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Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12D Inhibitor

  • Xiaolun Wang*
    Xiaolun Wang
    Mirati Therapeutics, San Diego, California 92121, United States
    *Email: [email protected]. Phone: 858-401-6730.
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  • Shelley Allen
    Shelley Allen
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
  • James F. Blake
    James F. Blake
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
  • Vickie Bowcut
    Vickie Bowcut
    Mirati Therapeutics, San Diego, California 92121, United States
  • David M. Briere
    David M. Briere
    Mirati Therapeutics, San Diego, California 92121, United States
  • Andrew Calinisan
    Andrew Calinisan
    Mirati Therapeutics, San Diego, California 92121, United States
  • Joshua R. Dahlke
    Joshua R. Dahlke
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
  • Jay B. Fell
    Jay B. Fell
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
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  • John P. Fischer
    John P. Fischer
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
  • Robin J. Gunn
    Robin J. Gunn
    Mirati Therapeutics, San Diego, California 92121, United States
  • Jill Hallin
    Jill Hallin
    Mirati Therapeutics, San Diego, California 92121, United States
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  • Jade Laguer
    Jade Laguer
    Mirati Therapeutics, San Diego, California 92121, United States
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  • J. David Lawson
    J. David Lawson
    Mirati Therapeutics, San Diego, California 92121, United States
  • James Medwid
    James Medwid
    Mirati Therapeutics, San Diego, California 92121, United States
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  • Brad Newhouse
    Brad Newhouse
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
  • Phong Nguyen
    Phong Nguyen
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
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  • Jacob M. O’Leary
    Jacob M. O’Leary
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
  • Peter Olson
    Peter Olson
    Mirati Therapeutics, San Diego, California 92121, United States
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  • Spencer Pajk
    Spencer Pajk
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
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  • Lisa Rahbaek
    Lisa Rahbaek
    Mirati Therapeutics, San Diego, California 92121, United States
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  • Mareli Rodriguez
    Mareli Rodriguez
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
  • Christopher R. Smith
    Christopher R. Smith
    Mirati Therapeutics, San Diego, California 92121, United States
  • Tony P. Tang
    Tony P. Tang
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
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  • Nicole C. Thomas
    Nicole C. Thomas
    Mirati Therapeutics, San Diego, California 92121, United States
  • Darin Vanderpool
    Darin Vanderpool
    Mirati Therapeutics, San Diego, California 92121, United States
  • Guy P. Vigers
    Guy P. Vigers
    Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
  • James G. Christensen
    James G. Christensen
    Mirati Therapeutics, San Diego, California 92121, United States
  • , and 
  • Matthew A. Marx*
    Matthew A. Marx
    Mirati Therapeutics, San Diego, California 92121, United States
    *Email: [email protected]. Phone: 858-332-3558.
Cite this: J. Med. Chem. 2022, 65, 4, 3123–3133
Publication Date (Web):December 10, 2021
https://doi.org/10.1021/acs.jmedchem.1c01688

Copyright © 2021 American Chemical Society. This publication is licensed under

CC-BY 4.0.
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Abstract

KRASG12D, the most common oncogenic KRAS mutation, is a promising target for the treatment of solid tumors. However, when compared to KRASG12C, selective inhibition of KRASG12D presents a significant challenge due to the requirement of inhibitors to bind KRASG12D with high enough affinity to obviate the need for covalent interactions with the mutant KRAS protein. Here, we report the discovery and characterization of the first noncovalent, potent, and selective KRASG12D inhibitor, MRTX1133, which was discovered through an extensive structure-based activity improvement and shown to be efficacious in a KRASG12D mutant xenograft mouse tumor model.

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Introduction

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Mutant KRAS has been recognized as an attractive drug target for the treatment of a number of cancers for many decades. (1−4) However, the high affinity of KRAS for GDP/GTP and the lack of any other apparent binding pocket have significantly hampered the development of KRAS inhibitors until recently. (1,4) The identification and subsequent clinical success of irreversible KRASG12C inhibitors that occupy the induced switch II pocket has been a very important breakthrough. (5−7) However, their inhibitory activity relies on a reactive warhead forming a stable covalent bond with the mutant Cys12. Conversely, KRASG12D, the most common mutation (33%) among KRAS mutant tumors, (8) lacks a reactive residue adjacent to the switch II pocket as evidenced in recent attempts, (9,10) and thus requires a novel approach toward the identification of selective inhibitors. A new class of KRAS inhibitors (for example, compound 2 in Figure 1) binding to a shallow pocket between switch I and II was reported (11−14) contemporaneously with the development of irreversible KRASG12C inhibitors; however, limited cellular activity was observed. Cyclic peptide KRASG12D inhibitors (for example, compound 3 in Figure 1) were also reported. (15−18) Although these peptides demonstrated better biochemical activity than the indole analogs illustrated by compound 2, their physicochemical properties limited their cellular potency. Recently, a bicyclic peptidyl Pan-RAS inhibitor with submicromolar cellular potency and in vivo efficacy was reported. (19−21)

Figure 1

Figure 1. KRASG12D inhibitors.

