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Background: Tuberculosis (TB) has been a challenging disease worldwide, especially for the neglected poor populations. Presently, there are approximately 2 billion people infected with TB worldwide and 10 million people in the world fell ill with active TB, leading to 1.5 million deaths. Introduction: The classic treatment is extensive and the drug- and multi-drug resistance of Mycobacterium tuberculosis has been a threat to the efficacy of the drugs currently used. Therefore, the rational design of new anti-TB candidates is urgently needed. Methods: With the aim of contributing to face this challenge, 78 compounds have been proposed based on SBDD (Structure-Based Drug Design) strategies applied to target the M. tuberculosis phosphopantetheine adenylyltransferase (MtPPAT) enzyme. Ligand-Based Drug Design (LBDD) strategies were also used for establishing Structure-Activity Relationships (SAR) and for optimizing the structures. MtPPAT is important for the biosynthesis of coenzyme A (CoA) and it has been studied recently toward the discovery of new inhibitors. Results: After docking simulations and enthalpy calculations, the interaction of selected compounds with MtPPAT was found to be energetically favorable. The most promising compounds were then synthesized and submitted to anti-M. tuberculosis and MtPPAT inhibition assays. Conclusion: One of the compounds synthesized (MCP163), showed the highest activity in both of these assays.

This study represents a systematic chemical and biological study of the rufomycin (RUF) class of cyclic heptapeptides, which our anti-TB drug discovery efforts have identified as potentially promising anti-TB agents that newly target the caseinolytic protein C1, ClpC1. Eight new RUF analogues, rufomycins NBZ1-NBZ8 (1-8), as well as five known peptides (9-13) were isolated and characterized from the Streptomyces atratus strain MJM3502. Advanced Marfey's and X-ray crystallographic analysis led to the assignment of the absolute configuration of the RUFs. Several isolates exhibited potent activity against both pathogens M. tuberculosis H37Rv and M. abscessus, paired with favorable selectivity (selectivity index >60), which collectively underscores the promise of the rufomycins as potential anti-TB drug leads.

Residual complexity (RC) involves the impact of subtle but critical structural and biological features on drug lead validation, including unexplained effects related to unidentified impurities. RC commonly plagues drug discovery efforts due to the inherent imperfections of chromatographic separation methods. The new diketopiperazine, rufomyazine (6), and the previously known antibiotic, rufomycin (7), represent a prototypical case of RC that (almost) resulted in the misassignment of biological activity. The case exemplifies that impurities well below the natural abundance of 13C (1.1%) can be highly relevant and calls for advanced analytical characterization of drug leads with extended molar dynamic ranges of >1:1,000 using qNMR and LC-MS. Isolated from an actinomycete strain, 6 was originally found to be active against Mycobacterium tuberculosis with a minimum inhibitory concentration (MIC) of 2 ��g/mL and high selectivity. As a part of lead validation, the dipeptide was synthesized and surprisingly found to be inactive. The initially observed activity was eventually attributed to a very minor contamination (0.24% [m/m]) with a highly active cyclic peptide (MIC ��� 0.02 ��M), subsequently identified as an analogue of 7. This study illustrates the serious implications RC can exert on organic chemistry and drug discovery, and what efforts are vital to improve lead validation and efficiency, especially in NP-related drug discovery programs.

New anti-tuberculosis (anti-TB) drugs are urgently needed to battle drug-resistant Mycobacterium tuberculosis strains and to shorten the current 6-12-month treatment regimen. In this work, we have continued the efforts to develop chalcone-based anti-TB compounds by using an in silico design and QSAR-driven approach. Initially, we developed SAR rules and binary QSAR models using literature data for targeted design of new heteroaryl chalcone compounds with anti-TB activity. Using these models, we prioritized 33 compounds for synthesis and biological evaluation. As a result, 10 heteroaryl chalcone compounds (4, 8, 9, 11, 13, 17-20, and 23) were found to exhibit nanomolar activity against replicating mycobacteria, low micromolar activity against nonreplicating bacteria, and nanomolar and micromolar against rifampin (RMP) and isoniazid (INH) monoresistant strains (rRMP and rINH) (

The TLC-based Generally Useful Estimate of Solvent Systems (GUESS) method was employed for countercurrent chromatography solvent system selection, in order to separate the three synthetic isomers: 3-O-methylpyridoxine, 4′-O-methylpyridoxine (ginkgotoxin) and 5′-O-methylpyridoxine. The Rf values of the three isomers indicated that ChMWat +2 (chloroformmethanol-water 10:5:5, v/v/v) was appropriate for the countercurrent chromatography. The isomer separation was highly selective and demonstrated that the TLC-based GUESS method can accelerate solvent system selection for countercurrent chromatography. Accordingly, the study re-emphasizes the practicality of TLC as a tool to facilitate the rapid development of new countercurrent and centrifugal partition chromatography methods for this solvent system. Purity and structure characterization of all samples was performed by quantitative 1H NMR.

