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Discovery of a High Affinity Adenosine A1/A3 Receptor Antagonist with a Novel 7-Amino-pyrazolo[3,4-d]pyridazine Scaffold

  • Anna Suchankova
    Anna Suchankova
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
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  • , 
  • Margarita Stampelou
    Margarita Stampelou
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
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  • , 
  • Klontiana Koutsouki
    Klontiana Koutsouki
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    Department of Nutrition & Dietetics, School of Health Sciences and Education, Harokopio University, 17671 Athens, Greece
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  • , 
  • Athanasios Pousias
    Athanasios Pousias
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
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  • , 
  • Lakshiv Dhingra
    Lakshiv Dhingra
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
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  • , 
  • Kerry Barkan
    Kerry Barkan
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
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  • , 
  • Nicole Pouli
    Nicole Pouli
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    More by Nicole Pouli
  • , 
  • Panagiotis Marakos
    Panagiotis Marakos
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    More by Panagiotis Marakos
  • , 
  • Roxane Tenta
    Roxane Tenta
    Department of Nutrition & Dietetics, School of Health Sciences and Education, Harokopio University, 17671 Athens, Greece
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  • , 
  • Antonios Kolocouris*
    Antonios Kolocouris
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0001-6110-1903
  • , 
  • Nikolaos Lougiakis*
    Nikolaos Lougiakis
    Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    *Email: [email protected]
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  • , and 
  • Graham Ladds*
    Graham Ladds
    Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0001-7320-9612
Cite this: ACS Med. Chem. Lett. 2022, 13, 6, 923–934
Publication Date (Web):May 31, 2022
https://doi.org/10.1021/acsmedchemlett.2c00052

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Here we describe the design and synthesis of pyrazolo[3,4-d]pyridazines as adenosine receptor (AR) ligands. We demonstrate that the introduction of a 3-phenyl group, together with a 7-benzylamino and 1-methyl group at the pyrazolopyridazine scaffold, generated the antagonist compound 10b, which displayed 21 nM affinity and a residence time of ∼60 min, for the human A1R, 55 nM affinity and a residence time of ∼73 min, for the human A3R and 1.7 μΜ affinity for the human A2BR while not being toxic. Strikingly, the 2-methyl analog of 10b, 15b, had no significant affinity. Docking calculations and molecular dynamics simulations of the ligands inside the orthosteric binding area suggested that the 2-methyl group in 15b hinders the formation of hydrogen bonding interactions with N6.55 which are considered critical for the stabilization inside the orthosteric binding cavity. We, therefore, demonstrate that 10a is a novel scaffold for the development of high affinity AR ligands. From the mutagenesis experiments the biggest effect was observed for the Y2717.46A mutation which caused an ∼10-fold reduction in the binding affinity of 10b.

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KEYWORDS:
Adenosine, a naturally occurring purine nucleoside, is the endogenous agonist of adenosine receptors (ARs). (1) ARs are G protein-coupled receptors (GPCRs) comprising four subtypes; A1, A2A, A2B, and A3. The A2A and A2B subtypes act synergistically with Gαs stimulating adenylyl cyclase and, therefore, increasing 3′,5′-cyclic adenosine monophosphate (cAMP) levels. In contrast, A1 and A3 receptor subtypes inhibit adenylyl cyclase and decrease cAMP levels by coupling to the Gi/o family of G proteins.
In the last two decades numerous heterocyclic compounds have been synthesized as AR ligands including xanthines and bi- or tricyclic fused heterocyclic analogues, e.g., purines, deazapurines, pyrazolopyridines, imidazotriazines, thienopyridazines, naphthyridines, pyridopyrimidines, and pyrazoloquinolines. (2−4)
Different therapeutic applications have been identified in preclinical and clinical studies for A1R antagonists as potassium-sparing diuretic agents with kidney-protecting properties, (2) treatments for chronic lung diseases such as asthma, (5,6) and possible use in Parkinson’s disease. (7)
A3R has been reported to be overexpressed in several types of cancer cells and is, thus, considered as a biological marker for tumors. (8) In a recent study, the potent and selective A3R antagonist LJ-1888 ((2R,3R,4S)-2-[2-chloro-6-(3-iodobenzylamino)-9H-purine-9-yl]tetrahydrothiophene-3,4-diol) blocked the development and attenuated the progression of renal interstitial fibrosis, (9) while A3R antagonists have demonstrated efficacy in eye pathologies by lowering intraocular pressure. (10)
While the binding mode of several agonists and antagonists at A1R has been revealed with X-ray crystallography or cryogenic electron microscopy, (11−13) the experimental structures for A3R and A2BR have, to date, not been resolved, and only homology models can be used for these AR subtypes.
By the repurposing of antiproliferative aromatic condensed nitrogen heterocycles, we previously identified nanomolar affinity pyrazolo[3,4-c]pyridine A1R/A3R antagonists. (14) It has been reported that non-xanthine pyrazolo derivatives that potently bind ARs are pyrazolo[4,3-d]pyrimidines, (3) pyrazolo[1,5-c]quinazolines, (15) pyrazolo[3,4-b]pyridines, (16,17) pyrazolo[3,4-b]pyridines, pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines, pyrazolo[3,4-c]- or -[4,3-c]quinolines, pyrazolo[4,3-d]pyrimidinones, pyrazolo[3,4-d]pyrimidines, and pyrazolo[1,5-a]pyridines. (18) After we previously identified the potent pyrazolo[3,4-c]pyridine A1R/A3R antagonists (14) and observed that certain substituted pyrazolo[3,4-b]pyridines had antagonistic potency against A3R or A1R, (16,17) we quantified the novel pyrazolo[3,4-d]pyridazine scaffold for activity at ARs. Here, we synthesized a series of new 3-alkyl- or 3-aryl-7-amino-pyrazolo-[3,4-d]pyridazine derivatives and determined their affinities against the different ARs using functional cAMP accumulation assays, fluorescent ligand displacement binding studies, and molecular dynamics (MD) simulations. (19,20) We identified the 21 nM A1R/55 nM A3R/<2 μΜ A2BR antagonist 1-methyl-3-phenyl-7-benzylaminopyrazolo[3,4-d]pyridazine (10b) as a lead compound. Strikingly, compound 15b, the 2-methyl congener of 10b, had lower affinity by >100-fold against 3AR subtypes since, we assumed, it cannot form hydrogen bonding interactions with N6.55 which are considered critical for stabilization inside the orthosteric binding cavity. Finally, as these new compounds present structural similarity to antiproliferative purine analogues, (21) we evaluated their cytotoxic potential against the human fibroblasts cell line (WI-38) and prostatic (PC-3) and colonic (HCT116) cancer cell lines.

Similarity Calculations

Searching the CHEMBL (22) database to determine if pyrazolo[3,4-d]pyridazine has been used as a scaffold for ligands binding to ARs, using a TanimotoCombo (Tc) (23) coefficient > 0.85, we did not find any pyrazolo[3,4-d]pyridazine derivatives with potency against ARs, suggesting that it is a novel ring system for the development of AR ligands. When we considered the amide 7-benzylamino-3-phenylpyrazolo[3,4-d]pyridazine, we found the 4-(2-phenethyl)amino 1-phenylethylpyrazolo[3,4-b]pyridine (Tc = 0.15) had been reported to bind A1R. (16,17) Thus, we proceeded with a structural activity relationship study around 7-benzylamino-3-phenyl pyrazolo[3,4-d]pyridazine and synthesized a series of 7-amino-pyrazolo[3,4-d]pyridazines for biological evaluation against ARs.

Chemistry

The synthesis of the target compounds was accomplished through the previously reported pyrazolecarboxylates 4a,b and 5a,b (Scheme 1). Briefly, commercial isopropylmethylketone (1a) or acetophenone (1b), was first converted to the ethyl 2,4-diketocarboxylates 2a and 2b, respectively, (24,25) which upon reaction with hydrazine monohydrate gave the pyrazolecarboxylates 3a,b. (26) These were methylated using methyl iodide in the presence of sodium hydride and provided the regioisomers 4a,b (27,28) and 5a,b, (28) respectively. Interestingly, when we used tetrahydrofuran as solvent in the place of dimethylformamide (DMF), we exclusively obtained the N1-methyl-5-carboxylate 4a isomer.

Scheme 1

Scheme 1. Synthesis of 4a,b and 5a,ba
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aReagents and conditions: (a) diethyl oxalate, NaH 60%, toluene dry, 50°C, 2 h; (b) NH2NH2 80%, EtOH, reflux, 90 min; (c) (i) NaH 60%, DMF dry, 0 °C, 15 min, (ii) CH3I, rt, 1 h.

Each of the isomeric pyrazoles 4a,b or 5a,b was subsequently treated with paraformaldehyde in the presence of a 33% HBr solution in acetic acid and was converted to the bromides 6a,b (Scheme 2) or 11a,b (Scheme 3), respectively. The bromomethyl group was then oxidized using N-methylmorpholine N-oxide to generate the carbaldehydes 7a,b (Scheme 2) and 12a,b (Scheme 3).

Scheme 2

Scheme 2. Synthesis of 10a–ca
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aReagents and conditions: (a) paraformaldehyde, 33% HBr in AcOH, 90 °C, 3.5 h; (b) Ν-methylmorpholine-Ν-oxide, MeCN dry, rt, 24 h; (c) NH2NH2 (80%), HCl 36%, EtOH, 90°C, 1 h; (d) POCl3, 110 °C, 2.5–8 h; (e) HNR1R2, EtOH, reflux, 2 h.

Scheme 3

Scheme 3. Synthesis of 15a–ca
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aReagents and conditions: (a) paraformaldehyde, 33% HBr in AcOH, 90 °C, 3.5 h; (b) Ν-methylmorpholine-Ν-oxide, MeCN dry, rt, 24 h; (c) NH2NH2 (80%), HCl 36%, EtOH, 90 °C, 1 h; (d) POCl3, 110 °C, 2.5–8 h; (e) HNR1R2, EtOH, reflux, 2 h.

The aldehydes 7a,b and 12a,b were then treated with hydrazine, and upon ring closure the pyrazolopyridazinones 8a,b and 13a,b were obtained. The pyridazinones reacted with phosphorus oxychloride to give the corresponding chloro derivatives 9a,b and 14a,b with suitable purity that they could be introduced to the next reaction. These crude products were then treated with benzylamine or morpholine to result in the target compounds 10a–c and 15a–c (Figures S1–S3).

Assessing Biological Activity of Pyrazolo[3,4-d]pyridazine Derivatives

cAMP Assays Assessing Activity at Adenosine Receptors

Having synthesized compounds 10a–c and 15a–c, we next tested their activity, as antagonists, against the different human AR subtypes using a single high concentration of the compound (1 μM) coadministered with NECA (5′-N-ethylcarboxamidoadenosine) in a cAMP accumulation assay (Figure 1A and B). Note that for A1R and A3R 10 μM forskolin was added since these are Gi/o-coupled receptors and reduce cAMP accumulation. (19,29) All compounds lacked efficacy at NECA-stimulated A2AR (even when tested at 10 μM) (Table S1). Compounds 10c, 15b, and 15c also lacked efficacy at the other 3AR subtypes, with 15a displaying weak efficacy only at A3R, while compounds 10a and 10b displayed activity at all 3ARs although this was only detectable for A2BR when a 10 μM concentration of the compound was used (Table S1). Based upon a single concentration of antagonist, we calculated the equilibrium dissociation constant (pKd) of each compound (Table 1). Of the compounds tested, 10b displayed the highest affinity at the different AR subtypes with greater selectivity toward A1R and A3R than A2BR. We next performed a more extensive Schild analysis using multiple doses of the most potent antagonist, 10b, only at A1R and A3R (Figure 1C). In both cases 10b acted as a competitive antagonist, generating a Schild slope that did not significantly differ from unity. Using the Schild plot, we calculated 10b’s affinity (pA2/pKb) to be 21 nM at A1R and 55 nM at A3R while only 1.7 μΜ at A2BR (Table S1).

Figure 1

Figure 1. Characterization of 7-amino-pyrazolo[3,4-d]pyridazines at human A1R and A3R. (A and B) Cells expressing either human A1R (A) or A3R (B) were exposed to 10 μM forskolin and stimulated with increasing concentrations of NECA for 30 min in the presence of a 1 μM concentration of the test compound, and the cAMP accumulation was quantified. (C) cAMP accumulation was measured as detailed in part A using multiple concentrations of 10b. Using pEC50 values, Schild regression analysis was conducted to calculate pA2/pKb values. All values are mean ± SEM expressed as percentage forskolin inhibition, relative to NECA. n ≥ 3 independent experimental repeats were performed in duplicate.

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Table 1. Chemical Structures, Antagonistic Potencies (pEC50 in the Presence of NECAa), and Affinities (pKib) of 7-Amino-pyrazolo[3,4-d]pyridazines 10a–c and 15a–c against A1R and A3R
a

Mean ± SEM; functional activities (pEC50 values of NECA in the presence of either 1 μM ligands or vehicle) as mean ± standard error of the mean (SEM) of at least three independent repeats, conducted in duplicate─values obtained from Figure 1.

b

Mean ± SEM; equilibrium binding affinities of the ligands measured with NanoBRET against Nluc-A3R or Nluc-A1R; NECA was used as positive control. (3)

#

Due to the high affinity of MRS1220, 10 nM was used to enable measurement of the full dose–response curve of NECA to determine pEC50.

Statistical significance compared to NECA was determined, at p < 0.05, through one-way ANOVA with Dunnett’s post-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Quantifying Binding Parameters Using a NanoBRET-Based Saturation Binding Assay

We next sought to independently verify the affinities determined using the Shield analysis by directly quantifying the potential antagonists’ binding to A1R and A3R using a previously described saturation nano-bioluminescence resonance energy transfer (NanoBRET) binding assay. (19) We determined the ability of all the compounds to displace the specific binding of CA200645, (30) a fluorescent antagonist of A3R and A1R, using Nluc-A3R expressing human embryonic kidney 293 (HEK293) and Nluc-A1R HEK293 cells (Figure 2 and Table 1). A2BR was not included in this analysis since the pKd values of 10a and 10b at A2BR were estimated to be below 1 μM (Figure 1 and Table 1). Consistent with the Schild analysis, compound 10b displayed the highest affinity at A1R and A3R (A1R, pKi = 7.95 ± 0.09; A3R, pKi = 7.89 ± 0.11). Of the remaining compounds, 10a displayed weak affinity at A3R (pKi, 6.42 ± 0.28), which agreed with the Schild regression estimate, but failed to fully displace CA200645 at A1R, making an estimate for its affinity unreliable. All the other compounds failed to displace CA200645 at A1R or A3R except for 15a and 15c, which did display some binding at A3R but, like 10a, also failed to fully displace CA200645 at the concentrations tested. Significantly, 15b, which contains an N-methyl substitution to 1-NH and 2-NMe compared to 1-NMe and 2-NH in 10b, failed to bind either AR subtype.

Figure 2

Figure 2. Inhibition of BRET between CA200645 at NLuc-A1R and Nluc-A3R by 10b and 10a. HEK293 cells expressing Nluc-A1R (A) or Nluc-A3R (B) were treated with 5 nM or 20 nM CA200645, respectively, enabling concentration-dependent decreases in the BRET ratio at 10 min to be determined with the response normalized to DMSO. Binding curves were fitted with the Cheng Prusoff equation built into GraphPad Prism 9.3 to enable estimates of the pKi. (19) Comparison of pKi values for A1R (C) and A3R (D) as determined via BRET binding. Each data point represents the mean ± SEM of at least three experiments performed in duplicate. The statistical significance compared to NECA was determined, at p < 0.05, through one-way ANOVA with Dunnett’s post-test (*, p < 0.05; ***, p < 0.001). #Compounds did not fully displace CA200645, so pKi values are estimates preventing statistical analysis.

