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Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses
Accepted Manuscript Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses Bernard Côté, Jason D. Burch, Ernest Asante-Appiah, Chris Bayly, Leanne Bédard, Marc Blouin, Louis-Charles Campeau, Elizabeth Cauchon, Manuel Chan, Amandine Chefson, Nathalie Coulombe, Wanda Cromlish, Smita Debnath, Denis Deschenes, Kristina Dupont-Gaudet, Jean-Pierre Falgueyret, Robert Forget, Sébastien Gagné, Danny Gauvreau, Melina Girardin, Sébastien Guiral, Eric Langlois, Chun Sing Li, Natalie Nguyen, Rob Papp, Serge Plamondon, Amélie Roy, Stéphanie Roy, Ria Seliniotakis, Miguel St-Onge, Stéphane Ouellet, Paul Tawa, Jean-François Truchon, Joe Vacca, Marc Wrona, Youwei Yan, Yves Ducharme PII: DOI: Reference:
S0960-894X(13)01454-6 http://dx.doi.org/10.1016/j.bmcl.2013.12.070 BMCL 21178
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
4 November 2013 16 December 2013 17 December 2013
Please cite this article as: Côté, B., Burch, J.D., Asante-Appiah, E., Bayly, C., Bédard, L., Blouin, M., Campeau, L-C., Cauchon, E., Chan, M., Chefson, A., Coulombe, N., Cromlish, W., Debnath, S., Deschenes, D., DupontGaudet, K., Falgueyret, J-P., Forget, R., Gagné, S., Gauvreau, D., Girardin, M., Guiral, S., Langlois, E., Li, C.S., Nguyen, N., Papp, R., Plamondon, S., Roy, A., Roy, S., Seliniotakis, R., St-Onge, M., Ouellet, S., Tawa, P., Truchon, J-F., Vacca, J., Wrona, M., Yan, Y., Ducharme, Y., Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.12.070
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Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses
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Bernard Côté∗, Jason D. Burch, Ernest Asante-Appiah, Chris Bayly, Leanne Bédard, Marc Blouin, Louis-Charles Campeau, Elizabeth Cauchon, Manuel Chan, Amandine Chefson, Nathalie Coulombe, Wanda Cromlish, Smita Debnath, Denis Deschenes, Kristina Dupont-Gaudet, Jean-Pierre Falgueyret, Robert Forget, Sébastien Gagné, Danny Gauvreau, Melina Girardin, Sébastien Guiral, Eric Langlois, Chun Sing Li, Natalie Nguyen, Rob Papp, Serge Plamondon, Amélie Roy, Stéphanie Roy, Ria Seliniotakis, Miguel St-Onge, Stéphane Ouellet, Paul Tawa, Jean-François Truchon, Joe Vacca, Marc Wrona, Youwei Yan and Yves Ducharme
Bioorganic & Medicinal Chemistry Letters jo u r n a l h o m e p a g e : w w w .e ls e v ie r .c o m
Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses ∗
Bernard Côté , Jason D. Burch, Ernest Asante-Appiah, Chris Bayly, Leanne Bédard, Marc Blouin, LouisCharles Campeau, Elizabeth Cauchon, Manuel Chan, Amandine Chefson, Nathalie Coulombe, Wanda Cromlish, Smita Debnath, Denis Deschenes, Kristina Dupont-Gaudet, Jean-Pierre Falgueyret, Robert Forget, Sébastien Gagné, Danny Gauvreau, Melina Girardin, Sébastien Guiral, Eric Langlois, Chun Sing Li, Natalie Nguyen, Rob Papp, Serge Plamondon, Amélie Roy, Stéphanie Roy, Ria Seliniotakis, Miguel StOnge, Stéphane Ouellet, Paul Tawa, Jean-François Truchon, Joe Vacca, Marc Wrona, Youwei Yana and Yves Ducharme Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Quebec, Canada H9H 3L1 a Merck Research Laboratories, 770 Sumneytown Pike, PO Box 4, West Point, PA, 19486-0004, United States
A R T IC LE IN F O
A B S TR AC T
Article history: Received Revised Accepted Available online
The optimization of a novel series of non- nucleoside reverse transcriptase inhibitors (NNRTI) led to the identification of pyridone 36. In cell cultures, this new NNRTI shows a superior potency profile against a range of wild type and clinically relevant, resistant mutant HIV viruses. The overall favorable preclinical pharmacokinetic profile of 36 led to the prediction of a once daily low dose regimen in human. NNRTI 36, now known as MK-1439, is currently in clinical development for the treatment of HIV infection.
Keywords: Non-nucleoside reverse transcriptase HIV triazolinone pyridone inhibitor
Highly active antiretroviral therapy (HAART) is the standard of care for the treatment of HIV infection.i Typically, this protocol recommends the combination of two nucleoside reversetranscriptase inhibitors (NRTIs) with either a non-nucleoside reverse-transcriptase inhibitor (NNRTI), a ritonavir-boosted protease inhibitor or an integrase inhibitor.ii NNRTI-based combinations have become first-line therapy mainly because of their demonstrated efficacies, convenient dosing regimen and relatively low toxicities.iii These inhibitors block the polymerase activity of the HIV reverse transcriptase by binding to an allosteric hydrophobic pocket adjacent to the active site.iv Efavirenz (1, Fig. 1) is a first generation NNRTI that has been conveniently co-formulated with NRTIs tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) as a once-aday fixed-dose combination (Atripla®). Although recommended for the therapy of treatment-naïve patients, efavirenz suffers from neurocognitive side effects,v teratogenicityvi and exacerbation of hyperlipidemia.vii Moreover, the low barrier to genetic resistance of first generation NNRTIs led to the emergence of resistant
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viruses bearing mutations K103N and Y181C in patients failing therapy. viii
Figure 1. Structures of marketed and lead NNRTIs
——— ∗ Corresponding author. Tel.: +1-514-924-1456; e-mail: [email protected]
Second generation NNRTIs etravirine (2) and rilpivirine (3) efficiently suppress the replication of the K103N resistant mutants as shown by an improved activity in cell culture assays (Table 1). Etravirine (200 mg, bid) is approved for use in treatment-experienced adult patients with multi-drug resistance.ix With an improved pharmacokinetic profile, the close analog rilpivirine (25 mg, qd) was recently approved for use in treatment-naïve patients. Phase III data reveal that at the 96-week point, a rilpivirine/truvada®x combination was better tolerated than efavirenz/truvada®.xi However, the virologic failure rate was twice as high for rilpivirine (14%) than it was for efavirenz (8%). For patients with viral load greater than 500,000 copies/mL, the response rate is 62% (rilpivirine) versus 81% (efavirenz). As a result, rilpivirine is not recommended for treating HIV patients with viral load >500,000 copies/mL. This difference in treatment durability could be explained by the much higher ratio of trough concentration over the antiviral activity for efavirenz versus rilpivirine. Pyridone 4 was recently identified as a novel NNRTI with an improved spectrum of antiviral activity against key mutant strains (Table 1).xii The related pyridone 5 was also found to have a similar in vitro profile. However, both analogs suffered from poor oral bioavailability or high inter-animal exposure variabilityxiii that limited their development potential. As poor patient compliance is a key element leading to drug resistance, optimization of the pharmacokinetic parameters becomes critical to access a simple dosing regimen that will maximize around-theclock viral suppression. In our effort to optimize this new NNRTI pyridone series, we therefore specifically aimed at developing a low dose, once daily drug that could be conveniently coformulated with other therapeutic agents. Table 1 Enzyme and Spread-of-virus inhibition of lead NNRTIs RTa SPREAD 50% NHSb IC50 (µM) IC95 (µM) Compound WTc WT K103N Y181C K103N/Y181C Efavirenz 0.002 0.039 1.4 0.09 3.2 Etravirine 0.001 0.033 0.044 0.24 0.59 Rilpivirine 0.001 0.036 0.044 0.12 0.37 4 0.003 0.008 0.012 0.02 0.07 5 0.003 0.017 0.024 0.06 0.07 a RT: Reverse transcriptase. ECL assay. Values are the geometric mean of at least two determinations. Assay protocols are detailed in Ref. xiv. b IC95 is defined as the concentration at which the Spread of the virus is inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref. xv. c WT: Wild type.
