ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1999, p. 2893–2897 0066-4804/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 12
Expanded-Spectrum Nonnucleoside Reverse Transcriptase Inhibitors Inhibit Clinically Relevant Mutant Variants of Human Immunodeficiency Virus Type 1 JEFFREY W. CORBETT,* SOO S. KO, JAMES D. RODGERS, SUSAN JEFFREY, LEE T. BACHELER, RONALD M. KLABE, SHARON DIAMOND, CHII-MING LAI, SHELLEY R. RABEL, JO ANNE SAYE, STEPHEN P. ADAMS, GEORGE L. TRAINOR, PAUL S. ANDERSON, AND SUSAN K. ERICKSON-VIITANEN DuPont Pharmaceuticals Co., Experimental Station, Wilmington, Delaware 19880-0500 Received 14 May 1999/Returned for modification 27 August 1999/Accepted 20 September 1999
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. asparagine is frequently observed (2). This has led to the designation of K103N as a “pan-class resistance mutation.” Efavirenz (SUSTIVA) is an NNRTI that is administered once a day and that has intrinsic potency at nanomolar levels. It has been demonstrated to have significant clinical activity, both in combination with protease inhibitors and in a proteasesparing regimen consisting of efavirenz plus two nucleoside RT inhibitors (10). In clinical trials in which efavirenz is combined with other highly active antiretroviral agents, viral load reduction is at least as significant and durable as that for any other anti-HIV compound tested to date (1, 10). Although the majority of patients receiving efavirenz-containing regimens have shown sustained antiviral responses, the sequences of more than 90% of virus from patients whose viral loads have rebounded after an initial response to drug have the K103N mutation (1). Following the appearance of virus with the K103N mutation, viruses with multiple mutations arise more slowly. Approximately 4 months after the initial viral load rebound, the double mutations K103N-V108I or K103N-P225H are observed in a large number of samples (1). Highly resistant virus with the double mutation K103N-L100I is much less prevalent. Considering the significant antiviral benefit apparent from ongoing clinical trials with efavirenz, development of additional NNRTIs which retain similar ease of administration and safety profiles but which have superior overall profiles against mutant viruses such as K103N, K103N-V108I, and K103N-P225H mutant viruses should provide even more powerful drugs that may serve as constituents of combination regimens and as components of salvage therapy when other treatment modalities have failed. We describe here the identification of four new NNRTI analogs that combine protein-adjusted potency toward wild-type viruses and viruses with multiple mutations with significant oral bioavailability, long half-lives in plasma, and satisfactory preclinical safety assessments. After the administration of single oral doses, we observed levels of free drug in the plasma of the rhesus monkey
New therapeutics for the management of human immunodeficiency virus (HIV) infection are needed in order to provide long-term inhibition of virus in infected individuals (4). The ongoing requirement for new therapeutics arises both from the emergence of drug-resistant strains resulting from continuing replication in the presence of regimens that do not completely suppress virus production and from acute HIV infection with drug-resistant strains (7). Current therapeutics are targeted to two essential enzymes of the virus, the aspartyl protease and the reverse transcriptase (RT). A large number of nucleoside inhibitors of the viral RT are approved for use for the treatment of HIV disease. These nucleoside RT inhibitors bind to the active site of RT and act as substrate decoys and chain terminators. Clinical studies have demonstrated the limited durabilities of these agents in certain combinations because of the appearance of drug-resistant mutant viruses. In the past several years, a second class of inhibitors of HIV RT has been described. These inhibitors, designated nonnucleoside RT inhibitors (NNRTIs), act by binding to an allosteric binding site which regulates enzyme activity rather than to the active site (5). In general, NNRTIs do not inhibit HIV type 2 (HIV-2) RT, which has significant amino acid alterations at this allosteric binding site. In clinical trials with NNRTIs as monotherapy (6) and in in vitro selection experiments (9), the rapid emergence of resistant virus with greatly decreased susceptibility to the particular NNRTI has been observed. Furthermore, in patients who fail treatment with antiretroviral regimens containing any currently available NNRTI, an RT mutation of the lysine at amino acid 103 to
* Corresponding author. Mailing address: DuPont Pharmaceuticals Co., Experimental Station, E500/4403A, P.O. Box 80500, Wilmington, DE 19880-0500. Phone: (302) 695-4823. Fax: (302) 695-9673. E-mail:
[email protected]. 2893
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FIG. 1. Synthesis of 4-alkynyl-3,4-dihydro-4-trifluoromethylquinazolin-2(1H)ones. (a) (i) TMSNCO, THF; (ii) TBAF-THF. (b) Toluene or xylenes at reflux. (c) Li-§-R, BF3 䡠 OEt2, THF.