We report herein the structure-based optimization of a series of molecules culminating in the discovery of MRTX1133 (Figure 1, compound 1), a potent, selective, noncovalent KRASG12D inhibitor with picomolar binding affinity, single digit nanomolar activity in cellular assays, and marked in vivo efficacy in tumor models harboring KRASG12D mutations. At the initiation of our KRASG12D-directed drug discovery activities, we were uncertain whether therapeutically meaningful KRASG12D inhibition could be achieved with a noncovalent inhibitor, as the kinetic study for an early KRASG12C irreversible inhibitor, ARS-853, revealed a modest KI of 200 μM. (22) Later reports on ARS-1620, sotorasib, and adagrasib also demonstrated reversible affinities in the μM range. (5,23) Thus, we anticipated that significant improvements to binding affinity would be needed to achieve potency necessary for requisite target inhibition in cells. Therefore, we focused on maximizing the contribution of each moiety of the inhibitor to increase affinity, expecting that greater occupancy of the induced switch II pocket would lead to sustained functional suppression of KRAS pathway signaling in cellular and tumor environments harboring the KRASG12D mutation sufficient for antitumor efficacy.

Results and Discussion

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Informed by our experience in discovering the KRASG12C inhibitor adagrasib (compound 4), (5,24) we synthesized compounds 5A and 5B with a pyrido[4,3-d]pyrimidine scaffold (25) with the anticipation that the protonated piperazine could interact productively with the mutant Asp12 side chain. Although compound 5A was inactive, analog 5B without the cyanomethyl group demonstrated a KD of 3.5 μM in a KRASG12D SPR assay with GDP-loaded KRASG12D (Figure 2). While the cyanomethyl group on the 3-position of the piperazine ring was found beneficial for irreversible KRASG12C inhibitors (5) by displacing a water molecule close to Gly10 and enhancing the reactivity of the acrylamide warhead, compounds 5A and 5B suggested that a different approach would be needed to enhance KRASG12D binding affinity in this region of the molecule for the pyrido[4,3-d]pyrimidine core. We also evaluated an analog of compound 5B with the tetrahydropyridopyrimidine core (5,24,26,27) present in adagrasib, and found it to be 5–10-fold less potent than 5B. Thus, the pyrido[4,3-d]pyrimidine scaffold was selected as a starting point for further optimization (Figure 2). The crystal structure of compound 5B with KRASG12D/GDP (Figure 3) revealed that its placement in the switch II pocket resembled the binding mode of adagrasib with KRASG12C (6UT0). (5) The hydrogen bond interaction of the 6-position nitrogen with Arg68 and the occupancy of the 8-fluorine in a small hydrophobic pocket may help explain why the pyrido[4,3-d]pyrimidine core is more potent than the corresponding tetrahydropyridopyrimidine. The three substituents of the pyrido[4,3-d]pyrimidine core interact extensively with the protein (Figure 3). The protonated piperazinyl group at the C4-position forms an ionic pair with the mutant Asp12, providing 10-fold selectivity over KRASWT. The piperazine twist-boat conformation (previously observed in the adagrasib/KRASG12C structure (5)) and its salt bridge with Asp12 are stabilized by an additional hydrogen bond to the carbonyl oxygen of Gly60. The conserved water molecule under the C2-substituent forms hydrogen bonds with Gly10 and Thr58 but has no apparent interaction with compound 5B, thus providing an opportunity for further exploration. The positively charged pyrrolidinyl moiety at the C2-position is well positioned for a favorable ionic interaction (28) with Glu62. Finally, the C7-naphthyl occupies a deep hydrophobic pocket as reported previously for adagrasib. (5,24) These three vectors were identified as opportunities to increase affinity for the KRASG12D protein.

Figure 2

Figure 2. Pyrido[4,3-d]pyrimidine analog.

Figure 3

Figure 3. X-ray structure of compound 5B with KRASG12D/GDP (7RT4).

Given the importance of the salt bridge with Asp12 to the binding and selectivity of this lead molecule, we began optimization of the series at the C4-position using the C7-naphthol substituent (Table 1) identified in our earlier KRASG12C effort. (24) The affinity of compound 6 with an unsubstituted piperazinyl group was 0.19 μM, while the homopiperazine analog 7 was 2-fold less potent. Both N-methylation (8) and the replacement of the secondary amino group with an oxygen (9) were deleterious. Moreover, compounds 8 and 9 lost selectivity over KRASWT (SPR KD = 6.2 and 0.56 μM, respectively). These observations further confirmed the importance of the ionized piperazine’s bifurcated interactions with Asp12 and Gly60 for both activity and specificity. Methyl-substitution of the piperazine (10 and 11) did not improve potency. However, rigidification (29) of bioactive conformation (30,31) in piperazine analogs 1215 increased affinity. Compound 15 with a [3.2.1]bicyclic diamino substituent had a KD of 0.8 nM and possessed greater than 200-fold selectivity for KRASG12D compared with KRASWT (KD = 182 nM). Consistent with the increased affinity, appreciable cell activity (pERK IC50 = 0.530 μM against AGS, a KRASG12D cell line) was also observed for 15. This provided early promise for the possibility of identifying an effective KRASG12D inhibitor. The X-ray structure of compound 15 with KRASG12D/GDP is shown in Figure 4. The two-carbon bridge of the bicyclic group occupies a small pocket, while one of the endo C-Hs forms a nonclassical hydrogen bond with the Gly10 carbonyl oxygen. (32,33) It also positions the charged secondary amine for optimal interactions with Asp12 and Gly60. At this point, we developed an HTRF assay with GDP-loaded KRASG12D using a probe based on compound 15 to increase screening throughput. The relative potency rankings of the HTRF assay aligned well with SPR affinity (Table 1), leading us to use the HTRF assay for subsequent compound profiling.
Table 1. Exploration of the C4-Position of Pyrido[4,3-d]pyrimidinea
a

& represents a diastereomeric mixture as the racemic amine was used for C4-substitution.