One of the striking elements that initially oriented our efforts to improve the oral bioavailability of the pyridone series was the poor aqueous solubilityxvi of 5 (0.2 ng/mL in water). This low solubility, as well as the high crystallinity of 5 (m.p. = 299oC), could be rationalized by the analysis of the solid-state x-ray structure of a close analog sharing the pyrazolopyridine moiety found in 5 (Fig. 2). We hypothesized that disruption of the strong donor/acceptor hydrogen bond motif of the pyrazolopyridine would weaken the crystal lattice and enhance the solubility.
Figure 2. Donor/acceptor hydrogen bonding of the pyrazolopyridine
To validate this strategy, two analogs were synthesized where the pyridine nitrogen of the pyrazolopyridine was either removed or shifted by one position (Table 2). The solubility was assessed by light scattering as a measure of the concentration threshold at which a DMSO solution of the test compounds precipitate in a PBS solution. As shown in table 2, both structural modifications found in pyridone 6 and 7 led to an improved aqueous solubility (5.2 µM and 12.0 µM respectively) and better oral bioavailability in rat (30% and 74%). This data set strongly suggested that the originally observed low solubility and low oral absorption were not inherent to the pyridone series. We therefore undertook the synthesis of a library of pyridone analogs to identify a pyrazolopyridine replacement using the intermediate hydroxypyridine 12a (Scheme 1). In parallel with this effort, SAR at the 4-position of the pyridone was also initiated using intermediate 12b (Scheme 2). Table 2 Solubility and oral rat bioavailability of substituted pyridone
RTa SPREAD 50% NHSb Sol c Rat PKd IC50 (µM) IC95 (µM) (µM) WTe WT K103N/Y181C F(%) Cl f 5 0.003 0.017 0.069 1.1 13 18 6 0.001 0.053 0.186 5.2 30 17 7 0.006 0.029 0.092 12.0 74 40 a RT: Reverse transcriptase. ECL assay. Values are the geometric mean of at least two determinations. Assay protocols are detailed in Ref. 14. b IC95 is defined as the concentration at which the Spread of the virus in inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref.15. c Sol: solubility threshold of the DMSO solution in PBS buffered solution measured by light scattering. d Average of 2 Sprague Dawley rat dosed at 5 mpk PO (methocel suspension) and 1 mpk IV 60% PEG200). eWT: Wild type. f Cl: clearance in mL/min/kg. Compd
The synthetic sequence to prepare the hydroxypyridine 12a is outlined in Scheme 1. Chloropyridine 8 was coupled with 2bromo-4-chlorophenol (9) under basic conditions to afford the biaryl ether 10 in 79% yield. This chloropyridine was then hydrolyzed to the hydroxypyridine 11 with potassium hydroxide in 71% yield. Finally, this intermediate was treated with zinc cyanide in the presence of a catalytic amount of Pd(PPh3)4 to provide the desired cyano derivative 12a. Scheme 2 describes the general approach to synthesize the different 4-substituted pyridones. The synthesis of the common intermediate 12b first involves the SNAr reaction of commercially available 2-iodo-4chlorophenol (14) with the nitropyridine-N-oxide 13 in 80% yield. The iodophenol was preferred to the previously used bromo analog in order to avoid the formation of a regioisomeric mixture during the cyanation reaction. Sequential treatment of the nitropyridine-N-oxide 15 with acetylbromide in acetic acid followed by the reaction of the crude material with phosphorus tribromide in chloroform provided the dibromopyridine 16 in 87% overall yield. Hydrolysis of the bromopyridine with potassium hydroxide (72% yield) followed by the quantitative cyanation of the iodoaryl moiety gave the 4-bromo-2hydroxypirydine 12b. This key intermediate was then fonctionnalized by taking advantage of the selective reactivity of the bromide. The final assemblage was completed by alkylating the hydroxypyridines 12b-f as describe before (see ref. 12) with
the protected 3-bromomethyl pyrazolopyridine followed by TFAmediated hydrolysis of the tert-butyl carboxylate.
Scheme 1. Reagents and conditions: (a) K2CO3 , NMP, 120o C; (b) KOH, tert-BuOH, 75o C; (c) Zn(CN)2 , Pd(PPh3)4 , DMF, 100o C.
Scheme 2. Reagents and conditions: (a) K2CO3 , DMF, 55o C; (b) AcBr, AcOH, 80oC; (c) PBr3, CHCl 3, 60oC; (d) KOH, tert-BuOH; (c) Zn(CN)2 , Pd(PPh3)4, DMF, 100o C; (f) K2CO3 , bromide, DMF, 55o C, then TFA.
The biological activity and the rat pharmacokinetic properties of pyrazolopyridines 17-21 were evaluated (Table 3). In general, most of the functional groups were very well tolerated with low nanomolar enzyme inhibition activity. In the Spread assay, bromo derivative 17 was identified as the most potent analog ever prepared in the pyridone series with an IC95 = 0.004 µM against the wild type virus and an IC95 = 0.021 µM against the double mutant K103N/Y181C. In cell culture, the antiviral potency of all these new analogs is superior to the benchmark NNRTIs efavirenz and rilpivirine. The key differentiation factor amongst them is their in vivo ADME properties. The CF3 substituted pyridone 5 presents a substantially longer elimination half-life (t1/2 = 7 h) in rats compared to its close analogs. Further studies in dogs (data not shown) corroborated this pharmacokinetic trend and the strong electron withdrawing CF3 group was therefore considered as optimal. The next series of pyridone analogs were synthesized by the alkylation of intermediate 12a with different heterocycle derivatives (Table 4). Using standard reaction conditions (K2CO3, DMF) a 48-membered library was rapidly built. The specific aim of this exercise was to identify compounds that would retain the antiviral activity of 5, and would present increased aqueous solubility and ultimately improved systemic exposure following oral administration in pre-clinical species. Inhibitors 22 to 25 are pyrazolopyridine derivatives that were substituted to increase the polarity of the compounds (22 and 23) or to reduce the basicity of the pyridine nitrogen (24 and 25). All four analogs were found to be potent, but suffered from poor rat pharmacokinetics.xvii We have previously shown in table 2 that the potent regioisomeric pyrazolopyridine 7 is well absorbed but eliminated too rapidly in
rats. With the fluorinated analog 26, we were able to extend the rat half-life to 4 hours with an oral bioavailability of 65% but unfortunately, a significant potency shift was observed in the cellular assay in the presence of 50% NHS (IC95 = 76 nM). Table 3 Biological activity and rat half-life of 4-substituted pyridones.
