(3-Amino-2-oxoalkyl)phosphonic acids and their analogues as novel inhibitors of D-alanine:D-alanine ligase

The dipeptide D-alanyl-D-alanine is an essential precursor of bacterial peptidoglycan; thus, blocking its formation is a possible target for the design of novel antibacterial agents. The synthesis of this dipeptide by bacterial D-alanine:D-alanine ligase requires ATP. In analogy with glutamine synthetase, we hypothesized a mechanism for this enzyme involving the intermediacy of D-alanyl phosphate. Several (3-amino-2-oxoalkyl)phosphonic acids and their analogues have been synthesized as possible inhibitory mimics of this proposed intermediate. The most active of them, (3(R)-amino-2-oxobutyl)phosphonic acid (8a) and the corresponding aza analogue (22), were effective ligase inhibitors although they had no significant antibacterial activity. The ligase inhibition of these compounds is consistent with an acyl phosphate displacement step in the mechanism of DAla-DAla ligase.     

Renin inhibitors. Design of angiotensinogen transition-state analogues containing novel. (2R,3R,4R,5S)-5-amino-3,4-dihydroxy-2-isopropyl-7-methyloctanoic acid

A highly stereoselective synthesis of 2(R)-[5(R)-[1(S)-[(tert-butyloxycarbonyl)amino]-3-methylbutyl]-2,2- dimethyl-4(R)-dioxolanyl]-3-methylbutanoic acid is described. This is a suitably protected carboxylic acid useful as an intermediate for the preparation of renin inhibitory peptides. Angiotensinogen analogues such as peptides IX and X that contain the dipeptide isostere (2R,3R,4R,5S)-5-amino-3,4-dihydroxy-2-isopropyl-7-methyloctanoic acid residue at the scissile site are shown to be potent inhibitors of human plasma renin. The glycol moiety in this novel acid, dihydroxyethylene isostere, is suggested to act as a transition-state analogue and mimics the tetrahedral intermediate formed during the enzyme-catalyzed hydrolysis of the peptidic bond.     

Cysteine derivatives as inhibitors for carboxypeptidase A: synthesis and structure-activity relationships

A series of cysteine (Cys) derivatives having an alkyl or arylalkyl moiety on the alpha-amino group of the amino acid have been synthesized as a novel type of inhibitor for carboxypeptidase A. These compounds are readily prepared starting with Cys in an optically active form. The structure-activity relationship study revealed that the inhibitors prepared from D-Cys are much more potent than the corresponding inhibitors obtained from L-Cys, and the most potent inhibitor in the series, (S)-1j with a K(i) value of 55 +/- 4 nM, is obtained by introducing a phenethyl moiety on the amino group of D-Cys. In comparison, the most active inhibitor in the series of 2-substituted 3-mercaptopropanoic acid is found to be 20, in which the phenyl ring is linked to the mercaptocarboxylic acid at the alpha-position with a methylene unit. A proposal that accounts for the different structural requirement for the maximum activity between the two series of inhibitors is provided.     

Anti-tyrosinase, antioxidant and antimicrobial activities of hydroxycinnamoylamides

Synthetic hydroxycinnamoylamides of amino acids (precursors of aromatic amines) were studied for their antioxidant activity in vitro by two antioxidant assay systems, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and inhibition of lipid peroxidation (LPO). Furthermore, these compounds were tested and compared with their corresponding cinnamoylamides of aromatic amines for their inhibitory activity using mushroom tyrosinase. In addition, five hydroxycinnamoyl amino acid amides were investigated for their antimicrobial effect. Structure–activity relationships analysis disclosed that the presence of catechol rest at amino acid or at benzene moieties of substituted cinnamic acid amides significantly scavenged DPPH radical and inhibited LPO. The results obtained by LPO clearly expressed the positive influence of indole moiety on the activity. Moreover, the existence of p-hydroxy substituted cinnamic acid moiety leads to better tyrosinase inhibition. Amongst the tested compounds, amides of p-coumaroyldopamine or tyramine and their corresponding amino acid precursors are the most potent tyrosinase inhibitors.     

