Novel Class Of Inhibitors Research Articles

A novel class of inhibitors for human steroid 5 alpha-reductase: phenoxybenzoic acid derivatives. I.

In a search for novel nonsteroidal inhibitors of human prostatic 5 alpha-reductase, we found a new series of phenoxybenzoic acid derivatives to be potent human prostatic 5 alpha-reductase inhibitors. Among them, 4-(biphenyl-4-yloxy)benzoic acid derivatives (2n, YM-31758), 2o and 2s showed more potent inhibitory activities than finasteride with IC50 values of 0.87, 0.67 and 0.56 nM, respectively. The optimized structures for the phenoxybenzoic acid derivatives 2d-2i were calculated by molecular modeling analysis, and the favorable distance between the carbon of the carboxyl group and the centroid of the phenyl group (benzene ring C) was found to be in the 9-11 A range.

2-Amino-3-substituted-6-[(E)-1-phenyl-2-(N-methylcarbamoyl)vinyl]imid azo[1,2-a]pyridines as a novel class of inhibitors of human rhinovirus: stereospecific synthesis and antiviral activity.

A series of 2-amino-3-substituted-6-[(E)-1-phenyl-2-(N-methylcarbamoyl)vinyl]+ ++imid azo[1,2-a]pyridines 1a-i, structurally related to Enviroxime and its analogous benzimidazoles, was designed and prepared for testing as antirhinovirus agents. The imidazo ring in this class of compounds was constructed starting from the aminopyridine after tosylation and subsequent treatment with the appropriate acetamides. The key steps in the synthesis include the development and use of a new Horner-Emmons reagent for the direct incorporation of methyl vinylcarboxamide. The reaction was stereospecific in the substrates 5a-f leading exclusively to the desired E-isomer and avoiding the use of reverse-phase preparative HPLC for the separation of both possible isomers before antiviral activity evaluation. The isopropylsulfonyl group, known as the best substituent at the 1-position in the benzimidazole SAR in terms of activity, was introduced in this new series of imidazo[1,2-a]pyridines via halogen-metal exchange and subsequent treatment with isopropyl isopropanethiolsulfonate. Compounds 1a-i were evaluated in plaque reduction assay and in a cytopathic effect assay. Compounds 1b-d,h exhibited a strong antirhinovirus activity, and no apparent cellular toxicity was visible. The substitution at the 3-position was required for activity. Surprisingly the isopropylsulfonyl in this family of compounds did not enhance the activity as in the case of benzimidazoles. Instead, compound 1i was 4 times less active than its phenyl and sulfide partners. The chemistry as well as the biological evaluation are discussed.

A New Structural Class of Proteasome Inhibitors that Prevent NF-κB Activation

The multicatalytic proteinase or proteasome is a highly conserved cellular structure that is responsible for the ATP-dependent proteolysis of many proteins involved in important regulatory cellular processes. We have identified a novel class of inhibitors of the chymotrypsin-like proteolytic activity of the 20S proteasome that exhibit ic 50 values ranging from 0.1 to 0.5 μg/mL (0.1 to 1 μM). In cell proliferation assays, these compounds inhibit growth with an ic 50 ranging from 5 to 10 μg/mL (10–20 μM). A representative member of this class of inhibitors was tested in other biological assays. CVT-634 (5-methoxy-1-indanone-3-acetyl-leu- d-leu-1-indanylamide) prevented lipopolysaccharide (LPS), tumor necrosis factor (TNF)-, and phorbol ester-induced activation of nuclear factor κB (NF-κB) in vitro by preventing signal-induced degradation of IκB-α. In these studies, the IκB-α that accumulated was hyperphosphorylated, indicating that CVT-634 did not inhibit IκB-α kinase, the enzyme responsible for signal-induced phosphorylation of IκB-α. In vivo studies indicated that CVT-634 prevented LPS-induced TNF synthesis in a murine macrophage cell line. In addition, in mice pretreated with CVT-634 at 25 and 50 mg/kg and subsequently treated with LPS, serum TNF levels were significantly lower (225 ± 59 and 83 ± 41 pg/mL, respectively) than in those mice that were treated only with LPS (865 ± 282 pg/mL). These studies suggest that specific inhibition of the chymotrypsin-like activity of the proteasome is sufficient to prevent signal-induced NF-κB activation and that the proteasome is a novel target for the identification of agents that may be useful in the treatment of diseases whose etiology is dependent upon the activation of NF-κB.