and the chimpanzee which have the potential for inhibition of K103N single and double mutant viruses. MATERIALS AND METHODS Chemical methods. Treatment of amino ketones 1a and 1b (11) with trimethylsilylisocyanate (TMSNCO) followed by tetrabutylamonium fluoride (TBAF) delivered aminols 2a and 2b, respectively (Fig. 1). Heating of 2a and 2b with 4-Å-pore-size molecular sieves in either toluene or xylenes at reflux effected the desired dehydration to afford the trifluoromethyl ketimines 3a and 3b, respectively. Aminol 2a, which contains a 5-halogen substituent, required higher temperatures to effect dehydration and was therefore dehydrated with xylenes as the solvent. Ketimines 3a and 3b were alkylated with lithiated acetylides in tetrahydrofuran (THF) in the presence of boron trifluoride diethyl etherate (BF3 䡠 OEt2) to give the desired quinazolinones. Additional experimental details will be published elsewhere. Biological assays. The ability of NNRTI analogs to inhibit the HIV-1 RT was assessed with purified, recombinant enzyme, radiolabeled dTTP, and a poly r(A) 䡠 oligo(dT)12–18 template-primer as substrate. The ability of NNRTI analogs to inhibit HIV replication in tissue culture was assessed with three different assay systems. The yield of infectious virus produced in 3-day acute infections by the HIV-1 (RF) strain in MT-2 cells was measured by a plaque assay of the culture fluid containing progeny virions as described previously (12). The antiviral activity was also determined by measurement of viral RNA accumulation in HIV-1 (RF)-infected MT-2 cells with biotinylated capture and alkaline phosphatasederivatized reporter oligonucleotides (3). In a third system, the effects of the analogs on the replication of recombinant viruses in the HXB2 or NL4-3 background was determined by measurement of viral p24 antigen levels from infected
MT-4 cells as described previously (8). In all assays, the concentration of compound which reduced the measured parameter by 90% was designated the IC90. The IC90s for laboratory wild-type strains were similar by the three assays. The IC90s of efavirenz for the RF strain were 1.7 ⫾ 0.5 nM by the RNA measurement assay (n ⫽ 38) and 2.4 ⫾ 0.5 nM by the plaque assay (n ⫽ 4), and the efavirenz IC90s for recombinant wild-type viruses determined by the p24 antigen detection assay were 2.9 ⫾ 0.3 nM for HXB2 (n ⫽ 13) and 3.0 ⫾ 1.3 nM for NL4-3 (n ⫽ 49). To estimate the effect of human plasma protein binding on antiviral efficacy, a functional assay and, in some cases, physical measurement of the extent of binding to serum proteins were used. In the functional assay, in vitro antiviral assays were conducted in the absence or presence of the two major components of human plasma, namely, human serum albumin and alpha-1-acid glycoprotein (antiviral shift assay). In the latter condition, the tissue culture medium contained final concentrations of 45 mg of human serum albumin per ml and 1 mg of alpha-1-acid glycoprotein per ml, concentrations of serum proteins likely found in the plasma of AIDS patients. The IC90s in the presence and absence of these added components were then compared and reported as the fold increase in IC90 observed, which is reported as the protein binding shift (PB shift). Dialysis and/or ultrafiltration was used to determine the percent free drug present in human serum or in tissue culture medium, which contains 5% fetal bovine serum. Pharmacokinetic studies. The pharmacokinetics of the analogs were investigated in the rhesus monkey and the chimpanzee. The compounds were administered orally to male rhesus monkeys at 10 mg/kg of body weight in a 0.5% aqueous methylcellulose suspension. Chimpanzees were dosed at 2 mg/ml from an oral suspension in aqueous Tang–1.0% methylcellulose suspension (50/50; vol/vol). Blood samples were collected and the concentration of the NNRTI analog was determined by liquid chromatography-mass spectroscopy-mass spectroscopy (LC/MS/MS) after liquid-liquid sample extraction. Pharmacokinetic parameters were calculated by noncompartmental methods. In vitro protein binding to human serum and to tissue culture medium was determined by LC/MS/MS after equilibrium dialysis or ultrafiltration.
