Joshua J.
Kennedy-Smith
*a,
Nidhi
Arora
a,
J. Roland
Billedeau
a,
Jennifer
Fretland
e,
Julie Q.
Hang
d,
Gabrielle M.
Heilek
c,
Seth F.
Harris
b,
Donald
Hirschfeld
a,
Hassan
Javanbakht
c,
Yu
Li
d,
Weiling
Liang
a,
Ralf
Roetz
a,
Mark
Smith
a,
Guoping
Su
c,
Judy M.
Suh
a,
Armando G.
Villaseñor
b,
Jeffrey
Wu
a,
Dennis
Yasuda
a,
Klaus
Klumpp
d and
Zachary K.
Sweeney
*a
aDepartment of Medicinal Chemistry, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304. E-mail: joshua.kennedy_smith@roche.com; sweeney.zachary@gene.com
bDepartment of Discovery Technologies, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304
cDepartment of Viral Disease Biology, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304
dDepartment of Viral Disease Biochemistry, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304
eDepartment of Non-Clinical Safety, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304
First published on 28th April 2010
New COMPOUND LINKS
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Download mol file of compoundpyridone non-nucleoside reverse transcriptase inhibitors (NNRTIs) were prepared and several flexible routes to this class of inhibitor were identified. These NNRTIs were active inhibitors of the replication of wild-type and NNRTI-resistant HIV. Structure-based drug design was used to optimize the activity of the compounds against NNRTI-resistant mutants. The co-crystal structure of inhibitor 2b in the NNRTI binding pocket of HIV reverse transcriptase (HIVRT) is also described.
We have recently described the discovery of a series of diaryl ether NNRTIs.6 These compounds are potent inhibitors of the wild-type virus and commonly observed NNRTI-resistant mutant viruses in vitro, and have promising pharmacokinetic profiles in several animal species. A fluorinated central aromatic ring is a common feature of these inhibitors (Figure 1, A), as these compounds were generally more potent inhibitors of polymerase activity than their non-fluorinated analogs. Structural analysis revealed that the fluorine atom engaged in weak hydrophobic interactions. We also speculated that the fluorine might affect the strength of a C–H hydrogen bond between the hydrogen atom meta to the fluorine (bold in Figure 1) and the Lys101 carbonyl oxygen of the viral reverse transcriptase.7 Most NNRTIs feature direct or water-mediated hydrogen bonding interactions with the carbonyl oxygen of Lys101.8
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Fig. 1 Diaryl ether (A) and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundpyridone aryl ether (B) NNRTIs. |
The diaryl ether NNRTIs A also contain a heterocyclic or amide functionality that engages in hydrogen-bonding interactions with the backbone of Lys103. This unit stabilizes an expanded NNRTI binding pocket and strongly influences the potency of the inhibitors against NNRTI-resistant viruses.6
These considerations led to the design of a series of COMPOUND LINKS
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Download mol file of compoundpyridone diaryl ether inhibitors that feature aliphatic or aromatic groups attached to C(5) or C(6) of the COMPOUND LINKS
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Download mol file of compoundpyridone ring (Figure 1, B). Such COMPOUND LINKS
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Download mol file of compoundpyridone inhibitors would be expected to interact with the Lys101 carbonyl group of HIVRT through a strong traditional hydrogen bonding interaction. Appropriate substitution of the pyridones should allow for stabilization of the expanded NNRTI binding pocket, potential interactions with the backbone amide of Lys103, and optimization of the physical properties of the inhibitors.
COMPOUND LINKS
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Download mol file of compoundPyridone NNRTIs have been known for over 20 years and the field has been reviewed.9 Furthermore, substituted 4-arylthio- and 4-aryloxypyridones have recently been reported in publications by the Tibotec/CNRS group.10,11 However, the structural diversity of the COMPOUND LINKS
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Download mol file of compoundpyridone NNRTI class is limited by the availability of synthetic precursors for the diversity-oriented preparation of substituted pyridines. As a consequence, most COMPOUND LINKS
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Download mol file of compoundpyridone NNRTIs contain a number of metabolically unstable functional groups (e.g. thioethers, alkylaromatics). In addition, there has been relatively little published data regarding the structure–activity relationships for this class of inhibitors. This report describes novel synthetic routes and the biological activity of diversely substituted COMPOUND LINKS
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Download mol file of compoundpyridone NNRTIs that have excellent antiviral properties.