Design and Synthesis of Novel Imidazole-Substituted Dipeptide Amides as Potent and Selective Inhibitors of Candida albicans MyristoylCoA:Protein N-Myristoyltransferase and Identification of Related Tripeptide Inhibitors with Mechanism-Based Antifungal Activity

A new class of antifungal agents has been discovered which exert their activity by blockade of myristoylCoA: protein N-myristoyltransferase (NMT; EC 2.1.3.97). Genetic experiments have established that NMT is needed to maintain the viability of Candida albicans and Cryptococcus neoformans,the two principal causes of systemic fungal infections in immunocompromised humans. Beginning with a weak octapeptide inhibitor ALYASKLS-NH2 (2, Ki = 15.3 +/- 6.4 microM), a series of imidazole-substituted Ser-Lys dipeptide amides have been designed and synthesized as potent and selective inhibitors of Candida albicans NMT. The strategy that led to these inhibitors evolved from the identification of those functional groups in the high-affinity octapeptide substrate GLYASKLS-NH2 1a necessary for tight binding, truncation of the C-terminus, replacement of the four amino acids at the N-terminus by a spacer group, and substitution of the glycine amino group with an N-linked 2-methylimidazole moiety. Initial structure-activity studies led to the identification of 31 as a potent and selective peptidomimetic inhibitor with an IC50 of 56 nM and 250-fold selectivity versus human NMT. 2-Methylimidazole as the N-terminal amine replacement in combination with a 4-substituted phenacetyl moiety imparts remarkable potency and selectivity to this novel class of inhibitors. The (S,S) stereochemistry of serine and lysine residues is critical for the inhibitory activity, since the (R,R) enantiomer 40 is 10(3)-fold less active than the (S,S) isomer 31. The inhibitory profile exhibited by this new class of NMT ligands is a function of the pKa of the imidazole substituent as illustrated by the benzimidazole analog 35 which is about 10-fold less potent than 31. The measured pKa (7.1 +/- 0.5) of 2-methylimidazole in 31 is comparable with the estimated pKa (approximately 8.0) of the glycyl residue in the high-affinity substrate 1a. Groups bulkier than methyl, such as ethyl, isopropyl, or iodo, at the imidazole 2-position have a detrimental effect on potency. Further refinement of 31 by grafting an alpha-methyl group at the benzylic position adjacent to the serine residue led to 61 with an IC50 of 40 nM. Subsequent chiral chromatography of 61 culminated in the discovery of the most potent Candida NMT inhibitor 61a reported to date with an IC50 of 20 nM and 400-fold selectivity versus the human enzyme. Both 31 and 61a are competitive inhibitors of Candida NMT with respect to the octapeptide substrate GNAASARR-NH2 with Ki(app) = 30 and 27 nM, respectively. The potency and selectivity displayed by these inhibitors are dependent upon the size and orientation of the alpha-substituent. An alpha-methyl group with the R configuration corresponding to the (S)-methyl-4-alanine in 2 confers maximum potency and selectivity. Structural modification of 31 and 61 by appending an (S)-carboxyl group beta to the cyclohexyl moiety provided the less potent tripeptide inhibitors 73a and 73b with an IC50 of 1.45 +/- 0.08 and 0.38 +/- 0.03 microM, respectively. However, these tripeptides (73a and 73b) exhibited a pronounced selectivity of 560- and 2200-fold versus the human NMT. More importantly 73a displayed fungistatic activity against C albicans with an EC50 of 51 +/- 17 microM in cell culture. Compound 73b also exhibited a similar antifungal activity. An Arf protein gel mobility shift assay for monitoring intracellular myristoylation revealed that a single dose of 200 microM of 73a or 73b produced < 50% reduction in Arf N-myristoylation, after 24 and 48 h, consistent with their fungistatic rather than fungicidal activity. In contrast, the enantiomer 73d which had an IC50 > 1000 microM against C. albicans NMT did not exhibit antifungal activity and produced no detectable reduction in Arf N-myristoylation in cultures of C. albicans. These studies confirm that the observed antifungal activity of 73a and 73b is due to the attenuation of NMT activity and that NMT represents an attractive tar

