TORKi) compounds.

Their efficacy as single agents in major solid tumors, however, has turned out to be limited. (20) This might be explained by the selective allosteric inhibition of TORC1, which (i) leaves mTORC2 intact and (ii) suppresses TORC1-dependent feedback loops. (21−24) Small molecule ATP-competitive TORKi compounds target both mTORC1 and mTORC2. Hence, these inhibitors efficiently block two branches of PI3K downstream signaling: (i) one via PKB/Akt to tuberous sclerosis complex (TSC) and Ras homolog enriched in brain (Rheb1) controlling TORC1 and (ii) the PtdIns(3,4,5)P₃-medited Sin1-dependent TORC2 activation, (25) explaining their robust antitumor efficacy. (26) However, by targeting the ATP-binding site of mTOR, TORKi compounds potentially inhibit other protein and lipid kinases at elevated doses, especially the structurally related PI3Ks. Thus, the development of highly selective TORKi is an ongoing challenge. Structurally distinct mTOR-selective inhibitors have been reported and evaluated in a wide range of malignancies. Among the most noteworthy examples, CC-223 (55), (27) sapanisertib (INK128, MLN0128, 56), (28) and vistusertib (AZD2014, 57) (29) have been explored in clinical trials and were found superior to rapalogs in terms of cytostatic and cytotoxic potential (see Figure 1 for chemical structures). (30)

Very recently, we have reported on PQR620 (58) (Figure 1), a highly selective and potent ATP-competitive mTOR inhibitor, targeting both mTORC1 and mTORC2 kinase activities. (31) PQR620 (58) had been discovered through chemical modification of PQR309 (1), a pan-PI3K and moderate mTOR inhibitor currently in clinical trials for the treatment of lymphoma and solid tumors. (32−35)

Here, we report the discovery of conformationally restricted compounds as a novel scaffold for the development of highly selective TORKi compounds. The design of conformationally constrained analogs is often used to minimize the entropic loss associated with target binding, to enhance the potency for the target, and to increase the selectivity. (36) This conformational restriction approach explores a novel chemical space and secured intellectual property. (37) An extensive structure–activity relationship (SAR) study led us to the identification of a highly selective and potent ATP-competitive inhibitor 12b. Compound 12b is orally bioavailable, metabolically stable and has a minimal brain permeability, an advantage in the treatment of systemic tumors. Our results disclose the tricyclic pyrimido-pyrrolo-oxazine moiety as an innovative scaffold for selectively targeting mTOR kinase.

Results and Discussion

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Rigidification Strategy

In our rigidification strategy, we started from PQR309 (1) and introduced a methylene bridge between one of the morpholines and the aromatic core (Figure 2). This led to the conversion of the triazine ring into a pyrimidine core. Moreover, we have removed the trifluoromethyl group from the 2-aminopyridine moiety (Figure 2) to alter lipophilicity, increase solubility, and change electronic and steric parameters. (38)

For the designed compounds, two different regioisomers exist, each with two enantiomers (Table 1, 2a /2b and 2c/2d). To evaluate the rigidification effect on compounds activity, compounds 2a–d were evaluated for in vitro binding [K_i for mTOR and PI3Kα (p110α)] and for PI3K/mTOR signaling in A2058 cells (IC₅₀ for phosphorylated S6 to detect mTORC1 activity, and protein kinase B (PKB/Akt) phosphorylation on Ser473 to detect mTORC2 activity). One regioisomer is more potent both in cells and in vitro, as highlighted by comparing 2a with 2c and 2b with 2d. Compound 2a was >9-fold more potent in cells and >11-fold more potent in vitro than 2c (Table 1).

Table 1. Conformationally Restricted Regioisomers 2a/2b and 2c/2d

	cellular assay, IC₅₀ [nM]^a		in vitro binding assay, K_i [nM]^b
compd	pPKB S473	pS6 S235/236	p110α	mTOR	selectivity K_i(p110α)/K_i(mTOR)	clogP^c
PQR309 (1)	139	205	17	62	0.27	3.11
2a	194	164	76	69	1.1	2.65
2b	118	96	432	24	18.3	2.65
2c	1871	1790	867	812	1.1	2.65
2d	1560	1857	541	1279	0.4	2.65

^{^a}

PKB phosphorylation on Ser473 and ribosomal S6 phosphorylation on Ser235/236 were analyzed in A2058 cells exposed to the indicated inhibitors and subsequent detection of phosphoproteins in an in-cell Western assay. Each experiment was performed with n = 2. The log IC₅₀ values and standard errors are reported in Table S7 in the Supporting Information.

^{^b}

Compounds were tested for the in vitro binding to the ATP-binding site of p110α and mTOR using a commercially available time-resolved FRET (TR-FRET) displacement assay (LanthaScreen). Each experiment was performed with n = 2. IC₅₀ values and log IC₅₀ values and standard errors are reported in Table S7 in the Supporting Information.

^{^c}

Marvin/JChem 16.10.17 was used for calculation of log P (partition coefficient) values.

Elucidation of Binding Modes to PI3K and mTOR

To explain activity differences of the regioisomers, we considered the possible interactions of the inhibitors within the ATP-binding site of the kinases. As previously reported for PQR309 (1) (33) and other morpholino-substituted PI3K inhibitors, (32,39) a crucial interaction for PI3K binding is the hydrogen bond formation in the hinge region between the backbone amide of Val882 residue in PI3Kγ (Val851 in PI3Kα) and the morpholine oxygen atom. Herein, we started from the conformationally restricted PQR309 (1) analogs (3a and 3b, Table 2) and replaced the morpholine with a piperidine (4a,b and 5a,b, Table 2) to investigate whether the methylene-bridged morpholine or the unrestricted morpholine points toward and interacts with the hinge region Val of the ATP-binding pocket. The obtained K_i values for PI3Kα suggested that the methylene-bridged morpholine is located in the solvent exposed region, since only the compounds with a methylene-bridged piperidine (4a and 4b) displayed a strong affinity for PI3Kα (K_i = 22 and 48 nM, respectively). The in vitro potency of 4a and 4b is comparable to that of the dimorpholine-substituted parental compounds (3a and 3b). On the contrary, replacement of the unrestricted morpholine with a piperidine (5a and 5b) led to a drop in PI3K binding affinity.

Table 2. Binding Mode: Morpholine vs Piperidine in Unrestricted and Restricted Positions

^{^a}

^{^b}

Compounds were tested for the in vitro binding to the ATP-binding site of p110α and mTOR using a commercially available time-resolved FRET (TR-FRET) displacement assay (LanthaScreen). Each experiment was performed with n = 2. IC₅₀ values, logIC₅₀ values, and standard errors are reported in Table S8.

We thus concluded that the unrestricted morpholine binds to the hinge region and establishes a H-bond with the Val backbone amide. We exploited the 2.48 Å resolution X-ray structure of PIKiN3 in PI3Kγ (see ref (32); PDB code 5JHB), and substituted PIKiN3 for compound 3a to confirm its binding mode. Energy minimization calculations of the resulting PI3Kγ-3a complex maintained important interactions such as H-bonds between the aminopyridine and Asp836/964 as well as the interaction of the oxygen atom of the unrestricted morpholine and the backbone amide of Val882 (as present in the parental structure with PIKiN3; Figure 3A). Moreover, the interaction of the core nitrogen of compound 3a with the structured water molecule allows stabilization of the inhibitor binding through a H-bond network. In PI3Kγ, this interaction had been proved to play a pivotal role in inhibitor binding (see ref (32); PDB code 5JHB).

Figure 3. (A) Docking of compound 3a (plum) into PI3Kγ (gray) starting from PDB code 5JHB (see ref (32)). Structural water molecules are shown in red, and water-mediated H-bonds are depicted as dashed black lines. (B) Docking of compound 2a (gold) and (C) compound 2b (green) into mTOR (turquoise) starting from PDB code 4JT6. The important features for mTOR selectivity are depicted in a ball and stick representation. (D) Docking of compound 2a (gold) and (E) compound 2b (green) into PI3Kα (gray) starting from PDB code 3ZIM. The exit vector from the restricted morpholine oxygen is shown as a black arrow.

Given the high homology of the ATP-binding pocket of class I PI3Ks and mTOR, a similar binding mode and orientation in mTOR and all PI3K isoforms can be assumed. Computational modeling studies were performed to elucidate the binding mode of the conformationally restricted compounds 2a and 2b in mTOR starting from the PI103-mTOR complex (PDB code 4JT6). In analogy to PI3K, the higher mTOR potency of the compounds bearing the unrestricted morpholine in the 4-position of the pyrimidine core (2a and 2b, Scheme 1B) could be derived from the interaction of a pyrimidine core nitrogen with a putative structured water molecule, even though the 3.6 Å structural resolution of mTOR did not reveal such structured water molecules. Thus, the position of N-1 of the pyrimidine core (see Scheme 1 for numbering) in the ATP-binding pocket is an essential parameter for mTOR affinity. The binding affinity data of compounds 2a–d suggest a similar structured water network in mTOR, which is only maintained by binding of regioisomer 2a and 2b but not with 2c and 2d (K_i2a, 2b = 69, 24 nM vs K_i2c, 2d = 812, 1279 nM). An additional stabilization of 2a/2b-mTOR complex is provided by hydrophobic interactions between the methylene bridge and Met2345 (Figure 3B and Figure 3C). The important features for mTOR selectivity are depicted in Figure 3B and Figure 3C in a ball and stick representation.

Within one regioisomer pair (2a and 2b), the (R)-enantiomer 2b displayed a stronger affinity for mTOR, together with an 18-fold selectivity for mTOR kinase over PI3Kα (K_i for mTOR = 24 nM and K_i for PI3Kα = 432 nM). In contrast, the (S)-enantiomer 2a is a dual mTOR/PI3Kα inhibitor (Table 1). The 6-fold difference in PI3K inhibitory activity between compound 2a and 2b (K_i for PI3Kα = 76 and 432 nM, respectively) might be related to the different orientation of the morpholine due to scaffold rigidification. In the (S)-configured compound 2a, the oxygen of the restricted morpholine points toward the amide side chain of Gln859 (4 Å) and a weak H-bond could stabilize the binding of 2a in PI3Kα (see exit vector in Figure 3D). In the (R)-tricyclic pyrimido-pyrrolo-oxazine 2b the morpholine oxygen is rotated by 1 Å with respect to the oxygen atom of 2a and the exit vector does not point toward Gln859 (see Figure 3E). Therefore, no H-bond can be established.

Chemistry

Procedures for library synthesis are depicted in Scheme 1. First, sulfamidates 33a and 33b were prepared (Scheme 1A). Typically, sulfamidates are made from chiral amino alcohols in several steps. (40) The 3-hydroxymethyl morpholines (34a and 34b) were synthesized by a chiral synthesis starting from serine enantiomers. As for 34a, the amino group of (R)-serine was protected with a benzyl group (compound 37b) since the ring closure has been described to proceed in higher yields when a protecting group is present. (41)N-Benzyl-(R)-serine (37b) underwent ring closure in the presence of chloroacetyl chloride to give (R)-4-benzyl-5-oxomorpholine-3-carboxylic acid (36b) in 71% yield. Reduction of 36b using borane–dimethyl sulfide complex in tetrahydrofuran (THF) gave intermediate 35a with an 82% yield. Hydrogenolysis of N-benzyl derivative (35a) over palladium on charcoal (Pd/C, H₂) yielded (S)-morpholin-3-ylmethanol (34a). Subsequent two-step cyclic sulfamidate formation was performed by treating the amino alcohol (34a) with thionyl chloride to give the corresponding cyclic sulfamidite, followed by oxidation using NaIO₄ and catalytic amounts of ruthenium(IV) oxide hydrate to give sulfamidate 33b in 52% yield. The (S)-enantiomer (33a) was synthesized from (S)-serine following the procedure described for 33b (Scheme 1A). The dichloropyrimidines (26–31) were prepared according to Scheme 1B starting from the commercially available 2,4,6-trichloropyrimidine. Morpholines M₀–M₅ were introduced as M_n substituents by nucleophilic aromatic substitution reaction. Following this procedure, both regioisomers were generated and separated by column chromatography. The desired regioisomers (27–31) were isolated in moderate to high yields (46–86%). The (S)-tricyclic intermediates (20a–25a) were synthesized by sequential alkylation and nucleophilic aromatic substitution reactions of dichloropyrimidines (26–31) with (R)-sulfamidate 33b using n-BuLi in THF at −78 °C and a catalytic amount of copper(I) iodide followed by acidic hydrolysis of the intermediary sulfaminic acid and ring closure mediated by base treatment. (42) The same procedure was followed for the preparation of (R)-tricyclic intermediates (20b–25b), starting from (S)-sulfamidate 33a. A subsequent palladium catalyzed Suzuki coupling between choloropyrimidine intermediates (20a–25a and 20b–25b) and protected 2-aminopyridine-5-boronic acid pinacol ester (38, see Supporting Information) afforded compounds 2a, 6a–10a, and 2b, 6b–10b in moderate to high yields (49–88%; Scheme 1B). In addition, the chlorine of intermediate 22b was displaced by heteroaryl moieties using Suzuki cross-coupling reaction with boronic acid pinacol esters to give compounds 11b–17b (Scheme 1B). For not commercially available boronic acid pinacol esters or corresponding bromo derivatives, the synthesis is reported in Schemes S1 and S2 in Supporting Information. The regioisomer bearing the unrestricted morpholine in the 2-position of the pyrimidine core (enantiomers 2c and 2d) was synthesized starting from dichloropyrimidine 32 (Scheme 1C) following the procedure described for 2a and 2b. The synthesis of the conformationally restricted PQR309 (1) analogs 3a and 3b (Table 2) is depicted in Scheme S3A in Supporting Information, and the synthetic procedure is analogous to that described for 2a and 2b. The procedure for the preparation of the compounds bearing a piperidine instead of a morpholine (4a,b and 5a,b, Table 2) is reported in Scheme S3B and Scheme S3C in Supporting Information.

Determination of Cellular Potency and PI3K vs mTOR Kinase Activities

In the search for novel mTOR inhibitors, we selected the regioisomer bearing the unrestricted morpholine in the 4-position of the pyrimidine core (Scheme 1B) and built up a structure–activity relationship (SAR) study to investigate (i) the influence of the stereogenic center on mTOR selectivity and (ii) the effect of substitution on the unrestricted morpholine. We prepared a series of compounds introducing different morpholine derivatives (M_n substituents, Scheme 1B), such as 3-methylmorpholine [M₁, (S); M₂, (R)], 8-oxa-3-azabicyclo[3.2.1]octane (M₃), 3-oxa-8-azabicyclo[3.2.1]octane (M₄), and 3,3-dimethylmorpholine (M₅). Morpholines with increased steric demand had already been shown to increase selectivity for mTOR over PI3K. (31,43) For each conformationally restricted compound both (S)- (2a, 6a–10a) and (R)-enantiomers (2b, 6b–10b) were synthesized and tested in kinase binding and cellular assays. For all the regioisomer pairs, the (R)-enantiomer had higher affinity for mTOR than the (S)-configured compound (Table 3), confirming the results of compounds 2a and 2b. Also the cellular potency of the (R)-configured compounds was 2- to 7-fold higher than that of their corresponding enantiomers (see Table 3). The introduction of sterically hindered morpholines in the hinge region of (R)-configured tricyclic pyrimido-pyrrolo-oxazines (6b–10b) did not affect the potency for mTOR but led to a significant improvement in mTOR selectivity. With the exception of compound 10b, the selectivity of (R)-configured tricyclic compounds for mTOR versus PI3Kα was higher than 35-fold [K_i(p110α)/K_i(mTOR) 6b = 43; 7b = 165; 8b = 53; 9b = 36]. The (S)-3-methylmorpholine (M₁) only slightly increased the compound potency and selectivity for mTOR if compared with the morpholine (M₀) [K_i for mTOR 2b = 24 nM; 6b = 9.1 nM; selectivity K_i(p110α)/K_i(mTOR) for 2b was 18-fold and for 6b 43-fold]. On the contrary, the presence of a (R)-3-methylmorpholine (M₂) on the (R)-configured tricyclic pyrimido-pyrrolo-oxazine scaffold yielded the highest selectivity of the series [K_i(p110α)/K_i(mTOR) for 7b was 165-fold]. In cells, compound 7b displayed good activity (IC₅₀ for phosphorylated PKB/Akt = 133 nM and for phosphorylated S6 = 61 nM) and was therefore selected for further optimization.

Table 3. SAR Study on the Unrestricted Morpholine of Tricyclic Pyrimido-pyrrolo-oxazines^d

^{^a}

^{^b}

Compounds were tested for the in vitro binding to the ATP-binding site of p110α and mTOR using a commercially available time-resolved FRET (TR-FRET) displacement assay (LanthaScreen). Each experiment was performed with n = 2. IC₅₀ values, log IC₅₀ values, and standard errors are reported in Table S7 in the Supporting Information.

^{^c}

Marvin/JChem 16.10.17 was used for calculation of log P (partition coefficient) values.

^{^d}

Chemical structures of M₀–M₅ are depicted in Scheme 1.

To further improve the potency and selectivity for mTOR and to modulate the physicochemical properties of compound 7b, we investigated the role of the aryl moiety (Table 4). First, we replaced the pyridine ring with a pyrimidine (11b) and a pyrazine (12b) to assess the effect of the polarity of the heteroaromatic ring. Afterward, we introduced a methyl (13b and 14b), a methoxy (15b), or a dimethoxymethyl substituent (16b and 17b) on the 2-amino-substituted aryl group. The presence of a pyrimidine or a pyrazine ring slightly increased mTOR potency, selectivity, and cellular activity (see comparison between 11b/12b and 7b, Table 4). Compound 13b, bearing a methyl substituent in position 4 of the 2-aminopyridine ring, had a moderate activity toward mTOR (K_i = 85 nM), and was poorly active in cells (IC₅₀ for phosphorylated PKB/Akt and for phosphorylated S6 was >800 nM, Table 4). In contrast, compound 14b, with a 3-methyl-substituted aminopyridine, maintained a moderate activity in cells (IC₅₀ for phosphorylated PKB/Akt was 488 nM and 289 nM for phosphorylated S6). The introduction of a methoxy (15b) and a dimethoxymethyl substituent (16b) on the pyridine ring reduced cellular potency and binding to both PI3Kα and mTOR. Compound 17b, bearing a dimethoxymethyl substituent on the pyrimidine ring, is a dual PI3Kα/mTOR inhibitor (K_i for p110α = 66 nM; K_i for mTOR = 34 nM) but showed a limited potency in cells (IC₅₀(pPKB) > 500 nM and IC₅₀(pS6) > 300 nM, Table 4). With respect to compound 7b, the introduction of an additional nitrogen on the heteroaromatic ring decreased the clogP value (compound 11b = 2.22 and 12b = 2.15), leading to compounds with predicted optimal physicochemical and ADME properties for oral drugs. (44) Moreover, the polar surface area (PSA) of > 90 Å of compounds 11b and 12b is indicative that they might not cross the blood–brain barrier (BBB). Limited brain uptake of mTOR inhibitors, normally used to treat systemic tumors, is an advantage because inhibition of the mTOR pathway in the brain could lead to neurological side effects.

Table 4. SAR Study on the Aryl Moiety of Tricyclic Pyrimido-pyrrolo-oxazines^d

^{^a}

^{^b}

Compounds were tested for the in vitro binding to the ATP-binding site of p110α and mTOR using a commercially available time-resolved FRET (TR-FRET) displacement assay (LanthaScreen). Each experiment was performed with n = 2. IC₅₀ values, log IC₅₀ values, and standard errors are reported in Table S7 in the Supporting Information.

^{^c}

Marvin/JChem 16.10.17 was used for calculation of log P (partition coefficient) values.

^{^d}

Chemical structures of Ar₀–Ar₇ are depicted in Scheme 1.

Pharmacological Parameters

To assess the oral availability and to determine BBB permeability, pharmacokinetic (PK) studies were carried out for compounds 7b and 12b. Compounds 7b and 12b were administered orally (5 mg/kg po) in male Sprague Dawley rats, and compound concentrations in plasma and brain were determined after a single dose (Figure 4A and Figure 4B, respectively; see also Tables S1–S4 in Supporting Information). Both compounds showed plasma exposure sufficient to efficiently inhibit mTOR kinase, and brain levels remined minimal. After 8 h, the total exposure AUC_0–8 was 5730 ng·h/mL in plasma and 104 ng·h/g in brain for compound 12b.

Figure 4. Plasma and brain concentration of (A) compound 7b and (B) compound **12b** after po dosing at 5 mg/kg in male Sprague Dawley rats. Stability of compound **11b** (5 μM) with primary hepatocytes from (C) mice (green) and rats (turquoise) and (D) dogs (red) and humans (black) (n = 2). All values are the mean ± SEM. Error bars are not shown when smaller than the symbols.

To predict the metabolic stability of our restricted scaffold, in vitro assays were performed for compound 11b, using hepatocyte cultures of different origin (CD-1 mice, Sprague Dawley rats, beagle dogs, and humans). Compound 11b was only minimally metabolized when incubated with mouse, rat, dog, and human hepatocytes, as indicated by 81.2%, 93.6%, 93.0%, and 93.2% remaining compound after 3 h of incubation (Figure 3C, Figure 3D, and Table S5 in Supporting Information). As compound 11b was highly stable across species, neither half-lives nor intrinsic clearance could be determined under the experimental conditions.

CYP450 Reactive Phenotyping for Compounds 7b, 11b, and 12b

CYP450 reactive phenotyping was performed to evaluate the involvement of different human hepatic CYP isoenzymes (CYP1A1 and CYP1A2) in the metabolism of compounds 7b, 11b, and 12b. All three compounds turned out to be moderate CYP1A1 substrates, as indicated by <77% parental compound remaining after 60 min of incubation (Table 5). Compounds 7b and 11b were partially stable toward CYP1A1 (44% and 51% compound remaining, respectively), while the lowest turnover was observed with compound 12b (77% remaining). A low metabolization by CYP1A2 was observed with 11b (73% remaining). On the other hand, compounds 7b and 12b remained stable (Table 5), indicating that they are not substrates of CYP1A2.

Table 5. CYP1A1 and CYP1A2 Reactive Phenotyping of 7b, 11b, and 12b (1 μM)^a

	remaining test item with cofactors		% remaining in corresponding negative control without cofactors
test item	mean %	SD	mean	SD	mean (%)_corr^b
CYP1A1
7b	44	2.0	109	3.7	35
11b	51	0.3	93	2.9	58
12b	77	0.08	96	4.5	81
CYP1A2
7b	98	0.6	94	0.1	104
11b	73	0.4	91	2.4	83
12b	92	0.2	91	0.8	101

^{^a}

Compounds remaining after 60 min of incubation with Supersomes at 25 pmol/mL (n = 2), calculation based on absolute amounts (nM).

^{^b}

mean (%)_corr = (100 – % remaining negative control) + % remaining sample.

On the basis of these results, compound 11b was further investigated to identify metabolites formed in vitro by CYP1A1 (Figure 5A) and CYP1A2 (Figure 5B). Upon incubation of 11b with human recombinant CYP1A1, the highest peak areas were observed for metabolite M2 (9.3% of total peak areas, hydrolysis of the 3R-methylmorpholinyl residue and oxidation to corresponding aldehyde) and its secondary products M5 (7.5%, introduction of a C–C double bond (desaturation) at the tricyclic backbone of M2; Figure 5C). Other minor metabolites were observed, highlighting that the preferred site of oxidative metabolism of compound 11b was the 3R-methyl-substituted morpholinyl moiety (Table S6 and Figures S1–S5 in Supporting Information). In CYP1A2 incubates, M2 was the only metabolite (0.3%) suggesting that compound 11b is not a substrate of CYP1A2.

Figure 5. Pie chart showing the percentage of compound **11b** and its metabolites after 60 min of incubation with human recombinant (A) CYP1A1 and (B) CYP1A2. (C) Major metabolites observed upon incubation of **11b** with CYP1A1.

Enzymatic Profiling and Determination of Selectivity

According to our SAR studies, compounds 7b, 11b, and 12b were the most potent and selective mTOR inhibitors showing high potency in cellular assays and good pharmacokinetic profile. Therefore, 7b, 11b, and 12b were chosen for further characterization using the KINOMEScan platform of DiscoverX (Table 6). DiscoverX KdELECT assays confirmed the excellent mTOR selectivity of compound 12b over class I and class III PI3K and PI4K: 12b was ∼450-fold more potent for mTOR than for class I PI3K isoforms. The selectivity of 12b for mTOR over PI3K exceeds competitor compounds such as CC223 (55) (45) (∼80×), INK128 (56) (∼40×), and AZD2104 (57) (∼230×) (Table 6). Moreover, compound 12b showed negligible off-target effects when screened against a DiscoverX scanMAX kinase assay panel containing a set of 468 protein and lipid kinases (Supporting Information, Figure S6 and Table S10). At a concentration of 10 μM, compound 12b and PQR620 (58) reached outstanding selectivity scores [S(10) was 0.005 for both compounds; see Supporting Information, Table S9], while the same value for INK128 (56) reached 0.14 (corresponding to 55 hits, Table S9).

Table 6. mTOR and Lipid Kinase Binding Constants of 7b, 11b, 12b, and Reference Compounds

	inhibitor binding constant,^aK_d [nM]
compd	mTOR	PI3Kα	PI3Kβ	PI3Kδ	PI3Kγ	PI4Kβ	VPS34	most sensitive PI3K/mTOR,^c fold selectivity
7b	34	610	4100	6200	8700	>30000	2100	17.9
11b	14	120	1400	1900	1800	>30000	980	8.6
12b	3.5	1600	7500	12000	11000	>30000	3200	457
CC223 (55)^b	28	2300	18000	6200	7100	39	2500	>80×
INK128 (56)^b	0.092	15	81	30	3.7	nd	8200	>40×
AZD2014 (57)^b	0.14	33	3300	1500	8400	>30000	23000	>230×
PQR620 (58)	0.27	1000	22000	23000	18000	>30000	2750	>3700×

^{^a}

Dissociation constants (K_d) were determined using ScanMax technology (DiscoveRx) with 11-point 3-fold serial dilutions of the indicated compounds. K_d is the mean value from experiments performed in duplicate and was calculated from standard dose–response curves using the Hill equation. nd = not determined.

^{^b}

Dissociation constants (K_d) of CC223 (55), INK128 (56), AZD2014 (57), and PQR620 (58) are from ref (31).

^{^c}

Fold selectivity: ratio of K_d of the most sensitive class I PI3K isoform (displayed in bold type) over K_d for mTOR.

Conclusion

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In summary, we have exploited a conformational restriction strategy to enlarge the chemical space around PQR309 (1), a pan-PI3K and moderate mTOR inhibitor. Our rigidification strategy allowed us to enhance potency and selectivity for mTOR. Molecular modeling elucidated interactions of the tricyclic pyrimido-pyrrolo-oxazine scaffold in the ATP-binding pocket of PI3Ks and mTOR and explained the difference in potency between the regioisomers. An extensive SAR study led to the discovery of compound 12b, with a selectivity for mTOR higher than competitor compounds such as CC223 (55), INK128 (56), and AZD2014 (57). The selectivity of PQR620 (58) for mTOR over PI3Ks exceeds that of 12b; however 12b explores a novel chemical space and paves the way for additional chemical modifications. A lead optimization process of the tricyclic pyrimido-pyrrolo-oxazine scaffold could lead to further improvement of the mTOR potency and selectivity of 12b. Compound 12b efficiently inhibited mTOR signaling in cells and showed plasma drug exposure after oral dosing that is expected to fully inhibit mTOR in vivo. The minimal brain permeability of compounds 7b and 12b suggests the tricyclic pyrimido-pyrrolo-oxazine as a novel scaffold for the development of highly selective and potent ATP-competitive mTOR inhibitors to be exploited for the treatment of systemic tumors. PQR620 (58) and the tricyclic pyrimido-pyrrolo-oxazine derivatives span along the different therapeutic applications of mTOR inhibitors. PQR620 (58) showed an excellent brain penetration and could be exploited in the treatment of neurological disorders. On the contrary, the conformationally restricted compounds have a limited brain uptake and could find an optimal application in the treatment of systemic cancers, with the advantage of avoiding neurological side effects. Our novel scaffold was also characterized for CYP450 related metabolism to assess the substrate specificity toward CYP1A1 and CYP1A2. The tricyclic pyrimido-pyrrolo-oxazines 7b, 11b, and 12b were metabolically stable, with compound 12b showing the lowest turnover in CYP450 reactive phenotyping. On the basis of its remarkable mTOR potency/selectivity and favorable pharmacokinetic profile, compound 12b represents a promising starting point for the development of a novel mTOR candidate in oncology.

Experimental Section

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General Information

Reagents were purchased at the highest commercial quality from Acros, Sigma-Aldrich, or Fluorochem and used without further purification. Solvents were purchased from Acros Organics in AcroSeal bottles over molecular sieves. Cross-coupling reactions were carried out under nitrogen atmosphere in anhydrous solvents, and glassware was oven-dried prior to use. Thin layer chromatography (TLC) plates were purchased from Merck KGaA (Polygram SIL/UV254, 0.2 mm silica with fluorescence indicator), and UV light (254 nm) was used to visualize the compounds. Column chromatographic purifications were performed on Merck KGaA silica gel (pore size 60 Å, 230–400 mesh particle size). Alternatively, flash chromatography was performed with Isco CombiFlash Companion systems using prepacked silica gel columns (40–60 μm particle size RediSep). ¹H, ¹⁹F, and ¹³C NMR spectra were recorded on a Bruker Avance 400 spectrometer. NMR spectra were obtained in deuterated solvents, such as CDCl₃, (CD₃)₂SO, or CD₃OD. The chemical shifts (δ values) are reported in ppm and corrected to the signal of the deuterated solvents (7.26 ppm (¹H NMR) and 77.16 ppm (¹³C NMR) for CDCl₃; 2.50 ppm (¹H NMR) and 39.52 ppm (¹³C NMR) for (CD₃)₂SO; and 3.31 ppm (¹H NMR) and 49.00 ppm (¹³C NMR) for CD₃OD). ¹⁹F NMR spectra are calibrated relative to CFCl₃ (δ = 0 ppm) as external standard. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), td (triplet of doublets), q (quartet), m (multiplet), br (broadened). Coupling constants, when given, are reported in hertz (Hz). High resolution mass spectra (HRMS) were recorded on a Bruker maxis 4G, high resolution ESI-QTOF. All analyses were carried out in positive ion mode and in MeOH + 0.1% formic acid as solvent. Sodium formate was used as calibration standard. MALDI-ToF mass spectra were obtained on a Voyager-De Pro measured in m/z. The chromatographic purity of final compounds was determined by high performance liquid chromatography (HPLC) analyses on an Ultimate 3000SD system from ThermoFisher with LPG-3400SD pump system, ACC-3000 autosampler and column oven, and DAD-3000 diode array detector. An Acclaim-120 C18 reversed-phase column from ThermoFisher was used as stationary phase. Gradient elution (5:95 for 0.2 min, 5:95 → 100:0 over 10 min, 100:0 for 3 min) of the mobile phase consisting of CH₃CN/MeOH:H₂O_(10:90) was used at a flow rate of 0.5 mL/min at 40 °C. The purity of all final compounds was >95%. Optical rotations ([α]_D²³) were measured on a PerkinElmer polarimeter 341 (in a cuvette (l = 1 dm)) at 23 °C at 589 nm.

General Procedure 1

A flask was charged with the corresponding boronic acid pinacol ester (1 equiv), N,N-dimethylformamide–dimethyl acetal (DMF–DMA, 1.3–1.5 equiv), and THF (5 mL). The mixture was stirred at 70 °C overnight. After completion of the reaction monitored by NMR, the solvents were evaporated and the compound was dried under vacuum.

General Procedure 2

To a solution of the desired morpholine (1.05 equiv) in DCM (approximately 1 mL/0.6 mmol) at 0 °C, N,N-diisopropylethylamine (2.1 equiv) was added, followed by 2,4,6-trichloropyrimidine (1.0 equiv). The reaction mixture was allowed to warm up to rt and was stirred overnight. The reaction mixture was washed with aqueous saturated NaHSO₄ solution (2×). The aqueous layer was extracted with DCM, and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel.

Using this procedure, both regioisomers were generated and separated by column chromatography. Only the characterization of the desired regioisomer is reported.

General Procedure 3

Under nitrogen atmosphere, a 1.6 M n-BuLi solution (1.0 mL, 1.52 mmol, 1.2 equiv) in THF (approximately 1 mL/1.5 mmol) was cooled down to −78 °C and a solution of the corresponding morpholine (1 equiv) in THF (approximately 1 mL/0.4 mmol) was added dropwise. The mixture was stirred at −78 °C for 35 min. CuI (0.05 equiv) and a solution of the corresponding sulfamidate (1–1.2 equiv) in THF (approximately 1 mL/0.5 mmol) were added. The mixture was stirred at −78 °C for 15 min, then allowed to warm to rt and stirred overnight. The reaction was quenched by addition of water. Conc HCl and methanol were added, and the mixture was heated at 45 °C for 4–6 h. The reaction mixture was cooled down to 0 °C, and a 6 M NaOH solution was added until pH 10 was obtained. The reaction mixture was stirred at rt for 1–16 h, then transferred to a separatory funnel. The layers were separated, and the aqueous layer was extracted with EtOAc (2×). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel.

