Discovery of novel phosphonate derivatives as hepatitis C virus NS3 protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.05.023

A novel class of phosphonate derivatives was designed to mimic the interaction of product-like carboxylate based inhibitors of HCV NS3 protease. A phosphonic acid (compound 2) was demonstrated to be a potent HCV NS3 protease inhibitor, and a potential can

Full text of DOI:10.1016/j.bmcl.2009.05.023

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3453–3457
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel phosphonate derivatives as hepatitis C virus NS3
protease inhibitors
*
X. Christopher Sheng , Hyung-Jung Pyun, Kleem Chaudhary , Jianying Wang, Edward Doerffler,
Melissa Fleury à, Darren McMurtrie §, Xiaowu Chen, William E. Delaney IV, Choung U. Kim
Gilead Sciences, Inc, 333 Lakeside Drive, Foster City, CA 94404, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 February 2009
Revised 4 May 2009
Accepted 6 May 2009
Available online 9 May 2009
A novel class of phosphonate derivatives was designed to mimic the interaction of product-like carbox-
ylate based inhibitors of HCV NS3 protease. A phosphonic acid (compound 2) was demonstrated to be a
potent HCV NS3 protease inhibitor, and a potential candidate for treating HCV infection. The syntheses
and preliminary biological evaluation of this phosphonate class of inhibitor are described.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HCV NS3 protease inhibitors
Phosphonate as caboxylate replacement
Hepatitis C virus (HCV) infection remains a significant public
health problem; an estimated 3% of the world population has been
infected.1 HCV infection is usually asymptomatic and frequently
becomes chronic which leads to a significantly increased risk of
developing hepatocellular carcinoma and liver cirrhosis. The cur-
There are generally two classes of NS3 protease inhibitor. One is
comprised of product based inhibitors which contain a P1 carbox-
ylic acid such as 1 (BILN-2061), or the carboxylate isostere acylsulf-
onamide, first disclosed by Bristol-Myers Squibb scientists.5 The
second class of NS3 inhibitors are so called serine traps. This class
rent standard of therapy is based on
a
-interferon (Peg-IntronÒ
contains an electrophilic carbonyl group, such as a
a-ketoamide,
and PegasysÒ) in combination with ribavirin. This existing therapy
is only partially effective with ꢀ50% of patients with HCV genotype
1 demonstrating sustained virological responses. Furthermore, the
current therapy is very poorly tolerated with ꢀ75% of patients
exhibiting systemic side-effects including flu-like symptoms,
hematological abnormalities and neuropsychiatric symptoms such
that treatment is deferred in the majority of patients due to mild
disease, a low chance of response or contra-indications.
In recent years, direct anti-viral drugs targeting the inhibition of
viral enzymes have shown promise in human clinical trials. One of
these viral targets is the protein product of the non structural gene
3 (NS3) which possesses both protease and helicase activities. So
far, there are several inhibitors targeting NS3 protease activity,
such as ciluprevir 1 (BILN-2061), telaprevir (VX-950) and bocepre-
vir (SCH 503034), that have shown impressive anti-viral activity in
man.2–4
represented by VX-950 and SCH 503034. This -ketoamide forms
a covalent bond with the active site serine residue in a reversible
fashion.
a
A major challenge to developing small molecule NS3 inhibitors
is the featureless and relatively solvent exposed active site. A
potent inhibitor requires a series of weak lipophilic and electro-
static interactions distributed along its contact with the protease.
In the case of product based inhibitors, the P1 carboxylate has been
shown to be crucial for potency; it helps to anchor the inhibitor
through H-bond interactions with the oxyanion hole.6
Phosphonates have been studied extensively in drug design as
biologically active compounds and as isosteric replacements for
carboxylates.7 In our HCV NS3 protease inhibitor project, we envi-
sioned that a pentavalent phosphonic acid would bind to the S1
oxyanion hole in a similar fashion as the carboxylate. Molecular
modeling studies suggested that a phosphonic acid would be capa-
ble of making extensive interactions with the oxyanion hole of HCV
protease. As shown in Figure 1, phosphonic acid 2 was expected to
maintain similar interactions with the His 57 side chain and the
Gly 137 backbone amide, as observed for BILN-2061. Furthermore,
the phosphonic acid might make additional H-bonds with the side
chains of Ser 139 and Lys 136, both of which are conserved active
site residues. Because of the potential for more extensive interac-
tions with the oxyanion hole, it was expected phosphonic acid 2
* Corresponding author. Tel.: +1 650 522 5922.
E-mail address: csheng@gilead.com (X.C. Sheng).
Present address: Novartis Institutes for BioMedical Research, Inc, 220 Massachu-
setts Avenue, Cambridge, MA 02139, USA.
à
Present address: Theravance, Inc, 901 Gateway Boulevard, South San Francisco,
CA 94080, USA.
§
Present address: Rigel Pharmaceuticals, Inc, 1180 Veterans Boulevard, South San
Francisco, CA 94080, USA.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.023