A new strategy for the analysis of natural products uses a combination of quantitative (1)H NMR (qHNMR) and adsorbent-free countercurrent separation (CS) methodology to establish a quantification method for ginkgotoxin (4'-O-methylpyridoxine) in Ginkgo biloba preparations. The target analyte was concentrated in a one-step CS process using the ChMWat +2 solvent system (CHCl3-MeOH-H2O, 10:5:5) and subsequently assayed by qHNMR. While commercial G. biloba seeds contained 59 μg of ginkgotoxin per seed, the compound was below the limit of detection (9 ppm) in a typical leaf extract. Due to the enrichment potential and loss-free operation of CS, the combination of CS and qHNMR is a generally suitable approach for threshold assays aimed at quantifying target compounds such as botanical negative markers at the low ppm level. As the proof of principle is demonstrated for relatively small CS capacities (20 mL, 1:40 loading) and modest NMR sensitivity (n = 16, 400 MHz, 5 mm RT probe), the approach can be adapted to quantification at the ppb level. The procedure enables the quantification of a botanical negative marker in the absence of identical reference material, which otherwise is a prerequisite for LC-based assays.

Methyl 3-azidomethyl-5-azido-benzoate (1b) and its corresponding hydroxymethyl reduction product (1a) have been utilized for the synthesis of valuable nonradioactive photoaffinity probes. Previous ...

Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistant mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.

The structure of a novel indigoid component was characterized by X-ray crystallography. This compound exhibited excellent anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv in whole cell culture showing a submicromolar minimum inhibitory concentration (MIC). A synthesis of this molecule was designed and carried out to produce sufficient material for further testing. The in vitro profile, structure, and first synthesis of this indigoid component is reported.

Benzothiadiazine inhibitors of the HCV NS5B RNA-dependent RNA polymerase are an important class of non-nucleoside inhibitors that have received considerable attention in the search for novel HCV therapeutics. Research in our laboratories has identified a novel series of tetracyclic benzothiadiazine inhibitors of HCV polymerase bearing a benzylamino substituent on the B-ring. Compounds in this series exhibit low-nanomolar activities in both genotypes 1a and 1b polymerase inhibition assays and subgenomic replicon assays. Optimization of pharmacokinetic properties in rat led to compound 30, which has good oral bioavailability (F = 56%) and a favorable tissue distribution drug profile, with high liver to plasma ratios. Compound 30 is a potent inhibitor in replicon assays, with EC(50) values of 10 and 6 nM against genotypes 1a and 1b, respectively.

A-790742 is a potent human immunodeficiency virus type 1 (HIV-1) protease inhibitor, with 50% effective concentrations ranging from 2 to 7 nM against wild-type HIV-1. The activity of this compound is lowered by approximately sevenfold in the presence of 50% human serum. A-790742 maintained potent antiviral activity against lopinavir-resistant variants generated in vitro as well as against a panel of molecular clones containing proteases derived from HIV-1 patient isolates with multiple protease mutations. During in vitro selection, A-790742 selected two primary mutations (V82L and I84V) along with L23I, L33F, K45I, A71V/A, and V77I in the pNL4-3 background and two other mutations (A71V and V82G) accompanied by M46I and L63P in the HIV-1 RF background. HIV-1 pNL4-3 clones with a single V82L or I84V mutation were phenotypically resistant to A-790742 and ritonavir. Taking these results together, A-790742 displays a favorable anti-HIV-1 profile against both the wild type and a large number of mutants resistant to other protease inhibitors. The selection of the uncommon V82L and V82G mutations in protease by A-790742 suggests the potential for an advantageous resistance profile with this protease inhibitor.

