Synthesis and anti-HIV activity of cyclic pyrimidine phosphonomethoxy nucleosides and their prodrugs: A comparison of phosphonates and corresponding nucleosides

Article abstract of DOI:10.1080/15257770701490126

Cyclic phosphonomethoxy pyrimidine nucleosides that are bioisosteres of the monophosphate metabolites of HIV reverse transcriptase (RT) inhibitors AZT, d4T, and ddC have been synthesized. The RT inhibitory activities of the phosphonates were reduced for both dideoxy (dd) and dideoxydidehydro (d4) analogs compared to the nucleosides. Bis-isopropyloxymethylcarbonyl (BisPOC) prodrugs were prepared on selected compounds and provided > 150-fold improvements in antiviral activity. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701490126

Nucleosides, Nucleotides, and Nucleic Acids, 26:573–577, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701490126
SYNTHESIS AND ANTI-HIV ACTIVITY OF CYCLIC PYRIMIDINE
PHOSPHONOMETHOXY NUCLEOSIDES AND THEIR PRODRUGS:
A COMPARISON OF PHOSPHONATES AND CORRESPONDING
NUCLEOSIDES
Richard L. Mackman, Lijun Zhang, Vidya Prasad, Constantine G. Boojamra,
James Chen, Janet Douglas, Deborah Grant, Genevieve Laflamme, Hon Hui,
Choung U. Kim, Jay Parrish, Antitsa D. Stoycheva, Swami Swaminathan,
KeYu Wang, and Tomas Cihlar
Gilead Sciences, Inc., Foster City, California, USA
2
Cyclic phosphonomethoxy pyrimidine nucleosides that are bioisosteres of the monophosphate
2
metabolites of HIV reverse transcriptase (RT) inhibitors AZT, d4T, and ddC have been synthesized.
The RT inhibitory activities of the phosphonates were reduced for both dideoxy (dd) and dideoxydide-
hydro (d4) analogs compared to the nucleosides. Bis-isopropyloxymethylcarbonyl (BisPOC) prodrugs
were prepared on selected compounds and provided >150-fold improvements in antiviral activity.
Keywords Phosphonate; HIV; antiviral; prodrugs
INTRODUCTION
The backbone of antiretroviral therapy usually includes the use of two
nucleoside and nucleotide HIV reverse transcriptase inhibitors (NRTIs and
NtRTIs, respectively) selected from a panel of eight drugs that include a
tenofovir prodrug (bisPOC PMPA, Viread, (1), Figure 1) as a unique nu-
cleotide analog.[1−3] Due to the emergence of resistance and drug related
toxicities, new N(t)RTIs with optimal resistance and toxicity profiles, along
with the convenience of once daily dosing regimens are highly desirable. In
an effort to identify new nucleoside phosphonate inhibitors of RT, we syn-
thesized phosphonomethoxy analogs (2–7) (Figure 1) of cyclic pyrimidine
nucleosides, for example, d4T, AZT, and ddC, compared and contrasted
their respective RT inhibitory activity, and evaluated the antiviral potency
and resistance profile of selected bisPOC prodrugs.
Address correspondence to Richard Mackman, Gilead Sciences, Inc., 333 Lakeside Drive, Foster
City, CA 94404. E-mail: rmackman@gilead.com
573

574
R. L. Mackman et al.
FIGURE 1 Structures of phosphonates (1--8).
SYNTHESIS
The phosphonomethoxy analogs of d4t, ddT, d4U, and ddU, analogs (2–
3), and (5–6), respectively (Figure 1), were prepared following procedures
similar to those reported earlier.[4] The phosphonomethoxy AZT analog (4)
was prepared from thymidine according to Scheme 1. The poor yield in the
transacetalization step likely is due to the prolonged reaction times, which
resulted in a mixture of products including some anomerization at the C1
center. However, none of the α-anomer at the newly created C4 anomeric
SCHEME 1 Synthesis of AZT phosphonate (4).

