Structure-based design, synthesis and evaluation of conformationally constrained cysteine protease inhibitors

The inhibition of cysteine proteases is being studied as a strategy to combat parasitic diseases such as Chagas' disease, leishmaniasis, and malaria. Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiologic agent of Chagas' disease. A crystal structure of cruzain, covalently inactivated by fluoromethyl ketone inhibitor 1 (Cbz-Phe-Ala-FMK), was used as a template to design potential inhibitors. Conformationally constrained gamma-lactams containing electrophilic aldehyde (12, 17, 18, 25, 26, and 29) or vinyl sulfone (43, 44, and 46) units were synthesized. Constrained lactam 26 had IC50 values of ca. 20 nM against the Leishmania major protease and ca. 50 nM versus falcipain, an important cysteine protease isolated from Plasmodium falciparum. However, all of the conformationally constrained inhibitors were weak inhibitors of cruzain, compared to unconstrained peptide aldehyde (e.g. 5 ) and vinyl sulfone inhibitors (e.g. 48, which proved to be an excellent inhibitor of cruzain with an apparent second order inhibition rate constant (k(inact)/Ki) of 634,000s(-1)M(-1). A significant reduction in activity was also observed with acyclic inhibitors 30 and 51 containing alpha-methyl phenylalanine residues at the P2 position. These data indicate that the pyrrolidinone ring, especially the quarternary center at P2, interferes with the normal substrate binding mode with cruzain, but not with falcipain or the leishmania protease.     

Cyclopropane-derived peptidomimetics. Design, synthesis, evaluation, and structure of novel HIV-1 protease inhibitors

Toward establishing the general efficacy of using trisubstituted cyclopropanes as peptide mimics to stabilize extended peptide structures, the cyclopropanes 20a-d were incorporated as replacements into 9-13, which are analogues of the known HIV-1 protease inhibitors 14 and 15. The syntheses of 20a-d commenced with the Rh2[5(S)-MEPY]4-catalyzed cyclization of the allylic diazoesters 16a-d to give the cyclopropyl lactones 17a-d in high enantiomeric excess. Opening of the lactone moiety using the Weinreb protocol and straightforward refunctionalization of the intermediate amides 18a-d gave 20a-d. A similar sequence of reactions was used to prepare the N-methyl-2-pyridyl analogue 28. Coupling of 20a-d and 28 with the known diamino diol 22 delivered 9-13. Pseudopeptides 9-12 were found to be competitive inhibitors of wild-type HIV-1 protease in biological assays having Kis of 0.31-0.35 nM for 9, 0.16-0.21 nM for 10, 0.47 nM for 11, and 0.17 nM for 12; these inhibitors were thus approximately equipotent to the known inhibitor 14(IC50 = 0.22 nM) from which they were derived. On the other hand 13 (Ki = 80 nM) was a weaker inhibitor than its analogue 15 (Ki = 0.11 nM). The solution structures of 9 and 10 were analyzed by NMR spectroscopy and simulated annealing procedures that included restraints derived from homo- and heteronuclear coupling constants and NOEs; because of the molecular symmetry of9 and 10, a special protocol to treat the NOE data was used. The final structure was checked by restrained and free molecular dynamic calculations using an explicit DMSO solvent box. The preferred solution conformations of 9 and 10 are extended structures that closely resemble the three-dimensional structure of 10 bound to HIV-1 protease as determined by X-ray crystallographic analysis of the complex. This work convincingly demonstrates that extended structures of peptides may be stabilized by the presence of substituted cyclopropanes that serve as peptide replacements. Moreover, the linear structure enforced in solution by the two cyclopropane rings in the pseudopeptides 9-12 appears to correspond closely to the biologically active conformation of the more flexible inhibitors 14 and 15. The present work, which is a combination of medicinal, structural, and quantum chemistry, thus clearly establishes that cyclopropanes may be used as structural constraints to reduce the flexibility of linear pseudopeptides and to help enforce the biologically active conformation of such ligands in solution.     

