6-Phenyl-1H-imidazo[4,5-c]pyridine-4-carbonitrile as cathepsin S inhibitors

6-Phenyl-1H-imidazo[4,5-c]pyridine-4-carbonitrile analogues were identified as potent and selective cathepsin S inhibitor against both purified enzyme and in human JY cell based cellular assays. This core has a very stable thio-trapping nitrile war-head in comparison with the well reported pyrimidine-2-carbonitrile cysteine cathepsin inhibitors. Compound 47 is also very potent in in vivo mouse spleenic Lip10 accumulation assays.     

Selective inhibition of human cathepsin S by 2,4,6-trisubstituted 1,3,5-triazine analogs

We report herein the synthesis and biological evaluation of a new series of 2,4,6-trisubstituted 1,3,5-triazines as reversible inhibitors of human cysteine cathepsins. The desired products bearing morpholine and N-Boc piperidine, respectively, were obtained in three to four steps from commercially available trichlorotriazine. Seventeen hitherto unknown compounds were evaluated in vitro against various cathepsins for their inhibitory properties. Among them, compound 7c (4-(morpholin-4-yl)-6-[4-(trifluoromethoxy)anilino]-1,3,5-triazine-2-carbonitrile) was identified as the most potent and selective inhibitor of cathepsin S (Ki??=??2??±??0.3?nM). Also 7c impaired the autocatalytic maturation of procathepsin S. Molecular docking studies support that 7c bound within the active site of cathepsin S, by interacting with Gly23, Cys25 and Trp26 (S1 subsite), with Asn67, Gly69 and Phe70 (S2 subsite) and with Gln19 (S1′ pocket).     

1,3-Benzoxazole-4-carbonitrile as a novel antifungal scaffold of β-1,6-glucan synthesis inhibitors

Synthesis and in vitro antifungal evaluations of 1,3-benzoxazole-7-carbonitrile 3, 1,3-benzoxazole-4-carbonitrile 4, benzofuran 5, benzoxazine 7, and benzimidazole 8 were reported. Among them, 1,3-benzoxazole-4-carbonitrile was found to be a superior scaffold structure with moderate growth inhibition against Candida species. 1,3-Benzoxazole-4-carbonitrile 6 showed potent activity against Candida species compared to 5-desmethyl compound 4 and triazolopyridine 2. Compound 6 was efficiently prepared from versatile intermediate 24, which possessed six different substituents on the benzene ring. Conversion of benzene 24 into various 1,3-benzoxazole derivatives such as 2-aliphatic 34, 2-amino 35, and lactone 38 was demonstrated.     

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design,synthesis and biochemical characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (K_i = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (K_i = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.     

Synthesis of proline analogues as potent and selective cathepsin S inhibitors

Cathepsin S is a potential target of autoimmune disease. A series of proline derived compounds were synthesized and evaluated as cathepsin S inhibitors. We discovered potent cathepsin S inhibitors through structure–activity relationship studies of proline analogues. In particular, compound 19-(S) showed promising in vitro/vivo pharmacological activities and properties as a selective cathepsin S inhibitor.     

Synthesis and biological evaluation of 4-styrylcoumarin derivatives as inhibitors of TNF-α and IL-6 with anti-tubercular activity

A series of 4-styrylcoumarin have been synthesized by Knoevenagel condensation between substituted 4-methylcoumarin-3-carbonitrile and different heterocyclic or aromatic aldehydes. 4-Methylcoumarin-3-carbonitrile has been synthesized by the base catalyzed reaction between substituted 2-hydroxyacetophenone and ethyl cyanoacetate. The structures of the newly synthesized compounds were confirmed by ¹H NMR, IR and mass spectral analysis. All the compounds were evaluated for their anti-inflammatory activity (against TNF-α and IL-6) and anti-tubercular activity. Compounds 6a, 6h and 6j exhibited promising activity against IL-6 with 72-87% inhibition and compound 6v showed potent activity against TNF-α with 73% inhibition at 10 μM concentration. Whereas compounds 6n, 6o, 6r and 6u showed very good anti-tubercular activity against Mycobacterium tuberculosis H37Rv strain at <6.25 μM.     