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Determining Kinetic Parameters of 10b Binding at A3R and A1R Using NanoBRET

We next investigated the real-time binding kinetics (19,30) of 10b at A3R and A1R using the NanoBRET binding method. Specifically, we quantified 10b’s ability to inhibit specific binding of CA200645 to Nluc-A3R and Nluc-A1R expressed in HEK293 cells. The kinetic parameters for CA200645 binding at Nluc-A3R were previously determined as Kon = 32.5 ± 0.28 × 105 M–1 min–1 and Koff = 0.025 ± 0.005 min–1 with a pKD of 10.11. Conversely the kinetics of CA200645 binding at Nluc-A1R were determined as Kon = 14.5 ± 0.4 × 105 M–1 min–1, Koff = 0.023 ± 0.001 min–1, and pKD = 7.80 ± 0.2 nM. (14) Applying these parameters into the “kinetics of competitive binding” model built into GraphPad Prism9.0, we were able to provide estimates of the kinetics of binding for 10b against A1R (Kon = 51.4 ± 0.26 × 105 M–1 min–1, Koff = 0.019 ± 0.003 min–1 with a pKD = 7.46 ± 0.1 and RT = 59.8 ± 12.7 min) and against the A3R, (Kon = 25.6 ± 0.1 × 105 M–1min–1, Koff = 0.0014 ± 0.002 min–1 with a pKD = 7.26 ± 0.05 and RT = 72.58 ± 8.8 min). None of the other compounds were analyzed using this method due to their extremely fast Koff rates (>min–1). For compound 10b there was an excellent agreement between pKD (Kon/Koff) of the compounds from the kinetics assays and the Schild analysis (pA2/pKb) and fair agreement (∼3.16-fold) with the saturation binding assays (pKi).

Simulations

Investigation of the Binding of the 7-Amino-pyrazolo[3,4-d]pyridazines to A1R and A3R

Having pharmacologically evaluated the different compounds, we then used molecular docking to provide insights into how they bind to the ARs. We docked 10a–10c into the orthosteric binding site of A1R and 10b and 15b into A2BR and A3R (the amino acid sequences of A1R, A3R, and A2BR in the orthosteric binding area are shown in Scheme S1) using ChemScore as the scoring function (31) with the highest score docking pose being inserted into a hydrated phosphatidylethanolamine bilayer. The complexes were subjected to 100 ns MD simulations with amber99sb, (32) and then, the MD simulations’ trajectory was analyzed (Table S2). The MD simulations showed that the 7-benzylamino-pyrazolo[3,4-d]pyridazine 10b substituted with N1Me and a 3-phenyl group formed a stable complex with all 3ARs with RMSDprotein values <2.1 Å. Starting from the same docking pose of 10b in A1R or A3R (Figure 3), the mean frame from MD simulations was close to the starting docking pose in A1R (RMSDlig = 1.21 Å) while in A3R (Figure S2) the ligand moved considerably into the cleft between the transmembrane (TM)3, TM5, and TM6 helices (RMSDlig = 4.88 Å). Thus, starting from the same binding pose for 10b, the MD simulations produced two different binding orientations at A1R and A3R. This is due to the fact that A1R has a broader binding area, expanded toward TM1 and TM2, compared to the other ARs, according to the X-ray structures of A1R in complex with antagonists. (11,12) A similar AR ligand reported in the literature is 4-(2-phenethyl)amino 1-phenylethyl pyrazolo[3,4-b]pyridine (Tc = 0.15), which binds with a similar docking pose to 10b to A1R. (16) We also docked a representative adenine derivative (N9-methyl,N6-benzyl adenine) to A1R and found a similar docking pose (Figure S3).

Figure 3

Figure 3. (A–C) 100 ns MD simulations of 10a–c inside the orthosteric binding area of A1R. (D) 100 ns MD simulations of 15b inside the orthosteric binding area of A1R. Starting structures are shown (docking pose), and representative frames from MD simulations, receptor–ligand interaction frequency histograms, and RMSD plots of proteins (RMSDprotein; blue plots) and ligand heavy atoms (RMSDligand; red plots) inside the orthosteric binding area of WT A1R or A3R. Bars are plotted only for residues with interaction frequencies ≥0.2. Color scheme: ligand = brown sticks, receptor = white cartoon and sticks, hydrogen bonding interactions = yellow (dashes or bars), π–π interactions = green (dashes or bars), hydrophobic interactions = gray, water bridges = blue. For the protein models of A1R in complex with 10a–c or 15b, the experimental structure of the inactive form of A1R in complex with an antagonist (PDB ID 5UEN (4)) was used.

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Inside the A1R orthosteric site, compound 10b formed hydrogen bonds through its pyrazole or pyridazine nitrogen donor groups to the amide side chain of N2546.5 or the imidazole side chain of H2787.43. Furthermore, 10b was stabilized in the orthosteric binding site through π–π interactions between its pyrazolo[3,4-d]pyridazine or phenyl rings with F1715.29, H2516.2, and W2476.48, respectively. The benzylamino group of 10b oriented toward the widened TM2 area in A1R, forming hydrophobic interactions with A662.61 and I692.64. Furthermore, 10b was found to bind deep in the pocket interacting with V873.32 and W2476.48 while 3-phenyl-pyrazole aligned close to the side chains of M1805.38 and L2506.1 (Figure 3A). In A3R, compound 10b was stabilized through formation of hydrogen bonding interactions with N2546.5 and H2787.43 and hydrophobic interactions with L903.32, L913.33, F1685.29, M1775.38, L2466.1, and I2687.39 (Figure S2B). Finally, the MD simulations for 10b (Figure S2A) in complex with A2BR (Figure S2) show weak hydrogen bond interactions with N2546.5.
Pharmacologically, compounds 10b and 15b differed considerably in their affinity to the ARs (Figures 1 and 2 and Table 1). Comparing MD simulations for 15b with 10b in the orthosteric binding area of A1R, A3R (and A2BR) shows that starting from a similar docking pose, the substitution from N1 Me and 2-NH (found in 10b) to N1H and N2 Me (in 15b) results in 15b failing to generate hydrogen bonds with N6.55 because of the steric repulsion between 2-methyl and the amide side chain of N6.55; for this reason also 15a and 15c were inactive (Figure S2). Although many ligands can have similar docking poses, subtle changes in the ligand substitution pattern can result in significant changes in binding, and this can be followed only with MD simulations. Considering the two active compounds, 10b and 10a, replacement of the 3-phenyl group (found in 10b) with a 3-isopropyl group (generating 10a) results in a remarkable reduction of affinity. This is due to 10a losing significant π–π interactions with H2516.2 and hydrophobic interactions with residues deeper in the binding site, e.g., W2476.48, L2506.1, and V873.32 (Figure 3). Finally, substitution of 10b’s 7-benzylamino by the more rigid morpholinyl group (found in 10c) resulted in reduced affinity to the ARs. The more rigid morpholino group in 10c repels F1715.29, so the ligand rotates and moves to the bottom of the binding area, losing hydrogen bonding interactions with N2546.5 and weakening its hydrophobic interaction with critical residues, e.g., F1715.29 and L2506.1 (Figure 3). With an accuracy of ∼±4 kcal mol–1, the MM-GBSA method (33,34) (Supporting Information) only provides an approximation when applied to structure–activity relationships for analogs in the same series. Nevertheless, the MM-GBSA binding free energy calculations for ligands 10a–c against A1R (Table S2), using the OPLS2005 force field (35,36) with a hydrophobic slab as an implicit membrane model and including the waters in the orthosteric binding area, predicted fairly well the stability of 10a–c in complex with A1R with binding free energy values (after neglecting entropy) ΔGeff = −94.50, −96.42, and −85.35 kcal mol–1.

Mutagenesis Experiments to Study 10b Binding to A1R

We have previously observed that mutation of residues that do not directly interact with the ligands (e.g., V5.30 for A3R, which is more than 4 Å apart from the ligand inside the orthosteric binding area) can, through allosteric interactions due to the plasticity of the binding area, significantly affect ligand affinity. (20,21,37) As such it is not always straightforward to determine the effects of a mutation on affinity properties. Despite this caveat, we next used mutational analysis combined with NanoBRET to determine the important residues required for 10b binding to A1R. The mutation of L2506.1A resulted in only a slight reduction of binding affinity for 10b (Table 2) despite the MD simulations suggesting that the ligand should be close enough to L2506.1 to enable hydrophobic interactions. It is possible that residues H2516.52 and W2476.48 could contribute to the stabilization of 10b with hydrophobic interactions even if L2506.1 is mutated to alanine. It is noteworthy that mutation of E1725.30 (which is also more than 4 Å apart from the ligand inside the orthosteric binding area) to alanine also did not significantly change the binding affinity (Table 2). This contrasts with our studies using 3-phenyl-7-anilinopyrazolo[3,4-c]pyridines which showed a 1.5-fold decrease in affinity due to the E1725.30A mutation. (14)
Table 2. Binding Affinities (pKi) for 10b Measured Using Saturation NanoBRET Binding with CA200645 as the Fluorescent Tracer against WT A1R and Mutant A1Rs
A1RpKiEffect on affinity
WT7.68 ± 0.11baseline
T913.36A7.68 ± 0.07no change
E1725.30 A7.34 ± 0.06no significant change
L2506.51 A7.57 ± 0.04no significant change
H2516.52A7.62 ± 0.06no significant change
S2677.42A7.86 ± 0.03no significant change
Y2717.46A6.99 ± 0.05∼10-fold reduction
In addition, mutation of H2516.2A has been reported to reduce antagonist affinity against A3R (20,21) although here it did not have any effect on 10b affinity at A1R. Other residues of interest to mutate were T913.36A and S2677.42A, which are deep in the orthosteric pocket. Interestingly, we found that mutation to alanine of these residues also did not have a significant effect on the binding affinity of 10b (Table 2). This is in contrast to our results for pyrazolo[3,4-c]pyridines which can interact directly with these residues. (9) The results for 10b suggested that it is positioned above pyrazolo[3,4-c]pyridines, (9) in the A1R pocket, and so unaffected by these mutations.
The biggest effect in this study was observed for the Y2717.46A A1R mutation, which caused a ∼10-fold reduction in the binding affinity of 10b (Table 2). This effect is in contrast to that observed previously for pyrazolo[3,4-c]pyridines (14) for which we showed that the Y2717.46A mutation caused a slight increase in binding affinity. Since the MD simulations showed contacts with H2787.43 and not Y2717.46, the Y2717.46A mutation in A1R might affect the binding of 10b through contact with H2787.43. We performed the MD simulations of 10b in complex with A1R-Y2717.46A and observed that the ligand loses its hydrogen bonding interactions with N2546.5, which might weaken its binding interactions with the orthosteric binding area (Figure S4).

Preliminary Toxicological Analysis of Pyrazolo[3,4-d]pyridazine Derivatives

Given the high affinity 10b displays for A1R and A3R, and thus the potential for it to be a scaffold for future compound development, we wanted to evaluate its antiproliferative nature as an early indicator of its toxicological profile. We therefore evaluated 10b, alongside the other compounds in this study, for cytotoxic activity against human fibroblasts (WI-38) and two cancer cell lines, namely the prostate cancer (PC-3) and colon cancer (HCT116) cell lines. Importantly, 10b alongside all the compounds proved to be not cytotoxic against the cell lines, with IC50 values >10 μM. The only compound that did display any cytotoxicity was 15b, which displayed moderate cytotoxicity against the PC-3 and HCT116 cell lines, showing IC50 values of 5.3 ± 0.1 μM against PC-3 cells and 4.15 ± 0.05 μM against HCT116 cells. As a result of these data, we are confident that 10b is noncytotoxic and can be progressed for further development as a dual A1R/A3R antagonist.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00052.

  • Scheme S1. Comparison of amino acid residue sequences of the binding area. Table S1. Chemical structures and antagonistic potencies of 7-amino-pyrazolo[3,4-d]pyridazines 10a–c and 15a–c against A2AR and A2BR. Table S2. Mean RMSD values for all compounds against A1R, A2AR, and A2BR and ΔGeff only for 10–10c against A1R. Figure S1. 1H and 13C NMR spectra of the target compounds. Figure S2. Results from the MD simulations of 10b and 15b against A3R and A2BR. Figure S3. Docking poses of 4-(2-phenethyl)amino-1-phenylethyl pyrazolo[3,4-b]pyridine and N9-methyl,N6-benzyl adenine to A1R. Figure S4. Representative frames from 100 ns MD simulations of 10b inside the orthosteric binding area of WT A1R and 10b inside mutant Y271A A1R and the receptor–ligand interaction frequency histogram and RMSD graphs of protein Ca and ligand heavy atoms. Information for the methods and synthetic protocols. (PDF)

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Author Information

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  • Corresponding Authors
    • Antonios Kolocouris - Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece;  Orcidhttps://orcid.org/0000-0001-6110-1903;  Email: [email protected]
    • Nikolaos Lougiakis - Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece;  Email: [email protected]
    • Graham Ladds - Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.;  Orcidhttps://orcid.org/0000-0001-7320-9612;  Email: [email protected]
  • Authors
    • Anna Suchankova - Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
    • Margarita Stampelou - Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    • Klontiana Koutsouki - Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece;  Department of Nutrition & Dietetics, School of Health Sciences and Education, Harokopio University, 17671 Athens, Greece;  Present Address: Cyprus Pharmaceutical Organization Limited, 11 King Paul A’ Street, 1096 Nicosia, Cyprus
    • Athanasios Pousias - Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    • Lakshiv Dhingra - Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
    • Kerry Barkan - Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, U.K.
    • Nicole Pouli - Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    • Panagiotis Marakos - Laboratory of Medicinal Chemistry, Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, 15771 Athens, Greece
    • Roxane Tenta - Department of Nutrition & Dietetics, School of Health Sciences and Education, Harokopio University, 17671 Athens, Greece
  • Author Contributions

    A.S., M.S., and K.K. contributed equally. A.K., N.L. and G.L. contributed equally. A.K., N.L., G.L., and M.S. conceived and designed the research; K.K. and A.P. synthesized and characterized the compounds in the P.M., N.P., and N.L. lab; A.S. and L.D. performed the mammalian assays in the G.L. lab; M.S. performed the simulations in the A.K. lab; K.K., A.P., M.S., A.S., N.L., P.M., A.K., and G.L. analyzed the data; A.K., P.M., N.P., N.L., and G.L. wrote the manuscript; A.K., G.L., N.L., and M.S. edited the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This research represents part of the master’s thesis of K.K. and part of the Ph.D. theses of A.S. and M.S. We gratefully acknowledge the support of Chiesi Hellas (A.K.), the Cambridge Trust (A.S.), the Leverhulme Trust (G.L.), and the BBSRC (G.L.). This work was supported by computational time granted from the Greek Research & Technology Network (GRNET) in the National HPC facility - ARIS - under project ID pr001007.