The Gabriel-Colman rearrangement in biological systems: design, synthesis and biological evaluation of phthalimide and saccharin derivatives as potential mechanism-based inhibitors of human leukocyte elastase, cathepsin G and proteinase 3

Metal ion-promoted binding of Saccharomyces cerevisiae alcohol dehydrogenase to Cibacron Blue 3GA was used for its isolation by affinity precipitation with Eudragit-bound dye. The yeast cells were broken and the cell debris was separated by flocculation with poly(ethylene imine). The supernatant containing the enzyme activity was mixed with Eudragit--Cibacron Blue in the presence of zinc ions. The precipitation of the affinity complex was induced by the addition of 50 mM CaCl2 and a subsequent increase in temperature to 40 degrees C. The enzyme was desorbed by treating the precipitate with iminodiacetic acid solution. The procedure resulted in about 66% recovery of enzyme activity with more than 20-fold purification. Recycling of Eudragit--dye for enzyme purification was also shown to be possible.     

The synthesis of symmetrical and unsymmetrical P1/P1' cyclic ureas as HIV protease inhibitors

Cyclic urea SD146, a potent HIV protease inhibitor bearing a flat resistance profile, possessed poor solubility and bioavailability, which precluded further development of the compound. In an effort to improve upon the pharmacokinetic profile of the compound, several analogs modified at the P1/P1' residues were prepared and evaluated. Several of those compounds displayed significant improvement of physical properties.     

Nonpeptide cyclic cyanoguanidines as HIV-1 protease inhibitors: synthesis, structure-activity relationships, and X-ray crystal structure studies

Comparison of the high-resolution X-ray structures of the native HIV-1 protease and its complexes with the inhibitors suggested that the enzyme flaps are flexible. The movement at the tip of the flaps could be as large as 7 A. On the basis of this observation, cyclic cyanoguanidines have been designed, synthesized, and evaluated as HIV-1 protease (PR) inhibitors. Cyclic cyanoguanidines were found to be very potent inhibitors of HIV-1 protease. The choice of cyclic cyanoguanidines over cyclic guanidines was based on the reduced basicity of the former. X-ray structure studies of the HIV PR complex with cyclic cyanoguanidine demonstrated that in analogy to cyclic urea, cyclic cyanoguanidines also displace the unique structural water molecule. The structure-activity relationship of the cyclic cyanoguanidines is compared with that of the corresponding cyclic urea analogues. The differences in binding constants of the two series of compounds have been rationalized using high-resolution X-ray structure information.     

Cyclic HIV protease inhibitors: design and synthesis of orally bioavailable, pyrazole P2/P2' cyclic ureas with improved potency

Highly potent HIV-1 protease (HIVPR) inhibitors have been designed and synthesized by introducing bidentate hydrogen-bonding oxime and pyrazole groups at the meta-position of the phenyl ring on the P2/P2' substituents of cyclic ureas. Nonsymmetrical cyclic ureas incorporating 3(1H)-pyrazolylbenzyl as P2 and hydrophilic functionalities as P2' show potent protease inhibition and antiviral activities against HIV and have good oral bioavailabilities. The X-ray structure of HIVPR.10A complex confirms that the two pyrazole rings of 10A form bidentate hydrogen bonds with the side-chain oxygen (C=O) and backbone nitrogen (N-H) of Asp30/30' of HIVPR.     

Stereoisomers of cyclic urea HIV-1 protease inhibitors: synthesis and binding affinities

We have synthesized stereoisomers of cyclic urea HIV-1 protease inhibitors to study the effect of varying configurations on binding affinities. Four different synthetic approaches were used to prepare the desired cyclic urea stereoisomers. The original cyclic urea synthesis using amino acid starting materials was used to prepare three isomers. Three additional isomers were prepared by synthetic routes utilizing L-tartaric acid and D-sorbitol as chiral starting materials. A stereoselective hydroxyl inversion of the cyclic urea trans-diol was used to prepare three additional isomers. In all 9 of the 10 possible cyclic urea stereoisomers were prepared, and their binding affinities are described.     