Inhibitors of hepatitis C virus (HCV) protease have shown marked antiviral activity in short-term clinical studies in HCV-infected individuals. The interaction of the investigational HCV protease inhibitors VX-950 and SCH 503034 with ritonavir, a potent inhibitor of cytochrome P450 3A, was studied in vitro and in vivo. In rat and human liver microsomes, the metabolism of VX-950 and SCH 503034 was strongly inhibited by the presence of 4 µM ritonavir. Upon co-dosing either VX-950 or SCH 503034 with ritonavir in rats, plasma exposure of the HCV protease inhibitors was increased by >15-fold, and plasma concentrations 8 h after dosing were increased by >50-fold. A human pharmacokinetic model of VX-950 co-administered with low-dose ritonavir suggested that improved efficacy and/or dosing convenience may be feasible by pharmacokinetic enhancement with ritonavir.

Our recent efforts at finding new anti-tuberculosis drug leads from actinomycetes have led to the discovery of ecumicin (ECU, MW 1599.22, MIC 0.16 uM) and; more recently, a rufomycin analogue (RUF-I, MW 1041.55, MIC 0.019 uM). Although both of these cyclic peptides target the currently unexploited chaperone protein ClpC1 ATPase complex, there is no observed cross resistance. In this study, the structures of RUF-I and other rufomycin analogues, heptapeptides, were elucidated by 1H, 13C, 2D NMR, MS and HiFSA. To understand the lack of cross resistance, clpC1 from spontaneously generated resistant clones from both leads were sequenced. This revealed several distinct single point mutations, suggesting these compounds bind differently to ClpC1. Checkerboard assay indicated a lack of antagonism between ECU and RUF-I and potential synergistic effects. Preliminary data indicates that ClpC1 could be a multifaceted drug target for multidrug resistant M. tuberculosis. Research pertaining to the peptide drug leads, associated analogues, and their mechanism of action against ClpC1 will be presented.

A new series of HIV protease inhibitors has been designed and synthesized based on the combination of the (R)-(hydroxyethylamino)sulfonamide isostere and the cyclic urea component of lopinavir. The series was optimized by replacing the 6-membered cyclic urea linker with an imidazolidine-2,4-dione which readily underwent N-alkylation to incorporate various methylene-linked heterocycle groups that bind favorably in site 3 of HIV protease. Significant improvements compared to lopinavir were seen in cell culture activity versus wild-type virus (pNL4-3) and the lopinavir-resistant mutant virus A17 (generated by in vitro serial passage of HIV-1 (pNL4-3) in MT-4 cells). Select imidazolidine-2,4-dione containing PIs were also more effective at inhibiting highly resistant patient isolates Pt1 and Pt2 than lopinavir. Pharmacokinetic data collected for compounds in this series varied considerably when coadministered orally in the rat with an equal amount of ritonavir (5 mg/kg each). The AUC values ranged from 0.144 to 12.33 microg h/mL.

As part of our efforts to identify potent HIV-1 protease inhibitors that are active against resistant viral strains, structural modification of the azacyclic urea (I) was undertaken by incorporating acyl groups as P1′ ligands. The extensive SAR study has yielded a series of N-acyl azacyclic ureas (II), which are highly potent against both wild-type and multiple PI-resistant viral strains.

A series of novel azacyclic urea HIV protease inhibitors bearing a benzenesulfonamide group at P1′ were synthesized utilizing a parallel synthesis method. Structural studies of early analogs bound in the enzyme active site were used to design more potent inhibitors. The effects of substituting the P1′ benzenesulfonyl group on antiviral activity and protein binding are described.

BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection. The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients. The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug. Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization. The 50% inhibitory concentrations of BILN 2061 for these colonies were 72- to 1,228-fold higher than that for the wild-type replicon. Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene. Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon. Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site. These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease.

The need for new therapies to treat systemic fungal infections continues to rise. Naturally occurring hexapeptide echinocandin B (1) has shown potent antifungal activity via its inhibition of the synthesis of beta-1,3 glucan, a key fungal cell wall component. Although this series of agents has been limited thus far based on their physicochemical characteristics, we have found that the synthesis of analogues bearing an aminoproline residue in the 'northwest' position imparts greatly improved water solubility (> 5 mg/mL). The synthesis and structure-activity relationships (SAR) based on whole cell and upon in vivo activity of the series of compounds are reported.