Sulfamide-based inhibitors for carboxypeptidase A. Novel type transition state analogue inhibitors for zinc proteases

N-Sulfamoylphenylalanine and its derivatives having varied alkyl groups on the terminal amino group were designed rationally as transition state analogue inhibitors for carboxypeptidase A (CPA) and synthesized. In CPA inhibitory assays the parent compound having the (S)-configuration, i.e., (S)-1a, showed potent inhibitory activity with the K(i) value of 0.64 microM. Its enantiomer was shown to be much less potent (K(i) = 470 microM). Introduction of an alkyl group such as methyl or isopropyl group on the terminal amino group of (S)-1a lowered the inhibitory potency drastically. Introduction of a methyl group on the internal amino group of (S)-1a also caused a drastic reduction of the inhibitory activity. The structure of the CPA x(S)-1a complex determined by single-crystal X-ray diffraction reveals that the sulfamoyl moiety interacts with the zinc ion and functional groups at the active site of CPA, which is reminiscent of the postulated stabilization mode of a tetrahedral transition state in the CPA-catalyzed hydrolysis of a peptide substrate. On the basis of the design rationale and the binding mode of (S)-1a to CPA shown by X-ray crystallographic analysis, the present inhibitors are inferred to be a novel type of transition state analogue inhibitor for CPA.     

Analogues of carbamyl aspartate as inhibitors of dihydroorotase: preparation of boronic acid transition-state analogues and a zinc chelator carbamylhomocysteine

Dihydroorotase (DHO) catalyzes the conversion of carbamyl aspartate (CA) to dihydroorotate (DO) in the de novo pyrimidine biosynthetic pathway. Few effective inhibitors of DHO have been reported, and thus blockade of this reaction has not been widely pursued as a strategy for development of antitumor agents. Utilizing two mechanism-based strategies, we have designed and prepared potential DHO inhibitor analogues of CA. One strategy replaced the gamma-carboxyl moiety of CA with a boronic acid. This substitution yields compounds which form stable charged tetrahedral intermediates and mimic the enzyme-substrate transition state. Preparation of the boronic acid analogues of CA and its carboxylic acid esters focused on a Curtius rearrangement as a key step following a malonic ester synthesis. This was followed by carbamoylation of the free amine under nonaqueous neutral conditions with Si(NCO)4. The ethyl ester was a competitive inhibitor of DHO with an apparent Ki of 5.07 microM, while the nonesterified analogue and the methyl ester were not effective inhibitors. None of the compounds were cytotoxic against L1210 cells in culture. An active-site-directed sulfhydryl-containing zinc chelator was also prepared. This analogue irreversibly inhibited the enzyme, but it also was ineffective in L1210 growth inhibition.     

Structure of the peptide antibiotic polypeptin.

Polypeptin, a basic peptide antibiotic isolated from Bacillus circulans, was separated into two components by countercurrent distribution. The two components, polypeptin A and polypeptin B, had identical amino acid compositions but varied in the structure of the hydroxy acid constituent attached to the alpha-amino group of the peptide chain. Polypeptin A contained 3-hydrosy-4-methylhexanoic acid and polypeptin B contained 3-hydrosy-5-methylhexanoic acid. T-HE STEROCHEMISTRY OF THESE HYDROXY ACIDS WAS NOT DETERMINED. Studies involving partial acid hydrolysis and chemical synthesis are consistent with the lactone sturcture for polypeptin A. Polypeptin B differs only in the position of the methyl group in the hydroxyacyl moiety.     

Synthesis and aldose reductase inhibitory activity of substituted 2-oxoquinoline-1-acetic acid derivatives

A number of 2-oxoquinoline-1-alkanoic acids that contain the N-acylglycine fragment found in several known inhibitors of aldose reductase were synthesized and tested in the rat lens assay. All of the target compounds were prepared by alkylation of the appropriate 2-oxoquinoline intermediates with a halo ester, followed by hydrolysis of the intermediate esters. In the rat lens assay, the 1-acetic acid derivatives 9a-e display the highest level of aldose reductase inhibitor activity with IC50 values of 0.45-6.0 microM. Modification of the 1-acetic acid moiety by esterification, substitution of an alpha-methyl group, or insertion of an additional methylene unit results in reduced inhibitory potency. Structure-activity data also suggests that both the benzene and 2-oxopyridine rings of 9a-e contribute substantially toward activity and that inhibitory potency is influenced by aromatic ring substituents.     