Figure 4

Figure 4. X-ray structure of compound 15 with KRASG12D/GDP (7RT1).

With a highly potent and selective C4 substitution in hand, we next focused on optimizing the C2 position of the core, which is summarized in Table 2. Deletion of the C2-pyrrolidinyl substitution in 15 yielded compound 16, which resulted in an almost 600-fold decrease in potency, confirming the importance of this moiety for affinity. Compounds 17 and 18 were designed to interact with Glu62 in a similar fashion to 15, but both only yielded a 42-fold increase in potency over 16, likely due to lack of rigidity (17) and suboptimal interaction geometry (18). The activity of compound 19 with a basic imidazole group was comparable to that of 17 or 18 but was less potent than the pyrrolidinyl analog 15. The focus of the C2-substituent design was then turned to conformationally constrained amines. While the activity of the bridged morpholine 20 was inferior, the pyrrolizidine analog 21 provided over a 2-fold increase in cellular potency compared to 15. Further optimization revealed the rigid 5,5-fused bicycle was preferred over the 6,5 system (22), and the basicity at this position is crucial for activity (23). Modification of the pyrrolizidine with a 2-fluoro substituent led to the discovery of the potent enantiomeric pair 24 and 25. The IC50 of eutomer 25 in the pERK inhibition cell assay was 24 nM, 11-fold more active than the parent compound 21. The absolute configuration of both enantiomers was confirmed by X-ray crystal structures with KRASG12D/GDP (Figure 5). As expected, the protonated pyrrolizidine forms a strong ionic interaction (N–O distances: 2.8 and 3.3 Å) with the negatively charged carboxylate of Glu62 (Figure 6). Interestingly, the 3-carbon of pyrrolizidine sits 3.4 Å from one of carboxylate oxygens suggesting a nonclassical hydrogen bond. (32,33) While the electron withdrawing nature of the fluorine atom likely reinforces both the ionic and nonclassical H-bond interactions in 25, the shift of the fluorine position reduces the polarization of the C5 proton in 24 and may explain the slight activity difference between the two enantiomers.
Table 2. Examination of the 2-Substitution of Pyrido[4,3-d]pyrimidinea
a

& represents racemic as the racemic amine was used for C4-substitution.

Figure 5

Figure 5. Overlay of the X-ray structures of compounds 24 (yellow, 7RT3) and 25 (cyan, 7RT2) with KRASG12D/GDP.

Figure 6

Figure 6. Interaction of 2-Fluoropyrrolizidine (25) with Glu62.

The C7-substituent of the pyrido[4,3-d]pyrimidine is buried deeply in the binding site, and a small hydrophobic pocket near the 8-position of the naphthyl (5) provided an opportunity for further optimization. For this exploration, the readily prepared naphthyl derivatives, rather than the more elaborated naphthols, were utilized. Simple naphthyl 8-substituents such as methyl, fluorine, or chlorine provided an 8- to 30-fold activity boost (Table 3, compounds 27, 28, and 29) over the unsubstituted 26 indicating that optimally filling this pocket may yield further improvement in activity. Furthermore, the substitutions also stabilized the preferred perpendicular conformation of the naphthyl ring. Extending the alkyl group from methyl to ethyl (30) provided another 7-fold increase in affinity, suggesting the terminal carbon favorably filled the hydrophobic space perpendicular to the 8-position of the naphthyl. However, a methoxy group at this position (31) was detrimental. Dihedral energy profiling (Figure 7) revealed that this was likely due to the terminal carbon preferring an approximately coplanar orientation to the naphthyl group, while the preferred conformation of the ethyl group of 108° is close to the modeled binding pose of 100°. The energy calculations predicted a 15-fold difference in binding affinity, consistent with the observed potency shift between 30 and 31. It has been recognized in our previously reported KRASG12C structures that a conserved water molecule makes hydrogen bond interactions with both Gly10 and Thr58, (5) and we envisioned that a hydrogen bond donor (HBD) from the 8-position of the naphthyl group might further stabilize this hydrogen bond network. The initial attempt with the introduction of a hydroxyl group (32) was unsuccessful. A careful examination suggested an 8-ethynyl substitution would not only fill the hydrophobic space well, but could also engage the conserved water molecule via a nonclassical HBD. (34,35) Indeed, the 8-ethynyl analog 33 was 120-fold more potent than the parent compound 26. Next, we turned our attention to a small pocket near to the side chains of Val9, Phe78, and Ile100. A fluorine atom at the 7-position of the naphthyl (34) was introduced to fill this space and provided a 13-fold increase in potency over unsubstituted 26. This effect was additive with the activity increases observed when combined with selected substituents at the 8-position as evidenced in the pairs of 27 vs 35 and 33 vs 36. The biochemical activity of compound 36 with the optimized 7-fluoro-8-ethynylnaphthyl was less than 2 nM and its IC50 in the cellular pERK assay was 24 nM, the most potent compound identified of the des-naphthol analogs. However, compound 36 demonstrated limited pERK inhibition in KRASG12D mutated tumor xenografts when intraperitoneally administered in vivo at the maximal tolerated dose (30 mg/kg, AUC0–24 = 9495 ng·h/mL) indicating further improvement of potency and/or physiochemical properties was needed for suitable levels of target inhibition. The X-ray structure of 36 in KRASG12D/GDP revealed that the 7-fluoro and 8-ethynyl substituents are nicely positioned in a hydrophobic pocket formed with Val9, Thr58, Phe78, Met72, Tyr96, and Ile100 (Figure 8). The conserved water molecule is hydrogen-bonded to two HBAs, the hydroxyl from Thr58 and the carbonyl oxygen from Gly10, and two HBDs, the alkynyl proton of the ligand and NH of Gly10, forming a well-organized hydrogen bond network (Figure 9a,b). These interactions allow the terminal alkynyl group to effectively bridge the lipophilic and polar regions of the switch II pocket. Reintroduction of the naphthol (37, 38) confirmed that the potency increases derived from 3- and 8-naphthyl substitutions are indeed combinable.
Table 3. Evaluation of the C7-Position of Pyrido[4,3-d]pyrimidine