A series of new and potent motilides, 8( R ),9( R )-9-deoxo-4''-deoxy-6,9-epoxyerythromycin A was designed, synthesized and evaluated for gastrointestinal prokinetic activity. These compounds were acid-stable with many demonstrating in vitro potency markedly superior to erythromycin.

Variation of the C-1 position of the pyridopyridone antibacterials shows a sensitivity to both size and electronic character of the substituent.

An EtOH extract of the polypore mushroom Fomitopsis officinalis afforded two new naturally occurring chlorinated coumarins, which were identified as the previously synthesized compounds 6-chloro-4-phenyl-2H-chromen-2-one (1) and ethyl 6-chloro-2-oxo-4-phenyl-2H-chromen-3-carboxylate (2). The structures of the two isolates were deduced by ab initio spectroscopic methods and confirmed by chemical synthesis. In addition, an analogue of each was synthesized as 7-chloro-4-phenyl-2H-chromen-2-one (3) and ethyl 7-chloro-2-oxo-4-phenyl-2H-chromen-3-carboxylate (4). All four compounds were characterized physicochemically, and their antimicrobial activity profiles revealed a narrow spectrum of activity with lowest MICs against the Mycobacterium tuberculosis complex.

Acid-promoted cyclization of alkoximinoacetanilides is developed to provide a convenient synthesis of a broad spectrum of isatins bearing lipophilic substituents.

The preparation of C-6′ ester analogs of fusacandin ( 2 ) is described leading to derivatives exhibiting improved in vitro activity in the presence of mouse serum.

The synthesis of 9-deoxotaxol (9) ,7,9-dideoxy and 7,9,10-trideoxygenated analogs is described starting from 13-acetyl-9(R)-dihydrobaccatin III.

C-3′-N-Acyl analogs of 9(R)-dihydrotaxol were synthesized from 7-triethylsilyl-9(R)-dihydrobaccatin III and the corresponding (3R,4S)-N-acyl-3-(1-ethoxyethoxy)-4-phenylazetidin-2-ones. The analogs were tested in a microtubule assembly assay, and in an invitro cytotoxicity assay. The highest activities observed were for the alkylcarbamate substitutions.

Reaction of the C-7 hydroryl group on the 9-dihydrotaxane skeleton with triflic anhydride causes a major skeletal rearrangement to occur leading to contraction of ring B. In addition the formation of a ring C-fused cyclopropane structure occurs. The requisite C-13 phenylisoserinate side chins are appended via an initial deacylation of the C-13 acetate followed by reacylation and deprotection. These rearranged compounds show very similar structural features with the parent 9-dihydrotaxane skeleton and also retain biological activity

The synthesis of 9-dihydrotaxol ( 8 ) is described via a novel deacylation reaction. This analog exhibits increased activity in the microtubule assay.

The two step synthesis of 1,3-benzodithiol-2-ylidene compounds such as 4 is described starting from various aromatic thiols 1 .

The preparation of 1,3,2-benzodithiazole S-oxide analogs exhibiting in vitro antifungal activity against several strains of Candida is described. For the preparation of derivatives bearing aromatic substituents, a novel electrophilic aromatic thiolation reaction was utilized which produced substituted aromatic 1,2-dithiol intermediates. The reactions of nucleophiles with the parent heterocyclic system have led to an efficient transamidation process which allows for the direct production of these analogs. The S-oxide bond exhibits poor stereochemical stability and has been found to epimerize under ambient conditions. The structure-activity data report that a side chain of greater than 10 carbons effects a loss in activity as does the placement of polar groups in this chain.

A series of new and potent motilides, 8( R ),9( R )-9-deoxo-4''-deoxy-6,9-epoxyerythromycin A was designed, synthesized and evaluated for gastrointestinal prokinetic activity. These compounds were acid-stable with many demonstrating in vitro potency markedly superior to erythromycin.

