Protease Inhibitors: Synthesis of L-Alanine Hydroxamate Sulfonylated Derivatives as Inhibitors of Clostridium Histolyticum Collagenase

L-alanine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with L-alanine, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group with hydroxylamine in the presence of carbodiimides. Other derivatives were obtained by reaction of N-benzyl-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by a similar conversion of the COOH to the CONHOH moiety. The obtained compounds were assayed as inhibitors of Clostridium histolyticum collage-nase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to the most potent ChC inhibitors were those involving perfluoroalkylsulfonyl-and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl-, or 1- and 2-naphthylsulfonyl among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P₂, and P₃ sites, in order to achieve tight binding to the enzyme.     

Protease inhibitors. Part 8: synthesis of potent Clostridium histolyticum collagenase inhibitors incorporating sulfonylated L-alanine hydroxamate moieties

A series of hydroxamates was prepared by reaction of alkyl/arylsulfonyl halides with N-2-chlorobenzyl-L-alanine, followed by conversion of the COOH moiety to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-2-chlorobenzyl-L-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a bacterial zinc metallo-peptidase which degrades triple helical collagen as well as a large number of synthetic peptides. The prepared hydroxamate derivatives proved to be 100-500 times more active collagenase inhibitors than the corresponding carboxylates. Substitution patterns leading to best ChC inhibitors (both for carboxylates as well as for the hydroxamates) were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethyl-phenylsulfonyl; as well as 1- and 2-naphthyl-, quinoline-8-yl- or substituted-arylsulfonylamidocarboxyl moieties among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P2' and P3' sites, in order to achieve tight binding to the enzyme. This study also proves that the 2-chlorobenzyl moiety, investigated here for the first time, is an efficient P2' anchoring moiety for obtaining potent ChC inhibitors.     

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating arylsulfonylureido and 5-dibenzo-suberenyl/suberyl moieties

Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respectively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.     

Protease inhibitors: synthesis of clostridium histolyticum collagenase inhibitors incorporating sulfonyl-L-alanine hydroxamate moieties

A series of hydroxamates was obtained by the reaction of N-(4-nitrobenzyl)-L-alanine with alkyl/arylsulfonyl halides, followed by conversion of the COOH group into CONHOH. Structurally-related compounds were prepared similarly by using arylsulfonyl isocyanates, aryl isocyanates or arylsulfenyl halides instead of the sulfonyl halides. Many of the new compounds showed nanomolar affinity for the bacterial collagenase isolated from the pathogen Clostridium histolyticum.     

Protease inhibitors: synthesis of matrix metalloproteinase and bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions

Matrix metalloproteinase (MMP)/bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions are reported. A series of compounds was prepared by reaction of arylsulfonyl isocyanates or arylsulfonyl halides with phenylalanyl-alanine, followed by coupling with 5-amino-2-mercapto-1,3,4-thiadiazole in the presence of carbodiimides. These new compounds were assayed as inhibitors of human MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from the anaerobe Clostridium histolyticum (ChC). The new derivatives proved to be powerful inhibitors of these metalloproteases, with activities in the low micromolar range for some of the target enzymes, depending on the substitution pattern at the arylsulfonyl(ureido) moieties.     

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating succinyl hydroxamate and iminodiacetic acid hydroxamate moieties

A series of succinyl hydroxamates/bishydroxamates as well as a new structural type of matrix metalloproteinase (MMP)/bacterial protease (BP) inhibitors, incorporating iminodiacetic (IDA) hydroxamate/bishydroxamate moieties, has been synthesized and tested for interaction with four vertebrate proteases, MMP-1, MMP-2, MMP-8 and MMP-9, and a BP, the collagenase isolated from Clostridium histolyticum (ChC). The new derivatives generally showed inhibition constants in the range of 8-62 nM against the five proteases mentioned above.     

