Biomedical subjects
Publications and source records attributed to L T Bacheler.
A series of 4,4-disubstituted quinolinones was prepared and evaluated as HIV-1 reverse transcriptase inhibitors. The C-3 substituted compound 9h displayed improved antiviral activity against clinically significant single (K103N) and double (K103N/L100I) mutant viruses.
A series of 4,1-benzoxazepinone analogues of efavirenz (Sustiva) as potent NNRTIs has been discovered. The cis-3-alkylbenzoxazepinones are more potent then the trans isomers and can be synthesized preferentially by a novel stereoselective cyclization. The best compounds are potent orally bioavailable inhibitors of both wild-type HIV-1 and its clinically relevant K103N mutant virus, but are highly protein-bound in human plasma.
Two series of efavirenz analogues have been developed: one in which the cyclopropane ring has been replaced by small heterocycles and another in which the entire acetylenic side chain has been replaced by alkyloxy groups. Several members of both series show equivalent potency to efavirenz against both wild-type virus and the key K103N mutant.
A series of 4-alkenyl and 4-alkynyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones were found to be potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) of human immunodeficiency virus type-1 (HIV-1). The 4-alkenyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones DPC 082 and DPC 083 and the 4-alkynyl-3, 4-dihydro-4-(trifluoromethyl)-2-(1H)-quinazolinones DPC 961 and DPC 963 were found to exhibit low nanomolar potency toward wild-type RF virus (IC(90) = 2.0, 2.1, 2.0, and 1.3 nM, respectively) and various single and many multiple amino acid substituted HIV-1 mutant viruses. The increased potency is combined with favorable plasma serum protein binding as demonstrated by improvements in the percent free drug in human plasma when compared to efavirenz: 3.0%, 2.0%, 1.5%, 2. 8%, and 0.2-0.5% for DPC 082, DPC 083, DPC 961, DPC 963, and efavirenz, respectively.
Efavirenz is a potent and selective nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Nucleotide sequence analyses of the protease and RT genes (coding region for amino acids 1 to 229) of multiple cloned HIV-1 genomes from virus found in the plasma of patients in phase II clinical studies of efavirenz combination therapy were undertaken in order to identify the spectrum of mutations in plasma-borne HIV-1 associated with virological treatment failure. A K103N substitution was the HIV-1 RT gene mutation most frequently observed among plasma samples from patients for whom combination therapy including efavirenz failed, occurring in at least 90% of cases of efavirenz-indinavir or efavirenz-zidovudine (ZDV)-lamivudine (3TC) treatment failure. V108I and P225H mutations were observed frequently, predominantly in viral genomes that also contained other nonnucleoside RT inhibitor (NNRTI) resistance mutations. L100I, K101E, K101Q, Y188H, Y188L, G190S, G190A, and G190E mutations were also observed. V106A, Y181C, and Y188C mutations, which have been associated with high levels of resistance to other NNRTIs, were rare in the patient samples in this study, both before and after exposure to efavirenz. The spectrum of mutations observed in cases of virological treatment failure was similar for patients initially dosed with efavirenz at 200, 400, or 600 mg once a day and for patients treated with efavirenz in combination with indinavir, stavudine, or ZDV-3TC. The proportion of patients carrying NNRTI resistance mutations, usually K103N, increased dramatically at the time of initial viral load rebound in cases of treatment failure after exposure to efavirenz. Viruses with multiple, linked NNRTI mutations, especially K103N-V108I and K103N-P225H double mutants, accumulated more slowly following the emergence of K103N mutant viruses.
The preparation of unsymmetrical cyclic ureas bearing novel biaryl indazoles as P2/P2' substituents was undertaken, utilizing a Suzuki coupling reaction as the key step. Compound 6i was equipotent to the lead compound of the series SE063.