Patients treated with highly active antiretroviral therapy may develop metabolic side effects such as hyperlipidemia, insulin resistance, lipoatrophy and lactic acidosis. The pathophysiology of these metabolic abnormalities is unknown, although some, e.g., lactic acidosis and lipoatrophy, are more associated with nucleoside use while protease inhibitors (PIs) have been shown to contribute to hyperlipidemia and insulin resistance. Identifying new PIs that are not associated with dyslipidemia has been hindered by the lack of mechanistic information and the unavailability of relevant animal models. In order to understand the molecular mechanism behind the hyperlipidemia associated with other protease inhibitors, and to develop a more effective, faster screen for compounds with this liability, we have analyzed expression profiles from PI-treated animals. Previously, we have shown that treatment of rats with ritonavir results in increases in the expression of proteasomal subunit genes in the liver. We show this increase is similar in rats treated with bortezomib, a proteasome inhibitor. In addition, we have treated rats with additional protease inhibitors, including atazanavir, which is associated with lower rates of lipid elevations in the clinic when administered in the absence of ritonavir. Our results indicate a strong correlation between proteasomal induction and lipid elevations, and have allowed us to develop a rapid screen for identifying novel PIs that do not induce the proteasome.

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2-P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.

We studied the synthesis, cleavage rates, and oral administration of prodrugs of the HIV protease inhibitors (PIs) lopinavir and ritonavir. Phosphate esters attached directly to the central hydroxyl groups of these PIs did not demonstrate enzyme-mediated cleavage in vitro and did not provide measurable plasma levels of the parent drugs in vivo. However, oxymethylphosphate (OMP) and oxyethylphosphate (OEP) prodrugs provided improved rates of cleavage, high levels of aqueous solubility, and high plasma levels of the parent drugs when dosed orally in rats and dogs. Dosing unformulated capsules containing the solid prodrugs led to plasma levels equivalent to those observed for dosing formulated solutions of the parent drugs. A direct synthetic process for the preparation of OMP and OEP prodrugs was developed, and the improved synthetic method may be applicable to the preparation of analogous soluble prodrugs of other drug classes with limited solubility.

A series of symmetry-based HIV protease inhibitors was designed and synthesized. Modification of the core regiochemistry and stereochemistry significantly affected the potency, metabolic stability, and oral bioavailability of the inhibitors, as did the variation of a pendent arylmethyl P3 group. Optimization led to the selection of two compounds, 10c (A-790742) and 9d (A-792611), for advancement to preclinical studies. Both compounds displayed low nanomolar potency against wild type HIV in the presence of human serum, low rates of metabolism in human liver microsomes, and high oral bioavailability in animal models. The compounds were examined in a preclinical model for the hyperbilirubinemia observed with some HIV PIs, and both exhibited less bilirubin elevation than comparator compounds. X-ray crystallographic analyses of the new cores were used to examine differences in their binding modes. The antiviral activity of the compounds against protease inhibitor resistant strains of HIV was also determined.

The hepatitis C virus (HCV) NS5B polymerase is essential for viral replication and has been a prime target for drug discovery research. Our efforts directed toward the discovery of HCV polymerase inhibitors resulted in the identification of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines 2 and 3. The most active compound displayed activity in genotypes 1a and 1b polymerase and replicon cell culture inhibition assays at subnanomolar and low nanomolar concentrations, respectively. It also displayed an excellent pharmacokinetic profile in rats, with a plasma elimination half-life after intravenous dosing of 4.5 h, oral bioavailability of 77%, and a peak liver concentration of 21.8 microg/mL.

Several ring-contracted analogues of the antitumor agent etoposide have been prepared. The synthesis of the simple indanyl system 3 is described along with two bicyclic systems of general structure 4 prepared through a stereoselective allylation of the keto-ester 6. A cis-fused lactone analogue 5, which is isomeric with the etoposide aglycone, has been synthesized via a dialkylation of the indene-2-carboxylate anion. Regiochemical and stereochemical results of these alkylations are described. The cytotoxicity of these derivatives toward several tumor cell lines is described and generally follows the structure-activity relationships known for the agent podophyllotoxin (2).

4,4-Dialkyl-1-hydroxy-3-oxo-3.4-dihydronaphthalene-3-yl benzothiadiazine derivatives were synthesized and evaluated as inhibitors of genotypes 1a and 1b HCV NS5B polymerase. A number of these compounds exhibited potent activity against genotypes 1a and 1b HCV polymerase in both enzymatic and cell culture activities. A representative compound also showed favorable pharmacokinetics in the rat.

The two step synthesis of 1,3-benzodithiol-2-ylidene compounds such as 4 is described starting from various aromatic thiols 1 .

