Pyrazole-containing pharmaceuticals: target, pharmacological activity, and their SAR studies

Fan Y., Zhang Y., Liu Y., Jiang H., Zhou Y., Tang C., Fan W.

As a receptor tyrosine kinase (RTK), tropomyosin receptor kinase (Trk) is a key drug target in solid tumors. However, the use of the First-generation Trk inhibitors was greatly restricted due to mutant drug resistance. Fortunately, the emergence of the Second-generation of Trk inhibitors has brought an effective solution to this mutant resistance, such as TPX-0005 (Repotrectinib). Here, we reported a series of pyrizolo[1,5-a]pyrimidine derivatives as the second-generation Trk inhibitors, and carried out the subsequent biological activity evaluation. Among them, the best compound 14h (IC50 = 1.40, 1.80 nM, against TrkA, TrkAG595R, respectively) and 14j (IC50 = 0.86, 6.92 nM, against TrkA, TrkAG595R, respectively) has a kinase activity comparable to TPX-0005, and 14j (IC50 = 350 nM against ALK) has a higher selectivity of Trk inhibition than TPX-0005, which may be of great significance for reducing toxicity.

Hoy S.M.

Palovarotene (Sohonos™) is an orally bioavailable selective retinoic acid receptor (RAR)γ agonist being developed by Ipsen for the reduction of heterotopic ossification (HO) formation in patients with fibrodysplasia ossificans progressiva (FOP). By binding to RARγ, palovarotene inhibits bone morphogenetic protein and SMAD 1/5/8 signalling: interfering with these pathways prevents chondrogenesis and ultimately HO by permitting normal muscle tissue repair or regeneration to occur. Palovarotene received its first approval on 21 January 2022 to reduce the formation of HO in adults and children aged 8 years and above for females and 10 years and above for males with FOP in Canada. This article summarizes the milestones in the development of palovarotene leading to this first approval.

Lee J., Choi N., Kim S., Jin M.S., Shen H., Kim Y.

N6A-methyladenosine (m6A) post-transcriptional modification, the most abundant internal RNA modification, is catalyzed by the METTL3-14 methyltransferase complex. Recently, attention has been drawn to the METTL3-14 complex regarding its significant roles in the pathogenesis of acute myeloid leukemia (AML), attracting the potential of novel therapeutic targets for the disease. Herein, we report the identification and characterization of eltrombopag as a selective allosteric inhibitor of the METTL3-14 complex. Eltrombopag exhibited selective inhibitory activity in the most active catalytic form of the METTL3-14 complex by direct binding, and the mechanism of inhibition was confirmed as a noncompetitive inhibition by interacting at a putative allosteric binding site in METTL3, which was predicted by cavity search and molecular docking studies. At a cellular level, eltrombopag displayed anti-proliferative effects in the relevant AML cell line, MOLM-13, in correlation with a reduction in m6A levels. Molecular mechanism studies of eltrombopag using m6A-seq analysis provided further evidence of its cellular function by determining the hypomethylation of leukemogenic genes in eltrombopag-treated MOLM-13 cells and the overlapping of the pattern with those of METTL3-knockdown MOLM-13 cells. In conclusion, eltrombopag was first disclosed as a functional METTL3-14 allosteric inhibitor in AML cells, which could be utilized for the further development of novel anti-AML therapy.

Mathis A., Sale M., Cornpropst M., Sheridan W.P., Ma S.C.

Hereditary angioedema (HAE) is an autosomal dominant disorder characterized by recurrent episodes of swelling of the skin, larynx, gastrointestinal tract, genitals, and extremities that can be disruptive to patient quality of life. Dysregulation of plasma kallikrein activity leads to increased production and accumulation of bradykinin in HAE and causes attacks of angioedema. Plasma kallikrein is a serine protease essential for the formation of bradykinin. Berotralstat is a potent, highly selective, orally bioavailable small-molecule plasma kallikrein inhibitor that has been approved to prevent attacks of HAE in adults and children 12 years of age and older. Population pharmacokinetic (PK) analyses were conducted to describe the PK of berotralstat (BCX7353; Orladeyo™ ) and to evaluate the covariates that may explain variability in PK. The PK of berotralstat were characterized by population PK modeling of data from 13 clinical studies and a total of 771 healthy subjects and patients with HAE. The PK profile was well described by a three-compartment model with first-order absorption including an absorption lag time and linear elimination. Among the covariates tested, the effects of bilirubin and food were found not to be clinically significant and were removed from the model. Covariate analysis indicated significant effects of dose on bioavailability and weight on berotralstat clearance and volume. Despite the covariate effect of weight, simulations in adolescents and adults who were underweight, low weight, and overweight demonstrated similar predicted exposures to those observed at therapeutic doses in a clinical trial. Therefore, no dose adjustment is required in these HAE patient subpopulations.