Figure 7

Figure 7. Preferred conformation of 8-substitutions in 30 and 31. Dihedral scan performed for 8-substituted ethyl (blue) and methoxy (orange) analogs in Spartan (36) using ωB97X-D/6-31G**. Ar represents the 8-fluoro-azaquinazoline ring which was held in the binding nascent pose during the scan

Figure 8

Figure 8. Residues around 7-fluoro-8-ethynylnaphthyl in the X-ray structure of compound 36 with KRASG12D/GDP.

Figure 9

Figure 9. (a,b) Hydrogen network around the conserved water molecule in the X-ray structure of compound 36 with KRASG12D/GDP (7RT5).

Finally, the combination of the optimized three substituents on the 2-, 4-, and 7-positions of pyrido[4,3-d]pyrimidine led to the discovery of MRTX1133, an exceptionally potent and selective KRASG12D inhibitor (Figure 10). MRTX1133 optimally fills the switch II pocket and extends three substituents to favorably interact with the protein (Figure 11), resulting in an estimated KD against KRASG12D of 0.2 pM. AlphaLISA data confirmed that binding of the inhibitor prevented SOS1-catalyzed nucleotide exchange and/or formation of the KRASG12D/GTP/RAF1 complex, thereby inhibiting mutant KRAS-dependent signal transduction. MRTX1133 inhibited ERK phosphorylation in the AGS cell line with an IC50 of 2 nM (see Table S2 for activity against a panel of KRASG12D cell lines). In a 2D viability assay, the IC50 of MRTX1133 was 6 nM against the same cell line, while demonstrating more than 500-fold selectivity against MKN1, a cell line which is dependent on KRAS for its growth and survival due to the amplification of wild-type KRAS (37) (Table 4).

Figure 10

Figure 10. Design of MRTX1133.

Figure 11

Figure 11. MRTX1133 with KRASG12D/GDP (7RPZ).

Table 4. In Vitro Profile of MRTX1133
assayactivity
KRASG12D KD (nM)∼0.0002a
AlphaLISA IC50 (nM)5
pERK AGS IC50 (nM)2
2D viability AGS (KRASG12D) IC50 (nM)6
2D viability MKN1 (KRASWT) IC50 (nM)>3000
a

The KD value generated is beyond what the instrument can accurately determine but is reported as an approximate value.

Intraperitoneal (IP) administration of MRTX1133 at 30 mg/kg in CD-1 mice (Figure 12) resulted in sustained plasma exposure exceeding the free-fraction-adjusted pERK IC50 value in the KRASG12D mutant Panc 04.03 cell line for approximately 8 h. Encouraged by this result, we evaluated the ability to modulate KRAS-dependent ERK phosphorylation in the Panc 04.03 xenograft tumor model at 30 mg/kg BID (IP) and observed 62% and 74% inhibition of pERK signal at 1 and 12 h after the second dose, respectively (Figure 13). An antitumor efficacy study in this model resulted in MRTX1133 dose-dependent antitumor activity with 94% growth inhibition observed at 3 mg/kg BID (IP) and tumor regressions of −62% and −73% observed at 10 and 30 mg/kg BID (IP), respectively (Figure 14). In contrast, no significant antitumor activity was observed in the non-KRASG12D tumor model MKN1 (data not shown).

Figure 12

Figure 12. PK Curve of MRTX1133 in CD-1 mouse.

Figure 13

Figure 13. pERK modulation in Panc 04.03 model after intraperitoneal administration of MRTX1133. *p-value with t test assuming equal variance <0.05. Statistical analysis of differences in mean pERK inhibition between vehicle and MRTX1133-treated cohorts was run using a two-tailed Student’s t test with equal variance in Excel (Microsoft; Redmond, WA).

Figure 14

Figure 14. Efficacy of MRTX1133 in Panc 04.03 model. **p-value with t test assuming equal variance <0.01. Statistical analysis of differences in mean tumor volume between vehicle and MRTX1133-treated cohorts was run using a two-tailed Student’s t test with equal variance in Excel (Microsoft; Redmond, WA).