BILN 2061 is a macrocyclic tripeptide inhibitor of hepatitis C virus NS3-4A protease that has shown efficacy in the clinic for treating patients infected with HCV. We have synthesized a P3 aza-peptide analog of a potent macrocyclic tripeptide inhibitor closely related to BILN 2061. This aza-derivative was found to be >2 orders of magnitude less active than the parent macrocycle in both isolated enzyme (HCV NS3-4A) and HCV subgenomic replicon assays. NMR studies of P3 aza-peptides revealed these compounds adopt a beta-turn conformation stabilized by an intramolecular H-bonding interaction. Molecular models of these structures indicate a D-like configuration of the P3 aza-residue. Thus, the configurationally undefined nature at P3 in the aza-peptide allows the compound to adopt an H-bond stabilized conformation that is substantially different from that necessary for tight binding to the active site of HCV NS3 protease.

The major objective of the antifungal drug program at Abbott Laboratories was to develop a non-toxic drug, superior to amphotercin B, which could be used to treat the life threatening fungal infections. One such drug candidate (I) originated from the highly functionalized antifungal natural product echinocandins [1,2]. It showed high antifungal activity with the least toxicity. The key components of this candidate are a cyclic hexapeptide core and a lipophilic side chain (R). An efficient synthesis of this cyclic hexapeptide has been developed for the purpose of preparing multigram quantities of the product for SAR studies. The cyclic hexapeptide is composed of (2S, 4S)-4-amino-proline (Amp), two threonines, 4-hydroxyproline, homotyrosine (hTyr) and ornithine. The two segments selected for synthesis were Boc-Orn-Thr-Hyp-OH (II) and Fmoc-hTyr-Thr-AzP-OMe (III). Strategically, these segments were selected because they contain C-terminal proline derivatives, known to be less sensitive to racemization during segment condensation. In the synthetic scheme the 4-amino-proline was introduced as 4-azido-proline (Azp). Homotyrosine, now it is commercially available, was prepared by literature procedure [3,4] and converted to Fmoc-hTyr-OH by a standard procedure. Boc-Azp-OMe was prepared starting from Boc-Hyp-OMe. Boc-Hyp-OMe was converted to mesyl derivative by treatment with MS-Cl/pyridine. The mesyl derivative was then converted to Boc-Azp-OMe by treatment with sodium azide in DMF. Now this derivative is also available commercially. Segment II was prepared by a stepwise approach starting from Hyp-OBzl. HypOBzl was coupled with Z-Thr-OH utilizing the standard EDC/HOBt method to yield ZThr-Hyp-OBzl as a solid material. This dipeptide on hydrogenation and coupling with Boc-Orn(Fmoc)-OSu gave crude segment II. The crude peptide was purified by silica gel chromatography to give pure crystalline peptide II. The synthesis of segment III was also used the stepwise approach starting from Azp-OMe. The dipeptide Z-Thr-Azp-OMe was obtained as a crystalline solid by coupling Boc-Thr-OH with Azp-OMe with EDC/HOBt. The Boc group was removed from this dipeptide and the resulting product coupled with Fmoc-hTyr-OH using EDC/HOBt yielding segment III as a solid material. The removal of Fmoc from III followed by coupling with segment II using EEDQ produced the crude linear hexapeptide, which was then purified by silica gel chromatography. The linear

A practical preclinical model for the hyperbilirubinemia produced by human immunodeficiency virus protease inhibitors has been developed. Indinavir and atazanavir produced significant hyperbilirubinemia, whereas amprenavir, the negative control, was indistinguishable from the ritonavir booster dose. This model was used to disqualify an exploratory protease inhibitor from development.

As a continuation of the recently communicated discovery of oximinoarylsulfonamides as potent inhibitors of HIV-1 aspartyl protease, compounds bearing pyridylmethyl substituents at P3 were designed and synthesized. Potent analogs in this series provided low single-digit nanomolar EC50 values against both wild-type HIV and resistant mutant virus (A17), attenuated some 3- to 12-fold in the presence of 50% human serum. Pharmacokinetic results for compounds in this series showed good to excellent exposure when co-administered orally with an equal amount of ritonavir (5mg/kg each) in the rat, with average AUC8 microg h/mL. Similar dosing in dog resulted in significantly lower plasma levels (average AUC2 microg h/mL). The 3-pyridylmethyl analog 30 gave the best overall exposure (rat AUC=7.1 microg h/mL and dog AUC=4.9 microg h/mL), however, this compound was found to be a potent inhibitor of cytochrome P450 3A (Ki=2.4 nM).