Pyrimidine Phosphomethoxy Nucleosides
575
center was isolated from the mixture. The bisPOC prodrugs were prepared
from the phosphonate diacids by treatment with chloromethylisopropyl car-
bonate in the presence of Hu¨nigs base.[5] D4CP analog (7) was prepared
from the phosphonate diester of (5) using typical procedures for conver-
sion of uridine to cytidine[6] followed by removal of the phosphonate es-
ter groups using TMSBr. The dd analog (8) was prepared from the phos-
phonate ester of (7) by catalytic reduction followed by TMSBr treatment.
Nucleosides were obtained from commercial sources and their respective
triphosphates from Trilink Biotechnologies, Inc. (San Diego, CA, USA) or
Chemcyte, Inc. (San Diego, CA, USA).
RESULTS AND DISCUSSION
Only d4T phosphonate (2) demonstrated antiviral activity below 200 µM
(Table 1). Poor antiviral activity can be the result of one or more of the
following factors; (1) poor permeation of the phosphonate diacid into the
cells; (2) poor metabolism to the diphospho-phosphonates; and (3) poor
inhibition of RT. To probe the latter, the diphosphates of (2, 3, 5, and 6)
were prepared and in each case, weaker RT activity than the corresponding
nucleoside triphosphates was observed (Table 1). Thus, the poor antiviral
effects are at least in part, due to the weaker RT inhibition properties of the
phosphonates. The weaker RT activity of the dd phosphonates (3) and (6)
compared to their respective d4 phosphonates (2) and (5) mirrors the trend
observed for the corresponding dd and d4 nucleoside analogs. This parallel
trend implies that the phosphonates likely have similar binding modes to
their nucleoside counterparts in the active site of RT.
TABLE 1 Anti-HIV RT activity and antiviral potency for nucleosides, phosphonates, and bisPoc
prodrugs
HIV RT IC50[µM]
WT HIV EC50[µM]
Nucleoside
Diphospho-phosphonate
Phosphonate
BisPOC
triphosphate
of (2–8)
Nucleoside
(2–8)
phosphonate of (2–8)
d4T
ddT
AZT
d4U
ddU
d4C
ddC
0.06 0.03
0.12 0.09
0.05 0.03
0.55 0.15
0.83 0.28
n.d.
0.39 0.17
5.4 2.1
n.d.
4.8 0.02
>200
0.16 0.12
>200
26 8.8
>200
>200
>200
>200
>200
>200
0.91 0.42
1.3 0.8
n.d.
2.8 0.6
>5
5.7
31
n.d.
n.d.
>200
2.6a
n.d.
n.d.
0.16 0.08
2.5 0.9
Cell based antiviral cytopathic assays were conducted in MT-2 cells infected with HIV-1, strain IIIb.
Antiviral effect was determined after 5 days of incubation with serial dilutions of tested compounds
using XTT-based cell viability method.[8] Enzyme inhibition assays were performed with recombinant
p66/p51 heterodimer RT.[9]
aData from.[10]

576
R. L. Mackman et al.
TABLE 2 Antiviral resistance profile represented as fold EC50 change relative to wild-type virus
Fold EC50 change relative to wild-type HIV-1
Compound
WT EC50[µM]
K65R
6 TAMsa
M184V
d4T
4.8 2.3
0.91 0.42
2.3 1.1
6.7 2.6
5.5 2.5
6.8 0.7
1.2 0.2
1.1 0.7b
BisPOC d4TP (2)
All values are the result of at least 2 experiments. Assays were conducted in MT-2 cells using HIV-1
HXB2 wild-type strain side-by-side with corresponding site directed recombinant mutant strains.
a6TAMs virus contains mutations M41L/D67N/K70R/L210W/T215Y/K219Q in HIV RT gene.
b M184V resistance determined for parent nucleoside phosphonate.
Given that several of the diphospho-phosphonate analogs were shown
to be RT inhibitors (Table 1), albeit weak inhibitors, the potential impact of
limited cellular uptake on anti-viral activity was then addressed by prepara-
tion of bisPOC prodrugs. The bisPOC prodrug of d4T phosphonate (2)
improved anti-HIV activity by 29-fold, and for ddT phosphonate (3) by
>150-fold respectively. Improvements in potency also were found for the
corresponding bisPOC prodrug of d4U phosphonate (5) thereby indicat-
ing that this prodrug strategy is broadly applicable toward improving the
antiviral potency of cyclic phosphonates, similar to that observed for Viread
(1).[3] It is noteworthy that the antiviral activity of bisPOC d4T phosphonate
is now several-fold improved over nucleoside d4T despite being a weaker in-
hibitor of RT. It, therefore, is likely that higher levels of active metabolites
are generated by d4T phosphonate (2) through superior metabolism prop-
erties and a longer intracellular half-life of the active species.[3]
The resistance profile of bisPOC (2) toward viruses containing six thymi-
dine analog mutations (6TAMs), K65R or M184V was evaluated and com-
pared to d4T (Table 2). A comparable resistance profile was observed to-
ward the M184V and 6TAMs, whereas a small increase was observed in the
K65R resistance. The close overlap of the resistance profiles further supports
the conclusion that the phosphonates and their corresponding nucleosides
interact with WT and mutant RT through similar binding modes.
CONCLUSION
Evaluation of several cyclic pyrimidine phosphonates reveals that the
phosphonates are ‘bioisosteres’ of their corresponding nucleoside phos-
phates but weaker RT inhibitors. Antiviral potency can be improved con-
siderably by the application of bisPOC prodrugs. The trends observed for
pyrimidine analogs have recently been extended to purine analogs lead-
ing to the discovery of the novel clinical candidate, 2ꢁF-d4A phosphonate,
GS9148.[7]