In order to confirm that the COMPOUND LINKS
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Download mol file of compoundpyridone ring would effectively replace the central phenyl ring of our diaryl ether NNRTIs, inhibitors 1, 2a and 2b were prepared (Fig. 2). Both COMPOUND LINKS
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Download mol file of compoundpyridone compounds 2 were found to strongly inhibit the polymerase activity of wild type HIV reverse transcriptase. Compounds 2 also prevented HIV-induced cell death in vitro.12 Finally, the COMPOUND LINKS
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Download mol file of compoundpyridone inhibitors were determined to have promising activity in assays assessing the ability of compounds to inhibit the polymerase activity of the NNRTI-resistant Lys103Asn and Tyr181Cys mutant enzymes. The comparable efficacy of compounds 1 and 2 encouraged us to further explore the effects of substitution of the COMPOUND LINKS
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Download mol file of compoundpyridone inhibitors. This optimization effort was assisted by a co-crystal structure of compound 2b and wild-type HIVRT, which confirmed the expected binding mode of our COMPOUND LINKS
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Download mol file of compoundpyridone inhibitors (Fig. 3).13,14
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Fig. 2 Structures of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundpyridone and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundphenyl amide inhibitors 1 and 2 and associated IC50s for inhibition of the wild type polymerase and wild-type HIVRT. |
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Fig. 3 Co-crystal structure of 2b in the NNRTI binding pocket (3FFI). |
In an effort to replace the amide linkage of 2 while maintaining the open NNRTI binding pocket, we prepared a series of COMPOUND LINKS
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Download mol file of compoundpyridone NNRTIs that contained aromatic groups attached to the C(6) carbon of the COMPOUND LINKS
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Download mol file of compoundpyridone (Table 1). This effort was enabled by the synthesis of the new, versatile aryl bromide intermediate 8. All of the compounds prepared in this exercise strongly inhibited the polymerase activity of the wild-type and NNRTI-resistant enzymes. Two important conclusions were drawn from this initial work. First, in contrast to our previous observations, fluorination of the central ring did not significantly influence the potency of the inhibitors. Second, while a wide variety of aromatic structures were accommodated in this flexible region of the NNRTI binding pocket, there appeared to be no significant advantage for hydrogen bond donor–acceptor interactions between the COMPOUND LINKS
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Download mol file of compoundpyridone inhibitors and the K103 amide backbone such as those found in initial compounds 2.
Compd | X | R1 | WT | K103Nb | Y181Cc |
---|---|---|---|---|---|
a IC50 in nM. See ref. 10 for assay details. b Experiment using the Lys103Asn mutant enzyme. c Experiment using the Tyr181Cys mutant enzyme. | |||||
3a | Cl | Ph | 8 | — | — |
3b | Cl | 2-Benzoxazole | 6 | 16 | 4 |
3c | Cl | 2-Pyridyl | 21 | 78 | 33 |
3d | Cl | 3-Pyridyl | 22 | 16 | 22 |
3e | Cl | 4-Pyridyl | 19 | 14 | 21 |
3f | Cl | 3-(Cl-phenyl) | 12 | 6 | 7 |
4a | Cl | Ph | 7 | — | — |
4b | Br | 3-(Cl-phenyl) | 17 | 8 | 8 |
4c | Br | 4-Pyridyl | 2 | 1 | 1 |
Analysis of the modeled structure of compound 4c in the expanded NNRTI binding pocket,6,15 suggested that inhibitors with an alkyl substituent at C(5) of the COMPOUND LINKS
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Download mol file of compoundpyridone ring might be able to effectively contact the conserved L234 and F227 residues. Synthesis of functionalized haloaromatics 16 and 18 allowed for the preparation of a series of compounds with aromatic groups attached to C(5) of the COMPOUND LINKS
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Download mol file of compoundpyridone ring. Testing in the biological assays revealed that benzyl-substituted compounds 5a and 6a could function as potent inhibitors of polymerase activity, and that the methyl substituent at C(6) modestly increased potency against NNRTI-resistant mutants (Table 2). Extension of the linkage between the aromatic group and the COMPOUND LINKS
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Download mol file of compoundpyridone ring provided a number of potent NNRTIs, including COMPOUND LINKS