General Procedure 4

Chloropyrimidine derivative (1.0 equiv) and boronic acid pinacol ester (1.0 equiv) were charged in a flask. Under nitrogen atmosphere, 1,4-dioxane (approximately 1 mL/0.2 mmol) was added, followed by an aqueous K₃PO₄ solution (2 equiv) and chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (XPhos Pd G2, 0.05 equiv). The resulting mixture was stirred at 95 °C for 2–16 h. After completion of the reaction monitored by TLC, a 3 M aqueous HCl solution (10 equiv) was added and the mixture was stirred at 60 °C for 3–16 h. A 2 M aqueous NaOH solution was added until pH 9–10 was obtained. The aqueous layer was extracted with EtOAc (3×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel.

General Procedure 5

Step 1

The corresponding di-Boc protected bromo derivative 44 or 49 (1.0 equiv), bis(pinacolato)diboron (1.5 equiv), and KOAc (3.0 equiv) were charged into a flask. Under nitrogen atmosphere, 1,4-dioxane (approximately 1 mL/0.2 mmol) was added, followed by [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl₂, 0.1 equiv). The reaction mixture was stirred at 100 °C for 2 h.

Step 2

After completion of the reaction monitored by TLC, the chloropyrimidine derivative 22b (200 mg, 0.65 mmol, 1 equiv) and K₃PO₄ (3.0 equiv) in water (approximately 1 mL/0.3 mmol) and chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (XPhos Pd G2, 0.05 equiv) were added. The reaction mixture was stirred at 95 °C for 2–16 h. A 15% NH₄Cl solution was added, and the aqueous layer was extracted with EtOAc (3×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel.

General Procedure 6

To a cold solution (0 °C) of the corresponding Boc-protected compound (1 equiv) in DCM (approximately 1 mL/0.1 mmol), trifluoroacetic acid (TFA, 40 equiv) was added dropwise. The reaction mixture was allowed to warm up to room temperature and was stirred for 1–3 h. After completion of the reaction monitored by TLC, a saturated NaHCO₃ solution was slowly added. The aqueous layer was extracted with DCM (3×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel.

(S)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (2a)

2a was prepared according to general procedure 4 from (S)-2-chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 20a (131 mg, 0.44 mmol, 1 equiv) and (E)-N,N-dimethyl-N′-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (121 mg, 0.44 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 2a as a colorless solid (102 mg, 0.29 mmol, 65%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.83 (d, J = 2.3 Hz, 1 H), 8.18 (dd, J = 8.7, 2.3 Hz, 1 H), 6.45 (d, J = 8.6 Hz, 1 H), 6.27 (br s, 2 H), 4.04 (dd, J = 13.5, 2.6 Hz, 1 H), 3.87 (ddt, J = 10.5, 8.8, 4.3 Hz, 1 H), 3.79–3.71 (m, 3 H), 3.68–3.54 (m, 7 H), 3.35–3.28 (m, 1 H), 3.22–3.10 (m, 3 H), 2.63 (dd, J = 15.3, 4.7 Hz, 1 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.71 (s, 1 C), 161.20 (s, 1 C), 160.90 (s, 1 C), 158.28 (s, 1 C), 148.91 (s, 1 C), 136.73 (s, 1 C), 122.60 (s, 1 C), 107.34 (s, 1 C), 93.55 (s, 1 C), 70.07 (s, 1 C), 66.64 (s, 2 C), 66.17 (s, 1 C), 56.91 (s, 1 C), 45.90 (s, 2 C), 42.07 (s, 1 C), 29.14 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₁₈H₂₃N₆O₂ 355.1877; found, 355.1880. HPLC (ACN with 0.1% TFA): t_R = 4.98 min (98.7% purity).

(R)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (2b)

2b was prepared as described for its (S)-enantiomer 2a starting from (R)-2-chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 20b (150 mg, 0.51 mmol, 1 equiv) and boronic acid pinacol ester 38 in a 76% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₁₈H₂₃N₆O₂ 355.1877; found, 355.1883. HPLC (ACN with 0.1% TFA): t_R = 4.95 min (97.4% purity).

(S)-5-(2-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-4-yl)pyridin-2-amine (2c)

2c was prepared according to general procedure 4 from (S)-4-chloro-2-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 20c (80 mg, 0.27 mmol, 1 equiv) and (E)-N,N-dimethyl-N′-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (81.5 mg, 0.30 mmol, 1.1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 2c as a colorless solid (45 mg, 0.13 mmol, 47%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.46 (d, J = 2.4 Hz, 1 H), 7.93 (dd, J = 8.7, 2.4 Hz, 1 H), 6.50 (d, J = 8.7 Hz, 1 H), 6.31 (br s, 2 H), 3.96–3.89 (m, 2 H), 3.81–3.75 (m, 2 H), 3.69–3.61 (m, 8 H), 3.34–3.27 (m, 1 H), 3.24–3.11 (m, 3 H), 2.62 (dd, J = 15.9, 4.5 Hz, 1 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 166.88 (s, 1 C), 161.13 (s, 1 C), 160.00 (s, 1 C), 152.84 (s, 1 C), 147.95 (s, 1 C), 136.12 (s, 1 C), 122.01 (s, 1 C), 107.22 (s, 1 C), 102.42 (s, 1 C), 70.07 (s, 1 C), 66.11 (s, 2 C), 65.54 (s, 1 C), 56.92 (s, 1 C), 44.23 (s, 2 C), 41.31 (s, 1 C), 27.75 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₁₈H₂₃N₆O₂ 355.1877; found, 355.1881. HPLC (ACN with 0.1% TFA): t_R = 3.57 min (99.8% purity).

(R)-5-(2-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-4-yl)pyridin-2-amine (2d)

2d was prepared as described for its (S)-enantiomer 2c starting from (R)-4-chloro-2-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 20d (100 mg, 0.34 mmol, 1 equiv) and boronic acid pinacol ester 38 in a 57% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₁₈H₂₃N₆O₂ 355.1877; found, 355.1880. HPLC (ACN with 0.1% TFA): t_R = 3.61 min (99.2% purity).

(S)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (3a)

3a was prepared according to general procedure 4 from (S)-2-chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 20a (544 mg, 1.83 mmol, 1.0 equiv) and boronic acid pinacol ester 50 (818 mg, 2.38 mmol, 1.3 equiv). Purification by column chromatography on silica gel (CH₂Cl₂/methanol 1:0 → 20:1) gave compound 3a as a colorless solid (503 mg, 1.19 mmol, 65%). ¹H NMR (400 MHz, CDCl₃): δ 8.62 (s, 1 H), 6.77 (s, 1 H), 4.75 (br s, 2 H), 4.10 (dd, J = 14.0, 2.5 Hz, 1 H), 4.01–3.93 (m, 1 H), 3.86–3.70 (m, 6 H), 3.70–3.58 (m, 4 H), 3.47 (td, J = 12.0, 2.8 Hz, 1 H), 3.35 (t, J = 11 Hz, 1 H), 3.27–3.17 (m, 2 H), 2.62 (dd, J = 15.0, 4.8 Hz, 1 H). ¹⁹F{¹H} NMR (376 MHz, CDCl₃): δ −60.2 (s, 3 F). ¹³C{¹H} NMR (101 MHz, CDCl₃): δ 167.70 (s, 1 C), 161.65 (s, 1 C), 158.91 (s, 1 C), 158.33 (s, 1 C), 152.29 (s, 1 C), 138.0 (q, J = 32 Hz, 1 C), 123.91–123.81 (m, 1 C), 123.1 (q, J = 274 Hz, 1 C), 105.2 (q, J = 5.6 Hz, 1 C), 93.59 (s, 1 C), 70.44 (s, 1 C), 66.96 (s, 2 C), 66.59 (s, 1 C), 56.97 (s, 1 C), 45.94 (s, 2 C), 42.05 (s, 1 C), 29.66 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₁₉H₂₂F₃N₆O₂ 423.1751; found, 423.1743. HPLC: t_R = 6.74 min (97.5% purity). (S)-enantiomer: [α]: [α]_D²⁰ = −13.2 (c = 2.0, CHCl₃).

(R)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (3b)

3b was prepared as described for its (S)-enantiomer 50a starting from (R)-2-chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 20b (552 mg, 1.86 mmol, 1.0 equiv) and boronic acid pinacol ester 50 (83 mg, 2.42 mmol, 1.3 equiv) in a 62% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₁₉H₂₂F₃N₆O₂ 423.1751; found, 423.1744. HPLC: t_R = 6.73 min (98.9% purity). (R)-enantiomer: [α]_D²⁰ = +13.5 (c = 1.6, CHCl₃).

(S)-5-(4-Morpholino-5,5a,6,7,8,9-hexahydropyrimido[5,4-b]indolizin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (4a)

4a was prepared according to general procedure 4 from intermediate 51a (150 mg, 0.51 mmol, 1.0 equiv) and boronic acid pinacol ester 50 (175 mg, 0.51 mmol, 1.0 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 3:2) gave compound 4a as a yellowish solid (90 mg, 0.21 mmol, 42%). ¹H NMR (400 MHz, DMSO): δ 8.42 (s, 1 H), 6.76 (s, 1 H), 6.67 (br s, 2 H), 4.05 (dd, J = 13, 4.2 Hz, 1 H), 3.67–3.49 (m, 9 H), 3.25 (dd, J = 15.0, 9.1 Hz, 1 H), 2.78–2.64 (m, 2 H), 1.83–1.67 (m, 2 H), 1.63–1.55 (m, 1 H), 1.51–1.37 (m, 1 H), 1.37–1.20 (m, 2 H). ¹⁹F{¹H} NMR (376 MHz, DMSO): δ −59.1 (s, 3 F). ¹³C{¹H} NMR (101 MHz, DMSO): δ 167.2 (s, 1 C), 160.7 (s, 1 C), 160.2 (s, 1 C), 157.5 (s, 1 C), 151.8 (s, 1 C), 135.7 (q, J = 31 Hz, 1 C), 123.3 (q, J = 274 Hz, 1 C), 121.2–121.1 (m, 1 C), 104.1 (q, J = 5.9 Hz, 1 C), 99.3 (s, 1 C), 66.1 (s, 2 C), 58.9 (s, 1 C), 45.5 (s, 2 C), 41.3 (s, 1 C), 33.6 (s, 1 C), 31.5 (s, 1 C), 24.4 (s, 1 C), 23.6 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₄F₃N₆O 421.1958; found, 421.1962. HPLC: t_R = 8.44 min (97.8% purity).

(R)-5-(4-Morpholino-5,5a,6,7,8,9-hexahydropyrimido[5,4-b]indolizin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (4b)

4b was prepared as described for its (S)-enantiomer 4a starting from 51b (150 mg, 0.51 mmol, 1.0 equiv) and boronic acid pinacol ester 50 (175 mg, 0.51 mmol, 1.0 equiv) in a 47% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₄F₃N₆O 421.1958; found, 421.1966. HPLC: t_R = 8.45 min (98.7% purity).

(S)-5-(4-(Piperidin-1-yl)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (5a)

5a was prepared as described for its (R)-enantiomer 5b starting from 52a (100 mg, 0.34 mmol, 1.0 equiv) and boronic acid pinacol ester 50 (128 mg, 0.37 mmol, 1.1 equiv) in a 75% yield. The spectroscopic data agree with those reported for the (R)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₄F₃N₆O 421.1958; found, 421.1966. HPLC: t_R = 8.55 min (98.3% purity).

(R)-5-(4-(Piperidin-1-yl)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (5b)

5b was prepared according to general procedure 4 from intermediate 52b (100 mg, 0.34 mmol, 1.0 equiv) and boronic acid pinacol ester 50 (128 mg, 0.38 mmol, 1.1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 3:2) gave compound 5b as a colorless solid (114 mg, 0.27 mmol, 80%). ¹H NMR (400 MHz, DMSO): δ 8.43 (s, 1 H), 6.76 (s, 1 H), 6.69 (br s, 2 H), 3.95–3.80 (m, 2 H), 3.77–3.67 (m, 2 H), 3.63–3.50 (m, 4 H), 3.29 (td, J = 12.0, 2.4 Hz, 1 H), 3.23–3.14 (m, 2 H), 3.08 (td, J = 13.0, 3.6 Hz, 1 H), 2.61 (dd, J = 15.0, 4.6 Hz, 1 H), 1.64–1.57 (m,. Two H), 1.53–1.44 (m, 4 H). ¹⁹F{¹H} NMR (376 MHz, DMSO): δ −58.9 (s, 3 F). ¹³C{¹H} NMR (101 MHz, DMSO): δ 167.10 (s, 1 C), 160.88 (s, 1 C), 160.18 (s, 1 C), 157.47 (s, 1 C), 151.81 (s, 1 C), 135.70 (q, J = 31 Hz, 1 C), 123.30 (q, J = 274 Hz, 1 C), 121.16 (s, 1 C), 104.12 (q, J = 5.7 Hz, 1 C), 92.47 (s, 1 C), 69.51 (s, 1 C), 65.62 (s, 1 C), 56.31 (s, 1 C), 45.88 (s, 2 C), 41.61 (s, 1 C), 29.11 (s, 1 C), 25.57 (s, 2 C), 24.43 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₄F₃N₆O 421.1958; found, 421.1963. HPLC: t_R = 8.55 min (98.9% purity).

5-((S)-4-((S)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (6a)

6a was prepared according to general procedure 4 from (S)-2-chloro-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 21a (84 mg, 0.27 mmol, 1 equiv) and (E)-N,N-dimethyl-N′-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (275 mg, 0.27 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 6a as a colorless solid (76 mg, 0.21 mmol, 76%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.82 (d, J = 2.2 Hz, 1 H), 8.17 (dd, J = 8.7, 2.3 Hz, 1 H), 6.45 (dd, J = 8.7, 0.8 Hz, 1 H), 6.28 (br s, 2 H), 4.36–4.30 (m, 1 H), 4.09–4.02 (m, 2 H), 3.93–3.84 (m, 2 H), 3.79–3.63 (m, 4 H), 3.48 (td, J = 11.7, 2.9 Hz, 1 H), 3.35–3.29 (m, 1 H), 3.26–3.10 (m, 4 H), 2.65 (dd, J = 15.3, 4.7 Hz, 1 H), 1.18 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.63 (s, 1 C), 161.19 (s, 1 C), 160.88 (s, 1 C), 157.80 (s, 1 C), 148.91 (s, 1 C), 136.70 (s, 1 C), 122.69 (s, 1 C), 107.33 (s, 1 C), 93.14 (s, 1 C), 70.86 (s, 1 C), 70.10 (s, 1 C), 66.98 (s, 1 C), 66.17 (s, 1 C), 56.77 (s, 1 C), 47.58 (s, 1 C), 42.02 (s, 1 C), 40.11 (s, 1 C), 29.18 (s, 1 C), 14.23 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₁₉H₂₅N₆O₂ 369.2034; found, 369.2033. HPLC (ACN with 0.1% TFA): t_R = 5.39 min (99.2% purity).

5-((R)-4-((S)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (6b)

6b was prepared as described for its (S,R)-enantiomer 7a starting from (R)-2-chloro-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 21b (120 mg, 0.39 mmol, 1 equiv) and boronic acid pinacol ester 38 in a 64% yield. The spectroscopic data are in agreement with those reported for the (S,R)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₁₉H₂₅N₆O₂ 369.2034; found, 369.2033. HPLC (ACN with 0.1% TFA): t_R = 5.37 min (98.9% purity).

5-((S)-4-((R)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (7a)

7a was prepared according to general procedure 4 from (S)-2-chloro-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 22a (84 mg, 0.27 mmol, 1 equiv) and (E)-N,N-dimethyl-N′-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (75 mg, 0.27 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 7a as a colorless solid (72 mg, 0.20 mmol, 74%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.82 (d, J = 2.4 Hz, 1 H), 8.17 (dd, J = 8.7, 2.3 Hz, 1 H), 6.45 (d, J = 8.7 Hz, 1 H), 6.27 (br s, 2 H), 4.35–4.30 (m, 1 H), 4.13–4.02 (m, 2 H), 3.92–3.81 (m, 2 H), 3.79–3.72 (m, 2 H), 3.69–3.61 (m, 2 H), 3.48 (td, J = 11.8, 2.9 Hz, 1 H), 3.36–3.29 (m, 1 H), 3.25–3.09 (m, 4 H), 2.57 (dd, J = 15.3, 4.7 Hz, 1 H), 1.21 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.25 (s, 1 C), 160.73 (s, 1 C), 160.40 (s, 1 C), 157.20 (s, 1 C), 148.43 (s, 1 C), 136.23 (s, 1 C), 122.22 (s, 1 C), 106.87 (s, 1 C), 92.50 (s, 1 C), 70.41 (s, 1 C), 69.53 (s, 1 C), 66.53 (s, 1 C), 65.68 (s, 1 C), 56.38 (s, 1 C), 47.12 (s, 1 C), 41.64 (s, 1 C), 28.84 (s, 1 C), 13.88 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₁₉H₂₅N₆O₂ 369.2034; found, 369.2034. HPLC (ACN with 0.1% TFA): t_R = 5.42 min (99.3% purity).

5-((R)-4-((R)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (7b)

7b was prepared as described for its (S,S)-enantiomer 6a starting from (R)-2-chloro-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 22b (150 mg, 0.48 mmol, 1 equiv) and boronic acid pinacol ester 38 in a 49% yield. The spectroscopic data are in agreement with those reported for the (S,S)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₁₉H₂₅N₆O₂ 369.2034; found, 369.2035. HPLC (ACN with 0.1% TFA): t_R = 5.35 min (99.6% purity).

5-[(9S)-6-{8-Oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (8a)

8a was prepared according to general procedure 4 from (9S)-4-chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene 23a (150 mg, 0.46 mmol, 1 equiv) and (E)-N,N-dimethyl-N′-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (128 mg, 0.46 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 8a as a colorless solid (118 mg, 0.31 mmol, 67%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.81 (dd, J = 2.3, 0.8 Hz, 1 H), 8.15 (dd, J = 8.6, 2.3 Hz, 1 H), 6.44 (dd, J = 8.7, 0.8 Hz, 1 H), 6.26 (br s, 2 H), 4.38–4.34 (m, 2 H), 4.09–3.95 (m, 3 H), 3.87–3.80 (m, 1 H), 3.77–3.69 (m, 2 H), 3.34–3.27 (m, 1 H), 3.22–3.05 (m, 5 H), 2.63 (dd, J = 15.3, 4.8 Hz, 1 H), 1.84–1.74 (m, 4 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.09 (s, 1 C), 160.72 (s, 1 C), 160.25 (s, 1 C), 158.61 (s, 1 C), 148.43 (s, 1 C), 136.26 (s, 1 C), 122.22 (s, 1 C), 106.86 (s, 1 C), 92.56 (s, 1 C), 73.16 (s, 1 C), 73.15 (s, 1 C), 69.60 (s, 1 C), 65.70 (s, 1 C), 56.38 (s, 1 C), 50.70 (s, 1 C), 50.48 (s, 1 C), 41.59 (s, 1 C), 28.84 (s, 1 C), 27.56 (s, 1 C), 27.52 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₅N₆O₂ 381.2034; found, 381.2033. HPLC (ACN with 0.1% TFA): t_R = 5.34 min (98.2% purity).

5-[(9R)-6-{8-Oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (8b)

8b was prepared as described for its (S)-enantiomer 8a starting from (9R)-4-chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene 23b (200 mg, 0.62 mmol, 1 equiv) and boronic acid pinacol ester 38 in a 86% yield. The spectroscopic data agree with those reported for the (S)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₅N₆O₂ 381.2034; found, 381.2036. HPLC (ACN with 0.1% TFA): t_R = 5.17 min (96.4% purity).

5-[(9S)-6-{3-Oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (9a)

9a was prepared according to general procedure 4 from (9S)-4-chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene 24a (150 mg, 0.46 mmol, 1 equiv) and (E)-N,N-dimethyl-N′-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (128 mg, 0.46 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 9a as a colorless solid (122 mg, 0.32 mmol, 69%). ¹H NMR (400 MHz, CDCl₃): δ 9.04 (d, J = 2.2 Hz, 1 H), 8.35 (dd, J = 8.6, 2.2 Hz, 1 H), 6.50 (d, J = 8.6 Hz, 1 H), 4.61–4.51 (m, 4 H), 4.20 (dd, J = 13.4, 2.8 Hz, 1 H), 4.01–3.93 (m, 1 H), 3.88–3.77 (m, 4 H), 3.58 (d, J = 10.7 Hz, 2 H), 3.50 (td, J = 11.7, 3.0 Hz, 1 H), 3.36–3.23 (m, 2 H), 3.09 (dd, J = 14.9, 9.5 Hz, 1 H), 2.48 (dd, J = 14.9, 4.9 Hz, 1 H), 2.12–1.97 (m, 4 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 166.92 (s, 1 C), 160.96 (s, 1 C), 160.72 (s, 1 C), 155.76 (s, 1 C), 148.43 (s, 1 C), 136.26 (s, 1 C), 122.23 (s, 1 C), 106.86 (s, 1 C), 93.44 (s, 1 C), 70.42 (s, 1 C), 70.38 (s, 1 C), 69.71 (s, 1 C), 65.74 (s, 1 C), 56.46 (s, 1 C), 55.11 (s, 1 C), 55.04 (s, 1 C), 41.51 (s, 1 C), 28.03 (s, 1 C), 26.52 (s, 1 C), 26.45 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₅N₆O₂ 381.2034; found, 381.2036. HPLC (ACN with 0.1% TFA): t_R = 5.39 min (97.2% purity).

5-[(9R)-6-{3-Oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (9b)

9b was prepared as described for its (S)-enantiomer 9a starting from (9S)-4-chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene 24b (200 mg, 0.62 mmol, 1 equiv) and boronic acid pinacol ester 38 in a 88% yield. The spectroscopic data agree with those reported for the (S)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₅N₆O₂ 381.2034; found, 381.2040. HPLC (ACN with 0.1% TFA): t_R = 5.35 min (96.7% purity).

(S)-5-(4-(3,3-Dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (10a)

10a was prepared according to general procedure 4 from (S)-2-chloro-4-(3,3-dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 25a (150 mg, 0.46 mmol, 1 equiv) and (E)-N,N-dimethyl-N′-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide 38 (127 mg, 0.46 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 3:7) gave compound 10a as a colorless solid (124 mg, 0.32 mmol, 70%). ¹H NMR (400 MHz, CDCl₃): δ 8.83 (d, J = 2.2 Hz, 1 H), 8.16 (dd, J = 8.7, 2.3 Hz, 1 H), 6.47 (d, J = 8.7 Hz, 1 H), 6.29 (br s, 2 H), 4.01 (dd, J = 13.4, 2.6 Hz, 1 H), 3.90–3.83 (m, 1 H), 3.79–3.65 (m, 4 H), 3.36–3.29 (m, 3 H), 3.20–3.04 (m, 4 H), 2.97 (dd, J = 16.0, 9.1 Hz, 1 H), 2.40 (dd, J = 15.9, 5.0 Hz, 1 H), 1.47 (s, 3 H), 1.43 (s, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.29 (s, 1 C), 160.72 (s, 1 C), 160.67 (s, 1 C), 159.98 (s, 1 C), 148.38 (s, 1 C), 136.03 (s, 1 C), 122.33 (s, 1 C), 107.05 (s, 1 C), 101.51 (s, 1 C), 77.82 (s, 1 C), 69.73 (s, 1 C), 67.12 (s, 1 C), 65.57 (s, 1 C), 56.91 (s, 1 C), 53.80 (s, 1 C), 43.68 (s, 1 C), 41.57 (s, 1 C), 27.77 (s, 1 C), 21.74 (s, 1 C), 20.72 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₇N₆O₂ 383.2190; found, 383.2192. HPLC (ACN with 0.1% TFA): t_R = 5.58 min (98.1% purity).

(R)-5-(4-(3,3-Dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (10b)

10b was prepared as described for its (S)-enantiomer 10a starting from (S)-2-chloro-4-(3,3-dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 25b (150 mg, 0.46 mmol, 1 equiv) and boronic acid pinacol ester 38 in a 88% yield. The spectroscopic data agree with those reported for the (S)-enantiomer. HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₇N₆O₂ 383.2190; found, 383.2193. HPLC (ACN with 0.1% TFA): t_R = 5.61 min (99.5% purity).

5-((R)-4-((R)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyrimidin-2-amine (11b)

Chloropyrimidine derivative 22b (100 mg, 0.32 mmol, 1.0 equiv) and 2-aminopyridine-5-boronic acid pinacol ester (71.2 mg, 0.32 mmol, 1.0 equiv) were charged in a flask. Under nitrogen atmosphere, 1,4-dioxane (2 mL) was added, followed by K₃PO₄ (137 mg, 0.65 mmol, 2 equiv) aqueous solution (1 mL) and chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (XPhos Pd G2, 12.7 mg, 0.016 mmol, 0.05 equiv). The resulting mixture was stirred at 95 °C overnight. After completion of the reaction monitored by TLC, a 15% NH₄Cl solution (15 mL) was added and the aqueous layer was extracted with EtOAc (3×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 11b as a colorless solid (100 mg, 0.27 mmol, 84%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.98 (s, 2 H), 7.00 (br s, 2 H), 4.35–4.29 (m, 1 H), 4.07–4.02 (m, 2 H), 3.92–3.85 (m, 2 H), 3.79–3.61 (m, 4 H), 3.46 (td, J = 11.7, 2.9 Hz, 1 H), 3.35–3.29 (m, 1 H), 3.26–3.09 (m, 4 H), 2.66 (dd, J = 15.4, 4.8 Hz, 1 H), 1.17 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.01 (s, 1 C), 164.01 (s, 1 C), 158.80 (s, 1 C), 157.70 (s, 2 C), 157.24 (s, 1 C), 120.30 (s, 1 C), 93.09 (s, 1 C), 70.37 (s, 1 C), 69.66 (s, 1 C), 66.50 (s, 1 C), 65.73 (s, 1 C), 56.28 (s, 1 C), 47.12 (s, 1 C), 41.54 (s, 1 C), 39.63 (s, 1 C), 28.71 (s, 1 C), 13.82 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₁₈H₂₄N₇O₂ 370.1986; found, 370.1991. HPLC: t_R = 6.09 min (98.7% purity).

5-((R)-4-((R)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyrazin-2-amine (12b)

Chloropyrimidine derivative 22b (200 mg, 0.65 mmol, 1.0 equiv), 39 (428 mg, 1.94 mmol, 3.0 equiv), and Cs₂CO₃ (421 mg, 1.29 mmol, 2.0 equiv) were charged in a flask. Under nitrogen atmosphere, THF (2 mL) was added, followed by [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl₂, 47.2 mg, 0.065 mmol, 0.1 equiv). The resulting mixture was stirred at reflux for 6 h. After completion of the reaction monitored by TLC, a 15% NH₄Cl solution (15 mL) was added and the aqueous layer was extracted with EtOAc (3×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 12b as a colorless solid (163 mg, 0.44 mmol, 68%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.78 (s, 1 H), 7.91 (s, 1 H), 6.73 (br s, 2 H), 4.36–4.30 (m, 1 H), 4.09–4.00 (m, 2 H), 3.92–3.85 (m, 2 H), 3.78–3.61 (m, 4 H), 3.46 (td, J = 11.7, 2.9 Hz, 1 H), 3.29–3.09 (m, 5 H), 2.67 (dd, J = 15.4, 4.6 Hz, 1 H), 1.17 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.44 (s, 1 C), 160.37 (s, 1 C), 157.39 (s, 1 C), 155.84 (s, 1 C), 142.83 (s, 1 C), 138.67 (s, 1 C), 130.87 (s, 1 C), 93.51 (s, 1 C), 70.37 (s, 1 C), 69.64 (s, 1 C), 66.52 (s, 1 C), 65.73 (s, 1 C), 56.27 (s, 1 C), 47.11 (s, 1 C), 41.60 (s, 1 C), 39.65 (s, 1 C), 28.71 (s, 1 C), 13.84 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₁₈H₂₄N₇O₂ 370.1986; found, 370.1986. HPLC: t_R = 5.53 min (98.1% purity).

4-Methyl-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (13b)

Step 1. (E)-N′-(5-Bromo-4-methylpyridin-2-yl)-N,N-dimethylformimidamide 40 (281 mg, 1.16 mmol, 3.0 equiv), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos, 55.4 mg, 0.12 mmol, 0.3 equiv), KOAc (342 mg, 3.48 mmol, 9.0 equiv), and bis(pinacolato)diboron (312 mg, 3.48 mmol, 9.0 equiv) were charged into a flask. Under nitrogen atmosphere, EtOH (11.6 mL) was added and the reaction mixture was heated at 80 °C. Chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (XPhos Pd G2, 45.7 mg, 0.058 mmol, 0.15 equiv) was added, and the reaction mixture was stirred at 80 °C for 2 h.

Step 2. Then the mixture was allowed to cool down to room temperature. The chloropyrimidine derivative 22b (120 mg, 0.39 mmol, 1.0 equiv), 1,4-dioxane (2 mL), and a 1.8 M aqueous K₂CO₃ solution (650 μL, 1,16 mmol, 3.0 equiv) were added. The reaction mixture was stirred at 95 °C for 3 h.

Step 3. After completion of the reaction, the mixture was allowed to cool down to room temperature, a 3 M HCl solution (1.3 mL, 3.87 mmol, 10.0 equiv) was added, and the reaction mixture was stirred at 80 °C overnight. A 2 M aqueous NaOH solution (15 mL) was added until pH 9–10 was obtained. The aqueous layer was extracted with EtOAc (3×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 13b as a colorless solid (46 mg, 0.12 mmol, 31%). ¹H NMR (400 MHz, CDCl₃): δ 8.64 (s, 1 H), 6.35 (s, 1 H), 4.57 (br s, 2 H), 4.34–4.27 (m, 1 H), 4.13 (dd, J = 13.4, 2.4 Hz, 1 H), 4.04–3.93 (m, 3 H), 3.85–3.71 (m, 4 H), 3.59 (td, J = 11.8, 2.9 Hz, 1 H), 3.47 (td, J = 11.7, 2.9 Hz, 1 H), 3.39–3.30 (m, 2 H), 3.28–3.17 (m, 2 H), 2.62 (dd, J = 14.9, 4.8 Hz, 1 H), 2.54 (s, 3 H), 1.28 (d, J = 6.8 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, CDCl₃): δ 167.57 (s, 1 C), 163.28 (s, 1 C), 158.42 (s, 1 C), 158.08 (s, 1 C), 150.47 (s, 1 C), 148.84 (s, 1 C), 126.18 (s, 1 C), 109.96 (s, 1 C), 92.79 (s, 1 C), 71.23 (s, 1 C), 70.57 (s, 1 C), 67.42 (s, 1 C), 66.68 (s, 1 C), 56.89 (s, 1 C), 48.07 (s, 1 C), 42.08 (s, 1 C), 40.43 (s, 1 C), 29.78 (s, 1 C), 22.04 (s, 1 C), 14.26 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₁₈H₂₃N₆O₂ 383.2190; found, 383.2191. HPLC (ACN with 0.1% TFA): t_R = 5.18 min (96.7% purity).

3-Methyl-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (14b)

14b was prepared according to general procedure 4 from (R)-2-chloro-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine 22b (120 mg, 0.39 mmol, 1 equiv) and (E)-N,N-dimethyl-N′-(3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide 41 (112 mg, 0.39 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 14b as a colorless solid (98 mg, 0.26 mmol, 66%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.70 (d, J = 2.1 Hz, 1 H), 8.01 (s, 1 H), 6.05 (br s, 2 H), 4.34–4.28 (m, 1 H), 4.09–4.02 (m, 2 H), 3.92–3.83 (m, 2 H), 3.78–3.62 (m, 4 H), 3.47 (td, J = 11.8, 2.9 Hz, 1 H), 3.30–3.28 (m, 1 H), 3.26–3.09 (m, 4 H), 2.64 (dd, J = 15.3, 4.7 Hz, 1 H), 2.08 (s, 3 H), 1.17 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.15 (s, 1 C), 160.53 (s, 1 C), 159.35 (s, 1 C), 157.36 (s, 1 C), 145.97 (s, 1 C), 135.92 (s, 1 C), 122.83 (s, 1 C), 114.54 (s, 1 C), 92.65 (s, 1 C), 70.41 (s, 1 C), 69.67 (s, 1 C), 66.54 (s, 1 C), 65.74 (s, 1 C), 56.29 (s, 1 C), 47.14 (s, 1 C), 41.57 (s, 1 C), 39.63 (s, 1 C), 28.74 (s, 1 C), 17.20 (s, 1 C), 13.77 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₁₈H₂₃N₆O₂ 383.2190; found, 383.2191. HPLC (ACN with 0.1% TFA): t_R = 5.67 min (99.2% purity).

4-Methoxy-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (15b)

Step 1. (tert-Butyl (5-bromo-4-methoxypyridin-2-yl)carbamate 42 (200 mg, 0.66 mmol, 1.0 equiv), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos, 31.5 mg, 0.066 mmol, 0.1 equiv), KOAc (194 mg, 1.98 mmol, 3.0 equiv), and bis(pinacolato)diboron (178 mg, 1.98 mmol, 3.0 equiv) were charged into a flask. Under nitrogen atmosphere, EtOH (6.6 mL) was added and the reaction mixture was heated at 60 °C. Chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (XPhos Pd G2, 26 mg, 0.033 mmol, 0.05 equiv) was added, and the reaction mixture was stirred at 80 °C for 3 h.