A research program targeted toward the identification of expanded-spectrum nonnucleoside reverse transcriptase inhibitors which possess increased potency toward K103N-containing mutant human immunodeficiency virus (HIV) and which maintain pharmacokinetics consistent with once-a-day dosing has resulted in the identification of the 4-cyclopropylalkynyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 961 and DPC 963 and the 4-cyclopropylalkenyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 082 and DPC 083 for clinical development. DPC 961, DPC 963, DPC 082, and DPC 083 all exhibit low-nanomolar potency toward wild-type virus, K103N and L100I single-mutation variants, and many multiply amino acid-substituted HIV type 1 mutants. This high degree of potency is combined with a high degree of oral bioavailability, as demonstrated in rhesus monkeys and chimpanzees, and with plasma serum protein binding that can result in significant free levels of drug.
Using the structural information gathered from the X-ray structures of various cyclic urea/HIVPR complexes, we designed and synthesized many nonsymmetrical P2/P2'-substituted cyclic urea analogues. Our efforts concentrated on using an indazole as one of the P2 substituents since this group imparted enzyme (Ki) potency as well as translation into excellent antiviral (IC90) potency. The second P2 substituent was used to adjust the physical and chemical properties in order to maximize oral bioavailability. Using this approach several very potent (IC90 11 nM) and orally bioavailable (F% 93-100%) compounds were discovered (21, 22). However, the resistance profiles of these compounds were inadequate, especially against the double (I84V/V82F) and ritonavir-selected mutant viruses. Further modification of the second P2 substituent in order to increase H-bonding interactions with the backbone atoms of residues Asp 29, Asp 30, and Gly 48 led to analogues with much better resistance profiles. However, these larger analogues were incompatible with the apparent molecular weight requirements for good oral bioavailability of the cyclic urea class of HIVPR inhibitors (MW < 610).
Resistance of HIV-1 to protease inhibitors has been associated with changes at residues Val82 and Ile84 of HIV-1 protease (HIV PR). Using both an enzyme assay with a peptide substrate and a cell-based infectivity assay, we examined the correlation between the inhibition constants for enzyme activity (Ki values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors. Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identified as conferring resistance during in vitro selection using a protease inhibitor. The mutant protease genes were expressed in Escherichia coli for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity. The inhibitors included saquinavir, indinavir, nelfinavir, 141W94, ritonavir (all in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3, P3' groups. The single mutations V82F and I84V caused changes with various inhibitors ranging from 0.3- to 86-fold in Ki and from 0.1- to 11-fold in IC90. Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for Ki (10-2000-fold) and for IC90 (0.7-377-fold). However, there were low correlations (r2 = 0.017-0.53) between the mutant/wild-type ratio of Ki values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiviral resistance) for each of the HIV proteases and the viruses containing the identical enzyme. Assessing enzyme resistance by "vitality values", which adjust the Ki values with the catalytic efficiencies (kcat/Km), caused no significant improvement in the correlation with antiviral resistance. Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of the antiviral effect in resistant viruses even when mutations are restricted to the protease gene.
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.
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.
Two series of cyclic ureas modified at the P1/P1' residue were prepared and evaluated for HIV protease inhibition and whole cell antiviral activity. Compounds 8b, 10 (3- and 4-pyridylmethyl analogs) and 6b (4-methoxy analog) showed significant improvement in antiviral activity relative to lead compounds DMP323 and DMP 450.
BACKGROUND: Recent clinical trials have demonstrated that HIV protease inhibitors are useful in the treatment of AIDS. It is necessary, however, to use HIV protease inhibitors in combination with other antiviral agents to inhibit the development of resistance. The daunting ability of the virus to rapidly generate resistant mutants suggests that there is an ongoing need for new HIV protease inhibitors with superior pharmacokinetic and efficacy profiles. In our attempts to design and select improved cyclic urea HIV protease inhibitors, we have simultaneously optimized potency, resistance profile, protein binding and oral bioavailability. RESULTS: We have discovered that nonsymmetrical cyclic ureas containing a 3-aminoindazole P2 group are potent inhibitors of HIV protease with excellent oral bioavailability. Furthermore, the 3-aminoindazole group forms four hydrogen bonds with the enzyme and imparts a good resistance profile. The nonsymmetrical 3-aminoindazoles DMP 850 and DMP 851 were selected as our next generation of cyclic urea HIV protease inhibitors because they achieve 8 h trough blood levels in dog, with a 10 mg/kg dose, at or above the protein-binding-adjusted IC90 value for the worst single mutant--that containing the Ile84-->Val mutation. CONCLUSIONS: In selecting our next generation of cyclic urea HIV protease inhibitors, we established a rigorous set of criteria designed to maximize chances for a sustained antiviral effect in HIV-infected individuals. As DMP 850 and DMP 851 provide plasma levels of free drug that are sufficient to inhibit wild-type HIV and several mutant forms of HIV, they could show improved ability to decrease viral load for clinically significant time periods. The ultimate success of DMP 850 and DMP 851 in clinical trials might depend on achieving or exceeding the oral bioavailability seen in dog.