Chemistry

The synthesis of compound 1 (Scheme 1) commenced with the chlorination of 7-chloro-8-fluoropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (25)39 through the treatment of POCl3 to afford 2,4,7-trichloro-8-fluoropyrido[4,3-d]pyrimidine 40. The C4 substituent was introduced by a selective SNAr with tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate followed by a second SNAr with ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methanol (25) to install C2 moiety. Suzuki coupling of 42 with ((2-fluoro-6-(methoxymethoxy)-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)ethynyl)triisopropylsilane (38) provided intermediate 43. The subsequent deprotections of the triisopropylsilyl and methoxymethyl ether groups resulted in the desired product 1.

Scheme 1

Scheme 1. Synthesis of Compound 1a

aReagents and conditions. (a) POCl3, N-ethyl-N-isopropylpropan-2-amine, 100 °C, 1 h; (b) tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate, N-ethyl-N-isopropylpropan-2-amine, CH2Cl2, −40 °C, 0.5 h, 42% (two steps); (c) ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methanol, N-ethyl-N-isopropylpropan-2-amine, dioxane, 95 °C, 41 h, 55%; (d) ((2-fluoro-6-(methoxymethoxy)-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)ethynyl)triisopropylsilane, Ad2nBuP-Pd-G3, K3PO4, THF, 60 °C, 3 h, 76%; (e) HCl in dioxane, 0 °C, 1 h, 56%; (f) CsF, DMF, 20 °C, 1 h, 56%.

Conclusion

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Through extensive structure-based drug design, MRTX1133 was identified as a noncovalent, potent, and selective inhibitor of KRASG12D. MRTX1133 suppresses KRASG12D signaling in cells and in vivo, and its antitumor benefit was demonstrated in a murine animal model. To the best of our knowledge, this is the first report in the literature of a small molecule inhibitor of KRASG12D that exhibits robust in vivo efficacy. These data support the potential for the advancement of an effective therapeutic against this “undruggable” target. The optimization process was facilitated by high-resolution X-ray crystal structures. In-depth binding mode analysis derived from cocrystal structures allowed the optimization of lipophilic contact of the inhibitor in the binding pocket and the identification of nonclassical hydrogen bonding and ion pair interactions, ultimately increasing selective binding affinity for KRASG12D by more than 1,000,000-fold relative to the initial hit 5B. MRTX1133 binds to the switch II pocket and inhibits the protein–protein interactions necessary for activation of the KRAS pathway. MRTX1133 not only possesses single-digit nM potency in a cellular proliferation assay, but also demonstrates tumor regressions in the Panc 04.03 xenograft model. A more comprehensive in vitro and in vivo pharmacological characterization of MRTX1133 will be disclosed in due course.

Experimental Section

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All chemicals were purchased from commercial suppliers and used as received unless otherwise indicated. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker Avance 400 MHz spectrometers. Chemical shifts are expressed in δ ppm and are calibrated to the residual solvent peak: proton (e.g., CDCl3, 7.27 ppm). Coupling constants (J), when given, are reported in hertz. Multiplicities are reported using the following abbreviations: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet (range of multiplet is given), br = broad signal, dt = doublet of triplets. Carbon nuclear magnetic resonance (13C NMR) spectra were recorded using a Bruker Avance HD spectrometer at 100 MHz. Chemical shifts are reported in parts per million (ppm) and are calibrated to the solvent peak: carbon (CDCl3, 77.23 ppm). All final compounds were purified by reverse-phase high-performance liquid chromatography (HPLC) or supercritical fluid chromatography (SFC). The purity for test compounds was determined by high-performance liquid chromatography (HPLC) on a LC-20AB Shimadzu instrument. HPLC conditions were as follows: Kinetex C18 4.6 × 50 mm, 5 μm, 10–80% ACN (0.0375% TFA) in water (0.01875% TFA), 4 min run, flow rate 1.5 mL/min, UV detection (λ = 220, 215, 254 nm) or XBridge C18, 2.1 × 50 mm, 5 μm, 10–80% ACN in water buffered with 0.025% ammonia, 4 min run, flow rate 0.8 mL/min, UV detection (λ = 220, 215, 254 nm). The mass spectra were obtained using liquid chromatography mass spectrometry (LC-MS) on a LCMS-2020 Shimadzu instrument using electrospray ionization (ESI in the positive mode). LCMS conditions were as follows: Kinetex EVO C18 30 × 2.1 mm, 5 μm, 5–95% ACN (0.0375% TFA) in water (0.01875% TFA), 1.5 min run, flow rate 1.5 mL/min, UV detection (λ = 220, 254 nm), or Kinetex EVO C18 2.1 × 30 mm, 5 μm, 5–95% ACN in water buffered with 0.025% ammonia, 1.5 min run, flow rate 1.5 mL/min, UV detection (λ = 220, 254 nm). High resolution mass measurements were carried out on an Agilent 1290LC and 6530Q-TOF series with ESI. Optical rotation data were recorded on an Anton Paar MCP500 [length = 1 dm, sodium lamp, λ (nm) = 589, temperature = 25 °C]. The SFC purity for test compounds was determined with a Shimadzu LC-30ADsf. All compounds are >95% pure by HPLC.