Step 2. Then the mixture was allowed to cool down to room temperature. The chloropyrimidine derivative 22b (102 mg, 0.33 mmol, 0.5 equiv) and K₃PO₄ (210 mg, 0.99 mmol, 1.5 equiv) in water (1.7 mL) were added. The reaction mixture was stirred at 80 °C for 3.5 h.

Step 3. After completion of the reaction monitored by TLC, a 15% NH₄Cl solution (10 mL) was added and the aqueous layer was extracted with EtOAc (3×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The crude was dissolved in 1,4-dioxane (1.6 mL), a 3 M HCl solution (0.7 mL, 1.98 mmol, 3.0 equiv) was added, and the reaction mixture was stirred at 80 °C for 3 h. A 2 M aqueous NaOH solution was added until pH 9–10 was obtained. The aqueous layer was extracted with EtOAc (3×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 15b as a colorless solid (58 mg, 0.15 mmol, 44%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.18 (s, 1 H), 6.04 (br s, 2 H), 6.02 (s, 1 H), 4.32–4.26 (m, 1 H), 4.00–3.90 (m, 2 H), 3.89–3.81 (m, 1 H), 3.75–3.69 (m, 2 H), 3.71 (s, 3 H), 3.67–3.58 (m, 2 H), 3.44 (td, J = 11.7, 2.9 Hz, 1 H), 3.30–3.25 (m, 2 H), 3.21–3.05 (m, 4 H), 2.64 (dd, J = 15.3, 4.7 Hz, 1 H), 1.16 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.05 (s, 1 C), 164.76 (s, 1 C), 161.73 (s, 1 C), 160.96 (s,1 C), 157.24 (s, 1 C), 150.54 (s, 1 C), 115.28 (s, 1 C), 92.45 (s, 1 C), 89.20 (s, 1 C), 70.45 (s, 1 C), 69.60 (s, 1 C), 66.51 (s, 1 C), 65.66 (s, 1 C), 56.24 (s, 1 C), 54.84 (s, 1 C), 47.03 (s, 1 C), 41.59 (s, 1 C), 39.70 (s, 1 C), 28.69 (s, 1 C), 13.76 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₂₀H₂₇N₆O₃ 399.2139; found, 399.2145. HPLC (ACN with 0.1% TFA): t_R = 4.28 min (95.3% purity).

4-(Dimethoxymethyl)-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (16b)

16b was prepared according to general procedure 6 from 18b (169 mg, 0.26 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 16b as a colorless solid (88 mg, 0.20 mmol, 76%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.41 (s, 1 H), 6.66 (s, 1 H), 6.45 (s, 1 H), 6.24 (br s, 2 H), 4.33–4.26 (m, 1 H), 3.99–3.94 (m, 2 H), 3.92–3.84 (m, 2 H), 3.79–3.72 (m, 2 H), 3.69–3.61 (m, 2 H), 3.46 (td, J = 11.7, 2.8 Hz, 1 H), 3.35–3.26 (m, 2 H), 3.24–3.10 (m, 3 H), 3.22 (s, 3 H), 3.16 (s, 3 H), 2.68 (dd, J = 15.3, 4.9 Hz, 1 H), 1.19 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 166.97 (s, 1 C), 162.33 (s, 1 C), 159.98 (s, 1 C), 157.32 (s, 1 C), 150.47 (s, 1 C), 145.81 (s, 1 C), 122.14 (s, 1 C), 104.47 (s, 1 C), 99.62 (s, 1 C), 92.62 (s, 1 C), 70.39 (s, 1 C), 69.65 (s, 1 C), 66.49 (s, 1 C), 65.68 (s, 1 C), 56.33 (s, 1 C), 53.71 (s, 1 C), 53.26 (s, 1 C), 47.11 (s, 1 C), 41.68 (s, 1 C), 39.79 (s, 1 C), 28.74 (s, 1 C), 13.88 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₂₂H₃₁N₆O₄ 443.2401; found, 443.2405. HPLC (ACN with 0.1% TFA): t_R = 5.19 min (99.5% purity).

4-(Dimethoxymethyl)-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyrimidin-2-amine (17b)

17b was prepared according to general procedure 6 from 19b (234 mg, 0.36 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 0:1) gave compound 17b as a colorless solid (122 mg, 0.28 mmol, 76%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.72 (s, 1 H), 6.97 (br s, 2 H), 6.36 (s, 1 H), 4.33–4.27 (m, 1 H), 4.01–3.85 (m, 4 H), 3.79–3.72 (m, 2 H), 3.68–3.60 (m, 2 H), 3.46 (td, J = 11.7, 2.8 Hz, 1 H), 3.36–3.29 (m, 1 H), 3.27 (s, 3 H), 3.25 (s, 3 H), 3.26–3.10 (m, 4 H), 2.68 (dd, J = 15.4, 4.9 Hz, 1 H), 1.18 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 166.95 (s, 1 C), 162.97 (s, 1 C), 162.76 (s, 1 C), 160.70 (s, 1 C), 160.57 (s, 1 C), 157.25 (s, 1 C), 120.35 (s, 1 C), 99.37 (s, 1 C), 92.84 (s, 1 C), 70.35 (s, 1 C), 69.66 (s, 1 C), 66.48 (s, 1 C), 65.69 (s, 1 C), 56.33 (s, 1 C), 53.94 (s, 1 C), 53.71 (s, 1 C), 47.13 (s, 1 C), 41.65 (s, 1 C), 39.68 (s, 1 C), 28.70 (s, 1 C), 13.96 (s, 1 C). HRMS (m/z): [M + H]⁺ calcd for C₂₁H₃₀N₇O₄ 444.2354; found, 444.2358. HPLC: t_R = 5.88 min (98.3% purity).

tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[4-(dimethoxymethyl)-5-[(9R)-6-[(3R)-3-methylmorpholin-4-yl]-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-yl]carbamate (18b)

18b was prepared according to general procedure 5 from 44 (289 mg, 0.65 mmol, 1.0 equiv) and 22b (200 mg, 0.65 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 1:1) gave compound 18b as a colorless solid (174 mg, 0.27 mmol, 42%). The product is a mixture of mono- and di-Boc protected derivative and was used for the next step without further purification. MALDI-MS: m/z = 643.813 ([M + H]⁺); 543.329 ([M – Boc + H]⁺).

tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[4-(dimethoxymethyl)-5-[(9R)-6-[(3R)-3-methylmorpholin-4-yl]-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyrimidin-2-yl]carbamate (19b)

19b was prepared according to general procedure 5 from 49 (289 mg, 0.65 mmol, 1.0 equiv) and 22b (200 mg, 0.65 mmol, 1 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 1:1) gave compound 19b as a yellowish solid (240 mg, 0.37 mmol, 57%). ¹H NMR (400 MHz, DMSO-d₆): δ 9.20 (s, 1 H), 6.36 (s, 1 H), 4.36–4.30 (m, 1 H), 4.05–3.08 (m, 4 H), 3.81–3.76 (m, 2 H), 3.70–3.62 (m, 2 H), 3.47 (td, J = 11.7, 2.9 Hz, 1 H), 3.33 (s, 3 H), 3.28 (s, 3 H), 3.39–3.14 (m, 5 H), 2.76 (dd, J = 15.6, 5.0 Hz, 1 H), 1.42 (s, 18 H), 1.21 (d, J = 6.8 Hz, 3 H). MALDI-MS: m/z = 644.445 ([M + H]⁺).

(S)-2-Chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (20a)

20a was prepared according to general procedure 3 from 4-(2,6-dichloropyrimidin-4-yl)morpholine 26 (653 mg, 2.79 mmol, 1 equiv) and (R)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33b (500 mg, 2.79 mmol, 1 equiv). Purification by column chromatography (cyclohexane/EtOAc 2:1 → 1:1) gave compound 20a as a colorless solid (550 mg, 1.85 mmol, 66%). ¹H NMR (400 MHz, CDCl₃): δ 4.01 (dd, J = 13.5, 2.9 Hz, 1 H), 3.95–3.87 (m, 1 H), 3.79–3.72 (m, 2 H), 3.68–3.65 (m, 4 H), 3.59–3.49 (m, 4 H), 3.38 (td, J = 11.7, 2.9 Hz, 1 H), 3.23 (t, J = 11.0 Hz, 1H), 3.18–3.09 (m, 2 H), 2.50 (dd, J = 15.0, 5.1 Hz, 1 H). ¹³C{¹H} NMR (101 MHz, CDCl₃): δ 167.84 (s, 1 C), 158.32 (s, 1 C), 158.24 (s, 1 C), 93.08 (s, 1 C), 70.49 (s, 1 C), 66.73 (s, 2 C), 66.58 (s, 1 C), 57.32 (s, 1 C), 45.81 (s, 2 C), 42.15 (s, 1 C), 29.06 (s, 1 C). MALDI-MS: m/z = 297.301 ([M + H]⁺). HPLC: t_R = 6.36 min (97.7% purity). (S)-enantiomer: [α_D] = +4.0 (CHCl₃, c = 1.2).

(R)-2-Chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (20b)

20b was prepared as described for its (S)-enantiomer 20a starting from (S)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33a (506 mg, 2.16 mmol, 1 equiv) in 73% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer. HPLC: t_R = 6.35 min (98.0% purity). (R)-enantiomer: [α_D] = −3.3 (CHCl₃, c = 1.5).

(S)-4-Chloro-2-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (20c)

20c was prepared according to general procedure 3 from 4-(4,6-dichloropyrimidin-2-yl)morpholine 32 (298 mg, 1.27 mmol, 1 equiv) and (R)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33b (228 mg, 1.27 mmol, 1 equiv). Purification by column chromatography (cyclohexane/EtOAc 2:1 → 1:1) gave compound 20c as a colorless solid (286 mg, 0.96 mmol, 76%). ¹H NMR (400 MHz, CDCl₃): δ 4.02–3.94 (m, 2 H), 3.89–3.87 (m, 1 H), 3.86–3.84 (m, 1 H), 3.75–3.70 (m, 8 H), 3.44 (td, J = 11.7, 3.0 Hz, 1 H), 3.26 (t, J = 11.0 Hz, 1 H), 3.25–3.19 (m, 1 H), 2.99 (dd, J = 16.1, 9.4 Hz, 1 H), 2.42 (dd, J = 16.1, 5.0 Hz, 1 H). ¹³C{¹H} NMR (101 MHz, CDCl₃): δ 166.64 (s, 1 C), 161.92 (s, 1 C), 151.94 (s, 1 C), 104.19 (s, 1 C), 71.07 (s, 1 C), 66.83 (s, 2 C), 66.40 (s, 1 C), 57.11 (s, 1 C), 44.54 (s, 2 C), 41.79 (s, 1 C), 26.35 (s, 1 C). MALDI-MS: m/z = 297.030 ([M + H]⁺). (S)-enantiomer: [α_D] = −61.0 (CHCl₃, c = 1.1).

(R)-4-Chloro-2-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (20d)

20d was prepared as described for its (S)-enantiomer 20c starting from (R)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33a (281 mg, 1.57 mmol, 1 equiv) in 24% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer. (R)-enantiomer: [α_D] = +56.2 (CHCl₃, c = 1.4).

(S)-2-Chloro-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (21a)

21a was prepared according to general procedure 3 from (S)-4-(2,6-dichloropyrimidin-4-yl)-3-methylmorpholine 27 (3.68 g, 14.92 mmol, 1 equiv) and (R)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33b (2.67 g, 14.92 mmol, 1 equiv). Purification by column chromatography (cyclohexane/ethyl acetate 1:0 → 3:2) gave compound 21a as a yellowish solid (853 mg, 2.75 mmol, 18%). ¹H NMR (400 MHz, DMSO-d₆): δ 4.23–4.18 (m, 1 H), 3.97–3.73 (m, 6 H), 3.66–3.57 (m, 2 H), 3.42 (td, J = 11.8, 3.0 Hz, 1 H), 3.34–3.29 (m, 1 H), 3.24–3.08 (m, 4 H), 2.68 (dd, J = 15.4, 5.1 Hz, 1 H), 1.17 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.87 (s, 1 C), 157.71 (s, 1 C), 157.48 (s, 1 C), 93.25 (s, 1 C), 70.13 (s, 1 C), 69.64 (s, 1 C), 66.27 (s, 1 C), 65.69 (s, 1 C), 56.71 (s, 1 C), 47.18 (s, 1 C), 41.55 (s, 1 C), 39.67 (s, 1 C), 28.07 (s, 1 C), 14.10 (s, 1 C). MALDI-MS: m/z = 311.109 ([M + H]⁺). HPLC: t_R = 7.03 min (96.5% purity).

(R)-2-Chloro-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (21b)

21b was prepared as described for its (S,R)-enantiomer 22a starting from (S)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33a (1.0 g, 5.58 mmol, 1 equiv) in 24% yield. The spectroscopic data are in agreement with those reported for the (S,R)-enantiomer.

(S)-2-Chloro-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (22a)

22a was prepared according to general procedure 3 from (R)-4-(2,6-dichloropyrimidin-4-yl)-3-methylmorpholine 28 (1.15 g, 4.65 mmol, 1 equiv) and (R)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33b (833 mg, 4.65 mmol, 1 equiv). Purification by column chromatography (cyclohexane/ethyl acetate 1:0 → 4:1) gave compound 22a as a yellowish solid (1.05 g, 3.39 mmol, 73%). ¹H NMR (400 MHz, DMSO-d₆): δ 4.20–4.14 (m, 1 H), 3.93–3.72 (m, 6 H), 3.63 (d, J = 11.1 Hz, 1 H), 3.56 (dd, J = 11.5, 3.1 Hz, 1 H), 3.41 (td, J = 11.8, 2.9 Hz, 1 H), 3.34–3.27 (m, 1 H), 3.25–3.06 (m, 4 H), 2.57 (dd, J = 15.4, 5.0 Hz, 1 H), 1.18 (d, J = 6.8 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.96 (s, 1 C), 157.72 (s, 1 C), 157.40 (s, 1 C), 93.14 (s, 1 C), 70.15 (s, 1 C), 69.55 (s, 1 C), 66.29 (s, 1 C), 65.66 (s, 1 C), 56.76 (s, 1 C), 47.23 (s, 1 C), 41.62 (s, 1 C), 39.56 (s, 1 C), 28.18 (s, 1 C), 14.21 (s, 1 C). MALDI-MS: m/z = 311.085 ([M + H]⁺). HPLC: t_R = 7.15 min (98.9% purity).

(R)-2-Chloro-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (22b)

22b was prepared as described for its (S,S)-enantiomer 21a starting from (S)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33a (1.0 g, 5.58 mmol, 1 equiv) in 55% yield. The spectroscopic data are in agreement with those reported for the (S,S)-enantiomer. HPLC: t_R = 7.24 min (99.6% purity).

(9S)-4-Chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene (23a)

23a was prepared according to general procedure 3 from 3-(2,6-dichloropyrimidin-4-yl)-8-oxa-3-azabicyclo[3.2.1]octane 29 (1.21 g, 4.65 mmol, 1 equiv) and (R)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33b (1 g, 5.58 mmol, 1.2 equiv). Purification by column chromatography (cyclohexane/ethyl acetate 1:0 → 3:2) gave compound 23a as a yellowish solid (1.06 g, 3.28 mmol, 71%). ¹H NMR (400 MHz, DMSO-d₆): δ 4.36–4.31 (m, 2 H), 3.92–3.71 (m, 6 H), 3.32–3.27 (m, 1 H), 3.24–3.19 (m, 2 H), 3.16–3.03 (m, 3 H), 2.65 (dd, J = 15.4, 5.0 Hz, 1 H), 1.83–1.69 (m, 4 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 167.75 (s, 1 C), 158.86 (s, 1 C), 157.52 (s, 1 C), 93.27 (s, 1 C), 72.89 (s, 1 C), 72.88 (s, 1 C), 69.59 (s, 1 C), 65.67 (s, 1 C), 56.75 (s, 1 C), 50.61 (s, 1 C), 50.42 (s, 1 C), 41.55 (s, 1 C), 28.20 (s, 1 C), 27.37 (s, 1 C), 27.34 (s, 1 C). MALDI-MS: m/z = 323.162 ([M + H]⁺). HPLC: t_R = 6.98 min (99.8% purity).

(9R)-4-Chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene (23b)

23b was prepared as described for its (S)-enantiomer 23a starting from (S)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33a (2.0 g, 11.17 mmol, 1 equiv) in 40% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer.

(9S)-4-Chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene (24a)

24a was prepared according to general procedure 3 from 8-(2,6-dichloropyrimidin-4-yl)-3-oxa-8-azabicyclo[3.2.1]octane 30 (1.21 g, 4.65 mmol, 1 equiv) and (R)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33b (1 g, 5.58 mmol, 1.2 equiv). Purification by column chromatography (cyclohexane/ethyl acetate 1:0 → 3:2) gave compound 24a as a colorless solid (893 mg, 2.77 mmol, 59%). ¹H NMR (400 MHz, DMSO-d₆): δ 4.38–4.33 (m, 2 H), 3.94–3.89 (m, 1 H), 3.80–3.71 (m, 3 H), 3.59 (dd, J = 10.8, 5.8 Hz, 2 H), 3.50 (d, J = 10.8 Hz, 2 H), 3.36–3.29 (m, 1 H), 3.23 (t, J = 11.0 Hz, 1 H), 3.14–3.03 (m, 2 H), 2.54–2.48 (m, 1 H), 1.93–1.85 (m, 4 H). MALDI-MS: m/z = 323.111 ([M + H]⁺). HPLC: t_R = 7.04 min (95.5% purity). ¹³C NMR spectroscopic data are not available due to insufficient solubility in standard deuterated solvents.

(9S)-4-Chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene (24b)

24b was prepared as described for its (S)-enantiomer 24a starting from (S)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33a (2.0 g, 11.17 mmol, 1 equiv) in 26% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer.

(S)-2-Chloro-4-(3,3-dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (25a)

25a was prepared according to general procedure 3 from 4-(2,6-dichloropyrimidin-4-yl)-3,3-dimethylmorpholine 31 (1.21 g, 4.65 mmol, 1 equiv) and (R)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33b (1 g, 5.58 mmol, 1.2 equiv). Purification by column chromatography (cyclohexane/ethyl acetate 1:0 → 4:1) gave compound 25a as a colorless solid (922 mg, 2.84 mmol, 61%). ¹H NMR (400 MHz, DMSO-d₆): δ 3.95–3.88 (m, 1 H), 3.81–3.73 (m, 3 H), 3.72–3.62 (m, 2 H), 3.35–3.27 (m, 4 H), 3.22–3.07 (m, 4 H), 2.99 (dd, J = 16.1, 9.5 Hz, 1 H), 2.42 (dd, J = 16.1, 5.3 Hz, 1 H), 1.36 (s, 3 H), 1.33 (s, 3 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 168.01 (s, 1 C), 160.49 (s, 1 C), 156.25 (s, 1 C), 102.21 (s, 1 C), 77.30 (s, 1 C), 69.71 (s, 1 C), 66.75 (s, 1 C), 65.51 (s, 1 C), 57.21 (s, 1 C), 54.29 (s, 1 C), 43.33 (s, 1 C), 41.56 (s, 1 C), 27.39 (s, 1 C), 21.71 (s, 1 C), 20.86 (s, 1 C). MALDI-MS: m/z = 325.036 ([M + H]⁺). HPLC: t_R = 8.19 min (99.1% purity).

(R)-2-Chloro-4-(3,3-dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (25b)

25b was prepared as described for its (S)-enantiomer 25a starting from (S)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33a (1.64 g, 9.16 mmol, 1.2 equiv) in 66% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer.

4-(2,6-Dichloropyrimidin-4-yl)morpholine (26)

26 was prepared according to the literature. (32)

(S)-4-(2,6-Dichloropyrimidin-4-yl)-3-methylmorpholine (27)

27 was prepared according to general procedure 2 from (S)-3-methylmorpholine (1.3 mL, 11.46 mmol, 1.05 equiv) and 2,4,6-trichloropyrimidine (1.25 mL, 10.92 mmol, 1.0 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 3:1) gave compound 27 as a colorless solid (1.96 g, 7.90 mmol, 72%). ¹H NMR (400 MHz, DMSO-d₆): δ 6.95 (s, 1 H), 4.49 (qd, J = 6.8, 2.9 Hz, 1 H), 4.13 (dd, J = 13.7, 2.8 Hz, 1 H), 3.90 (dd, J = 11.5, 3.8 Hz, 1 H), 3.69 (d, J = 11.5 Hz, 1 H), 3.56 (dd, J = 11.6, 3.2 Hz, 1 H), 3.44–3.37 (m, 1 H), 3.25–3.18 (m, 1 H), 1.21 (d, J = 6.8 Hz, 3 H). The spectroscopic data are in agreement with those reported for the (R)-enantiomer.

(R)-4-(2,6-Dichloropyrimidin-4-yl)-3-methylmorpholine (28)

28 was prepared according to general procedure 2 from (R)-3-methylmorpholine (4 g, 39.54 mmol, 1.05 equiv) and 2,4,6-trichloropyrimidine (4.55 mL, 39.54 mmol, 1.0 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 3:2) gave compound 28 as a colorless solid (6.94 g, 28.10 mmol, 71%). ¹H NMR (400 MHz, DMSO-d₆): δ 7.00 (s, 1 H), 4.46–4.32 (m, 1 H), 4.10–3.97 (m, 1 H), 3.91 (ddt, J = 11.5, 4.0, 1.1 Hz, 1 H), 3.70 (d, J = 11.6 Hz, 1 H), 3.57 (dd, J = 11.7, 3.0 Hz, 1 H), 3.43 (td, J = 11.9, 3.0 Hz, 1 H), 3.21 (td, J = 13.0, 3.8 Hz, 1 H), 1.21 (d, J = 6.7 Hz, 3 H). ¹³C{¹H} NMR (101 MHz, CDCl₃): δ 163.06 (s, 1 C), 160.71 (s, 1 C), 160.06 (s, 1 C), 99.90 (s, 1 C), 70.70 (s, 1 C), 66.56 (s, 1 C), 47.94 (s, 1 C), 39.72 (s, 1 C), 14.09 (s, 1 C). The spectroscopic data are in agreement with those reported for the (S)-enantiomer.

3-(2,6-Dichloropyrimidin-4-yl)-8-oxa-3-azabicyclo[3.2.1]octane (29)

29 was prepared according to general procedure 2 from 8-oxa-3-azabicyclo[3.2.1]octane·HCl (2.5 g, 16.70 mmol, 1.05 equiv) and 2,4,6-trichloropyrimidine (1.83 mL, 15.90 mmol, 1.0 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 4:1) gave compound 29 as a colorless solid (3.57 g, 13.73 mmol, 86%). ¹H NMR (400 MHz, DMSO-d₆): δ 6.94 (s, 1 H), 4.47–4.36 (m, 2 H), 4.25–4.04 (m, 1 H), 3.82–3.60 (m, 1 H), 3.26–2.95 (m, 2 H), 1.90–1.74 (m, 2 H), 1.73–1.55 (m, 2 H). ¹³C{¹H} NMR (101 MHz, CDCl₃): δ 164.65 (s, 1 C), 160.53 (s, 1 C), 159.83 (s, 1 C), 99.79 (s, 1 C), 73.33 (s, 2 C), 50.30 (s, 2 C), 27.79 (s, 2 C).

8-(2,6-Dichloropyrimidin-4-yl)-3-oxa-8-azabicyclo[3.2.1]octane (30)

30 was prepared according to general procedure 2 from 3-oxa-8-azabicyclo[3.2.1]octane.HCl (2.5 g, 16.70 mmol, 1.05 equiv) and 2,4,6-trichloropyrimidine (1.83 mL, 15.90 mmol, 1.0 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 4:1) gave compound 30 as a colorless solid (3.33 g, 12.81 mmol, 80%). ¹H NMR (400 MHz, DMSO-d₆): δ 6.98 (s, 1 H), 4.68–4.62 (m, 1 H), 4.52–4.45 (m, 1 H), 3.61–3.53 (m, 4 H), 2.02–1.87 (m, 4 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 160.52 (s, 1 C), 159.36 (s, 1 C), 159.22 (s, 1 C), 102.25 (s, 1 C), 71.21 (s, 2 C), 55.97 (s, 1 C), 55.71 (s, 1 C), 26.95 (s, 1 C), 26.17 (s, 1 C).

4-(2,6-Dichloropyrimidin-4-yl)-3,3-dimethylmorpholine (31)

31 was prepared according to general procedure 2 from 3,3-dimethylmorpholine (4.0 g, 34.73 mmol, 1.05 equiv) and 2,4,6-trichloropyrimidine (3.8 mL, 33.08 mmol, 1.0 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 9:1) gave compound 31 as a colorless solid (4.19 g, 15.99 mmol, 46%). ¹H NMR (400 MHz, DMSO-d₆): δ 7.04 (s, 1 H), 3.81 (t, J = 5.5 Hz, 2 H), 3.59 (t, J = 5.7 Hz, 2 H), 3.46 (s, 2 H), 1.44 (s, 6 H). ¹³C{¹H} NMR (101 MHz, DMSO-d₆): δ 165.40 (s, 1 C), 159.38 (s, 1 C), 157.59 (s, 1 C), 104.26 (s, 1 C), 75.62 (s, 1 C), 65.76 (s, 1 C), 57.14 (s, 1 C), 42.65 (s, 1 C), 22.57 (s, 2 C).

4-(4,6-Dichloropyrimidin-2-yl)morpholine (32)

32 was prepared according to the literature. (32)

(S)-Tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-Dioxide (33a)

33a was prepared as described for its (R)-enantiomer 33b starting from (R)-morpholin-3-ylmethanol 34b (0.685 g, 5.85 mmol, 1 equiv) in 64% yield. The spectroscopic data are in agreement with those reported for the (R)-enantiomer. (S)-enantiomer: [α_D] = +53.8 (CHCl₃, c = 0.75).

(R)-Tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-Dioxide (33b)

A solution of thionyl chloride (2.8 g, 1.7 mL, 23.3 mmol, 1.95 equiv) in DCM (1.7 mL) was added dropwise to a cooled (−5 °C) solution of imidazole (4.9 g, 71.7 mmol, 6 equiv) in DCM (30 mL), and the temperature was kept at −5 °C. The cooling bath was removed, and the reaction mixture was stirred for 45 min while allowing it to warm to rt. The mixture was cooled down to −10 °C, and a solution of 34a (1.4 g, 11.9 mmol, 1 equiv) in DCM (12 mL) was added dropwise. The mixture was stirred at 0 °C for 2 h. Water (30 mL) was added, and the layers were separated. The organic layer was washed with half-concentrated brine (30 mL) and cooled to 5 °C. A NaIO₄ solution (7.7 g, 35.9 mmol) in water (75 mL) was added, followed by RuO₂–H₂O (16 mg). The mixture was allowed to warm up to rt and stirred at rt overnight. The layers were separated, and the organic layer was filtered through a silica gel column eluting with DCM. The solvent was removed under reduced pressure to give compound 33b (1.1 g, 61.4 mmol, 52%) as a colorless solid. ¹H NMR (400 MHz, CDCl₃): δ 4.59 (dd, J = 8.1, 6.4 Hz, 1 H), 4.31 (dd, J = 9.3, 8.1 Hz, 1 H), 4.02 (dd, J = 11.6, 3.4 Hz, 1 H), 3.88 (dt, J = 11.9, 3.6 Hz, 1 H), 3.85–3.79 (m, 1 H), 3.74 (ddd, J = 11.9, 8.9, 3.1 Hz, 1 H), 3.61 (dd, J = 11.6, 7.8 Hz, 1 H), 3.39–3.33 (m, 1 H), 3.15 (ddd, J = 12.2, 8.9, 3.4 Hz, 1 H). ¹³C{¹H} NMR (101 MHz, CDCl₃): δ 69.85 (s, 1 C), 66.52 (s, 1 C), 64.65 (s, 1 C), 54.21 (s, 1 C), 43.43 (s, 1 C). MALDI-MS: m/z = 180.032 ([M + H]⁺). (R)-enantiomer: [α_D] = −42.8 (CHCl₃, c = 0.65).

(S)-Morpholin-3-ylmethanol (34a)

A solution of 35a (14 g, 67.4 mmol) in methanol (200 mL) was placed in a Parr apparatus and degassed with argon. Palladium on carbon (10 wt %, 1.5 g) was added, and the mixture was stirred under H₂ at 2.8 bar for 48 h. The resulting mixture was filtered through Celite, washed with MeOH, and concentrated under reduced pressure to give compound 34a (8.0 g, 68.3 mmol, 99%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃): δ 3.81–3.80 (m, 1 H), 3.79–3.76 (m, 1 H), 3.60–3.47 (m, 4 H), 3.38–3.32 (m, 1 H), 3.00–2.91 (m, 4 H). The spectroscopic data are consistent with previous literature reports. (41)

(R)-Morpholin-3-ylmethanol (34b)

34b was prepared as described for its (S)-enantiomer 34a starting from (R)-(4-benzylmorpholin-3-yl)methanol 35b (5.2 g, 25.1 mmol, 1 equiv) in 96% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer.

(S)-(4-Benzylmorpholin-3-yl)methanol (35a)

To a stirred solution of 36b (20.6 g, 87.5 mmol, 1 equiv) in THF (320 mL), triethylamine (10.6 g, 14.7 mL, 105.1 mmol) was added. The solution was cooled to 0 °C, and borane–dimethyl sulfide complex (10 M in THF, 50 mL, 524.0 mmol) was slowly added. The reaction mixture was heated up to 65 °C for 5 h. After cooling to rt, the reaction was quenched by addition of water (60 mL). The reaction mixture was stirred at rt overnight. The organic solvents were removed under reduced pressure, and the residue was diluted with aqueous NaOH solution (20% v/v, 2×). The aqueous layer was extracted with EtOAc (2×), and the organic layer was washed with an aqueous 1 M HCl solution (2×). The combined aqueous fractions were then basified to pH 14 by addition of solid NaOH and re-extracted with EtOAc (3×). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduce pressure to give compound 35a (15.0 g, 72.4 mmol, 82%) as a clear oil. ¹H NMR (400 MHz, CDCl₃): δ 7.53–7.26 (m, 5 H), 4.13 (d, J = 13.3 Hz, 1 H), 3.96 (dd, J = 11.5, 4.5 Hz, 1 H), 3.84 (ddd, J = 11.5, 3.8, 1.1 Hz, 1 H), 3.75 (td, J = 3.3, 1.1 Hz, 1 H), 3.73 (td, J = 3.3, 1.1 Hz, 1 H), 3.67 (dd, J = 11.6, 9.2 Hz, 1 H), 3.56–3.49 (m, 2 H), 2.73 (dt, J = 12.0, 2.9 Hz, 1 H), 2.59–2.54 (m, 1 H), 2.52 (br s, 1 H), 2.34 (ddd, J = 12.0, 10.0, 3.3 Hz, 1 H). The spectroscopic data are consistent with previous literature reports. (41)

(R)-(4-Benzylmorpholin-3-yl)methanol (35b)

35b was prepared as described for its (S)-enantiomer 35a starting from (S)-4-benzyl-5-oxomorpholine-3-carboxylic acid 36a (7.4 g, 31.5 mmol, 1 equiv) in 81% yield. The spectroscopic data are in agreement with those reported for the (S)-enantiomer.

(S)-4-Benzyl-5-oxomorpholine-3-carboxylic Acid (36a)

36a was prepared as described for its (R)-enantiomer 36b starting from N-benzyl-S-serine 37a (12.2 g, 62.5 mmol, 1 equiv) in 51% yield. The spectroscopic data agree with those reported for the (R)-enantiomer.

(R)-4-Benzyl-5-oxomorpholine-3-carboxylic Acid (36b)

To a solution of N-benzyl-R-serine 37b (25.6 g, 131.1 mmol, 1 equiv) and K₂CO₃ (36.2 g, 262.2 mmol, 2 equiv) in THF/water (150 mL/150 mL) at 0 °C, a chloroacetyl chloride (17.8 g, 12.5 mL, 157.4 mmol, 1.2 equiv) solution in THF (13 mL) was added dropwise. The mixture was stirred at 0 °C for 1 h. Solid NaOH (15.72 g, 393 mmol, 3 equiv) was added, and the mixture was stirred at 5 °C for 2 h. After completion of the reaction, cyclohexane was added and vigorously stirred. The layers were separated and the basic aqueous layer was acidified to pH 1 with conc HCl. The mixture was kept in the fridge overnight and the solid was filtered off, washed with cold water, and dried under vacuum to give compound 36b (21.8 g, 92.7 mmol, 71%) as a colorless solid. ¹H NMR (400 MHz, DMSO-d₆): δ 7.37–7.26 (m, 5 H), 5.26 (d, J = 15.4 Hz, 1 H), 4.19–4.12 (m, 3 H), 3.96–3.90 (m, 2 H), 3.83 (d, J = 15.4 Hz, 1 H). The spectroscopic data are consistent with previous literature reports. (41)

N-Benzyl-(S)-serine (37a)

37a was prepared as described for its (R) enantiomer 37b starting from (S)-serine (29.5 g, 280.7 mmol, 1 equiv) in 63% yield. The spectroscopic data are in agreement with those reported for the (R) enantiomer.