Compd
X
RTa IC50 (µM) WT
c
Spread 50% NHSb IC95 (µM) WT
K103N/Y181C
Rat t1/2 (h)d
4 Cl 0.003 0.008 0.069 2.2 5 CF3 0.003 0.017 0.069 7.0 17 Br 0.002 0.004 0.021 1.7 18 CH3 0.007 0.012 0.096 0.8 19 CF2 CH3 0.005 0.014 0.044 1.3 20 SCH3 0.009 0.015 0.033 1.5 21 c-Pr 0.007 0.012 0.11 1.5 a RT: Reverse transcriptase. ECL assay. Values are the geometric mean of at least two determinations. Assay protocols are detailed in Ref. 14. b IC95 is defined as the concentration at which the Spread of the virus in inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref.15. c WT: Wild type. d Average of 2 Sprague Dawley rats dosed at 1 mpk IV (60% PEG200).
Greater structural diversity was explored with the following heterocycles. Despite their good intrinsic potency against the enzyme, the large lipophilic quinoline 27 and benzoisoxazole 28
showed largely shifted potencies in the Spread assay. The incorporation of small heterocyclic rings to produce inhibitors such as 4-methylthiazole 29, generally resulted in significantly increased aqueous solubility. While this finding was true for most small rings, the nature of the heterocycle and its substitution pattern had a tremendous impact on the antiviral potency. While substitution with a methyl group at the 4-position seems to assure enzyme activity in the thiazole series (29 and 31 vs 30), this observation did not translate well for the pyrazole 32 (IC50 = 140 nM), the imidazole 33 (IC50 = 300 nM) and the triazole 34 (IC50 = 43 nM). In the triazolinone series though, methylation at the 4position as described in Scheme 3, was found to be beneficial with a 5-fold increase in enzyme potency (35 vs 36). Conversely, the slight increase in steric hindrance of the 4-ethyl substituted triazolone 37 negatively impacted the cell culture activity (IC95 > 125 nM). Table 4. Potency and solubility of alkylated pyridones
a
RT: Reverse transcriptase. ECL assay. Values are the geometric mean of at least two determinations. Assay protocols are detailed in Ref. 14. b IC95 is defined as the concentration at which the Spread of the virus in inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref. 15. c Sol: solubility threshold of the DMSO solution in PBS buffered solution measured by light scattering.
In addition to the 4-methyl substitution of the triazolinone, one of the key structural features contributing to the good antiviral potency of inhibitor 36xviii is the presence of the N-NH motif that was also present in the pyrazolopyridine series. In an analogous biaryl ether series, Sweeney et al. have described a series of heterocycles sharing this structural feature.xix Based on crystal structure analysis with HIV-RT, they highlighted the role of the N-NH acceptor-donor motif that engages a pair of hydrogen-bonds with the K103 backbone of the RT enzyme. This type of interaction was also observed in the binding of pyridone 36 with HIV-RT in the structural elucidation of a co-crystal by X-ray, as shown in Figure 3.
Y Figure 3. X-ray crystal structure of compound 36 bound to HIV-RT. The wild-type RT complex structure has been deposited in the Protein Data Bank under accession code 4NCG.
As seen in Table 5, inhibitor 36 presents an exceptional in vitro profile against clinically relevant resistant viruses. Compared to efavirenz, pyridone 36 is 33-fold more active against K103N and 59-fold more potent against the double mutant K103N/Y181C. Compared to rilpivirine, inhibitor 36 is respectively 5 and 7-fold more potent against the mutant viruses Y181C and K103N/Y181C respectively. Table 5 Antiviral activity of pyridone 36 in cell culture SPREAD IC95 (µM) with 50% NHSa Compound WT K103N Y181C K103N/Y181C 36 0.019 0.042 0.025 0.054 Efavirenz 0.039 1.4 0.089 3.2 Rilpivirine 0.036 0.044 0.120 0.37 a IC95 is defined as the concentration at which the Spread of the virus in inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref. 15.
In addition to the above mutant substituted viruses, pyridone 36 was tested against a panel of 15 mutant viruses (Monogram Biosciences) and compared to efavirenz, rilpivirine and lead inhibitor 5.
bioavailability of our new NNRTI confers a 16-fold increase in systemic exposure (AUCN = 3.9 µM*h). In dog, the same net result is also observed with a 26-fold improvement in exposure. In this case, a combination of low clearance (Cl = 0.36 mL/min/kg) and better oral absorption (F = 52%) is responsible for the high observed AUCN of 44 µM*h. The overall favorable pharmacokinetic profile of 36 in preclinical species, combined to a low metabolic turnover (<10%) in human liver microsomes and hepatocytesxx lead to the prediction of a once daily low dose regimen in human.
Fold Change (vs. Control)
700 600 500 400 300 200 100 0
Efavirenz Rilpivirine Compd 5 Compd 36
Mutant viruses Figure 4. Fold change in activity (IC50 versus the control CNDO virus) of NNRTIs in a panel of 15 mutant viruses (Monogram Biosciences). A maximum cut-off of 625-fold is shown. The data for the inhibitors are presented as follows: efavirenz (purple), rilpivirine (green), compound 5 (red), compound 36 (blue).
As indicated in Figure 4, efavirenz was less active against a number of the mutant viruses in contrast to the second-generation inhibitor, rilpivirine. Pyridone 36 maintained the potent antiviral activity of compound 5 and demonstrated a robust activity against the mutant viruses. It was less active against the rare mutants harboring the Y188L and V106A/G109A/F227L amino acid changes for which two or more nucleotide changes are required. Additional profiling of the antiviral activity of compound 36 on a panel of 96 mutant viruses shows a similar robust activity (to be reported in a future publication). As shown before, the solubility threshold of compound 36 (45 µM) is significantly increased compared to the original lead pyridone 5 (1.0 µM) in water. At both pH 4 and 8, the equilibrium solubility of 36 is 6 µg/mL in comparison to <0.1 µg/mL for 5. Solubility in fasted simulated intestinal fluid (FaSSIF) went up from 1.3 µg/mL for pyrazolopyridine 5 to 130 µg/mL for the crystalline triazolinone 36. These improved physicochemical properties correlate with a superior pharmacokinetic profile for 36 in rats and dogs as presented in table 6.