RESULTS AND DISCUSSION Analogs were assessed for inhibition of HIV-1 RT in an in vitro enzyme assay (8) and for their ability to inhibit the wildtype RF strain of HIV-1 (3), as shown in Table 1. In addition, an initial indication of the influence of plasma protein binding on antiviral efficacy was determined by the antiviral shift assay. Table 1 shows that racemic quinazolinones with a variety of halide substitutions at X and alkyl side chains at R are potent inhibitors of the enzyme and, as a consequence, of virus replication. See Fig. 2 for a generic structure of the compounds described in Table 1. All analogs were more potent than nevirapine or delavirdine. Most analogs had antiviral potencies similar to that of efavirenz, with compound 4 appearing to be potentially more potent (racemates contain only 50% of the correct enantiomer). When the effect of human plasma protein binding was considered, which was estimated by applying the PB shift to the observed IC90, several analogs appeared to be
TABLE 1. In vitro biological activities of 4-alkynyl-4-trifluoromethyl-3,4-dihydro-2(1H)-quinazolinones Compound
X
R
4 5 6 7 Efavirenz 8 9 10 11 12 13 Nevirapine Delavirdine
5,6-diF 5,6-diF 5,6-diF 5,6-diF
Ethyl Cyclopropyl i-Propyl 2-Pyridyl
5,6-diF 6-Cl 6-Cl 6-Cl 6-Cl 6-Cl
Phenyl Cyclopropyl Ethyl i-Propyl Phenyl 2-Pyridyl
Enzyme IC50 (nM)
IC90 (nM) for wild typea
PB shift
PB-adjusted IC90 (nM) for wild type
74 ⫾ 27 74 ⫾ 35 91 ⫾ 13 68 ⫾ 17 47 ⫾ 25 181 ⫾ 83 111 ⫾ 34 110 ⫾ 61 281 ⫾ 105 277 ⫾ 94 129 ⫾ 36 4,848 ⫾ 1,739 422 ⫾ 92
1.5 2.1 2.1 ⫾ 1.4 2.0 1.7 ⫾ 0.5 6.2 2.7 ⫾ 0.6 3.3 3.0 ⫾ 0.2 7.1 3.4 50 ⫾ 10 37 ⫾ 9
6.9 9 9.8 12 16.5 7 15 25 30 12 23 2 38
10 19 21 24 28 43 41 83 90 86 79 100 1,406
a Antiviral activity against the wild type was determined by measurement of viral RNA via oligonucleotide capture from MT-2 cells acutely infected with the RF strain of HIV-1 after 3 days. The data represent the means ⫾ standard deviations for two to six independent determinations.