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Download mol file of compoundpyridine-containing compounds that had acceptable thermodynamic aqueous solubility.16 A model of compound 6c in the NNRTI binding pocket is shown in Fig. 4.17,18
Compd | X | R2 | WT | K103Nb | Y181Cc |
---|---|---|---|---|---|
a IC50 in nM. See ref. 10 for assay details. b Experiment using the Lys103Asn mutant enzyme. c Experiment using the Tyr181Cys mutant enzyme. | |||||
5a | Br | Ph | 34 | 71 | 41 |
5b | Br | CH2O(3-pyridyl) | 11 | 16 | 8 |
5c | Br | CH2O(4-pyridyl) | 10 | 14 | 8 |
5d | Br | OCH2(4-pyridyl) | 21 | 13 | 11 |
6a | Br | Ph | 10 | 7 | 8 |
6b | Cl | CH2OPh | 6 | 5 | 6 |
6c | Cl | (CH2)2(3-pyridyl) | 4 | 2 | 3 |
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Fig. 4 Modeled structure of inhibitor 6c in the NNRTI binding pocket. |
COMPOUND LINKS
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Download mol file of compoundPyridone NNRTIs that strongly inhibited polymerase activity were advanced into cellular assays that measure the ability of the NNRTIs to inhibit HIV-replication in MT4 cells (Table 3). Efavirenz, which was used as a standard in this assay, had significantly reduced potency in experiments conducted either in the presence of additional human serum or with the NNRTI-resistant K103N virus. Compound 4c had activity comparable to efavirenz in assays with the wild-type virus and was a much stronger inhibitor of the K103N mutant. Inhibitor 6c was approximately 3-fold more active than efavirenz in the serum-shifted assay and strongly inhibited both NNRTI-resistant viruses.
Cmpd | WT | WT+s | K103N | Y181C |
---|---|---|---|---|
a IC50 in nM. See ref. 10 for assay details. WT+s refers to experiments run in the presence of 40% human serum. K103N and Y181C refer to experiments using the Lys103Asn mutant virus or the Tyr181Cys mutant virus, respectively. | ||||
efv | 2 | 18 | 65 | 3 |
4c | 5 | 23 | 5 | 11 |
5d | 7 | 10 | 13 | 19 |
6a | 6 | 32 | 16 | 29 |
6b | 4 | 13 | 24 | 11 |
6c | 2 | 5 | 2 | 6 |
The synthesis of 2a began with compound 719 (Scheme 1). Reduction of the nitro group, selective bromination, and a Sandmeyer reaction provided the flexible intermediate 8. Installation of the COMPOUND LINKS
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Download mol file of compoundacetic acid functionality in 9 was then accomplished by Negishi coupling of the COMPOUND LINKS
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Download mol file of compoundbromide, followed by selective deprotection of the tert-butyl ester with TFA. Standard EDC coupling and removal of the COMPOUND LINKS
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Download mol file of compoundmethyl ether with TMSI provided the target amide 2a.
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Scheme 1 Reagents and conditions: (a) Fe, NH4Cl, H2O, EtOH, 100 °C, 97%. (b) NBS, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDMF, 0 °C, 97%. (c) tBuONO, LiCl, CuCl2, MeCN, 60 °C, 64%. (d) 2-tBu-2-oxoethylzinc chloride, Pd(PtBu3)2, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounddioxane, 25 °C, 75%. (e) TFA, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDCM, 25 °C, 99%. (f) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundTetrahydroisoquinoline, EDCI, HOBT, DMAP, DIPEA, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDMF, 99%. (g) TMSCl, NaI, MeCN, 25 °C, 46%. |
Compounds 3 were also prepared using intermediate 8 (Scheme 2). For example, inhibitors 3a and 3g were produced by Negishi coupling of the commercially available COMPOUND LINKS
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Download mol file of compoundphenethylzinc bromide reagents followed by deprotection with TMSI. The heteroaryl compounds 3c–3e could be prepared by Sonogashira coupling, alkyne hydrogenation, and deprotection.
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Scheme 2 Reagents and conditions: (a) ArCH2CH2ZnCl, Pd(PPtBu3)2, dioxane, 25 °C. (b) ArCCH, CuI, Cl2Pd(PPh3)2, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundTEA, 25 °C, then H2, Pd/C, 25 °C. (c) TMSCl, NaI, MeCN, 25 °C. |
The synthesis of compounds 4 commenced from aryl ether 1120 (Scheme 3). Regioselective COMPOUND LINKS
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Download mol file of compoundhydrazine addition followed by oxidative halogenation21 provided brominated intermediate 12. Installation of the phenethyl and heteroaryl groups as above, conversion of the COMPOUND LINKS
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Download mol file of compoundfluoropyridine to a COMPOUND LINKS
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Download mol file of compoundpyridone, and halogenation provided the desired COMPOUND LINKS
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Download mol file of compoundpyridone inhibitors.
