Further SAR studies on novel small molecule inhibitors of the hepatitis C (HCV) NS5B polymerase

Article abstract of DOI:10.1016/S0960-894X(03)00670-X

Herein, we describe the structure-activity relationship (SAR) of N,N-disubstituted phenylalanine series of NS5B polymerase inhibitors of hepatitis C. The NS5B polymerase inhibitory activity of the most active compound exhibited an IC₅₀ of 2.7 μ

Full text of DOI:10.1016/S0960-894X(03)00670-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3341–3344
Further SAR Studies on Novel Small Molecule Inhibitors of the
Hepatitis C (HCV) NS5BPolymerase
T. Jagadeeswar Reddy,* Laval Chan, Nathalie Turcotte, Melanie Proulx, Oswy Z. Pereira,
Sanjoy K. Das, Arshad Siddiqui, Wuyi Wang, Carl Poisson, Constantin G. Yannopoulos,
Darius Bilimoria, Lucille L’Heureux, Hicham M. A. Alaoui and Nghe Nguyen-Ba
Shire BioChem Inc., 275 Armand-Frappier, Laval, Quebec, Canada H7V 4A7
Received 13 February 2003; accepted 9 May 2003
Abstract—Herein, we describe the structure–activity relationship (SAR) of N,N-disubstituted phenylalanine series of NS5B poly-
merase inhibitors of hepatitis C. The NS5B polymerase inhibitory activity of the most active compound exhibited an IC50 of
2.7 mM.
# 2003 Elsevier Ltd. All rights reserved.
It is estimated that 170 million people worldwide are
infected with Hepatitis C virus (HCV) including 4 mil-
lion in the United States. Within 20 years of infection,
about 4–5% of them will develop cirrhosis and hepato-
cellular carcinoma, often resulting in death.1 The
recommended current treatment, interferon a (IFN-a,
2a or 2b or a polyethylene glycol conjugate) in combi-
nation with ribavirin provides a sustained viral response
in only 54–56% of the treated patients. However, for
patients infected with HCV genotype 1a/b, the pre-
dominant genotype found in the USA, Japan and parts
of Europe, the response is at best 42–46% and, further-
more, treatment is expensive and side effects can be
severe.2 The lack of an effective and well-tolerated
treatment has therefore spurred intense research efforts
to develop affordable, oral and novel anti-HCV agents.
protease, NS5B polymerase inhibitors started appearing
recently in the patent literature.6 Only, NS5B poly-
merase inhibitor (JTK-002/JTK-003) from Japan
Tobacco Inc.7 has been reported to be undergoing
Phase II clinical trials.
We have recently reported the identification of a novel
class of HCV NS5B polymerase inhibitors (e.g., 1).8
These N,N-disubstituted phenylalanine analogues dis-
played not only potent inhibition of HCV polymerase
but also selectivity over human polymerases.8 Further-
more, X-ray crystal structures of these inhibitors bound
to NS5B polymerase enzyme revealed an allosteric site
in the thumb region8c about 30 A from the catalytic site
and about 10 A from the recently identified rGTP
binding site.9 As part of ongoing research on Hepatitis
C programme,8a herein we disclose the detailed struc-
ture–activity relationship (SAR) of the benzamide
portion of 1.
HCV is a 9.6-kb positive strand RNA virus of the Fla-
viviridae, genus hepacivirus which encodes a 3011–3033
amino acid polyprotein variable in several genotypes.3
This polyprotein is further processed into various
structural (core, E1 and E2, p7) and non-structural
(NS2, NS3, NS4A, NS4B, NS5A and NS5B) viral pro-
teins by host and viral proteases.4 The NS3 chymo-
trypsin-like protease and the NS5B RNA dependent
RNA polymerase (RdRp) are probably the most stud-
ied targets for anti-HCV therapy as they are crucial for
viral replication.5 Despite intense research effort on NS3
The test compounds (e.g., 1) were synthesized according
to Scheme 1. Reductive amination of the C-protected
l-phenylalanine with m-bromobenzaldehyde using ZnCl2
and NaCNBH3 in MeOH at room temperature gave the
*Corresponding author. Tel.: +1-450-978-7820; fax: +1-450-978-
7777; e-mail: treddy@ca.shire.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00670-X