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FIG. 2. Generic structure of racemic compounds described in Table 1.
more potent than efavirenz. In a comparison of similar pairs of analogs, 5,6-difluoro substituents were found to confer improvements in potency compared to the potencies of 6-chlorosubstituted compounds, and small groups on the alkyne are generally favored over large groups such as phenyl. We next examined the abilities of the new analogs to inhibit replication of mutant virus carrying the amino acid substitution K103N or L100I (Table 2). K103N is the most prevalent mutation observed in vivo in patients who have failed treatment with NNRTI-containing regimens (1, 2), and the L100I mutation is observed in in vitro selection experiments (3). The superior potencies of the new analogs became quite clear: 6 of 10 analogs assayed as the racemates had potencies at least twice that of efavirenz against the virus with the K103N mutation. On the basis of these encouraging findings, compounds 5, 6, and 9 were synthesized in gram quantities, and the enantiomers were separated by chiral high-performance liquid chromatography. The active isomers of these compounds were designated DPC 961, DPC 963, and compound 14, respectively (Fig. 3). The stereochemistry of DPC 961 was determined from a single crystal X ray, and the absolute stereochemistry of DPC 963 and compound 14 were inferred from the antiviral and enzyme data (Table 3), with the undesired enantiomers exhibiting virtually no activity (data not shown). Previous work (13, 14) has shown that when the stereochemistry of the quaternary center is inverted in the quinazolinones or the benzoxaxinones, the compounds are devoid of activity. Compared to efavirenz, DPC 961, DPC 963, and compound 14 showed 6.4-, 5.8-, and 7.1-fold greater potencies, respectively, toward the K103N mutant virus.
TABLE 2. Resistance profiles of 4-alkynyl-4-trifluoromethyl3,4-dihydro-2(1H)-quinazolinones Compound
X
R
5 4 6 9 11 10 Efavirenz 7 13 8 12 Delavirdine Nevirapine
5,6-diF 5,6-diF 5,6-diF 6-Cl 6-Cl 6-Cl
Cyclopropyl Ethyl i-Propyl Cyclopropyl i-Propyl Ethyl
5,6-diF 6-Cl 5,6-diF 6-Cl
2-Pyridyl 2-Pyridyl Phenyl Phenyl
IC90 (nM)a K103N mutant
L100I mutant
13 14 14 22 22 26 64 ⫾ 24 109 140 ⫾ 2.0 191 250 1,000 ⫾ 270 5,100 ⫾ 830
12 18 10 18 28 NA 77 ⫾ 26 37 57 ⫾ 8.0 256 365 NA NA
a Antiviral activity was determined by measurement of p24 antigen production in MT-4 cells infected with recombinant mutant viruses in the HXB2 or NL4-3 backgrounds. The data represent the means ⫾ standard deviations for two to six independent determinations. NA, not available.
FIG. 3. Absolute stereochemistries of DPC 961, DPC 963, compound 14, and efavirenz.
All three analogs were dosed orally as combined suspensions in rhesus monkeys, and only very low levels of compound 14 were observed in blood. Compound 14 was assessed for its propensity to undergo metabolism by liver homogenate fractions. Preliminary experiments indicated that the i-propyl analog of compound 14 was rapidly degraded compared to the rate of degradation of efavirenz, and compound 14 was eliminated from further consideration. However, DPC 961 and DPC 963 were present at significant levels in the blood of rhesus monkeys, even several hours after dosing. Following the administration of a single oral dose of 2 mg (per kg) each of DPC 961, DPC 963, and efavirenz, combined in a dosing vehicle, the maximum concentrations reached were 1.01, 0.44, and 0.6 M, respectively. After 24 h, measurable levels of DPC 961 (0.11 M) were detected in plasma, while DPC 963 and efavirenz were not present at detectable levels in plasma, suggesting that the half-life of DPC 961 may be longer than that of DPC 963 or efavirenz. On the basis of the results presented above, we prepared the corresponding olefin analogs of DPC 961 and DPC 963 since previous SAR around efavirenz suggested that olefins had a slightly improved resistance profile. DPC 083 and DPC 082 were synthesized by lithium aluminum hydride reductions of DPC 961 and DPC 083, respectively (Fig. 4).