Preparation of Compound 1 (MRTX1133) (38)

2,4,7-Trichloro-8-fluoropyrido[4,3-d]pyrimidine (40)

A mixture of 7-chloro-8-fluoropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (5 g, 23 mmol, 1.0 equiv) and N-ethyl-N-isopropylpropan-2-amine (20 mL, 115 mmol, 5.0 equiv) in POCl3 (50 mL, 535 mmol, 23 equiv) was stirred at 100 °C for 1 h. Then, the mixture was concentrated under vacuum to give crude title compound (6.5 g) as yellow oil, which was used in the next step without further purification.

tert-Butyl (1R,5S)-3-(2,7-dichloro-8-fluoropyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (41)

To a solution of 2,4,7-trichloro-8-fluoropyrido[4,3-d]pyrimidine (6.5 g, crude) and N-ethyl-N-isopropylpropan-2-amine (26.9 mL, 154 mmol) in dichloromethane (20 mL) was added tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate (4.9 g, 23 mmol) at −40 °C. The reaction was stirred at −40 °C for 0.5 h. Then, 20 mL water was added to the reaction, and the mixture was extracted with dichloromethane (2 × 20 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under vacuum to afford the title compound as yellow solid (4 g, two steps 41% yield). LCMS [ESI, M+1]: 428.

(1R,5S)-tert-Butyl 3-(7-chloro-8-fluoro-2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (42)

To a solution of (1R,5S)-tert-butyl 3-(2,7-dichloro-8-fluoropyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (53.8 g, 126 mmol, 1 equiv), ((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a-yl)methanol (20 g, 126 mmol, 1 equiv), and N-ethyl-N-isopropylpropan-2-amine (40.6 g, 314 mmol, 2.5 equiv) in dioxane (160 mL) was added 4 Å molecular sieve (20 g) at 20 °C. The suspension was stirred at 95 °C under N2 for 41 h. The reaction was cooled to 60 °C and filtered. The filter cake was washed with ethyl acetate (0.1 L × 2) and dichloromethane (0.1 L). The filtrate was concentrated under reduced pressure to give a residue. The residue was dispersed in DMF (0.12 L), and the mixture was stirred at 60 °C for 1.5 h. The mixture was filtered, and the filter cake was washed with petroleum ether (0.1 L × 3) to give a solid. The solid was dried under reduced pressure to afford the title compound (43.6 g, 88% purity, 55% yield) as white solid; LCMS [ESI, M+1]: 551.2; 1H NMR (400 MHz, CDCl3) δ 8.72 (s, 1H), 5.28 (d, J = 54.4 Hz, 1H), 4.56–4.43 (m, 2H), 4.42–4.30 (m, 2H), 4.28–4.22 (m, 1H), 4.18–4.10 (m, 1H), 3.77–3.56 (m, 3H), 3.32–3.08 (m, 3H), 3.02–2.93 (m, 1H), 2.34–2.09 (m, 3H), 2.01–1.90 (m, 4H), 1.69 (d, J = 7.8 Hz, 2H), 1.52 (s, 9H).

(1R,5S)-tert-Butyl-3-(8-fluoro-7-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (43)

To a mixture of (1R,5S)-tert-butyl 3-(7-chloro-8-fluoro-2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (55 g, 87.8 mmol, 88% purity, 1.0 equiv) and K3PO4 (1.5 M, 176 mL, 3.0 equiv) in THF (275 mL) were added Ad2nBuP-Pd-G3 (6.4 g, 8.78 mmol, 0.1 equiv), and ((2-fluoro-6-(methoxymethoxy)-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)ethynyl)triisopropylsilane (49.5 g, 96.6 mmol, 1.1 equiv) in one portion under N2.The mixture was stirred at 60 °C for 3 h. After completion, the mixture was concentrated under reduced pressure to give a residue. The residue was purified with column chromatography (Al2O3, petroleum ether/ethyl acetate = 20/1 to 5/1) to give a crude product. Then, it was purified by reversed-phase HPLC (C18, 0.1% FA in water, 0–100% MeCN) to afford the title compound (63.3 g, 95% purity, 76% yield) as yellow solid; LCMS [ESI, M+1]: 901.4; 1H NMR (400 MHz, methanol-d4) δ 8.99 (d, J = 2.1 Hz, 1H), 7.87 (dd, J = 5.7, 9.1 Hz, 1H), 7.56 (d, J = 2.6 Hz, 1H), 7.30 (t, J = 8.9 Hz, 1H), 7.22 (t, J = 2.3 Hz, 1H), 5.29–5.10 (m, 3H), 4.82 (br t, J = 11.2 Hz, 1H), 4.38–4.06 (m, 5H), 3.79 (br dd, J = 6.1, 11.9 Hz, 1H), 3.43–3.35 (m, 4H), 3.18–3.05 (m, 3H), 2.91 (dt, J = 5.6, 9.5 Hz, 1H), 2.23–2.00 (m, 3H), 1.91–1.56 (m, 7H), 1.43 (s, 9H), 0.85–0.74 (m, 18H), 0.44 (q, J = 7.5 Hz, 3H).