Carbonic anhydrase inhibitors: inhibition of isozymes I, II and IV by sulfamide and sulfamic acid derivatives

Sulfamide and sulfamic acid are the simplest compounds containing the SO2NH2 moiety, responsible for binding to the Zn(II) ion within carbonic anhydrase (CA, EC 4.2.1.1) active site, and thus acting as inhibitors of the many CA isozymes presently known. Here we describe two novel classes of CA inhibitors obtained by derivatizations of the lead molecules mentioned above. The new compounds, possessing the general formula RSO2NH-SO2X (X = OH, NH2), were obtained by reaction of sulfamide or sulfamic acid with alkyl/arylsulfonyl halides or arylsulfonyl isocyanates. A smaller series of derivatives has been obtained by reaction of aromatic aldehydes with sulfamide, leading to Schiff bases of the type ArCH = NSO2NH2. All the new compounds act as strong inhibitors of isozymes I, II and IV of carbonic anhydrase. Their mechanism of CA inhibition is also discussed based on electronic spectroscopic measurements on adducts with the Co(II)-substituted enzyme. These experiments led to the conclusion that the new inhibitors are directly coordinated (in a monodentate manner) to the metal ion within the enzyme active site, similarly to the classical inhibitors, the aromatic/heterocyclic sulfonamides.     

Protease Inhibitors: Synthesis of Bacterial Collagenase and Matrix Metalloproteinase Inhibitors Incorporating Succinyl Hydroxamate and Iminodiacetic Acid Hydroxamate Moieties

A series of succinyl hydroxamates/bishydroxamates as well as a new structural type of matrix metalloproteinase (MMP)/bacterial protease (BP) inhibitors, incorporating iminodiacetic (IDA) hydroxamate/bishydroxamate moieties, has been synthesized and tested for interaction with four vertebrate proteases, MMP-1, MMP-2, MMP-8 and MMP-9, and a BP, the collagenase isolated from Clostridium histolyticum (ChC). The new derivatives generally showed inhibition constants in the range of 8-62 nM against the five proteases mentioned above.     

Carbonic anhydrase inhibitors: inhibition of cytosolic/tumor-associated isoforms I, II, and IX with iminodiacetic carboxylates/hydroxamates also incorporating benzenesulfonamide moieties

The synthesis of a new class of sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs), also possessing carboxylate/hydroxamate moieties in their molecule, is reported. These compounds may act on dual antitumor targets, the tumor-associated CA isozymes (CA IX) and some matrix metalloproteinases (MMPs). The compounds were prepared by an original method starting from iminodiacetic acid, and assayed as inhibitors of three isozymes, hCA I, II (cytosolic), and IX (transmembrane). The new derivatives showed weak inhibitory activity against isozyme I (K(I)s in the range of 95-8300 nM), were excellent to moderate CA II inhibitors (K(I)s in the range of 8.4-65 nM), and very good and selective CA IX inhibitors (K(I)s in the range of 3.8-26 nM). The primary sulfonamide moiety is a better zinc-binding group in the design of CAIs as compared to the carboxylate/hydroxamate one, but the presence of hydroxamate functionalities in the molecule of CAIs leads to selectivity for the tumor-associated isozyme IX over the ubiquitous, cytosolic isoform II.     

Protease inhibitors, part 13: Specific, weakly basic thrombin inhibitors incorporating sulfonyl dicyandiamide moieties in their structure

A series of compounds has been prepared by reaction of dicyandiamide with alkyl/arylsulfonyl halides as well as arylsulfonylisocyanates to locate a lead for obtaining weakly basic thrombin inhibitors with sulfonyldicyandiamide moieties as the S1 anchoring group. The detected lead was sulfanilyl-dicyandiamide (K1 of 3 microM against thrombin, and 15 microM against trypsin), which has been further derivatized at the 4-amino group by incorporating arylsulfonylureido as well as amino acyl/dipeptidyl groups protected at the amino terminal moiety with benzyloxycarbonyl or tosylureido moieties. The best compound obtained (ts-D-Phe-Pro-sulfanilyl-dicyandiamide) showed inhibition constants of 9 nM against thrombin and 1400 nM against trypsin. pKa measurements showed that the new derivatives reported here do indeed possess a reduced basicity, with the pKa of the modified guanidine moieties in the range 7.9-8.3 pKa units. Molecular mechanics calculations showed that the preferred tautomeric form of these compounds is of the type ArSO2N=C(NH2) NH-CN, probably allowing for the formation of favorable interaction between this new anchoring group and the active site amino acid residue Asp 189, critical for substrate/inhibitor binding to this type of serine protease. Thus, the main finding of the present paper is that the sulfonyldicyandiamide group may constitute an interesting alternative for obtaining weakly basic, potent thrombin inhibitors, which bind with less affinity to trypsin.     