Selective Pneumocystis carinii dihydrofolate reductase inhibitors: design, synthesis, and biological evaluation of new 2,4-diamino-5-substituted-furo[2,3-d]pyrimidines

Nonclassical antifolates, 2,4-diamino-5-substituted-furo[2, 3-d]pyrimidines 3-12 with bridge region variations of C8-S9, C8-N9, and C8-O9 and 1-naphthyl, 2-naphthyl, 2-phenoxyphenyl, 4-phenoxyphenyl, and 2-biphenyl side chains were synthesized as phenyl ring appended analogues of previously reported 2, 4-diamino-5-(anilinomethyl)furo[2,3-d]pyrimidines. The phenyl ring appended analogues were designed to specifically interact with Phe69 of dihydrofolate reductase (DHFR) from Pneumocystis carinii (pc) to afford selective inhibitors of pcDHFR. Additional substituted phenyl side chains which include 2,5-dichloro, 3,4-dichloro, 3,4,5-trichloro, 3-methoxy, and 2,5-dimethoxy analogues 13-17 were also synthesized. The compounds were prepared by nucleophilic displacement of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine(2) with the appropriate thiol, amine, or naphthol. Compound 2 was obtained from 2,4-diamino-6-hydroxypyrimidine and 1, 3-dichloroacetone. The compounds were evaluated as inhibitors against DHFR from P. carinii, Toxoplasma gondii, and rat liver. Two analogues, 2,4-diamino-5-[(2'-naphthylthio)methyl]furo[2, 3-d]pyrimidine (5) and 2,4-diamino-5-[(2'-phenylanilino)methyl]furo[2,3-d]pyrimidine (11) showed significant selectivity and potency for pcDHFR compared to trimethoprim. The X-ray crystal structure of 5 with pcDHFR was also carried out, which corroborated the design rationale and indicated a hydrophobic interaction of the naphthalene ring of 5 and Phe69 of pcDHFR which is responsible, in part, for the more than 18-fold selectivity of 5 for pcDHFR as compared with rat liver DHFR.     

Protein structure-based design, synthesis, and biological evaluation of 5-thia-2,6-diamino-4(3H)-oxopyrimidines: potent inhibitors of glycinamide ribonucleotide transformylase with potent cell growth inhibition

The design, synthesis, biochemical, and biological evaluation of a novel series of 5-thia-2,6-diamino-4(3H)-oxopyrimidine inhibitors of glycinamide ribonucleotide transformylase (GART) are described. The compounds were designed using the X-ray crystal structure of human GART. The monocyclic 5-thiapyrimidinones were synthesized by coupling an alkyl thiol with 5-bromo-2, 6-diamino-4(3H)-pyrimidinone, 20. The bicyclic compounds were prepared in both racemic and diastereomerically pure forms using two distinct synthetic routes. The compounds were found to have human GART KiS ranging from 30 microM to 2 nM. The compounds inhibited the growth of both L1210 and CCRF-CEM cells in culture with potencies down to the low nanomolar range and were found to be selective for the de novo purine biosynthesis pathway. The most potent inhibitors had 2,5-disubstituted thiophene rings attached to the glutamate moiety. Placement of a methyl substituent at the 4-position of the thiophene ring to give compounds 10, 18, and 19 resulted in inhibitors with significantly decreased mFBP affinity.     

Cyclic HIV-1 protease inhibitors derived from mannitol: synthesis, inhibitory potencies, and computational predictions of binding affinities