TABLE 3. Activities of resolved quinazolinonesa Compound
14 DPC 961 DPC 963
IC90 (nM) K103N mutant
Wild type
Enzyme IC50 (nM)
9 10 ⫾ 3.2 11 ⫾ 4.8
1.6 2.0 ⫾ 0.7 1.3 ⫾ 0.6
15 ⫾ 8 31 ⫾ 8 18 ⫾ 5
a Wild-type virus was assessed in MT-2 cells by oligonucleotide capture of viral RNA after 3 days of infection. K103N mutant variant was used to infect MT-4 cells, and viral p24 antigen levels were measured after 3 days. The data represent the means ⫾ standard deviations for two to six independent determinations.
a Wild-type virus was assessed in MT-2 cells by oligonucleotide capture of viral RNA after 3 days of infection. K103N single and double mutant variants were used to infect MT-4 cells, and viral p24 antigen levels were measured after 3 days. The amounts of the L100I mutant were assessed in MT-2 cells by determination of the yield of infectious virus progeny by plaque assay. The data represent the means ⫾ standard deviations for two to six independent determinations.
292 660 340 330 1,990 95 55 23 17 84 117 35 12 13 65 11 11 3.1 4.0 17 1.1 0.8 0.6 0.5 0.5 53 39 31 37 27.2 550 ⫾ 400 1,690 ⫾ 160 1,100 ⫾ 160 890 ⫾ 90 7,300 ⫾ 5,000 180 ⫾ 29 140 ⫾ 92 73 ⫾ 18 46 ⫾ 2.2 310 ⫾ 130 21 ⫾ 9.2 27 ⫾ 11 10 ⫾ 3.2 11 ⫾ 4.8 64 ⫾ 24 2.0 ⫾ 0.2 2.1 ⫾ 0.8 2.0 ⫾ 0.7 1.3 ⫾ 0.6 1.7 ⫾ 0.5 DPC 082 DPC 083 DPC 961 DPC 963 Efavirenz
K103N
L100I
220 ⫾ 140 90 ⫾ 6.6 38 ⫾ 4 34 ⫾ 5.4 240 ⫾ 68
K103NL100I K103NP225H K103NV108I K103N Wild type
Intrinsic (free drug) potency (IC90 [nM])
% Free drug in tissue culture medium K103NL100I K103NP225H Measured potency (IC90 [nM])
K103NV108I Wild type (HIV-1 RF) NNRTI
The virologic profile and protein binding data show that the olefin analogs are as potent as DPC 961 or DPC 963, and all are either as potent or more potent than efavirenz (Table 4). Importantly, this potency improvement is maintained against both single- and double-mutation variant viruses containing K103N. In particular, DPC 963 is sevenfold more potent against the clinically important K103N-V108I and K103NP225H double mutants. The four analogs maintain the high degree of potency exhibited by efavirenz against other common mutations induced by NNRTIs, such as Y181C and V106A (data not shown). Because only free drug is available for diffusion into cells and inhibition of the RT, the extent of binding of NNRTIs to components in the tissue culture medium was determined to obtain the intrinsic potency of each compound. We define the intrinsic potency as the concentration of free drug required for 90% inhibition of various types of HIV-1. Table 4 shows that the expanded-spectrum NNRTIs have intrinsic potencies against mutant variants of HIV that are up to sixfold greater than that of efavirenz. To maintain suppression of virus replication, the free drug concentration in the plasma must exceed the intrinsic potency. Table 4 also shows that the percent binding of the expanded-spectrum NNRTIs by proteins in human plasma is substantially less than that of efavirenz. Taken together, the data suggest that if the bioavailabilities of these four NNRTIs are equivalent to that of efavirenz in humans, then they would likely provide improved suppression of replication of mutant virus strains. Table 5 shows the results of oral dosing of individual suspensions of DPC 961, DPC 963, DPC 082, and DPC 083 in rhesus monkeys at 10 mg/kg and in chimpanzees at 2 mg/kg. Data for efavirenz are included for comparison. Similar to efavirenz, the half-life in these