(1R,5S)-tert-Butyl-3-(7-(8-ethynyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate

To a solution of (1R,5S)-tert-butyl 3-(8-fluoro-7-(7-fluoro-3-(methoxymethoxy)-8-((triisopropylsilyl)ethynyl)naphthalen-1-yl)-2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (42.0 g, 46.6 mmol, 1.0 equiv) in DMF (160 mL) was added CsF (70.8 g, 466 mmol, 17.2 mL, 10 equiv) in one portion under N2. The mixture was stirred at 20 °C for 1 h. After completion, the mixture was purified with reversed-phase HPLC (C18, 0.1% FA in water, 0–100% MeCN) to afford the title compound (30.3 g, 87% yield) as yellow solid (44); LCMS [ESI, M+1]: 745.2.

4-(4-((1R,5S)-3,8-Diazabicyclo[3.2.1]octan-3-yl)-8-fluoro-2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrido[4,3-d]pyrimidin-7-yl)-5-ethynyl-6-fluoronaphthalen-2-ol (1)

To a mixture of (1R,5S)-tert-butyl 3-(7-(8-ethynyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (40.0 g, 53.7 mmol, 1.0 equiv) and MeCN (240 mL) was added HCl/dioxane (4 M, 240 mL, 17.9 equiv) in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 1 h. After completion, the mixture was concentrated under reduced pressure to give a residue, and the residue was basified with NaHCO3 solution (pH = 8). The mixture was extracted with MeOH and DCM twice. The combined organic phases were dried with anhydrous Na2SO4, filtered, and concentrated in vacuum to give the crude product. Then it was purified with reverse-phase HPLC (C18, 0.1% FA in water, 0–100% MeCN). The desired fractions were combined, basified with NaHCO3 (pH = 8), and extracted with MeOH and DCM twice. The combined organic phases were dried with anhydrous Na2SO4, filtered, and concentrated in vacuum to give a residue. The residue was then purified with another reverse-phase HPLC [column: Phenomenex luna C18 250 mm × 100 mm × 10 μm, mobile phase: A: water (0.1% TFA), B: ACN, B %: 12–38% 23 min]. The desired fractions were combined, basified with NaHCO3 (pH = 8), and extracted with MeOH and DCM twice. The combined organic phases were dried with anhydrous Na2SO4, filtered, and concentrated in vacuum to give a residue. The residue was then lyophilized to afford the title compound (18.2 g, 56% yield, 99.7% purity) as yellow solid; LCMS [ESI, M+1]: 601.3; SFC: >99% ee, Chiralpak IC-3 50 × 4.6 mm I.D., 3 μm column A: CO2, B: MeOH (0.05% DEA), 3 mL/min, 220 nm, tR: 1.762 min; [α]D (25 °C): + 10.143° (c = 0.38 g/100 mL, MeOH); 1H NMR (400 MHz, methanol-d4) δ 9.02 (s, 1H), 7.87 (dd, J = 5.8, 9.1 Hz, 1H), 7.39–7.30 (m, 2H), 7.23 (d, J = 2.5 Hz, 1H), 5.42–5.23 (m, 1H), 4.70–4.55 (m, 2H), 4.3–4.20 (m, 2H), 3.80–3.62 (m, 4H), 3.36 (dd, J = 0.9, 6.9 Hz, 1H), 3.32–3.14 (m, 3H), 3.03 (dt, J = 5.7, 9.5 Hz, 1H), 2.41–2.11 (m, 3H), 2.07–1.97 (m, 2H), 1.96–1.75 (m, 5H); 13C NMR (100 MHz, methanol-d4) δ 166.42, 165.78, 165.58, 163.11, 155.84, 155.82, 153.92, 151.37, 150.58, 150.46, 146.94, 146.79, 145.23, 145.17, 134.80, 134.39, 131.70, 131.61, 127.26, 124.36, 117.29, 117.03, 113.25, 112.44, 105.68, 105.52, 99.91, 98.17, 90.13, 90.09, 90.07, 76.36, 74.93, 74.36, 61.60, 61.41, 58.40, 56.47, 56.40, 56.32, 56.28, 55.78, 55.75, 43.82, 43.62, 37.35, 28.74, 28.66, 26.45. HRMS(ESI+) calcd for C33H32F3N6O2+ (M+H+) 601.2533, found 601.2533.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01688.

  • Synthetic experimental procedures for compound 5 to 38; NMR spectra and HPLC trace of final Compounds; HTRF, SPR, AlphaLISA, and cellular assay protocols; in vivo PK/PD and TGI studies (PDF)

  • Molecular formula strings (CSV)

Accession Codes

PDB code: 7RPZ, 7RT1, 7RT2, 7RT3, 7RT4, 7RT5.

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Author Information

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  • Corresponding Authors
  • Authors
    • Shelley Allen - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • James F. Blake - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • Vickie Bowcut - Mirati Therapeutics, San Diego, California 92121, United States
    • David M. Briere - Mirati Therapeutics, San Diego, California 92121, United States
    • Andrew Calinisan - Mirati Therapeutics, San Diego, California 92121, United States
    • Joshua R. Dahlke - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • Jay B. Fell - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • John P. Fischer - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • Robin J. Gunn - Mirati Therapeutics, San Diego, California 92121, United States
    • Jill Hallin - Mirati Therapeutics, San Diego, California 92121, United States
    • Jade Laguer - Mirati Therapeutics, San Diego, California 92121, United States
    • J. David Lawson - Mirati Therapeutics, San Diego, California 92121, United States
    • James Medwid - Mirati Therapeutics, San Diego, California 92121, United States
    • Brad Newhouse - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • Phong Nguyen - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • Jacob M. O’Leary - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • Peter Olson - Mirati Therapeutics, San Diego, California 92121, United States
    • Spencer Pajk - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • Lisa Rahbaek - Mirati Therapeutics, San Diego, California 92121, United States
    • Mareli Rodriguez - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • Christopher R. Smith - Mirati Therapeutics, San Diego, California 92121, United States
    • Tony P. Tang - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • Nicole C. Thomas - Mirati Therapeutics, San Diego, California 92121, United States
    • Darin Vanderpool - Mirati Therapeutics, San Diego, California 92121, United States
    • Guy P. Vigers - Pfizer Boulder Research & Development, Boulder, Colorado 80301, United States
    • James G. Christensen - Mirati Therapeutics, San Diego, California 92121, United States
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    Authors will release the atomic coordinates and experimental data upon article publication.
    The authors declare no competing financial interest.

