Synthesis and evaluation of optically pure dioxolanes as inhibitors of hepatitis C virus RNA replication

Article abstract of DOI:10.1016/j.bmcl.2003.09.008

A series of optically pure 1,3-dioxolane nucleoside mimics was synthesized by a synthetic route that allowed incorporation of a 5R-methyl substituent from commercially available starting materials. The pyrrolo[2,3-d]pyrimidine heterocycle was chosen as a

Full text of DOI:10.1016/j.bmcl.2003.09.008

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4455–4458
Synthesis and Evaluation of Optically Pure Dioxolanes as
Inhibitors of Hepatitis C Virus RNA Replication
Sanjib Bera,a Leila Malik,a Balkrishen Bhat,a Steven S. Carroll,b Malcolm MacCoss,c
David B. Olsen,b Joanne E. Tomassinib and Anne B. Eldrupa,*
aDepartment of Medicinal Chemistry, Isis Pharmaceuticals, Carlsbad, CA 92008, USA
bDepartment of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 19 June 2003; revised 3 September 2003; accepted 4 September 2003
Abstract—A series of optically pure 1,3-dioxolane nucleoside mimics was synthesized by a synthetic route that allowed incor-
poration of a 5R-methyl substituent from commercially available starting materials. The pyrrolo[2,3-d]pyrimidine heterocycle was
chosen as a substitute for the purine derivative. Coupling of the pyrrolo[2,3-d]pyrimidine and the dioxolane was performed under
solid–liquid phase transfer conditions. The ability to inhibit HCV RNA replication was assessed in a cell based subgenomic replicon
assay. None of the described compounds displayed significant anti-HCV activity.
# 2003 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is the pathogen associated with
the majority of chronic hepatitis infections world wide
and is a major cause of liver disease and transplanta-
tions. The currently recommended antiviral therapy
consists of interferon alpha and ribavirin, a treatment
that leads to only moderate sustained response rates.
HCV encodes a series of viral proteins: the NS2/3
autoprotease, the NS3 serine protease and NTPase/
helicase, and NS5B, the RNA dependent RNA poly-
merase (RdRp).1 The polymerase is essential to viral
replication and proliferation, and hence represents a
valid drug discovery target. Non-nucleoside inhibitors
(NNIs) and nucleoside inhibitors (NIs) of virally enco-
ded polymerases have been validated for other targets,
for example, HIV. While a range of NNI inhibitors of
HCV RNA replication have been reported,2ꢀ5 few NIs
have been identified.6ꢀ10
sence of a 20-OH/20-O-methyl is strictly necessary for
activity in a cell based assay.
Nucleosides where the 30 carbon has been replaced by
a heteroatom such as oxygen or sulfur, have been
demonstrated to be effective in cancer and viral chemo-
therapy.11ꢀ18 For example, (ꢀ)-l-b-1,3-oxathionyl cyto-
sine (3TC, Lamivudine) 4 is an effective anti-HIV and
anti-HBV agent while (ꢀ)-l-b-1,3-dioxanyl cytosine 5
(O-ddC) has demonstrated anti-tumor,19 anti-HIV and
anti-HBV activity15 (Fig. 2). In the purine series, 1,3-
dioxanylguanine 617,20 has demonstrated significant
anti-HIV activity. Furthermore, dideoxynucleosides
containing a 4-aminopyrrolo[2,3-d]pyrimidine moiety
have been demonstrated to display increased chemical and
enzymatic stability21 relative to the corresponding purine
derivatives. Based on the above findings, we decided to
Some of the NS5B NIs described have encompassed
alteration of the 20 position, either in the form of a 20-O-
methyl6 or a 20-C-methyl modification6ꢀ8 (Fig. 1). Other
reports9,10 have indicated dioxolane based nucleoside
triphosphates to be inhibitors of HCV RdRp mediated
RNA synthesis. Hence, it remains unclear if the pre-
*Corresponding author. Tel.: +1-760-603-3852; fax: +1-760-603-
4654; e-mail: aeldrup@isisph.com
Figure 1. Examples of known HCV polymerase inhibitors, modified at
the C2 or O2 position.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.008