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X.C. Sheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3453–3457
S
S
N
H
N
NH
O
O
N
O
O
N
O
N
O
H
N
H
N
OH
OH
N
H
N
P
H
N
OH
N
O
O
O
O
O
O
O
O
1
2
Figure 1. Proposed phosphonate NS3 protease inhibitor. Model of phosphonic inhibitor 2 bound to HCV protease. Atoms are colored according to atom type, except for
carbons, which is colored white for inhibitor and cyan for the oxyanion hole residues of the protease, respectively. Yellow dashes indicate potential H-bonds, green cartoons
represent HCV protease.
would bind the enzyme stronger than its carboxylic acid
counterpart.
presence of cesium hydroxide and a phase transfer catalyst pro-
vided the corresponding phosphonate 4. After the resulting imine
was hydrolyzed, chiral resolution provided the optically pure
It was our concern that the strong acidity of phosphonic acids
may interfere with membrane permeability. Further analysis of
modeling results suggested that one of the hydroxyl groups of the
phosphonic acid is solvent exposed and could accommodate a large
ester moiety. It is therefore possible to reduce the polarity of phos-
phonic acid analogs by ester formation, without a loss in potency.
In this Letter, we wish to report the results of our research.
The P1 amino phosphonate was prepared in good overall yield
starting from the commercially available intermediate 3 (Scheme 1).
Condensation of the [(benzylidene-amino)-methyl]-phosphonic
acid diethyl ester 3 with trans-1,4-dibromo-but-2-ene in the
amine salt 6 as the salt of dibenzoyl-L
-tartaric acid.8 A simple
biphasic extraction was used to free the amine salt to yield the free
amine 7 in quantitative yield.
The synthesis of phosphonate NS3 inhibitors utilizing amine 7 is
outlined in Scheme 2.
The P2 aminothiazole 8 was reacted with hydroxyl proline 9
under Mitsunobu conditions to give ether 10. Protecting group
removal, followed by peptide coupling with tert-butyl leucine
and subsequent saponification provided the carboxylic acid dipep-
tide 11. Coupling with amino phosphonate 7 proceeded smoothly
O
P
OEt
O
P
OEt
O
P
OEt
a
b
Ph
N
OEt
OEt
OEt
N
H2N
Ph
3
4
5
O
P
OEt
O
P
OEt
H2N
H2N
PhOCO
PhOCO
COOH
c
OEt
OEt
d
COOH
6
7
Scheme 1. Synthesis of P1 amino phosphonate. Reagents and conditions: (a) 1,4-dibromo-but-2-ene, CsOH, BnEt3NCl, CH2Cl2, reflux 24 h; (b) 1 N aq HCl, rt 3 h, 40% for two
steps; (c) dibenzoyl- -tartaric acid, CH3CN, 40%; (d) NaHCO3, CH2Cl2, 100%.
L