N-Benzyl-(R)-serine (37b)

To a stirred solution of (R)-serine (25 g, 237.9 mmol, 1 equiv) in aqueous 2 M NaOH (120 mL), benzaldehyde (24.7 g, 24.5 mL, 233.1 mmol, 1 equiv) was added. The reaction mixture was stirred at rt for 30 min before cooling to 5 °C. NaBH₄ (2.7 g, 71.4 mmol, 0.3 equiv) was added portionwise while keeping the temperature below 10 °C. After addition, the reaction mixture was allowed to warm up to rt and stirred 1 h. Additional benzaldehyde (24.7 g, 24.5 mL, 233.1 mmol, 1 equiv) was added, and the reaction mixture was stirred at rt for 30 min. The reaction mixture was cooled to 5 °C, and further NaBH₄ (2.7 g, 71.4 mmol, 0.3 equiv) was added portionwise while a temperature of 5–10 °C was maintained. After completion of the addition, the reaction mixture was stirred at rt for 2 h. The reaction mixture was then extracted with ether (3×), and the aqueous phase was cooled down to 0 °C and acidified to pH 5 with conc HCl. The resulting white precipitate was filtered off, washed with water, and dried under reduced pressure to give compound 37b (25.8 g, 132.2 mmol, 55%). ¹H NMR (400 MHz, DMSO-d₆): δ 7.45–7.30 (m, 5 H), 4.04–3.92 (m, 2 H), 3.70–3.61 (m, 3 H), 3.17 (t, J = 5.8 Hz, 1 H). The spectroscopic data are consistent with previous literature reports. (41)

(E)-N,N-Dimethyl-N′-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide (38)

38 was prepared according to general procedure 1 from 2-aminopyridine-5-boronic acid pinacol ester (500 mg, 2.27 mmol, 1 equiv) and N,N-dimethylformamide–dimethyl acetal (DMF–DMA, 395 μL, 2.95 mmol, 1.3 equiv). Compound 38 was obtained as a beige solid (538 mg, 1.96 mmol, 86%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.57 (s, 1 H), 8.41 (dd, J = 2.0, 0.9 Hz, 1 H), 7.75 (dd, J = 8.1, 2.0 Hz, 1 H), 6.78 (dd, J = 8.0, 0.9 Hz, 1 H), 3.11 (s, 3 H), 3.00 (s, 3 H), 1.29 (s, 12 H).

5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazin-2-amine (39)

2-Amino-5-bromopyrazine (1.50 g, 8.77 mmol, 1 equiv), bis(pinacolato)diboron (3.29 g, 12.95 mmol, 1.5 equiv), and KOAc (2.53 g, 25.82 mmol, 3.0 equiv) were charged in a flask. Under nitrogen atmosphere, 1,4-dioxane (20 mL) and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl₂, 605 mg, 0.86 mmol, 0.1 equiv) were added. The reaction mixture was stirred at 105 °C for 3.5 h. After completion of the reaction monitored by TLC, the mixture was filtered through Celite and the solvents were evaporated under reduced pressure. Methyl tert-butyl ether (MTBE) was added, and the solid was filtered off. Compound 39 was obtained as a light brownish solid (1.23 g, 5.57 mmol, 64%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.11 (d, J = 1.5 Hz, 1 H), 7.99 (d, J = 1.5 Hz, 1 H), 6.81 (br s, 2 H), 1.27 (s, 12 H).

(E)-N′-(5-Bromo-4-methylpyridin-2-yl)-N,N-dimethylformimidamide (40)

40 was prepared according to general procedure 1 from 5-bromo-4-methylpyridin-2-amine (8.06 g, 43.10 mmol, 1 equiv) and DMF–DMA (6.92 mL, 51.70 mmol, 1.2 equiv). Compound 40 was obtained as a beige solid (7.6 g, 31.52 mmol, 73%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.69 (s, 1 H), 8.54 (s, 1 H), 7.14 (s, 1 H), 3.38 (s, 3 H), 3.37 (s, 3 H), 2.62 (s, 3 H).

(E)-N,N-Dimethyl-N′-(3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)formimidamide (41)

41 was prepared according to general procedure 1 from 3-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine (500 mg, 2.13 mmol, 1 equiv) and DMF–DMA (430 μL, 3.20 mmol, 1.5 equiv). Compound 41 was obtained as a brownish solid (407 mg, 1.41 mmol, 66%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.51 (s, 1 H), 8.26 (s, 1 H), 7.62 (s, 1 H), 3.11 (s, 3 H), 3.03 (s, 3 H), 2.19 (s, 3 H), 1.28 (s, 12 H).

tert-Butyl (5-Bromo-4-methoxypyridin-2-yl)carbamate (42)

To a solution of Boc anhydride (Boc₂O, 4.73 g, 21.67 mmol, 2.2 equiv) in THF (20 mL) at 0 °C, 5-bromo-4-methoxypyridin-2-amine (2.0 g, 9.85 mmol, 1 equiv) was added. Then, 4-dimethylaminopyridine (DMAP, 241 mg, 1.97 mmol, 0.2 equiv) was added portionwise. The reaction mixture was stirred overnight while it was allowed to warm up to room temperature. After completion of the reaction monitored by TLC, the solvent was evaporated and DCM (200 mL) was added. The organic layer was washed with NH₄Cl 15% solution (2×), and the combined aqueous layers were extracted with DCM (1×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Purification by column chromatography (cyclohexane/EtOAc 1:0 → 1:4) gave compound 42 as a colorless solid (767 mg, 2.53 mmol, 26%). ¹H NMR (400 MHz, DMSO-d₆): δ 9.97 (s, 1 H), 8.22 (s, 1 H), 7.61 (s, 1 H), 3.92 (s, 3 H), 1.48 (s, 9 H).

5-Bromo-4-(dimethoxymethyl)pyridin-2-amine (43)

To a solution of 4-(dimethoxymethyl)pyridin-2-amine (2.0 g, 11.89 mmol, 1.0 equiv) in 2-methyltetrahydrofuran (24 mL) at 0 °C, N-bromosuccinimide (NBS, 2.23 g, 12.48 mmol, 1.05 equiv) was added portionwise. The reaction mixture was stirred at 0 °C for 1 h and then at rt overnight. After completion of the reaction monitored by TLC, the reaction mixture was washed with a Na₂CO₃ 8% solution (3×). The combined aqueous layers were extracted with 2-methyltetrahydrofuran (1×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Purification by column chromatography (cyclohexane/EtOAc 1:0 → 4:1) gave compound 43 as a beige solid (2.585 g, 10.46 mmol, 88%). ¹H NMR (400 MHz, DMSO-d₆): δ 7.98 (s, 1 H), 6.65 (s, 1 H), 6.24 (br s, 2 H), 5.28 (s, 1 H), 3.29 (s, 6 H).

tert-Butyl N-[5-Bromo-4-(dimethoxymethyl)pyridin-2-yl]-N-[(tert-butoxy)carbonyl]carbamate (44) and tert-Butyl N-[5-Bromo-4-(dimethoxymethyl)pyridin-2-yl]carbamate (45)

To a solution of Boc anhydride (Boc₂O, 5.02 g, 22.98 mmol, 2.2 equiv) in THF (26 mL) at 0 °C, 5-bromo-4-(dimethoxymethyl)pyridin-2-amine 43 (2.58 g, 10.44 mmol, 1 equiv) was added. Then, 4-dimethylaminopyridine (DMAP, 256 mg, 2.09 mmol, 0.2 equiv) was added portionwise. The reaction mixture was stirred overnight while it was allowed to warm up to room temperature. After completion of the reaction monitored by TLC, the reaction mixture was washed with NH₄Cl 15% solution (2×) and the organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Purification by column chromatography (cyclohexane/EtOAc 1:0 → 0:1) gave compound 44 as a yellowish oil (1.286 g, 2.87 mmol, 27%) and 45 as a white solid (2.154 g, 6.21 mmol, 59%). 44: ¹H NMR (400 MHz, DMSO-d₆): δ 8.66 (s, 1 H), 7.45 (s, 1 H), 5.51 (s, 1 H), 3.33 (s, 6 H), 1.40 (s, 18 H). 45: ¹H NMR (400 MHz, DMSO-d₆): δ 8.70 (s, 1 H), 7.51 (s, 1 H), 5.51 (s, 1 H), 3.33 (s, 6 H), 1.38 (s, 9 H).

(E)-4-(Dimethylamino)-1,1-dimethoxybut-3-en-2-one (46)

46 was prepared as previously reported in the literature. (46) A solution of methylglyoxal dimethyl acetal (26 mL, 211.6 mmol, 1.0 equiv) and DMF–DMA (37 mL, 275.1 mmol, 1.3 equiv) was stirred at 100 °C overnight. The reaction mixture was concentrated under reduced pressure. Compound 46 was obtained as a brown oil (35.7 g, 206.1 mmol, 75%) and used for the next step without further purification.

4-(Dimethoxymethyl)pyrimidin-2-amine (47)

Compound 46 (35.7 g, 206.1 mmol, 1.0 equiv) was dissolved in ethanol (780 mL). Potassium carbonate (71.2 g, 512.3 mmol, 2.5 equiv) and guanidine hydrochloride (23.6 g, 247.3 mmol, 1.2 equiv) were added, and the resulting suspension was heated to reflux overnight. After completion of the reaction monitored by TLC, ethanol was evaporated under reduced pressure. The residue was stirred with water for 6 h, filtered, and dried under vacuum to afford compound 47. The filtrate was extracted with dichloromethane (3×) and the combined organic layers were dried over anhydrous Na₂SO₄ sodium sulfate and concentrated under reduced pressure to give compound 47 as a brown solid (24.8 g, 146.6 mmol, 71%). ¹H NMR (400 MHz, CDCl₃): δ 8.34 (d, J = 5.0 Hz, 1 H), 6.84 (d, J = 5.0 Hz, 1 H), 5.28 (br s, 1 H), 5.14 (s, 1 H), 4.76 (br s, 1 H), 3.39 (s, 6 H).

5-Bromo-4-(dimethoxymethyl)pyrimidin-2-amine (48)

To a solution of 4-(dimethoxymethyl)pyrimidin-2-amine 47 (5.0 g, 29.58 mmol, 1.0 equiv) in ACN (150 mL), NBS (5.53 g, 31.06 mmol, 1.05 equiv) was added portionwise. The reaction mixture was stirred at 70 °C overnight. After completion of the reaction monitored by TLC, ACN was evaporated. The solid was dissolved in DCM (250 mL), and the organic layer was washed with a Na₂CO₃ 8% solution (3×). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The product was recrystallized from dichloromethane/heptanes to obtain compound 48 as a beige solid (6.50 g, 26.20 mmol, 89%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (s, 1 H), 7.02 (br s, 2 H), 5.27 (s, 1 H), 3.35 (s, 6 H).

tert-Butyl N-[5-Bromo-4-(dimethoxymethyl)pyrimidin-2-yl]-N-[(tert-butoxy)carbonyl]carbamate (49)

To a solution of Boc anhydride (Boc₂O, 12.59 g, 57.66 mmol, 2.2 equiv) in THF (65 mL) at 0 °C, 5-bromo-4-(dimethoxymethyl)pyrimidin-2-amine 48 (6.50 g, 26.21 mmol, 1 equiv) was added. Then, DMAP (640 mg, 5.24 mmol, 0.2 equiv) was added portionwise. The reaction mixture was stirred overnight while it was allowed to warm up to room temperature. After completion of the reaction monitored by TLC, the reaction mixture was washed with a 0.1 M HCl solution (2×) and the organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Recrystallization from heptanes gave compound 49 as a beige solid (10.71 g, 23.91 mmol, 91%). ¹H NMR (400 MHz, DMSO-d₆): δ 9.12 (s, 1 H), 5.54 (s, 1 H), 3.39 (s, 6 H), 1.40 (s, 18 H).

(E)-N,N-Dimethyl-N′-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4-(trifluoromethyl)pyridin-2-yl)formimidamide (50)

50 was prepared according to the literature. (33)

(S)-4-(2-Chloro-5,5a,6,7,8,9-hexahydropyrimido[5,4-b]indolizin-4-yl)morpholine (51a)

51a was prepared as described for its (R)-enantiomer 51b starting from (S)-hexahydro[1,2,3]oxathiazolo[3,4-a]pyridine 1,1-dioxide 53a (507 mg, 2.86 mmol, 1.2 equiv) in 43% yield. The spectroscopic data are in agreement with those reported for the (R)-enantiomer.

(R)-4-(2-Chloro-5,5a,6,7,8,9-hexahydropyrimido[5,4-b]indolizin-4-yl)morpholine (51b)

51b was prepared according to general procedure 3 from 4-(2,6-dichloropyrimidin-4-yl)morpholine (702 mg, 3.00 mmol, 1.0 equiv) and (R)-hexahydro-[1,2,3]oxathiazolo[3,4-a]pyridine 1,1-dioxide 53b (638 mg, 3.60 mmol, 1.2 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 9:1) gave compound 51b as a colorless solid (324 mg, 1.10 mmol, 36%). ¹H NMR (400 MHz, CDCl₃): δ 3.91 (dd, J = 13.0, 4.2 Hz, 1 H), 3.68–3.57 (m, 5 H), 3.52–3.44 (m, 4 H), 3.24 (dd, J = 15.0, 9.5 Hz, 1 H), 2.75 (td, J = 13.0, 3.2 Hz, 1 H), 2.66 (dd, J = 15.0, 6.4 Hz, 1 H), 1.82–1.67 (m, 2 H), 1.63–1.55 (m, 1 H), 1.50–1.36 (m, 1 H), 1.35–1.20 (m, 2 H). ¹³C{¹H} NMR (101 MHz, DMSO): δ 167.84 (s, 1 C), 157.66–157.53 (m, 2 C), 93.32 (s, 1 C), 65.99 (s, 2 C), 59.15 (s, 1 C), 45.34 (s, 2 C), 41.29 (s, 1 C), 32.60 (s, 1 C), 31.62 (s, 1 C), 24.43 (s, 1 C), 23.35 (s, 1 C). MALDI-MS: m/z = 295.267 ([M + H]⁺).

(S)-2-Chloro-4-(piperidin-1-yl)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (52a)

52a was prepared as described for its (R)-enantiomer 52b starting from (R)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33b in 70% yield. The spectroscopic data are in agreement with those reported for the (R)-enantiomer.

(R)-2-Chloro-4-(piperidin-1-yl)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (52b)

52b was prepared according to general procedure 3 from dichloropyrimidine 54 (200 mg, 0.86 mmol, 1.0 equiv) and (S)-tetrahydro-3H-[1,2,3]oxathiazolo[4,3-c][1,4]oxazine 1,1-dioxide 33a (185 mg, 1.03 mmol, 1.2 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 9:1) gave compound 52b as a colorless solid (168 mg, 0.57 mmol, 66%). ¹H NMR (400 MHz, CDCl₃): δ 4.03–3.88 (m, 2 H), 3.79 (ddd, J = 16.0, 11.0, 3.8 Hz, 2 H), 3.60–3.53 (m, 4 H), 3.44 (td, J = 12.0, 2.9 Hz, 1 H), 3.29 (t, J = 11.0 Hz, 1 H), 3.22–3.12 (m, 2 H), 2.54 (dd, J = 15.0, 5.0 Hz, 1 H), 1.69–1.61 (m, 2 H), 1.61–1.53 (m, 4 H). ¹³C{¹H} NMR (101 MHz, CDCl₃): δ 168.44 (s, 1 C), 158.98 (s, 1 C), 158.49 (s, 1 C), 92.49 (s, 1 C), 70.55 (s, 1 C), 66.72 (s, 1 C), 57.32 (s, 1 C), 46.69 (s, 2 C), 42.11 (s, 1 C), 29.55 (s, 1 C), 26.16 (s, 2 C), 24.84 (s, 1 C). MALDI-MS: m/z = 295.082 ([M + H]⁺).

(S)-Hexahydro-[1,2,3]oxathiazolo[3,4-a]pyridine 1,1-Dioxide (53a)

53a was prepared as described for its (R)-enantiomer 53b in a 74% yield. ¹H NMR (400 MHz, CDCl₃): δ 4.57 (dd, J = 7.7, 5.8 Hz, 1 H), 4.18 (dd, J = 9.9, 7.8 Hz, 1 H), 3.59–3.51 (m, 1 H), 3.50–3.40 (m, 1 H), 2.76 (ddd, J = 12.0, 11.0, 3.1 Hz, 1 H), 1.96–1.86 (m, 2 H), 1.86–1.78 (m, 1 H), 1.69–1.57 (m, 1 H), 1.50–1.27 (m, 2 H). The spectroscopic data are consistent with those reported in literature. (47)

(R)-Hexahydro-[1,2,3]oxathiazolo[3,4-a]pyridine 1,1-Dioxide (53b)

To a solution of imidazole (1.77 g, 26.0 mmol, 6.0 equiv) in DCM (18 mL), a solution of SOCl₂ (610 μL, 8.36 mmol, 1.9 equiv) in DCM (6 mL) was added dropwise. The resulting colorless suspension was stirred at rt for 1 h. Then, a solution of (R)-piperidin-2-ylmethanol (500 mg, 4.34 mmol, 1.0 equiv) in DCM (800 μL) was added dropwise at −10 °C. The reaction mixture was allowed to warm up to rt and stirred at rt for 1.5 h. After completion of the reaction monitored by TLC, deionized H₂O (35 mL) was added and the layers were separated. The organic layer was washed with brine (15 mL) and used in the next step. Under vigorous stirring, a solution of RuO₂–H₂O (5.80 mg, 43.6 μmol, 0.01 equiv) and NaIO₄ (2.38 g, 11.1 mmol, 2.6 equiv) in deionized H₂O (24 mL) was added. The reaction mixture was stirred at rt for 30 min. After completion of the reaction, the layers were separated and the organic layer was filtered through a pad of silica gel (12 g) (eluent: DCM). The solvent was evaporated to give compound 53b as a colorless liquid (638 mg, 3.60 mmol, 83%). ¹H NMR (400 MHz, CDCl₃): δ 4.57 (dd, J = 7.8, 5.8 Hz, 1 H), 4.18 (dd, J = 9.9, 7.7 Hz, 1 H), 3.59–3.52 (m, 1 H), 3.50–3.40 (m, 1 H), 2.76 (ddd, J = 12.0, 11.0, 3.1 Hz, 1 H), 1.96–1.86 (m, 2 H), 1.86–1.78 (m, 1 H), 1.69–1.56 (m, 1 H), 1.51–1.27 (m, 2 H).

2,4-Dichloro-6-(piperidin-1-yl)pyrimidine (54)

54 was prepared according to general procedure 2 from 2,4,6-trichloropyrimidine (627 μL, 5.45 mmol, 1.0 equiv) and piperidine (566 μL, 5.72 mmol, 1.0 equiv). Purification by column chromatography on silica gel (cyclohexane/ethyl acetate 1:0 → 4:1) gave compound 54 as a colorless solid (849 mg, 3.66 mmol, 67%). ¹H NMR (400 MHz, CDCl₃): δ 6.39 (s, 1 H), 3.79–3.36 (m, 4 H), 1.74–1.66 (m, 2 H), 1.66–1.57 (m, 4 H). ¹³C{¹H} NMR (101 MHz, CDCl₃): δ 162.82 (s, 1 C), 160.31 (s, 1 C), 160.02 (s, 1 C), 99.66 (s, 1 C), 45.81 (br s, 2 C), 25.61 (s, 2 C), 24.41 (s, 1 C).

Structure Modeling of PI3K and mTOR Kinase Complexes

The coordinates of PIKiN3 in PI3Kγ complex (PDB code 5JHB, resolution of 2.48 Å), CNX-1351 in PI3Kα (PDB code 3ZIM, resolution of 2.85 Å), and mTOR kinase bound to PI103 (PDB code 4JT6; 3.6 Å) were used as starting points to dock molecules into the ATP-binding sites. Ligands in crystal structures were manually replaced using Maestro 11.1, and energy minimization was carried out. Further measurements and figures were generated in Maestro 11.1 and Chimera UCSF.

Determination of Inhibitor Dissociation Constants

Dissociation constants of compounds (K_i) for p110α and mTOR were determined by commercial LanthaScreen (Life Technologies) and evaluated as described in ref (33). Briefly, AlexaFluor647-labeled kinase tracer 314 (no. PV6087) with a K_d of 2.2 nM was used at 20 nM for p110α and at a final concentration of 10 nM for mTOR (K_d of 19 nM). Recombinant N-terminally (His)₆-tagged p110α was detected with biotinylated anti-(His)₆-tag antibody (2 nM, no. PV6089) and LanthaScreen Eu-Steptavidin (2 nM, no. PV5899); N-terminal GST fused to truncated mTOR (amino acids 1360–2549; no. PR8683B) was detected with a LanthaScreen Eu-labeled anti-GST antibody (2 nM, no. PV5594). The p110α assay buffer was composed of 50 mM HEPES, pH 7.5, 10 mM MgCl₂, 1 mM EGTA, and 0.01% (v/v) Brij-35, and the mTOR assay buffer contained 50 mM HEPES; 5 mM MgCl₂; 1 mM EGTA; 0.01% Pluronic F-127. Further details and calculations are explained in ref (32).

Kinome Profiling

The inhibitory capacity and selectivity of compound were determined using the ScanMax platform provided by DiscoverX. (48) In short binding of immobilized ligand to DNA-tagged kinases competed with 10 μM compound. The amount of kinase bound to the immobilized ligand was measured by quantitative PCR of the respective DNA tags and is given as percentage of control. Binding constants of compounds for kinases of interest were determined by competing the immobilized ligand kinase interactions with an 11-point 3-fold serial dilution of compound starting from 30 μM and subsequent quantitative PCR of DNA tags. Binding constants were calculated by a standard dose–response curve using the Hill equation (with Hill slope set to −1):

Selectivity cores (49) were calculated as S = number of hits/number of tested kinases (excluding mutant variants), where S35, S10, S1 were calculated using %Ctrl as a potency threshold (35, 10, 1%); for example S(35) = (number of nonmutant kinases with %Ctrl < 35)/(number of nonmutant kinases tested).

Cellular PI3K and mTOR Signaling

Downstream signals emerging from mTORC2 (phosphorylation of Ser473 of PKB/Akt; rabbit polyclonal antibody from Cell Signaling Technology (CST), no. 4058) and mTORC1 (phosphorylation of Ser235/236 on the ribosomal protein S6; rabbit monoclonal antibody from CST, no. 4856) were measured in in-cell Western assays plating 2 × 10⁴ A2058 cells/well in 96-well plates (Cell Carrier, PerkinElmer) for 24 h (37 °C, 5% CO₂) before exposing cells for 1 h to inhibitors or DMSO. Then, cells were fixed (4% PFA in PBS for 30 min at rt), blocked (1% BSA/0.1% Triton X-100/5% goat serum in PBS for 30 min, RT), and stained with CST primary antibodies (1:500). Tubulin staining (mouse anti-α-tubulin, 1:2000, Sigma no. T9026) was assessed as internal standard. Secondary antibody [IRDye680-conjugated goat anti-mouse, and IRDye800-conjugated goat anti-rabbit antibodies (LICOR no. 926-68070 and no. 926-32211), both 1:500] fluorescence was detected on an Odyssey CLx infrared imaging scanner (LICOR). Remaining phospho-protein signals were normalized to cellular tubulin and related to DMSO controls. ICW anaylsis and determination of IC₅₀ were done as described in ref (33).

Pharmacokinetic Studies

Male Sprague Dawley rats (8 weeks old at delivery) were purchased from Janvier Labs (France). The animals were housed in a temperature-controlled room (20–24 °C) and maintained in a 12h light/12h dark cycle. Food and water were available ad libitum throughout the duration of the study. Formulations of compounds 7b and 12b were prepared by weighing the test items into glass vials and dissolving them by addition of Captisol (40% w/w in water) and water for injection in a proportion of 50% and 35% of the final desired volume. The pH was adjusted to 3 with 0.2 M HCl, and finally, the volume was completed with water for injection. The formulations were stirred continuously until application to the animals. At each time point (30 min, 2 h, 4 h, and 8 h), three rats were anesthetized with isoflurane and 1 mL blood was collected, via heart puncture, in tubes containing lithium-heparin. After blood sampling, the rats were euthanized and brain, liver, and skin were collected. Blood samples were stored on dry ice until centrifugation at 6000 rpm (10 min, 4 °C). Plasma supernatants and tissue samples were kept at −80 °C until being assayed. The calibration standards and quality controls were prepared in duplicates. A volume of 50 μL of unknown samples, zero samples, and blanks was spiked with 6 μL of DMSO. After 10 min of equilibration, a volume of 100 μL of acetonitrile containing the internal standard (diazepam, 300 ng/mL) was added to each calibration standard, QC, zero sample, and unknown sample, while a volume of 100 μL of plain acetonitrile was added to all blanks. Samples were vigorously shaken and centrifuged for 10 min at 6000g and 20 °C. The particle free supernatant was diluted 1 + 1 with water. An aliquot was transferred to 200 μL sampler vials and subsequently subjected to LC–MS. The HPLC system consisted of an Accela U-HPLC pump and an Accela autosampler (Thermo Fisher Scientific, USA). Mass spectrometry was performed on an Exactive mass spectrometer (Orbitrap technology with accurate mass) equipped with a heated electrospray (H-ESI 2) interface (Thermo Fisher Scientific, USA) connected to a PC running the standard software Xcalibur 2.1.

Ethics Statement. All experimental procedures were approved by and conducted in accordance with the regulations of the local animal welfare authorities (Landesamt für Gesundheit and Verbraucherschutz, Abteilung Lebensmittel- and Veterinärwesen, Saarbrücken).

Hepatocyte Stability Assay

Primary hepatocytes from mouse (CD-1), rat (Sprague Dawley, SD), dog (Beagle), and human were used. Assays were performed using cryopreserved hepatocytes in suspension. Hepatocytes were thawed according to the instructions of the supplier before seeding in 48-well cell culture plates at a density of 200 000 cells/well in 225 μL of incubation medium consisting of WME (Williams medium E) supplemented with 2 mM l-glutamine and 25 mM HEPES. Stock solution of compound 11b was prepared with 10 mM in DMSO. A working solution was obtained by dilution of the stock solution in DMSO (first step) and in incubation medium (second step) resulting in a concentration of 10-fold higher strength (50 μM) than the final intended test concentration (5 μM) and a solvent content of 5% DMSO.

Positive control incubations were performed using 7-ethoxycoumarin as substrate. A 10 mM stock solution in acetonitrile (ACN) was further diluted in ACN (first step) and in incubation medium (second step) to give a working solution in 10% ACN and of 10-fold higher strength than the final intended incubation concentration (5 μM). To 225 μL of cell suspension, 25 μL of the 10-fold concentrated working solution of test or reference item was added, resulting in a final test concentration of 5 μM for compound 11b or 7-ethoxycoumarin, respectively, with final solvent concentrations of 0.5% DMSO (11b) or 1% ACN (7-ethoxycoumarin). For analysis, samples were taken after 0, 15, 60, 90, and 180 min of incubation for 11b and after 0, 60, and 180 min for reference item. The sample preparation was performed afterward by protein precipitation using ACN (200 μL of cell suspension plus 200 μL of ACN/ISTD3). After centrifugation (5 min, 4800g), the particle-free supernatants were diluted with one volume of water and were analyzed by LC–MS. Negative controls were performed to exclude nonmetabolic degradation processes; i.e., the finding that the concentrations remained stable over the investigated time suggests that the decrease of the parent compound was mainly due to metabolism. Negative control incubations were performed in line with all experiments using incubation medium in absence of hepatocytes.

For quantitative analysis of compound 11b in samples from primary human hepatocytes, the HPLC system consisted of a LC pump Surveyor Plus and an autosampler Surveyor Plus (Thermo-Fisher, USA). Mass spectrometry was performed on a TSQ Quantum Discovery Max triple quadrupole mass spectrometer equipped with an electrospray ion source (ESI) (Thermo Fisher Scientific, USA) connected to a PC running the standard software Xcalibur 2.0.7.

CYP Reactive Phenotyping with Human Recombinant CYP1A1 and CYP1A2 Isoenzymes

Human recombinant isoenzymes from insect cells infected by baculovirus and containing cDNA of a single human CYP isoenzyme (Supersomes, Corning) were used. The test item stock solutions were diluted in DMSO/H₂O (1:8, v/v) to obtain 50-fold concentrated working solutions (solvent content 12.5% DMSO/87.5% H₂O) for CYP1A1 and CYP1A2. The test compound concentration applied in the CYP reactive phenotyping assay was 1 μM in the presence of 0.25% DMSO. The assays were performed in duplicate using human recombinant enzymes systems from Corning (BD Gentest P450 high throughput inhibitor screening kits). The cofactor mix, containing the NADP⁺-regenerating system, was prepared according to the instructions of the manufacturer. For CYP1A1 and CYP1A2, 4 μL of the 50-fold concentrated working solution was added to 96 μL of cofactor mix. Cofactor mix and test item were pipetted into the respective wells of a prewarmed 96-well plate and prewarmed for 10 min on a shaker with fitted heating block. The reactions were initiated by addition of 100 μL of prewarmed enzyme mix. By default, the final protein concentration of all CYP isoenzymes was 25 pmol/mL. Incubations with a final volume of 200 μL were performed at 37 °C. After 0 and 60 min (30 min for positive control substrates), the reactions were stopped by the addition of 200 μL of stop solution, i.e., ACN containing the internal standard. Two control groups were run in parallel for every assay: positive controls (PC, n = 2) using specific probe substrates for each CYP isoform as reference compounds (CYP1A1 = melatonin and CYP1A2 = phenacetin) to prove the quality of the enzyme activity of the used batches and a negative control (NC, n = 2), which were performed without cofactors and glucose 6-phosphate dehydrogenase to ensure that the potential loss of parent compound is due to CYP-mediated metabolism.

For quantitative analysis of compounds 7b, 11b, and 12b, LC–MS systems were used. (i) LC–MS: Accela U-HPLC pump and an Accela autosampler (Thermo Fisher Scientific, USA) connected to an Exactive mass spectrometer (Orbitrap with accurate mass (Thermo Fisher Scientific, USA)); data handling with the standard software Xcalibur 2.1. (ii) LC–HRMS: Accela U-HPLC pump and an Accela Open autosampler (Thermo Fisher Scientific, USA) connected to an Q-Exactive mass spectrometer (Orbitrap); data handling with the standard software Xcalibur 2.2. (iii) LC–MS: Surveyor MS Plus HPLC (Thermo Electron) HPLC system connected to a TSQ Quantum Discovery Max (Thermo Electron) triple quadrupole mass spectrometer equipped with an electrospray (ESI) or APCI interface (Thermo Fisher Scientific, USA); connected to a PC running the standard software Xcalibur 2.0.7.

The pump flow rate was set to 600 μL/min, and the analytes were separated on a Kinetex phenylhexyl analytical column 2.6 μm, 50 mm × 2.1 mm (Phenomenex, Germany).

Metabolite Identification of Compound 11b

CYP1A1 and CYP1A2 incubates were screened for the presence of potential metabolites using LC–HRMS Q-Exactive (Thermo Scientific) instrument, combining high-performance quadrupole precursor selection with high resolution (up to 140 000) and accurate mass detection (Orbitrap). The full scan accurate mass analysis of suspected metabolites was confirmed or refused using three primary tools: (i) screening for prototypical phase I metabolites, (ii) predictive chemical formula and corresponding mass error analysis, and (iii) confirmation by product ion scan. The putative metabolites were identified based on the test item fragmentation pattern of compound 11b and its corresponding characteristic fragments.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00972.

Syntheses of bromo derivative 44 (Scheme S1), bromo derivative 49 (Scheme S2), and compounds 3a–5a and 3b–5b (Scheme S3); plasma concentration of 7b after a single po dose of 5 mg/kg in rats (Table S1); brain concentration of 7b after a single po dose of 5 mg/kg in rats (Table S2); plasma concentration of 12b after a single po dose of 5 mg/kg in rats (Table S3); brain concentration of 12b after a single po dose of 5 mg/kg in rats (Table S4); stability of compound 11b (5 μM) in primary mouse, rat, dog, and human hepatocytes (Table S5); CYP1A1 and CYP1A2 metabolites identification of 11b (Table S6); proposed metabolic pathway for CYP-dependent metabolism of 11b (Figure S1); chromatogram of compound 11b incubated with CYP1A1 (60 min) (Figure S2); chromatogram of compound 11b incubated with CYP1A1 (0 min) (Figure S3); chromatogram of compound 11b incubated with CYP1A2 (60 min) (Figure S4); chromatogram of compound 11b incubated with CYP1A2 (0 min) (Figure S5); activity data and standard errors of final compounds (Table S7); activity data and standard errors of compounds for modeling (Table S8); TREEspot data visualization of KINOMEScan interactions of compound 12b, PQR620, and INK128 (Figure S6); selectivity profile calculated from KinomeScan data (Table S9); kinase interactions (KINOMEscan data) (Table S10); ¹H NMR, ¹³C{¹H} NMR, and NSI-HRMS spectra; HPLC chromatograms; chemical structures of final compounds and intermediates (PDF)
Compound 3a-PI3Kγ (PDB)
Compound 2a-mTOR (PDB)
Compound 2b-mTOR (PDB)
Compound 2a-PI3Kα (PDB)
Compound 2b-PI3Kα (PDB)
Molecular formula strings and some data (CSV)

Accession Codes

PDB code 5JHB was used for docking of compound 3a into PI3Kγ. PDB code 4JT6 was used for docking of compounds 2a and 2b into mTOR kinase. PDB code 3ZIM was used for docking of compounds 2a and 2b into PI3Kα.

