[d4U]-butyne-[HI-236] as a non-cleavable, bifunctional NRTI/NNRTI HIV-1 reverse-transcriptase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2007.01.107

The synthesis of bifunctional compound 10 consisting of d4U joined at C-5 to a butynyl spacer attached to HI-236 is reported using a Sonogashira coupling as a key step. As a non-cleavable bifunctional HIV inhibitor incorporating an NRTI with an NNRTI, 10 shows good inhibitory activity (EC₅₀ = 250 nM) against HIV (IIIB) replication in MT-2 cell culture, which is eight times greater than that of d4T and between those of the two component drugs.

Full text of DOI:10.1016/j.bmcl.2007.01.107

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2614–2617
[d4U]-butyne-[HI-236] as a non-cleavable, bifunctional
NRTI/NNRTI HIV-1 reverse-transcriptase inhibitor
a,
a
b
Roger Hunter,a,* Clare I. Muhanji, Ian Hale, Christopher M. Bailey,
*
Aravind Basavapathrunib and Karen S. Andersonb
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
bDepartment of Pharmacology, School of Medicine, Yale University, New Haven, CT 06520, USA
Received 1 January 2007; revised 30 January 2007; accepted 31 January 2007
Available online 4 February 2007
Abstract—The synthesis of bifunctional compound 10 consisting of d4U joined at C-5 to a butynyl spacer attached to HI-236 is
reported using a Sonogashira coupling as a key step. As a non-cleavable bifunctional HIV inhibitor incorporating an NRTI with
an NNRTI, 10 shows good inhibitory activity (EC50 = 250 nM) against HIV (IIIB) replication in MT-2 cell culture, which is eight
times greater than that of d4T and between those of the two component drugs.
Ó 2007 Elsevier Ltd. All rights reserved.
The use of combination therapy (HAART) for HIV is
designed to prevent the development of resistant HIV
strains by effectively suppressing viral replication, thus
denying HIV the opportunity to produce new muta-
tions.1 Non-nucleoside reverse transcriptase inhibitors
(NNRTIs) act allosterically by slowing down the chem-
ical step catalysed by the HIV reverse transcriptase (RT)
enzyme, and this retardation allows the two-step binding
of the nucleoside analogues (NRTI) to come to equilib-
rium leading to tighter binding of the nucleotide.2 These
studies suggested that the nucleotide triphosphate and
non-nucleoside inhibitors could simultaneously bind to
their respective NRTI and NNRTI sites in RT and that
there was communication between the two sites. The
inhibition at both target sites. In principle, such a
bifunctional inhibitor would utilize the additive binding
energies from the interactions at each site, translating
into very tight binding.3b
Such NRTI/NNRTI double-drugs have used the C-50,
C-5 or N-3 positions of the nucleosides for attachment
of the linker. The first examples were reported by the
Camarasa4a group in 1995 and incorporated a TSAO
derivative linked to AZT via a polymethylene spacer be-
tween N-3 on each drug. Subsequent work by these
authors extended to incorporating d4T as a nucleoside
as well as derivatising to form phosphoramidate4b dou-
ble-drugs. Foscarnet was also evaluated as an NNRTI
option.4c Subsequently, the Laduree group reported
´
˚
close proximity (10–15 A) of the NRTI/NNRTI binding
sites has inspired several groups to synthesize bifunc-
tional heterodimers containing one of each class of drug
separated by a spacer following an original suggestion
by Nanni.3a The heterodimers (double-drugs) reported
fall into two distinct classes, with the more common of
the two joining the two drugs via a cleavable linker in
the expectation that both drugs will be released into
the cell cytoplasm, resulting in synergistic inhibitory ef-
fects. The other class involves incorporation of a non-
cleavable linker in the hope of achieving concurrent
the synthesis and activity of both non-cleavable5a,b and
cleavable systems5c in which the former type as a
[d4T]-spacer-[imidazo{1,5-b}pyridazine] system was
found to be the most active. Similarly, Pedersen6 recent-
ly reported the synthesis and antiviral activities of dou-
ble-prodrugs incorporating d4T as an S-acyl-2-thioethyl
(SATE) 50-phosphate ester linked to N-3 of MKC-442
(a HEPT derivative) via a cleavable p-hydroxybenzoyl
linker. These double-drugs displayed good anti-HIV
activity which was attributed to the d4T part of the mol-
ecule, Figure 1.