Discovery of novel cathepsin inhibitors with potent anti-metastatic effects in breast cancer cells

It is still challenging to determine the potential targets of natural products, which is essential for further drug research and development. Due to its novel mechanism of action of inducing autophagy effects in breast cancer cells, asperphenamate has received our considerable attention. However, its unknown target inevitably impedes further study. In our previous work, the target enzyme of asperphenamate was predicted as cathepsin by the natural product consensus pharmacophore strategy. Then, asperphenamate and its three derivatives were chosen to study in detail by molecular docking calculations with AutoDock 4 suite. The docking results showed the three derivatives interacted more tightly with either cathepsin L or cathepsin S than with asperphenamate. The ortho-benzyloxyl phenylacetyl derivative 1 and p-toluenesulfonyl derivative 3 showed similar interactions with cathepsin L and adopted a better geometric shape within the binding pocket than did the N-CBZ-piperidyl analog 2. On the other hand, compound 2 formed more hydrogen bonds than 1 and 3 to make it tightly bind within cathepsin S. The cathepsin inhibitory activity assay verified the molecular simulation results. Compound 2 was remarkably less active than 1 and 3 against cathepsin L. However, compound 2 showed the strongest potency against cathepsin S with IC₅₀ of 13.12 ± 0.29 μM. Considering that cathepsin S plays a vital role in the process of metastasis in breast cancer cells, the inhibitory effect of 2 on migration and invasion was further studied in human breast cancer MDA-MB-231 cells by wound healing and transwell chamber assays. The results illustrated that 2 exhibited an apparent inhibitory ability to the metastasis of MDA-MB-231 cells. Next, 2 will be chosen as a lead compound to develop novel double functional chemotherapeutic agents with both novel mechanisms of action against apoptosis-resistant cancer cells, such as inducing autophagy and inhibiting cancer metastasis.     

Structural basis for reversible and irreversible inhibition of human cathepsin L by their respective dipeptidyl glyoxal and diazomethylketone inhibitors

Cathepsin L plays a key role in many pathophysiological conditions including rheumatoid arthritis, tumor invasion and metastasis, bone resorption and remodeling. Here we report the crystal structures of two analogous dipeptidyl inhibitor complexes which inhibit human cathepsin L in reversible and irreversible modes, respectively. To-date, there are no crystal structure reports of complexes of proteases with their glyoxal inhibitors or complexes of cathepsin L and their diazomethylketone inhibitors. These two inhibitors – inhibitor 1, an α-keto-β-aldehyde and inhibitor 2, a diazomethylketone, have different groups in the S1 subsite. Inhibitor 1 [Z-Phe-Tyr (OBut)-COCHO], with a K_i of 0.6 nM, is the most potent, reversible, synthetic peptidyl inhibitor of cathepsin L reported to-date. The structure of the inhibitor 1 complex was refined up to 2.2 Å resolution. The structure of the complex of the inhibitor 2 [Z-Phe-Tyr (t-Bu)-diazomethylketone], an irreversible inhibitor that can inactivate cathepsin L at μM concentrations, was refined up to 1.76 Å resolution. These two inhibitors have substrate-like interactions with the active site cysteine (Cys25). Inhibitor 1 forms a tetrahedral hemithioacetal adduct, whereas the inhibitor 2 forms a thioester with Cys25. The inhibitor 1 β-aldehyde group is shown to make a hydrogen bond with catalytic His163, whereas the ketone carbonyl oxygen of the inhibitor 2 interacts with the oxyanion hole. tert-Butyl groups of both inhibitors are found to make several non-polar contacts with S′ subsite residues of cathepsin L. These studies, combined with other complex structures of cathepsin L, reveal the structural basis for their potency and selectivity.     