Scheme 3. Reagents and conditions: (a) K2CO3 , DMF, -10oC; (b) MeI or EtI, K2CO3, DMF.
Although the clearance of 36 (Cl = 5.4 mL/min/kg) is lower compared to 5 (15 mL/min/kg) in rats, the half-life of 36 is slightly shorter due to its smaller volume of distribution (Vd). Nevertheless, this lower Vd combined with the good rat
i. (a) Hirschel, B.; Francioli, P. N. Engl. J. Med. 1998, 338, 906. (b) Schneider, M. F. ; et al. AIDS, 2005, 19, 2009. ii. Hammer, S. M.; et al. JAMA, 2006, 296, 827. iii. Fokunang, C. N.; Hitchcock, J.; Spence, F.; Tembe-Fokunang, E. A.; Burkhardt, J.; Levy, L.; George, C. Int. J. Pharmacol. 2006, 2(1), 152. iv. (a) Sluis-Cremer, N.; Temiz, N. A.; Bahar, I. Curr. HIV Res. 2004, 2(4), 323. (b) Hopkins, A. L.; Ren, J.; Esnouf, R. M.; Willcox, B. E.; Jones, E. Y.;
With a broad spectrum of antiviral activity against clinically relevant mutant viruses, analogs from the new pyridone series clearly meet a mandatory criterion for next generation NNRTIs. Unfortunately, poor oral bioavailability in preclinical species was a key liability originally associated with this series. Using a simple synthetic approach, we rapidly built a library of inhibitors that led to the identification of pyridone 36. In cell culture, this new inhibitor has a superior profile against the wild type and K103N/Y181C viruses compared with benchmark compounds efavirenz, etravirine and rilpivirine. The increased aqueous solubility of pyridone 36 translated into a significant improvement of pharmacokinetic parameters in preclinical species. NNRTI 36, now known as MK-1439, is currently in clinical development for the treatment of HIV infection.
Table 6 Comparative pharmacokinetic parameters of pyridones 5 and 36 in rat and dog Species Parameters 5 36 Vd (L/kg)
8.8
Cl (mL/min/kg)
15
5.4
t1/2 (hr)
7
4.4
Rata
F (%)
2.3
15
57
AUCN (µM*h)c
0.25
3.9
Vd (L/kg)
1.5
1.2
Cl (mL/min/kg)
1.8
0.36
t1/2 (hr)
10
37
F (%)
9
52
Dogb
AUCN (µM*h) 1.7 44 Average of 2 Sprague Dawley rats for each doses. 5 was dosed IV at 1 mpk (80%PEG200) and PO at 5 mpk (Ball-milled, 0.5% methocel). 36 was dosed IV at 1 mpk (60%PEG200) and PO at 5 mpk (Ball-milled, 0.5% methocel). b Average of 2 Beagle dogs for each doses. 5 was dosed IV at 0.5 mpk (80%PEG200) and PO at 1 mpk (Ball-milled, 0.5% methocel). 36 was dosed IV at 0.5 mpk (60%PEG200) and PO at 1 mpk (Ball-milled, 10% Tween). c Normalized for 1 mpk from 0-24h. a
Acknowledgements The authors wish to thank Neville Anthony, Christopher Burgey and Ming-Tain Lai for reviewing the manuscript. References and notes
Ross, C.; Miyasaka, T.; Walker, R. T.; Tanaka, H.; Stammers, D. K.; Stuart, D. I. J. Med. Chem. 1996, 39, 1589. v. Kenedi, C. A.; Goforth, H. W. AIDS Behav. 2011, 15, 1803. vi. Hsu, H. E.; Rydzak, C. E.; Cotich, K. L.; Wang, B.; Sax, P. E.; Losina, E.; Freedberg, K. A.; Goldie, S.J.; Lu, Z.; Walensky, R. P. HIV Med. 2011, 12(2), 97. vii. Fontas, E.; van Leth, F.; Sabin, C. A.; Friis-Møller, N.; Rickenbach, M.; d’Arminio Monforte, A.; Kirk, O.; Dupon, M.; Morfeldt, L.; Mateu, S.;
Petoumenos, K.; El-Sadr, W.; de Wit, S.; Lundgren, J. D.; Pradier, C.; Reiss, P. J. Infect. Dis. 2004, 189, 1056. viii. Tambuyzer, L.; Azijn, H.; Rimsky, L. T.; Vingerhoets, J.; Lecocq, P.; Kraus, G.; Picchio, G.; de Bethune, M.-P. Antiviral Ther. 2009, 14(1), 103. ix. Schiller, D. S.; Youssef-Bessler, M. Clin. Res. 2009, 31(4), 692. x. Truvada® is a fixed dose combination of 300 mg of tenofovir disoproxil fumarate and 200 mg of emtricitabine. xi. Sanford, M. Drugs 2012, 72(4), 525. xii. Gomez, R.; Jolly, S.; Williams, T.; Tucker, T.; Tynebor, R.; Vacca, J.; McGaughey, G.; Lai, M.T.; Felock, P.; Munshi, V.; DeStefano, D.; Touch, S.; Miller, M.; Yan, Y.; Sanchez, R.; Liang, Y.; Paton, B.; Wan, B.L.; Anthony, N. Bioorg. Med. Chem. Lett. 2011, 21, 7344. xiii. For analog 4, F = 57% in rat at 10 mpk as a methocel suspension but the exposure varied from 1.5 µM*h up to 38 µM*h. For analog 5, F = 15% in rat at 5 mpk as a methocel suspension. In dog F = 9% at 1 mpk as a methocel suspension. xiv. Saggar, S. A.; Sisko, J. T.; Tucker, T. J.; Tynebor, R. M.; Su, D.-S.; Anthony, N. J. U.S. Patent Appl. US 2007/021442 A1, 2007. xv. Vacca, J. P.; Dorsey, B. D.; Schleif, W. A.; Levin, R. B.; McDaniel, S. L.; Darke, P. D.; Zugay, J.; Quintero, J. C.; Blahy, O. M.; Roth, E.; Sardana, V. V.; Schlabac, A. J.; Graham, P. I.; Condra, J. H.; Gotlib, L.; Holloway, M. K.; Lin, J.; Chen, I.-W.; Vastag, K.; Ostovic, D.; Anderson, P. S.; Emini, E. E.; Huff, J. R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4096. xvi. Aqueous equilibrium solubility was evaluated by stirring excess solid in water at room temperature for 24h in the absence of light. The solubility was measured by HPLC. xvii. For analog 24, F = 0% in rat. For analog 25, F = 15% in rat. xviii. 36: m.p. 280 oC. 1H NMR (500 MHz): δ 3.11 (s, 3H), 5.16 (s, 2H), 6.67 (d, 1H), 7.53 (s, 1H), 7.61 (s, 1H), 7.75 (s, 1H), 7.88 (d, 1H), 11.68 (s, 1H). xix. Sweeney, Z. K.; Acharya, S.; Briggs, A.; Dunn, J. P.; Elworthy, T. R.; Fretland, J.; Giannetti, A. M. Bioorg. Med. Chem. Lett. 2008, 18, 4348. xx. Microsomes: 0.5 mg/mL protein with 1 µM cpd + 1 mM NADPH, incubated for 30 min at 37 oC; hepatocytes: 106 cells + 1 µM cpd, incubated for 60 min at 37 oC.