The objective of the present study was to synthesize and
evaluate the anti-HIV activity of bifunctional com-
pounds of the general formula [d4U]-spacer-[HI-236]
in the search for evidence of synergism between the
Keywords: HIV RT bifunctional inhibitor; NRTI and NNRTI double-
drug; HIV Heterodimer; Sonogashira coupling.
*
Corresponding authors. Tel.: +27 21 650 2544; fax: +27 21 689
7499; e-mail: Roger.Hunter@uct.ac.za
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.107

R. Hunter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2614–2617
2615
O
N
O
O
N
N
(CH2)n
N
HN
Camarasa
O
O
O P
O
O
O
N
O
O
TBSO
H2N
HO
[TSAO-T]-spacer-[AZT]
S
O
O
O
O
O OTBS
N3
S
O
N
O
O
N
O
(CH2)
N
O
O
N
HN
N
H
n N
H
N
Ph
O
N
HO
Laduree
[d4T]-spacer-[imidazo{1,5-b}pyridazine]
O
O
Pedersen
[d4T]-spacer-[MKC-442]
Figure 1. Structures of known NRTI/NNRTI double-drugs.
two drug sites. A biologically non-cleavable butynyl
spacer was chosen for the prototype, Figure 2. HI-236
was selected due to its potency against wild-type and
NNRTI-resistant HIV-1 strains,7 while d4T is currently
used to treat HIV. Regarding the attachment points for
the linker, on the NRTI side, the pyrimidine C-5 posi-
tion was selected in view of its anticipated low interfer-
ence with base pairing.8 On the NNRTI side,
attachment to the C-2 phenolic oxygen (see Fig. 2 for
numbering) was chosen in view of structural studies
reported by Uckun and co-workers,7 which revealed
that the C-2 phenolic oxygen substituent of HI-236 is
located beneath the ethyl linker and points into a size-
able hydrophobic space. Attachment of the linker to
the C-5 position of the nucleoside was thought to be
accessible via a Sonogashira9 palladium cross-coupling
reaction of a terminal alkyne with the 5-iodo nucleoside.
During the course of this study, C-5 tethered heterodi-
OH
OBn
NH2
d
OBn
CHO
a, b, c
NHBoc
1
2
e
OMe
O
OMe
OMe
OH
H
NHBoc
NHBoc
f
3
4
OMe
OMe
Scheme 1. Reagents and conditions: (a) BnBr, K2CO3, EtOH, reflux,
16 h, (95%); (b) CH3NO2, NH4OAc, 70 °C, 14 h, (75%); (c) LiAlH4,
THF, reflux, 4 h; (d) (Boc)2O, Et3N, CH3CN, rt, overnight, (76%, 2
steps); (e) H2, Pd/C, EtOH, rt, 5 h, (66%); (f) 3-butynyl-1-tosylate (6
equiv), K2CO3 (1.5 equiv), CH3CN, reflux, 5 d, (70%).
3 in good yield (mp: 116–117 °C; m/z HRMS (EI)
267.1433, C14H21NO4 requires m/z 267.1471), which was
alkylated with 3-butynyl-1-tosylate (6 equiv)11 using
K2CO3 (1.5 equiv) in acetonitrile at reflux for 5 days to
produce 4 in 70% yield after chromatography (mp: 76–
77 °C; m/z HRMS(EI) 319.1776, C18H25NO4 requires
m/z 319.1784).
mers were reported5c by the Laduree group in which a
´
Sonogashira coupling strategy was demonstrated.