Abbreviations

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ARs

adenosine receptors

BRET

bioluminescence resonance energy transfer

GPCRs

G protein-coupled receptors

HEK

human embryonic kidney

MD

molecular dynamics

NECA

5′-N-ethylcarboxamidoadenosine

PDB

Protein Data Bank

RMSD

root-mean-square deviation

Tc

TanimotoCombo

tm

mixing time

TM

transmembrane

References

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    Adenosine in the normal kidney significantly elevates in response to cellular damage. The renal A3 adenosine receptor (A3AR) is up-regulated under stress, but the therapeutic effects of A3AR antagonists on chronic kidney disease are not fully understood. The present study examd. the effect of LJ-1888 [(2R,3R,4S)-2-[2-chloro-6-(3-iodobenzylamino)-9H-purine-9-yl]-tetrahydrothiophene-3,4-diol], a newly developed potent, selective, species-independent, and orally active A3AR antagonist, on unilateral ureteral obstruction (UUO)-induced renal fibrosis. Pretreatment with LJ-1888 inhibited UUO-induced fibronectin and collagen I up-regulation in a dose-dependent manner. Masson's trichrome staining confirmed that LJ-1888 treatment effectively reduced UUO-induced interstitial collagen accumulation. Furthermore, delayed administration of LJ-1888 showed an equiv. therapeutic effect on tubulointerstitial fibrosis to that of losartan. Small-interfering A3AR transfection effectively inhibited transforming growth factor-β1 (TGF-β1)-induced fibronectin and collagen I up-regulation in proximal tubular cells similar to LJ-1888, confirming that the renoprotective effect of LJ-1888 resulted from A3AR blockade. UUO- or TGF-β1-induced c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation decreased significantly after LJ-1888 administration. A3AR blockade reduced UUO- or TGF-β1-induced up-regulation of lysyl oxidase, which induces crosslinking of extracellular matrix, suggesting that LJ-1888 may also regulate extracellular matrix accumulation via post-translational regulation. In conclusion, the present data demonstrate that the A3AR antagonist, LJ-1888, blocked the development and attenuated the progression of renal fibrosis, and they suggest that LJ-1888 may become a new therapeutic modality for renal interstitial fibrosis.
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    Wang, Z.; Do, C. W.; Avila, M. Y.; Peterson-Yantorno, K.; Stone, R. A.; Gao, Z.-G.; Joshi, B.; Besada, P.; Jeong, L. S.; Jacobson, K. A.; Civan, M. M. Nucleoside-Derived Antagonists to A3 Adenosine Receptors Lower Mouse Intraocular Pressure and Act across Species. Exp. Eye Res. 2010, 90, 146– 154,  DOI: 10.1016/j.exer.2009.10.001
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    10
    Nucleoside-derived antagonists to A3 adenosine receptors lower mouse intraocular pressure and act across species
    Wang, Zhao; Do, Chi-Wai; Avila, Marcel Y.; Peterson-Yantorno, Kim; Stone, Richard A.; Gao, Zhan-Guo; Joshi, Bhalchandra; Besada, Pedro; Jeong, Lak-Shin; Jacobson, Kenneth A.; Civan, Mortimer M.
    Experimental Eye Research (2010), 90 (1), 146-154CODEN: EXERA6; ISSN:0014-4835. (Elsevier B.V.)
    The purpose of the study was to det. whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the 5 agonist derivs., LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other 4 compds., consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, 5 derivs. of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivs. of the highly-selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivs. are promising A3 antagonists for study in multiple animal models.
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  11. 11
    Glukhova, A.; Thal, D. M.; Nguyen, A. T.; Vecchio, E. A.; Jörg, M.; Scammells, P. J.; May, L. T.; Sexton, P. M.; Christopoulos, A. Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity. Cell 2017, 168, 867– 877,  DOI: 10.1016/j.cell.2017.01.042
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    11
    Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity
    Glukhova, Alisa; Thal, David M.; Nguyen, Anh T.; Vecchio, Elizabeth A.; Jorg, Manuela; Scammells, Peter J.; May, Lauren T.; Sexton, Patrick M.; Christopoulos, Arthur
    Cell (Cambridge, MA, United States) (2017), 168 (5), 867-877.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The adenosine A1 receptor (A1-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We detd. a 3.2 Å crystal structure of the A1-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A2A receptor (A2A-AR) structure. Mutational and computational anal. of A1-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a mol. basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.
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  12. 12
    Cheng, R. K. Y.; Segala, E.; Robertson, N.; Deflorian, F.; Doré, A. S.; Errey, J. C.; Fiez-Vandal, C.; Marshall, F. H.; Cooke, R. M. Structures of Human A1 and A2A Adenosine Receptors with Xanthines Reveal Determinants of Selectivity. Structure 2017, 25, 1275– 1285,  DOI: 10.1016/j.str.2017.06.012
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    12
    Structures of Human A1 and A2a Adenosine Receptors with Xanthines Reveal Determinants of Selectivity
    Cheng, Robert K. Y.; Segala, Elena; Robertson, Nathan; Deflorian, Francesca; Dore, Andrew S.; Errey, James C.; Fiez-Vandal, Cedric; Marshall, Fiona H.; Cooke, Robert M.
    Structure (Oxford, United Kingdom) (2017), 25 (8), 1275-1285.e4CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
    The adenosine A1 and A2a receptors belong to the purinergic family of G protein-coupled receptors, and regulate diverse functions of the cardiovascular, respiratory, renal, inflammation, and CNS. Xanthines such as caffeine and theophylline are weak, non-selective antagonists of adenosine receptors. Here we report the structure of a thermostabilized human A1 receptor at 3.3 Å resoln. with PSB36, an A1-selective xanthine-based antagonist. This is compared with structures of the A2a receptor with PSB36 (2.8 Å resoln.), caffeine (2.1 Å), and theophylline (2.0 Å) to highlight features of ligand recognition which are common across xanthines. The structures of A1R and A2aR were analyzed to identify the differences that are important selectivity determinants for xanthine ligands, and the role of T2707.35 in A1R (M2707.35 in A2aR) in conferring selectivity was confirmed by mutagenesis. The structural differences confirmed to lead to selectivity can be utilized in the design of new subtype-selective A1R or A2aR antagonists.
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  13. 13
    Draper-Joyce, C. J.; Khoshouei, M.; Thal, D. M.; Liang, Y.-L.; Nguyen, A. T. N.; Furness, S. G. B.; Venugopal, H.; Baltos, J.-A.; Plitzko, J. M.; Danev, R.; Baumeister, W.; May, L. T.; Wootten, D.; Sexton, P. M.; Glukhova, A.; Christopoulos, A. Structure of the Adenosine-Bound Human Adenosine A1 Receptor-Gi Complex. Nature 2018, 558, 559– 563,  DOI: 10.1038/s41586-018-0236-6
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    13
    Structure of the adenosine-bound human adenosine A1 receptor-Gi complex
    Draper-Joyce, Christopher J.; Khoshouei, Maryam; Thal, David M.; Liang, Yi-Lynn; Nguyen, Anh T. N.; Furness, Sebastian G. B.; Venugopal, Hariprasad; Baltos, Jo-Anne; Plitzko, Jurgen M.; Danev, Radostin; Baumeister, Wolfgang; May, Lauren T.; Wootten, Denise; Sexton, Patrick M.; Glukhova, Alisa; Christopoulos, Arthur
    Nature (London, United Kingdom) (2018), 558 (7711), 559-563CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein detd. by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound β2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides mol. insights into receptor and G-protein selectivity.
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  14. 14
    Stampelou, M.; Suchankova, A.; Tzortzini, E.; Dhingra, L.; Barkan, K.; Lougiakis, N.; Marakos, P.; Pouli, N.; Ladds, G.; Kolocouris, A. Novel Pyrazolo[3,4-c]Pyridine Antagonists with Nanomolar Affinity for A1/A3 Adenosine Receptors: Binding Kinetics and Exploration of Their Binding Profile Using Mutagenesis Experiments, MD Simulations and TI/MD Calculations. ChemRxiv 2021,  DOI: 10.26434/chemrxiv-2021-j18mg-v3 .
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  15. 15
    Catarzi, D.; Colotta, V.; Varano, F.; Poli, D.; Squarcialupi, L.; Filacchioni, G.; Varani, K.; Vincenzi, F.; Borea, P. A.; Dal Ben, D.; Lambertucci, C.; Cristalli, G. Pyrazolo[1,5-c]Quinazoline Derivatives and Their Simplified Analogues as Adenosine Receptor Antagonists: Synthesis, Structure-Affinity Relationships and Molecular Modeling Studies. Bioorg. Med. Chem. 2013, 21, 283– 294,  DOI: 10.1016/j.bmc.2012.10.031
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    15
    Pyrazolo[1,5-c]quinazoline derivatives and their simplified analogues as adenosine receptor antagonists: Synthesis, structure-affinity relationships and molecular modeling studies
    Catarzi, Daniela; Colotta, Vittoria; Varano, Flavia; Poli, Daniela; Squarcialupi, Lucia; Filacchioni, Guido; Varani, Katia; Vincenzi, Fabrizio; Borea, Pier Andrea; Dal Ben, Diego; Lambertucci, Catia; Cristalli, Gloria
    Bioorganic & Medicinal Chemistry (2013), 21 (1), 283-294CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
    A no. of 5-oxo-pyrazolo[1,5-c]quinazolines, bearing at position-2 the (hetero)aryl moiety but also a carboxylate group, were designed as hA3 AR antagonists. This study produced some interesting compds. endowed with good hA3 receptor affinity and high selectivity, being totally inactive at all the other AR subtypes. In contrast, the corresponding 5-ammino derivs. do not bind or bind with very low affinity at the hA3 AR, the only exception being the 5-N-benzoyl compd. I that shows a hA3Ki value in the high μ-molar range. Evaluation of the synthetic intermediates led to the identification of some 5(3)-(2-aminophenyl)-3(5)-(hetero)arylpyrazoles II [R = Ph, 4-MeOC6H4, 2-furyl, 2-thienyl, CO2Et] with modest affinity but high selectivity toward the hA3 AR subtype. Mol. docking of the herein reported tricyclic and simplified derivs. was carried out to depict their hypothetical binding mode to our model of hA3 receptor.
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  16. 16
    Manetti, F.; Schenone, S.; Bondavalli, F.; Brullo, C.; Bruno, O.; Ranise, A.; Mosti, L.; Menozzi, G.; Fossa, P.; Trincavelli, M. L.; Martini, C.; Martinelli, A.; Tintori, C.; Botta, M. Synthesis and 3D QSAR of new pyrazolo[3,4-b]pyridines: potent and selective inhibitors of A1 adenosine receptors. J. Med. Chem. 2005, 48, 7172– 7185,  DOI: 10.1021/jm050407k
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    16
    Synthesis and 3D QSAR of New Pyrazolo[3,4-b]pyridines: Potent and Selective Inhibitors of A1 Adenosine Receptors
    Manetti, Fabrizio; Schenone, Silvia; Bondavalli, Francesco; Brullo, Chiara; Bruno, Olga; Ranise, Angelo; Mosti, Luisa; Menozzi, Giulia; Fossa, Paola; Trincavelli, Maria Letizia; Martini, Claudia; Martinelli, Adriano; Tintori, Cristina; Botta, Maurizio
    Journal of Medicinal Chemistry (2005), 48 (23), 7172-7185CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A no. of 4-(amino)pyrazolo[3,4-b]pyridine-5-carboxylic acid esters were synthesized and evaluated for their binding affinity at the A1, A2A, and A3 adenosine receptors (AR), in bovine cortical membranes, as well as for their affinity toward human A1AR (hA1AR) (A1 purinoceptor antagonists). Some of the new compds. were characterized by a high affinity and selectivity toward the A1 receptor subtype, showing a significant improvement in comparison with other pyrazolo-pyridines previously reported in the literature. In particular two compds., both of them bearing a [(p-methoxyphenyl)ethyl]amino side chain presented Ki values of 6 and 7 nM, resp. To rationalize the relationship between structure and affinity of the novel compds., a 3D QSAR model was also generated starting from compds. belonging to different classes of known A1AR antagonists.
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  17. 17
    Tuccinardi, T.; Zizzari, A. T.; Brullo, C.; Daniele, S.; Musumeci, F.; Schenone, S.; Trincavelli, M. L.; Martini, C.; Martinelli, A.; Giorgi, G.; Botta, M. Substituted pyrazolo[3,4-b]pyridines as human A1 adenosine antagonists: Developments in understanding the receptor stereoselectivity. Org. Biomol. Chem. 2011, 9, 4448– 4455,  DOI: 10.1039/c0ob01064b
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    17
    Substituted pyrazolo[3,4-b]pyridines as human A1 adenosine antagonists: Developments in understanding the receptor stereoselectivity
    Tuccinardi, Tiziano; Zizzari, Alessandra Tania; Brullo, Chiara; Daniele, Simona; Musumeci, Francesca; Schenone, Silvia; Trincavelli, Maria Letizia; Martini, Claudia; Martinelli, Adriano; Giorgi, Gianluca; Botta, Maurizio
    Organic & Biomolecular Chemistry (2011), 9 (12), 4448-4455CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    A1 adenosine receptor antagonists have been proposed to possess an interesting range of potential therapeutic applications. The authors have already reported the synthesis and the biol. characterization of a family of pyrazolo[3,4-b]pyridine derivs. as A1 adenosine ligands endowed with an antagonistic profile. In the present work, the authors report the LC sepn. of enantiomers of the authors' most active A1 antagonists together with the detn. of their abs. configuration by x-ray crystal structure anal. Biol. assays confirmed a different activity for the two enantiomers, with the R one showing the higher human A1AR affinity. The authors also developed a homol. model of this receptor subtype to suggest a binding disposition of the ligands into the hA1AR. All of the obtained data suggest that the compd.'s chirality plays a key role in A1 affinity.
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  18. 18
    Cheong, S. L.; Venkatesan, G.; Paira, P.; Jothibasu, R.; Mandel, A. L.; Federico, S.; Spalluto, G.; Pastorin, G. Pyrazolo Derivatives as Potent Adenosine Receptor Antagonists: An Overview on the Structure-Activity Relationships. Int. J. Med. Chem. 2011, 480652,  DOI: 10.1155/2011/480652
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    18
    Pyrazolo derivatives as potent adenosine receptor antagonists: an overview on the structure-activity relationships
    Cheong, Siew Lee; Venkatesan, Gopalakrishnan; Paira, Priyankar; Jothibasu, Ramasamy; Mandel, Alexander Laurence; Federico, Stephanie; Spalluto, Giampiero; Pastorin, Giorgia
    International Journal of Medicinal Chemistry (2011), (), 480652, 15 pp.CODEN: IJMCCH; ISSN:2090-2077. (Hindawi Publishing Corp.)
    A review. In the past few decades, medicinal chem. research towards potent and selective antagonists of human adenosine receptors (namely, A1, A2A, A2B, and A3) has been evolving rapidly. These antagonists are deemed therapeutically beneficial in several pathol. conditions including neurol. and renal disorders, cancer, inflammation, and glaucoma. Up to this point, many classes of compds. have been successfully synthesized and identified as potent human adenosine receptor antagonists. In this paper, an overview of the structure-activity relationship (SAR) profiles of promising nonxanthine pyrazolo derivs. is reported and discussed. We have emphasized the SAR for some representative structures such as pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines; pyrazolo-[3,4-c] or -[4,3-c]quinolines; pyrazolo-[4,3-d]pyrimidinones; pyrazolo-[3,4-d]pyrimidines and pyrazolo-[1,5-a]pyridines. This overview not only clarifies the structural requirements deemed essential for affinity towards individual adenosine receptor subtypes, but it also sheds light on the rational design and optimization of existing structural templates to allow us to conceive new, more potent adenosine receptor antagonists.
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  19. 19
    Barkan, K.; Lagarias, P.; Stampelou, M.; Stamatis, D.; Hoare, S.; Safitri, D.; Klotz, K.-N.; Vrontaki, E.; Kolocouris, A.; Ladds, G. Pharmacological Characterisation of Novel Adenosine A3 Receptor Antagonists. Sci. Rep. 2020, 10, 20781,  DOI: 10.1038/s41598-020-74521-y
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    19
    Pharmacological characterization of novel adenosine A3 receptor antagonists
    Barkan, Kerry; Lagarias, Panagiotis; Stampelou, Margarita; Stamatis, Dimitrios; Hoare, Sam; Safitri, Dewi; Klotz, Karl-Norbert; Vrontaki, Eleni; Kolocouris, Antonios; Ladds, Graham
    Scientific Reports (2020), 10 (1), 20781CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but crit. task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Pos. hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was detd. using Schild anal. (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the arom. nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by mol. dynamic simulations combined with Mol. Mechanics-Poisson Boltzmann Surface Area calcns. identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 μM) competitive antagonist. Finally, we introduce a model that enables ests. of the equil. binding affinity for rapidly disassocg. compds. from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacol. characterization of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.
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  20. 20
    Lagarias, P.; Barkan, K.; Tzortzini, E.; Stampelou, M.; Vrontaki, E.; Ladds, G.; Kolocouris, A. Insights to the Binding of a Selective Adenosine A3 Receptor Antagonist Using Molecular Dynamic Simulations, MM-PBSA and MM-GBSA Free Energy Calculations, and Mutagenesis. J. Chem. Inf. Model. 2019, 59, 5183– 5197,  DOI: 10.1021/acs.jcim.9b00751
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    20
    Insights to the Binding of a Selective Adenosine A3 Receptor Antagonist Using Molecular Dynamic Simulations, MM-PBSA and MM-GBSA Free Energy Calculations, and Mutagenesis
    Lagarias, Panagiotis; Barkan, Kerry; Tzortzini, Eva; Stampelou, Margarita; Vrontaki, Eleni; Ladds, Graham; Kolocouris, Antonios
    Journal of Chemical Information and Modeling (2019), 59 (12), 5183-5197CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
    Adenosine A3 receptor (A3R) is a promising drug target cancer and for a no. of other conditions like inflammatory diseases, including asthma and rheumatoid arthritis, glaucoma, chronic obstructive pulmonary disease, and ischemic injury. Currently, there is no exptl. detd. structure of A3R. We explored the binding profile of O4-{[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl}-2-methyl-1,3-thiazole-4-carbohydroximamide (K18), which is a new specific and competitive antagonist at the orthosteric binding site of A3R. MD simulations and MM-GBSA calcns. of the WT A3R in complex with K18 combined with in vitro mutagenic studies show that the most plausible binding conformation for the dichlorophenyl group of K18 is oriented toward trans-membrane helixes (TM) 5, 6 and reveal important residues for binding. Further, MM-GBSA calcns. distinguish mutations that reduce or maintain or increase antagonistic activity. Our studies show that selectivity of K18 toward A3R is defined not only by direct interactions with residues within the orthosteric binding area but also by remote residues playing a significant role. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid, K18 maintained antagonistic potency, in agreement with our previous results obtained for agonists binding profile investigation. Mutation of the direct interacting residue L903.32 in the low region and the remote L2647.35 in the middle/upper region to alanine increases antagonistic potency, suggesting an empty space in the orthosteric area available for increasing antagonist potency. These results approve the computational model for the description of K18 binding at A3R, which we previously performed for agonists binding to A3R, and the design of more effective antagonists based on K18.
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  21. 21
    Papastathopoulos, A.; Lougiakis, N.; Kostakis, I. K.; Marakos, P.; Pouli, N.; Pratsinis, H.; Kletsas, D. New Bioactive 5-Arylcarboximidamidopyrazolo[3,4-c]Pyridines: Synthesis, Cytotoxic Activity, Mechanistic Investigation and Structure-Activity Relationships. Eur. J. Med. Chem. 2021, 218, 113387,  DOI: 10.1016/j.ejmech.2021.113387
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    21
    New bioactive 5-arylcarboximidamidopyrazolo[3,4-c]pyridines: Synthesis, cytotoxic activity, mechanistic investigation and structure-activity relationships
    Papastathopoulos, Athanasios; Lougiakis, Nikolaos; Kostakis, Ioannis K.; Marakos, Panagiotis; Pouli, Nicole; Pratsinis, Harris; Kletsas, Dimitris
    European Journal of Medicinal Chemistry (2021), 218 (), 113387CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    In this study, a series of novel substituted pyrazolo[3,4-c]pyridin-5-ylamidines I [R = Me, (4-methoxyphenyl)methyl; R1 = H, (3,4,5-trimethoxyphenyl)aminyl, cyclohexylaminyl, morpholin-4-yl; R2 = H, Ph; R3 = H, CF3; R4 = H, 4-methylpiperazin-1-yl] and II was synthesized and their cytotoxicity against three cancer cell lines (MDA-MB-231, HT-1080, PC-3), as well as a human normal cell line (AG01523) was evaluated. A no. of derivs. I and II could strongly reduce cancer cells proliferation and exhibit apoptotic induction capability, while reasonable structure-activity relationships could be extd. Certain analogs were endowed with low toxicity against normal cells. Cell cycle anal. revealed that most of the active compds. induced a G0/G1 arrest of HT-1080 cells. Moreover, the potential mechanisms of the cytotoxic activity of the promising compds. I and II were investigated in HT-1080 cells, upon study of their effects on the phosphorylation of Akt, ERK and p38 MAPK. Most of the active derivs. inhibit phosphorylation of Akt and ERK and/or induce p38 MAPK phosphorylation, providing a potential indication on the mode of action of this class.
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  22. 22
    Bento, A. P.; Gaulton, A.; Hersey, A.; Bellis, L. J.; Chambers, J.; Davies, M.; Krüger, F. A.; Light, Y.; Mak, L.; McGlinchey, S.; Nowotka, M.; Papadatos, G.; Santos, R.; Overington, J. P. The ChEMBL Bioactivity Database: An Update. Nucleic Acids Res. 2014, 42, D1083– D1090,  DOI: 10.1093/nar/gkt1031
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    22
    The ChEMBL bioactivity database: an update
    Bento, A. Patricia; Gaulton, Anna; Hersey, Anne; Bellis, Louisa J.; Chambers, Jon; Davies, Mark; Krueger, Felix A.; Light, Yvonne; Mak, Lora; McGlinchey, Shaun; Nowotka, Michal; Papadatos, George; Santos, Rita; Overington, John P.
    Nucleic Acids Research (2014), 42 (D1), D1083-D1090CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    ChEMBL is an open large-scale bioactivity database (https://www.ebi.ac.uk/chembl), previously described in the 2012 Nucleic Acids Research Database Issue. Since then, a variety of new data sources and improvements in functionality have contributed to the growth and utility of the resource. In particular, more comprehensive tracking of compds. from research stages through clin. development to market is provided through the inclusion of data from United States Adopted Name applications; a new richer data model for representing drug targets has been developed; and a no. of methods have been put in place to allow users to more easily identify reliable data. Finally, access to ChEMBL is now available via a new Resource Description Framework format, in addn. to the web-based interface, data downloads and web services.
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    Hawkins, P. C. D.; Skillman, A. G.; Nicholls, A. Comparison of Shape-Matching and Docking as Virtual Screening Tools. J. Med. Chem. 2007, 50, 74– 82,  DOI: 10.1021/jm0603365