*Graphical Abstract (for review)
Cl
CN O O F3C
N
CH3 N O N NH
(MK-1439)
S0960-894X(13)01454-6 http://dx.doi.org/10.1016/j.bmcl.2013.12.070 BMCL 21178
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
4 November 2013 16 December 2013 17 December 2013
Please cite this article as: Côté, B., Burch, J.D., Asante-Appiah, E., Bayly, C., Bédard, L., Blouin, M., Campeau, L-C., Cauchon, E., Chan, M., Chefson, A., Coulombe, N., Cromlish, W., Debnath, S., Deschenes, D., DupontGaudet, K., Falgueyret, J-P., Forget, R., Gagné, S., Gauvreau, D., Girardin, M., Guiral, S., Langlois, E., Li, C.S., Nguyen, N., Papp, R., Plamondon, S., Roy, A., Roy, S., Seliniotakis, R., St-Onge, M., Ouellet, S., Tawa, P., Truchon, J-F., Vacca, J., Wrona, M., Yan, Y., Ducharme, Y., Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.12.070
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Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses
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Bernard Côté∗, Jason D. Burch, Ernest Asante-Appiah, Chris Bayly, Leanne Bédard, Marc Blouin, Louis-Charles Campeau, Elizabeth Cauchon, Manuel Chan, Amandine Chefson, Nathalie Coulombe, Wanda Cromlish, Smita Debnath, Denis Deschenes, Kristina Dupont-Gaudet, Jean-Pierre Falgueyret, Robert Forget, Sébastien Gagné, Danny Gauvreau, Melina Girardin, Sébastien Guiral, Eric Langlois, Chun Sing Li, Natalie Nguyen, Rob Papp, Serge Plamondon, Amélie Roy, Stéphanie Roy, Ria Seliniotakis, Miguel St-Onge, Stéphane Ouellet, Paul Tawa, Jean-François Truchon, Joe Vacca, Marc Wrona, Youwei Yan and Yves Ducharme
Bioorganic & Medicinal Chemistry Letters jo u r n a l h o m e p a g e : w w w .e ls e v ie r .c o m
Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses ∗
Bernard Côté , Jason D. Burch, Ernest Asante-Appiah, Chris Bayly, Leanne Bédard, Marc Blouin, LouisCharles Campeau, Elizabeth Cauchon, Manuel Chan, Amandine Chefson, Nathalie Coulombe, Wanda Cromlish, Smita Debnath, Denis Deschenes, Kristina Dupont-Gaudet, Jean-Pierre Falgueyret, Robert Forget, Sébastien Gagné, Danny Gauvreau, Melina Girardin, Sébastien Guiral, Eric Langlois, Chun Sing Li, Natalie Nguyen, Rob Papp, Serge Plamondon, Amélie Roy, Stéphanie Roy, Ria Seliniotakis, Miguel StOnge, Stéphane Ouellet, Paul Tawa, Jean-François Truchon, Joe Vacca, Marc Wrona, Youwei Yana and Yves Ducharme Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Quebec, Canada H9H 3L1 a Merck Research Laboratories, 770 Sumneytown Pike, PO Box 4, West Point, PA, 19486-0004, United States
A R T IC LE IN F O
A B S TR AC T
Article history: Received Revised Accepted Available online
The optimization of a novel series of non- nucleoside reverse transcriptase inhibitors (NNRTI) led to the identification of pyridone 36. In cell cultures, this new NNRTI shows a superior potency profile against a range of wild type and clinically relevant, resistant mutant HIV viruses. The overall favorable preclinical pharmacokinetic profile of 36 led to the prediction of a once daily low dose regimen in human. NNRTI 36, now known as MK-1439, is currently in clinical development for the treatment of HIV infection.
Keywords: Non-nucleoside reverse transcriptase HIV triazolinone pyridone inhibitor
Highly active antiretroviral therapy (HAART) is the standard of care for the treatment of HIV infection.i Typically, this protocol recommends the combination of two nucleoside reversetranscriptase inhibitors (NRTIs) with either a non-nucleoside reverse-transcriptase inhibitor (NNRTI), a ritonavir-boosted protease inhibitor or an integrase inhibitor.ii NNRTI-based combinations have become first-line therapy mainly because of their demonstrated efficacies, convenient dosing regimen and relatively low toxicities.iii These inhibitors block the polymerase activity of the HIV reverse transcriptase by binding to an allosteric hydrophobic pocket adjacent to the active site.iv Efavirenz (1, Fig. 1) is a first generation NNRTI that has been conveniently co-formulated with NRTIs tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) as a once-aday fixed-dose combination (Atripla®). Although recommended for the therapy of treatment-naïve patients, efavirenz suffers from neurocognitive side effects,v teratogenicityvi and exacerbation of hyperlipidemia.vii Moreover, the low barrier to genetic resistance of first generation NNRTIs led to the emergence of resistant
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viruses bearing mutations K103N and Y181C in patients failing therapy. viii
Figure 1. Structures of marketed and lead NNRTIs
——— ∗ Corresponding author. Tel.: +1-514-924-1456; e-mail: [email protected]
Second generation NNRTIs etravirine (2) and rilpivirine (3) efficiently suppress the replication of the K103N resistant mutants as shown by an improved activity in cell culture assays (Table 1). Etravirine (200 mg, bid) is approved for use in treatment-experienced adult patients with multi-drug resistance.ix With an improved pharmacokinetic profile, the close analog rilpivirine (25 mg, qd) was recently approved for use in treatment-naïve patients. Phase III data reveal that at the 96-week point, a rilpivirine/truvada®x combination was better tolerated than efavirenz/truvada®.xi However, the virologic failure rate was twice as high for rilpivirine (14%) than it was for efavirenz (8%). For patients with viral load greater than 500,000 copies/mL, the response rate is 62% (rilpivirine) versus 81% (efavirenz). As a result, rilpivirine is not recommended for treating HIV patients with viral load >500,000 copies/mL. This difference in treatment durability could be explained by the much higher ratio of trough concentration over the antiviral activity for efavirenz versus rilpivirine. Pyridone 4 was recently identified as a novel NNRTI with an improved spectrum of antiviral activity against key mutant strains (Table 1).xii The related pyridone 5 was also found to have a similar in vitro profile. However, both analogs suffered from poor oral bioavailability or high inter-animal exposure variabilityxiii that limited their development potential. As poor patient compliance is a key element leading to drug resistance, optimization of the pharmacokinetic parameters becomes critical to access a simple dosing regimen that will maximize around-theclock viral suppression. In our effort to optimize this new NNRTI pyridone series, we therefore specifically aimed at developing a low dose, once daily drug that could be conveniently coformulated with other therapeutic agents. Table 1 Enzyme and Spread-of-virus inhibition of lead NNRTIs RTa SPREAD 50% NHSb IC50 (µM) IC95 (µM) Compound WTc WT K103N Y181C K103N/Y181C Efavirenz 0.002 0.039 1.4 0.09 3.2 Etravirine 0.001 0.033 0.044 0.24 0.59 Rilpivirine 0.001 0.036 0.044 0.12 0.37 4 0.003 0.008 0.012 0.02 0.07 5 0.003 0.017 0.024 0.06 0.07 a RT: Reverse transcriptase. ECL assay. Values are the geometric mean of at least two determinations. Assay protocols are detailed in Ref. xiv. b IC95 is defined as the concentration at which the Spread of the virus is inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref. xv. c WT: Wild type.