Pan S., Zhang L., Luo X., Nan J., Yang W., Bin H., Li Y., Huang Q., Wang T., Pan Z., Mu B., Wang F., Tian C., Liu Y., Li L., et. al.

Tropomyosin receptor kinases (TrkA, TrkB, and TrkC) are attractive therapeutic targets for multiple cancers. Two first-generation small-molecule Trks inhibitors, larotrectinib and entrectinib, have just been approved to use clinically. However, the drug-resistance mutations of Trks have already emerged, which calls for new-generation Trks inhibitors. Herein, we report the structural optimization and structure-activity relationship studies of 6,6-dimethyl-4-(phenylamino)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one derivatives as a new class of pan-Trk inhibitors. The prioritized compound 11g exhibited low nanomolar IC50 values against TrkA, TrkB, and TrkC and various drug-resistant mutants. It also showed good kinase selectivity. 11g displayed excellent in vitro antitumor activity and strongly suppressed Trk-mediated signaling pathways in intact cells. In in vivo studies, compound 11g exhibited good antitumor activity in BaF3-TEL-TrkA and BaF3-TEL-TrkCG623R allograft mouse models without exhibiting apparent toxicity. Collectively, 11g could be a promising lead compound for drug discovery targeting Trks and deserves further investigation.

Veenstra J.A.

This work was initiated because an old publication suggested that electrocoagulation of four paraldehyde fuchsin positive cells in the brain of Locusta migratoria might produce a diuretic hormone, the identity of which remains unknown, since none of the antisera to the various putative Locusta diuretic hormones recognizes these cells. The paraldehyde fuchsin positive staining suggests a peptide with a disulfide bridge and the recently identified Locusta calcitonins have both a disulfide bridge and are structurally similar to calcitonin-like diuretic hormone. In situ hybridization and antisera raised to calcitonin-A and -B were used to show where these peptides are expressed in Locusta. Calcitonin-A is produced by neurons and neuroendocrine cells that were previously shown to be immunoreactive to an antiserum to pigment dispersing factor (PDF). The apparent PDF-immunoreactivity in these neurons and neuroendocrine cells is due to crossreactivity with the calcitonin-A precursor. As confirmed by both an PDF-precursor specific antiserum and in situ hybridisation, those calcitonin-A expressing cells do not express PDF. Calcitonin B is expressed by numerous enteroendocrine cells in the midgut as well as the midgut caeca. A guinea pig antiserum to calcitonin A seemed quite specific as it recognized only the calcitonin A expressing cells. However, rabbit antisera to calcitonin-A and-B both crossreacted with neuroendocrine cells in the brain that produce ACP (AKH/corazonin-related peptide), this is almost certainly due to the common C-terminal dipeptide SPamide that is shared between Locusta calcitonin-A, calcitonin-B and ACP.

Baatallah N., Elbahnsi A., Mornon J., Chevalier B., Pranke I., Servel N., Zelli R., Décout J., Edelman A., Sermet-Gaudelus I., Callebaut I., Hinzpeter A.