Introduction of the 4-(4-bromophenyl)benzenesulfonyl group to hydrazide analogs of Ilomastat leads to potent gelatinase B (MMP-9) inhibitors with improved selectivity

Hydrazide derivatives of Ilomastat, carrying either aryl groups or distinct alkyl and arylsulfonyl moieties were synthesized and evaluated for their MMP inhibitory activity. Potent and selective MMP-9 inhibition (IC(50)=3 nM) was observed for compound 3m (arylsulfonyl group: 4-(4-Br-C6H4)-C6H4-SO(2)-). Interaction with the S2 enzyme subsite is mainly responsible for the inhibitory properties of this derivative as confirmed by molecular docking computation.     

Anthranilate sulfonamide hydroxamate TACE inhibitors. Part 2: SAR of the acetylenic P1' group

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups was explored. In particular, compound 4t bearing a butynyloxy P1' moiety has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and oral activity in an in vivo model of TNF-alpha production.     

N-Sulfonyl hydroxamate derivatives as inhibitors of class II fructose-1,6-diphosphate aldolase

Dihydroxyacetone-phosphate and phosphonate derivatives were synthesized bearing a N-sulfonyl hydroxamate moiety. The phosphate derivatives represent competitive inhibitors for the class II-FBP aldolase catalyzed reaction, while the phosphonate isosteres are comparatively weaker inhibitors.     

Carbonic anhydrase inhibitors. N-cyanomethylsulfonamides--a new zinc binding group in the design of inhibitors targeting cytosolic and membrane-anchored isoforms

A series of N-cyanomethyl aromatic sulfonamides and bis-sulfonamides was prepared by reaction of arylsulfonyl halides with aminoacetonitrile. The obtained derivatives incorporated various aryl moieties, such as 4-halogeno/alkyl/aryl/nitro-substituted-phenyl, pentafluorophenyl or 2-naphthyl. Moderate inhibitory activity was detected for some compounds against the cytosolic human isoform II of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA II, with inhibition constants of 90, 180 and 560n M for the 4-nitrophenyl-, 4-iodophenyl- and pentafluorophenyl-N-cyanomethylsulfonamides, respectively. Other derivatives acted as weak inhibitors of isoforms hCA I (KIs of 720 nM-45 microM), hCA II (KIs of 1000-9800 nM) and hCA IX (KIs of 900-10200 nM). Thus, the N-cyanomethylsulfonamide zinc binding group is less effective than the sulfonamide, sulfamate or sulfamide ones for the design of effective CA inhibitors.     

Macrocyclic hydroxamate inhibitors of matrix metalloproteinases and TNF-alpha production.

Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.     

Derivatives of antineoplastic antibiotics of the daunorubicin series containing methylurea or nitrosomethylurea residues

Derivatives of antitumour anthracycline antibiotics containing N-methylurea moiety in the carbohydrate ring were obtained by the interaction of methyl isocyanate with daunorubicin, doxorubicin, carminomycin and daunorubicin derivatives, substituted at C-13 or C-14 positions. N-Nitrosation of these compounds yielded modified anthracycline antibiotics containing the N-methyl-N-nitrosourea substituent at C-3' position. Alkaline degradation of these derivatives produced, through corresponding isocyanates cyclic 3'-N,4'-carbonylderivatives. In these anthracycline derivatives with sugar cycles conjugated with oxazoline-2-ones the predominant conformations of sugar ring has changed from 1C4 to 4C1, 2,5B, or B0,3 (shown by 1H NMR spectroscopy). It was demonstrated, both in vitro and in vivo, that introduction of methylurea or cytotoxic methylnitrosourea moieties does not potentiate antimicrobial, cytotoxic or antitumour properties of these compounds.     