Gem-dialkyl succinic acids: a novel class of inhibitors for carboxypeptidases.

gem-Dimethylsuccinic acid and its higher homolog, 2-methyl-2-ethylsuccinic acid (MESA) are highly potent inhibitors of both carboxypeptidase A (CPA) and B. The inhibition constant of MESA for CPA (0.11 microM for the racemic mixture) is remarkable considering the relatively simple structure of the compound. The molecular feature which is crucial for high affinity binding to both carboxypeptidases appears to be the nonpolar gem-dialkyl locus. The structure of the complex between MESA and CPA has been determined by X-ray crystallography to 2.0 A resolution and shows the R enantiomer of the inhibitor to be bound in a generally substrate-like manner. The carboxymethyl group is coordinated to the Zn ion in the active site, and the gem-dialkyl locus corresponds in position to the alpha-carbon of the C-terminal amino acid in a peptide substrate. The methyl group of the inhibitor occupies a cavity in the enzyme which is apparently not filled upon substrate-binding. We postulate that this cavity (the alpha-methyl hole) is designed to allow the proximal Glu-270 residue to undergo a critical movement during catalysis. The hydrophobic nature of the above cavity may play a role in modulating the reactivity of this residue. These results suggest that similar cenophilic(empty-loving) inhibitors may be found for other enzymes.

19-nor-10-azasteroids: a novel class of inhibitors for human steroid 5alpha-reductases 1 and 2.

Steroid 5alpha-reductase is a system of two isozymes (5alphaR-1 and 5alphaR-2) which catalyzes the NADPH-dependent reduction of testosterone to dihydrotestosterone in many androgen sensitive tissues and which is related to several human endocrine diseases such as benign prostatic hyperplasia (BPH), prostatic cancer, acne, alopecia, pattern baldness in men and hirsutism in women. The discovery of new potent and selective 5alphaR inhibitors is thus of great interest for pharmaceutical treatment of these diseases. The synthesis of a novel class of inhibitors for human 5alphaR-1 and 5alphaR-2, having the 19-nor-10-azasteroid skeleton, is described. The inhibitory potency of the 19-nor-10-azasteroids was determined in homogenates of human hypertrophic prostates toward 5alphaR-2 and in DU-145 human prostatic adenocarcinoma cells toward 5alphaR-1, in comparison with finasteride (IC50 = 3 nM for 5alphaR-2 and approximately 42 nM for 5alphaR-1), a drug which is currently used for BPH treatment. The inhibition potency was dependent on the type of substituent at position 17 and on the presence and position of the unsaturation in the A and C rings. delta9(11)-19-Nor-10-azaandrost-4-ene-3,17-dione (or 10-azaestra-4,9(11)-diene-3,17-dione) (4a) and 19-nor-10-azaandrost-4-ene-3,17-dione (5) were weak inhibitors of 5alphaR-2 (IC50 = 4.6 and 4.4 microM, respectively) but more potent inhibitors of 5alphaR-1 (IC50 = 263 and 299 nM, respectively), whereas 19-nor-10-aza-5alpha-androstane-3,17-dione (7) was inactive for both the isoenzymes. The best result was achieved with the 9:1 mixture of delta9(11)- and delta8(9)-17beta-(N-tert-butylcarbamoyl)-19-nor-10-aza-4- androsten-3-one (10a,b) which was a good inhibitor of 5alphaR-1 and 5alphaR-2 (IC50 = 127 and 122 nM, respectively), with a potency very close to that of finasteride. The results of ab initio calculations suggest that the inhibition potency of 19-nor-10-azasteroids could be directly related to the nucleophilicity of the carbonyl group in the 3-position.

Effects of protein tyrosine kinase inhibitors on the replication of herpes simplex virus and the phosphorylation of viral proteins.