Various synthetic disconnections were investigated, and
eventually it became clear that the thiourea moiety needed
to be introduced last. Thus, the key alkyne 4 was
synthesized in six steps starting from commercially avail-
able 2-hydroxy-5-methoxybenzaldehyde (Scheme 1),
beginning with chemistry described by Glennon10 and
coworkers to afford amine 1. To assist with isolation
and subsequent elaboration, 1 was protected as its Boc-
carbamate with di-tert-butyldicarbonate and triethyl-
amine in acetonitrile at room temperature to give 2 in
76% yield (over 2 steps) after purification by column
chromatography (mp: 103–104 °C). Hydrogenolytic
debenzylation of 2 in the presence of 10% palladium-
on-carbon in ethanol at room temperature led to phenol
The Sonogashira coupling partner 6 was synthesized by
iodinating 50-O-benzoyl d4U 5, which was prepared in
four steps from uridine according to the Bristol-Myers
Squibb procedure for producing d4T from 5-methyluri-
dine.12 Iodination of 5 was accomplished using elemen-
tal iodine and ceric ammonium nitrate (CAN)13 in
acetonitrile at 60 °C in 80% yield (Scheme 2) to afford
6, which underwent Sonogashira coupling with alkyne
4 to yield the desired coupled product 7 in 69% yield.
With 7 in hand, benzoyl deprotection, Boc removal
S
N
H
NH
Br
S
N
O
N
O
N
O
N
N
H
NH
Br
HN
HN
O
O
O
2
O
HO
HO
O
O
O
[d4U]-butyne-[HI-236]
HI-236
Figure 2. Structures of d4T, HI-236 and [d4U]-butyne-[HI-236].
d4T

2616
R. Hunter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2614–2617
NHBoc
O
N
O
O
N
O
N
I
HN
HN
HN
O
Br
S
O
BzO
O
O
BzO
BzO
a
b
O
N
N
H
N
O
O
c, d
N
8
7
S
5
6
N
H
NH
Br
S
N
O
N
O
1''''
N
N
H
NH
2'''
2'''''
HN
O
O
O
1'''
4''
5
O
1''
HN3
BzO
4'''
O
e
1
5'''''
O
O
5'
N
Br
HO
O
1'
3'
9
10
Scheme 2. Reagents and conditions: (a) I2, CAN, CH3CN, 60 °C, 1 h, (80%); (b) alkyne 4, (PPh3)4Pd, CuI, Et3N, DMF, THF, rt, 4 h, (69%); (c)
CF3COOH, CH2Cl2, 0 °C; (d) EtN(i–Pr)2 followed by 8, THF, rt/overnight, (60% for 2 steps); (e) NaOMe (cat), MeOH, 0 °C, 20 min, (51%).
Table 1. Inhibition and cytotoxicity assaya results for d4T, HI-236, 10,
11 and 12 against HIV -1 (IIIB) in MT-2 cell culture
and thiourea installation remained for completion of the
synthesis, and a number of permutations were tried out.
Cytotoxicityc IC50 (lM)
TId
b
Eventually, it was established that debenzoylation as the
final step worked best, as the protected alkylated d4U
moiety appeared to be less prone to acid-catalysed ano-
meric cleavage with the trifluoroacetic acid used to
deprotect the Boc group. Thus deprotection of the Boc
group of 7 using trifluoroacetic acid in methylene chlo-
ride at 0 °C furnished the crude amine as a trifluoroace-
Compound
EC50 (lM)
d4T
HI-236
10
2
>100
>1
17
>50
>24
68
0.042
0.25
0.023
120
11
12e
>1
360
>44
3
a Ref. 16; MOI was 0.1.
b Effective concentration that inhibits viral-mediated T-cell death by
50% and as an average of three results.
c Concentration that kills 50% of the T-cells and as an average of three
results.
tate salt. Processing involved adding Hunig’s base to
¨
liberate the amine, and condensing directly with thiocar-
bonyl derivative 87 at rt overnight in THF. This con-
trasted with the much harsher conditions used by
Sahlberg and co-workers14 of heating in DMF at
100 °C.
d In vitro therapeutic index (IC50/EC50).
e See Ref. 17.
In this fashion, bifunctional benzoate 9 was produced in
60% for the two steps. Final deprotection with catalytic
methoxide in methanol furnished the target 10 in 51%
yield and in an acceptable level of purity15 after
chromatography.