Protein misfolding is involved in a large number of diseases, among which cystic fibrosis. Complex intra- and inter-domain folding defects associated with mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, among which p.Phe508del (F508del), have recently become a therapeutical target. Clinically approved correctors such as VX-809, VX-661, and VX-445, rescue mutant protein. However, their binding sites and mechanisms of action are still incompletely understood. Blind docking onto the 3D structures of both the first membrane-spanning domain (MSD1) and the first nucleotide-binding domain (NBD1), followed by molecular dynamics simulations, revealed the presence of two potential VX-809 corrector binding sites which, when mutated, abrogated rescue. Network of amino acids in the lasso helix 2 and the intracellular loops ICL1 and ICL4 allosterically coupled MSD1 and NBD1. Corrector VX-445 also occupied two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. Binding of both correctors on MSD1 enhanced the allostery between MSD1 and NBD1, hence the increased efficacy of the corrector combination. These correctors improve both intra-domain folding by stabilizing fragile protein–lipid interfaces and inter-domain assembly via distant allosteric couplings. These results provide novel mechanistic insights into the rescue of misfolded proteins by small molecules.

Murray B.W., Rogers E., Zhai D., Deng W., Chen X., Sprengeler P.A., Zhang X., Graber A., Reich S.H., Stopatschinskaja S., Solomon B., Besse B., Drilon A.

Abstract NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to TRK inhibitors (larotrectinib and entrectinib) but often mutate, limiting the durability of response for NTRK+ patients. Next-generation inhibitors with compact macrocyclic structures (repotrectinib and selitrectinib) were designed to avoid resistance mutations. Head-to-head potency comparisons of TRK inhibitors and molecular characterization of binding interactions are incomplete, obscuring a detailed understanding of how molecular characteristics translate to potency. Larotrectinib, entrectinib, selitrectinib, and repotrectinib were characterized using cellular models of wild-type TRKA/B/C fusions and resistance mutant variants with a subset evaluated in xenograft tumor models. Crystal structures were determined for repotrectinib bound to TRKA (wild-type, solvent-front mutant). TKI-naïve and pretreated case studies are presented. Repotrectinib was the most potent inhibitor of wild-type TRKA/B/C fusions and was more potent than selitrectinib against all tested resistance mutations, underscoring the importance of distinct features of the macrocycle structures. Cocrystal structures of repotrectinib with wild-type TRKA and the TRKAG595R SFM variant elucidated how differences in macrocyclic inhibitor structure, binding orientation, and conformational flexibility affect potency and mutant selectivity. The SFM crystal structure revealed an unexpected intramolecular arginine sidechain interaction. Repotrectinib caused tumor regression in LMNA–NTRK1 xenograft models harboring GKM, SFM, xDFG, and GKM + SFM compound mutations. Durable responses were observed in TKI-naïve and -pretreated patients with NTRK+ cancers treated with repotrectinib (NCT03093116). This comprehensive analysis of first- and second-generation TRK inhibitors informs the clinical utility, structural determinants of inhibitor potency, and design of new generations of macrocyclic inhibitors.

Liu R., Kang Y., Chen L.

Soluble guanylate cyclase (sGC) is the receptor for nitric oxide (NO) in human. It is an important validated drug target for cardiovascular diseases. sGC can be pharmacologically activated by stimulators and activators. However, the detailed structural mechanisms, through which sGC is recognized and positively modulated by these drugs at high spacial resolution, are poorly understood. Here, we present cryo-electron microscopy structures of human sGC in complex with NO and sGC stimulators, YC-1 and riociguat, and also in complex with the activator cinaciguat. These structures uncover the molecular details of how stimulators interact with residues from both β H-NOX and CC domains, to stabilize sGC in the extended active conformation. In contrast, cinaciguat occupies the haem pocket in the β H-NOX domain and sGC shows both inactive and active conformations. These structures suggest a converged mechanism of sGC activation by pharmacological compounds. Soluble guanylate cyclase (sGC) is a validated drug target for cardiovascular diseases. Here, the authors report structures of human sGC in complex with NO and sGC stimulators or activator, providing insight into the mechanism of sGC activation by pharmacological compounds.

Luo Z., Wang L., Fu Z., Shuai B., Luo M., Hu G., Chen J., Sun J., Wang J., Li J., Chen S., Zhang Y.