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S. Bera et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4455–4458
For the synthesis of the 5-methyl-1,3-dioxolane nucleo-
side analogues, the methyl (4R,5S)-2,2,5-trimethyl-1,3-
dioxolane-4-carboxylate 15 was used as the starting
material. Treatment of 15 with aldehyde 9 resulted in a
2R,S-diastereoisomeric mixture of the carboxy methyl-
ester derivatives 16 in favor of the 2R-isomer. Saponi-
fication of the methyl ester with LiOH, followed by
acidification, afforded the carboxylic acid derivatives 17
and 18, which were easily separable by silica gel column
chromatography. The structure of the major isomer 17
was determined by NOE. Treatment of 17 with
Pb(OAc)4 and pyridine in acetonitrile gave an anomeric
mixture of the acetates 19 (Scheme 2).
Figure 2. Examples of dioxolane and oxathiolane based antiviral and
anti-neoplastic nucleosides.
Attempted Vorbruggen coupling22 of the acetate 14
with in situ silylated 4-chloropyrrolo[2,3-d]pyrimidine in
the presence of trimethylsilyl trifluoromethane-sulfonate
was ineffective and resulted in poor yield. However, in a
more general approach, in situ formation of the dioxo-
lanyl bromide 20a was accomplished from the acetate 14
using bromotrimethylsilane (TMSBr). The thus formed
bromide was reacted with 4-chloropyrrolo[2,3-d]pyr-
imidine under solid–liquid phase transfer conditions, in the
presence of tris-[2-(2-methoxyethoxy)ethyl]amine (TDA-1)
and KOH21,23 in acetonitrile to afford a separable mix-
ture (2:3) of a- and b-nucleoside derivatives 21a and 22a
in 38% overall yield.24 Under similar conditions, cou-
pling of the 2-amino-4-chloropyrrolo[2,3-d]pyrimidine
derivative gave a mixture of the nucleosides 21b and 22b
as a 1:1.4 mixture in 39% overall yield (Scheme 3).
Amination of the nucleoside derivatives 21a and 22a
separately, with saturated methanolic ammonia, was
followed by deprotection of the benzyl group with Pd/C
and ammonium formate in refluxing methanol to furnish
the desired nucleosides 23a and 24a.25
Figure 3. Target compounds. R can be H or methyl.
synthesize and evaluate the dioxolane nucleosides of
general structure 7 and 8 (Fig. 3).
Chemistry
The derivatives 7 were synthesized via transketalization
of the known 2-benzyloxy acetaldehyde 9 and commer-
cially available methyl-(R)-2,2-dimethyl-1,3-dioxalane-
4-carboxylate 10 in the presence of p-TSA to give the
1,3-dioxolane derivative 11 as an inseparable 1:1
mixture of diastereomers. Saponification of the methyl
esters 11 with LiOH in THF, followed by acidification
with aqueous H2SO4, gave a mixture of dioxolane car-
boxylic acids 12 and 13, which were separated by silica
gel column chromatography (dichloromethane/acetic
acid, 98:2). The stereochemistry of the carboxylic acid
derivatives 12 was confirmed by NOE. Oxidative dec-
arboxylation of the cis carboxylic acid derivative 12
with Pb(OAc)4 in acetonitrile in the presence of pyridine
gave the key acetate intermediate 14, as a diastereo-
isomeric mixture (Scheme 1).
For the synthesis of the 5-methyl dioxolane derivatives,
acetate 19 was treated with TMSBr in dichloromethane
to give the bromo derivative 20b, which on condensa-
tion with 4-chloropyrrolo[2,3-d]pyrimidine in the pre-
sence of TDA-1 and KOH in acetonitrile afforded a
mixture of 21c and 22c (1:1.5) in 32% overall yield.
Treatment of 21c and 22c separately with methanolic
ammonia followed by hydrogenolysis with Pd(OH)2 on
Scheme 1. Reagents and conditions: (i) p-TsOH, toluene, 80 ꢁC, 1 h;
(ii) (a) 1 M aqueous LiOH/THF (1:1), (b) 1 N HCl or H2SO4 (pH 2–
3); (iii) Pb(OAc)4, acetonitrile, pyridine.
Scheme 2. Reagents and conditions: (i) p-TsOH, toluene, 80 ꢁC, 1 h;
(ii) (a) 1 M aqueous LiOH/THF (1:1), (b) 1 N HCl or H2SO4 (pH 2–
3); (iii) Pb(OAc)4, acetonitrile, pyridine.