Carbonic Anhydrase Inhibitors: Inhibition of Isozymes I,II and IV by Sulfamide and Sulfamic Acid Derivatives

Abstract

Sulfamide and sulfamic acid are the simplest compounds containing the SO₂NH₂ moiety, responsible for binding to the Zn(II) ion within carbonic anhydrase (CA, EC 4.2.1.1) active site, and thus acting as inhibitors of the many CA isozymes presently known. Here we describe two novel classes of CA inhibitors obtained by derivatizations of the lead molecules mentioned above. The new compounds, possessing the general formula RSO₂NH-SO₂X (X = OH, NH₂), were obtained by reaction of sulfamide or sulfamic acid with alkyl/arylsulfonyl halides or aryl-sulfonyl isocyanates. A smaller series of derivatives has been obtained by reaction of aromatic aldehydes with sulfamide, leading to Schiff bases of the type ArCH=NSO₂NH₂. All the new compounds act as strong inhibitors of isozymes I, II and IV of carbonic anhydrase. Their mechanism of CA inhibition is also discussed based on electronic spectroscopic measurements on adducts with the Co(II)-substituted enzyme. These experiments led to the conclusion that the new inhibitors are directly coordinated (in a monodentate manner) to the metal ion within the enzyme active site, similarly to the classical inhibitors, the aromatic/heterocyclic sulfonamides.     

Synthesis and biological evaluation of esters of 16-formyl-17-methoxy-dehydroepiandrosterone derivatives as inhibitors of 5α-reductase type 2

In this study, we investigated the in vitro effect of 16-formyl-17-methoxy dehydroepiandrosterone derivatives on the activity of 5α-reductase type 2 (5α-R2) obtained from human prostate. The activity of different concentrations of these derivatives was determined for the conversion of labelled testosterone to dihydrotestosterone. The results indicated that an aliphatic ester moiety at the C-3 position of these derivatives increases their in vitro potency as inhibitors of 5α-R2 activity compared to finasteride®, which is considered to be a potent inhibitor of 5α-R2. In this case, the augmentation of the lipophilicity of these dehydroepiandrosterone derivatives increased their potency as inhibitors of 5α-R2. However, the presence of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings as the cycloaliphatic ester moiety at C-3 of the formyl methoxy dehydroepiandrosterone scaffold did not inhibit the activity of this enzyme. This may be due to the presence of steric factors between the enzyme and the spatial structure of these derivatives.     

Carbonic anhydrase and matrix metalloproteinase inhibitors. Inhibition of human tumor-associated isozymes IX and cytosolic isozyme I and II with sulfonylated hydroxamates

A series of sulfonylated hydroxamates were synthesized and evaluated as dual inhibitors of both human carbonic anhydrases (hCAs) and matrix metalloproteinases (MMPs), two metalloenzyme families involved in carcinogenesis and tumor invasion processes. The new derivatives were tested on three CA isozymes, the cytosolic isozymes I and II, and the transmembrane, tumor-associated isozyme IX, and also on human gelatinases (MMP-2 and MMP-9). Some of the new derivatives proved to be potent and selective inhibitors of CA II, but only compounds 3b and 6b, devoid of the arylsulfonyl moiety, proved to have a better inhibitory activity on hCA IX than on hCA I and II, in the micromolar range.     

Protease inhibitors: Part 4. Synthesis of weakly basic thrombin inhibitors incorporating pyridinium-sulfanilylaminoguanidine moieties

Three series of derivatives have been prepared by reaction of sulfanilylaminoguanidine with pyrylium salts, with the pyridinium derivatives of glycine and with the pyridinium derivatives of beta-alanine, respectively. The new compounds were assayed as inhibitors of two serine proteases, thrombin and trypsin. The study showed that in contrast to the leads, possessing KI's around 100-300 nM against thrombin, and 450-1420 nM against trypsin, respectively, the new derivatives showed inhibition constants in the range of 15-50 nM against thrombin, whereas their affinity for trypsin remained relatively low. Derivatives of beta-alanine were more active than the corresponding glycine derivatives, which in turn were more inhibitory than the pyridinium derivatives of sulfanilylaminoguanidine possessing the same substitution pattern at the pyridinium ring. Thus, the present study proposes two novel approaches for the preparation of high affinity, specific thrombin inhibitors: a novel S1 anchoring moiety in the already large family of arginine/amidine-based inhibitors, i.e., the SO2NHNHC(=NH)NH2 group, and novel non-peptidomimetic scaffolds obtained by incorporating alkyl-/aryl-substituted-pyridinium moieties in the hydrophobic binding site(s). The first one is important for obtaining bioavailable thrombin inhibitors, devoid of the high basicity of the commonly used arginine/amidine-based inhibitors, whereas the second one may lead to improved water solubility of such compounds.