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    Comparison of Shape-Matching and Docking as Virtual Screening Tools
    Hawkins, Paul C. D.; Skillman, A. Geoffrey; Nicholls, Anthony
    Journal of Medicinal Chemistry (2007), 50 (1), 74-82CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    Ligand docking is a widely used approach in virtual screening. In recent years a large no. of publications have appeared in which docking tools are compared and evaluated for their effectiveness in virtual screening against a wide variety of protein targets. These studies have shown that the effectiveness of docking in virtual screening is highly variable due to a large no. of possible confounding factors. Another class of method that has shown promise in virtual screening is the shape-based, ligand-centric approach. Several direct comparisons of docking with the shape-based tool ROCS have been conducted using data sets from some of these recent docking publications. The results show that a shape-based, ligand-centric approach is more consistent than, and often superior to, the protein-centric approach taken by docking.
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  24. 24
    Pei, Y.; Wickham, B. O. S. Regioselective Syntheses of 3-Aminomethyl-5-Substituted Isoxazoles: A Facile and Chemoselective Reduction of Azide to Amine by Sodium Borohydride Using 1,3-Propanedithiol as a Catalyst. Tetrahedron Lett. 1993, 34, 7509– 7512,  DOI: 10.1016/S0040-4039(00)60386-6
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    Regioselective syntheses of 3-Aminomethyl-5-substituted isoxazoles: a facile and chemoselective reduction of azide to amine by sodium borohydride using 1,3-propanedithiol as a catalyst
    Pei, Yazhong; Wickham, Barbara O. S.
    Tetrahedron Letters (1993), 34 (47), 7509-12CODEN: TELEAY; ISSN:0040-4039.
    A series of isoxazole azides I (R = Me, Ph, i-Pr; R1 = N3) were prepd. and reduced selectively to isoxazole amines I (R1 = NH2) in quant. yield by sodium borohydride using 1,3-propanedithiol as a catalyst.
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  25. 25
    Fadnavis, N. W.; Radhika, K. R. Enantio- and Regiospecific Reduction of Ethyl 4-Phenyl-2,4-Dioxobutyrate with Baker’s Yeast: Preparation of (R)-HPB Ester. Tetrahedron Asymmetry 2004, 15, 3443– 3447,  DOI: 10.1016/j.tetasy.2004.09.007
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    Enantio- and regiospecific reduction of ethyl 4-phenyl-2,4-dioxobutyrate with baker's yeast: preparation of (R)-HPB ester
    Fadnavis, Nitin W.; Radhika, Kasiraman R.
    Tetrahedron: Asymmetry (2004), 15 (21), 3443-3447CODEN: TASYE3; ISSN:0957-4166. (Elsevier B.V.)
    Et 2,4-dioxo-4-phenylbutyrate, obtained by condensation of acetophenone with di-Et oxalate, is reduced enantio- and regiospecifically by baker's yeast in a diisopropyl ether/water two-phase system to give (-)-Et (R)-2-hydroxy-4-oxo-4-phenylbutyrate with an 98% ee in 80% isolated yield. This (hydroxy)keto ester was hydrogenated over Pd-C to obtain (-)-Et (R)-2-hydroxy-4-phenylbutyrate (HPB ester), an important intermediate for the synthesis of ACE inhibitors. Prolonged contact of the redn. product with baker's yeast produced 3-phenyl-3-oxopropanol in 90% yield.
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  26. 26
    Dang, T. T.; Dang, T. T.; Fischer, C.; Görls, H.; Langer, P. Synthesis of Pyrazole-3-Carboxylates and Pyrazole-1,5-Dicarboxylates by One-Pot Cyclization of Hydrazone Dianions with Diethyl Oxalate. Tetrahedron 2008, 64, 2207– 2215,  DOI: 10.1016/j.tet.2007.12.024
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    Synthesis of pyrazole-3-carboxylates and pyrazole-1,5-dicarboxylates by one-pot cyclization of hydrazone dianions with diethyl oxalate
    Dang, Tung T.; Dang, Tuan T.; Fischer, Christine; Goerls, Helmar; Langer, Peter
    Tetrahedron (2008), 64 (9), 2207-2215CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)
    The one-pot cyclization of hydrazone dianions with di-Et oxalate allows a convenient synthesis of pyrazole-3-carboxylates and pyrazole-1,5-dicarboxylates. Under the condition of toluene as solvent and p-TsOH as acid, pyrazole-3-carboxylates are obtained; under the condition of CH2Cl2 as solvent and TFA as acid, pyrazole-1,5-dicarboxylates are obtained.
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    Yan, Z.; Liu, A.; Huang, M.; Liu, M.; Pei, H.; Huang, L.; Yi, H.; Liu, W.; Hu, A. Design, Synthesis, DFT Study and Antifungal Activity of the Derivatives of Pyrazolecarboxamide Containing Thiazole or Oxazole Ring. Eur. J. Med. Chem. 2018, 149, 170– 181,  DOI: 10.1016/j.ejmech.2018.02.036
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    Design, synthesis, DFT study and antifungal activity of the derivatives of pyrazolecarboxamide containing thiazole or oxazole ring
    Yan, Zhongzhong; Liu, Aiping; Huang, Mingzhi; Liu, Minhua; Pei, Hui; Huang, Lu; Yi, Haibo; Liu, Weidong; Hu, Aixi
    European Journal of Medicinal Chemistry (2018), 149 (), 170-181CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    To discover new pyrazolecarboxamide analogs with broad spectrum and high activity, a class of new compds. of pyrazole carboxamide derivs. contg. thiazole or oxazole ring I (R1 = H, 4-Me, 2-Cl, etc.; R2 = H, Cl, Me; R3 = Me, Et, i-Pr, cyclopropyl; R4 = H, Et, Me, Ph; Z = S, O) was designed by scaffold hopping and bioisosterism, and 36 pyrazole carboxamide derivs. with antifungal activity were synthesized. Those compds. I were evaluated against five phytopathogenic fungi, Gibberella zeae, Phytophythora capsici, Sclerotonia sclerotiorum, Erysiphe graminis and Puccinia sorghi. The results indicated that most of the compds. displayed good fungicidal activities, esp. against E. graminis. Theor. calcns. were carried out at the B3LYP/6-31G (d, p) level and the full geometry optimization was carried out using the 6-31G (d, p) basis set, and the frontier orbital energy, at. net charges and, mol. docking were discussed, and the structure-activity relationships were also studied.
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  28. 28
    Schmidt, A.; Habeck, T.; Kindermann, M. K.; Nieger, M. New Pyrazolium-Carboxylates as Structural Analogues of the Pseudo-Cross-Conjugated Betainic Alkaloid Nigellicine. J. Org. Chem. 2003, 68, 5977– 5982,  DOI: 10.1021/jo0344337
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    28
    New Pyrazolium-carboxylates as Structural Analogues of the Pseudo-Cross-Conjugated Betainic Alkaloid Nigellicine
    Schmidt, Andreas; Habeck, Tobias; Kindermann, Markus Karl; Nieger, Martin
    Journal of Organic Chemistry (2003), 68 (15), 5977-5982CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
    Pyrazolium-3-carboxylates were examd. as relatives of the betainic alkaloid nigellicine and as new examples of the sparsely populated class of heterocyclic pseudo-cross-conjugated mesomeric betaines. The title compds. were prepd. in a 4-step procedure starting from EtO2CCOCH:CROH [R = Me, Ph] which were cyclized with substituted hydrazines. The resulting isomeric pyrazole esters were sepd. and quaternized with di-Me sulfate in the presence of nitrobenzene to pyrazolium esters. Sapon. was best accomplished in dild. sulfuric acid, which resulted in the formation of the pseudo-cross-conjugated mesomeric betaines in one step. Protonation to the corresponding carboxylic acids required the treatment of the betaines with tetrafluoroboric acid in dichloromethane. The effect of neg. solvatochromism proves the charge sepn. in the ground state of the mols. X-ray crystallog. analyses, semiempirical calcns., and ESI mass spectrometric measurements were performed to gain knowledge about the phenomenon of pseudo-cross-conjugation.
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  29. 29
    Knight, A.; Hemmings, J. L.; Winfield, I.; Leuenberger, M.; Frattini, E.; Frenguelli, B. G.; Dowell, S. J.; Lochner, M.; Ladds, G. Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity. J. Med. Chem. 2016, 59, 947– 964,  DOI: 10.1021/acs.jmedchem.5b01402
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    29
    Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity
    Knight, Anthony; Hemmings, Jennifer L.; Winfield, Ian; Leuenberger, Michele; Frattini, Eugenia; Frenguelli, Bruno G.; Dowell, Simon J.; Lochner, Martin; Ladds, Graham
    Journal of Medicinal Chemistry (2016), 59 (3), 947-964CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A series of N6-bicyclic and N6-(2-hydroxy)cyclopentyl derivs. of adenosine were synthesized as novel A1R agonists and their A1R/A2R selectivity assessed using a simple yeast screening platform. We obsd. that the most selective, high potency ligands were achieved through N6-adamantyl substitution in combination with 5'-N-ethylcarboxamido or 5'-hydroxymethyl groups. In addn., we detd. that 5'-(2-fluoro)thiophenyl derivs. all failed to generate a signaling response despite showing an interaction with the A1R. Some selected compds. were also tested on A1R and A3R in mammalian cells revealing that four of them are entirely A1R-selective agonists. By using in silico homol. modeling and ligand docking, we provide insight into their mechanisms of recognition and activation of the A1R. We believe that given the broad tissue distribution, but contrasting signaling profiles, of adenosine receptor subtypes, these compds. might have therapeutic potential.
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  30. 30
    Stoddart, L. A.; Vernall, A. J.; Denman, J. L.; Briddon, S. J.; Kellam, B.; Hill, S. J. Fragment Screening at Adenosine-A3 Receptors in Living Cells Using a Fluorescence-Based Binding Assay. Chem. Biol. 2012, 19, 1105– 1115,  DOI: 10.1016/j.chembiol.2012.07.014
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    Fragment Screening at Adenosine-A3 Receptors in Living Cells Using a Fluorescence-Based Binding Assay
    Stoddart, Leigh A.; Vernall, Andrea J.; Denman, Jessica L.; Briddon, Stephen J.; Kellam, Barrie; Hill, Stephen J.
    Chemistry & Biology (Oxford, United Kingdom) (2012), 19 (9), 1105-1115CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
    G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is detd. in the correct cellular environment and preferably using an unmodified receptor. The authors developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to det. binding affinity consts. of competing ligands at human adenosine-A1 and -A3 receptors. This method was validated as a tool to screen a library of low mol. wt. fragments, and identified a hit with submicromolar binding affinity (KD). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A3 receptor. This technol. represents a significant advance that will allow the detn. of ligand and fragment affinities at receptors in their native membrane environment.
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    Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee, R. P. Empirical Scoring Functions: I. The Development of a Fast Empirical Scoring Function to Estimate the Binding Affinity of Ligands in Receptor Complexes. J. Comput. Aided. Mol. Des. 1997, 11, 425– 445,  DOI: 10.1023/A:1007996124545
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    Empirical scoring functions: I. The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes
    Eldridge, Matthew D.; Murray, Christopher W.; Auton, Timothy R.; Paolini, Gaia V.; Mee, Roger P.
    Journal of Computer-Aided Molecular Design (1997), 11 (5), 425-445CODEN: JCADEQ; ISSN:0920-654X. (Kluwer Academic Publishers)
    This paper describes the development of a simple empirical scoring function designed to est. the free energy of binding for a protein-ligand complex when the 3D structure of the complex is known or can be approximated. The function uses simple contact terms to est. lipophilic and metal-ligand binding contributions, a simple explicit form for hydrogen bonds and a term which penalizes flexibility. The coeffs. of each term are obtained using a regression based on 82 ligand-receptor complexes for which the binding affinity is known. The function reproduces the binding affinity of the complexes with a cross-validated error of 8.68 kJ/mol. Tests on internal consistency indicate that the coeffs. obtained are stable to changes in the compn. of the training set. The function is also tested on two test sets contg. a further 20 and 10 complexes, resp. The deficiencies of this type of function are discussed and it is compared to approaches by other workers.
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    Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Comparison of Multiple Amber Force Fields and Development of Improved Protein Backbone Parameters. Proteins Struct. Funct. Genet. 2006, 65, 712– 725,  DOI: 10.1002/prot.21123
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    Comparison of multiple Amber force fields and development of improved protein backbone parameters
    Hornak, Viktor; Abel, Robert; Okur, Asim; Strockbine, Bentley; Roitberg, Adrian; Simmerling, Carlos
    Proteins: Structure, Function, and Bioinformatics (2006), 65 (3), 712-725CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
    The ff94 force field that is commonly assocd. with the Amber simulation package is one of the most widely used parameter sets for biomol. simulation. After a decade of extensive use and testing, limitations in this force field, such as over-stabilization of α-helixes, were reported by the authors and other researchers. This led to a no. of attempts to improve these parameters, resulting in a variety of "Amber" force fields and significant difficulty in detg. which should be used for a particular application. The authors show that several of these continue to suffer from inadequate balance between different secondary structure elements. In addn., the approach used in most of these studies neglected to account for the existence in Amber of two sets of backbone .vphi./ψ dihedral terms. This led to parameter sets that provide unreasonable conformational preferences for glycine. The authors report here an effort to improve the .vphi./ψ dihedral terms in the ff99 energy function. Dihedral term parameters are based on fitting the energies of multiple conformations of glycine and alanine tetrapeptides from high level ab initio quantum mech. calcns. The new parameters for backbone dihedrals replace those in the existing ff99 force field. This parameter set, which the authors denote ff99SB, achieves a better balance of secondary structure elements as judged by improved distribution of backbone dihedrals for glycine and alanine with respect to PDB survey data. It also accomplishes improved agreement with published exptl. data for conformational preferences of short alanine peptides and better accord with exptl. NMR relaxation data of test protein systems.
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    Massova, I.; Kollman, P. A. Combined molecular mechanical and continuum solvent approach (MM-PBSA/GBSA) to predict ligand binding. Per. Drug Discovery Design 2000, 18, 113– 135,  DOI: 10.1023/A:1008763014207
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    Combined molecular mechanical and continuum solvent approach (MM-PBSA/GBSA) to predict ligand binding
    Massova, Irina; Kollman, Peter A.
    Perspectives in Drug Discovery and Design (2000), 18 (Hydrophobicity and Solvation in Drug Design, Pt. II), 113-135CODEN: PDDDEC; ISSN:0928-2866. (Kluwer Academic Publishers)
    Significant progress has been achieved in computational methods to treat solvent effects in recent years. Among various techniques, the continuum solvent approach appears to be practically promising because it can be used to calc. reliable interaction and solvation energies in complex systems. A computational scanning mutagenesis method, one of such new approaches, has been recently developed. It combines the mol. mech. and continuum solvent approaches and allows one to identify the "hot spots" in binding interfaces from a single trajectory of a wild type complex. Such techniques can be also used as a tool to optimize the interacting species for the binding, or as a ranking procedure in high throughput screening.
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    Wang, E.; Sun, H.; Wang, J.; Wang, Z.; Liu, H.; Zhang, J. Z. H.; Hou, T. End-Point Binding Free Energy Calculation with MM/PBSA and MM/GBSA: Strategies and Applications in Drug Design. Chem. Rev. 2019, 119, 9478– 9508,  DOI: 10.1021/acs.chemrev.9b00055
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    34
    End-point binding free energy calculation with MM/PBSA and MM/GBSA: strategies and applications in drug design
    Wang, Ercheng; Sun, Huiyong; Wang, Junmei; Wang, Zhe; Liu, Hui; Zhang, John Z. H.; Hou, Tingjun
    Chemical Reviews (Washington, DC, United States) (2019), 119 (16), 9478-9508CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Mol. mechanics Poisson-Boltzmann surface area (MM/PBSA) and mol. mechanics generalized Born surface area (MM/GBSA) are arguably very popular methods for binding free energy prediction since they are more accurate than most scoring functions of mol. docking and less computationally demanding than alchem. free energy methods. MM/PBSA and MM/GBSA have been widely used in biomol. studies such as protein folding, protein-ligand binding, protein-protein interaction, etc. In this review, methods to adjust the polar solvation energy and to improve the performance of MM/PBSA and MM/GBSA calcns. are reviewed and discussed. The latest applications of MM/GBSA and MM/PBSA in drug design are also presented. This review intends to provide readers with guidance for practically applying MM/PBSA and MM/GBSA in drug design and related research fields.
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    Kaminski, G.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides. Phys. Chem. B 2001, 105 (28), 6474– 6487,  DOI: 10.1021/jp003919d
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    35
    Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides
    Kaminski, George A.; Friesner, Richard A.; Tirado-Rives, Julian; Jorgensen, William L.
    Journal of Physical Chemistry B (2001), 105 (28), 6474-6487CODEN: JPCBFK; ISSN:1089-5647. (American Chemical Society)
    We present results of improving the OPLS-AA force field for peptides by means of refitting the key Fourier torsional coeffs. The fitting technique combines using accurate ab initio data as the target, choosing an efficient fitting subspace of the whole potential-energy surface, and detg. wts. for each of the fitting points based on magnitudes of the potential-energy gradient. The av. energy RMS deviation from the LMP2/cc-pVTZ(-f)//HF/6-31G** data is reduced by ∼40% from 0.81 to 0.47 kcal/mol as a result of the fitting for the electrostatically uncharged dipeptides. Transferability of the parameters is demonstrated by using the same alanine dipeptide-fitted backbone torsional parameters for all of the other dipeptides (with the appropriate side-chain refitting) and the alanine tetrapeptide. Parameters of nonbonded interactions have also been refitted for the sulfur-contg. dipeptides (cysteine and methionine), and the validity of the new Coulombic charges and the van der Waals σ's and ε's is proved through reproducing gas-phase energies of complex formation heats of vaporization and densities of pure model liqs. Moreover, a novel approach to fitting torsional parameters for electrostatically charged mol. systems has been presented and successfully tested on five dipeptides with charged side chains.
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    Shivakumar, D.; Williams, J.; Wu, Y.; Damm, W.; Shelley, J.; Sherman, W. Prediction of Absolute Solvation Free Energies using Molecular Dynamics Free Energy Perturbation and the OPLS Force Field. J. Chem. Theory Comput. 2010, 6, 1509– 19,  DOI: 10.1021/ct900587b
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    Prediction of Absolute Solvation Free Energies using Molecular Dynamics Free Energy Perturbation and the OPLS Force Field
    Shivakumar, Devleena; Williams, Joshua; Wu, Yujie; Damm, Wolfgang; Shelley, John; Sherman, Woody
    Journal of Chemical Theory and Computation (2010), 6 (5), 1509-1519CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    The accurate prediction of protein-ligand binding free energies is a primary objective in computer-aided drug design. The solvation free energy of a small mol. provides a surrogate to the desolvation of the ligand in the thermodn. process of protein-ligand binding. Here, we use explicit solvent mol. dynamics free energy perturbation to predict the abs. solvation free energies of a set of 239 small mols., spanning diverse chem. functional groups commonly found in drugs and drug-like mols. We also compare the performance of abs. solvation free energies obtained using the OPLS_2005 force field with two other commonly used small mol. force fields - general AMBER force field (GAFF) with AM1-BCC charges and CHARMm-MSI with CHelpG charges. Using the OPLS_2005 force field, we obtain high correlation with exptl. solvation free energies (R2 = 0.94) and low av. unsigned errors for a majority of the functional groups compared to AM1-BCC/GAFF or CHelpG/CHARMm-MSI. However, OPLS_2005 has errors of over 1.3 kcal/mol for certain classes of polar compds. We show that predictions on these compd. classes can be improved by using a semiempirical charge assignment method with an implicit bond charge correction.
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    Stamatis, D.; Lagarias, P.; Barkan, K.; Vrontaki, E.; Ladds, G.; Kolocouris, A. Structural Characterization of Agonist Binding to an A3 Adenosine Receptor through Biomolecular Simulations and Mutagenesis Experiments. J. Med. Chem. 2019, 62, 8831– 8846,  DOI: 10.1021/acs.jmedchem.9b01164
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    37
    Structural Characterization of Agonist Binding to an A3 Adenosine Receptor through Biomolecular Simulations and Mutagenesis Experiments
    Stamatis, Dimitrios; Lagarias, Panagiotis; Barkan, Kerry; Vrontaki, Eleni; Ladds, Graham; Kolocouris, Antonios
    Journal of Medicinal Chemistry (2019), 62 (19), 8831-8846CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    The adenosine A3 receptor (A3R) binds adenosine and is a drug target against cancer cell proliferation. Currently, there is no exptl. structure of A3R. Here, we have generated a mol. model of A3R in complex with two agonists, the nonselective 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranuronamide (NECA) and the selective 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA). Mol. dynamics simulations of the wild-type A3R in complex with both agonists, combined with in vitro mutagenic studies revealed important residues for binding. Further, mol. mechanics-generalized Born surface area calcns. were able to distinguish mutations that reduce or negate agonistic activity from those that maintained or increased the activity. Our studies reveal that selectivity of IB-MECA toward A3R requires not only direct interactions with residues within the orthosteric binding area but also with remote residues. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid or alanine, the activity of IB-MECA increased by making new van der Waals contacts with TM5. This result may have implications in the design of new A3R agonists.
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  1. Barbara Preti, Anna Suchankova, Giuseppe Deganutti, Michele Leuenberger, Kerry Barkan, Iga Manulak, Xianglin Huang, Sabrina Carvalho, Graham Ladds, Martin Lochner. Discovery and Structure–Activity Relationship Studies of Novel Adenosine A1 Receptor-Selective Agonists. Journal of Medicinal Chemistry 2022, 65 (21) , 14864-14890. https://doi.org/10.1021/acs.jmedchem.2c01414
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  • Abstract