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T. J. Reddy et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3341–3344
secondary amine in 70–80% yield. Acylation of the sec-
ondary amine was carried out using acid chloride with
N-methylmorpholine (NMM) in CH2Cl2 in the presence
of a catalytic amount of DMAP to provide the amides
in 70–90% yields. Finally, deprotection of methyl esters
with LiOH in dioxane and tert-butyl esters with TFA/
CH2Cl2 (1:2, v/v) generated the test compounds in 80–90%
yields.10
80 ꢀC followed by hydrolysis of the resultant ester gave
the desired urea (entry 19). Sulfonamide (entry 20) was
synthesized from compound 3 and the corresponding
sulfonyl chloride as described in Scheme 1.
As shown in Table 1, removal of the two chlorine atoms
resulted in a significant loss of inhibitory activity (entry
2). A single chloro substituent (entries 3–5) or 2,4,6-tri-
substitution is marginally tolerated (entry 7). There is,
however, a clear preference for substituents both at
ortho and para positions (entry 1). We have also exam-
ined other 2,4-disubstiuted benzamide analogues
(entries 8–10). It appears that the only substitution tol-
erated at the para position is either chloro or methyl.
CuI catalyzed C–N coupling11 of 2a and 2,4-dichloro-
bromobenzene in DMF followed by selective N-alkyl-
ation of the resultant amino acid 4b with m-bromobenzyl
bromide gave compound 5b (Scheme 2, Table 2). Reac-
tion of 2,4-dichlorophenyl-isocyanate and 3 in DCE at
Scheme 2. Reagents and conditions: For 5a: (a) 2b, 2,4-dichloro-
benzaldehyde, MS4 A , CH2Cl2, rt, 16 h, and NaBH4, MeOH, 0 ꢀC, 3
h, 50%; (b) (i) Cs2CO3, m-bromobenzylbromide, DMF, rt, 16 h, 45 ꢀC,
5 h, 45%; (ii) LiOH, THF–MeOH–H2O (3:2:1, v/v), rt, 2 h, 74%; For
5b: (a) 2a, 2,4-dichlorobromobenzene, CuI, K2CO3, DMF, 110 ꢀC, 17
h, 32%; (b) NaH, m-bromobenzylbromide, THF, 0 ꢀC, rt, 24%.
Scheme 1. Reagents and conditions: (a) m-bromobenzaldehyde,
NaCNBH3, THF, rt, 4 h; (b) acid chloride, NMM, cat DMAP,
CH2Cl2, rt, 16 h; (c) for R=Me, LiOH, H2O, 1,4-dioxane, rt; for
R=But, TFA–CH2Cl2, rt, 4 h.
Table 1. Effect of benzamide substituents on NS5B polymerase activity
a
a
a
Entry
1
R
Poly IC50
Entry
7
R
Poly IC50
Entry
1312
R
Poly IC50
3.5
13
5.6
2
3
4
5
6
>50
31
8
9
43
14
15
16
11
10.7
5.9
31
5.0
2.7
30
10
11
12
10.6
30
3.7
aThe inhibitory effect (IC50, mM) of test compounds on polymerization activity was performed using a Flashplate scintillation proximity assay.8a,13