Acknowledgments

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We would like to thank the following teams/people for their valuable contributions to this work. IDSU chemistry team (WuXi AppTec in Wuhan, China): Tao Guo, Duan Liu, Feng Zhao, and Pan Hu. CSU chemistry team (WuXi AppTec in Wuhan, China): Rongfeng Zhao, Shaojun Song, Wenbing Ruan. Biofizik, Inc.: Simon Bergqvist. Pfizer Boulder Research & Development: Matthew Martinson, Alex Bergstrom, Francis Sullivan. Structure Based Design, Inc.: Frank Han. WuXi Biology team (WuXi AppTec in Shanghai, China): Peipei Xu, Yingjie Li, and Chuanxiu Yang. Part of the X-ray crystallography work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16M detector on 24-ID-E is funded by a NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

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  • Abstract

    Figure 1

    Figure 1. KRASG12D inhibitors.

    Figure 2

    Figure 2. Pyrido[4,3-d]pyrimidine analog.

    Figure 3

    Figure 3. X-ray structure of compound 5B with KRASG12D/GDP (7RT4).

    Figure 4

    Figure 4. X-ray structure of compound 15 with KRASG12D/GDP (7RT1).

    Figure 5

    Figure 5. Overlay of the X-ray structures of compounds 24 (yellow, 7RT3) and 25 (cyan, 7RT2) with KRASG12D/GDP.

    Figure 6

    Figure 6. Interaction of 2-Fluoropyrrolizidine (25) with Glu62.

    Figure 7

    Figure 7. Preferred conformation of 8-substitutions in 30 and 31. Dihedral scan performed for 8-substituted ethyl (blue) and methoxy (orange) analogs in Spartan (36) using ωB97X-D/6-31G**. Ar represents the 8-fluoro-azaquinazoline ring which was held in the binding nascent pose during the scan

    Figure 8

    Figure 8. Residues around 7-fluoro-8-ethynylnaphthyl in the X-ray structure of compound 36 with KRASG12D/GDP.

    Figure 9

    Figure 9. (a,b) Hydrogen network around the conserved water molecule in the X-ray structure of compound 36 with KRASG12D/GDP (7RT5).

    Figure 10

    Figure 10. Design of MRTX1133.

    Figure 11

    Figure 11. MRTX1133 with KRASG12D/GDP (7RPZ).

    Figure 12

    Figure 12. PK Curve of MRTX1133 in CD-1 mouse.

    Figure 13

    Figure 13. pERK modulation in Panc 04.03 model after intraperitoneal administration of MRTX1133. *p-value with t test assuming equal variance <0.05. Statistical analysis of differences in mean pERK inhibition between vehicle and MRTX1133-treated cohorts was run using a two-tailed Student’s t test with equal variance in Excel (Microsoft; Redmond, WA).

    Figure 14

    Figure 14. Efficacy of MRTX1133 in Panc 04.03 model. **p-value with t test assuming equal variance <0.01. Statistical analysis of differences in mean tumor volume between vehicle and MRTX1133-treated cohorts was run using a two-tailed Student’s t test with equal variance in Excel (Microsoft; Redmond, WA).

    Scheme 1

    Scheme 1. Synthesis of Compound 1a

    aReagents and conditions. (a) POCl3, N-ethyl-N-isopropylpropan-2-amine, 100 °C, 1 h; (b) tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate, N-ethyl-N-isopropylpropan-2-amine, CH2Cl2, −40 °C, 0.5 h, 42% (two steps); (c) ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methanol, N-ethyl-N-isopropylpropan-2-amine, dioxane, 95 °C, 41 h, 55%; (d) ((2-fluoro-6-(methoxymethoxy)-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)ethynyl)triisopropylsilane, Ad2nBuP-Pd-G3, K3PO4, THF, 60 °C, 3 h, 76%; (e) HCl in dioxane, 0 °C, 1 h, 56%; (f) CsF, DMF, 20 °C, 1 h, 56%.

  • References

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  • Supporting Information

    Supporting Information

    ARTICLE SECTIONS
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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01688.

    • Synthetic experimental procedures for compound 5 to 38; NMR spectra and HPLC trace of final Compounds; HTRF, SPR, AlphaLISA, and cellular assay protocols; in vivo PK/PD and TGI studies (PDF)

    • Molecular formula strings (CSV)

    Accession Codes

    PDB code: 7RPZ, 7RT1, 7RT2, 7RT3, 7RT4, 7RT5.


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    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

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