We present several novel P1/P1' substituents that can replace the characteristic benzyl P1/P1' moiety of the cyclic urea based HIV protease inhibitor series. These substituents typically provide 5-10-fold improvements in binding affinity compared to the unsubstituted benzyl analogs. The best substituent was the 3,4-(ethylenedioxy)benzyl group. Proper balancing of the molecule's lipophilicity facilitated the transfer of this improved binding affinity into a superior cellular antiviral activity profile. Several analogs were evaluated further for protein binding and resistance liabilities. Compound 18 (IC90 = 8.7 nM) was chosen for oral bioavailability studies based on its log P and solubility profile. A 10 mg/kg dose in dogs provided modest bioavailability with Cmax = 0.22 microg/mL. X-ray crystallographic analysis of two analogs revealed several interesting features responsible for the 3,4-(ethylenedioxy)benzyl-substituted analog's potency: (1) Comparing the two complexes revealed two distinct binding modes for each P1/P1' substituent; (2) The ethylenedioxy moieties are within 3.6 A of Pro 81 providing additional van der Waals contacts missing from the parent structure; (3) The enzyme's Arg 8 side chain moves away from the P1 substituent to accommodate the increased steric volume while maintaining a favorable hydrogen bond distance between the para oxygen substituent and the guanidine NH.
Cyclic urea amides, a novel series of HIV-1 protease (HIV PR) inhibitors, have increased activity against drug-resistant mutants of the HIV PR. The design strategy for these inhibitors is based on the hypotheses that (i) the hydrogen-bonding interactions between the inhibitor and the protease backbone will remain constant for wild-type and mutant enzymes and (ii) inhibitors which are capable of forming many nonbonded interactions, distributed throughout the active site, will experience a lower percent change in binding energy as a result of mutation in the target enzyme than those that form fewer interactions by partial occupation of the active site. The cyclic urea amide, SD146, forms 14 hydrogen bonds and 191 van der Waals contacts to HIV PR. SD146 is a very potent antiviral agent (IC90 = 5.1 nM) against wild-type HIV and maintains the same or improved level of high potency against a range of mutant strains of HIV with resistance to a wide variety of HIV protease inhibitors.
High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure-activity relationships of C2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and "unbound" (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2' pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure-activity relationships, and molecular recognition of this series of C2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2' is also discussed.
A series of novel P1/P1'-substituted cyclic urea-based HIV-1 protease inhibitors was prepared. Three different synthetic schemes were used to assemble these compounds. The first approach uses amino acid-based starting materials and was originally used to prepare DMP 323. The other two approaches use L-tartaric acid or L-mannitol as the starting material. The required four contiguous R,S,S,R centers of the cyclic urea scaffold are introduced using substrate control methodology. Each approach has specific advantages based on the desired P1/P1' substituent. Designing analogs based on the enzyme's natural substrates provided compounds with reduced activity. Attempts at exploiting hydrogen bond sites in the S1/S1' pocket, suggested by molecular modeling studies, were not fruitful. Several analogs had better binding affinity compared to our initial leads. Modulating the compound's physical properties led to a 10-fold improvement in translation resulting in better overall antiviral activity.
BACKGROUND: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds. RESULTS: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg-1 DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC90 suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response. CONCLUSIONS: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.