Pyrimidine Phosphomethoxy Nucleosides
577
REFERENCES
1. Gallant, J.E. The ideal nucleoside/nucleotide backbone. J. AIDS 2004, 37(supp 1), S44–S51.
2. Chapman, T.M.; McGavin, J.K.; Noble, S. Tenofovir disoproxil fumarate. Drugs 2003, 63(15), 1597–
1608.
3. Robbins, B.L.; Srinivas, R.V.; Kim, C.; Bischofberger, N.; Fridland, A. Anti-human immunodeficiency
virus activity and cellular metabolism of a potential prodrug of the acyclic nucleoside phospho-
nate 9-R-(2-phosphonomethoxypropyl)adenine (PMPA), Bis(isopropyloxymethylcarbonyl)PMPA.
Antimicrob. Agents Chemother. 1998, 42(3), 612–617.
4. Kim, C.U.; Luh, B.Y.; Martin, J.C. Regiospecific and highly stereoselective electrophilic addition
to furanoid glycals: synthesis of phosphonate nucleotide analogs with potent activity against HIV.
J. Org. Chem. 1991, 56, 2642–2647.
5. Arimilli, M.N.; Kim, C.U.; Dougherty, J.; Mulato, A.; Oliyai, R.; Shaw, J.P.; Cundy, K.C.; Bischofberger,
N. Synthesis, in vitro biological evaluation and oral bioavailability of 9-[2-(phosphonomethoxy)
propyl]adenine (PMPA) prodrugs. Antiviral Chem. Chemother. 1997, 8, 557–564.
6. Wang, P.; Gullen, B.; Newton, M.G.; Cheng, Y.-C.; Schinazi, R.F.; Chu, C.K. Asymmetric synthesis
and antiviral activities of L-carbocyclic 2ꢁ,3ꢁ-didehydro-2ꢁ,3ꢁ-dideoxy and 2ꢁ,3ꢁ-Dideoxy Nucleosides.
J. Med. Chem. 1999, 42, 3390–3399.
7. Mackman, R.; Boojamra, C.; Chen, J.; Douglas, J.; Grant, D.; Kim, C.; Lin, K.Y.; Markevitch, D.;
Prasad, V.; Ray, A.; Cihlar, T. In Discovery of GS9148, a Novel Nucleotide HIV Reverse Transcriptase (RT)
Inhibitor. Presented at the 19th International Conference on Antiviral Research, San Juan, Puerto
Rico, 2006, Abstr. 37.
8. Weislow, O.S.; Kiser. R.; Fine, D.L.; Bader, J.; Shoemaker, R.H.; Boyd, M.R. New soluble-formazan as-
say for HIV-1 cytopathic effects: application to high-flux screening of synthetic and natural products
for AIDS-antiviral activity. J. Natl. Cancer Inst. 1989, 81, 577–586.
9. White, K.L.; Chen, J.M.; Feng, J.Y.; Margot, N.A.; Ly, J.K.; Ray, A.S.; Macarthur, H.L.; McDermott,
M.J.; Swaminathan, S.; Miller, M.D. The K65R reverse transcriptase mutation in HIV-1 reverses the
excision phenotype of zidovudine resistance mutations. Antivir Ther. 2006, 11, 155–163.
10. Parikh, U.M.; Koontz, D.L.; Chu, C.K.; Schinazi, R.F.; Mellors, J.W. In vitro activity of structurally
diverse nucleoside analogs against human immunodeficiency virus type 1 with the K65R mutation
in reverse transcriptase. Antimicrob. Agents Chemother. 2005, 49, 1139–1144.