Aberrant alterations of rearranged during transfection (RET) have been identified as actionable drivers of multiple cancers, including thyroid carcinoma and lung cancer. Currently, several approved multikinase inhibitors such as vandetanib and cabozantinib demonstrate clinical activity in patients with RET-rearranged or RET-mutant cancers. However, the observed response rates are only modest and the ‘off-target’ toxicities resulted from the inhibition of other kinases is also a concern. Herein, we designed and synthesized a series of RET inhibitors based on the structure of selective RET inhibitor BLU-667 and investigated their biological activities. We identified compound 9 as a novel potent and selective RET inhibitor with improved drug-like properties. Compound 9 exhibits a selective inhibitory profile with an inhibitory concentration 50 (IC 50 ) of 1.29 nM for RET and 1.97 (RET V804M) or 0.99 (RET M918T) for mutant RETs. The proliferation of Ba/F3 cells transformed with NSCLC related KIF5B-RET fusion was effectively suppressed by compound 9 (IC 50 = 19 nM). Additionally, compound 9 displayed less ‘off-target’ effects than BLU-667. In mouse xenograft models, compound 9 repressed tumor growth driven by KIF5B-RET-Ba/F3 cells in a dose-dependent manner. Based on its exceptional kinase selectivity, good potency and high exposure in tumor tissues, compound 9 represents a promising lead for the discovery of RET directed therapeutic agents and the study of RET-driven tumor biology.

Kotian P.L., Wu M., Vadlakonda S., Chintareddy V., Lu P., Juarez L., Kellogg-Yelder D., Chen X., Muppa S., Chambers-Wilson R., Davis Parker C., Williams J., Polach K.J., Zhang W., Raman K., et. al.

Hereditary angioedema (HAE) is a rare and potentially life-threatening disease that affects an estimated 1 in 50 000 individuals worldwide. Until recently, prophylactic HAE treatment options were limited to injectables, a burdensome administration route that has driven the need for an oral treatment. A substantial body of evidence has shown that potent and selective plasma kallikrein inhibitors that block the generation of bradykinin represent a promising approach for the treatment of HAE. Berotralstat (BCX7353, discovered by BioCryst Pharmaceuticals using a structure-guided drug design strategy) is a synthetic plasma kallikrein inhibitor that is potent and highly selective over other structurally related serine proteases. This once-daily, small-molecule drug is the first orally bioavailable prophylactic treatment for HAE attacks, having successfully completed a Phase III clinical trial (meeting its primary end point) and recently receiving the U.S. Food and Drug Administration's approval for the prophylactic treatment of HAE attacks in patients 12 years and older.

Rue K., Raptis R.G.

The structure of 4-chloro-1H-pyrazole, C3H3ClN2, at 170 K has orthorhombic (Pnma) symmetry and is isostructural to its bromo analogue. Data were collected at low temperature since 4-chloro-1H-pyrazole sublimes when subjected to the localized heat produced by X-rays. The structure displays intermolecular N—H...N hydrogen bonding and the packing features a trimeric molecular assembly bisected by a mirror plane (m normal to b) running through one chlorine atom, one carbon atom and one N—N bond. The asymmetric unit therefore consists of one and one-half 4-chloro-1H-pyrazole molecules. Thus, the N—H proton is crystallographically disordered over two positions of 50% occupancy each.

Henriques-Abreu M., Serrano C.

Liu Z., Yu P., Dong L., Wang W., Duan S., Wang B., Gong X., Ye L., Wang H., Tian J.

The neurotrophic receptor tyrosine kinase (NTRK) genes including NTRK1, NTRK2, and NTRK3 encode the tropomyosin receptor kinase (Trk) proteins TrkA, TrkB, and TrkC, respectively. So far, two TRK inhibitors, larotrectinib sulfate (LOXO-101 sulfate) and entrectinib (NMS-E628, RXDX-101), have been approved for clinical use in 2018 and 2019, respectively. To overcome acquired resistance, next-generation Trk inhibitors such as selitrectinib (LOXO-195) and repotrectinib (TPX-0005) have been developed and exhibit effectiveness to induce remission in patients with larotrectinib treatment failure. Herein, we report the identification and optimization of a series of macrocyclic compounds as potent pan-Trk (WT and MT) inhibitors that exhibited excellent physiochemical properties and good oral pharmacokinetics. Compound 10 was identified via optimization from the aspects of chemistry and pharmacokinetic properties, which showed good activity against wild and mutant TrkA/TrkC in in vitro and in vivo studies.