1-Methyl-1H-pyrrole-2-carbonitrile containing tetrahydronaphthalene derivatives as non-steroidal progesterone receptor antagonists

Non-steroidal 1-methyl-1H-pyrrole-2-carbonitrile containing tetrahydronaphthalenes and acyclic derivatives were evaluated as novel series of progesterone receptor (PR) antagonists using the T47D cell alkaline phosphatase assay. Moderate to potent PR antagonists were achieved with these scaffolds. Several compounds (e.g., 15 and 20) demonstrated low nanomolar PR antagonist potency and good selectivity versus other steroid receptors.     

The alkenic unreactivity of mono- and bi-cyclic derivatives of 3,6-dihydro-2-(methylthio)-2H-thiopyran S,S,S′,S′-tetraoxide

The inertness of the alkenic bond towards electrophilic additions in 3-exocyano-3-(methylthio)-2-thiabicyclo[2.2.1]hept-5-ene S,S,S′,S′-tetraoxide (5), 3,6-dihydro-2-(methylthio)-2H-thiopyran-2-carbonitrile S,S,S′,S′-tetraoxide (3), and 2-(acetamidomethyl)-3,6-dihydro-2-(methylthio)-2H-thiopyran S,S,S′,S′-tetraoxide (4) is attributed to the “supra-annular effect” and field effects. Conformational analysis of a pentadeuterated derivative of 4 (10) is reported. On the basis of the 220-MHz ¹H n.m.r.-spectral data of 10, the compound was concluded to adopt the ⁰H₂ conformation in chloroform solution.     

‘3 + 1’ mixed-ligand oxorhenium(V) complexes and their inhibition of the cysteine proteases cathepsin B and cathepsin K

The synthesis of several new oxorhenium(V) complexes containing the ‘3 + 1’ mixed-ligand donor set, ReO(SXS)(SR) (where X = S, O, N(R′); R = alkyl, aryl, heterocylce; R′ = H, alkyl, aryl), is described. The X-ray structure for four of these complexes ReO(SN(Ph)S)(SPh) (6), ReO(SN(CH₂CH₂NMe₂)S)(SPhOMe-p) (10), ReO(SOS)(SPh) (29) and ReO(SOS)(SPhNO₂-p) (30) was determined. The inhibitory activity of all of the oxorhenium(V) complexes reported herein was evaluated against the cysteine proteases cathepsin B and K in vitro. Compound 25, ReO(SSS)(S-4py) · HCl, was the best inhibitor of the series against cathepsin B with an IC₅₀ of 10 nM. Several of the complexes exhibited specificity for cathepsin B over K, suggesting that oxorhenium(V) complexes can be designed to be enzyme specific. The results described in this paper show that the oxorhenium(V) ‘3 + 1’ complexes are potent inhibitors of cathepsin B and K, constituting promising potential for the treatment of cancer and osteoporosis, respectively.     

Synthesis and kinetic evaluation of ethyl acrylate and vinyl sulfone derived inhibitors for human cysteine cathepsins

A series of inhibitors targeting human cathepsins have been designed and synthesized following a combinatorial approach. The compounds bear an α,β-unsaturated phenyl vinyl sulfone or ethyl acrylate warhead and a peptidomimetic portion aligned to the non-primed binding region. Biochemical evaluation toward four human cathepsins was carried out and the kinetic characterization confirmed an irreversible mode of inhibition. Compound 6c combining the most advantageous building blocks for cathepsin S inhibition was identified as a potent cathepsin S inactivator exhibiting a second-order rate constant of 30600?M^?1?s^?1.     