The molecular characteristics of midbrain dopamine (DA) neurons have been extensively studied in Parkinson's disease (PD). No such studies of the characteristics of midbrain DA neurons in Alzheimer's disease (AD) or Alzheimer's disease with parkinsonism (AD/Park) have been published. We examined the levels of tyrosine hydroxylase (TH) protein, and the expression of TH and dopamine transporter (DAT) mRNAs, in midbrain neurons of PD, AD, and AD/Park cases. In PD, the loss of TH protein in the ventral tier of the substantia nigra pars compacta (SNpc) of the PD group in accompanied by severe losses in the number of neurons that express TH mRNA and DAT mRNA (74% loss). Remaining neurons show a shift to higher concentrations of TH mRNA but a shift to lower concentrations of DAT mRNA per cell. Hence, there is evidence that compensation in the remaining neurons can elevate concentrations of TH mRNA and lower DAT mRNA. Alternatively, there may be a predilection for a loss of neurons with high levels of DAT mRNA and low TH mRNA levels within the SNpc of PD cases. There was no change in TH protein but an elevation of TH mRNA concentrations per neuron without any change in concentrations of DAT mRNA in the AD group. The AD/Park group did not exhibit changes in the level of TH protein, but showed a small loss (26%) of neurons in the SNpc and a greater loss in other regions of the midbrain (43-53%). Remaining DA neurons showed a marked shift to lower concentrations of DAT mRNA per neuron and a nonsignificant shift in cellular concentration of TH mRNA to higher levels. This is consistent with our previous work showing that with AD/Park there is a significant reduction in the number of DAT sites located on DA terminals in the striatum, but the midbrain neurons have not died. Our results indicate that the differential regulation of mRNAs encoding TH and DAT is similar in the parkinsonian disorders (PD and AD/Park) even though the degree of cell death is very different. This might suggest that compensatory events occur in these DA neurons in AD/Park that are similar to those in PD and that result in differential effects on mRNAs encoding TH and DAT proteins.     

3-Alkanoyl-5-hydroxymethyl tetronic acid homologues and resistomycin: new inhibitors of HIV-1 protease. I. Fermentation, isolation and biological activity

In the course of a screening program for HIV-1 protease inhibiting activity, six new homologues of 3-alkanoyl-5-hydroxymethyl tetronic acids (1 approximately 6) and the known natural product resistomycin (7) were isolated from cultures of the Actinomycete strain DSM 7357. The substituted tetronic acids belong to a recently described structural class of secondary metabolites. The HIV-1 activity of resistomycin (7) has not been reported before.     

Structure-based design and synthesis of substituted 2-butanols as nonpeptidic inhibitors of HIV protease: secondary amide series

The design, synthesis, and crystallographic analysis of protein-inhibitor complexes is described for a novel series of nonpeptidic HIV protease (HIV Pr)inhibitors. Beginning with a cocrystal structure of a Phe-Pro peptidomimetic bound to the HIV Pr, design was initiated that resulted in the substituted 2-butanol compound 8 as the lead compound (Ki = 24.5 microM, racemic mixture). Modifications on the initial compound were then made on the basis of its cocrystal structure with HIV Pr and inhibition data, resulting in compounds with enhanced potency against the enzyme (compound 18, Ki = 0.48 microM). These inhibitors were found to bind to the enzyme essentially as predicted on the basis of the original design hypothesis. Stereospecific synthesis of individual enantiomers confirmed the prediction of a binding preference for the S alcohol stereochemistry. Modest antiviral activity was demonstrated for several of the more potent HIV Pr inhibitors in a HIV-1 infected CEM-SS cell line.     

Design, synthesis, and evaluation of tetrahydropyrimidinones as an example of a general approach to nonpeptide HIV protease inhibitors

Re-examination of the design of the cyclic urea class of HIV protease (HIVPR) inhibitors suggests a general approach to designing novel nonpeptide cyclic HIVPR inhibitors. This process involves the inversion of the stereochemical centers of the core transition-state isostere of the linear HIVPR inhibitors and cyclization of the resulting core using an appropriate cyclizing reagent. As an example, this process is applied to the diamino alcohol class of HIVPR inhibitors to give tetrahydropyrimidinones. Conformational analysis of the tetrahydropyrimidinones and modeling of its interaction with the active site of HIVPR suggested modifications which led to very potent inhibitors of HIVPR (24 with a Ki = 0.018 nM). The X-ray crystallographic structure of the complex of 24 with HIVPR confirms the analysis and modeling predictions. The example reported in this study and other examples that are cited indicate that this process may be generally applicable to other linear inhibitors.     