Braun L.M., Zeiser R.

Mao Q., Zhang P., Lin F., Fu X., Zhang B., Liu C., Liu Z., Chen X., Dai X., Yue X., Shi X., Pang J., Wang S.

Vashisht K., Sethi P., Ramasamy S.K., Bansal A., Dar M.O., Singh M., Alkhanjaf A.A., Ibrahim A.A., Umar A., Kumar R., Baskoutas S.

Kahk N.M., Mohamed F.E., Abdelhakeem M.M., Bakr R.B.

Gholivand K., Barzegari A., Ghorbani-Anarkooli M., Malekshah R.E., Pourbeiranvand S.

Garg A., Dureja D., Vijeata A., Chaudhary G.R., Berry S., Chaudhary S., Bhalla A.

Nagesh Khadri M.J., Shashikumar U., Tsai P., Manjappa K.B., Ponnusamy V.K., Khanum S.A.

AbstractNitrogen, oxygen, and sulfur‐containing heterocyclic compounds are broadly established as key scaffolds with potential chemical and biological properties. Benzimidazole, indole, benzoxazole, benzothiazole, pyrazole, and thiophene analogs are significant heterocyclic motifs that bear almost all pharmacological activities. Anti‐inflammatory activity is one of the most commonly associated biological activities with these heterocycles and thus has attracted the interest of researchers to synthesize and study them in‐depth. The present review highlights mainly the various synthetic methodologies utilized to prepare these diverse heterocycles and their associated pharmacological properties limited to anti‐inflammatory activities. Further, this review will provide a comprehensive idea of the methodologies used to prepare biologically active nitrogen, oxygen, and sulfur‐bearing heterocycles and possibly invoke new thoughts in the search for rational designs for developing more promising anti‐inflammatory agents.

Valapil D.G., Devabattula G., Barahdia A.S., Godugu C., Shankaraiah N.

Lee Y., Ahn S., Jung E., Lim Y., Koh D., Bae D., Shin S.Y.

Noori Z., Solà M., Viñas C., Teixidor F., Poater J.

A new series of o-carborane-fused pyrazoles has been recently successfully synthesized. This fusion was expected to create a hybrid 3D/2D aromatic system, combining the 3D aromaticity of o-carborane with the 2D aromaticity of pyrazole. However, while the boron cage retains its aromatic character, the pyrazole’s aromaticity is lost. As a result, rather than forming o-carborane-fused pyrazoles, the synthesis yielded o-carborane-fused pyrazolines, which are non-aromatic. The limited overlap between the π molecular orbitals (MOs) of the planar heterocycle and the n + 1 MOs of the carborane prevents significant electronic delocalization between the two fused components. This contrasts with the fusion of pyrazole and benzene to form indazole, where both rings maintain their 2D aromaticity. Our findings demonstrate that the peripheral σ-aromaticity of carborane and the π-aromaticity of the heterocycle are orthogonal, making a true 3D/2D aromatic system unachievable. The carborane is highly aromatic, generating highly negative NICS values (−25 to −30 ppm). We have observed that these high NICS values extend to fused rings, leading to incorrect estimations of aromaticity. Therefore, relying solely on NICS can be misleading, and other computational indicators, along with experimental or structural data, should be used to accurately assess aromaticity.

Gazieva G.A., Evdokimenkova Y.B., Soboleva N.O.

Abdulaziz F., Jamal A., Faizi M.S., Peedikakkal A.M., Hussain A., Al-Busaidi I.J., Dege N., Alenezi K.M., Haque A.

Nitrogen-containing heterocycles play a crucial role in drug design and discovery. Pyrazole derivatives have garnered attention due to their unique and intriguing features. This study investigates the structure and properties of two pyrazole-4-carbaldehyde derivatives (3 and 4). We report the single crystal structure of one of the intermediate compounds, 1-(1-(4-bromophenyl)ethylidene)-2-(2-chlorophenyl)hydrazine (2), and a final compound, 1-(2-chlorophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde (3). We analysed the non-covalent interactions within the crystal using Hirshfeld surface analysis and 2D fingerprint plots. We also used density functional theory (DFT) at the B3LYP/6–311G(d,p) level to optimise the structures and understand electronic properties, transitions and vibrational frequencies. To predict the anti-inflammatory potentials and selectivity of 3 and 4, molecular docking and molecular dynamics (MD) simulation studies against COX1 (PDB ID: 1EQG) and COX2 (PDB ID: 1CX2) receptors were conducted. Overall, this study sheds light on the structural, chemical, and biochemical features of two pyrazole-4-carbaldehyde derivatives.