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    Scheme 1

    Scheme 1. Synthesis of 4a,b and 5a,ba
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    aReagents and conditions: (a) diethyl oxalate, NaH 60%, toluene dry, 50°C, 2 h; (b) NH2NH2 80%, EtOH, reflux, 90 min; (c) (i) NaH 60%, DMF dry, 0 °C, 15 min, (ii) CH3I, rt, 1 h.

    Scheme 2

    Scheme 2. Synthesis of 10a–ca
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    aReagents and conditions: (a) paraformaldehyde, 33% HBr in AcOH, 90 °C, 3.5 h; (b) Ν-methylmorpholine-Ν-oxide, MeCN dry, rt, 24 h; (c) NH2NH2 (80%), HCl 36%, EtOH, 90°C, 1 h; (d) POCl3, 110 °C, 2.5–8 h; (e) HNR1R2, EtOH, reflux, 2 h.

    Scheme 3

    Scheme 3. Synthesis of 15a–ca
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    aReagents and conditions: (a) paraformaldehyde, 33% HBr in AcOH, 90 °C, 3.5 h; (b) Ν-methylmorpholine-Ν-oxide, MeCN dry, rt, 24 h; (c) NH2NH2 (80%), HCl 36%, EtOH, 90 °C, 1 h; (d) POCl3, 110 °C, 2.5–8 h; (e) HNR1R2, EtOH, reflux, 2 h.

    Figure 1

    Figure 1. Characterization of 7-amino-pyrazolo[3,4-d]pyridazines at human A1R and A3R. (A and B) Cells expressing either human A1R (A) or A3R (B) were exposed to 10 μM forskolin and stimulated with increasing concentrations of NECA for 30 min in the presence of a 1 μM concentration of the test compound, and the cAMP accumulation was quantified. (C) cAMP accumulation was measured as detailed in part A using multiple concentrations of 10b. Using pEC50 values, Schild regression analysis was conducted to calculate pA2/pKb values. All values are mean ± SEM expressed as percentage forskolin inhibition, relative to NECA. n ≥ 3 independent experimental repeats were performed in duplicate.

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    Figure 2

    Figure 2. Inhibition of BRET between CA200645 at NLuc-A1R and Nluc-A3R by 10b and 10a. HEK293 cells expressing Nluc-A1R (A) or Nluc-A3R (B) were treated with 5 nM or 20 nM CA200645, respectively, enabling concentration-dependent decreases in the BRET ratio at 10 min to be determined with the response normalized to DMSO. Binding curves were fitted with the Cheng Prusoff equation built into GraphPad Prism 9.3 to enable estimates of the pKi. (19) Comparison of pKi values for A1R (C) and A3R (D) as determined via BRET binding. Each data point represents the mean ± SEM of at least three experiments performed in duplicate. The statistical significance compared to NECA was determined, at p < 0.05, through one-way ANOVA with Dunnett’s post-test (*, p < 0.05; ***, p < 0.001). #Compounds did not fully displace CA200645, so pKi values are estimates preventing statistical analysis.

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    Figure 3

    Figure 3. (A–C) 100 ns MD simulations of 10a–c inside the orthosteric binding area of A1R. (D) 100 ns MD simulations of 15b inside the orthosteric binding area of A1R. Starting structures are shown (docking pose), and representative frames from MD simulations, receptor–ligand interaction frequency histograms, and RMSD plots of proteins (RMSDprotein; blue plots) and ligand heavy atoms (RMSDligand; red plots) inside the orthosteric binding area of WT A1R or A3R. Bars are plotted only for residues with interaction frequencies ≥0.2. Color scheme: ligand = brown sticks, receptor = white cartoon and sticks, hydrogen bonding interactions = yellow (dashes or bars), π–π interactions = green (dashes or bars), hydrophobic interactions = gray, water bridges = blue. For the protein models of A1R in complex with 10a–c or 15b, the experimental structure of the inactive form of A1R in complex with an antagonist (PDB ID 5UEN (4)) was used.

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  • References

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    This article references 37 other publications.