Aromatase inhibitors: synthesis, biological activity, and binding mode of azole-type compounds

The enantiomers of the potent nonsteroidal inhibitor of aromatase fadrozole hydrochloride 3 have been separated and their absolute configuration determined by X-ray crystallography. On the basis of a molecular modeling comparison of the active enantiomer 4 and one of the most potent steroidal inhibitors reported to date, (19R)-10-thiiranylestr-4-ene-3,17-dione, 7, a model describing the relative binding modes of the azole-type and steroidal inhibitors of aromatase at the active site of the enzyme is proposed. It is suggested that the cyanophenyl moiety present in the most active azole inhibitors partially mimics the steroid backbone of the natural substrate for aromatase, androst-4-ene-3,17-dione, 1. The synthesis and biological testing of novel analogues of 3 used to define the accessible and nonaccessible volumes to ligands in the model of the active site of aromatase are reported.     

Synthesis and evaluation of paclitaxel C7 derivatives: solution phase synthesis of combinatorial libraries

A novel and efficient two-step, automated solution phase synthesis of a 26-membered combinatorial chemistry library of paclitaxel C7 esters was accomplished using the HP 7686 Solution Phase Synthesizer. Results of combinatorial synthesis, purification, analysis, and biological evaluation are described.     

Design and synthesis of new potent C2-symmetric HIV-1 protease inhibitors. Use of L-mannaric acid as a peptidomimetic scaffold

A study on the use of derivatized carbohydrates as C2-symmetric HIV-1 protease inhibitors has been undertaken. L-Mannaric acid (6) was bis-O-benzylated at C-2 and C-5 and subsequently coupled with amino acids and amines to give C2-symmetric products based on C-terminal duplication. Potent HIV protease inhibitors, 28 Ki = 0.4 nM and 43 Ki = 0.2 nM, have been discovered, and two synthetic methodologies have been developed, one whereby these inhibitors can be prepared in just three chemical steps from commercially available materials. A remarkable increase in potency going from IC50 = 5000 nM (23) to IC50 = 15 nM (28) was observed upon exchanging -COOMe for -CONHMe in the inhibitor, resulting in the net addition of one hydrogen bond interaction between each of the two -NH- groups and the HIV protease backbone (Gly 48/148). The X-ray crystal structures of 43 and of 48 have been determined (Figures 5 and 6), revealing the binding mode of these inhibitors which will aid further design.     

Characterization of the three-dimensional solution structure of human profilin: 1H, 13C, and 15N NMR assignments and global folding pattern

Human profilin is a 15-kDa protein that plays a major role in the signaling pathway leading to cytoskeletal rearrangement. Essentially complete assignment of the 1H, 13C, and 15N resonances of human profilin have been made by analysis of multidimensional, double- and triple-resonance nuclear magnetic resonance (NMR) experiments. The deviation of the 13C alpha and 13C beta chemical shifts from their respective random coil values were analyzed and correlate well with the secondary structure determined from the NMR data. Twenty structures of human profilin were refined in the program X-PLOR using a total of 1186 experimentally derived conformational restraints. The structures converged to a root mean squared distance deviation of 1.5 A for the backbone atoms. The resultant conformational ensemble indicates that human profilin is an alpha/beta protein comprised of a seven-stranded, antiparallel beta-sheet and three helices. The secondary structure elements for human profilin are quite similar to those found in Acanthamoeba profilin I [Archer, S. J., Vinson, V. K., Pollard, T. D., & Torchia, D. A. (1993), Biochemistry 32, 6680-6687], suggesting that the three-dimensional structure of Acanthamoeba profilin I should be analogous to that determined here for human profilin. The structure determination of human profilin has facilitated the sequence alignment of lower eukaryotic and human profilins and provides a framework upon which the various functionalities of profilin can be explored. At least one element of the actin-binding region of human profilin is an alpha-helix. Two mechanisms by which phosphatidylinositol 4,5-bisphosphate can interfere with actin-binding by human profilin are proposed.