The effect of protein tyrosine kinase (PTK) inhibitors on the replication of herpes simplex virus (HSV) was examined. Tyrphostins AG17, AG213, AG490, and AG555, and herbimycin A all inhibited the plaque formation of HSV type 1 (HSV-1) in Vero cells, but AG17, AG490, and AG555 exhibited a more selective antiviral effect. In the presence of 0.4 microM AG17, the virus production 24 h after infection was decreased to 7.7% of the untreated control level. Even if the treatment was started 12 h after the initiation of infection, the viral titer was reduced by 82.4%, compared with the untreated control level. In HSV-1-infected cells ICPs 6, 17/18, 19/20, and 25 were tyrosine-phosphorylated proteins. The synthesis and phosphorylation of these proteins were inhibited by AG17, and suppression of ICP 19/20, which were identified as the UL47 gene products, was the greatest. In contrast, the in vitro autophosphorylation of viral proteins was not affected by this PTK inhibitor. These results indicate that tyrphostin may represent a novel class of inhibitors of HSV-1, and that the viral proteins which have phosphorylated tyrosine residues and are suppressed by AG17 most significantly are the products of the UL47 gene, the tegument proteins VP13/14.

Novel, selective mechanism-based inhibitors of the herpes proteases

A novel class of inhibitor of the herpes proteases acting upon the catalytic apparatus by forming covalent complexes are described. Two new families of inhibitor, the spirocyclopropyl oxazolones and the benzylidine N-sulphonyloxyimidazolones, have been shown to be submicromolar inhibitors of HSV-2 and HCMV proteases which are selective relative to a panel of standard serine proteases.

Phenylamino-pyrimidine (PAP) — derivatives: a new class of potent and highly selective PDGF-receptor autophosphorylation inhibitors

Phenylamino-pyrimidines represent a novel class of inhibitors of the PDGF-receptor autophosphorylation with a high degree of selectivity versus other tyrosine and serine/threonine kinases. Optimum activity of ca 10 nM (IC50) was observed when the phenylamino-group which is attached to the pyrimidine carries a benzamide-moiety with a lipophilic substituent in 4-position.

Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L

Peptidyl vinyl sulphones are a novel class of extremely potent and specific cysteine protease inhibitors. They are highly active against the therapeutically important cathepsins O2, S and L. The highest kinact/K1 values exceed 10(7)M(-1) x s(-1) for cathepsin S and 10(5)M(-1) x s(-1) for cathepsins O2 and L. To study the primary specificity site of the novel human cathepsin O2 and the effectiveness of this novel class of inhibitors, a series of peptidyl vinyl sulphones with variations in the P2 residue was synthesized. Leucine in the P2 position was proven to be the most effective residue for cathepsin O2 and also for cathepsins S and L. Cathepsins O2 and S share a decreased accessibility towards P2 hydrophobic non-branched residues such as aminohexanoic acid (norleucine), methionine and oxidized methionine, but are distinguished by their different affinity towards phenylalanine in the P2 position. In contrast, cathepsin S accepts a broader range of hydrophobic residues in its S2 subsite than cathepsins O2 and L. The primary specificity-determining subsite pocket S2 in cathepsin O2 appears to be spatially more restricted than those of cathepsins S and L.

Phenylamino-pyrimidine (PAP) derivatives: a new class of potent and selective inhibitors of protein kinase C (PKC).

Phenylamino-pyrimidines represent a novel class of inhibitors of the protein kinase C with a high degree of selectivity versus other serine/threonine and tyrosine kinases. Steady state kinetic analysis of N-(3-[1-imidazolyl]-phenyl-4-(3-pyridyl)-2-pyrimidinamine (5), which showed potent inhibitory activity, revealed competitive kinetics relative to ATP. The adjacent H-bond acceptor of the pyrimidine moiety next to an H-bond donor of the phenylamine was found to be crucial for inhibitory activity. N-(3-Nitro-phenyl)-4-(3-pyridyl)-2-pyrimidinamine (7) preferentially inhibited PKC-alpha (IC50 = 0.79 microM) and not the other subtypes tested. The inhibition constants of PKC-alpha and the antiproliferative effect on T24 human bladder carcinoma cells showed a qualitative correlation, although with some exceptions.