Synthesis and matched molecular pair analysis of covalent reversible inhibitors of the cysteine protease CPB

Cysteine protease B (CPB) can be targeted by reversible covalent inhibitors that could serve as antileishmanial compounds. Here, sixteen dipeptidyl nitrile derivatives were synthesized, tested against CPB, and analyzed using matched molecular pairs to determine the effects of stereochemistry and p-phenyl substitution on enzyme inhibition. The compound (S)-2-(((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)amino)-N-(1-cyanocyclopropyl)-3-phenylpropanamide (5) was the most potent CPB inhibitor (pKi = 6.82), which was also selective for human cathepsin B (pKi < 5). The inversion of the stereochemistry from S to R was more detrimental to potency when placed at the P2 position than at P3. The p-Br derivatives were more potent than the p-CH₃ and p-OCH₃ derivatives, probably due to intermolecular interactions with the S3 subsite.     

Cytotoxic palladium complexes of bioreductive quinoxaline N1,N4-dioxide prodrugs

Four new palladium(II) complexes with the formula Pd(L)₂, where L are quinoxaline-2-carbonitrile N¹,N⁴-dioxide derivatives, were synthesized as a contribution to the chemistry and pharmacology of metal compounds with this class of pharmacologically interesting bioreductive prodrugs. Compounds were characterized by elemental, conductometric and thermogravimetric analyses, fast atom bombardment mass spectrometry (FAB-MS) and electronic, Fourier transform infrared (FTIR) and ¹H-nuclear magnetic resonance spectroscopies. The complexes were subjected to cytotoxic evaluation on V79 cells in hypoxic and aerobic conditions. In addition, a preliminary study on interaction with plasmid DNA in normoxia was performed. Complexes showed different in vitro biological behavior depending on the nature of the substituent on the quinoxaline ring. Pd(L1)₂ and Pd(L2)₂, where L1 is 3-aminoquinoxaline-2-carbonitrile N¹,N⁴-dioxide and L2 is 3-amino-6(7)-methylquinoxaline-2-carbonitrile N¹,N⁴-dioxide, showed non selective cytotoxicity, being cytotoxic either in hypoxic or in aerobic conditions. On the other hand, Pd(L3)₂, where L3 is 3-amino-6(7)-chloroquinoxaline-2-carbonitrile N¹,N⁴-dioxide, resulted in vitro more potent cytotoxin in hypoxia (P = 5.0 μM) than the corresponding free ligand (P = 9.0 μM) and tirapazamine (P = 30.0 μM), the first bioreductive cytotoxic drug introduced into clinical trials. In addition, it showed a very good selective cytotoxicity in hypoxic conditions, being non-cytotoxic in normoxia. Its hypoxic cytotoxicity relationship value, HCR, was of the same order than those of other hypoxia selective cytotoxins (i.e., Mitomycine C, Misonidazole and the N-oxide RB90740). Interaction of the complexes with plasmid DNA in normoxia showed dose dependent ability to relax the negative supercoiled forms via different mechanisms. Pd(L2)₂ introduced a scission event in supercoiled DNA yielding the circular relaxed form. Meanwhile, both Pd(L1)₂ and Pd(L3)₂ produced the loss of negative supercoils rendering a family of topoisomers with reduced electrophoretic mobility. Pd(L3)₂ showed a more marked effect than Pd(L1)₂. Indeed, for the highest doses assayed, Pd(L3)₂ was even able to introduce positive supercoils on the plasmid DNA.     

Selective inhibitors of tumor progression loci-2 (Tpl2) kinase with potent inhibition of TNF-α production in human whole blood

Tpl2 (cot/MAP3K8) is an upstream kinase of MEK in the ERK pathway. It plays an important role in Tumor Necrosis Factor-α (TNF-α) production and signaling. We have discovered that 8-halo-4-(3-chloro-4-fluoro-phenylamino)-6-[(1H-[1,2,3]triazol-4-ylmethyl)-amino]-quinoline-3-carbonitriles (4) are potent inhibitors of this enzyme. In order to improve the inhibition of TNF-α production in LPS-stimulated human blood, a series of analogs with a variety of substitutions around the triazole moiety were studied. We found that a cyclic amine group appended to the triazole ring could considerably enhance potency, aqueous solubility, and cell membrane permeability. Optimization of these cyclic amine groups led to the identification of 8-chloro-4-(3-chloro-4-fluorophenylamino)-6-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methylamino)quinoline-3-carbonitrile (34). In a LPS-stimulated rat inflammation model, compound 34 showed good efficacy in inhibiting TNF-α production.     