One of the striking elements that initially oriented our efforts to improve the oral bioavailability of the pyridone series was the poor aqueous solubilityxvi of 5 (0.2 ng/mL in water). This low solubility, as well as the high crystallinity of 5 (m.p. = 299oC), could be rationalized by the analysis of the solid-state x-ray structure of a close analog sharing the pyrazolopyridine moiety found in 5 (Fig. 2). We hypothesized that disruption of the strong donor/acceptor hydrogen bond motif of the pyrazolopyridine would weaken the crystal lattice and enhance the solubility.
Figure 2. Donor/acceptor hydrogen bonding of the pyrazolopyridine
To validate this strategy, two analogs were synthesized where the pyridine nitrogen of the pyrazolopyridine was either removed or shifted by one position (Table 2). The solubility was assessed by light scattering as a measure of the concentration threshold at which a DMSO solution of the test compounds precipitate in a PBS solution. As shown in table 2, both structural modifications found in pyridone 6 and 7 led to an improved aqueous solubility (5.2 µM and 12.0 µM respectively) and better oral bioavailability in rat (30% and 74%). This data set strongly suggested that the originally observed low solubility and low oral absorption were not inherent to the pyridone series. We therefore undertook the synthesis of a library of pyridone analogs to identify a pyrazolopyridine replacement using the intermediate hydroxypyridine 12a (Scheme 1). In parallel with this effort, SAR at the 4-position of the pyridone was also initiated using intermediate 12b (Scheme 2). Table 2 Solubility and oral rat bioavailability of substituted pyridone
RTa SPREAD 50% NHSb Sol c Rat PKd IC50 (µM) IC95 (µM) (µM) WTe WT K103N/Y181C F(%) Cl f 5 0.003 0.017 0.069 1.1 13 18 6 0.001 0.053 0.186 5.2 30 17 7 0.006 0.029 0.092 12.0 74 40 a RT: Reverse transcriptase. ECL assay. Values are the geometric mean of at least two determinations. Assay protocols are detailed in Ref. 14. b IC95 is defined as the concentration at which the Spread of the virus in inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref.15. c Sol: solubility threshold of the DMSO solution in PBS buffered solution measured by light scattering. d Average of 2 Sprague Dawley rat dosed at 5 mpk PO (methocel suspension) and 1 mpk IV 60% PEG200). eWT: Wild type. f Cl: clearance in mL/min/kg. Compd
The synthetic sequence to prepare the hydroxypyridine 12a is outlined in Scheme 1. Chloropyridine 8 was coupled with 2bromo-4-chlorophenol (9) under basic conditions to afford the biaryl ether 10 in 79% yield. This chloropyridine was then hydrolyzed to the hydroxypyridine 11 with potassium hydroxide in 71% yield. Finally, this intermediate was treated with zinc cyanide in the presence of a catalytic amount of Pd(PPh3)4 to provide the desired cyano derivative 12a. Scheme 2 describes the general approach to synthesize the different 4-substituted pyridones. The synthesis of the common intermediate 12b first involves the SNAr reaction of commercially available 2-iodo-4chlorophenol (14) with the nitropyridine-N-oxide 13 in 80% yield. The iodophenol was preferred to the previously used bromo analog in order to avoid the formation of a regioisomeric mixture during the cyanation reaction. Sequential treatment of the nitropyridine-N-oxide 15 with acetylbromide in acetic acid followed by the reaction of the crude material with phosphorus tribromide in chloroform provided the dibromopyridine 16 in 87% overall yield. Hydrolysis of the bromopyridine with potassium hydroxide (72% yield) followed by the quantitative cyanation of the iodoaryl moiety gave the 4-bromo-2hydroxypirydine 12b. This key intermediate was then fonctionnalized by taking advantage of the selective reactivity of the bromide. The final assemblage was completed by alkylating the hydroxypyridines 12b-f as describe before (see ref. 12) with
the protected 3-bromomethyl pyrazolopyridine followed by TFAmediated hydrolysis of the tert-butyl carboxylate.
Scheme 1. Reagents and conditions: (a) K2CO3 , NMP, 120o C; (b) KOH, tert-BuOH, 75o C; (c) Zn(CN)2 , Pd(PPh3)4 , DMF, 100o C.
Scheme 2. Reagents and conditions: (a) K2CO3 , DMF, 55o C; (b) AcBr, AcOH, 80oC; (c) PBr3, CHCl 3, 60oC; (d) KOH, tert-BuOH; (c) Zn(CN)2 , Pd(PPh3)4, DMF, 100o C; (f) K2CO3 , bromide, DMF, 55o C, then TFA.
The biological activity and the rat pharmacokinetic properties of pyrazolopyridines 17-21 were evaluated (Table 3). In general, most of the functional groups were very well tolerated with low nanomolar enzyme inhibition activity. In the Spread assay, bromo derivative 17 was identified as the most potent analog ever prepared in the pyridone series with an IC95 = 0.004 µM against the wild type virus and an IC95 = 0.021 µM against the double mutant K103N/Y181C. In cell culture, the antiviral potency of all these new analogs is superior to the benchmark NNRTIs efavirenz and rilpivirine. The key differentiation factor amongst them is their in vivo ADME properties. The CF3 substituted pyridone 5 presents a substantially longer elimination half-life (t1/2 = 7 h) in rats compared to its close analogs. Further studies in dogs (data not shown) corroborated this pharmacokinetic trend and the strong electron withdrawing CF3 group was therefore considered as optimal. The next series of pyridone analogs were synthesized by the alkylation of intermediate 12a with different heterocycle derivatives (Table 4). Using standard reaction conditions (K2CO3, DMF) a 48-membered library was rapidly built. The specific aim of this exercise was to identify compounds that would retain the antiviral activity of 5, and would present increased aqueous solubility and ultimately improved systemic exposure following oral administration in pre-clinical species. Inhibitors 22 to 25 are pyrazolopyridine derivatives that were substituted to increase the polarity of the compounds (22 and 23) or to reduce the basicity of the pyridine nitrogen (24 and 25). All four analogs were found to be potent, but suffered from poor rat pharmacokinetics.xvii We have previously shown in table 2 that the potent regioisomeric pyrazolopyridine 7 is well absorbed but eliminated too rapidly in
rats. With the fluorinated analog 26, we were able to extend the rat half-life to 4 hours with an oral bioavailability of 65% but unfortunately, a significant potency shift was observed in the cellular assay in the presence of 50% NHS (IC95 = 76 nM). Table 3 Biological activity and rat half-life of 4-substituted pyridones.