Terms & Conditions

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Author Information

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Corresponding Author
- Matthias P. Wymann - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland; http://orcid.org/0000-0003-3349-4281; Email: [email protected]
Authors
- Chiara Borsari - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland; http://orcid.org/0000-0002-4688-8362
- Denise Rageot - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland; http://orcid.org/0000-0002-2833-5481
- Alix Dall’Asen - PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
- Thomas Bohnacker - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
- Anna Melone - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
- Alexander M. Sele - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland; http://orcid.org/0000-0002-4903-7934
- Eileen Jackson - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
- Jean-Baptiste Langlois - Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland
- Florent Beaufils - PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
- Paul Hebeisen - PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
- Doriano Fabbro - PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
- Petra Hillmann - PIQUR Therapeutics AG, Hochbergerstrasse 60, 4057 Basel, Switzerland
Notes
The authors declare the following competing financial interest(s): A.D.A., F.B., P.He., P.Hi., and D.F. are current or past employees of PIQUR Therapeutics AG, Basel; and P.He., D.F., and M.P.W. are shareholders of PIQUR Therapeutics AG.

Acknowledgments

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We thank A. Pfaltz, J. Füglistaler, C. Meyer, J. Schwarte, and E. Teillet for advice, discussions, and contributions to synthetic efforts, and we thank S. Bünger for technical assistance. This work was supported by the Swiss Commission for Technology and Innovation (CTI) by PFLS-LS Grants 14032.1, 15811.2, and 17241.1; the Stiftung für Krebsbekämpfung Grant 341; and Swiss National Science Foundation Grants 310030_153211 and 316030_133860 (to M.P.W.).

Abbreviations Used

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mTOR	mechanistical (or mammalian) target of rapamycin
TORC1	mTOR complex 1
TORC2	mTOR complex 2
PI3K	phosphoinositide 3-kinase
PKB	protein kinase B/Akt
S6RP	ribosomal protein S6
S6K	p70 S6 kinase
VPS34	vacuolar protein sorting 34 (the class III PI3K)
TORKi	mTOR kinase inhibitor
PK	pharmacokinetic
TR-FRET	time-resolved Förster resonance energy transfer

References

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This article references 49 other publications.