X.C. Sheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3453–3457
3455
S
N
H
N
O
N
O
S
H
OH
N
O
N
a
N
OMe
+
N
Boc
O
OH
OMe
N
9
8
10
Boc
O
S
N
S
N
H
N
H
N
O
N
O
O
N
O
b, c, d
e
O
H
N
OEt
OEt
OH
N
N
P
H
N
H
N
O
O
O
O
O
O
O
O
12
11
g
f
S
N
S
H
N
H
N
O
N
O
N
N
O
O
O
P
O
H
N
H
N
OEt
OH
OH
N
N
O
P
H
H
N
N
OH
O
O
O
O
O
O
O
13
2
Scheme 2. Synthesis of phosphonic acids. Reagents and conditions: (a) DIAD, PPh3, THF, 70%; (b) HCl/dioxane, methylene chloride, 2 h; (c) tert-butyl leucine, HATU, DMF,
Hunig’s base, 81% for steps b and c; (d) LiOH, THF:H2O:MeOH, rt, 2 h, 100%; (e) aminophosphonate 7, HATU, NMM, methylene chloride, 12 h, 70%; (f) TMSI, CH3CN, 0 °C to rt,
4 h, 80%; (g) NaI, pyridine, reflux, 20 h, 85%.
to provide fully protected phosphonate tripeptide 12. To obtain the
phosphonic acid 2, the phosphonate diethyl ester was hydrolyzed
with TMSI in acetonitrile. The mono phosphonic acid 13 was
obtained by a selective hydrolysis using NaI in pyridine.
cellular activity of phosphonate mono-esters is strongly influ-
enced by the nature of the alkyl group (in this case, an electron
withdrawing trifluoroethyl group).
The phosphonate inhibitors were evaluated in a biochemical
assay using the catalytic domain of the NS3 protein and NS4A as
a cofactor. Compound 2 showed very potent inhibition of NS3 with
an IC50 = 0.9 nM. This IC50 is better than the corresponding acid 14
(IC50 3 nM). The cellular activity of compound 2 in Huh-7 replicon
cells was 92 nM, about 15-fold higher when compared to the car-
boxylic acid compound 14. We suspected that the reduced activity
in replicon cells was due to the highly polar phosphonic acid moi-
ety. A Caco-2 permeability assay of compound 2 indeed showed
low permeability.9
Table 1
Activities of phosphonate analogs
S
N
H
N
O
N
O
H
N
R
N
H
N
Hoping to reduce the hydrophilicity of the phosphonic acid
functionality, we evaluated the mono acidic compound 13. Unex-
pectedly, compound 13 showed compromised potency both in
the biochemical assay and in the replicon assay compared to
O
O
O
O
a
a
b
Compounds
R
IC50
EC50
CC50
compound 2 (eightfold higher IC50 and fourfold higher EC50
,
respectively). To elucidate whether the reduced activity of com-
pound 13 was due to a steric or an electronic effect of the OEt
moiety, we synthesized the trifluoroethyl (TFE) mono acid 15.
The IC50 of the TFE mono-ester remains almost unchanged as
the OEt analog (6 nM), but the replicon activity was improved
eightfold (Table 1). These results clearly demonstrated that the
14
2
13
15
CO2H
PO(OH)2
PO(OH)(OEt)
3
0.9
7
6
92
360
48
>50,000
>50,000
>50,000
>50,000
PO(OH)(OTFE)
6
a
All values are given in nM.
Cytotoxicity was measured in Huh-7 cells.
b