Phosphorus amino acid analogues as inhibitors of leucine aminopeptidase

A variety of phosphorus amino acid and dipeptide analogues have been synthesized and evaluated as inhibitors of the metalloenzyme leucine aminopeptidase from porcine kidney. Two phosphonate dipeptides were found to be modest inhibitors of the enzyme (8e, Ki = 58 microM; 8h, Ki = 340 microM). The phosphinic acid (17-OH) and phosphinamide (17-NH2) analogues related to bestatin were prepared by condensation of the phosphinate amino acid derivative 11, via a trivalent phosphonite ester 12, with leucine isocyanate derivatives 13. These compounds also proved to be unexceptional in their inhibition of LAP (17-O-, Ki = 56 microM; 17-NH2, Ki = 40 microM). A series of simple (alpha-aminoalkyl)phosphonic acid and -phosphinic acids were also evaluated, and the most potent inhibitors were found to be the phosphonic acid analogues of L-Leu and L-Phe [R)-3e, Ki = 0.23 microM; (R)-3h, Ki = 0.42 microM). Slow-binding behavior was observed for (R)-3e (kon = 400 +/- 55 M-1 s-1) and (R)-3h (kon = 445 +/- 50 M-1 s-1). The phosphinic acid analogues of Leu and Phe are 100-fold less potent than the phosphonate derivatives. The fact that tetrahedral phosphorus analogues are less potent inhibitors of LAP than they are of other zinc peptidases suggests that the mechanism of LAP may be fundamentally different than that of the latter enzymes.     

Development of peptidomimetics with a vinyl sulfone warhead as irreversible falcipain-2 inhibitors

This paper describes the synthesis of a new class of peptidomimetic cysteine protease inhibitors based on a 1,4-benzodiazepine scaffold and on an electrophilic vinyl sulfone moiety. The former was introduced internally to a peptide sequence that mimics the fragment D-Ser-Gly; the latter was built on the P1-P1' site and reacts as a classical "Michael acceptor", leading to an alkylated enzyme by irreversible addition of the thiol group of the active site cysteine. The introduction of the vinyl sulfone moiety has been accomplished by olefin cross-metathesis, a powerful tool for the formation of carbon-carbon double bonds. New compounds 2-3 have been proven to be potent and selective inhibitors of falcipain-2, a cysteine protease isolated from Plasmodium falciparum, displaying antiplasmodial activity.     

N-(Phosphonoacetyl)amino phosphonates. Phosphonate analogues of N-(phosphonoacetyl)-L-aspartic acid (PALA)

Michaelis-Arbuzov reaction of N-(chloroacetyl)amino phosphonic acids or their esters, followed by acidolysis, gives moderate yields of N-(phosphonoacetyl) derivatives of a variety of (aminoalkyl)phosphonic acids, including analogues of the cytostatic agent PALA, in which the alpha- or beta-carboxylic groups in the aspartate moiety are replaced by a PO3H2 function. Assay of cytostatic activity with human KB cell lines indicates that the substitution of any of the COOH groups in PALA with PO3H2 results in total loss of cytostatic activity. No activity was observed also in the case of other [N-(phosphonoacetyl)amino]alkylphosphonic acids described in this report.     

Isolation of dealanylalahopcin, a new amino acid, and its biological activity

A new amino acid, dealanylalahopcin, was isolated from a culture filtrate of Streptomyces albulus subsp. ochragerus; it was also formed by the enzymatic hydrolysis of alahopcin using microbial alpha-amino acid ester hydrolase. The amino acid was obtained as colorless needles and its molecular formula is C6H10N2O5. It showed very weak antibacterial activity against Gram-positive and Gram-negative bacteria, and weak inhibitory activity against the collagen prolylhydroxylase.     