S. Bera et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4455–4458
4457
Biological Evaluation
Compounds 23, 24, 26 were evaluated as inhibitors of
HCV replication in a cell-based, subgenomic replicon
assay as previously described.6 None of the described
compounds displayed significant inhibition of HCV
RNA replication (EC50>50 mM). Based on previous
art, these compounds are likely inhibitors of HBV
polymerase and HIV reverse transcriptase. Work is in
progress to evaluate anti-HBV/anti-HIV activity.
Results and Discussion
Synthetic routes to a series of pyrrolo[2,3-d]pyrimidin-7-
yl)-1,3-dioxolanes were devised using phase transfer
conditions to effect coupling of either (2R)-2-benzyl-
oxymethyl-4-acetoxy-1,3-dioxolane or (2R)-2-benzyl-
oxymethyl-4-acetoxy-(5R)-methyl-1,3-dioxolane to the
appropriate 4-chloro-1H-pyrrolo[2,3-d]pyrimidine. Com-
pounds were evaluated as inhibitors of HCV RNA
replication in a cell based assay and found to be inac-
tive. In principle, the inactivity of these compounds in
the subgenomic replicon assay could reflect lack of
uptake and/or metabolism to the corresponding triphos-
phate. However, compounds of similar structure are
known inhibitors of viral and human polymerases indi-
cating a level of uptake and metabolism sufficient to
elicit activity. Hence it appears more likely that the basis
for the observed inactivity is inability to elicit recogni-
tion from the HCV RdRp. If so, this would point to the
20-hydroxy or 20-methoxy as essential pharmacophores
for recognition by the HCV RdRp. Given the fact that
HCV polymerase utilizes ribonucleoside triphosphates
as substrates for the incorporation of ribonucleoside
monophosphates against an RNA template, recognition
of the 20 position is likely to be highly refined. Work is
in progress to determine if the corresponding triphos-
phates are substrates and/or inhibitors of the HCV
RdRp.
References and Notes
Scheme 3. Reagents and conditions: (i) TMSBr, dichloromethane,
0 ꢁC to room temperature; (ii) 4-chloropyrrolo[2,3-d]pyrimidine or 2-
amino-4-chloropyrrolo[2,3-d]pyrimidine, TDA-1, KOH, acetonitrile;
(iii) methanolic ammonia, 80 ꢁC; (iv) ammonium formate, methanol,
reflux; (v) 2 M NaOH/dioxane (1:1), 2-mercaptoethanol, 100 ꢁC; (vi)
Pd(OH)2, cyclohexene/ethanol (1:2), 80 ꢁC, 2 h (26a) or Pd/C, ammo-
nium formate, methanol, reflux (26b).
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(dd, J=5.6, 9.2 Hz, 1H), 4.32 (dd, J=3.2, 9.2 Hz, 1H), 3.61 (d,
1
J=3.2 Hz, 2H); H NMR (MeOH-d4) of 24a: d 8.08 (s, 1H),
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7.40 (d, J=3.6 Hz, 1H), 6.59 (m, 2H), 5.09 (t, J=3.2 Hz), 4.36
(dd, J=2, 9.6 Hz, 1H), 4.24 (dd, J=5.6, 9.6 Hz, 1H), 3.73 (d,
J=3.2 Hz, 2H).
26. 1H NMR (200 MHz, methanol-d4) of 23b: d 8.27 (s, 1H),
7.65 (d, J=3.8 Hz, 1H), 6.95 (d, J=3.8 Hz, 1H), 6.12 (d,
J=5.4 Hz, 1H), 5.54 (t, J=3.2 Hz, 1H), 4.58 (m, 1H), 3.67 (d,
1
J=3.2 Hz, 2H), 1.44 (d, J=6.4 Hz, 3H); H NMR (200 MHz,
methanol-d4) of 24b: d 8.26 (s, 1H), 7.92 (d, J=3.7 Hz, 1H),
6.89 (d, J=3.7 Hz, 1H), 6.64 (d, J=4.8 Hz, 1H), 5.10 (t,
J=2.6 Hz, 1H), 4.36 (m, 1H), 3.73 (d, J=2.6 Hz, 2H), 0.92 (d,
J=6.2 Hz, 3H).
27. 1H NMR (D2O+methanol-d4) of 26a: d 6.82 (d, J=3.8
Hz, 1H), 6.28 (d, J=3.8 Hz, 1H), 6.20 (m, 1H), 4.94 (t, J=3.1
Hz, 1H), 4.23 (dd, J=2, 9.8 Hz, 1H), 4.07 (dd, J=5.8, 9.8 Hz,
1H), 3.55 (d, J=2.2 Hz, 2H); 1H NMR (CD3CN) of 26b: d
6.97 (d, J=3.8 Hz, 1H), 6.34 (d, J=3.8 Hz, 1H), 6.27 (d,
J=5.2 Hz, 1H), 5.16 (bs, 2H), 5.02 (t, J=3.2 Hz, 1H), 4.35 (m,
1H), 3.79 (m, 2H), 0.97 (d, J=6.2 Hz, 3H).
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muganathan, K.; Cannon, D. L.; Alves, A. J.; Jeong, L. S.;