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      A review. The accumulation of evidence implicating a role for adenosine in the pathogenesis of asthma has led to investigations into all adenosine receptor subtypes as potential therapeutic targets for the treatment of asthma. Selective A1 receptor antagonists are currently in preclin. development since adenosine has been shown exptl. to mediate various features of asthma through this receptor such as bronchoconstriction, mucus secretion and inflammation. The A2A receptor is expressed on most inflammatory cells implicated in asthma, and as A2A stimulation activates adenylate cyclase and consequently elevates cAMP, selective A2A receptor agonists have now reached clin. development. However, initial reports concerning their efficacy are inconclusive. A2B receptor antagonists are also under investigation based on the rationale that inhibiting the effects of adenosine on mast cells would be beneficial, in addn. to other reported pro-inflammatory effects mediated by the A2B receptor on cells such as airway smooth muscle, epithelial cells and fibroblasts. While the effects in pre-clin. models are promising, their efficacy in the clin. setting has also yet to be reported. Finally, adenosine A3 receptor stimulation has been demonstrated to mediate inhibitory effects on eosinophils since it also elevates cAMP. However, some exptl. reports suggest that A3 antagonists mediate anti-inflammatory effects, thus the rationale for A3 receptor ligands as therapeutic agents remains to be detd. In conclusion, establishing the precise role of adenosine in the pathogenesis of asthma and developing appropriate subtype selective agonists/antagonists represents an exciting opportunity for the development of novel therapeutics for the treatment of asthma.
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      Cohen, S.; Fishman, P. Targeting the A3 Adenosine Receptor to Treat Cytokine Release Syndrome in Cancer Immunotherapy. Drug Des. Devel. Ther. 2019, 13, 491– 497,  DOI: 10.2147/DDDT.S195294
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      Cancer patients undergoing immunotherapy may develop cytokine release syndrome (CRS), an inflmmatory cytokine storm condition, followed by neurotoxic manifestations and may be life-threatening. The current treatments for CRS successfully reduce the inflmmatory response but may limit the anticancer effect of the given immunotherapy and fail to overcome the neurotoxic adverse events. Adenosine, a ubiquitous purine nucleoside, induces a plethora of effects in the body via its binding to four adenosine receptors A1, A2a, A2b, and the A3. Highly selective agonists to the A3 adenosine receptor act as inhibitors of proinflmmatory cytokines, possess robust anti-inflmmatory and anticancer activity, and concomitantly, induce neuroprotective effects. Piclidenoson and namodenoson belong to this group of compds., are effective upon oral administration, show an excellent safety profie in human clin. studies, and therefore, may be considered as drug candidates to treat CRS. In this article, the detailed anti-inflmmatory characteristics of these compds. and the rationale to use them as drugs to combat CRS are described.
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      Lee, J.; Hwang, I.; Lee, J. H.; Lee, H. W.; Jeong, L.-S.; Ha, H. The Selective A3AR Antagonist LJ-1888 Ameliorates UUO-Induced Tubulointerstitial Fibrosis. Am. J. Pathol. 2013, 183, 1488– 1497,  DOI: 10.1016/j.ajpath.2013.07.010
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      Adenosine in the normal kidney significantly elevates in response to cellular damage. The renal A3 adenosine receptor (A3AR) is up-regulated under stress, but the therapeutic effects of A3AR antagonists on chronic kidney disease are not fully understood. The present study examd. the effect of LJ-1888 [(2R,3R,4S)-2-[2-chloro-6-(3-iodobenzylamino)-9H-purine-9-yl]-tetrahydrothiophene-3,4-diol], a newly developed potent, selective, species-independent, and orally active A3AR antagonist, on unilateral ureteral obstruction (UUO)-induced renal fibrosis. Pretreatment with LJ-1888 inhibited UUO-induced fibronectin and collagen I up-regulation in a dose-dependent manner. Masson's trichrome staining confirmed that LJ-1888 treatment effectively reduced UUO-induced interstitial collagen accumulation. Furthermore, delayed administration of LJ-1888 showed an equiv. therapeutic effect on tubulointerstitial fibrosis to that of losartan. Small-interfering A3AR transfection effectively inhibited transforming growth factor-β1 (TGF-β1)-induced fibronectin and collagen I up-regulation in proximal tubular cells similar to LJ-1888, confirming that the renoprotective effect of LJ-1888 resulted from A3AR blockade. UUO- or TGF-β1-induced c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation decreased significantly after LJ-1888 administration. A3AR blockade reduced UUO- or TGF-β1-induced up-regulation of lysyl oxidase, which induces crosslinking of extracellular matrix, suggesting that LJ-1888 may also regulate extracellular matrix accumulation via post-translational regulation. In conclusion, the present data demonstrate that the A3AR antagonist, LJ-1888, blocked the development and attenuated the progression of renal fibrosis, and they suggest that LJ-1888 may become a new therapeutic modality for renal interstitial fibrosis.
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      Wang, Zhao; Do, Chi-Wai; Avila, Marcel Y.; Peterson-Yantorno, Kim; Stone, Richard A.; Gao, Zhan-Guo; Joshi, Bhalchandra; Besada, Pedro; Jeong, Lak-Shin; Jacobson, Kenneth A.; Civan, Mortimer M.
      Experimental Eye Research (2010), 90 (1), 146-154CODEN: EXERA6; ISSN:0014-4835. (Elsevier B.V.)
      The purpose of the study was to det. whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the 5 agonist derivs., LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other 4 compds., consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, 5 derivs. of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivs. of the highly-selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivs. are promising A3 antagonists for study in multiple animal models.
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    11. 11
      Glukhova, A.; Thal, D. M.; Nguyen, A. T.; Vecchio, E. A.; Jörg, M.; Scammells, P. J.; May, L. T.; Sexton, P. M.; Christopoulos, A. Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity. Cell 2017, 168, 867– 877,  DOI: 10.1016/j.cell.2017.01.042
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      11
      Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity
      Glukhova, Alisa; Thal, David M.; Nguyen, Anh T.; Vecchio, Elizabeth A.; Jorg, Manuela; Scammells, Peter J.; May, Lauren T.; Sexton, Patrick M.; Christopoulos, Arthur
      Cell (Cambridge, MA, United States) (2017), 168 (5), 867-877.e13CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The adenosine A1 receptor (A1-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We detd. a 3.2 Å crystal structure of the A1-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A2A receptor (A2A-AR) structure. Mutational and computational anal. of A1-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a mol. basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.
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    12. 12
      Cheng, R. K. Y.; Segala, E.; Robertson, N.; Deflorian, F.; Doré, A. S.; Errey, J. C.; Fiez-Vandal, C.; Marshall, F. H.; Cooke, R. M. Structures of Human A1 and A2A Adenosine Receptors with Xanthines Reveal Determinants of Selectivity. Structure 2017, 25, 1275– 1285,  DOI: 10.1016/j.str.2017.06.012
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      12
      Structures of Human A1 and A2a Adenosine Receptors with Xanthines Reveal Determinants of Selectivity
      Cheng, Robert K. Y.; Segala, Elena; Robertson, Nathan; Deflorian, Francesca; Dore, Andrew S.; Errey, James C.; Fiez-Vandal, Cedric; Marshall, Fiona H.; Cooke, Robert M.
      Structure (Oxford, United Kingdom) (2017), 25 (8), 1275-1285.e4CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
      The adenosine A1 and A2a receptors belong to the purinergic family of G protein-coupled receptors, and regulate diverse functions of the cardiovascular, respiratory, renal, inflammation, and CNS. Xanthines such as caffeine and theophylline are weak, non-selective antagonists of adenosine receptors. Here we report the structure of a thermostabilized human A1 receptor at 3.3 Å resoln. with PSB36, an A1-selective xanthine-based antagonist. This is compared with structures of the A2a receptor with PSB36 (2.8 Å resoln.), caffeine (2.1 Å), and theophylline (2.0 Å) to highlight features of ligand recognition which are common across xanthines. The structures of A1R and A2aR were analyzed to identify the differences that are important selectivity determinants for xanthine ligands, and the role of T2707.35 in A1R (M2707.35 in A2aR) in conferring selectivity was confirmed by mutagenesis. The structural differences confirmed to lead to selectivity can be utilized in the design of new subtype-selective A1R or A2aR antagonists.
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    13. 13
      Draper-Joyce, C. J.; Khoshouei, M.; Thal, D. M.; Liang, Y.-L.; Nguyen, A. T. N.; Furness, S. G. B.; Venugopal, H.; Baltos, J.-A.; Plitzko, J. M.; Danev, R.; Baumeister, W.; May, L. T.; Wootten, D.; Sexton, P. M.; Glukhova, A.; Christopoulos, A. Structure of the Adenosine-Bound Human Adenosine A1 Receptor-Gi Complex. Nature 2018, 558, 559– 563,  DOI: 10.1038/s41586-018-0236-6
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      13
      Structure of the adenosine-bound human adenosine A1 receptor-Gi complex
      Draper-Joyce, Christopher J.; Khoshouei, Maryam; Thal, David M.; Liang, Yi-Lynn; Nguyen, Anh T. N.; Furness, Sebastian G. B.; Venugopal, Hariprasad; Baltos, Jo-Anne; Plitzko, Jurgen M.; Danev, Radostin; Baumeister, Wolfgang; May, Lauren T.; Wootten, Denise; Sexton, Patrick M.; Glukhova, Alisa; Christopoulos, Arthur
      Nature (London, United Kingdom) (2018), 558 (7711), 559-563CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein detd. by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound β2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides mol. insights into receptor and G-protein selectivity.
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    14. 14
      Stampelou, M.; Suchankova, A.; Tzortzini, E.; Dhingra, L.; Barkan, K.; Lougiakis, N.; Marakos, P.; Pouli, N.; Ladds, G.; Kolocouris, A. Novel Pyrazolo[3,4-c]Pyridine Antagonists with Nanomolar Affinity for A1/A3 Adenosine Receptors: Binding Kinetics and Exploration of Their Binding Profile Using Mutagenesis Experiments, MD Simulations and TI/MD Calculations. ChemRxiv 2021,  DOI: 10.26434/chemrxiv-2021-j18mg-v3 .
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    15. 15
      Catarzi, D.; Colotta, V.; Varano, F.; Poli, D.; Squarcialupi, L.; Filacchioni, G.; Varani, K.; Vincenzi, F.; Borea, P. A.; Dal Ben, D.; Lambertucci, C.; Cristalli, G. Pyrazolo[1,5-c]Quinazoline Derivatives and Their Simplified Analogues as Adenosine Receptor Antagonists: Synthesis, Structure-Affinity Relationships and Molecular Modeling Studies. Bioorg. Med. Chem. 2013, 21, 283– 294,  DOI: 10.1016/j.bmc.2012.10.031
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      15
      Pyrazolo[1,5-c]quinazoline derivatives and their simplified analogues as adenosine receptor antagonists: Synthesis, structure-affinity relationships and molecular modeling studies
      Catarzi, Daniela; Colotta, Vittoria; Varano, Flavia; Poli, Daniela; Squarcialupi, Lucia; Filacchioni, Guido; Varani, Katia; Vincenzi, Fabrizio; Borea, Pier Andrea; Dal Ben, Diego; Lambertucci, Catia; Cristalli, Gloria
      Bioorganic & Medicinal Chemistry (2013), 21 (1), 283-294CODEN: BMECEP; ISSN:0968-0896. (Elsevier B.V.)
      A no. of 5-oxo-pyrazolo[1,5-c]quinazolines, bearing at position-2 the (hetero)aryl moiety but also a carboxylate group, were designed as hA3 AR antagonists. This study produced some interesting compds. endowed with good hA3 receptor affinity and high selectivity, being totally inactive at all the other AR subtypes. In contrast, the corresponding 5-ammino derivs. do not bind or bind with very low affinity at the hA3 AR, the only exception being the 5-N-benzoyl compd. I that shows a hA3Ki value in the high μ-molar range. Evaluation of the synthetic intermediates led to the identification of some 5(3)-(2-aminophenyl)-3(5)-(hetero)arylpyrazoles II [R = Ph, 4-MeOC6H4, 2-furyl, 2-thienyl, CO2Et] with modest affinity but high selectivity toward the hA3 AR subtype. Mol. docking of the herein reported tricyclic and simplified derivs. was carried out to depict their hypothetical binding mode to our model of hA3 receptor.
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    16. 16
      Manetti, F.; Schenone, S.; Bondavalli, F.; Brullo, C.; Bruno, O.; Ranise, A.; Mosti, L.; Menozzi, G.; Fossa, P.; Trincavelli, M. L.; Martini, C.; Martinelli, A.; Tintori, C.; Botta, M. Synthesis and 3D QSAR of new pyrazolo[3,4-b]pyridines: potent and selective inhibitors of A1 adenosine receptors. J. Med. Chem. 2005, 48, 7172– 7185,  DOI: 10.1021/jm050407k
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      16
      Synthesis and 3D QSAR of New Pyrazolo[3,4-b]pyridines: Potent and Selective Inhibitors of A1 Adenosine Receptors
      Manetti, Fabrizio; Schenone, Silvia; Bondavalli, Francesco; Brullo, Chiara; Bruno, Olga; Ranise, Angelo; Mosti, Luisa; Menozzi, Giulia; Fossa, Paola; Trincavelli, Maria Letizia; Martini, Claudia; Martinelli, Adriano; Tintori, Cristina; Botta, Maurizio
      Journal of Medicinal Chemistry (2005), 48 (23), 7172-7185CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A no. of 4-(amino)pyrazolo[3,4-b]pyridine-5-carboxylic acid esters were synthesized and evaluated for their binding affinity at the A1, A2A, and A3 adenosine receptors (AR), in bovine cortical membranes, as well as for their affinity toward human A1AR (hA1AR) (A1 purinoceptor antagonists). Some of the new compds. were characterized by a high affinity and selectivity toward the A1 receptor subtype, showing a significant improvement in comparison with other pyrazolo-pyridines previously reported in the literature. In particular two compds., both of them bearing a [(p-methoxyphenyl)ethyl]amino side chain presented Ki values of 6 and 7 nM, resp. To rationalize the relationship between structure and affinity of the novel compds., a 3D QSAR model was also generated starting from compds. belonging to different classes of known A1AR antagonists.
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    17. 17
      Tuccinardi, T.; Zizzari, A. T.; Brullo, C.; Daniele, S.; Musumeci, F.; Schenone, S.; Trincavelli, M. L.; Martini, C.; Martinelli, A.; Giorgi, G.; Botta, M. Substituted pyrazolo[3,4-b]pyridines as human A1 adenosine antagonists: Developments in understanding the receptor stereoselectivity. Org. Biomol. Chem. 2011, 9, 4448– 4455,  DOI: 10.1039/c0ob01064b
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      17
      Substituted pyrazolo[3,4-b]pyridines as human A1 adenosine antagonists: Developments in understanding the receptor stereoselectivity
      Tuccinardi, Tiziano; Zizzari, Alessandra Tania; Brullo, Chiara; Daniele, Simona; Musumeci, Francesca; Schenone, Silvia; Trincavelli, Maria Letizia; Martini, Claudia; Martinelli, Adriano; Giorgi, Gianluca; Botta, Maurizio
      Organic & Biomolecular Chemistry (2011), 9 (12), 4448-4455CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      A1 adenosine receptor antagonists have been proposed to possess an interesting range of potential therapeutic applications. The authors have already reported the synthesis and the biol. characterization of a family of pyrazolo[3,4-b]pyridine derivs. as A1 adenosine ligands endowed with an antagonistic profile. In the present work, the authors report the LC sepn. of enantiomers of the authors' most active A1 antagonists together with the detn. of their abs. configuration by x-ray crystal structure anal. Biol. assays confirmed a different activity for the two enantiomers, with the R one showing the higher human A1AR affinity. The authors also developed a homol. model of this receptor subtype to suggest a binding disposition of the ligands into the hA1AR. All of the obtained data suggest that the compd.'s chirality plays a key role in A1 affinity.
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    18. 18
      Cheong, S. L.; Venkatesan, G.; Paira, P.; Jothibasu, R.; Mandel, A. L.; Federico, S.; Spalluto, G.; Pastorin, G. Pyrazolo Derivatives as Potent Adenosine Receptor Antagonists: An Overview on the Structure-Activity Relationships. Int. J. Med. Chem. 2011, 480652,  DOI: 10.1155/2011/480652
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      18
      Pyrazolo derivatives as potent adenosine receptor antagonists: an overview on the structure-activity relationships
      Cheong, Siew Lee; Venkatesan, Gopalakrishnan; Paira, Priyankar; Jothibasu, Ramasamy; Mandel, Alexander Laurence; Federico, Stephanie; Spalluto, Giampiero; Pastorin, Giorgia