CycloSal-BVDUMP pronucleotides: how to convert an antiviral-inactive nucleoside analogue into a bioactive compound against EBV

Novel cycloSal-BVDUMP triesters 2-4 5-[(E)-2-bromovinyl]-2'-deoxyuridine (BVDU, 1) have been studied with regard to their potential anti-EBV activity. In addition to the 3'-unmodified cycloSal-BVDUMP triesters 2a-f, the 3'-hydroxyl function has been esterified with different aliphatic carboxylic acids (3a-g) and alpha-amino acids having natural and nonnatural Calpha-configuration (4a-m). In addition to the synthesis of these compounds, different physicochemical properties of the new derivatives will be reported, i.e., lipophilicity and hydrolysis behavior. It could be proven that the monophosphate BVDUMP and not 3',5'-cyclic BVDUMP was delivered from most of the compounds by chemical hydrolysis in phosphate buffers at pH 6.8 and 7.3 as well as P3HR-1 cell extracts. Finally, the new compounds were tested for their anti-EBV activity. As a result, the prototype compounds and particularly triesters 2c,d exhibited pronounced anti-EBV activity making these compounds promising candidates for further development. However, the 3'-ester derivatives were devoid of any antiviral activity while the 3'-aminoacyl derivatives showed an antiviral activity dependent upon the amino acid and the Calpha-configuration     

Eurystatins A and B, new prolyl endopeptidase inhibitors. II. Physico-chemical properties and structure determination.

The structures of eurystatins A and B, new prolyl endopeptidase inhibitors, have been elucidated by chemical degradation and spectral studies. They have in common a unique 13-membered cyclic peptide core composed of L-leucine, L-ornithine and (S)-3-amino-2-oxobutyric acid, and differ from each other in the alpha,beta-unsaturated fatty acid attached to the alpha-amino moiety of the ornithine.     

5-对甲苯磺酰氨基酞酰亚胺衍生物的设计合成及α-糖苷酶抑制活性

目的寻找具有新型结构的OL-糖苷酶抑制剂类降血糖药物。方法N-取代-5-对甲苯磺酰氨基酞酰亚胺类目标化合物（I1～I8）以邻苯二甲酰亚胺为起始物,经硝化后,通过烃化反应在亚胺氮上引入含芳基的不同取代结构,再经还原及磺酰化反应合成;N-取代结构的苯环上含有酚羟基的目标化合物（Ⅱ1～Ⅱ3）采用乙酰化保护后再进行还原及磺酰化、去保护基等步骤制得;目标化合物Ⅱ。和Ⅱ。的合成是以5一硝基酞酰亚胺为原料,先与甲醛反应,在亚胺氮上引入羟甲基,再与酚性结构进行偶联反应引入相应取代基,最后经还原及磺酰化制得。采用4-硝基苯-“-D-吡喃葡萄糖苷反应体系对目标化合物的a一糖苷酶抑制活性进行评价;采用计算机辅助药物设计软件进行分子Docking分析。结果合成了13个未见报道的新化合物,其结构经MS及1H—NMR谱确证。所有目标化合物对酵母a-糖苷酶均表现出明显的抑制活性,其IC50值小于阳性药物阿卡波糖（Acar）;Docking分析显示,5-对甲苯磺酰氨基酞酰亚胺与酶活性催化位点的氨基酸残基能形成多个氢键,N-取代结构以疏水作用或氢键与酶分子产生相互作用而影响酶活性。结论5-对甲苯磺酰氨基酞酰亚胺结构是产生α-糖苷酶抑制活性的有利结构,亚胺氮原子上取代结构对抑酶活性有重要影响,此结果得到分子对接分析的支持。     

Phosphono Analogues of Glutathione as New Inhibitors of Glutathione S-Transferases