Compd
X
RTa IC50 (µM) WT
c
Spread 50% NHSb IC95 (µM) WT
K103N/Y181C
Rat t1/2 (h)d
4 Cl 0.003 0.008 0.069 2.2 5 CF3 0.003 0.017 0.069 7.0 17 Br 0.002 0.004 0.021 1.7 18 CH3 0.007 0.012 0.096 0.8 19 CF2 CH3 0.005 0.014 0.044 1.3 20 SCH3 0.009 0.015 0.033 1.5 21 c-Pr 0.007 0.012 0.11 1.5 a RT: Reverse transcriptase. ECL assay. Values are the geometric mean of at least two determinations. Assay protocols are detailed in Ref. 14. b IC95 is defined as the concentration at which the Spread of the virus in inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref.15. c WT: Wild type. d Average of 2 Sprague Dawley rats dosed at 1 mpk IV (60% PEG200).
Greater structural diversity was explored with the following heterocycles. Despite their good intrinsic potency against the enzyme, the large lipophilic quinoline 27 and benzoisoxazole 28
showed largely shifted potencies in the Spread assay. The incorporation of small heterocyclic rings to produce inhibitors such as 4-methylthiazole 29, generally resulted in significantly increased aqueous solubility. While this finding was true for most small rings, the nature of the heterocycle and its substitution pattern had a tremendous impact on the antiviral potency. While substitution with a methyl group at the 4-position seems to assure enzyme activity in the thiazole series (29 and 31 vs 30), this observation did not translate well for the pyrazole 32 (IC50 = 140 nM), the imidazole 33 (IC50 = 300 nM) and the triazole 34 (IC50 = 43 nM). In the triazolinone series though, methylation at the 4position as described in Scheme 3, was found to be beneficial with a 5-fold increase in enzyme potency (35 vs 36). Conversely, the slight increase in steric hindrance of the 4-ethyl substituted triazolone 37 negatively impacted the cell culture activity (IC95 > 125 nM). Table 4. Potency and solubility of alkylated pyridones
a
RT: Reverse transcriptase. ECL assay. Values are the geometric mean of at least two determinations. Assay protocols are detailed in Ref. 14. b IC95 is defined as the concentration at which the Spread of the virus in inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref. 15. c Sol: solubility threshold of the DMSO solution in PBS buffered solution measured by light scattering.
In addition to the 4-methyl substitution of the triazolinone, one of the key structural features contributing to the good antiviral potency of inhibitor 36xviii is the presence of the N-NH motif that was also present in the pyrazolopyridine series. In an analogous biaryl ether series, Sweeney et al. have described a series of heterocycles sharing this structural feature.xix Based on crystal structure analysis with HIV-RT, they highlighted the role of the N-NH acceptor-donor motif that engages a pair of hydrogen-bonds with the K103 backbone of the RT enzyme. This type of interaction was also observed in the binding of pyridone 36 with HIV-RT in the structural elucidation of a co-crystal by X-ray, as shown in Figure 3.
Y Figure 3. X-ray crystal structure of compound 36 bound to HIV-RT. The wild-type RT complex structure has been deposited in the Protein Data Bank under accession code 4NCG.
As seen in Table 5, inhibitor 36 presents an exceptional in vitro profile against clinically relevant resistant viruses. Compared to efavirenz, pyridone 36 is 33-fold more active against K103N and 59-fold more potent against the double mutant K103N/Y181C. Compared to rilpivirine, inhibitor 36 is respectively 5 and 7-fold more potent against the mutant viruses Y181C and K103N/Y181C respectively. Table 5 Antiviral activity of pyridone 36 in cell culture SPREAD IC95 (µM) with 50% NHSa Compound WT K103N Y181C K103N/Y181C 36 0.019 0.042 0.025 0.054 Efavirenz 0.039 1.4 0.089 3.2 Rilpivirine 0.036 0.044 0.120 0.37 a IC95 is defined as the concentration at which the Spread of the virus in inhibited by >95% in the presence of 50% NHS (normal human serum). Assay protocols are detailed in Ref. 15.
In addition to the above mutant substituted viruses, pyridone 36 was tested against a panel of 15 mutant viruses (Monogram Biosciences) and compared to efavirenz, rilpivirine and lead inhibitor 5.
bioavailability of our new NNRTI confers a 16-fold increase in systemic exposure (AUCN = 3.9 µM*h). In dog, the same net result is also observed with a 26-fold improvement in exposure. In this case, a combination of low clearance (Cl = 0.36 mL/min/kg) and better oral absorption (F = 52%) is responsible for the high observed AUCN of 44 µM*h. The overall favorable pharmacokinetic profile of 36 in preclinical species, combined to a low metabolic turnover (<10%) in human liver microsomes and hepatocytesxx lead to the prediction of a once daily low dose regimen in human.
Fold Change (vs. Control)
700 600 500 400 300 200 100 0
Efavirenz Rilpivirine Compd 5 Compd 36
Mutant viruses Figure 4. Fold change in activity (IC50 versus the control CNDO virus) of NNRTIs in a panel of 15 mutant viruses (Monogram Biosciences). A maximum cut-off of 625-fold is shown. The data for the inhibitors are presented as follows: efavirenz (purple), rilpivirine (green), compound 5 (red), compound 36 (blue).
As indicated in Figure 4, efavirenz was less active against a number of the mutant viruses in contrast to the second-generation inhibitor, rilpivirine. Pyridone 36 maintained the potent antiviral activity of compound 5 and demonstrated a robust activity against the mutant viruses. It was less active against the rare mutants harboring the Y188L and V106A/G109A/F227L amino acid changes for which two or more nucleotide changes are required. Additional profiling of the antiviral activity of compound 36 on a panel of 96 mutant viruses shows a similar robust activity (to be reported in a future publication). As shown before, the solubility threshold of compound 36 (45 µM) is significantly increased compared to the original lead pyridone 5 (1.0 µM) in water. At both pH 4 and 8, the equilibrium solubility of 36 is 6 µg/mL in comparison to <0.1 µg/mL for 5. Solubility in fasted simulated intestinal fluid (FaSSIF) went up from 1.3 µg/mL for pyrazolopyridine 5 to 130 µg/mL for the crystalline triazolinone 36. These improved physicochemical properties correlate with a superior pharmacokinetic profile for 36 in rats and dogs as presented in table 6.
Scheme 3. Reagents and conditions: (a) K2CO3 , DMF, -10oC; (b) MeI or EtI, K2CO3, DMF.