Odeh D.M., Odeh M.M., Hafez T.S., Hassan A.S.

Heterocyclic compounds, especially those containing the pyrazole moiety, are highly significant in organic chemistry and possess remarkable and diverse biological properties. The 5-aminopyrazole derivatives are key starting materials for the synthesis of numerous bioactive compounds such as pyrazolopyridine, pyrazolopyrimidine, pyrazoloquinazoline, and pyrazolotriazine derivatives. Many compounds inspired by the 5-aminopyrazole derivatives possess a wide spectrum of biological activities and medicinal applications such as antioxidants, anticancer agents, enzyme inhibitors, antimicrobials, and anti-tuberculosis activities. This review summarizes the recently reported synthesis methods and biological activities of fused pyrazole and pyrazole-based derivatives based on 5-aminopyrazole compounds within the last 5 years (2020 to present). One of the important goals of this review is to illustrate future strategies for the design, development, and utilization of pyrazole products as potent drugs.

Bavishi A., Vala H., Radadiya A., Swami S., Thakrar S., Sarkar D., Shah A.

AbstractThis study reports the synthesis, characterization, and docking analysis of 21 novel compounds, including asymmetric dihydropyridines (4a–4o) and chalcones (6a–6f), derived from coupling 1,3‐(substituted)‐diphenyl‐1H‐pyrazole‐4‐carbaldehyde with active methylene compounds and 3‐acetyl‐4‐hydroxycoumarin, respectively. Structural confirmation was achieved through 1H and 13C NMR, IR, and mass spectrometry. Biological screening against mycobacterium tuberculosis H37Ra identified compounds 4b, 4d, 4f, 4g , 4i, 4k, 6e, and 6f as significant antitubercular agents. Antibacterial evaluation at 30 µg/mL showed selective inhibition of gram‐positive bacteria, with compound 6e active against Staphylococcus aureus and Bacillus subtilis, and 6f showing activity specifically against S. aureus. Docking studies indicated effective InhA binding (ΔG = −3.52 to −7.27 kcal/mol) and inhibition constants ranging from 2640 to 4.71 µM. Key binding interactions with residues TYR156 and ILE192 were observed, enhancing affinity, particularly for compound 6e. The SAR analysis emphasized the enhanced antitubercular potency of 2‐chloroacetyl and 2,4‐dichlorobenzoyl substitutions in the dihydropyridine series, while in the chalcone series, specific substitutions, such as the 3‐nitro substitution in 6e, enhance antitubercular and antimicrobial potency due to stronger hydrogen bonding and increased polar surface area, while the 4‐fluoro substitution in 6f confers selective activity against S. aureus.

Dong M., Wang C., Li L., Chen Y., Zeng J., Lv J., Liao J., Wang H., Gu S., Chen F.

AbstractA tandem system comprising cyclization reaction of diazo compound and cross‐coupling of the other one has been developed. This provides facile access to pyrazoles and there derivatives with high levels of regioselectivity in moderate to good yields. This process uniquely utilizes CF3CH2OTf both as a solvent and a catalyst under mild conditions. Mechanistic studies provide insights into the intricate sequence involved in the formation of pyrazoles and further selective cross‐coupling with another diazo compound.

Shi H., Sun F., Wu J., He J., Sun Y., Du Y.

AbstractThe reaction of a series of pyrazole derivatives with DMSO/ ‐DMSO‐d6 and thionyl chloride conveniently afforded the 4‐thiolated pyrazole products under metal‐free conditions. This C−H functionalization/elelctrophilic thiolation process is postulated to involve the in situ generation of the electrophilic hypochlorothioite via interrupted Pummerer reaction, followed with the electrophilic methylthiolation of pyrazole skeleton.