Ligand based design and synthesis of pyrazole based derivatives as selective COX-2 inhibitors

The design and synthesis of novel pyrazole based derivatives has been carried out using the ligand based approach like pharmacophore and QSAR modelling of reported pyrazoles from the available literature to investigate the chemical features that are essential for the design of selective and potent COX-2 inhibitors. Both pharmacophore and QSAR models with good statistical parameters were selected for the design of the lead molecule. Also by exploiting the chemical structures of selective and marketed COX-2 inhibitors, celecoxib and SC-558 were used in designing the molecules which are used in the treatment of inflammation and related disorders. The therapeutic action of the Non-Steroidal Anti-inflammatory Agents (NSAIDs) is based primarily on the COX-2 inhibition. With this background we have synthesized some azomethine derivatives of 3-methyl-1-substituted-4-phenyl-6-[{(1E)-phenylmethylene}amino]-1,4-dihydro pyrano[2,3-c]pyrazole-5-carbonitrile 6(a-o) and were characterized by ¹HNMR, ¹³CNMR and Mass spectral techniques. All the synthesized pyrazole derivatives were tested for in vitro membrane stability property in both COX-1 & COX-2 inhibition studies and in vivo anti-inflammatory activity by carrageenan induced rat paw edema model. Among them, compound 6k showed very good activity by in vivo anti-inflammatory activity with 0.8575 mmol/kg as ED₅₀. Similarly compounds 6m, 6o, 6i and 6h exhibited comparable anti-inflammatory activity to standard drugs. Also the active compounds were further screened for ulcerogenic activity and were found be safer with less ulcer index compared to the marketed drugs like aspirin, ibuprofen and celecoxib.     

SAR studies of differently functionalized chalcones based hydrazones and their cyclized derivatives as inhibitors of mammalian cathepsin B and cathepsin H

Cathepsins have emerged as potential drug targets for melanoma therapy and engrossed attention of researchers for development and evaluation of cysteine cathepsin inhibitors as cancer therapeutics. In this direction, we have designed, synthesized, and assayed in vitro a small library of 30 low molecular weight functionalized analogs of chalcone hydrazones for evaluating structure–activity relationship aspects and inhibitory potency against cathepsin B and H. The maximum inhibitory effect was exerted by chalcone hydrazones, which are open chain analogues followed by their cyclized derivatives, pyrazolines and pyrazoles. All the synthesized compounds were established as reversible inhibitors of these enzymes. Cathepsin B was selectively inhibited by the compounds in each series. Compounds 1d, 2d and 4d were recognized as most potent inhibitors of cathepsin B in this study with K_i values of 0.042 μM, 0.053 μM and 0.131 μM whereas 1b (K_i = 1.111 μM), 2b (K_i = 1.174 μM) and 4b (K_i = 1.562 μM) inhibited cathepsin H activity effectively. And, preeminent cathepsin B inhibitors were –NO₂ functionalized however, –Cl substituted moieties were the most persuasive inhibitors for cathepsin H among all the designed compounds. Molecular docking studies performed using iGemdock provided valuable insights.     