      International Journal of Medicinal Chemistry (2011), (), 480652, 15 pp.CODEN: IJMCCH; ISSN:2090-2077. (Hindawi Publishing Corp.)
      A review. In the past few decades, medicinal chem. research towards potent and selective antagonists of human adenosine receptors (namely, A1, A2A, A2B, and A3) has been evolving rapidly. These antagonists are deemed therapeutically beneficial in several pathol. conditions including neurol. and renal disorders, cancer, inflammation, and glaucoma. Up to this point, many classes of compds. have been successfully synthesized and identified as potent human adenosine receptor antagonists. In this paper, an overview of the structure-activity relationship (SAR) profiles of promising nonxanthine pyrazolo derivs. is reported and discussed. We have emphasized the SAR for some representative structures such as pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines; pyrazolo-[3,4-c] or -[4,3-c]quinolines; pyrazolo-[4,3-d]pyrimidinones; pyrazolo-[3,4-d]pyrimidines and pyrazolo-[1,5-a]pyridines. This overview not only clarifies the structural requirements deemed essential for affinity towards individual adenosine receptor subtypes, but it also sheds light on the rational design and optimization of existing structural templates to allow us to conceive new, more potent adenosine receptor antagonists.
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    19. 19
      Barkan, K.; Lagarias, P.; Stampelou, M.; Stamatis, D.; Hoare, S.; Safitri, D.; Klotz, K.-N.; Vrontaki, E.; Kolocouris, A.; Ladds, G. Pharmacological Characterisation of Novel Adenosine A3 Receptor Antagonists. Sci. Rep. 2020, 10, 20781,  DOI: 10.1038/s41598-020-74521-y
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      19
      Pharmacological characterization of novel adenosine A3 receptor antagonists
      Barkan, Kerry; Lagarias, Panagiotis; Stampelou, Margarita; Stamatis, Dimitrios; Hoare, Sam; Safitri, Dewi; Klotz, Karl-Norbert; Vrontaki, Eleni; Kolocouris, Antonios; Ladds, Graham
      Scientific Reports (2020), 10 (1), 20781CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but crit. task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Pos. hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was detd. using Schild anal. (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the arom. nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by mol. dynamic simulations combined with Mol. Mechanics-Poisson Boltzmann Surface Area calcns. identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 μM) competitive antagonist. Finally, we introduce a model that enables ests. of the equil. binding affinity for rapidly disassocg. compds. from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacol. characterization of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.
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    20. 20
      Lagarias, P.; Barkan, K.; Tzortzini, E.; Stampelou, M.; Vrontaki, E.; Ladds, G.; Kolocouris, A. Insights to the Binding of a Selective Adenosine A3 Receptor Antagonist Using Molecular Dynamic Simulations, MM-PBSA and MM-GBSA Free Energy Calculations, and Mutagenesis. J. Chem. Inf. Model. 2019, 59, 5183– 5197,  DOI: 10.1021/acs.jcim.9b00751
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      20
      Insights to the Binding of a Selective Adenosine A3 Receptor Antagonist Using Molecular Dynamic Simulations, MM-PBSA and MM-GBSA Free Energy Calculations, and Mutagenesis
      Lagarias, Panagiotis; Barkan, Kerry; Tzortzini, Eva; Stampelou, Margarita; Vrontaki, Eleni; Ladds, Graham; Kolocouris, Antonios
      Journal of Chemical Information and Modeling (2019), 59 (12), 5183-5197CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      Adenosine A3 receptor (A3R) is a promising drug target cancer and for a no. of other conditions like inflammatory diseases, including asthma and rheumatoid arthritis, glaucoma, chronic obstructive pulmonary disease, and ischemic injury. Currently, there is no exptl. detd. structure of A3R. We explored the binding profile of O4-{[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl}-2-methyl-1,3-thiazole-4-carbohydroximamide (K18), which is a new specific and competitive antagonist at the orthosteric binding site of A3R. MD simulations and MM-GBSA calcns. of the WT A3R in complex with K18 combined with in vitro mutagenic studies show that the most plausible binding conformation for the dichlorophenyl group of K18 is oriented toward trans-membrane helixes (TM) 5, 6 and reveal important residues for binding. Further, MM-GBSA calcns. distinguish mutations that reduce or maintain or increase antagonistic activity. Our studies show that selectivity of K18 toward A3R is defined not only by direct interactions with residues within the orthosteric binding area but also by remote residues playing a significant role. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid, K18 maintained antagonistic potency, in agreement with our previous results obtained for agonists binding profile investigation. Mutation of the direct interacting residue L903.32 in the low region and the remote L2647.35 in the middle/upper region to alanine increases antagonistic potency, suggesting an empty space in the orthosteric area available for increasing antagonist potency. These results approve the computational model for the description of K18 binding at A3R, which we previously performed for agonists binding to A3R, and the design of more effective antagonists based on K18.
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    21. 21
      Papastathopoulos, A.; Lougiakis, N.; Kostakis, I. K.; Marakos, P.; Pouli, N.; Pratsinis, H.; Kletsas, D. New Bioactive 5-Arylcarboximidamidopyrazolo[3,4-c]Pyridines: Synthesis, Cytotoxic Activity, Mechanistic Investigation and Structure-Activity Relationships. Eur. J. Med. Chem. 2021, 218, 113387,  DOI: 10.1016/j.ejmech.2021.113387
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      21
      New bioactive 5-arylcarboximidamidopyrazolo[3,4-c]pyridines: Synthesis, cytotoxic activity, mechanistic investigation and structure-activity relationships
      Papastathopoulos, Athanasios; Lougiakis, Nikolaos; Kostakis, Ioannis K.; Marakos, Panagiotis; Pouli, Nicole; Pratsinis, Harris; Kletsas, Dimitris
      European Journal of Medicinal Chemistry (2021), 218 (), 113387CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      In this study, a series of novel substituted pyrazolo[3,4-c]pyridin-5-ylamidines I [R = Me, (4-methoxyphenyl)methyl; R1 = H, (3,4,5-trimethoxyphenyl)aminyl, cyclohexylaminyl, morpholin-4-yl; R2 = H, Ph; R3 = H, CF3; R4 = H, 4-methylpiperazin-1-yl] and II was synthesized and their cytotoxicity against three cancer cell lines (MDA-MB-231, HT-1080, PC-3), as well as a human normal cell line (AG01523) was evaluated. A no. of derivs. I and II could strongly reduce cancer cells proliferation and exhibit apoptotic induction capability, while reasonable structure-activity relationships could be extd. Certain analogs were endowed with low toxicity against normal cells. Cell cycle anal. revealed that most of the active compds. induced a G0/G1 arrest of HT-1080 cells. Moreover, the potential mechanisms of the cytotoxic activity of the promising compds. I and II were investigated in HT-1080 cells, upon study of their effects on the phosphorylation of Akt, ERK and p38 MAPK. Most of the active derivs. inhibit phosphorylation of Akt and ERK and/or induce p38 MAPK phosphorylation, providing a potential indication on the mode of action of this class.
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    22. 22
      Bento, A. P.; Gaulton, A.; Hersey, A.; Bellis, L. J.; Chambers, J.; Davies, M.; Krüger, F. A.; Light, Y.; Mak, L.; McGlinchey, S.; Nowotka, M.; Papadatos, G.; Santos, R.; Overington, J. P. The ChEMBL Bioactivity Database: An Update. Nucleic Acids Res. 2014, 42, D1083– D1090,  DOI: 10.1093/nar/gkt1031
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      22
      The ChEMBL bioactivity database: an update
      Bento, A. Patricia; Gaulton, Anna; Hersey, Anne; Bellis, Louisa J.; Chambers, Jon; Davies, Mark; Krueger, Felix A.; Light, Yvonne; Mak, Lora; McGlinchey, Shaun; Nowotka, Michal; Papadatos, George; Santos, Rita; Overington, John P.
      Nucleic Acids Research (2014), 42 (D1), D1083-D1090CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      ChEMBL is an open large-scale bioactivity database (https://www.ebi.ac.uk/chembl), previously described in the 2012 Nucleic Acids Research Database Issue. Since then, a variety of new data sources and improvements in functionality have contributed to the growth and utility of the resource. In particular, more comprehensive tracking of compds. from research stages through clin. development to market is provided through the inclusion of data from United States Adopted Name applications; a new richer data model for representing drug targets has been developed; and a no. of methods have been put in place to allow users to more easily identify reliable data. Finally, access to ChEMBL is now available via a new Resource Description Framework format, in addn. to the web-based interface, data downloads and web services.
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    23. 23
      Hawkins, P. C. D.; Skillman, A. G.; Nicholls, A. Comparison of Shape-Matching and Docking as Virtual Screening Tools. J. Med. Chem. 2007, 50, 74– 82,  DOI: 10.1021/jm0603365
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      23
      Comparison of Shape-Matching and Docking as Virtual Screening Tools
      Hawkins, Paul C. D.; Skillman, A. Geoffrey; Nicholls, Anthony
      Journal of Medicinal Chemistry (2007), 50 (1), 74-82CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      Ligand docking is a widely used approach in virtual screening. In recent years a large no. of publications have appeared in which docking tools are compared and evaluated for their effectiveness in virtual screening against a wide variety of protein targets. These studies have shown that the effectiveness of docking in virtual screening is highly variable due to a large no. of possible confounding factors. Another class of method that has shown promise in virtual screening is the shape-based, ligand-centric approach. Several direct comparisons of docking with the shape-based tool ROCS have been conducted using data sets from some of these recent docking publications. The results show that a shape-based, ligand-centric approach is more consistent than, and often superior to, the protein-centric approach taken by docking.
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    24. 24
      Pei, Y.; Wickham, B. O. S. Regioselective Syntheses of 3-Aminomethyl-5-Substituted Isoxazoles: A Facile and Chemoselective Reduction of Azide to Amine by Sodium Borohydride Using 1,3-Propanedithiol as a Catalyst. Tetrahedron Lett. 1993, 34, 7509– 7512,  DOI: 10.1016/S0040-4039(00)60386-6
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      Regioselective syntheses of 3-Aminomethyl-5-substituted isoxazoles: a facile and chemoselective reduction of azide to amine by sodium borohydride using 1,3-propanedithiol as a catalyst
      Pei, Yazhong; Wickham, Barbara O. S.
      Tetrahedron Letters (1993), 34 (47), 7509-12CODEN: TELEAY; ISSN:0040-4039.
      A series of isoxazole azides I (R = Me, Ph, i-Pr; R1 = N3) were prepd. and reduced selectively to isoxazole amines I (R1 = NH2) in quant. yield by sodium borohydride using 1,3-propanedithiol as a catalyst.
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    25. 25
      Fadnavis, N. W.; Radhika, K. R. Enantio- and Regiospecific Reduction of Ethyl 4-Phenyl-2,4-Dioxobutyrate with Baker’s Yeast: Preparation of (R)-HPB Ester. Tetrahedron Asymmetry 2004, 15, 3443– 3447,  DOI: 10.1016/j.tetasy.2004.09.007
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      Enantio- and regiospecific reduction of ethyl 4-phenyl-2,4-dioxobutyrate with baker's yeast: preparation of (R)-HPB ester
      Fadnavis, Nitin W.; Radhika, Kasiraman R.
      Tetrahedron: Asymmetry (2004), 15 (21), 3443-3447CODEN: TASYE3; ISSN:0957-4166. (Elsevier B.V.)
      Et 2,4-dioxo-4-phenylbutyrate, obtained by condensation of acetophenone with di-Et oxalate, is reduced enantio- and regiospecifically by baker's yeast in a diisopropyl ether/water two-phase system to give (-)-Et (R)-2-hydroxy-4-oxo-4-phenylbutyrate with an 98% ee in 80% isolated yield. This (hydroxy)keto ester was hydrogenated over Pd-C to obtain (-)-Et (R)-2-hydroxy-4-phenylbutyrate (HPB ester), an important intermediate for the synthesis of ACE inhibitors. Prolonged contact of the redn. product with baker's yeast produced 3-phenyl-3-oxopropanol in 90% yield.
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    26. 26
      Dang, T. T.; Dang, T. T.; Fischer, C.; Görls, H.; Langer, P. Synthesis of Pyrazole-3-Carboxylates and Pyrazole-1,5-Dicarboxylates by One-Pot Cyclization of Hydrazone Dianions with Diethyl Oxalate. Tetrahedron 2008, 64, 2207– 2215,  DOI: 10.1016/j.tet.2007.12.024
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      26
      Synthesis of pyrazole-3-carboxylates and pyrazole-1,5-dicarboxylates by one-pot cyclization of hydrazone dianions with diethyl oxalate
      Dang, Tung T.; Dang, Tuan T.; Fischer, Christine; Goerls, Helmar; Langer, Peter
      Tetrahedron (2008), 64 (9), 2207-2215CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)
      The one-pot cyclization of hydrazone dianions with di-Et oxalate allows a convenient synthesis of pyrazole-3-carboxylates and pyrazole-1,5-dicarboxylates. Under the condition of toluene as solvent and p-TsOH as acid, pyrazole-3-carboxylates are obtained; under the condition of CH2Cl2 as solvent and TFA as acid, pyrazole-1,5-dicarboxylates are obtained.
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    27. 27
      Yan, Z.; Liu, A.; Huang, M.; Liu, M.; Pei, H.; Huang, L.; Yi, H.; Liu, W.; Hu, A. Design, Synthesis, DFT Study and Antifungal Activity of the Derivatives of Pyrazolecarboxamide Containing Thiazole or Oxazole Ring. Eur. J. Med. Chem. 2018, 149, 170– 181,  DOI: 10.1016/j.ejmech.2018.02.036
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      27
      Design, synthesis, DFT study and antifungal activity of the derivatives of pyrazolecarboxamide containing thiazole or oxazole ring
      Yan, Zhongzhong; Liu, Aiping; Huang, Mingzhi; Liu, Minhua; Pei, Hui; Huang, Lu; Yi, Haibo; Liu, Weidong; Hu, Aixi
      European Journal of Medicinal Chemistry (2018), 149 (), 170-181CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      To discover new pyrazolecarboxamide analogs with broad spectrum and high activity, a class of new compds. of pyrazole carboxamide derivs. contg. thiazole or oxazole ring I (R1 = H, 4-Me, 2-Cl, etc.; R2 = H, Cl, Me; R3 = Me, Et, i-Pr, cyclopropyl; R4 = H, Et, Me, Ph; Z = S, O) was designed by scaffold hopping and bioisosterism, and 36 pyrazole carboxamide derivs. with antifungal activity were synthesized. Those compds. I were evaluated against five phytopathogenic fungi, Gibberella zeae, Phytophythora capsici, Sclerotonia sclerotiorum, Erysiphe graminis and Puccinia sorghi. The results indicated that most of the compds. displayed good fungicidal activities, esp. against E. graminis. Theor. calcns. were carried out at the B3LYP/6-31G (d, p) level and the full geometry optimization was carried out using the 6-31G (d, p) basis set, and the frontier orbital energy, at. net charges and, mol. docking were discussed, and the structure-activity relationships were also studied.
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    28. 28
      Schmidt, A.; Habeck, T.; Kindermann, M. K.; Nieger, M. New Pyrazolium-Carboxylates as Structural Analogues of the Pseudo-Cross-Conjugated Betainic Alkaloid Nigellicine. J. Org. Chem. 2003, 68, 5977– 5982,  DOI: 10.1021/jo0344337
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      28
      New Pyrazolium-carboxylates as Structural Analogues of the Pseudo-Cross-Conjugated Betainic Alkaloid Nigellicine
      Schmidt, Andreas; Habeck, Tobias; Kindermann, Markus Karl; Nieger, Martin
      Journal of Organic Chemistry (2003), 68 (15), 5977-5982CODEN: JOCEAH; ISSN:0022-3263. (American Chemical Society)
      Pyrazolium-3-carboxylates were examd. as relatives of the betainic alkaloid nigellicine and as new examples of the sparsely populated class of heterocyclic pseudo-cross-conjugated mesomeric betaines. The title compds. were prepd. in a 4-step procedure starting from EtO2CCOCH:CROH [R = Me, Ph] which were cyclized with substituted hydrazines. The resulting isomeric pyrazole esters were sepd. and quaternized with di-Me sulfate in the presence of nitrobenzene to pyrazolium esters. Sapon. was best accomplished in dild. sulfuric acid, which resulted in the formation of the pseudo-cross-conjugated mesomeric betaines in one step. Protonation to the corresponding carboxylic acids required the treatment of the betaines with tetrafluoroboric acid in dichloromethane. The effect of neg. solvatochromism proves the charge sepn. in the ground state of the mols. X-ray crystallog. analyses, semiempirical calcns., and ESI mass spectrometric measurements were performed to gain knowledge about the phenomenon of pseudo-cross-conjugation.