S-Alkyl-l-thiocitrullines: POTENT STEREOSELECTIVE INHIBITORS OF NITRIC OXIDE SYNTHASE WITH STRONG PRESSOR ACTIVITY IN VIVO

Nitric oxide synthase catalyzes the oxidation of a guanidino nitrogen of L-arginine to nitric oxide with concomitant formation of citrulline. Enzyme activity is inhibited by a variety of N omega-monosubstituted L-arginine analogs including N omega-alkyl-, N omega-amino-, and N omega-nitro-L-arginine derivatives. We report here that both constitutive and inducible isoforms of nitric oxide synthase are strongly inhibited by S-alkyl-L-thiocitrullines (N delta-(S-alkyl)isothioureido-L-ornithines) with n-alkyl groups of one to three carbons. These compounds represent a novel class of inhibitors and are the most potent nitric oxide synthase-inhibiting amino acids described to date. Inhibition is reversible, stereoselective, and competitive with L-arginine. Spectral studies show no direct interaction of inhibitor sulfur with heme iron, a result in contrast to that seen previously with the parent compound, L-thiocitrulline. The S-alkyl-L-thiocitrullines have strong pressor activity in normotensive control rats; S-methyl-L-thiocitrulline reverses hypotension in a rat model of septic peritonitis and in dogs administered endotoxin. These latter findings suggest that the inhibitors may have therapeutic utility in treating hypotension due to the overproduction of nitric oxide.

Characterization of the Antiviral Activity of Highly Substituted Pyrroles: A Novel Class of Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitor

As a result of mass screening of the Parke-Davis Pharmaceutical compound library for inhibitors of HIV-1 reverse transcriptase (RT) activity, a novel class of inhibitor, the pyrroles, was identified. Subsequently, a series of analogues was screened for inhibitory activity against HIV-1 and some structure-activity relationships were identified. Further characterization of the most potent pyrrole involved comparing its effects in peripheral blood lymphocytes (PBLs) with its effects in transformed cell lines; the pyrrole had the same efficacy (EC50 = approximately 2 μM) but was less toxic in PBLs (IC50 = 175 μM) than in the cell lines CEM-SS and MT-2 (IC50 = 60-70 μM). The pyrrole was active against a strain of HIV-1 resistant to AZT (strain G9106) but lost activity against both HIV-2 (strain ROD) and a strain of HIV-1 resistant to a non-nucleoside reverse transcriptase inhibitor (the pyridinone-resistant strain A17). Moreover, in direct enzymatic testing against HIV-1 RT purified from virus particles and against RT expressed recombinantly, the pyrrole showed potent inhibitory activity. We conclude that the pyrroles present a novel class of HIV-1 non-nucleoside reverse transcriptase inhibitor.

Structure of HIV-1 protease with KNI-272: a transition state mimetic inhibitor containing allophenylnorstatine.

Inhibitors of human immunodeficiency virus type 1 protease (HIV PR) are the subject of wide interest for the development of therapeutic drugs against AIDS [1–4]. The crystal structures of numerous HIV PR/inhibitor complexes have been solved to aid the process of inhibitor design and to rationalize the structure-activity relationships (SAR) for various classes of inhibitors [5–7]. Incorporation of allophenylnorstatine (APNS) [(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] into peptidomimetics can lead to highly potent inhibitors of HIV PR [8]. However, no structure of APNS containing inhibitor complexes has been reported. Recently, KNI-272, a conformationally-constrained inhibitor containing APNS (Figure la), has been shown to possess good anti-HIV activity [9]. As a step towards understanding the high affinity of this novel class of inhibitors, we determined the three dimensional structure of KNI-272 bound to HIV PR.

Dianilinophthalimides: Potent and Selective, ATP-Competitive Inhibitors of the EGF-Receptor Protein Tyrosine Kinase

Dianilinophthalimides represent a novel class of inhibitors of the EGF-receptor protein tyrosine kinase with a high degree of selectivity versus other tyrosine and serine/threonine kinases. Steady-state kinetic analysis of compound 3, which showed potent inhibitory activity, revealed competitive type kinetics relative to ATP. Despite a highly symmetrical structure of compound 3, X-ray studies revealed an unsymmetrical propeller-shaped conformation of the molecule which differs clearly from that of the constitutionally related staurosporine aglycons. These conformational differences may explain the reversal of the selectivity profile of compound 3 relative to the staurosporine aglycons. In cellular assays compounds 3 and 4 have been shown to inhibit EGF-induced receptor autophosphorylation, c-fos induction and EGF-dependent proliferation of Balb/c MK cells. This inhibition was selective as compounds had no effect on PDGF-induced receptor autophosphorylation and c-fos induction. Furthermore, compound 3 showed potent antitumor activity in vivo at well-tolerated doses.