species is consistent with the possibility of once-daily dosing. By extrapolation of the data for a 2-mg/kg oral dose of each of the four analogs in chimpanzees, the concentrations in plasma at 24 h for a 10-mg/kg dose (close to the efavirenz dosage of 600 mg once daily used clinically) were 4.7 to 12.7 M. The levels of all four compounds were higher than those of efavirenz observed previously. The free drug levels present at 24 h were then estimated from the data on the drug levels in chimpanzee plasma and the levels of human serum protein binding (the levels of serum protein binding of DPC 083, for example, in human and chimpanzee serum are identical). The data show that although the properties of alkyne and olefin analogs are quite different (alkynes are more potent, olefins are less highly plasma protein bound and have better pharmacokinetics), the levels of free drug in plasma for all four analogs would correlate with significant inhibition of replication of HIV of both the wild-type
TABLE 4. Summary of properties of expanded-spectrum NNRTIsa
FIG. 4. Synthesis of DPC 082 and DPC 083. (a) LiAlH4,1,2-dichlorobenzene, THF.
3.0 2.0 1.5 2.8 0.21–0.54
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5.9 ⫾ 3.8 11 ⫾ 6.8 13 ⫾ 7.9 8.0 ⫾ 5.7 120 ⫾ 30
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TABLE 5. Pharmacokinetic properties of expanded spectrum NNRTIs NNRTI
DPC 082 DPC 083 DPC 961 DPC 963 Efavirenz a b
Concn (M) at 24 h ina: Rhesus monkey
Chimpanzee
0.64 6.84 0.38 0.22 0.36
4.7 7.6 12.7 6.2 2.7
t1/2 (h)
Free drug concn (nM) at 24 h
t1/2 (h)
5.9 24 4.8 4.8 NAb
141 152 191 174 6–15
14.2 85.7 76.0 19.6 NA
Ratio of trough level of free drug in chimpanzees: IC90 of free drug Wild type
K103N
K103N-V108I
K103N-P225H
K103N-L100I
128 190 318 348 12–30
13 14 62 44 0.4–0.9
1.2 4.3 16 13 0.09–0.2
1.5 2.8 8.3 10 0.07–0.2
0.5 0.2 0.6 0.5 0.003–0.008
Each drug was administered to rhesus monkeys orally at 10 mg/kg. Data for chimpanzees, which were given 2 mg/kg, are extrapolated from data for rhesus monkeys. NA, not available.
and the mutant varieties. From the ratio of trough levels of free drug in chimpanzees to the IC90 of free drug (intrinsic potency), it was demonstrated that the four NNRTI analogs will adequately inhibit the K103N mutant and the clinically relevant double mutants K103N-V108I and K103N-P225H, provided that the levels in humans are similar to those observed in chimpanzees. Conclusions. Taken together, the expanded-spectrum NNRTIs described here show significant improvements in their overall protein binding-adjusted resistance profiles relative to those of all currently marketed NNRTIs. The overall profiles of each of the four NNRTIs indicate that levels in blood capable of causing inhibition of many mutant forms of HIV observed in patients who have failed treatment with NNRTI-containing regimens are achievable with once-a-day dosing, even though the improvements in intrinsic potency over that of efavirenz are in some cases modest. The ability of these compounds to inhibit the mutant viruses arises from a combination of greater intrinsic potency, increased levels of free drug, and improved pharmacokinetic properties. Our goal is to conduct phase I studies with the cohort of compounds and select the superior candidate for further study. Results from these studies will be reported when the data become available.
6. 7. 8.
9.
10.
11.
12.
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