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Motzer, Robert J.; Escudier, Bernard; Oudard, Stephane; Hutson, Thomas E.; Porta, Camillo; Bracarda, Sergio; Gruenwald, Viktor; Thompson, John A.; Figlin, Robert A.; Hollaender, Norbert; Urbanowitz, Gladys; Berg, William J.; Kay, Andrea; Lebwohl, David; Ravaud, Alain
Lancet (2008), 372 (9637), 449-456CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
Everolimus (RAD001) is an orally administered inhibitor of the mammalian target of rapamycin (mTOR), a therapeutic target for metastatic renal cell carcinoma. We did a phase III, randomized, double-blind, placebo-controlled trial of everolimus in patients with metastatic renal cell carcinoma whose disease had progressed on vascular endothelial growth factor-targeted therapy. Patients with metastatic renal cell carcinoma which had progressed on sunitinib, sorafenib, or both, were randomly assigned in a two to one ratio to receive everolimus 10 mg once daily (n=272) or placebo (n=138), in conjunction with best supportive care. Randomisation was done centrally via an interactive voice response system using a validated computer system, and was stratified by Memorial Sloan-Kettering Cancer Center prognostic score and previous anticancer therapy, with a permuted block size of six. The primary endpoint was progression-free survival, assessed via a blinded, independent central review. The study was designed to be terminated after 290 events of progression. Anal. was by intention to treat. This study is registered with, no. All randomized patients were included in efficacy analyses. The results of the second interim anal. indicated a significant difference in efficacy between arms and the trial was thus halted early after 191 progression events had been obsd. (101 [37%] events in the everolimus group, 90 [65%] in the placebo group; hazard ratio 0.30, 95% CI 0.22-0.40, p<0.0001; median progression-free survival 4.0 [95% CI 3.7-5.5] vs 1.9 [1.8-1.9] months). Stomatitis (107 [40%] patients in the everolimus group vs 11 [8%] in the placebo group), rash (66 [25%] vs six [4%]), and fatigue (53 [20%] vs 22 [16%]) were the most commonly reported adverse events, but were mostly mild or moderate in severity. Pneumonitis (any grade) was detected in 22 (8%) patients in the everolimus group, of whom eight had pneumonitis of grade 3 severity. Treatment with everolimus prolongs progression-free survival relative to placebo in patients with metastatic renal cell carcinoma that had progressed on other targeted therapies. Novartis Oncol.
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Jerusalem, G.; Rorive, A.; Collignon, J. Use of mTOR inhibitors in the treatment of breast cancer: an evaluation of factors that influence patient outcomes. Breast Cancer: Targets Ther. 2014, 6, 43– 57, DOI: 10.2147/BCTT.S38679
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Use of mTOR inhibitors in the treatment of breast cancer: an evaluation of factors that influence patient outcomes
Jerusalem, Guy; Rorive, Andree; Collignon, Joelle
Breast Cancer: Targets and Therapy (2014), 6 (), 43-57, 15CODEN: BCTTA9; ISSN:1179-1314. (Dove Medical Press Ltd.)
A review. Many systemic treatment options are available for advanced breast cancer, including endocrine therapy, chemotherapy, anti-human epidermal growth factor receptor 2 (HER2) therapy, and other targeted agents. Recently, everolimus, a mammalian target of rapamycin (mTOR) inhibitor, combined with exemestane, an aromatase inhibitor, has been approved in Europe and the USA for patients suffering from estrogen receptor-pos., HER2-neg. advanced breast cancer previously treated by a nonsteroidal aromatase inhibitor, based on the results of BOLERO-2 (Breast cancer trials of OraL EveROlimus). This study showed a statistically significant and clin. meaningful improvement in median progression-free survival. Results concerning the impact on overall survival are expected in the near future. This clin. oriented review focuses on the use of mTOR inhibitors in breast cancer. Results reported with first-generation mTOR inhibitors (ridaforolimus, temsirolimus, everolimus) are discussed. The current and potential role of mTOR inhibitors is reported according to breast cancer subtype (estrogen receptor-pos. HER2-neg., triple-neg., and HER2-pos. ER-pos./neg. disease). Everolimus is currently being evaluated in the adjuvant setting in high-risk estrogen receptor-pos., HER2-neg. early breast cancer. Continuing mTOR inhibition or alternatively administering other drugs targeting the phosphatidylinositol-3-kinase/protein kinase B-mTOR pathway after progression on treatments including an mTOR inhibitor is under evaluation. Potential biomarkers to select patients showing a more pronounced benefit are reviewed, but we are not currently using these biomarkers in routine practice. Subgroup anal. of BOLERO 2 has shown that the benefit is consistent in all subgroups and that it is impossible to select patients not benefiting from addn. of everolimus to exemestane. Side effects and impact on quality of life are other important issues discussed in this review. Second-generation mTOR inhibitors and dual mTOR-phosphatidylinositol-3-kinase inhibitors are currently being evaluated in clin. trials.
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André, F.; O’Regan, R.; Ozguroglu, M.; Toi, M.; Xu, B.; Jerusalem, G.; Masuda, N.; Wilks, S.; Arena, F.; Isaacs, C.; Yap, Y.-S.; Papai, Z.; Lang, I.; Armstrong, A.; Lerzo, G.; White, M.; Shen, K.; Litton, J.; Chen, D.; Zhang, Y.; Ali, S.; Taran, T.; Gianni, L. Everolimus for women with trastuzumab-resistant, HER2-positive, advanced breast cancer (BOLERO-3): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Oncol. 2014, 15 (6), 580– 591, DOI: 10.1016/S1470-2045(14)70138-X
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Everolimus for women with trastuzumab-resistant, HER2-positive, advanced breast cancer (BOLERO-3): a randomised, double-blind, placebo-controlled phase 3 trial
Andre, Fabrice; O'Regan, Ruth; Ozguroglu, Mustafa; Toi, Masakazu; Xu, Binghe; Jerusalem, Guy; Masuda, Norikazu; Wilks, Sharon; Arena, Francis; Isaacs, Claudine; Yap, Yoon-Sim; Papai, Zsuzsanna; Lang, Istvan; Armstrong, Anne; Lerzo, Guillermo; White, Michelle; Shen, Kunwei; Litton, Jennifer; Chen, David; Zhang, Yufen; Ali, Shyanne; Taran, Tetiana; Gianni, Luca
Lancet Oncology (2014), 15 (6), 580-591CODEN: LOANBN; ISSN:1470-2045. (Elsevier Ltd.)
Disease progression in patients with HER2-pos. breast cancer receiving trastuzumab might be assocd. with activation of the PI3K/Akt/mTOR intracellular signalling pathway. We aimed to assess whether the addn. of the mTOR inhibitor everolimus to trastuzumab might restore sensitivity to trastuzumab. In this randomised, double-blind, placebo-controlled, phase 3 trial, we recruited women with HER2-pos., trastuzumab-resistant, advanced breast carcinoma who had previously received taxane therapy. Eligible patients were randomly assigned (1:1) using a central patient screening and randomization system to daily everolimus (5 mg/day) plus weekly trastuzumab (2 mg/kg) and vinorelbine (25 mg/m2) or to placebo plus trastuzumab plus vinorelbine, in 3-wk cycles, stratified by previous lapatinib use. The primary endpoint was progression-free survival (PFS) by local assessment in the intention-to-treat population. We report the final anal. for PFS; overall survival follow-up is still in progress. This trial is registered with ClinicalTrials.gov, no. NCT01007942.Between Oct 26, 2009, and May 23, 2012, 569 patients were randomly assigned to everolimus (n=284) or placebo (n=285). Median follow-up at the time of anal. was 20·2 mo (IQR 15·0-27·1). Median PFS was 7·00 mo (95% CI 6·74-8·18) with everolimus and 5·78 mo (5·49-6·90) with placebo (hazard ratio 0·78 [95% CI 0·65-0·95]; p=0·0067). The most common grade 3-4 adverse events were neutropenia (204 [73%] of 280 patients in the everolimus group vs 175 [62%] of 282 patients in the placebo group), leucopenia (106 [38%] vs 82 [29%]), anemia (53 [19%] vs 17 [6%]), febrile neutropenia (44 [16%] vs ten [4%]), stomatitis (37 [13%] vs four [1%]), and fatigue (34 [12%] vs 11 [4%]). Serious adverse events were reported in 117 (42%) patients in the everolimus group and 55 (20%) in the placebo group; two on-treatment deaths due to adverse events occurred in each group. The addn. of everolimus to trastuzumab plus vinorelbine significantly prolongs PFS in patients with trastuzumab-resistant and taxane-pretreated, HER2-pos., advanced breast cancer. The clin. benefit should be considered in the context of the adverse event profile in this population. Novartis Pharmaceuticals Corporation.
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Peterson, M. E. Management of adverse events in patients with hormone receptor-positive breast cancer treated with everolimus: observations from a phase III clinical trial. Supportive care in cancer: official journal of the Multinational Association of Supportive Care in Cancer 2013, 21 (8), 2341– 2349, DOI: 10.1007/s00520-013-1826-3
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Management of adverse events in patients with hormone receptor-positive breast cancer treated with everolimus: observations from a phase III clinical trial
Peterson Mary E
Supportive care in cancer : official journal of the Multinational Association of Supportive Care in Cancer (2013), 21 (8), 2341-9 ISSN:.
Everolimus is a mammalian target of rapamycin (mTOR) inhibitor approved for the treatment of advanced renal cell carcinoma, pancreatic neuroendocrine tumors, subependymal giant cell astrocytoma associated with tuberous sclerosis complex, renal angiomyolipoma and tuberous sclerosis complex, and, in combination with exemestane, for hormone receptor-positive HER2-negative advanced breast cancer after failure of treatment with letrozole or anastrozole. Results from the phase III BOLERO-2 trial demonstrated that everolimus in combination with exemestane provided significant clinical benefit to patients with advanced hormone receptor-positive breast cancer. Although everolimus is generally well tolerated, as with most therapies administered in an advanced cancer setting, drug-related adverse events (AEs) inevitably occur. Most common AEs observed in the everolimus studies include stomatitis, rash, infection, noninfectious pneumonitis, and hyperglycemia. Clinical awareness and early identification of such AEs by oncology nurses are essential to dosing (interruptions, reduction, and treatment discontinuation); quality of life; and, ultimately, patient outcomes. Because everolimus has already been shown to significantly improve clinical efficacy in patients with advanced breast cancer, a proactive approach to the practical management of AEs associated with this mTOR inhibitor as well as other most common AEs observed in this patient population has been reviewed and outlined here.
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Santulli, G.; Totary-Jain, H. Tailoring mTOR-based therapy: molecular evidence and clinical challenges. Pharmacogenomics 2013, 14 (12), 1517– 1526, DOI: 10.2217/pgs.13.143
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Tailoring mTOR-based therapy: molecular evidence and clinical challenges
Santulli, Gaetano; Totary-Jain, Hana
Pharmacogenomics (2013), 14 (12), 1517-1526CODEN: PARMFL; ISSN:1462-2416. (Future Medicine Ltd.)
A review. The mTOR signaling pathway integrates inputs from a variety of upstream stimuli to regulate diverse cellular processes including proliferation, growth, survival, motility, autophagy, protein synthesis and metab. The mTOR pathway is dysregulated in a no. of human pathologies including cancer, diabetes, obesity, autoimmune disorders, neurol. disease and aging. Ongoing clin. trials testing mTOR-targeted treatments no. in the hundreds and underscore its therapeutic potential. To date mTOR inhibitors are clin. approved to prevent organ rejection, to inhibit restenosis after angioplasty, and to treat several advanced cancers. In this review we discuss the continuously evolving field of mTOR pharmacogenomics, as well as highlight the emerging efforts in identifying diagnostic and prognostic markers, including miRNAs, in order to assess successful therapeutic responses.
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Meng, L. H.; Zheng, X. F. Toward rapamycin analog (rapalog)-based precision cancer therapy. Acta Pharmacol. Sin. 2015, 36 (10), 1163– 1169, DOI: 10.1038/aps.2015.68
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Toward rapamycin analog (rapalog)-based precision cancer therapy
Meng, Ling-hua; Zheng, X. F. Steven
Acta Pharmacologica Sinica (2015), 36 (10), 1163-1169CODEN: APSCG5; ISSN:1671-4083. (Nature Publishing Group)
Rapamycin and its analogs (rapalogs) are the first generation of mTOR inhibitors, which have the same mol. scaffold, but different physiochem. properties. Rapalogs are being tested in a wide spectrum of human tumors as both monotherapy and a component of combination therapy. Among them, temsirolimus and everolimus have been approved for the treatment of breast and renal cancer. However, objective response rates with rapalogs in clin. trials are modest and variable. Identification of biomarkers predicting response to rapalogs, and discovery of drug combinations with improved efficacy and tolerated toxicity are crit. to moving this class of targeted therapeutics forward. This review focuses on the aberrations in the PI3K/mTOR pathway in human tumor cells or tissues as predictive biomarkers for rapalog efficacy. Recent results of combinational therapy using rapalogs and other anticancer drugs are documented. With the rapid development of next-generation genomic sequencing and precision medicine, rapalogs will provide greater benefits to cancer patients.
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Feldman, M. E.; Apsel, B.; Uotila, A.; Loewith, R.; Knight, Z. A.; Ruggero, D.; Shokat, K. M. Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol. 2009, 7 (2), e1000038, DOI: 10.1371/journal.pbio.1000038
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Kang, S. A.; Pacold, M. E.; Cervantes, C. L.; Lim, D.; Lou, H. J.; Ottina, K.; Gray, N. S.; Turk, B. E.; Yaffe, M. B.; Sabatini, D. M. mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin. Science 2013, 341 (6144), 1236566, DOI: 10.1126/science.1236566
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mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin
Kang Seong A; Pacold Michael E; Cervantes Christopher L; Lim Daniel; Lou Hua Jane; Ottina Kathleen; Gray Nathanael S; Turk Benjamin E; Yaffe Michael B; Sabatini David M
Science (New York, N.Y.) (2013), 341 (6144), 1236566 ISSN:.
The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs life span in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin, as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals.
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Jacinto, E.; Loewith, R.; Schmidt, A.; Lin, S.; Ruegg, M. A.; Hall, A.; Hall, M. N. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat. Cell Biol. 2004, 6 (11), 1122– 8, DOI: 10.1038/ncb1183
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Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive
Jacinto, Estela; Loewith, Robbie; Schmidt, Anja; Lin, Shuo; Rueegg, Markus A.; Hall, Alan; Hall, Michael N.
Nature Cell Biology (2004), 6 (11), 1122-1128CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth. In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2. A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals. Here, the authors report that a mammalian counterpart of TORC2 (mTORC2) also exists. The mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor. Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton. The mTORC2 is not upstream of the mTORC1 effector S6K. Thus, two distinct TOR complexes constitute a primordial signaling network conserved in eukaryotic evolution to control the fundamental process of cell growth.
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O’Reilly, K. E.; Rojo, F.; She, Q. B.; Solit, D.; Mills, G. B.; Smith, D.; Lane, H.; Hofmann, F.; Hicklin, D. J.; Ludwig, D. L.; Baselga, J.; Rosen, N. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006, 66 (3), 1500– 1508, DOI: 10.1158/0008-5472.CAN-05-2925
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mTOR Inhibition Induces Upstream Receptor Tyrosine Kinase Signaling and Activates Akt
O'Reilly, Kathryn E.; Rojo, Fredi; She, Qing-Bai; Solit, David; Mills, Gordon B.; Smith, Debra; Lane, Heidi; Hofmann, Francesco; Hicklin, Daniel J.; Ludwig, Dale L.; Baselga, Jose; Rosen, Neal
Cancer Research (2006), 66 (3), 1500-1508CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin deriv., RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity.
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Liu, P.; Gan, W.; Chin, Y. R.; Ogura, K.; Guo, J.; Zhang, J.; Wang, B.; Blenis, J.; Cantley, L. C.; Toker, A.; Su, B.; Wei, W. PtdIns(3,4,5)P3-dependent activation of the mTORC2 kinase complex. Cancer Discovery 2015, 5 (11), 1194– 1209, DOI: 10.1158/2159-8290.CD-15-0460
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PtdIns(3,4,5)P3-dependent activation of the mtorc2 kinase complex
Liu, Pengda; Gan, Wenjian; Chin, Y. Rebecca; Ogura, Kohei; Guo, Jianping; Zhang, Jinfang; Wang, Bin; Blenis, John; Cantley, Lewis C.; Toker, Alex; Su, Bing; Wei, Wenyi
Cancer Discovery (2015), 5 (11), 1194-1209CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)
MTOR serves as a central regulator of cell growth and metab. by forming two distinct complexes, mTORC1 and mTORC2. Although mechanisms of mTORC1 activation by growth factors and amino acids have been extensively studied, the upstream regulatory mechanisms leading to mTORC2 activation remain largely elusive. Here, we report that the pleckstrin homol. (PH) domain of SIN1, an essential and unique component of mTORC2, interacts with the mTOR kinase domain to suppress mTOR activity. More importantly, PtdIns(3,4,5)P3, but not other PtdInsPn species, interacts with SIN1-PH to release its inhibition on the mTOR kinase domain, thereby triggering mTORC2 activation. Mutating crit. SIN1 residues that mediate PtdIns(3,4,5)P3 interaction inactivates mTORC2, whereas mTORC2 activity is pathol. increased by patient-derived mutations in the SIN1-PH domain, promoting cell growth and tumor formation. Together, our study unravels a PI3K-dependent mechanism for mTORC2 activation, allowing mTORC2 to activate AKT in a manner that is regulated temporally and spatially by PtdIns(3,4,5)P3. Significance: The SIN1-PH domain interacts with the mTOR kinase domain to suppress mTOR activity, and PtdIns(3,4,5)P3 binds the SIN1-PH domain to release its inhibition on the mTOR kinase domain, leading to mTORC2 activation. Cancer patient-derived SIN1-PH domain mutations gain oncogenicity by loss of suppressing mTOR activity as a means to facilitate tumorigenesis. Cancer Discov; 5(11); 1194-209. ©2015 AACR. See related commentary by Yuan and Guan, p. 1127. This article is highlighted in the In This Issue feature, p.1111.
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Nowak, P.; Cole, D. C.; Brooijmans, N.; Bursavich, M. G.; Curran, K. J.; Ellingboe, J. W.; Gibbons, J. J.; Hollander, I.; Hu, Y.; Kaplan, J.; Malwitz, D. J.; Toral-Barza, L.; Verheijen, J. C.; Zask, A.; Zhang, W. G.; Yu, K. Discovery of potent and selective inhibitors of the mammalian target of rapamycin (mTOR) kinase. J. Med. Chem. 2009, 52 (22), 7081– 7089, DOI: 10.1021/jm9012642
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Discovery of Potent and Selective Inhibitors of the Mammalian Target of Rapamycin (mTOR) Kinase
Nowak, Pawel; Cole, Derek C.; Brooijmans, Natasja; Curran, Kevin J.; Ellingboe, John W.; Gibbons, James J.; Hollander, Irwin; Hu, Yong Bo; Kaplan, Joshua; Malwitz, David J.; Toral-Barza, Lourdes; Verheijen, Jeroen C.; Zask, Arie; Zhang, Wei-Guo; Yu, Ker
Journal of Medicinal Chemistry (2009), 52 (22), 7081-7089CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metab., and angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our compd. collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phosphoinositide 3-kinase (PI3K). Optimization of compd. 5a, employing an mTOR homol. model based on an X-ray crystal structure of closely related PI3Kγ led to the discovery of 6-(1H-indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent and selective mTOR inhibitor (mTOR IC50 = 9 nM; PI3Kα IC50 = 1962 nM). Compd. 5u selectively inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with potential for development for cancer therapy.
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Jin, Z.; Niu, H.; Wang, X.; Zhang, L.; Wang, Q.; Yang, A. Preclinical study of CC223 as a potential anti-ovarian cancer agent. Oncotarget 2017, 8 (35), 58469– 58479, DOI: 10.18632/oncotarget.17753
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Preclinical study of CC223 as a potential anti-ovarian cancer agent
Jin Zhenzhen; Niu Huanfu; Wang Xuenan; Wang Qin; Yang Aijun; Zhang Lei
Oncotarget (2017), 8 (35), 58469-58479 ISSN:.
Aberrant activation of mTOR contributes to ovarian cancer progression. CC223 is a novel and potent mTOR kinase inhibitor. The current study tested its activity against human ovarian cancer cells. We showed that CC223, at nM concentrations, inhibited survival and proliferation of established/primary human ovarian cancer cells. Further, significant apoptosis activation was observed in CC223-treated ovarian cancer cells. CC223 disrupted assembly of mTOR complex 1 (mTORC1) and mTORC2 in SKOV3 cells. Meanwhile, activation of mTORC1 and mTORC2 was almost completely blocked by CC223. Intriguingly, restoring mTOR activation by introduction of a constitutively-active Akt1 only partially inhibited CC223-induced cytotoxicity in SKOV3 cells. Further studies showed that CC223 inhibited sphingosine kinase 1 (SphK1) activity and induced reactive oxygen species (ROS) production in SKOV3 cells. At last, oral administration of CC223 potently inhibited SKOV3 xenografted tumor growth in nude mice. The results of this study imply that CC223 could be further studied as a potential anti-ovarian cancer agent.
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Slotkin, E. K.; Patwardhan, P. P.; Vasudeva, S. D.; de Stanchina, E.; Tap, W. D.; Schwartz, G. K. MLN0128, an ATP-competitive mTOR kinase inhibitor with potent in vitro and in vivo antitumor activity, as potential therapy for bone and soft-tissue sarcoma. Mol. Cancer Ther. 2015, 14 (2), 395– 406, DOI: 10.1158/1535-7163.MCT-14-0711
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MLN0128, an ATP-Competitive mTOR Kinase Inhibitor with Potent In Vitro and In Vivo Antitumor Activity, as Potential Therapy for Bone and Soft-Tissue Sarcoma
Slotkin, Emily K.; Patwardhan, Parag P.; Vasudeva, Shyamprasad D.; de Stanchina, Elisa; Tap, William D.; Schwartz, Gary K.
Molecular Cancer Therapeutics (2015), 14 (2), 395-406CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that exists in two complexes (mTORC1 and mTORC2) and integrates extracellular and intracellular signals to act as a master regulator of cell growth, survival, and metab. The PI3K/AKT/mTOR prosurvival pathway is often dysregulated in multiple sarcoma subtypes. First-generation allosteric inhibitors of mTORC1 (rapalogues) have been extensively tested with great preclin. promise, but have had limited clin. utility. Here, we report that MLN0128, a second-generation, ATP-competitive, pan-mTOR kinase inhibitor, acts on both mTORC1 and mTORC2 and has potent in vitro and in vivo antitumor activity in multiple sarcoma subtypes. In vitro, MLN0128 inhibits mTORC1/2 targets in a concn.-dependent fashion and shows striking antiproliferative effect in rhabdomyosarcoma (RMS), Ewing sarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, osteosarcoma, and liposarcoma. Unlike rapamycin, MLN0128 inhibits phosphorylation of 4EBP1 and NDRG1 as well as prevents the reactivation of pAKT that occurs via neg. feedback release with mTORC1 inhibition alone. In xenograft models, MLN0128 treatment results in suppression of tumor growth with two dosing schedules (1 mg/kg daily and 3 mg/kg b.i.d. t.i.w.). At the 3 mg/kg dosing schedule, MLN0128 treatment results in significantly better tumor growth suppression than rapamycin in RMS and Ewing sarcoma models. In addn., MLN0128 induces apoptosis in models of RMS both in vitro and in vivo. Results from our study strongly suggest that MLN0128 treatment should be explored further as potential therapy for sarcoma. Mol Cancer Ther; 14(2); 395-406. ©2014 AACR.
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Pike, K. G.; Malagu, K.; Hummersone, M. G.; Menear, K. A.; Duggan, H. M.; Gomez, S.; Martin, N. M.; Ruston, L.; Pass, S. L.; Pass, M. Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: the discovery of AZD8055 and AZD2014. Bioorg. Med. Chem. Lett. 2013, 23 (5), 1212– 1216, DOI: 10.1016/j.bmcl.2013.01.019
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Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: The discovery of AZD8055 and AZD2014
Pike, Kurt G.; Malagu, Karine; Hummersone, Marc G.; Menear, Keith A.; Duggan, Heather M. E.; Gomez, Sylvie; Martin, Niall M. B.; Ruston, Linette; Pass, Sarah L.; Pass, Martin
Bioorganic & Medicinal Chemistry Letters (2013), 23 (5), 1212-1216CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
The optimization of a potent and highly selective series of dual mTORC1 and mTORC2 inhibitors is described. An initial focus on improving cellular potency while maintaining or improving other key parameters, such as aq. soly. and margins over hERG IC50, led to the discovery of the clin. candidate AZD8055. Further optimization, particularly aimed at reducing the rate of metab. in human hepatocyte incubations, resulted in the discovery of the clin. candidate AZD2014.
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Lee, J. S.; Vo, T. T.; Fruman, D. A. Targeting mTOR for the treatment of B cell malignancies. Br. J. Clin. Pharmacol. 2016, 82 (5), 1213– 1228, DOI: 10.1111/bcp.12888
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Targeting mTOR for the treatment of B cell malignancies
Lee, Jong-Hoon Scott; Vo, Thanh-Trang; Fruman, David A.
British Journal of Clinical Pharmacology (2016), 82 (5), 1213-1228CODEN: BCPHBM; ISSN:1365-2125. (Wiley-Blackwell)
Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions as a key regulator of cell growth, division and survival. Many haematol. malignancies exhibit elevated or aberrant mTOR activation, supporting the launch of numerous clin. trials aimed at evaluating the potential of single agent mTOR-targeted therapies. While promising early clin. data using allosteric mTOR inhibitors (rapamycin and its derivs., rapalogs) have suggested activity in a subset of haematol. malignancies, these agents have shown limited efficacy in most contexts. Whether the efficacy of these partial mTOR inhibitors might be enhanced by more complete target inhibition is being actively addressed with second generation ATP-competitive mTOR kinase inhibitors (TOR-KIs), which have only recently entered clin. trials. However, emerging preclin. data suggest that despite their biochem. advantage over rapalogs, TOR-KIs may retain a primarily cytostatic response. Rather, combinations of mTOR inhibition with other targeted therapies have demonstrated promising efficacy in several preclin. models. This review investigates the current status of rapalogs and TOR-KIs in B cell malignancies, with an emphasis on emerging preclin. evidence of synergistic combinations involving mTOR inhibition.
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Rageot, D.; Bohnacker, T.; Melone, A.; Langlois, J. B.; Borsari, C.; Hillmann, P.; Sele, A. M.; Beaufils, F.; Zvelebil, M.; Hebeisen, P.; Loscher, W.; Burke, J.; Fabbro, D.; Wymann, M. P. Discovery and preclinical characterization of 5-[4,6-Bis({3-oxa-8-azabicyclo[3.2.1]octan-8-yl})-1,3,5-triazin-2-yl]-4-(difluoro methyl)pyridin-2-amine (PQR620), a highly potent and selective mTORC1/2 inhibitor for cancer and neurological disorders. J. Med. Chem. 2018, 61 (22), 10084– 10105, DOI: 10.1021/acs.jmedchem.8b01262
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Discovery and Preclinical Characterization of 5-[4,6-Bis({3-oxa-8-azabicyclo[3.2.1]octan-8-yl})-1,3,5-triazin-2-yl]-4-(difluoromethyl)pyridin-2-amine (PQR620), a Highly Potent and Selective mTORC1/2 Inhibitor for Cancer and Neurological Disorders
Rageot, Denise; Bohnacker, Thomas; Melone, Anna; Langlois, Jean-Baptiste; Borsari, Chiara; Hillmann, Petra; Sele, Alexander M.; Beaufils, Florent; Zvelebil, Marketa; Hebeisen, Paul; Loscher, Wolfgang; Burke, John; Fabbro, Doriano; Wymann, Matthias P.
Journal of Medicinal Chemistry (2018), 61 (22), 10084-10105CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. I is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. I showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague-Dawley mice, max. concn. (Cmax) in plasma and brain was reached after 30 min, with a half-life (t1/2) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of I inhibited tumor growth significantly. Moreover, I attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, I inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.
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Bohnacker, T.; Prota, A. E.; Beaufils, F.; Burke, J. E.; Melone, A.; Inglis, A. J.; Rageot, D.; Sele, A. M.; Cmiljanovic, V.; Cmiljanovic, N.; Bargsten, K.; Aher, A.; Akhmanova, A.; Diaz, J. F.; Fabbro, D.; Zvelebil, M.; Williams, R. L.; Steinmetz, M. O.; Wymann, M. P. Deconvolution of Buparlisib’s mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention. Nat. Commun. 2017, 8, 14683, DOI: 10.1038/ncomms14683
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Deconvolution of Buparlisib's mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention
Bohnacker Thomas; Beaufils Florent; Melone Anna; Rageot Denise; Sele Alexander M; Cmiljanovic Vladimir; Cmiljanovic Natasa; Wymann Matthias P; Prota Andrea E; Bargsten Katja; Steinmetz Michel O; Burke John E; Inglis Alison J; Williams Roger L; Aher Amol; Akhmanova Anna; Diaz J Fernando; Fabbro Doriano; Zvelebil Marketa
Nature communications (2017), 8 (), 14683 ISSN:.
BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.
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Beaufils, F.; Cmiljanovic, N.; Cmiljanovic, V.; Bohnacker, T.; Melone, A.; Marone, R.; Jackson, E.; Zhang, X.; Sele, A.; Borsari, C.; Mestan, J.; Hebeisen, P.; Hillmann, P.; Giese, B.; Zvelebil, M.; Fabbro, D.; Williams, R. L.; Rageot, D.; Wymann, M. P. 5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a potent, brain-penetrant, orally bioavailable, pan-class I PI3K/mTOR inhibitor as clinical candidate in oncology. J. Med. Chem. 2017, 60 (17), 7524– 7538, DOI: 10.1021/acs.jmedchem.7b00930
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5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology
Beaufils, Florent; Cmiljanovic, Natasa; Cmiljanovic, Vladimir; Bohnacker, Thomas; Melone, Anna; Marone, Romina; Jackson, Eileen; Zhang, Xuxiao; Sele, Alexander; Borsari, Chiara; Mestan, Jurgen; Hebeisen, Paul; Hillmann, Petra; Giese, Bernd; Zvelebil, Marketa; Fabbro, Doriano; Williams, Roger L.; Rageot, Denise; Wymann, Matthias P.
Journal of Medicinal Chemistry (2017), 60 (17), 7524-7538CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclin. characterization of compd. 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concns. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was obsd. for the structurally related 4 (BKM120, buparlisib). Compd. 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compd. 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compd.'s safety profile, identifies 1 as a clin. candidate with a broad application range in oncol., including treatment of brain tumors or CNS metastasis. Compd. 1 is currently in phase II clin. trials for advanced solid tumors and refractory lymphoma.
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Tarantelli, C.; Gaudio, E.; Arribas, A. J.; Kwee, I.; Hillmann, P.; Rinaldi, A.; Cascione, L.; Spriano, F.; Bernasconi, E.; Guidetti, F.; Carrassa, L.; Pittau, R. B.; Beaufils, F.; Ritschard, R.; Rageot, D.; Sele, A.; Dossena, B.; Rossi, F. M.; Zucchetto, A.; Taborelli, M.; Gattei, V.; Rossi, D.; Stathis, A.; Stussi, G.; Broggini, M.; Wymann, M. P.; Wicki, A.; Zucca, E.; Cmiljanovic, V.; Fabbro, D.; Bertoni, F. PQR309 is a novel dual PI3K/mTOR inhibitor with preclinical antitumor activity in lymphomas as a single agent and in combination therapy. Clin. Cancer Res. 2018, 24 (1), 120– 129, DOI: 10.1158/1078-0432.CCR-17-1041
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PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy
Tarantelli, Chiara; Gaudio, Eugenio; Arribas, Alberto J.; Kwee, Ivo; Hillmann, Petra; Rinaldi, Andrea; Cascione, Luciano; Spriano, Filippo; Bernasconi, Elena; Guidetti, Francesca; Carrassa, Laura; Pittau, Roberta Bordone; Beaufils, Florent; Ritschard, Reto; Rageot, Denise; Sele, Alexander; Dossena, Barbara; Rossi, Francesca Maria; Zucchetto, Antonella; Taborelli, Monica; Gattei, Valter; Rossi, Davide; Stathis, Anastasios; Stussi, Georg; Broggini, Massimo; Wymann, Matthias P.; Wicki, Andreas; Zucca, Emanuele; Cmiljanovic, Vladimir; Fabbro, Doriano; Bertoni, Francesco
Clinical Cancer Research (2018), 24 (1), 120-129CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacol. inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clin. evaluation, in preclin. lymphoma models. Exptl. Design: This study included preclin. in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation expts. on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors. Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was assocd. with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib. Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compd. that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120-9. ©2017 AACR.
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Wicki, A.; Brown, N.; Xyrafas, A.; Bize, V.; Hawle, H.; Berardi, S.; Cmiljanovic, N.; Cmiljanovic, V.; Stumm, M.; Dimitrijevic, S.; Herrmann, R.; Pretre, V.; Ritschard, R.; Tzankov, A.; Hess, V.; Childs, A.; Hierro, C.; Rodon, J.; Hess, D.; Joerger, M.; von Moos, R.; Sessa, C.; Kristeleit, R. First-in human, phase 1, dose-escalation pharmacokinetic and pharmacodynamic study of the oral dual PI3K and mTORC1/2 inhibitor PQR309 in patients with advanced solid tumors (SAKK 67/13). Eur. J. Cancer 2018, 96, 6– 16, DOI: 10.1016/j.ejca.2018.03.012
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First-in human, phase 1, dose-escalation pharmacokinetic and pharmacodynamic study of the oral dual PI3K and mTORC1/2 inhibitor PQR309 in patients with advanced solid tumors (SAKK 67/13)
Wicki, Andreas; Brown, Nicholas; Xyrafas, Alexandros; Bize, Vincent; Hawle, Hanne; Berardi, Simona; Cmiljanovic, Natasa; Cmiljanovic, Vladimir; Stumm, Michael; Dimitrijevic, Sasa; Herrmann, Richard; Pretre, Vincent; Ritschard, Reto; Tzankov, Alexandar; Hess, Viviane; Childs, Alexa; Hierro, Cinta; Rodon, Jordi; Hess, Dagmar; Joerger, Markus; von Moos, Roger; Sessa, Cristiana; Kristeleit, Rebecca
European Journal of Cancer (2018), 96 (), 6-16CODEN: EJCAEL; ISSN:0959-8049. (Elsevier Ltd.)
PQR309 is an orally bioavailable, balanced pan-phosphatidylinositol-3-kinase (PI3K), mammalian target of rapamycin (mTOR) C1 and mTORC2 inhibitor. This is an accelerated titrn., 3 D 3 dose-escalation, open-label phase Itrial of continuous once-daily (OD) PQR309 administration to evaluate the safety, pharmacokinetics (PK) and pharmacodynamics in patients with advanced solid tumors. Primary objectives were to det. the max. tolerated dose (MTD) and recommended phase 2 dose (RP2D).Twenty-eight patients were included in six dosing cohorts and treated at a daily PQR309 dose ranging from 10 to 150 mg. Common adverse events (AEs; ≥30% patients) included fatigue, hyperglycemia, nausea, diarrhea, constipation, rash, anorexia and vomiting. Grade (G) 3 or 4 drug-related AEs were seen in 13 (46%) and three (11%) patients, resp. Dose-limiting toxicity (DLT) was obsd. in two patients at 100 mg OD (>14-d interruption in PQR309 due to G3 rash, G2 hyperbilirubinemia, G4 suicide attempt; dose redn. due to G3 fatigue, G2 diarrhoea, G4 transaminitis) and one patient at 80 mg (G3 hyperglycemia >7 d). PK shows fast absorption (Tmax 1-2 h) and dose proportionality for Cmax and area under the curve. A partial response in a patient with metastatic thymus cancer, 24% disease vol. redn. in a patient with sinonasal cancer and stable disease for more than 16 wk in a patient with clear cell Bartholin's gland cancer were obsd.The MTD and RP2D of PQR309 is 80 mg of orally OD. PK is dose-proportional. PD shows PI3K pathway phosphoprotein downregulation in paired tumor biopsies. Clin. activity was obsd. in patients with and without PI3K pathway dysregulation.
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Fang, Z.; Song, Y.; Zhan, P.; Zhang, Q.; Liu, X. Conformational restriction: an effective tactic in “follow-on”-based drug discovery. Future Med. Chem. 2014, 6 (8), 885– 901, DOI: 10.4155/fmc.14.50
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Conformational restriction: an effective tactic in 'follow-on'-based drug discovery
Fang, Zengjun; Song, Yu'ning; Zhan, Peng; Zhang, Qingzhu; Liu, Xinyong
Future Medicinal Chemistry (2014), 6 (8), 885-901CODEN: FMCUA7; ISSN:1756-8919. (Future Science Ltd.)
A review. The conformational restriction (rigidification) of a flexible ligand has often been a commonly used strategy in drug design, as it can minimize the entropic loss assocd. with the ligand adopting a preferred conformation for binding, which leads to enhanced potency for a given physiol. target, improved selectivity for isoforms and reduced the possibility of drug metab. Therefore, the application of conformational restriction strategy is a core aspect of drug discovery and development that is widely practiced by medicinal chemists either deliberately or subliminally. The present review will highlight current representative examples and a brief overview on the rational design of conformationally restricted agents as well as discuss its advantages over the flexible counterparts.
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Cmiljanovic, V.; Hebeisen, P.; Jackson, E.; Beaufils, F.; Bohnacker, T.; Wymann, M. P. Conformationally Restricted PI3K and mTOR Inhibitors. Patent WO2015049369, 2015.
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Leroux, F. R.; Manteau, B.; Vors, J. P.; Pazenok, S. Trifluoromethyl ethers--synthesis and properties of an unusual substituent. Beilstein J. Org. Chem. 2008, 4 (13), DOI: 10.3762/bjoc.4.13 .
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Burger, M. T.; Pecchi, S.; Wagman, A.; Ni, Z. J.; Knapp, M.; Hendrickson, T.; Atallah, G.; Pfister, K.; Zhang, Y.; Bartulis, S.; Frazier, K.; Ng, S.; Smith, A.; Verhagen, J.; Haznedar, J.; Huh, K.; Iwanowicz, E.; Xin, X.; Menezes, D.; Merritt, H.; Lee, I.; Wiesmann, M.; Kaufman, S.; Crawford, K.; Chin, M.; Bussiere, D.; Shoemaker, K.; Zaror, I.; Maira, S. M.; Voliva, C. F. Identification of NVP-BKM120 as a potent, selective, orally bioavailable class I PI3 Kinase inhibitor for treating cancer. ACS Med. Chem. Lett. 2011, 2 (10), 774– 779, DOI: 10.1021/ml200156t
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Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer
Burger, Matthew T.; Pecchi, Sabina; Wagman, Allan; Ni, Zhi-Jie; Knapp, Mark; Hendrickson, Thomas; Atallah, Gordana; Pfister, Keith; Zhang, Yanchen; Bartulis, Sarah; Frazier, Kelly; Ng, Simon; Smith, Aaron; Verhagen, Joelle; Haznedar, Joshua; Huh, Kay; Iwanowicz, Ed; Xin, Xiaohua; Menezes, Daniel; Merritt, Hanne; Lee, Isabelle; Wiesmann, Marion; Kaufman, Susan; Crawford, Kenneth; Chin, Michael; Bussiere, Dirksen; Shoemaker, Kevin; Zaror, Isabel; Maira, Sauveur-Michel; Voliva, Charles F.
ACS Medicinal Chemistry Letters (2011), 2 (10), 774-779CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
Phosphoinositide-3-kinases (PI3Ks) are important oncol. targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clin. trials for the treatment of cancer.
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Rousseau, J. F.; Chekroun, I.; Ferey, V.; Labrosse, J. R. Concise preparation of a stable cyclic sulfamidate intermediate in the synthesis of a enantiopure chiral active diamine derivative. Org. Process Res. Dev. 2015, 19 (4), 506– 513, DOI: 10.1021/op500264v
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Concise Preparation of a Stable Cyclic Sulfamidate Intermediate in the Synthesis of a Enantiopure Chiral Active Diamine Derivative
Rousseau, Jean-Francois; Chekroun, Isaac; Ferey, Vincent; Labrosse, Jean Robert
Organic Process Research & Development (2015), 19 (4), 506-513CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
A potentially scalable route to the nonracemic antipsychotic candidate SSR 504374 I was developed using the stereoselective substitution reaction of nonracemic sulfamidate II with 2-chloro-3-trifluoromethylbenzamide as the key step. II was prepd. in seven steps from 2-benzoylpyridine by chemo- and diastereoselective hydrogenation, sepn. of the desired racemic erythro diastereomer, resoln. with di-p-toluoyl-(+)-tartaric acid, formation of a sulfamidite, and oxidn. at sulfur with RuCl3 and sodium hypochlorite; a workup for the sulfamidite oxidn. using isopropanol was developed to avoid darkening of the intermediate due to oxidn. by RuO2 and RuO4 left in the sulfamidate product.
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Brown, G. R.; Foubister, A. J.; Wright, B. Chiral synthesis of 3-substituted morpholines via serine enantiomers and reductions of 5-oxomorpholine-3-carboxylates. J. Chem. Soc., Perkin Trans. 1 1985, 1, 2577– 2580, DOI: 10.1039/p19850002577
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Hebeisen, P.; Alker, A.; Buerkler, M. Iterative one pot reactions of a chiral sulfamidate with 2,4,6-trichloropyridine: regiocontrolled synthesis of linear and angular chiral dipyrrolidino pyridines. Heterocycles 2012, 85 (1), 65– 72, DOI: 10.3987/COM-11-12360
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Iterative one pot reactions of a chiral sulfamidate with 2,4,6-trichloropyridine: Regiocontrolled synthesis of linear and angular chiral dipyrrolidino pyridines
Hebeisen, Paul; Alker, Andre; Buerkler, Markus
Heterocycles (2012), 85 (1), 65-72CODEN: HTCYAM; ISSN:0385-5414. (Japan Institute of Heterocyclic Chemistry)
The product of the ring opening of a chiral sulfamidate with the 3-lithiopyridine species obtained by deprotonation of 2,4,6-trichloropyridine with BuLi was deprotonated again in situ with BuLi and reacted with a 2nd equiv. of the sulfamidate furnishing a bis(β-aminoethyl)pyridine deriv., which could be cyclized regioselectively to linear or angular chiral dipyrrolidinopyridines.
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Zask, A.; Kaplan, J.; Verheijen, J. C.; Richard, D. J.; Curran, K.; Brooijmans, N.; Bennett, E. M.; Toral-Barza, L.; Hollander, I.; Ayral-Kaloustian, S.; Yu, K. Morpholine derivatives greatly enhance the selectivity of mammalian target of rapamycin (mTOR) inhibitors. J. Med. Chem. 2009, 52 (24), 7942– 7945, DOI: 10.1021/jm901415x
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Morpholine Derivatives Greatly Enhance the Selectivity of Mammalian Target of Rapamycin (mTOR) Inhibitors
Zask, Arie; Kaplan, Joshua; Verheijen, Jeroen C.; Richard, David J.; Curran, Kevin; Brooijmans, Natasja; Bennett, Eric M.; Toral-Barza, Lourdes; Hollander, Irwin; Ayral-Kaloustian, Semiramis; Yu, Ker
Journal of Medicinal Chemistry (2009), 52 (24), 7942-7945CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
Dramatic improvements in mTOR-targeting selectivity were achieved by replacing morpholine in pyrazolopyrimidine inhibitors with bridged morpholines. Analogs I [R1 = (R)- or (S)-3-methyl-4-morpholinyl, 2-methyl-4-morpholinyl, 8-oxa-3-azabicyclo[3.2.1]octan-3-yl, etc.; R2 = Me, Et, cyclopropyl, FCH2CH2, 3-pyridyl, 4-pyridyl; R3 = F3CCH2, 1-methoxycarbonyl-4-piperidinyl, 1-ethoxycarbonyl-4-piperidinyl, 3-pyridylmethyl] with subnanomolar mTOR IC50 values and up to 26000-fold selectivity vs. PI3Kα were prepd. Chiral morpholines gave inhibitors whose enantiomers had different selectivity and potency profiles. Mol. modeling suggests that a single amino acid difference between PI3K and mTOR (Phe961Leu) accounts for the profound selectivity seen by creating a deeper pocket in mTOR that can accommodate bridged morpholines.
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Thomas, V. H.; Bhattachar, S.; Hitchingham, L.; Zocharski, P.; Naath, M.; Surendran, N.; Stoner, C. L.; El-Kattan, A. The road map to oral bioavailability: an industrial perspective. Expert Opin. Drug Metab. Toxicol. 2006, 2 (4), 591– 608, DOI: 10.1517/17425255.2.4.591
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The road map to oral bioavailability: an industrial perspective
Thomas, V. Hayden; Bhattachar, Shobha; Hitchingham, Linda; Zocharski, Philip; Naath, Maryanne; Surendran, Narayanan; Stoner, Chad L.; El-Kattan, Ayman
Expert Opinion on Drug Metabolism & Toxicology (2006), 2 (4), 591-608CODEN: EODMAP; ISSN:1742-5255. (Informa Healthcare)
A review. Optimization of oral bioavailability is a continuing challenge for the pharmaceutical and biotechnol. industries. The no. of potential drug candidates requiring in vivo evaluation has significantly increased with the advent of combinatorial chem. In addn., drug discovery programs are increasingly forced into more lipophilic and lower soly. chem. space. To aid in the use of in vitro and in silico tools as well as reduce the no. of in vivo studies required, a team-based discussion tool is proposed that provides a road map' to guide the selection of profiling assays that should be considered when optimizing oral bioavailability. This road map divides the factors that contribute to poor oral bioavailability into two interrelated categories: absorption and metab. This road map provides an interface for cross discipline discussions and a systematic approach to the experimentation that drives the drug discovery process towards a common goal - acceptable oral bioavailability using minimal resources in an acceptable time frame.
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Mortensen, D. S.; Fultz, K. E.; Xu, S.; Xu, W.; Packard, G.; Khambatta, G.; Gamez, J. C.; Leisten, J.; Zhao, J.; Apuy, J.; Ghoreishi, K.; Hickman, M.; Narla, R. K.; Bissonette, R.; Richardson, S.; Peng, S. X.; Perrin-Ninkovic, S.; Tran, T.; Shi, T.; Yang, W. Q.; Tong, Z.; Cathers, B. E.; Moghaddam, M. F.; Canan, S. S.; Worland, P.; Sankar, S.; Raymon, H. K. CC-223, a potent and selective inhibitor of mTOR kinase: in vitro and in vivo characterization. Mol. Cancer Ther. 2015, 14 (6), 1295– 1305, DOI: 10.1158/1535-7163.MCT-14-1052
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CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization
Mortensen, Deborah S.; Fultz, Kimberly E.; Xu, Shuichan; Xu, Weiming; Packard, Garrick; Khambatta, Godrej; Gamez, James C.; Leisten, Jim; Zhao, Jingjing; Apuy, Julius; Ghoreishi, Kamran; Hickman, Matt; Narla, Rama Krishna; Bissonette, Rene; Richardson, Samantha; Peng, Sophie X.; Perrin-Ninkovic, Sophie; Tran, Tam; Shi, Tao; Yang, Wen Qing; Tong, Zeen; Cathers, Brian E.; Moghaddam, Mehran F.; Canan, Stacie S.; Worland, Peter; Sankar, Sabita; Raymon, Heather K.
Molecular Cancer Therapeutics (2015), 14 (6), 1295-1305CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
MTOR is a serine/threonine kinase that regulates cell growth, metab., proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are crit. mediators of the PI3K-AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogs, allosteric inhibitors that target only the mTORC1 complex, have shown some clin. activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclin. characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematol. and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematol. cancer cell lines. Correlative anal. revealed that IRF4 expression level assocs. with resistance, whereas mTOR pathway activation seems to assoc. with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223-treated tumors suggests that the obsd. antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clin. trials. Mol Cancer Ther; 14(6); 1295-305. ©2015 AACR.
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Nosik, P. S.; Ryabukhin, S. V.; Artamonov, O. S.; Grygorenko, O. O. Synthesis of trans-disubstituted pyrazolylcyclopropane building blocks. Monatsh. Chem. 2016, 147 (9), 1629– 1636, DOI: 10.1007/s00706-016-1726-6
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Synthesis of trans-disubstituted pyrazolylcyclopropane building blocks
Nosik, Pavel S.; Ryabukhin, Sergey V.; Artamonov, Oleksiy S.; Grygorenko, Oleksandr O.
Monatshefte fuer Chemie (2016), 147 (9), 1629-1636CODEN: MOCMB7; ISSN:0026-9247. (Springer-Verlag GmbH)
Diastereoselective synthesis of trans-disubstituted pyrazolylcyclopropane building blocks (i.e. carboxylic acids and amines) is described starting from easily available pyrazolecarbaldehydes. The key step of the synthesis was Corey-Chaikowsky cyclopropanation of the corresponding α,β-unsatd. Weinreb amides. The title compds. were prepd. in four or six steps and 32-60 and 17-40 % overall yields, resp., on up to 50 g scale. The building blocks obtained are good starting points for the design of lead-like libraries of peptidomimetic drugs.
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Zhang, L.; Luo, S.; Mi, X.; Liu, S.; Qiao, Y.; Xu, H.; Cheng, J. P. Combinatorial synthesis of functionalized chiral and doubly chiral ionic liquids and their applications as asymmetric covalent/non-covalent bifunctional organocatalysts. Org. Biomol. Chem. 2008, 6 (3), 567– 576, DOI: 10.1039/B713843A
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Combinatorial synthesis of functionalized chiral and doubly chiral ionic liquids and their applications as asymmetric covalent/non-covalent bifunctional organocatalysts
Zhang, Long; Luo, Sanzhong; Mi, Xueling; Liu, Song; Qiao, Yupu; Xu, Hui; Cheng, Jin-Pei
Organic & Biomolecular Chemistry (2008), 6 (3), 567-576CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
A facile combinatorial strategy was developed for the construction of libraries of functionalized chiral ionic liqs. (FCILs) including doubly chiral ionic liqs. and bis-functional chiral ionic liqs. These FCIL libraries have the potential to be used as asym. catalysts or chiral ligands. As an example, novel asym. bifunctional catalysts were developed by simultaneously incorporating functional groups onto the cation and anion. The resultant bis-functionalized CILs showed significantly improved stereoselectivity over the mono-functionalized parent CILs.
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Fabian, M. A.; Biggs, W. H.; Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lélias, J. M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 2005, 23, 329– 336, DOI: 10.1038/nbt1068
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A small molecule-kinase interaction map for clinical kinase inhibitors
Fabian, Miles A.; Biggs, William H.; Treiber, Daniel K.; Atteridge, Corey E.; Azimioara, Mihai D.; Benedetti, Michael G.; Carter, Todd A.; Ciceri, Pietro; Edeen, Philip T.; Floyd, Mark; Ford, Julia M.; Galvin, Margaret; Gerlach, Jay L.; Grotzfeld, Robert M.; Herrgard, Sanna; Insko, Darren E.; Insko, Michael A.; Lai, Andiliy G.; Lelias, Jean-Michel; Mehta, Shamal A.; Milanov, Zdravko V.; Velasco, Anne Marie; Wodicka, Lisa M.; Patel, Hitesh K.; Zarrinkar, Patrick P.; Lockhart, David J.
Nature Biotechnology (2005), 23 (3), 329-336CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
Kinase inhibitors show great promise as a new class of therapeutics. Here the authors describe an efficient way to det. kinase inhibitor specificity by measuring binding of small mols. to the ATP site of kinases. The authors have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clin. development, against a panel of 119 protein kinases. The authors find that specificity varies widely and is not strongly correlated with chem. structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. The authors also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small mol.-protein interaction map for clin. compds. across a large no. of related proteins.
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Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127– 132, DOI: 10.1038/nbt1358
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A quantitative analysis of kinase inhibitor selectivity
Karaman, Mazen W.; Herrgard, Sanna; Treiber, Daniel K.; Gallant, Paul; Atteridge, Corey E.; Campbell, Brian T.; Chan, Katrina W.; Ciceri, Pietro; Davis, Mindy I.; Edeen, Philip T.; Faraoni, Raffaella; Floyd, Mark; Hunt, Jeremy P.; Lockhart, Daniel J.; Milanov, Zdravko V.; Morrison, Michael J.; Pallares, Gabriel; Patel, Hitesh K.; Pritchard, Stephanie; Wodicka, Lisa M.; Zarrinkar, Patrick P.
Nature Biotechnology (2008), 26 (1), 127-132CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biol. consequences of multikinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. The authors present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global anal. of the results, the authors introduce the concept of a selectivity score as a general tool to quantify and differentiate the obsd. interaction patterns. The authors further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.
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Abstract
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Figure 1
Figure 1. Chemical structures of rapamycin and rapalogs and a selection of ATP-competitive mTOR kinase inhibitor (TORKi) compounds.
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Figure 2
Figure 2. Strategy for the development of mTOR selective inhibitors starting from PQR309 (1): rigidification strategy (red dotted lines) and removal of trifluoromethyl group from the 2-aminopyridine moiety (blue).
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Figure 3
Figure 3. (A) Docking of compound 3a (plum) into PI3Kγ (gray) starting from PDB code 5JHB (see ref (32)). Structural water molecules are shown in red, and water-mediated H-bonds are depicted as dashed black lines. (B) Docking of compound 2a (gold) and (C) compound 2b (green) into mTOR (turquoise) starting from PDB code 4JT6. The important features for mTOR selectivity are depicted in a ball and stick representation. (D) Docking of compound 2a (gold) and (E) compound 2b (green) into PI3Kα (gray) starting from PDB code 3ZIM. The exit vector from the restricted morpholine oxygen is shown as a black arrow.
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Scheme 1
Scheme 1. ^c
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^aPrepared according to ref (32).
^bPrepared according to procedure vii. After the reaction, the two regioisomers (26 and 32) were separated by column chromatography.
^c(A) Reagents and conditions: (i) (1) benzaldehyde, 2 M NaOH, rt, 30 min; (2) NaBH₄, 5 °C → rt, 1 h; (ii) (1) chloroacetyl chloride, K₂CO₃, THF/H₂O, 0 °C, 1 h; (2) NaOH, 5 °C, 2 h; (iii) borane–dimethyl sulfide complex, Et₃N, THF, 0 °C → 65 °C, 5 h; (iv) Pd/C, H₂, 2.8 bar, 48 h; (v) thionyl chloride, imidazole, DCM, −5 °C → rt → 0 °C, 2 h; (vi) ruthenium(IV) oxide hydrate, NaIO₄, rt, o/n. (B) Reagents and conditions: (vii) morpholine derivative (M_n–H), DIPEA, DCM, 0 °C → rt, o/n; (viii) (1) n-BuLi, CuI, −78 °C → rt, o/n; (2) HCl conc, MeOH, 45 °C, 4–6 h; (3) NaOH, H₂O, rt, 1–16 h; (ix) (1) boronic ester 38 or 41, XPhosPdG2 (cat.), K₃PO₄, dioxane/H₂O, 95 °C, 2–16 h; (2) HCl, dioxane/H₂O, 60 °C, 3–16 h (for 2a, 2b, 2c, 2d, 6a, 6b, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, and 14b); (x) 2-aminopyridine-5-boronic acid pinacol ester, XPhosPdG2 (cat.), K₃PO₄, dioxane/H₂O, 95 °C (for 11b), o/n; (xi) boronic ester 39, Pd(dppf)Cl₂ (cat.), CsCO₃, THF, Δ, o/n (for 12b); (xii) (1) boronic ester generated in situ, XPhosPdG2 (cat.), K₃PO₄, dioxane/H₂O, 95 °C, 3–3.5 h; (2) HCl, 80 °C, o/n (for 13b and 15b); (xiii) (1) boronic ester generated in situ, XPhosPdG2 (cat.), K₃PO₄, dioxane/H₂O, 95 °C, 2–16 h (isolated intermediates 18b and 19b); (2) 18b or 19b, TFA, DCM, 0 °C → rt, 1–3 h (for 16b and 17b).
Figure 4
Figure 4. Plasma and brain concentration of (A) compound 7b and (B) compound 12b after po dosing at 5 mg/kg in male Sprague Dawley rats. Stability of compound 11b (5 μM) with primary hepatocytes from (C) mice (green) and rats (turquoise) and (D) dogs (red) and humans (black) (n = 2). All values are the mean ± SEM. Error bars are not shown when smaller than the symbols.
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Figure 5
Figure 5. Pie chart showing the percentage of compound 11b and its metabolites after 60 min of incubation with human recombinant (A) CYP1A1 and (B) CYP1A2. (C) Major metabolites observed upon incubation of 11b with CYP1A1.
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  A review. Signaling lipids such as eicosanoids, phosphoinositides, sphingolipids and fatty acids control important cellular processes, including cell proliferation, apoptosis, metab. and migration. Extracellular signals from cytokines, growth factors and nutrients control the activity of a key set of lipid-modifying enzymes: phospholipases, prostaglandin synthase, 5-lipoxygenase, phosphoinositide 3-kinase, sphingosine kinase and sphingomyelinase. These enzymes and their downstream targets constitute a complex lipid signaling network with multiple nodes of interaction and cross-regulation. Imbalances in this network contribute to the pathogenesis of human disease. Although the function of a particular signaling lipid is traditionally studied in isolation, this review attempts a more integrated overview of the key role of these signaling lipids in inflammation, cancer and metabolic disease, and discusses emerging strategies for therapeutic intervention.
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2. 2
  Yang, H.; Rudge, D. G.; Koos, J. D.; Vaidialingam, B.; Yang, H. J.; Pavletich, N. P. mTOR kinase structure, mechanism and regulation. Nature 2013, 497 (7448), 217– 223, DOI: 10.1038/nature12122
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  mTOR kinase structure, mechanism and regulation
  Yang, Haijuan; Rudge, Derek G.; Koos, Joseph D.; Vaidialingam, Bhamini; Yang, Hyo J.; Pavletich, Nikola P.
  Nature (London, United Kingdom) (2013), 497 (7448), 217-223CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
  The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase-related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a complex of truncated mTOR and mammalian lethal with SEC13 protein 8 (mLST8) with an ATP transition state mimic (MgF3-) and with ATP-site inhibitors (Torin2, PP242, and PI-103). The structures reveal an intrinsically active kinase conformation, with catalytic residues and a catalytic mechanism remarkably similar to canonical protein kinases. The active site is highly recessed owing to the FKBP12-rapamycin-binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. MTOR-activating mutations map to the structural framework that holds these elements in place, indicating that the kinase is controlled by restricted access. In vitro biochem. shows that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. Rapamycin-FKBP12 inhibits the kinase by directly blocking substrate recruitment and by further restricting active-site access. The structures also reveal active-site residues and conformational changes that underlie inhibitor potency and specificity.
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3. 3
  Sarbassov, D. D.; Guertin, D. A.; Ali, S. M.; Sabatini, D. M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005, 307 (5712), 1098– 1101, DOI: 10.1126/science.1106148
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  Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex
  Sarbassov, Dos D.; Guertin, David A.; Ali, Siraj M.; Sabatini, David M.
  Science (Washington, DC, United States) (2005), 307 (5712), 1098-1101CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. We show that in Drosophila and human cells the target of rapamycin (TOR) kinase and its assocd. protein rictor are necessary for Ser473 phosphorylation and that a redn. in rictor or mammalian TOR (mTOR) expression inhibited an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation.
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4. 4
  Feng, J.; Park, J.; Cron, P.; Hess, D.; Hemmings, B. A. Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J. Biol. Chem. 2004, 279 (39), 41189– 41196, DOI: 10.1074/jbc.M406731200
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  Identification of a PKB/Akt Hydrophobic Motif Ser-473 Kinase as DNA-dependent Protein Kinase
  Feng, Jianhua; Park, Jongsun; Cron, Peter; Hess, Daniel; Hemmings, Brian A.
  Journal of Biological Chemistry (2004), 279 (39), 41189-41196CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), resp. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and assocd. with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a ∼10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.
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5. 5
  Wymann, M. P.; Marone, R. Phosphoinositide 3-kinase in disease: timing, location, and scaffolding. Curr. Opin. Cell Biol. 2005, 17 (2), 141– 149, DOI: 10.1016/j.ceb.2005.02.011
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  Phosphoinositide 3-kinase in disease: timing, location, and scaffolding
  Wymann, Matthias P.; Marone, Romina
  Current Opinion in Cell Biology (2005), 17 (2), 141-149CODEN: COCBE3; ISSN:0955-0674. (Elsevier Ltd.)
  A review. When PI3Ks are deregulated by aberrant surface receptors or modulators, accumulation of PtdIns(3,4,5)P3 leads to increased cell growth, proliferation and contact-independent survival. The PI3K/PKB/TOR axis controls protein synthesis and growth, while PtdIns(3,4,5)P3-mediated activation of Rho GTPases directs cell motility. PI3K activity has been linked to the formation of tumors, metastasis, chronic inflammation, allergy and cardiovascular disease. Although increased PtdIns(3,4,5)P3 is a well-established cause of disease, it is seldom known which PI3K isoform is implied. Recent work has demonstrated that PI3Kγ contributes to the control of cAMP levels in the cardiac system, where the protein acts as a scaffold, but not as a lipid kinase.
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6. 6
  Bozulic, L.; Hemmings, B. A. PIKKing on PKB: regulation of PKB activity by phosphorylation. Curr. Opin. Cell Biol. 2009, 21 (2), 256– 261, DOI: 10.1016/j.ceb.2009.02.002
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  PIKKing on PKB: regulation of PKB activity by phosphorylation
  Bozulic, Lana; Hemmings, Brian A.
  Current Opinion in Cell Biology (2009), 21 (2), 256-261CODEN: COCBE3; ISSN:0955-0674. (Elsevier B.V.)
  A review. Protein kinase B (PKB)/Akt kinase is a key regulator of a wide range of cellular processes including growth, proliferation and survival. PKB is clearly a crucial signaling mol. and extensive research efforts aim to understand its regulation and action. Recent studies of the regulation of PKB activity by hydrophobic motif phosphorylation have yielded several exciting findings about members of the phosphatidylinositol 3-kinase (PI3K)-like family of kinases (PIKKs) acting as PKB regulators. Mammalian target of rapamycin complex 2 (mTORC2) and DNA-dependent protein kinase (DNA-PK) can both phosphorylate Ser-473 and activate PKB. This review concerns PKB regulation by mTORC2 and DNA-PK in a stimulus-dependent and context-dependent manner and the possible implications of this for PKB activity, substrate specificity, and therapeutic intervention.
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7. 7
  Magnuson, B.; Ekim, B.; Fingar, D. C. Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem. J. 2012, 441 (1), 1– 21, DOI: 10.1042/BJ20110892
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  Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks
  Magnuson, Brian; Ekim, Bilgen; Fingar, Diane C.
  Biochemical Journal (2012), 441 (1), 1-21CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)