Diol sulfonamides: A potent and novel class of inhibitors of human renin

The syntheses and pharmacological activity of a series of diol sulfonamides which function as inhibitors of human renin are described. The most potent compound in this series, compound 20 (SQ 33,800), is a subnanomolar inhibitor of human renin (IC 50 = 0.35 × 10 −9 M).

Peptide glyoxals: a novel class of inhibitor for serine and cysteine proteinases.

A series of novel synthetic dipeptides, containing a C-terminal glyoxal grouping (-COCHO), have been tested as inhibitors against typical members of the serine- and cysteine-proteinase families. For example, the sequences benzyloxycarbonyl (Cbz)-Pro-Phe-CHO (I) and Cbz-Phe-Ala-CHO (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and cathepsin B respectively, have been found to be potent reversible inhibitors of their respective target proteinase. Thus I was found to inhibit chymotrypsin with a Ki of approximately 0.8 microM, whereas II exhibits a Ki of approximately 80 nm against cathepsin B. These Ki values are some 10-fold and 3-fold lower than those reported for the corresponding peptide-aldehyde inhibitors of chymotrypsin and cathepsin B upon which the peptidyl-glyoxals were fashioned. Unexpectedly, the sequence Cbz-Pro-Ala-CHO, which was designed to inhibit elastase-like proteinases, exhibited no inhibitory activity towards porcine pancreatic elastase, even when used at concentrations as high as 200 microM.

Staurosporine: A Prototype of a Novel Class of Inhibitors of Tumor Cell Invasion?

Staurosporine: A Prototype of a Novel Class of Inhibitors of Tumor Cell Invasion?

Inhibition of the bovine branched-chain 2-oxo acid dehydrogenase complex and its kinase by arylidenepyruvates

A novel class of inhibitors for the branched-chain 2-oxo acid dehydrogenase (BCOAD) complex has been synthesized and studied. The sodium salts of arylidenepyruvates: e.g., furfurylidenepyruvate (compound I), 4-(3-thienyl)-2-oxo-3-butenoate (compound II), cinnamalpyruvate (compound III) and 4-(2-thienyl)-2-oxo-3-butenoate (compound IV) inhibit the overall and kinase reactions of the BCOAD complex from bovine liver. Inhibitions of the overall reaction occur at the decarboxylase (E1) step as determined by a spectrophotometric assay with 2,6-dichlorophenolindophenol as an electron acceptor. Inhibition of the E1 reaction by compound I ( K i = 0.5 μ M ) is competitive, whereas inhibitions by compounds II ( K i = 150 μ M ) and III ( K i =500μ M ) are non-competitive with respect to the substrate 2-oxoisovalerate. The K m value for 2-oxoisovalerate is 6.7 μM as measured by the E1 assay. Inhibition of the E1 step by compounds I, II and III are reversible at low inhibitor concentrations based on the Michaelis-Menten kinetics observed. By comparison, compound I does not significantly inhibit pyruvate and 2-oxoglutarate dehydrogenase complexes. The arylidenepyruvates (compounds I, II and IV) inhibit the BCOAD kinase reaction in a manner similar to the substrate 2-oxo acids. The inhibition of the kinase reaction by compound I is non-competitive with respect to ATP, with an apparent K i value of 4.5 mM. The results suggest that arylidenepyruvates may be useful probes for elucidating the reaction mechanisms of the BCOAD complex and its kinase.

Hebal extracts and their components as a novel class of inhibitors for HIV-reverse transcriptase : K. Ono. Laboratory of Viral Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan

Hebal extracts and their components as a novel class of inhibitors for HIV-reverse transcriptase : K. Ono. Laboratory of Viral Oncology, Aichi Cancer Center Research Institute, Nagoya, Japan