Phosphono-analogues of glutathione containing the O=P(OR)₂ moiety in place of the cysteinyl residue CH₂SH 1a–1d were prepared by solution phase peptide synthesis. Benzyl, benzyloxycarbonyl, and tert-butyl protecting groups were used to mask the individual amino acid functional groups. The formation of peptide bonds was achieved by the usual peptide synthesis via activation of carboxylic functions with cyclohexylcarbodiimide and subsequent reaction with free amino groups. The thus obtained, fully-protected peptides were each purified by normal phase column chromatography. Deprotection was accomplished by hydrogenolysis and by treatment with HBr/acetic acid yielding the desired phosphonic acid diester 1a–1d . The inhibition of the glutathione conjugation of 1-chloro-2,4-dinitrobenzene by human placental glutathione S-transferase was studied by determining the IC₅₀ values of the new glutathione analogues. The IC₅₀ values were 291 μM, 139 μM, 64 μM, and 21 μM for the dimethyl, diethyl, diisopropyl, and di-n-butyl esters, respectively. The results clearly show that the formal substitution of the glutathione thiol function by phosphonic acid esters leads to a new class of glutathione S-transferase inhibitors. Further investigations directed at the question of whether or not these glutathione analogues are suitable for a modulation in chemotherapy are in progress.     

新型抑制破骨细胞生成的化合物合成与活性研究

目的　寻找有抑制破骨细胞活性的苄膦酸型先导化合物。方法　用类似Arbuzov型反应合成α-烷氨苄膦酸化合物。以体外培养大鼠骨髓液中的破骨细胞样细胞(osteoclast-likecells,OLC)生成为模型,筛选上述合成产物,化合物结构经MS或¹HNMR得到证明。结果　得到10个α-烷氨苄膦酸化合物,均为新化合物。生物活性筛选表明,化合物2,8及9对体外大鼠的OLC的生成有显著抑制活性(P<0.01)。结论　增强分子的芳香性及亲脂性(9)可提高抑制生成破骨细胞的活性。     

Total synthesis and biological activities of (+)-sulfamisterin (AB5366) and its analogues

The first total synthesis of (+)-sulfamisterin (AB5366), a naturally occurring alpha-substituted alpha-amino acid derivative possessing a sulfonated hydroxy function, is described. Overman rearrangement of an allylic trichloroacetimidate derived from D-tartrate effectively generated the tetrasubstituted carbon containing a nitrogen substituent. Construction of the amino acid moiety and sulfonation of the hydroxy group, followed by deprotection completed the total synthesis, which fully confirmed the proposed absolute structure of the natural product. The possible stereoisomers of (+)-sulfamisterin and their desulfonated derivatives were also synthesized. Biological assessment of all synthetic compounds revealed that natural (+)-sulfamisterin and its 3-epimer as well as their desulfonated derivatives possessing 2S-configuration strongly inhibit the serine palmitoyl transferase both in vitro and in vivo, whereas compounds with 2R-configuration were found to show much weaker inhibitory activity.     

Retro-inverso concept applied to the complete inhibitors of enkephalin-degrading enzymes

Peptide retro-inverso modification was applied to the complete hydroxamate inhibitors of the three zinc metallopeptidases (neutral endopeptidase 24-11 (NEP, EC 3.4.24.11), aminopeptidase N (APN, EC 3.4.11.2), and a dipeptidylaminopeptidase (DAP) involved in the in vitro enkephalin degradation by brain tissues. Compounds corresponding to the general formula RN(OH)CO(CH2)nCH(CH2Ph)NHCOCH(R')COOH (n = 0, 1) were synthesized. In the first series of inhibitors (n = 0), the "retro-inverso" modification induced a large decrease in inhibitory potency for NEP as compared to that of the parent compounds. In contrast, the presence of a methylene group between the hydroxamate and CH alpha in the second series (n = 1) led to derivatives with inhibitory potencies in the nanomolar range, similar to their analogues with a natural amide bond. On the other hand, the retro-inverso modification led to a slight improvement in the inhibition of DAP and APN, in the first series of inhibitors, while the inverse result occurred in the second series. Thus, compounds containing an alpha-amino acid moiety in P'1 position behave as weak inhibitors of the three enzymes, with IC50 values in the micromolar range, and compounds bearing a beta-amino acid moiety in the same position are more specific than the parent compounds for NEP inhibition.