  A review. The ribosomal protein S6K (S6 kinase) represents an extensively studied effector of the TORC1 [TOR (target of rapamycin) complex 1], which possesses important yet incompletely defined roles in cellular and organismal physiol. TORC1 functions as an environmental sensor by integrating signals derived from diverse environmental cues to promote anabolic and inhibit catabolic cellular functions. mTORC1 (mammalian TORC1) phosphorylates and activates S6K1 and S6K2, whose first identified substrate was rpS6 (ribosomal protein S6), a component of the 40S ribosome. Studies over the past decade have uncovered a no. of addnl. S6K1 substrates, revealing multiple levels at which the mTORC1-S6K1 axis regulates cell physiol. The results thus far indicate that the mTORC1-S6K1 axis controls fundamental cellular processes, including transcription, translation, protein and lipid synthesis, cell growth/size and cell metab. In the present review we summarize the regulation of S6Ks, their cellular substrates and functions, and their integration within rapidly expanding mTOR (mammalian TOR) signalling networks. Although our understanding of the role of mTORC1-S6K1 signalling in physiol. remains in its infancy, evidence indicates that this signalling axis controls, at least in part, glucose homeostasis, insulin sensitivity, adipocyte metab., body mass and energy balance, tissue and organ size, learning, memory and aging. As dysregulation of this signalling axis contributes to diverse disease states, improved understanding of S6K regulation and function within mTOR signalling networks may enable the development of novel therapeutics.
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8. 8
  Laplante, M.; Sabatini, D. M. mTOR signaling in growth control and disease. Cell 2012, 149 (2), 274– 293, DOI: 10.1016/j.cell.2012.03.017
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  8
  mTOR signaling in growth control and disease
  Laplante, Mathieu; Sabatini, David M.
  Cell (Cambridge, MA, United States) (2012), 149 (2), 274-293CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
  A review. The mechanistic target of rapamycin (mTOR) signaling pathway senses and integrates a variety of environmental cues to regulate organismal growth and homeostasis. The pathway regulates many major cellular processes and is implicated in an increasing no. of pathol. conditions, including cancer, obesity, type 2 diabetes, and neurodegeneration. Here, we review recent advances in our understanding of the mTOR pathway and its role in health, disease, and aging. We further discuss pharmacol. approaches to treat human pathologies linked to mTOR deregulation.
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9. 9
  Saxton, R. A.; Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 2017, 168 (6), 960– 976, DOI: 10.1016/j.cell.2017.02.004
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  9
  mTOR signaling in growth, metabolism, and disease
  Saxton, Robert A.; Sabatini, David M.
  Cell (Cambridge, MA, United States) (2017), 168 (6), 960-976CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
  A review. The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metab. with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiol. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.
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10. 10
  Vivanco, I.; Sawyers, C. L. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat. Rev. Cancer 2002, 2 (7), 489– 501, DOI: 10.1038/nrc839
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  The phosphatidylinositol 3-Kinase-AKT pathway in human cancer
  Vivanco, Igor; Sawyers, Charles L.
  Nature Reviews Cancer (2002), 2 (7), 489-501CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
  A review. One signal that is overactivated in a wide range of tumor types is the prodn. of a phospholipid, phosphatidylinositol (3,4,5) trisphosphate, by phosphatidylinositol 3-kinase (PI3K). This lipid and the protein kinase that is activated by it, AKT, trigger a cascade of responses, from cell growth and proliferation to survival and motility, that drive tumor progression. Small-mol. therapeutics that block PI3K signaling might deal a severe blow to cancer cells by blocking many aspects of the tumor-cell phenotype.
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11. 11
  Marone, R.; Cmiljanovic, V.; Giese, B.; Wymann, M. P. Targeting phosphoinositide 3-kinase: moving towards therapy. Biochim. Biophys. Acta, Proteins Proteomics 2008, 1784 (1), 159– 185, DOI: 10.1016/j.bbapap.2007.10.003
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  Targeting phosphoinositide 3-kinase-Moving towards therapy
  Marone, Romina; Cmiljanovic, Vladimir; Giese, Bernd; Wymann, Matthias P.
  Biochimica et Biophysica Acta, Proteins and Proteomics (2008), 1784 (1), 159-185CODEN: BBAPBW; ISSN:1570-9639. (Elsevier Ltd.)
  A review. Phosphoinositide 3-kinases (PI3K) orchestrate cell responses including mitogenic signaling, cell survival and growth, metabolic control, vesicular trafficking, degranulation, cytoskeletal rearrangement and migration. Deregulation of the PI3K pathway occurs by activating mutations in growth factor receptors or the PIK3CA locus coding for PI3Kα, by loss of function of the lipid phosphatase and tensin homolog deleted in chromosome ten (PTEN/MMAC/TEP1), by the up-regulation of protein kinase B (PKB/Akt), or the impairment of the tuberous sclerosis complex (TSC1/2). All these events are linked to growth and proliferation, and have thus prompted a significant interest in the pharmaceutical targeting of the PI3K pathway in cancer. Genetic targeting of PI3Kγ (p110γ) and PI3Kδ (p110δ) in mice has underlined a central role of these PI3K isoforms in inflammation and allergy, as they modulate chemotaxis of leukocytes and degranulation in mast cells. Proof-of-concept mols. selective for PI3Kγ have already successfully alleviated disease progress in murine models of rheumatoid arthritis and lupus erythematosus. As targeting PI3K moves forward to therapy of chronic, non-fatal disease, safety concerns for PI3K inhibitors increase. Many of the present inhibitor series interfere with target of rapamycin (TOR), DNA-dependent protein kinase (DNA-PKcs) and activity of the ataxia telangiectasia mutated gene product (ATM). Here we review the current disease-relevant knowledge for isoform-specific PI3K function in the above mentioned diseases, and review the progress of > 400 recent patents covering pharmaceutical targeting of PI3K. Currently, several drugs targeting the PI3K pathway have entered clin. trials (phase I) for solid tumors and suppression of tissue damage after myocardial infarction (phases I,II).
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12. 12
  Wymann, M. PI3Ks—Drug Targets in Inflammation and Cancer. In Phosphoinositides I: Enzymes of Synthesis and Degradation; Balla, T., Wymann, M., York, J. D., Eds.; Springer: Dordrecht, The Netherlands, 2012; pp 111– 181.
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13. 13
  Choi, J.; Chen, J.; Schreiber, S. L.; Clardy, J. Structure of the FKBP1 2-rapamycin complex interacting with the binding domain of human FRAP. Science 1996, 273 (5272), 239– 242, DOI: 10.1126/science.273.5272.239
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  Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP
  Choi, Jungwon; Chen, Jie; Schreiber, Stuart L.; Clardy, Jon
  Science (Washington, D. C.) (1996), 273 (5272), 239-242CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  Rapamycin, a potent immunosuppressive agent, binds two proteins: the FK506-binding protein (FKBP12) and the FKBP-rapamycin-assocd. protein (FRAP). A crystal structure of the ternary complex of human FKBP12, rapamycin, and the FKBP12-rapamycin-binding (FRB) domain of human FRAP at a resoln. of 2.7 angstroms revealed the two proteins bound together as a result of the ability of rapamycin to occupy two different hydrophobic binding pockets simultaneously. The structure shows extensive interactions between rapamycin and both proteins, but fewer interactions between the proteins. The structure of the FRB domain of FRAP clarifies both rapamycin-independent and -dependent effects obsd. for mutants of FRAP and its homologs in the family of proteins related to the ataxia-telangiectasis mutant gene product, and it illustrates how a small cell-permeable mol. can mediate protein dimerization.
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14. 14
  Hudes, G.; Carducci, M.; Tomczak, P.; Dutcher, J.; Figlin, R.; Kapoor, A.; Staroslawska, E.; Sosman, J.; McDermott, D.; Bodrogi, I.; Kovacevic, Z.; Lesovoy, V.; Schmidt-Wolf, I. G. H.; Barbarash, O.; Gokmen, E.; O’Toole, T.; Lustgarten, S.; Moore, L.; Motzer, R. J. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N. Engl. J. Med. 2007, 356 (22), 2271– 2281, DOI: 10.1056/NEJMoa066838
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  Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma
  Hudes, Gary; Carducci, Michael; Tomczak, Piotr; Dutcher, Janice; Figlin, Robert; Kapoor, Anil; Staroslawska, Elzbieta; Sosman, Jeffrey; McDermott, David; Bodrogi, Istvan; Kovacevic, Zoran; Lesovoy, Vladimir; Schmidt-Wolf, Ingo G. H.; Barbarash, Olga; Gokmen, Erhan; O'Toole, Timothy; Lustgarten, Stephanie; Moore, Laurence; Motzer, Robert J.
  New England Journal of Medicine (2007), 356 (22), 2271-2281CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
  Interferon alfa is widely used for metastatic renal-cell carcinoma but has limited efficacy and tolerability. Temsirolimus, a specific inhibitor of the mammalian target of rapamycin kinase, may benefit patients with this disease. In this multicenter, phase 3 trial, we randomly assigned 626 patients with previously untreated, poor-prognosis metastatic renal-cell carcinoma to receive 25 mg of i.v. temsirolimus weekly, 3 million U of interferon alfa (with an increase to 18 million U) s.c. three times weekly, or combination therapy with 15 mg of temsirolimus weekly plus 6 million U of interferon alfa three times weekly. The primary end point was overall survival in comparisons of the temsirolimus group and the combination-therapy group with the interferon group. RESULTS Patients who received temsirolimus alone had longer overall survival (hazard ratio for death, 0.73; 95% confidence interval [CI], 0.58 to 0.92; P = 0.008) and progression-free survival (P < 0.001) than did patients who received interferon alone. Overall survival in the combination-therapy group did not differ significantly from that in the interferon group (hazard ratio, 0.96; 95% CI, 0.76 to 1.20; P = 0.70). Median overall survival times in the interferon group, the temsirolimus group, and the combination-therapy group were 7.3, 10.9, and 8.4 mo, resp. Rash, peripheral edema, hyperglycemia, and hyperlipidemia were more common in the temsirolimus group, whereas asthenia was more common in the interferon group. There were fewer patients with serious adverse events in the temsirolimus group than in the interferon group (P = 0.02). As compared with interferon alfa, temsirolimus improved overall survival among patients with metastatic renal-cell carcinoma and a poor prognosis. The addn. of temsirolimus to interferon did not improve survival. (ClinicalTrials.gov no., NCT00065468.).
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15. 15
  Motzer, R. J.; Escudier, B.; Oudard, S.; Hutson, T. E.; Porta, C.; Bracarda, S.; Grünwald, V.; Thompson, J. A.; Figlin, R. A.; Hollaender, N.; Urbanowitz, G.; Berg, W. J.; Kay, A.; Lebwohl, D.; Ravaud, A. Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet 2008, 372 (9637), 449– 456, DOI: 10.1016/S0140-6736(08)61039-9
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  15
  Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomized, placebo-controlled phase III trial
  Motzer, Robert J.; Escudier, Bernard; Oudard, Stephane; Hutson, Thomas E.; Porta, Camillo; Bracarda, Sergio; Gruenwald, Viktor; Thompson, John A.; Figlin, Robert A.; Hollaender, Norbert; Urbanowitz, Gladys; Berg, William J.; Kay, Andrea; Lebwohl, David; Ravaud, Alain
  Lancet (2008), 372 (9637), 449-456CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
  Everolimus (RAD001) is an orally administered inhibitor of the mammalian target of rapamycin (mTOR), a therapeutic target for metastatic renal cell carcinoma. We did a phase III, randomized, double-blind, placebo-controlled trial of everolimus in patients with metastatic renal cell carcinoma whose disease had progressed on vascular endothelial growth factor-targeted therapy. Patients with metastatic renal cell carcinoma which had progressed on sunitinib, sorafenib, or both, were randomly assigned in a two to one ratio to receive everolimus 10 mg once daily (n=272) or placebo (n=138), in conjunction with best supportive care. Randomisation was done centrally via an interactive voice response system using a validated computer system, and was stratified by Memorial Sloan-Kettering Cancer Center prognostic score and previous anticancer therapy, with a permuted block size of six. The primary endpoint was progression-free survival, assessed via a blinded, independent central review. The study was designed to be terminated after 290 events of progression. Anal. was by intention to treat. This study is registered with, no. All randomized patients were included in efficacy analyses. The results of the second interim anal. indicated a significant difference in efficacy between arms and the trial was thus halted early after 191 progression events had been obsd. (101 [37%] events in the everolimus group, 90 [65%] in the placebo group; hazard ratio 0.30, 95% CI 0.22-0.40, p<0.0001; median progression-free survival 4.0 [95% CI 3.7-5.5] vs 1.9 [1.8-1.9] months). Stomatitis (107 [40%] patients in the everolimus group vs 11 [8%] in the placebo group), rash (66 [25%] vs six [4%]), and fatigue (53 [20%] vs 22 [16%]) were the most commonly reported adverse events, but were mostly mild or moderate in severity. Pneumonitis (any grade) was detected in 22 (8%) patients in the everolimus group, of whom eight had pneumonitis of grade 3 severity. Treatment with everolimus prolongs progression-free survival relative to placebo in patients with metastatic renal cell carcinoma that had progressed on other targeted therapies. Novartis Oncol.
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16. 16
  Jerusalem, G.; Rorive, A.; Collignon, J. Use of mTOR inhibitors in the treatment of breast cancer: an evaluation of factors that influence patient outcomes. Breast Cancer: Targets Ther. 2014, 6, 43– 57, DOI: 10.2147/BCTT.S38679
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  16
  Use of mTOR inhibitors in the treatment of breast cancer: an evaluation of factors that influence patient outcomes
  Jerusalem, Guy; Rorive, Andree; Collignon, Joelle
  Breast Cancer: Targets and Therapy (2014), 6 (), 43-57, 15CODEN: BCTTA9; ISSN:1179-1314. (Dove Medical Press Ltd.)
  A review. Many systemic treatment options are available for advanced breast cancer, including endocrine therapy, chemotherapy, anti-human epidermal growth factor receptor 2 (HER2) therapy, and other targeted agents. Recently, everolimus, a mammalian target of rapamycin (mTOR) inhibitor, combined with exemestane, an aromatase inhibitor, has been approved in Europe and the USA for patients suffering from estrogen receptor-pos., HER2-neg. advanced breast cancer previously treated by a nonsteroidal aromatase inhibitor, based on the results of BOLERO-2 (Breast cancer trials of OraL EveROlimus). This study showed a statistically significant and clin. meaningful improvement in median progression-free survival. Results concerning the impact on overall survival are expected in the near future. This clin. oriented review focuses on the use of mTOR inhibitors in breast cancer. Results reported with first-generation mTOR inhibitors (ridaforolimus, temsirolimus, everolimus) are discussed. The current and potential role of mTOR inhibitors is reported according to breast cancer subtype (estrogen receptor-pos. HER2-neg., triple-neg., and HER2-pos. ER-pos./neg. disease). Everolimus is currently being evaluated in the adjuvant setting in high-risk estrogen receptor-pos., HER2-neg. early breast cancer. Continuing mTOR inhibition or alternatively administering other drugs targeting the phosphatidylinositol-3-kinase/protein kinase B-mTOR pathway after progression on treatments including an mTOR inhibitor is under evaluation. Potential biomarkers to select patients showing a more pronounced benefit are reviewed, but we are not currently using these biomarkers in routine practice. Subgroup anal. of BOLERO 2 has shown that the benefit is consistent in all subgroups and that it is impossible to select patients not benefiting from addn. of everolimus to exemestane. Side effects and impact on quality of life are other important issues discussed in this review. Second-generation mTOR inhibitors and dual mTOR-phosphatidylinositol-3-kinase inhibitors are currently being evaluated in clin. trials.
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17. 17
  André, F.; O’Regan, R.; Ozguroglu, M.; Toi, M.; Xu, B.; Jerusalem, G.; Masuda, N.; Wilks, S.; Arena, F.; Isaacs, C.; Yap, Y.-S.; Papai, Z.; Lang, I.; Armstrong, A.; Lerzo, G.; White, M.; Shen, K.; Litton, J.; Chen, D.; Zhang, Y.; Ali, S.; Taran, T.; Gianni, L. Everolimus for women with trastuzumab-resistant, HER2-positive, advanced breast cancer (BOLERO-3): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Oncol. 2014, 15 (6), 580– 591, DOI: 10.1016/S1470-2045(14)70138-X
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  17
  Everolimus for women with trastuzumab-resistant, HER2-positive, advanced breast cancer (BOLERO-3): a randomised, double-blind, placebo-controlled phase 3 trial
  Andre, Fabrice; O'Regan, Ruth; Ozguroglu, Mustafa; Toi, Masakazu; Xu, Binghe; Jerusalem, Guy; Masuda, Norikazu; Wilks, Sharon; Arena, Francis; Isaacs, Claudine; Yap, Yoon-Sim; Papai, Zsuzsanna; Lang, Istvan; Armstrong, Anne; Lerzo, Guillermo; White, Michelle; Shen, Kunwei; Litton, Jennifer; Chen, David; Zhang, Yufen; Ali, Shyanne; Taran, Tetiana; Gianni, Luca
  Lancet Oncology (2014), 15 (6), 580-591CODEN: LOANBN; ISSN:1470-2045. (Elsevier Ltd.)
  Disease progression in patients with HER2-pos. breast cancer receiving trastuzumab might be assocd. with activation of the PI3K/Akt/mTOR intracellular signalling pathway. We aimed to assess whether the addn. of the mTOR inhibitor everolimus to trastuzumab might restore sensitivity to trastuzumab. In this randomised, double-blind, placebo-controlled, phase 3 trial, we recruited women with HER2-pos., trastuzumab-resistant, advanced breast carcinoma who had previously received taxane therapy. Eligible patients were randomly assigned (1:1) using a central patient screening and randomization system to daily everolimus (5 mg/day) plus weekly trastuzumab (2 mg/kg) and vinorelbine (25 mg/m2) or to placebo plus trastuzumab plus vinorelbine, in 3-wk cycles, stratified by previous lapatinib use. The primary endpoint was progression-free survival (PFS) by local assessment in the intention-to-treat population. We report the final anal. for PFS; overall survival follow-up is still in progress. This trial is registered with ClinicalTrials.gov, no. NCT01007942.Between Oct 26, 2009, and May 23, 2012, 569 patients were randomly assigned to everolimus (n=284) or placebo (n=285). Median follow-up at the time of anal. was 20·2 mo (IQR 15·0-27·1). Median PFS was 7·00 mo (95% CI 6·74-8·18) with everolimus and 5·78 mo (5·49-6·90) with placebo (hazard ratio 0·78 [95% CI 0·65-0·95]; p=0·0067). The most common grade 3-4 adverse events were neutropenia (204 [73%] of 280 patients in the everolimus group vs 175 [62%] of 282 patients in the placebo group), leucopenia (106 [38%] vs 82 [29%]), anemia (53 [19%] vs 17 [6%]), febrile neutropenia (44 [16%] vs ten [4%]), stomatitis (37 [13%] vs four [1%]), and fatigue (34 [12%] vs 11 [4%]). Serious adverse events were reported in 117 (42%) patients in the everolimus group and 55 (20%) in the placebo group; two on-treatment deaths due to adverse events occurred in each group. The addn. of everolimus to trastuzumab plus vinorelbine significantly prolongs PFS in patients with trastuzumab-resistant and taxane-pretreated, HER2-pos., advanced breast cancer. The clin. benefit should be considered in the context of the adverse event profile in this population. Novartis Pharmaceuticals Corporation.
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  Peterson, M. E. Management of adverse events in patients with hormone receptor-positive breast cancer treated with everolimus: observations from a phase III clinical trial. Supportive care in cancer: official journal of the Multinational Association of Supportive Care in Cancer 2013, 21 (8), 2341– 2349, DOI: 10.1007/s00520-013-1826-3
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  18
  Management of adverse events in patients with hormone receptor-positive breast cancer treated with everolimus: observations from a phase III clinical trial
  Peterson Mary E
  Supportive care in cancer : official journal of the Multinational Association of Supportive Care in Cancer (2013), 21 (8), 2341-9 ISSN:.
  Everolimus is a mammalian target of rapamycin (mTOR) inhibitor approved for the treatment of advanced renal cell carcinoma, pancreatic neuroendocrine tumors, subependymal giant cell astrocytoma associated with tuberous sclerosis complex, renal angiomyolipoma and tuberous sclerosis complex, and, in combination with exemestane, for hormone receptor-positive HER2-negative advanced breast cancer after failure of treatment with letrozole or anastrozole. Results from the phase III BOLERO-2 trial demonstrated that everolimus in combination with exemestane provided significant clinical benefit to patients with advanced hormone receptor-positive breast cancer. Although everolimus is generally well tolerated, as with most therapies administered in an advanced cancer setting, drug-related adverse events (AEs) inevitably occur. Most common AEs observed in the everolimus studies include stomatitis, rash, infection, noninfectious pneumonitis, and hyperglycemia. Clinical awareness and early identification of such AEs by oncology nurses are essential to dosing (interruptions, reduction, and treatment discontinuation); quality of life; and, ultimately, patient outcomes. Because everolimus has already been shown to significantly improve clinical efficacy in patients with advanced breast cancer, a proactive approach to the practical management of AEs associated with this mTOR inhibitor as well as other most common AEs observed in this patient population has been reviewed and outlined here.
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19. 19
  Santulli, G.; Totary-Jain, H. Tailoring mTOR-based therapy: molecular evidence and clinical challenges. Pharmacogenomics 2013, 14 (12), 1517– 1526, DOI: 10.2217/pgs.13.143
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  19
  Tailoring mTOR-based therapy: molecular evidence and clinical challenges
  Santulli, Gaetano; Totary-Jain, Hana
  Pharmacogenomics (2013), 14 (12), 1517-1526CODEN: PARMFL; ISSN:1462-2416. (Future Medicine Ltd.)
  A review. The mTOR signaling pathway integrates inputs from a variety of upstream stimuli to regulate diverse cellular processes including proliferation, growth, survival, motility, autophagy, protein synthesis and metab. The mTOR pathway is dysregulated in a no. of human pathologies including cancer, diabetes, obesity, autoimmune disorders, neurol. disease and aging. Ongoing clin. trials testing mTOR-targeted treatments no. in the hundreds and underscore its therapeutic potential. To date mTOR inhibitors are clin. approved to prevent organ rejection, to inhibit restenosis after angioplasty, and to treat several advanced cancers. In this review we discuss the continuously evolving field of mTOR pharmacogenomics, as well as highlight the emerging efforts in identifying diagnostic and prognostic markers, including miRNAs, in order to assess successful therapeutic responses.
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20. 20
  Meng, L. H.; Zheng, X. F. Toward rapamycin analog (rapalog)-based precision cancer therapy. Acta Pharmacol. Sin. 2015, 36 (10), 1163– 1169, DOI: 10.1038/aps.2015.68
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  20
  Toward rapamycin analog (rapalog)-based precision cancer therapy
  Meng, Ling-hua; Zheng, X. F. Steven
  Acta Pharmacologica Sinica (2015), 36 (10), 1163-1169CODEN: APSCG5; ISSN:1671-4083. (Nature Publishing Group)
  Rapamycin and its analogs (rapalogs) are the first generation of mTOR inhibitors, which have the same mol. scaffold, but different physiochem. properties. Rapalogs are being tested in a wide spectrum of human tumors as both monotherapy and a component of combination therapy. Among them, temsirolimus and everolimus have been approved for the treatment of breast and renal cancer. However, objective response rates with rapalogs in clin. trials are modest and variable. Identification of biomarkers predicting response to rapalogs, and discovery of drug combinations with improved efficacy and tolerated toxicity are crit. to moving this class of targeted therapeutics forward. This review focuses on the aberrations in the PI3K/mTOR pathway in human tumor cells or tissues as predictive biomarkers for rapalog efficacy. Recent results of combinational therapy using rapalogs and other anticancer drugs are documented. With the rapid development of next-generation genomic sequencing and precision medicine, rapalogs will provide greater benefits to cancer patients.
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21. 21
  Feldman, M. E.; Apsel, B.; Uotila, A.; Loewith, R.; Knight, Z. A.; Ruggero, D.; Shokat, K. M. Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol. 2009, 7 (2), e1000038, DOI: 10.1371/journal.pbio.1000038
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  There is no corresponding record for this reference.
22. 22
  Kang, S. A.; Pacold, M. E.; Cervantes, C. L.; Lim, D.; Lou, H. J.; Ottina, K.; Gray, N. S.; Turk, B. E.; Yaffe, M. B.; Sabatini, D. M. mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin. Science 2013, 341 (6144), 1236566, DOI: 10.1126/science.1236566
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  22
  mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin
  Kang Seong A; Pacold Michael E; Cervantes Christopher L; Lim Daniel; Lou Hua Jane; Ottina Kathleen; Gray Nathanael S; Turk Benjamin E; Yaffe Michael B; Sabatini David M
  Science (New York, N.Y.) (2013), 341 (6144), 1236566 ISSN:.
  The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs life span in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin, as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals.
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23. 23
  Jacinto, E.; Loewith, R.; Schmidt, A.; Lin, S.; Ruegg, M. A.; Hall, A.; Hall, M. N. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat. Cell Biol. 2004, 6 (11), 1122– 8, DOI: 10.1038/ncb1183
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  23
  Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive
  Jacinto, Estela; Loewith, Robbie; Schmidt, Anja; Lin, Shuo; Rueegg, Markus A.; Hall, Alan; Hall, Michael N.
  Nature Cell Biology (2004), 6 (11), 1122-1128CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
  The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth. In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2. A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals. Here, the authors report that a mammalian counterpart of TORC2 (mTORC2) also exists. The mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor. Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton. The mTORC2 is not upstream of the mTORC1 effector S6K. Thus, two distinct TOR complexes constitute a primordial signaling network conserved in eukaryotic evolution to control the fundamental process of cell growth.
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24. 24
  O’Reilly, K. E.; Rojo, F.; She, Q. B.; Solit, D.; Mills, G. B.; Smith, D.; Lane, H.; Hofmann, F.; Hicklin, D. J.; Ludwig, D. L.; Baselga, J.; Rosen, N. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006, 66 (3), 1500– 1508, DOI: 10.1158/0008-5472.CAN-05-2925
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  24
  mTOR Inhibition Induces Upstream Receptor Tyrosine Kinase Signaling and Activates Akt
  O'Reilly, Kathryn E.; Rojo, Fredi; She, Qing-Bai; Solit, David; Mills, Gordon B.; Smith, Debra; Lane, Heidi; Hofmann, Francesco; Hicklin, Daniel J.; Ludwig, Dale L.; Baselga, Jose; Rosen, Neal
  Cancer Research (2006), 66 (3), 1500-1508CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
  Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin deriv., RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity.
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25. 25
  Liu, P.; Gan, W.; Chin, Y. R.; Ogura, K.; Guo, J.; Zhang, J.; Wang, B.; Blenis, J.; Cantley, L. C.; Toker, A.; Su, B.; Wei, W. PtdIns(3,4,5)P3-dependent activation of the mTORC2 kinase complex. Cancer Discovery 2015, 5 (11), 1194– 1209, DOI: 10.1158/2159-8290.CD-15-0460
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  25
  PtdIns(3,4,5)P3-dependent activation of the mtorc2 kinase complex
  Liu, Pengda; Gan, Wenjian; Chin, Y. Rebecca; Ogura, Kohei; Guo, Jianping; Zhang, Jinfang; Wang, Bin; Blenis, John; Cantley, Lewis C.; Toker, Alex; Su, Bing; Wei, Wenyi
  Cancer Discovery (2015), 5 (11), 1194-1209CODEN: CDAIB2; ISSN:2159-8274. (American Association for Cancer Research)
  MTOR serves as a central regulator of cell growth and metab. by forming two distinct complexes, mTORC1 and mTORC2. Although mechanisms of mTORC1 activation by growth factors and amino acids have been extensively studied, the upstream regulatory mechanisms leading to mTORC2 activation remain largely elusive. Here, we report that the pleckstrin homol. (PH) domain of SIN1, an essential and unique component of mTORC2, interacts with the mTOR kinase domain to suppress mTOR activity. More importantly, PtdIns(3,4,5)P3, but not other PtdInsPn species, interacts with SIN1-PH to release its inhibition on the mTOR kinase domain, thereby triggering mTORC2 activation. Mutating crit. SIN1 residues that mediate PtdIns(3,4,5)P3 interaction inactivates mTORC2, whereas mTORC2 activity is pathol. increased by patient-derived mutations in the SIN1-PH domain, promoting cell growth and tumor formation. Together, our study unravels a PI3K-dependent mechanism for mTORC2 activation, allowing mTORC2 to activate AKT in a manner that is regulated temporally and spatially by PtdIns(3,4,5)P3. Significance: The SIN1-PH domain interacts with the mTOR kinase domain to suppress mTOR activity, and PtdIns(3,4,5)P3 binds the SIN1-PH domain to release its inhibition on the mTOR kinase domain, leading to mTORC2 activation. Cancer patient-derived SIN1-PH domain mutations gain oncogenicity by loss of suppressing mTOR activity as a means to facilitate tumorigenesis. Cancer Discov; 5(11); 1194-209. ©2015 AACR. See related commentary by Yuan and Guan, p. 1127. This article is highlighted in the In This Issue feature, p.1111.
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26. 26
  Nowak, P.; Cole, D. C.; Brooijmans, N.; Bursavich, M. G.; Curran, K. J.; Ellingboe, J. W.; Gibbons, J. J.; Hollander, I.; Hu, Y.; Kaplan, J.; Malwitz, D. J.; Toral-Barza, L.; Verheijen, J. C.; Zask, A.; Zhang, W. G.; Yu, K. Discovery of potent and selective inhibitors of the mammalian target of rapamycin (mTOR) kinase. J. Med. Chem. 2009, 52 (22), 7081– 7089, DOI: 10.1021/jm9012642
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  26
  Discovery of Potent and Selective Inhibitors of the Mammalian Target of Rapamycin (mTOR) Kinase
  Nowak, Pawel; Cole, Derek C.; Brooijmans, Natasja; Curran, Kevin J.; Ellingboe, John W.; Gibbons, James J.; Hollander, Irwin; Hu, Yong Bo; Kaplan, Joshua; Malwitz, David J.; Toral-Barza, Lourdes; Verheijen, Jeroen C.; Zask, Arie; Zhang, Wei-Guo; Yu, Ker
  Journal of Medicinal Chemistry (2009), 52 (22), 7081-7089CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
  The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metab., and angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our compd. collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phosphoinositide 3-kinase (PI3K). Optimization of compd. 5a, employing an mTOR homol. model based on an X-ray crystal structure of closely related PI3Kγ led to the discovery of 6-(1H-indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent and selective mTOR inhibitor (mTOR IC50 = 9 nM; PI3Kα IC50 = 1962 nM). Compd. 5u selectively inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with potential for development for cancer therapy.
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27. 27
  Jin, Z.; Niu, H.; Wang, X.; Zhang, L.; Wang, Q.; Yang, A. Preclinical study of CC223 as a potential anti-ovarian cancer agent. Oncotarget 2017, 8 (35), 58469– 58479, DOI: 10.18632/oncotarget.17753
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  27
  Preclinical study of CC223 as a potential anti-ovarian cancer agent
  Jin Zhenzhen; Niu Huanfu; Wang Xuenan; Wang Qin; Yang Aijun; Zhang Lei
  Oncotarget (2017), 8 (35), 58469-58479 ISSN:.
  Aberrant activation of mTOR contributes to ovarian cancer progression. CC223 is a novel and potent mTOR kinase inhibitor. The current study tested its activity against human ovarian cancer cells. We showed that CC223, at nM concentrations, inhibited survival and proliferation of established/primary human ovarian cancer cells. Further, significant apoptosis activation was observed in CC223-treated ovarian cancer cells. CC223 disrupted assembly of mTOR complex 1 (mTORC1) and mTORC2 in SKOV3 cells. Meanwhile, activation of mTORC1 and mTORC2 was almost completely blocked by CC223. Intriguingly, restoring mTOR activation by introduction of a constitutively-active Akt1 only partially inhibited CC223-induced cytotoxicity in SKOV3 cells. Further studies showed that CC223 inhibited sphingosine kinase 1 (SphK1) activity and induced reactive oxygen species (ROS) production in SKOV3 cells. At last, oral administration of CC223 potently inhibited SKOV3 xenografted tumor growth in nude mice. The results of this study imply that CC223 could be further studied as a potential anti-ovarian cancer agent.
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28. 28
  Slotkin, E. K.; Patwardhan, P. P.; Vasudeva, S. D.; de Stanchina, E.; Tap, W. D.; Schwartz, G. K. MLN0128, an ATP-competitive mTOR kinase inhibitor with potent in vitro and in vivo antitumor activity, as potential therapy for bone and soft-tissue sarcoma. Mol. Cancer Ther. 2015, 14 (2), 395– 406, DOI: 10.1158/1535-7163.MCT-14-0711
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  28
  MLN0128, an ATP-Competitive mTOR Kinase Inhibitor with Potent In Vitro and In Vivo Antitumor Activity, as Potential Therapy for Bone and Soft-Tissue Sarcoma
  Slotkin, Emily K.; Patwardhan, Parag P.; Vasudeva, Shyamprasad D.; de Stanchina, Elisa; Tap, William D.; Schwartz, Gary K.
  Molecular Cancer Therapeutics (2015), 14 (2), 395-406CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
  The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that exists in two complexes (mTORC1 and mTORC2) and integrates extracellular and intracellular signals to act as a master regulator of cell growth, survival, and metab. The PI3K/AKT/mTOR prosurvival pathway is often dysregulated in multiple sarcoma subtypes. First-generation allosteric inhibitors of mTORC1 (rapalogues) have been extensively tested with great preclin. promise, but have had limited clin. utility. Here, we report that MLN0128, a second-generation, ATP-competitive, pan-mTOR kinase inhibitor, acts on both mTORC1 and mTORC2 and has potent in vitro and in vivo antitumor activity in multiple sarcoma subtypes. In vitro, MLN0128 inhibits mTORC1/2 targets in a concn.-dependent fashion and shows striking antiproliferative effect in rhabdomyosarcoma (RMS), Ewing sarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, osteosarcoma, and liposarcoma. Unlike rapamycin, MLN0128 inhibits phosphorylation of 4EBP1 and NDRG1 as well as prevents the reactivation of pAKT that occurs via neg. feedback release with mTORC1 inhibition alone. In xenograft models, MLN0128 treatment results in suppression of tumor growth with two dosing schedules (1 mg/kg daily and 3 mg/kg b.i.d. t.i.w.). At the 3 mg/kg dosing schedule, MLN0128 treatment results in significantly better tumor growth suppression than rapamycin in RMS and Ewing sarcoma models. In addn., MLN0128 induces apoptosis in models of RMS both in vitro and in vivo. Results from our study strongly suggest that MLN0128 treatment should be explored further as potential therapy for sarcoma. Mol Cancer Ther; 14(2); 395-406. ©2014 AACR.
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  Pike, K. G.; Malagu, K.; Hummersone, M. G.; Menear, K. A.; Duggan, H. M.; Gomez, S.; Martin, N. M.; Ruston, L.; Pass, S. L.; Pass, M. Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: the discovery of AZD8055 and AZD2014. Bioorg. Med. Chem. Lett. 2013, 23 (5), 1212– 1216, DOI: 10.1016/j.bmcl.2013.01.019
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  29
  Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: The discovery of AZD8055 and AZD2014
  Pike, Kurt G.; Malagu, Karine; Hummersone, Marc G.; Menear, Keith A.; Duggan, Heather M. E.; Gomez, Sylvie; Martin, Niall M. B.; Ruston, Linette; Pass, Sarah L.; Pass, Martin
  Bioorganic & Medicinal Chemistry Letters (2013), 23 (5), 1212-1216CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
  The optimization of a potent and highly selective series of dual mTORC1 and mTORC2 inhibitors is described. An initial focus on improving cellular potency while maintaining or improving other key parameters, such as aq. soly. and margins over hERG IC50, led to the discovery of the clin. candidate AZD8055. Further optimization, particularly aimed at reducing the rate of metab. in human hepatocyte incubations, resulted in the discovery of the clin. candidate AZD2014.
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30. 30
  Lee, J. S.; Vo, T. T.; Fruman, D. A. Targeting mTOR for the treatment of B cell malignancies. Br. J. Clin. Pharmacol. 2016, 82 (5), 1213– 1228, DOI: 10.1111/bcp.12888
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  Targeting mTOR for the treatment of B cell malignancies
  Lee, Jong-Hoon Scott; Vo, Thanh-Trang; Fruman, David A.
  British Journal of Clinical Pharmacology (2016), 82 (5), 1213-1228CODEN: BCPHBM; ISSN:1365-2125. (Wiley-Blackwell)
  Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions as a key regulator of cell growth, division and survival. Many haematol. malignancies exhibit elevated or aberrant mTOR activation, supporting the launch of numerous clin. trials aimed at evaluating the potential of single agent mTOR-targeted therapies. While promising early clin. data using allosteric mTOR inhibitors (rapamycin and its derivs., rapalogs) have suggested activity in a subset of haematol. malignancies, these agents have shown limited efficacy in most contexts. Whether the efficacy of these partial mTOR inhibitors might be enhanced by more complete target inhibition is being actively addressed with second generation ATP-competitive mTOR kinase inhibitors (TOR-KIs), which have only recently entered clin. trials. However, emerging preclin. data suggest that despite their biochem. advantage over rapalogs, TOR-KIs may retain a primarily cytostatic response. Rather, combinations of mTOR inhibition with other targeted therapies have demonstrated promising efficacy in several preclin. models. This review investigates the current status of rapalogs and TOR-KIs in B cell malignancies, with an emphasis on emerging preclin. evidence of synergistic combinations involving mTOR inhibition.
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  Rageot, D.; Bohnacker, T.; Melone, A.; Langlois, J. B.; Borsari, C.; Hillmann, P.; Sele, A. M.; Beaufils, F.; Zvelebil, M.; Hebeisen, P.; Loscher, W.; Burke, J.; Fabbro, D.; Wymann, M. P. Discovery and preclinical characterization of 5-[4,6-Bis({3-oxa-8-azabicyclo[3.2.1]octan-8-yl})-1,3,5-triazin-2-yl]-4-(difluoro methyl)pyridin-2-amine (PQR620), a highly potent and selective mTORC1/2 inhibitor for cancer and neurological disorders. J. Med. Chem. 2018, 61 (22), 10084– 10105, DOI: 10.1021/acs.jmedchem.8b01262
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  Discovery and Preclinical Characterization of 5-[4,6-Bis({3-oxa-8-azabicyclo[3.2.1]octan-8-yl})-1,3,5-triazin-2-yl]-4-(difluoromethyl)pyridin-2-amine (PQR620), a Highly Potent and Selective mTORC1/2 Inhibitor for Cancer and Neurological Disorders
  Rageot, Denise; Bohnacker, Thomas; Melone, Anna; Langlois, Jean-Baptiste; Borsari, Chiara; Hillmann, Petra; Sele, Alexander M.; Beaufils, Florent; Zvelebil, Marketa; Hebeisen, Paul; Loscher, Wolfgang; Burke, John; Fabbro, Doriano; Wymann, Matthias P.
  Journal of Medicinal Chemistry (2018), 61 (22), 10084-10105CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
  Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. I is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. I showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague-Dawley mice, max. concn. (Cmax) in plasma and brain was reached after 30 min, with a half-life (t1/2) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of I inhibited tumor growth significantly. Moreover, I attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, I inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.
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  Bohnacker, T.; Prota, A. E.; Beaufils, F.; Burke, J. E.; Melone, A.; Inglis, A. J.; Rageot, D.; Sele, A. M.; Cmiljanovic, V.; Cmiljanovic, N.; Bargsten, K.; Aher, A.; Akhmanova, A.; Diaz, J. F.; Fabbro, D.; Zvelebil, M.; Williams, R. L.; Steinmetz, M. O.; Wymann, M. P. Deconvolution of Buparlisib’s mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention. Nat. Commun. 2017, 8, 14683, DOI: 10.1038/ncomms14683
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  Deconvolution of Buparlisib's mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention
  Bohnacker Thomas; Beaufils Florent; Melone Anna; Rageot Denise; Sele Alexander M; Cmiljanovic Vladimir; Cmiljanovic Natasa; Wymann Matthias P; Prota Andrea E; Bargsten Katja; Steinmetz Michel O; Burke John E; Inglis Alison J; Williams Roger L; Aher Amol; Akhmanova Anna; Diaz J Fernando; Fabbro Doriano; Zvelebil Marketa
  Nature communications (2017), 8 (), 14683 ISSN:.
  BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.
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  Beaufils, F.; Cmiljanovic, N.; Cmiljanovic, V.; Bohnacker, T.; Melone, A.; Marone, R.; Jackson, E.; Zhang, X.; Sele, A.; Borsari, C.; Mestan, J.; Hebeisen, P.; Hillmann, P.; Giese, B.; Zvelebil, M.; Fabbro, D.; Williams, R. L.; Rageot, D.; Wymann, M. P. 5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a potent, brain-penetrant, orally bioavailable, pan-class I PI3K/mTOR inhibitor as clinical candidate in oncology. J. Med. Chem. 2017, 60 (17), 7524– 7538, DOI: 10.1021/acs.jmedchem.7b00930
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  5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology
  Beaufils, Florent; Cmiljanovic, Natasa; Cmiljanovic, Vladimir; Bohnacker, Thomas; Melone, Anna; Marone, Romina; Jackson, Eileen; Zhang, Xuxiao; Sele, Alexander; Borsari, Chiara; Mestan, Jurgen; Hebeisen, Paul; Hillmann, Petra; Giese, Bernd; Zvelebil, Marketa; Fabbro, Doriano; Williams, Roger L.; Rageot, Denise; Wymann, Matthias P.
  Journal of Medicinal Chemistry (2017), 60 (17), 7524-7538CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
  Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclin. characterization of compd. 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concns. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was obsd. for the structurally related 4 (BKM120, buparlisib). Compd. 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compd. 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compd.'s safety profile, identifies 1 as a clin. candidate with a broad application range in oncol., including treatment of brain tumors or CNS metastasis. Compd. 1 is currently in phase II clin. trials for advanced solid tumors and refractory lymphoma.
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  Tarantelli, C.; Gaudio, E.; Arribas, A. J.; Kwee, I.; Hillmann, P.; Rinaldi, A.; Cascione, L.; Spriano, F.; Bernasconi, E.; Guidetti, F.; Carrassa, L.; Pittau, R. B.; Beaufils, F.; Ritschard, R.; Rageot, D.; Sele, A.; Dossena, B.; Rossi, F. M.; Zucchetto, A.; Taborelli, M.; Gattei, V.; Rossi, D.; Stathis, A.; Stussi, G.; Broggini, M.; Wymann, M. P.; Wicki, A.; Zucca, E.; Cmiljanovic, V.; Fabbro, D.; Bertoni, F. PQR309 is a novel dual PI3K/mTOR inhibitor with preclinical antitumor activity in lymphomas as a single agent and in combination therapy. Clin. Cancer Res. 2018, 24 (1), 120– 129, DOI: 10.1158/1078-0432.CCR-17-1041
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  PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy
  Tarantelli, Chiara; Gaudio, Eugenio; Arribas, Alberto J.; Kwee, Ivo; Hillmann, Petra; Rinaldi, Andrea; Cascione, Luciano; Spriano, Filippo; Bernasconi, Elena; Guidetti, Francesca; Carrassa, Laura; Pittau, Roberta Bordone; Beaufils, Florent; Ritschard, Reto; Rageot, Denise; Sele, Alexander; Dossena, Barbara; Rossi, Francesca Maria; Zucchetto, Antonella; Taborelli, Monica; Gattei, Valter; Rossi, Davide; Stathis, Anastasios; Stussi, Georg; Broggini, Massimo; Wymann, Matthias P.; Wicki, Andreas; Zucca, Emanuele; Cmiljanovic, Vladimir; Fabbro, Doriano; Bertoni, Francesco
  Clinical Cancer Research (2018), 24 (1), 120-129CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)
  Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacol. inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clin. evaluation, in preclin. lymphoma models. Exptl. Design: This study included preclin. in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation expts. on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors. Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was assocd. with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib. Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compd. that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120-9. ©2017 AACR.
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  Wicki, A.; Brown, N.; Xyrafas, A.; Bize, V.; Hawle, H.; Berardi, S.; Cmiljanovic, N.; Cmiljanovic, V.; Stumm, M.; Dimitrijevic, S.; Herrmann, R.; Pretre, V.; Ritschard, R.; Tzankov, A.; Hess, V.; Childs, A.; Hierro, C.; Rodon, J.; Hess, D.; Joerger, M.; von Moos, R.; Sessa, C.; Kristeleit, R. First-in human, phase 1, dose-escalation pharmacokinetic and pharmacodynamic study of the oral dual PI3K and mTORC1/2 inhibitor PQR309 in patients with advanced solid tumors (SAKK 67/13). Eur. J. Cancer 2018, 96, 6– 16, DOI: 10.1016/j.ejca.2018.03.012
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  First-in human, phase 1, dose-escalation pharmacokinetic and pharmacodynamic study of the oral dual PI3K and mTORC1/2 inhibitor PQR309 in patients with advanced solid tumors (SAKK 67/13)
  Wicki, Andreas; Brown, Nicholas; Xyrafas, Alexandros; Bize, Vincent; Hawle, Hanne; Berardi, Simona; Cmiljanovic, Natasa; Cmiljanovic, Vladimir; Stumm, Michael; Dimitrijevic, Sasa; Herrmann, Richard; Pretre, Vincent; Ritschard, Reto; Tzankov, Alexandar; Hess, Viviane; Childs, Alexa; Hierro, Cinta; Rodon, Jordi; Hess, Dagmar; Joerger, Markus; von Moos, Roger; Sessa, Cristiana; Kristeleit, Rebecca
  European Journal of Cancer (2018), 96 (), 6-16CODEN: EJCAEL; ISSN:0959-8049. (Elsevier Ltd.)
  PQR309 is an orally bioavailable, balanced pan-phosphatidylinositol-3-kinase (PI3K), mammalian target of rapamycin (mTOR) C1 and mTORC2 inhibitor. This is an accelerated titrn., 3 D 3 dose-escalation, open-label phase Itrial of continuous once-daily (OD) PQR309 administration to evaluate the safety, pharmacokinetics (PK) and pharmacodynamics in patients with advanced solid tumors. Primary objectives were to det. the max. tolerated dose (MTD) and recommended phase 2 dose (RP2D).Twenty-eight patients were included in six dosing cohorts and treated at a daily PQR309 dose ranging from 10 to 150 mg. Common adverse events (AEs; ≥30% patients) included fatigue, hyperglycemia, nausea, diarrhea, constipation, rash, anorexia and vomiting. Grade (G) 3 or 4 drug-related AEs were seen in 13 (46%) and three (11%) patients, resp. Dose-limiting toxicity (DLT) was obsd. in two patients at 100 mg OD (>14-d interruption in PQR309 due to G3 rash, G2 hyperbilirubinemia, G4 suicide attempt; dose redn. due to G3 fatigue, G2 diarrhoea, G4 transaminitis) and one patient at 80 mg (G3 hyperglycemia >7 d). PK shows fast absorption (Tmax 1-2 h) and dose proportionality for Cmax and area under the curve. A partial response in a patient with metastatic thymus cancer, 24% disease vol. redn. in a patient with sinonasal cancer and stable disease for more than 16 wk in a patient with clear cell Bartholin's gland cancer were obsd.The MTD and RP2D of PQR309 is 80 mg of orally OD. PK is dose-proportional. PD shows PI3K pathway phosphoprotein downregulation in paired tumor biopsies. Clin. activity was obsd. in patients with and without PI3K pathway dysregulation.
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  Fang, Z.; Song, Y.; Zhan, P.; Zhang, Q.; Liu, X. Conformational restriction: an effective tactic in “follow-on”-based drug discovery. Future Med. Chem. 2014, 6 (8), 885– 901, DOI: 10.4155/fmc.14.50
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  Conformational restriction: an effective tactic in 'follow-on'-based drug discovery
  Fang, Zengjun; Song, Yu'ning; Zhan, Peng; Zhang, Qingzhu; Liu, Xinyong
  Future Medicinal Chemistry (2014), 6 (8), 885-901CODEN: FMCUA7; ISSN:1756-8919. (Future Science Ltd.)
  A review. The conformational restriction (rigidification) of a flexible ligand has often been a commonly used strategy in drug design, as it can minimize the entropic loss assocd. with the ligand adopting a preferred conformation for binding, which leads to enhanced potency for a given physiol. target, improved selectivity for isoforms and reduced the possibility of drug metab. Therefore, the application of conformational restriction strategy is a core aspect of drug discovery and development that is widely practiced by medicinal chemists either deliberately or subliminally. The present review will highlight current representative examples and a brief overview on the rational design of conformationally restricted agents as well as discuss its advantages over the flexible counterparts.
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  Cmiljanovic, V.; Hebeisen, P.; Jackson, E.; Beaufils, F.; Bohnacker, T.; Wymann, M. P. Conformationally Restricted PI3K and mTOR Inhibitors. Patent WO2015049369, 2015.
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  Leroux, F. R.; Manteau, B.; Vors, J. P.; Pazenok, S. Trifluoromethyl ethers--synthesis and properties of an unusual substituent. Beilstein J. Org. Chem. 2008, 4 (13), DOI: 10.3762/bjoc.4.13 .
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  Burger, M. T.; Pecchi, S.; Wagman, A.; Ni, Z. J.; Knapp, M.; Hendrickson, T.; Atallah, G.; Pfister, K.; Zhang, Y.; Bartulis, S.; Frazier, K.; Ng, S.; Smith, A.; Verhagen, J.; Haznedar, J.; Huh, K.; Iwanowicz, E.; Xin, X.; Menezes, D.; Merritt, H.; Lee, I.; Wiesmann, M.; Kaufman, S.; Crawford, K.; Chin, M.; Bussiere, D.; Shoemaker, K.; Zaror, I.; Maira, S. M.; Voliva, C. F. Identification of NVP-BKM120 as a potent, selective, orally bioavailable class I PI3 Kinase inhibitor for treating cancer. ACS Med. Chem. Lett. 2011, 2 (10), 774– 779, DOI: 10.1021/ml200156t
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  Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer
  Burger, Matthew T.; Pecchi, Sabina; Wagman, Allan; Ni, Zhi-Jie; Knapp, Mark; Hendrickson, Thomas; Atallah, Gordana; Pfister, Keith; Zhang, Yanchen; Bartulis, Sarah; Frazier, Kelly; Ng, Simon; Smith, Aaron; Verhagen, Joelle; Haznedar, Joshua; Huh, Kay; Iwanowicz, Ed; Xin, Xiaohua; Menezes, Daniel; Merritt, Hanne; Lee, Isabelle; Wiesmann, Marion; Kaufman, Susan; Crawford, Kenneth; Chin, Michael; Bussiere, Dirksen; Shoemaker, Kevin; Zaror, Isabel; Maira, Sauveur-Michel; Voliva, Charles F.
  ACS Medicinal Chemistry Letters (2011), 2 (10), 774-779CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)
  Phosphoinositide-3-kinases (PI3Ks) are important oncol. targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clin. trials for the treatment of cancer.
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  Rousseau, J. F.; Chekroun, I.; Ferey, V.; Labrosse, J. R. Concise preparation of a stable cyclic sulfamidate intermediate in the synthesis of a enantiopure chiral active diamine derivative. Org. Process Res. Dev. 2015, 19 (4), 506– 513, DOI: 10.1021/op500264v
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  Concise Preparation of a Stable Cyclic Sulfamidate Intermediate in the Synthesis of a Enantiopure Chiral Active Diamine Derivative
  Rousseau, Jean-Francois; Chekroun, Isaac; Ferey, Vincent; Labrosse, Jean Robert
  Organic Process Research & Development (2015), 19 (4), 506-513CODEN: OPRDFK; ISSN:1083-6160. (American Chemical Society)
  A potentially scalable route to the nonracemic antipsychotic candidate SSR 504374 I was developed using the stereoselective substitution reaction of nonracemic sulfamidate II with 2-chloro-3-trifluoromethylbenzamide as the key step. II was prepd. in seven steps from 2-benzoylpyridine by chemo- and diastereoselective hydrogenation, sepn. of the desired racemic erythro diastereomer, resoln. with di-p-toluoyl-(+)-tartaric acid, formation of a sulfamidite, and oxidn. at sulfur with RuCl3 and sodium hypochlorite; a workup for the sulfamidite oxidn. using isopropanol was developed to avoid darkening of the intermediate due to oxidn. by RuO2 and RuO4 left in the sulfamidate product.
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  Brown, G. R.; Foubister, A. J.; Wright, B. Chiral synthesis of 3-substituted morpholines via serine enantiomers and reductions of 5-oxomorpholine-3-carboxylates. J. Chem. Soc., Perkin Trans. 1 1985, 1, 2577– 2580, DOI: 10.1039/p19850002577
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  Hebeisen, P.; Alker, A.; Buerkler, M. Iterative one pot reactions of a chiral sulfamidate with 2,4,6-trichloropyridine: regiocontrolled synthesis of linear and angular chiral dipyrrolidino pyridines. Heterocycles 2012, 85 (1), 65– 72, DOI: 10.3987/COM-11-12360
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  Iterative one pot reactions of a chiral sulfamidate with 2,4,6-trichloropyridine: Regiocontrolled synthesis of linear and angular chiral dipyrrolidino pyridines
  Hebeisen, Paul; Alker, Andre; Buerkler, Markus
  Heterocycles (2012), 85 (1), 65-72CODEN: HTCYAM; ISSN:0385-5414. (Japan Institute of Heterocyclic Chemistry)
  The product of the ring opening of a chiral sulfamidate with the 3-lithiopyridine species obtained by deprotonation of 2,4,6-trichloropyridine with BuLi was deprotonated again in situ with BuLi and reacted with a 2nd equiv. of the sulfamidate furnishing a bis(β-aminoethyl)pyridine deriv., which could be cyclized regioselectively to linear or angular chiral dipyrrolidinopyridines.
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  Zask, A.; Kaplan, J.; Verheijen, J. C.; Richard, D. J.; Curran, K.; Brooijmans, N.; Bennett, E. M.; Toral-Barza, L.; Hollander, I.; Ayral-Kaloustian, S.; Yu, K. Morpholine derivatives greatly enhance the selectivity of mammalian target of rapamycin (mTOR) inhibitors. J. Med. Chem. 2009, 52 (24), 7942– 7945, DOI: 10.1021/jm901415x
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  Morpholine Derivatives Greatly Enhance the Selectivity of Mammalian Target of Rapamycin (mTOR) Inhibitors
  Zask, Arie; Kaplan, Joshua; Verheijen, Jeroen C.; Richard, David J.; Curran, Kevin; Brooijmans, Natasja; Bennett, Eric M.; Toral-Barza, Lourdes; Hollander, Irwin; Ayral-Kaloustian, Semiramis; Yu, Ker
  Journal of Medicinal Chemistry (2009), 52 (24), 7942-7945CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
  Dramatic improvements in mTOR-targeting selectivity were achieved by replacing morpholine in pyrazolopyrimidine inhibitors with bridged morpholines. Analogs I [R1 = (R)- or (S)-3-methyl-4-morpholinyl, 2-methyl-4-morpholinyl, 8-oxa-3-azabicyclo[3.2.1]octan-3-yl, etc.; R2 = Me, Et, cyclopropyl, FCH2CH2, 3-pyridyl, 4-pyridyl; R3 = F3CCH2, 1-methoxycarbonyl-4-piperidinyl, 1-ethoxycarbonyl-4-piperidinyl, 3-pyridylmethyl] with subnanomolar mTOR IC50 values and up to 26000-fold selectivity vs. PI3Kα were prepd. Chiral morpholines gave inhibitors whose enantiomers had different selectivity and potency profiles. Mol. modeling suggests that a single amino acid difference between PI3K and mTOR (Phe961Leu) accounts for the profound selectivity seen by creating a deeper pocket in mTOR that can accommodate bridged morpholines.
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  Thomas, V. H.; Bhattachar, S.; Hitchingham, L.; Zocharski, P.; Naath, M.; Surendran, N.; Stoner, C. L.; El-Kattan, A. The road map to oral bioavailability: an industrial perspective. Expert Opin. Drug Metab. Toxicol. 2006, 2 (4), 591– 608, DOI: 10.1517/17425255.2.4.591
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  The road map to oral bioavailability: an industrial perspective
  Thomas, V. Hayden; Bhattachar, Shobha; Hitchingham, Linda; Zocharski, Philip; Naath, Maryanne; Surendran, Narayanan; Stoner, Chad L.; El-Kattan, Ayman
  Expert Opinion on Drug Metabolism & Toxicology (2006), 2 (4), 591-608CODEN: EODMAP; ISSN:1742-5255. (Informa Healthcare)
  A review. Optimization of oral bioavailability is a continuing challenge for the pharmaceutical and biotechnol. industries. The no. of potential drug candidates requiring in vivo evaluation has significantly increased with the advent of combinatorial chem. In addn., drug discovery programs are increasingly forced into more lipophilic and lower soly. chem. space. To aid in the use of in vitro and in silico tools as well as reduce the no. of in vivo studies required, a team-based discussion tool is proposed that provides a road map' to guide the selection of profiling assays that should be considered when optimizing oral bioavailability. This road map divides the factors that contribute to poor oral bioavailability into two interrelated categories: absorption and metab. This road map provides an interface for cross discipline discussions and a systematic approach to the experimentation that drives the drug discovery process towards a common goal - acceptable oral bioavailability using minimal resources in an acceptable time frame.
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  Mortensen, D. S.; Fultz, K. E.; Xu, S.; Xu, W.; Packard, G.; Khambatta, G.; Gamez, J. C.; Leisten, J.; Zhao, J.; Apuy, J.; Ghoreishi, K.; Hickman, M.; Narla, R. K.; Bissonette, R.; Richardson, S.; Peng, S. X.; Perrin-Ninkovic, S.; Tran, T.; Shi, T.; Yang, W. Q.; Tong, Z.; Cathers, B. E.; Moghaddam, M. F.; Canan, S. S.; Worland, P.; Sankar, S.; Raymon, H. K. CC-223, a potent and selective inhibitor of mTOR kinase: in vitro and in vivo characterization. Mol. Cancer Ther. 2015, 14 (6), 1295– 1305, DOI: 10.1158/1535-7163.MCT-14-1052
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  CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization
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  Molecular Cancer Therapeutics (2015), 14 (6), 1295-1305CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
  MTOR is a serine/threonine kinase that regulates cell growth, metab., proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are crit. mediators of the PI3K-AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogs, allosteric inhibitors that target only the mTORC1 complex, have shown some clin. activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclin. characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematol. and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematol. cancer cell lines. Correlative anal. revealed that IRF4 expression level assocs. with resistance, whereas mTOR pathway activation seems to assoc. with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223-treated tumors suggests that the obsd. antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clin. trials. Mol Cancer Ther; 14(6); 1295-305. ©2015 AACR.
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  Diastereoselective synthesis of trans-disubstituted pyrazolylcyclopropane building blocks (i.e. carboxylic acids and amines) is described starting from easily available pyrazolecarbaldehydes. The key step of the synthesis was Corey-Chaikowsky cyclopropanation of the corresponding α,β-unsatd. Weinreb amides. The title compds. were prepd. in four or six steps and 32-60 and 17-40 % overall yields, resp., on up to 50 g scale. The building blocks obtained are good starting points for the design of lead-like libraries of peptidomimetic drugs.
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  A facile combinatorial strategy was developed for the construction of libraries of functionalized chiral ionic liqs. (FCILs) including doubly chiral ionic liqs. and bis-functional chiral ionic liqs. These FCIL libraries have the potential to be used as asym. catalysts or chiral ligands. As an example, novel asym. bifunctional catalysts were developed by simultaneously incorporating functional groups onto the cation and anion. The resultant bis-functionalized CILs showed significantly improved stereoselectivity over the mono-functionalized parent CILs.
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  Nature Biotechnology (2005), 23 (3), 329-336CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
  Kinase inhibitors show great promise as a new class of therapeutics. Here the authors describe an efficient way to det. kinase inhibitor specificity by measuring binding of small mols. to the ATP site of kinases. The authors have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clin. development, against a panel of 119 protein kinases. The authors find that specificity varies widely and is not strongly correlated with chem. structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. The authors also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small mol.-protein interaction map for clin. compds. across a large no. of related proteins.
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  Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biol. consequences of multikinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. The authors present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global anal. of the results, the authors introduce the concept of a selectivity score as a general tool to quantify and differentiate the obsd. interaction patterns. The authors further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.
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Supporting Information
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00972.
- Syntheses of bromo derivative 44 (Scheme S1), bromo derivative 49 (Scheme S2), and compounds 3a–5a and 3b–5b (Scheme S3); plasma concentration of 7b after a single po dose of 5 mg/kg in rats (Table S1); brain concentration of 7b after a single po dose of 5 mg/kg in rats (Table S2); plasma concentration of 12b after a single po dose of 5 mg/kg in rats (Table S3); brain concentration of 12b after a single po dose of 5 mg/kg in rats (Table S4); stability of compound 11b (5 μM) in primary mouse, rat, dog, and human hepatocytes (Table S5); CYP1A1 and CYP1A2 metabolites identification of 11b (Table S6); proposed metabolic pathway for CYP-dependent metabolism of 11b (Figure S1); chromatogram of compound 11b incubated with CYP1A1 (60 min) (Figure S2); chromatogram of compound 11b incubated with CYP1A1 (0 min) (Figure S3); chromatogram of compound 11b incubated with CYP1A2 (60 min) (Figure S4); chromatogram of compound 11b incubated with CYP1A2 (0 min) (Figure S5); activity data and standard errors of final compounds (Table S7); activity data and standard errors of compounds for modeling (Table S8); TREEspot data visualization of KINOMEScan interactions of compound 12b, PQR620, and INK128 (Figure S6); selectivity profile calculated from KinomeScan data (Table S9); kinase interactions (KINOMEscan data) (Table S10); ¹H NMR, ¹³C{¹H} NMR, and NSI-HRMS spectra; HPLC chromatograms; chemical structures of final compounds and intermediates (PDF)
- Compound 3a-PI3Kγ (PDB)
- Compound 2a-mTOR (PDB)
- Compound 2b-mTOR (PDB)
- Compound 2a-PI3Kα (PDB)
- Compound 2b-PI3Kα (PDB)
- Molecular formula strings and some data (CSV)
Accession Codes
PDB code 5JHB was used for docking of compound 3a into PI3Kγ. PDB code 4JT6 was used for docking of compounds 2a and 2b into mTOR kinase. PDB code 3ZIM was used for docking of compounds 2a and 2b into PI3Kα.