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    29. 29
      Knight, A.; Hemmings, J. L.; Winfield, I.; Leuenberger, M.; Frattini, E.; Frenguelli, B. G.; Dowell, S. J.; Lochner, M.; Ladds, G. Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity. J. Med. Chem. 2016, 59, 947– 964,  DOI: 10.1021/acs.jmedchem.5b01402
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      29
      Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity
      Knight, Anthony; Hemmings, Jennifer L.; Winfield, Ian; Leuenberger, Michele; Frattini, Eugenia; Frenguelli, Bruno G.; Dowell, Simon J.; Lochner, Martin; Ladds, Graham
      Journal of Medicinal Chemistry (2016), 59 (3), 947-964CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A series of N6-bicyclic and N6-(2-hydroxy)cyclopentyl derivs. of adenosine were synthesized as novel A1R agonists and their A1R/A2R selectivity assessed using a simple yeast screening platform. We obsd. that the most selective, high potency ligands were achieved through N6-adamantyl substitution in combination with 5'-N-ethylcarboxamido or 5'-hydroxymethyl groups. In addn., we detd. that 5'-(2-fluoro)thiophenyl derivs. all failed to generate a signaling response despite showing an interaction with the A1R. Some selected compds. were also tested on A1R and A3R in mammalian cells revealing that four of them are entirely A1R-selective agonists. By using in silico homol. modeling and ligand docking, we provide insight into their mechanisms of recognition and activation of the A1R. We believe that given the broad tissue distribution, but contrasting signaling profiles, of adenosine receptor subtypes, these compds. might have therapeutic potential.
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    30. 30
      Stoddart, L. A.; Vernall, A. J.; Denman, J. L.; Briddon, S. J.; Kellam, B.; Hill, S. J. Fragment Screening at Adenosine-A3 Receptors in Living Cells Using a Fluorescence-Based Binding Assay. Chem. Biol. 2012, 19, 1105– 1115,  DOI: 10.1016/j.chembiol.2012.07.014
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      30
      Fragment Screening at Adenosine-A3 Receptors in Living Cells Using a Fluorescence-Based Binding Assay
      Stoddart, Leigh A.; Vernall, Andrea J.; Denman, Jessica L.; Briddon, Stephen J.; Kellam, Barrie; Hill, Stephen J.
      Chemistry & Biology (Oxford, United Kingdom) (2012), 19 (9), 1105-1115CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
      G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is detd. in the correct cellular environment and preferably using an unmodified receptor. The authors developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to det. binding affinity consts. of competing ligands at human adenosine-A1 and -A3 receptors. This method was validated as a tool to screen a library of low mol. wt. fragments, and identified a hit with submicromolar binding affinity (KD). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A3 receptor. This technol. represents a significant advance that will allow the detn. of ligand and fragment affinities at receptors in their native membrane environment.
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    31. 31
      Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee, R. P. Empirical Scoring Functions: I. The Development of a Fast Empirical Scoring Function to Estimate the Binding Affinity of Ligands in Receptor Complexes. J. Comput. Aided. Mol. Des. 1997, 11, 425– 445,  DOI: 10.1023/A:1007996124545
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      31
      Empirical scoring functions: I. The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes
      Eldridge, Matthew D.; Murray, Christopher W.; Auton, Timothy R.; Paolini, Gaia V.; Mee, Roger P.
      Journal of Computer-Aided Molecular Design (1997), 11 (5), 425-445CODEN: JCADEQ; ISSN:0920-654X. (Kluwer Academic Publishers)
      This paper describes the development of a simple empirical scoring function designed to est. the free energy of binding for a protein-ligand complex when the 3D structure of the complex is known or can be approximated. The function uses simple contact terms to est. lipophilic and metal-ligand binding contributions, a simple explicit form for hydrogen bonds and a term which penalizes flexibility. The coeffs. of each term are obtained using a regression based on 82 ligand-receptor complexes for which the binding affinity is known. The function reproduces the binding affinity of the complexes with a cross-validated error of 8.68 kJ/mol. Tests on internal consistency indicate that the coeffs. obtained are stable to changes in the compn. of the training set. The function is also tested on two test sets contg. a further 20 and 10 complexes, resp. The deficiencies of this type of function are discussed and it is compared to approaches by other workers.
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    32. 32
      Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Comparison of Multiple Amber Force Fields and Development of Improved Protein Backbone Parameters. Proteins Struct. Funct. Genet. 2006, 65, 712– 725,  DOI: 10.1002/prot.21123
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      32
      Comparison of multiple Amber force fields and development of improved protein backbone parameters
      Hornak, Viktor; Abel, Robert; Okur, Asim; Strockbine, Bentley; Roitberg, Adrian; Simmerling, Carlos
      Proteins: Structure, Function, and Bioinformatics (2006), 65 (3), 712-725CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
      The ff94 force field that is commonly assocd. with the Amber simulation package is one of the most widely used parameter sets for biomol. simulation. After a decade of extensive use and testing, limitations in this force field, such as over-stabilization of α-helixes, were reported by the authors and other researchers. This led to a no. of attempts to improve these parameters, resulting in a variety of "Amber" force fields and significant difficulty in detg. which should be used for a particular application. The authors show that several of these continue to suffer from inadequate balance between different secondary structure elements. In addn., the approach used in most of these studies neglected to account for the existence in Amber of two sets of backbone .vphi./ψ dihedral terms. This led to parameter sets that provide unreasonable conformational preferences for glycine. The authors report here an effort to improve the .vphi./ψ dihedral terms in the ff99 energy function. Dihedral term parameters are based on fitting the energies of multiple conformations of glycine and alanine tetrapeptides from high level ab initio quantum mech. calcns. The new parameters for backbone dihedrals replace those in the existing ff99 force field. This parameter set, which the authors denote ff99SB, achieves a better balance of secondary structure elements as judged by improved distribution of backbone dihedrals for glycine and alanine with respect to PDB survey data. It also accomplishes improved agreement with published exptl. data for conformational preferences of short alanine peptides and better accord with exptl. NMR relaxation data of test protein systems.
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    33. 33
      Massova, I.; Kollman, P. A. Combined molecular mechanical and continuum solvent approach (MM-PBSA/GBSA) to predict ligand binding. Per. Drug Discovery Design 2000, 18, 113– 135,  DOI: 10.1023/A:1008763014207
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      Combined molecular mechanical and continuum solvent approach (MM-PBSA/GBSA) to predict ligand binding
      Massova, Irina; Kollman, Peter A.
      Perspectives in Drug Discovery and Design (2000), 18 (Hydrophobicity and Solvation in Drug Design, Pt. II), 113-135CODEN: PDDDEC; ISSN:0928-2866. (Kluwer Academic Publishers)
      Significant progress has been achieved in computational methods to treat solvent effects in recent years. Among various techniques, the continuum solvent approach appears to be practically promising because it can be used to calc. reliable interaction and solvation energies in complex systems. A computational scanning mutagenesis method, one of such new approaches, has been recently developed. It combines the mol. mech. and continuum solvent approaches and allows one to identify the "hot spots" in binding interfaces from a single trajectory of a wild type complex. Such techniques can be also used as a tool to optimize the interacting species for the binding, or as a ranking procedure in high throughput screening.
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    34. 34
      Wang, E.; Sun, H.; Wang, J.; Wang, Z.; Liu, H.; Zhang, J. Z. H.; Hou, T. End-Point Binding Free Energy Calculation with MM/PBSA and MM/GBSA: Strategies and Applications in Drug Design. Chem. Rev. 2019, 119, 9478– 9508,  DOI: 10.1021/acs.chemrev.9b00055
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      34
      End-point binding free energy calculation with MM/PBSA and MM/GBSA: strategies and applications in drug design
      Wang, Ercheng; Sun, Huiyong; Wang, Junmei; Wang, Zhe; Liu, Hui; Zhang, John Z. H.; Hou, Tingjun
      Chemical Reviews (Washington, DC, United States) (2019), 119 (16), 9478-9508CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Mol. mechanics Poisson-Boltzmann surface area (MM/PBSA) and mol. mechanics generalized Born surface area (MM/GBSA) are arguably very popular methods for binding free energy prediction since they are more accurate than most scoring functions of mol. docking and less computationally demanding than alchem. free energy methods. MM/PBSA and MM/GBSA have been widely used in biomol. studies such as protein folding, protein-ligand binding, protein-protein interaction, etc. In this review, methods to adjust the polar solvation energy and to improve the performance of MM/PBSA and MM/GBSA calcns. are reviewed and discussed. The latest applications of MM/GBSA and MM/PBSA in drug design are also presented. This review intends to provide readers with guidance for practically applying MM/PBSA and MM/GBSA in drug design and related research fields.
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    35. 35
      Kaminski, G.; Friesner, R. A.; Tirado-Rives, J.; Jorgensen, W. L. Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides. Phys. Chem. B 2001, 105 (28), 6474– 6487,  DOI: 10.1021/jp003919d
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      35
      Evaluation and Reparametrization of the OPLS-AA Force Field for Proteins via Comparison with Accurate Quantum Chemical Calculations on Peptides
      Kaminski, George A.; Friesner, Richard A.; Tirado-Rives, Julian; Jorgensen, William L.
      Journal of Physical Chemistry B (2001), 105 (28), 6474-6487CODEN: JPCBFK; ISSN:1089-5647. (American Chemical Society)
      We present results of improving the OPLS-AA force field for peptides by means of refitting the key Fourier torsional coeffs. The fitting technique combines using accurate ab initio data as the target, choosing an efficient fitting subspace of the whole potential-energy surface, and detg. wts. for each of the fitting points based on magnitudes of the potential-energy gradient. The av. energy RMS deviation from the LMP2/cc-pVTZ(-f)//HF/6-31G** data is reduced by ∼40% from 0.81 to 0.47 kcal/mol as a result of the fitting for the electrostatically uncharged dipeptides. Transferability of the parameters is demonstrated by using the same alanine dipeptide-fitted backbone torsional parameters for all of the other dipeptides (with the appropriate side-chain refitting) and the alanine tetrapeptide. Parameters of nonbonded interactions have also been refitted for the sulfur-contg. dipeptides (cysteine and methionine), and the validity of the new Coulombic charges and the van der Waals σ's and ε's is proved through reproducing gas-phase energies of complex formation heats of vaporization and densities of pure model liqs. Moreover, a novel approach to fitting torsional parameters for electrostatically charged mol. systems has been presented and successfully tested on five dipeptides with charged side chains.
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    36. 36
      Shivakumar, D.; Williams, J.; Wu, Y.; Damm, W.; Shelley, J.; Sherman, W. Prediction of Absolute Solvation Free Energies using Molecular Dynamics Free Energy Perturbation and the OPLS Force Field. J. Chem. Theory Comput. 2010, 6, 1509– 19,  DOI: 10.1021/ct900587b
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      36
      Prediction of Absolute Solvation Free Energies using Molecular Dynamics Free Energy Perturbation and the OPLS Force Field
      Shivakumar, Devleena; Williams, Joshua; Wu, Yujie; Damm, Wolfgang; Shelley, John; Sherman, Woody
      Journal of Chemical Theory and Computation (2010), 6 (5), 1509-1519CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      The accurate prediction of protein-ligand binding free energies is a primary objective in computer-aided drug design. The solvation free energy of a small mol. provides a surrogate to the desolvation of the ligand in the thermodn. process of protein-ligand binding. Here, we use explicit solvent mol. dynamics free energy perturbation to predict the abs. solvation free energies of a set of 239 small mols., spanning diverse chem. functional groups commonly found in drugs and drug-like mols. We also compare the performance of abs. solvation free energies obtained using the OPLS_2005 force field with two other commonly used small mol. force fields - general AMBER force field (GAFF) with AM1-BCC charges and CHARMm-MSI with CHelpG charges. Using the OPLS_2005 force field, we obtain high correlation with exptl. solvation free energies (R2 = 0.94) and low av. unsigned errors for a majority of the functional groups compared to AM1-BCC/GAFF or CHelpG/CHARMm-MSI. However, OPLS_2005 has errors of over 1.3 kcal/mol for certain classes of polar compds. We show that predictions on these compd. classes can be improved by using a semiempirical charge assignment method with an implicit bond charge correction.
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    37. 37
      Stamatis, D.; Lagarias, P.; Barkan, K.; Vrontaki, E.; Ladds, G.; Kolocouris, A. Structural Characterization of Agonist Binding to an A3 Adenosine Receptor through Biomolecular Simulations and Mutagenesis Experiments. J. Med. Chem. 2019, 62, 8831– 8846,  DOI: 10.1021/acs.jmedchem.9b01164
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      37
      Structural Characterization of Agonist Binding to an A3 Adenosine Receptor through Biomolecular Simulations and Mutagenesis Experiments
      Stamatis, Dimitrios; Lagarias, Panagiotis; Barkan, Kerry; Vrontaki, Eleni; Ladds, Graham; Kolocouris, Antonios
      Journal of Medicinal Chemistry (2019), 62 (19), 8831-8846CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      The adenosine A3 receptor (A3R) binds adenosine and is a drug target against cancer cell proliferation. Currently, there is no exptl. structure of A3R. Here, we have generated a mol. model of A3R in complex with two agonists, the nonselective 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranuronamide (NECA) and the selective 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide (IB-MECA). Mol. dynamics simulations of the wild-type A3R in complex with both agonists, combined with in vitro mutagenic studies revealed important residues for binding. Further, mol. mechanics-generalized Born surface area calcns. were able to distinguish mutations that reduce or negate agonistic activity from those that maintained or increased the activity. Our studies reveal that selectivity of IB-MECA toward A3R requires not only direct interactions with residues within the orthosteric binding area but also with remote residues. Although V1695.30 is considered to be a selectivity filter for A3R binders, when it was mutated to glutamic acid or alanine, the activity of IB-MECA increased by making new van der Waals contacts with TM5. This result may have implications in the design of new A3R agonists.
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    • Scheme S1. Comparison of amino acid residue sequences of the binding area. Table S1. Chemical structures and antagonistic potencies of 7-amino-pyrazolo[3,4-d]pyridazines 10a–c and 15a–c against A2AR and A2BR. Table S2. Mean RMSD values for all compounds against A1R, A2AR, and A2BR and ΔGeff only for 10–10c against A1R. Figure S1. 1H and 13C NMR spectra of the target compounds. Figure S2. Results from the MD simulations of 10b and 15b against A3R and A2BR. Figure S3. Docking poses of 4-(2-phenethyl)amino-1-phenylethyl pyrazolo[3,4-b]pyridine and N9-methyl,N6-benzyl adenine to A1R. Figure S4. Representative frames from 100 ns MD simulations of 10b inside the orthosteric binding area of WT A1R and 10b inside mutant Y271A A1R and the receptor–ligand interaction frequency histogram and RMSD graphs of protein Ca and ligand heavy atoms. Information for the methods and synthetic protocols. (PDF)


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