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X.C. Sheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3453–3457
S
H
N
S
H
N
O
N
O
N
O
N
O
N
a, b
c, d
OMe
N
OMe
N
BocHN
Boc
O
O
O
10
16
S
N
H
N
S
N
H
N
O
N
O
N
O
O
O
e, f
O
P
H
N
H
N
OH
OH
OEt
OEt
N
P
N
H
N
O
BocHN
O
O
O
O
O
18
17
Scheme 3. Synthesis of macrocyclic phosphonic acid. Reagents and conditions: (a) HCl/dioxane, methylene chloride, 2 h; (b) 2-tert-butoxycarbonylamino-non-8-enoic acid,
HATU, DMF, Hunig’s base, 60% for two steps; (c) LiOH, THF:H2O:MeOH, rt, 2 h, 100%; (d) aminophosphonate 7, HATU, NMM, methylene chloride, 12 h, 60%; (e) Grubb’s
catalyst (G1), methylene chloride, reflux 14 h, 20%; (f) TMSI, CH3CN, rt, 4 h, 80%.
Encouraged by these promising results, we then explored the
macrocyclic phosphonate 18. Scheme 3 depicts the synthesis of
compound 18 starting from ether 10. The Boc protection group of
10 was removed under acidic condition, condensation of resulting
amine salt with 2-tert-butoxycarbonylamino-non-8-enoic acid
yielded dipeptide 16. Hydrolysis of methyl ester with aq LiOH pro-
vided acid which was coupled with amine 7 using HATU and NMM.
The resulting intermediate 17 was cyclized with Grubb’s G1 cata-
lyst to give the macrocyclic phosphonate diethyl ester in 20% yield.
Subsequent hydrolysis with TMSI as described before provided tar-
get phosphonic acid 18.
With the promising phosphonic acid NS3 inhibitor in hand, the
pharmacokinetic properties of the phosphonate 2 were evaluated
in both rat and dog. Although the intravenous plasma exposure
(measured as AUC), half-lives and clearance in both species were
generally good, the oral bioavailability was rather low. The poor
oral bioavailability of the phosphonate 2 was attributed to the
low permeability of the highly charged phosphonate anion under
physiological conditions. One way to overcome the low membrane
permeability of the phosphonate was a pro-drug approach. As seen
in tenofovir and adefovir, ester formation with acyloxymethyl and
alkoxy-carbonyloxymethyl delivered the active agents to systemic
circulation by enhancing intestinal absorption.10 A search of liter-
ature for possible alkoxy-carbonyloxymethyl ester prodrugs indi-
cated that the most straight forward examples for phosphonates
were the bis-(ethoxycarbonylmethoxy) ester and the bis-(cyclo-
propylmethoxycarbonylmethoxy) ester. The synthesis of these
prodrugs is described in Scheme 4. The activities of these prodrugs
are listed in Table 2.
The pharmacokinetic properties of phosphonate prodrug 19
were evaluated in dogs to determine if the prodrug approach im-
proved the oral bioavailability. After intravenous administration
of 1.1 mg/kg of compound 19 in dogs, the apparent systemic clear-
ance was high relative to the liver blood flow (1.7 L/h/kg); a signif-
icant amount of the mono ester was detected as the major
metabolite. The parent compound 2 was also observed. Oral
administration at 2.7 mg/kg gave F% = 1 of the parent compound.
Another prodrug, compound 20 showed a similar profile in dog
PK studies.
Macrocyclic phosphonic acid 18 indeed exhibits very potent
activity against HCV NS3 protease with an IC50 = 1 nM and an
EC50 = 5 nM.
Table 2
Activities of phosphonate prodrug analogs
S
N
H
N
O
N
O
O
P
H
N
OR2
OR1
N
H
N
O
O
O
O
Further optimization efforts to improve oral absorption of the
phosphonate, as well as anti-viral activities, will be reported in
the future.
In summary, a novel class of phosphonate derivatives as
potent NS3 protease inhibitors was identified. These inhibitors
have been shown to have nanomolar activities in biochemical
and replicon assays. Phosphonate compound 2 was shown to
be even more potent than the corresponding carboxylate 14 in
the enzyme assay.
R1
R2
IC50
EC50
a
a
Compounds
O
O
19
280
85
39
O
O
O
O
O
O
O
O
O
O
20
41
a
All values are given in nM.

X.C. Sheng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3453–3457
3457
S
N
H
N
S
N
H
N
O
N
O
O
N
O
R
O
O
O
O
P
O
P
H
N
O
H
N
OH
OH
N
N
H
N
O
H
O
O
N
O
O
O
O
O
O
R
O
O
O
19, R = Et
20, R = CH2-c-Pr
2
Scheme 4. Synthesis of phosphonate prodrugs. Et3N, DMP, carbonic acid chloromethyl ester ethyl ester, 50 °C, 22 h, 22% for compound 19. Et3N, DMF, TBAI, carbonic acid
chloromethyl ester cyclopropylmethyl ester, 70 °C, 5 h, 18% for compound 20.
5. a Campbell, J. A.; Good, A. C. WO Patent 2003/099316 A1.; b Wang, X. A.; Sun,
Acknowledgments
L.-Q.; Sit, S.-Y.; Sin, N.; Scola, P. M.; Hewawasam, P.; Good, A. C.; Chen, Y.;
Campbell, J. A. WO Patent 2003/099274 A1.; c Ripka, A.; Campbell, J. A.; Good,
The authors thank Mr. Scott Hluhanich, Ms. Huiling Yang and
Ms. Margaret Robinson for determining biological activities. The
authors also thank Drs. Chris Yang and Rowchanak Pakdaman for
the formulation and P.K. study of key compounds.
A. C.; Scola, P. M.; Sin, N.; Venables, B. WO Patent 2004/032827 A2.;
Campbell, J. A.; D’Andrea, S. V.; Good, A.; Li, J.; McPhee, F.; Ripka, A.; Scola, P.
M.; Tu, Y. WO Patent 2004/043339 A2.
d
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