Discovery of a novel cathepsin inhibitor with dual autophagy-inducing and metastasis-inhibiting effects on breast cancer cells

Drug resistance and cancer cells metastasis have been the leading causes of chemotherapy failure and cancer-associated death in breast cancer patients. In present, various active molecules either exhibiting novel mechanism of action such as inducing autophagy or inhibiting metastasis have been developed to address these problems. However, the compounds exhibiting such dual functions have rarely been described. Previous work in our group showed that TSA, as a synthetic analog of asperphenamate, induced autophagic cell death in breast cancer cells instead of apoptosis. Furthermore, the target enzyme of TSA was predicted to be cathepin L (Cat L) by natural product consensus pharmacophore strategy. Accumulated evidences have shown that cathepsins are closely associated with migration and invasion of breast cancer cells. It seemed likely that TSA-like molecules may possess the dual functions of inducing autophagy and inhibiting metastasis. Therefore, sixty optically active derivatives were firstly designed and synthesized by replacing the A-ring moiety of TSA with other substituted-phenyl sulfonyl groups. Further cathepsin inhibitory activity assay showed that (S, S) and (S, R) isomers displayed no activity against four kinds of cathepsins (L, S, K, B), while all derivatives tested were inactive toward K and B subtypes. Compound 6a with meta-bromo substituent displayed the greatest inhibitory activity, and its inhibitory capability against Cat L and S was 3.9 and 11.5-fold more potent than that of TSA, respectively. Molecular docking also exhibited that 6a formed more hydrogen bonds or π-π contacts with Cat L or S than TSA. In order to determine whether 6a could play dual roles, its anti-cancer mechanism was further investigated. On the one hand, MDC staining experiment and western blotting analysis validated that 6a can induce autophagy in MDA-MB-231 cells. On the other hand, its metastatic inhibitory ability was also confirmed by wound healing and transwell chamber experiment.     

3-Thiomorpholin-8-oxo-8H-acenaphtho [1,2-b] pyrrole-9-carbonitrile (S1) derivatives as pan-Bcl-2-inhibitors of Bcl-2, Bcl-xL and Mcl-1

Based on the binding mode of our previously discovered dual inhibitor of Bcl-2 and Mcl-1, 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (3, S1), a library of 9-substituted 3 derivatives was synthesized to further probe the p4 pocket of the two targets. By NMR, structure–activity relationship study, and site-directed mutation, compound 6d (3-(4-aminophenylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-3-phenyl)propylamine) was identified to span p2–p4 pockets of Mcl-1, Bcl-2 and Bcl-x_L, and then exhibited 9- to 35-fold better affinity to the three targets than 3 (IC₅₀ = 10, 20 and 18 nM, respectively), which led to greater activity in induction of apoptosis in multiple cancer cell lines. Different contribution of p4 pocket to binding Bcl-2 and Mcl-1 was also investigated by plotting the potency and the HAC of the derivatives.     

Di-substituted pyridinyl aminohydantoins as potent and highly selective human β-secretase (BACE1) inhibitors

The identification of highly selective small molecule di-substituted pyridinyl aminohydantoins as β-secretase inhibitors is reported. The more potent and selective analogs demonstrate low nanomolar potency for the BACE1 enzyme as measured in a FRET assay, and exhibit comparable activity in a cell-based (ELISA) assay. In addition, these pyridine-aminohydantoins are highly selectivity (>500×) against the other structurally related aspartyl proteases BACE2, cathepsin D, pepsin and renin.Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on the previously reported aminohydantoin 3a. We have taken advantage of the amino acid difference between the BACE1 and BACE2 at the S2′ pocket (BACE1 Pro₇₀ changed to BACE2 Lys₈₆) to build ligands with >500-fold selectivity against BACE2. The addition of large substituents on the targeted ligand at the vicinity of this aberration has generated a steric conflict between the ligand and these two proteins, thus impacting the ligand’s affinity and selectivity. These ligands have also shown an exceptional selectivity against cathepsin D (>5000-fold) as well as the other aspartyl proteases mentioned. One of the more potent compounds (S)-39 displayed an IC₅₀ value for BACE1 of 10 nM, and exhibited cellular activity with an EC₅₀ value of 130 nM in the ELISA assay.