T. J. Reddy et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3341–3344
3343
Table 2. Effect of nitrogen-functionalized group on NS5B poly-
merase
A SAR study on the benzamide portion of a newly
identified anti-NS5B polymerase lead is described. A
hydrophobic chlorine atom at the para position of the
benzamide is a requisite for activity. Having an extra
hydrophobic atom (Cl, Me, Br, I) at the ortho position
enhances potency whereas substitution at the meta
position diminishes the activity. Of all the N-function-
alities examined, the amide was found optimal.
a
a
Entry
1
R
Poly IC50 Entry
R
Poly IC50
Acknowledgements
3.5
19
20
31
We thank Martine Hamel for assistance in the initial
optimization of flashplate assay.
17
18
26
31
References and Notes
15
1. (a) Memon, M. I.; Memon, M. A. J. Viral. Hepat. 2002, 9,
84. (b) Moradpour, D.; Cerny, A.; Heim, M. H.; Blum, H. E.
Swiss Med Weekly 2001, 131, 291.
2. Ideo, G.; Bellobuono, A. Curr. Pharm. Des. 2002, 8, 959.
´
aThe inhibitory effect (IC50, mM) of test compounds on polymerization
activity was performed using a Flashplate scintillation proximity
assay.8a,13
3. (a) Wilkinson, T. Curr. Opin. Invest. Drugs 2001, 2, 1516.
(b) Orland, J. R., Wright, T. L., Cooper, S. Hepatology 2001,
Feb, 321.
4. Rosenberg, S. J. Mol. Biol. 2002, 313, 451.
5. (a) Dymock, B. W. Emerg. Drugs 2001, 6, 13. (b) HCV
RNA-dependent RNA polymerase as a target for antiviral
development: (i) Lai, M. M. C. Antiviral Chem. Chemother.
2001, 12, 143 (ii) Walker, M. P.; Hong, Z. Curr. Opin. Pharm.
2002, 2, 1471. (c) For a review on Non-structural protein
inhibitors see: Beaulieu, P. L.; Llinas-Brunet, M. Curr. Med.
Chem.: Anti-Infect. Agents 2002, 1, 163.
6. (a) For a review on recent patent literature, see: Zhang, X.
Idrugs 2002, 5, 154. (b) For recent paper on NS5B polymerase,
see: Dhanak, D.; Duffy, K. J.; Johnston, V. K.; Lin-Goerke,
J.; Darcy, M.; Shaw, A. N.; Gu, B.; Silverman, C.; Gates,
A. T.; Nonnemacher, M. R.; Earnshaw, D. L.; Casper, D. J.;
Kaura, A.; Baker, A.; Greenwood, C.; Gutshall, L. L.; Maley,
D.; DelVecchio, A.; Macarron, R.; Hofmann, G. A.; Alnoah,
Z.; Cheng, H.-Y.; Chan, G.; Khandekar, S.; Keenan, R. M.;
Sarisky, R. T. J. Biol. Chem. 2002, 277, 38322.
7. Information reported in Iddb3 from Current Drugs Ltd.
8. (a) For optimization of N-benzyl portion of 1 see: Chan, L.;
Reddy, T. J.; Proulx, M.; Das, S. K.; Pereira, O.; Wang, W.;
Siddiqui, A.; Yannopoulos, C.; Turcotte, N.; Drouin, A.;
Alaouii, H.; Bethel, R. C.; Lucile, H.; Nguyen-Ba, N. J. Med.
Chem. 2003, 46, 1283. (b) Ismaili, H.; Moulay, A.; Cheng, Y.-
X.; Lavallee, J.-F.; Siddiqui, A.; Storer, R. WO0160315, 2001
and USPatent 0019363, 2002. (c) Wang, M.; Ng, K. K.-S.;
Cherney, M. M.; Chan, L.; Yannopoulos, C. G.; Bedard, J.;
Morin, N.; Nguyen-Ba, N.; Bethell, R. C.; James, M. N. G. J.
Biol. Chem. 2003, 278, 9489.
For example, significant loss of activity was observed
for small substituents, such as fluoro (entry 8), while
approximately 2- to 3-fold-reduced potency were
observed for dimethoxy and dimethyl benzamides,
respectively (entries 9 and 10). A basic nitrogen in the
dichlorobenzamide ring is also detrimental to activity
(8-fold loss, entry 11).
Based on these observations it appeared that the para
substituent is important for activity while having an
extra substituent at the ortho position enhances potency.
Thus, apparent additive effect of an extra ortho sub-
stituent on the polymerase activity was examined next
(entries 12–16). Polymerase inhibitory activity was
either slightly decreased or maintained with a variety of
functional groups at the ortho position (entries 12–16).
The ortho position seems to be quite tolerant since
moderately bulky groups (Cl, Me, Br, and I) increase
the potency (entries 1 and 12–16).
The effect of a non-amidic link to phenylalanine nitro-
gen on NS5B polymerase activity was also examined
(Table 2). Removal of the amide functionality resulted
in an 8-fold loss of activity (entry 17). Direct linkage of
the 2,4-dichlorophenyl moiety also diminishes the
activity (entry 18). The activity was considerably
decreased by replacing the 2,4-dichlorobenzamide with
other functional groups such as urea and sulfonamide
(entries 19 and 20). The mechanism by which this com-
pound inhibits NS5B polymerase is being actively
examined. Further optimization of 1 is in progress and
will be reported in due course.
9. (a) Bressanelli, S.; Tomei, L.; Rey, F. A.; De Francesco, R.
J. Virol. 2002, 76, 3482 (rGTP binding site). (b) During pre-
paration of manuscript a patent on X-ray co-crstallization of
inhibitors of an allosteric site of NS5B polymerase has
appeared: Love, R. A.; Yu, X.; Diehl, W.; Hickey, M. J.;
Parge, H. E.; Gao, J.; Fuhrman, S.; E. P. Patent 1256628 CAN
137:194827. Curr. Med. Chem.: Anti-Infect. Agents 2002, 1,
163.
1
10. All the compounds in this study were characterized by H
NMR and MSand purity was assessed by HPLC. The 1H
NMR spectra of test compounds also indicated the presence of
numerous rotamers.

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T. J. Reddy et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3341–3344
11. Ma, D.; Zhang, Y.; Yao, J.; Wu, S.; Tao, F. J. Am. Chem.
Soc. 1998, 120, 12459.
12. 2-Ethyl-4-chlorobenzoic acid was prepared from methyl 2-
bromo-4-chlorobenzoic acid via a three step sequence employ-
ing vinyl Stille coupling, hydrogenation, and hydrolysis.
13. Earnshaw D. L.; Pope A. J. FlashPlateTM scintillation
proximity assays for characterization and screening of DNA
polymerase, primase, and helicase activities: J. Biomol. Screen
2001, 6, 39. IC50 was calculated from a single experiment using
an 11 serial 2-fold dilution (0.05–50 mM). Data were con-
sidered reliable only when the IC50 value of a positive internal
control was within standard deviation range.