Although the clearance of 36 (Cl = 5.4 mL/min/kg) is lower compared to 5 (15 mL/min/kg) in rats, the half-life of 36 is slightly shorter due to its smaller volume of distribution (Vd). Nevertheless, this lower Vd combined with the good rat
i. (a) Hirschel, B.; Francioli, P. N. Engl. J. Med. 1998, 338, 906. (b) Schneider, M. F. ; et al. AIDS, 2005, 19, 2009. ii. Hammer, S. M.; et al. JAMA, 2006, 296, 827. iii. Fokunang, C. N.; Hitchcock, J.; Spence, F.; Tembe-Fokunang, E. A.; Burkhardt, J.; Levy, L.; George, C. Int. J. Pharmacol. 2006, 2(1), 152. iv. (a) Sluis-Cremer, N.; Temiz, N. A.; Bahar, I. Curr. HIV Res. 2004, 2(4), 323. (b) Hopkins, A. L.; Ren, J.; Esnouf, R. M.; Willcox, B. E.; Jones, E. Y.;
With a broad spectrum of antiviral activity against clinically relevant mutant viruses, analogs from the new pyridone series clearly meet a mandatory criterion for next generation NNRTIs. Unfortunately, poor oral bioavailability in preclinical species was a key liability originally associated with this series. Using a simple synthetic approach, we rapidly built a library of inhibitors that led to the identification of pyridone 36. In cell culture, this new inhibitor has a superior profile against the wild type and K103N/Y181C viruses compared with benchmark compounds efavirenz, etravirine and rilpivirine. The increased aqueous solubility of pyridone 36 translated into a significant improvement of pharmacokinetic parameters in preclinical species. NNRTI 36, now known as MK-1439, is currently in clinical development for the treatment of HIV infection.
Table 6 Comparative pharmacokinetic parameters of pyridones 5 and 36 in rat and dog Species Parameters 5 36 Vd (L/kg)
8.8
Cl (mL/min/kg)
15
5.4
t1/2 (hr)
7
4.4
Rata
F (%)
2.3
15
57
AUCN (µM*h)c
0.25
3.9
Vd (L/kg)
1.5
1.2
Cl (mL/min/kg)
1.8
0.36
t1/2 (hr)
10
37
F (%)
9
52
Dogb
AUCN (µM*h) 1.7 44 Average of 2 Sprague Dawley rats for each doses. 5 was dosed IV at 1 mpk (80%PEG200) and PO at 5 mpk (Ball-milled, 0.5% methocel). 36 was dosed IV at 1 mpk (60%PEG200) and PO at 5 mpk (Ball-milled, 0.5% methocel). b Average of 2 Beagle dogs for each doses. 5 was dosed IV at 0.5 mpk (80%PEG200) and PO at 1 mpk (Ball-milled, 0.5% methocel). 36 was dosed IV at 0.5 mpk (60%PEG200) and PO at 1 mpk (Ball-milled, 10% Tween). c Normalized for 1 mpk from 0-24h. a
Acknowledgements The authors wish to thank Neville Anthony, Christopher Burgey and Ming-Tain Lai for reviewing the manuscript. References and notes
Ross, C.; Miyasaka, T.; Walker, R. T.; Tanaka, H.; Stammers, D. K.; Stuart, D. I. J. Med. Chem. 1996, 39, 1589. v. Kenedi, C. A.; Goforth, H. W. AIDS Behav. 2011, 15, 1803. vi. Hsu, H. E.; Rydzak, C. E.; Cotich, K. L.; Wang, B.; Sax, P. E.; Losina, E.; Freedberg, K. A.; Goldie, S.J.; Lu, Z.; Walensky, R. P. HIV Med. 2011, 12(2), 97. vii. Fontas, E.; van Leth, F.; Sabin, C. A.; Friis-Møller, N.; Rickenbach, M.; d’Arminio Monforte, A.; Kirk, O.; Dupon, M.; Morfeldt, L.; Mateu, S.;
Petoumenos, K.; El-Sadr, W.; de Wit, S.; Lundgren, J. D.; Pradier, C.; Reiss, P. J. Infect. Dis. 2004, 189, 1056. viii. Tambuyzer, L.; Azijn, H.; Rimsky, L. T.; Vingerhoets, J.; Lecocq, P.; Kraus, G.; Picchio, G.; de Bethune, M.-P. Antiviral Ther. 2009, 14(1), 103. ix. Schiller, D. S.; Youssef-Bessler, M. Clin. Res. 2009, 31(4), 692. x. Truvada® is a fixed dose combination of 300 mg of tenofovir disoproxil fumarate and 200 mg of emtricitabine. xi. Sanford, M. Drugs 2012, 72(4), 525. xii. Gomez, R.; Jolly, S.; Williams, T.; Tucker, T.; Tynebor, R.; Vacca, J.; McGaughey, G.; Lai, M.T.; Felock, P.; Munshi, V.; DeStefano, D.; Touch, S.; Miller, M.; Yan, Y.; Sanchez, R.; Liang, Y.; Paton, B.; Wan, B.L.; Anthony, N. Bioorg. Med. Chem. Lett. 2011, 21, 7344. xiii. For analog 4, F = 57% in rat at 10 mpk as a methocel suspension but the exposure varied from 1.5 µM*h up to 38 µM*h. For analog 5, F = 15% in rat at 5 mpk as a methocel suspension. In dog F = 9% at 1 mpk as a methocel suspension. xiv. Saggar, S. A.; Sisko, J. T.; Tucker, T. J.; Tynebor, R. M.; Su, D.-S.; Anthony, N. J. U.S. Patent Appl. US 2007/021442 A1, 2007. xv. Vacca, J. P.; Dorsey, B. D.; Schleif, W. A.; Levin, R. B.; McDaniel, S. L.; Darke, P. D.; Zugay, J.; Quintero, J. C.; Blahy, O. M.; Roth, E.; Sardana, V. V.; Schlabac, A. J.; Graham, P. I.; Condra, J. H.; Gotlib, L.; Holloway, M. K.; Lin, J.; Chen, I.-W.; Vastag, K.; Ostovic, D.; Anderson, P. S.; Emini, E. E.; Huff, J. R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4096. xvi. Aqueous equilibrium solubility was evaluated by stirring excess solid in water at room temperature for 24h in the absence of light. The solubility was measured by HPLC. xvii. For analog 24, F = 0% in rat. For analog 25, F = 15% in rat. xviii. 36: m.p. 280 oC. 1H NMR (500 MHz): δ 3.11 (s, 3H), 5.16 (s, 2H), 6.67 (d, 1H), 7.53 (s, 1H), 7.61 (s, 1H), 7.75 (s, 1H), 7.88 (d, 1H), 11.68 (s, 1H). xix. Sweeney, Z. K.; Acharya, S.; Briggs, A.; Dunn, J. P.; Elworthy, T. R.; Fretland, J.; Giannetti, A. M. Bioorg. Med. Chem. Lett. 2008, 18, 4348. xx. Microsomes: 0.5 mg/mL protein with 1 µM cpd + 1 mM NADPH, incubated for 30 min at 37 oC; hepatocytes: 106 cells + 1 µM cpd, incubated for 60 min at 37 oC.
*Graphical Abstract (for review)
Cl
CN O O F3C
N
CH3 N O N NH
(MK-1439)