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A Conformational Restriction Strategy for the Identification of a Highly Selective Pyrimido-pyrrolo-oxazine mTOR Inhibitor

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Abstract

Introduction

Figure 1

Results and Discussion

Rigidification Strategy

Figure 2

Elucidation of Binding Modes to PI3K and mTOR

Figure 3

Scheme 1

Chemistry

Determination of Cellular Potency and PI3K vs mTOR Kinase Activities

Pharmacological Parameters

Figure 4

CYP450 Reactive Phenotyping for Compounds 7b, 11b, and 12b

Figure 5

Enzymatic Profiling and Determination of Selectivity

Conclusion

Experimental Section

General Information

General Procedure 1

General Procedure 2

General Procedure 3

General Procedure 4

General Procedure 5

Step 1

Step 2

General Procedure 6

(S)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (2a)

(R)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (2b)

(S)-5-(2-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-4-yl)pyridin-2-amine (2c)

(R)-5-(2-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-4-yl)pyridin-2-amine (2d)

(S)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (3a)

(R)-5-(4-Morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (3b)

(S)-5-(4-Morpholino-5,5a,6,7,8,9-hexahydropyrimido[5,4-b]indolizin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (4a)

(R)-5-(4-Morpholino-5,5a,6,7,8,9-hexahydropyrimido[5,4-b]indolizin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (4b)

(S)-5-(4-(Piperidin-1-yl)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (5a)

(R)-5-(4-(Piperidin-1-yl)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (5b)

5-((S)-4-((S)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (6a)

5-((R)-4-((S)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (6b)

5-((S)-4-((R)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (7a)

5-((R)-4-((R)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (7b)

5-[(9S)-6-{8-Oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (8a)

5-[(9R)-6-{8-Oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (8b)

5-[(9S)-6-{3-Oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (9a)

5-[(9R)-6-{3-Oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (9b)

(S)-5-(4-(3,3-Dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (10a)

(R)-5-(4-(3,3-Dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (10b)

5-((R)-4-((R)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyrimidin-2-amine (11b)

5-((R)-4-((R)-3-Methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyrazin-2-amine (12b)

4-Methyl-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (13b)

3-Methyl-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (14b)

4-Methoxy-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (15b)

4-(Dimethoxymethyl)-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyridin-2-amine (16b)

4-(Dimethoxymethyl)-5-((R)-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazin-2-yl)pyrimidin-2-amine (17b)

tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[4-(dimethoxymethyl)-5-[(9R)-6-[(3R)-3-methylmorpholin-4-yl]-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-yl]carbamate (18b)

tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[4-(dimethoxymethyl)-5-[(9R)-6-[(3R)-3-methylmorpholin-4-yl]-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-trien-4-yl]pyrimidin-2-yl]carbamate (19b)

(S)-2-Chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (20a)

(R)-2-Chloro-4-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (20b)

(S)-4-Chloro-2-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (20c)

(R)-4-Chloro-2-morpholino-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (20d)

(S)-2-Chloro-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (21a)

(R)-2-Chloro-4-((S)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (21b)

(S)-2-Chloro-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (22a)

(R)-2-Chloro-4-((R)-3-methylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (22b)

(9S)-4-Chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-triene (23a)

(9R)-4-Chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-triene (23b)

(9S)-4-Chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-triene (24a)

(9S)-4-Chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.02,7]trideca-2(7),3,5-triene (24b)

(S)-2-Chloro-4-(3,3-dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (25a)

(R)-2-Chloro-4-(3,3-dimethylmorpholino)-5a,6,8,9-tetrahydro-5H-pyrimido[5′,4′:4,5]pyrrolo[2,1-c][1,4]oxazine (25b)

4-(2,6-Dichloropyrimidin-4-yl)morpholine (26)

(S)-4-(2,6-Dichloropyrimidin-4-yl)-3-methylmorpholine (27)

(R)-4-(2,6-Dichloropyrimidin-4-yl)-3-methylmorpholine (28)

3-(2,6-Dichloropyrimidin-4-yl)-8-oxa-3-azabicyclo[3.2.1]octane (29)

8-(2,6-Dichloropyrimidin-4-yl)-3-oxa-8-azabicyclo[3.2.1]octane (30)

5-[(9S)-6-{8-Oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (8a)

5-[(9R)-6-{8-Oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (8b)

5-[(9S)-6-{3-Oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (9a)

5-[(9R)-6-{3-Oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-amine (9b)

tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[4-(dimethoxymethyl)-5-[(9R)-6-[(3R)-3-methylmorpholin-4-yl]-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyridin-2-yl]carbamate (18b)

tert-Butyl N-[(tert-Butoxy)carbonyl]-N-[4-(dimethoxymethyl)-5-[(9R)-6-[(3R)-3-methylmorpholin-4-yl]-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-trien-4-yl]pyrimidin-2-yl]carbamate (19b)

(9S)-4-Chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene (23a)

(9R)-4-Chloro-6-{8-oxa-3-azabicyclo[3.2.1]octan-3-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene (23b)

(9S)-4-Chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene (24a)

(9S)-4-Chloro-6-{3-oxa-8-azabicyclo[3.2.1]octan-8-yl}-11-oxa-1,3,5-triazatricyclo[7.4.0.0^2,7]trideca-2(7),3,5-triene (24b)