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Scheme 3 Reagents and conditions: (a) H2NNH2, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundTHF, 25 °C, then Br2, CHCl3, 60 °C, 38%. (b) ArCH2CH2ZnCl, Pd(PtBu3)2, dioxane, 25 °C. (c) ArCCH, CuI, Cl2Pd(PPh3)2, TEA, 25 °C, then H2, Pd/C, 25 °C. (d) BnOH, NaH, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundTHF, 25 °C. (e) TFA, 50 °C. (f) NXS, MeCN, 25–60 °C. |
In order access compounds 5, known COMPOUND LINKS
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Download mol file of compoundpyridone 1422 was converted in two steps to COMPOUND LINKS
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Download mol file of compoundbenzyl ether COMPOUND LINKS
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Download mol file of compound15. Reduction to the COMPOUND LINKS
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Download mol file of compoundaniline and conversion to the COMPOUND LINKS
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Download mol file of compoundiodide then provided key intermediate 16. This intermediate could be selectively functionalized to provide a range of C(5) substituted COMPOUND LINKS
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Download mol file of compoundpyridone NNRTIs (Scheme 4).
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Scheme 4 Reagents and conditions: (a) Ag2CO3, BnBr, PhH, 60 °C, 44%. (b) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3-Chloro-5-cyanophenol, K2CO3, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDMF, 50 °C, 55% (two steps). (c) Fe, NH4Cl, H2O, EtOH, 100 °C, 97%. (d) tBuONO, CH2I2, 60 °C, 48%. (e) BnZnBr, Pd(PtBu3)2, dioxane, 25 °C, 75%. (f) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundAllyltributyltin, Pd(PPh3)4, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDMF, 100 °C. (g) OsO4, NMO, NaIO4, H2O, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundTHF, 25 °C, then NaBH4, MeOH, 25 °C. (h) ArOH, DIAD, PPh3, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundTHF, 0–25 °C. (i) TFA, DCM, 25 °C. |
The culmination of our chemistry efforts led to the synthesis of compounds 6 (Scheme 5). Key intermediate 18 was prepared from known compound 1723 in 5 steps. Either a Stille/Mitsunobu or Grubbs/Suzuki24 sequence followed by deprotection allowed for the preparation of 6b–6c.
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Scheme 5 Reagents and conditions: (a) POCl3, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundbenzyltriethylammonium chloride, MeCN, 40 °C. (b) Ag2CO3, MeI, PhH, 60 °C. (c) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound3-Chloro-5-cyanophenol, K2CO3, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDMF, 50 °C, 55% (two steps). (d) Fe, NH4Cl, H2O, EtOH, 100 °C, 97%. (e) tBuONO, LiCl, CuCl2, MeCN, 60 °C, 64%. (f) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundAllyltributyltin, Pd(PPh3)4, PhMe, 100 °C. (g) OsO4, NMO, NaIO4, H2O, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundTHF, 25 °C, then NaBH4, MeOH, 25 °C. (h) ArOH, DIAD, PPh3, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundTHF, 0 °C to 25 °C. (i) Propenylpinacolatoborane, Grubbs II, DCM, 50 °C. (j) ArI, Pd(PPh3)4, K2CO3, H2O, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDME, 90 °C. (k) H2, PtO2, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundTHF, 25 °C. (l) TMSCl, NaI, MeCN, 25 °C. |
In this communication we have described novel synthetic routes to polysubstituted COMPOUND LINKS
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Download mol file of compoundpyridone NNRTIs. These syntheses provide for facile exploration of the C(3), C(5) and C(6) substituents of the COMPOUND LINKS
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Download mol file of compoundpyridone ring, and permit the introduction of a wider variety of aryl ethers than previously reported approaches. The COMPOUND LINKS
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Download mol file of compoundpyridone NNRTIs described herein are active inhibitors of HIV-induced cell death in experiments employing both wild-type and common NNRTI-resistant viruses, and X-ray crystallography and molecular modeling permitted the determination of the binding modes of the inhibitors in the NNRTI binding pocket. We hope that these compounds and their synthetic precursors will enable the identification of new medicines for the treatment of HIV infection.
Footnote |
† Electronic supplementary information (ESI) available: Crystallographic data for the co-crystal structure of 2b and HIV-RT. See DOI: 10.1039/c0md00009d |
This journal is © The Royal Society of Chemistry 2010 |