Laduree’s work17 showed that elongating d4T at C-5
with a hydroxybutynyl spacer (compound 12) results
in a drastic reduction in activity (see Table 1). Interest-
ingly, our current study showed that in elongating the
NNRTI, the butynylated HI-236 11 was found to be
twice as active as the parent HI-236. The inhibitory
activity of the bifunctional compound 10 as an NRTI-
spacer-NNRTI non-cleavable inhibitor is 250 nM. This
level of inhibition is eight times greater than that of
d4T in this system, and lies between that of HI-236
and d4T, indicating our prototype may be a promising
lead for this type of inhibitor. Studies are underway to
establish if the NRTI and NNRTI moieties of 10 are in-
deed simultaneously binding in their respective target
sites. The rather potent EC50 result for 10 together with
the previous results from compound 1217 argues that at
least a portion of the potential binding interactions for
the NRTI sites are being realized. Work is currently in
progress to evaluate the influence of changing the spacer
as well as derivatising to the C-50 phosphoramidate.18
The inhibition of viral replication in HIV-infected cells
of the prototype bifunctional compound 10 together
with HI-236, d4T, and the truncated constituent of
bifunctional 10, compound 11– see Figure 3– were mea-
sured against HIV-1 (IIIB) replication in MT-2 cell cul-
tures using an MTT assay.16 The other truncated
component of 10, compound 12, has already been eval-
17
´
uated by Laduree. The results of inhibition are shown
in Table 1.
An examination of the inhibitory properties for these
compounds offers insight into the design of a prototyp-
ical bifunctional compound. Earlier studies from
O
OH
S
HN
N
H
NH
O
O
N
N
HO
Acknowledgments
O
O
11
12
Br
We thank the National Research Foundation of South
Africa, the Third World Organization for Women in
Figure 3. Truncated components of bifunctional 10.

R. Hunter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2614–2617
2617
9. Agrofoglio, L. A.; Gillaizeau, I.; Saito, Y. Chem. Rev.
2003, 103, 1875.
Science and the University of Cape Town for financial
support. We also thank the National Institute of Health
for support of GM49551 to K.S.A.
10. Glennon, R. A.; Liebowitz, S. M.; Leming-Doot, D.;
Rosecrans, J. A. J. Med. Chem. 1980, 23, 990.
11. Deng, B. L.; Hartman, T. L.; Buckheit, R. W., Jr.;
Pannecouque, C.; De Clercq, E.; Fanwick, P. E.; Cush-
man, M. J. Med. Chem. 2005, 48, 6140.
Supplementary data
12. Chen, B. C.; Quinlan, S. L.; Stark, D. R.; Reid, G.; Audia,
V. H.; George, J. G.; Eisenreich, E.; Brundidge, S. P.;
Racha, S.; Spector, R. H. Tetrahedron Lett. 1995, 36,
7957.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.01.107.
13. Asakura, J.; Robins, M. J. J. Org. Chem. 1990, 55,
4928.
14. Bell, F. W.; Cantrell, A. S.; Ho¨gberg, M.; Jaskunas, S. R.;
References and notes
Johansson, N. G.; Jordan, C. L.; Kinnick, M. D.; Lind,
¨
1. (a) Stevens, M.; De Clercq, E.; Balzarini, J. Med. Res. Rev.
2006, 26, 595; (b) De Clercq, E. J. Med. Chem. 2005, 48, 1297.
2. (a) Spence, R.; Kati, W.; Anderson, K. S.; Johnson, K. A.
Science 1995, 267, 988; (b) Rittinger, K.; Divita, G.;
Goody, R. S. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8046;
(c) Basavapathruni, A.; Anderson, K. S. Curr. Pharm.
Des. 2006, 12, 1857.
3. (a) Nanni, R. G.; Ding, J.; Jacobo-Molina, A.; Hughes, S.
H.; Arnold, E. Perspect. Drug Discov. Des. 1993, 1, 129;
(b) Shuker, S.; Hajduk, P.; Meadow, R.; Fesik, S. Science
1996, 274, 1531.
´
P., ; Morin, J. M., Jr.; Noreen, R.; Oberg, B.; Palkowitz, J.
A.; Parrish, C. A.; Pranc, P.; Sahlberg, C.; Ternansky, R.
J.; Vasileff, R. T.; Vrang, L.; West, S. J.; Zhang, H.; Zhou,
X-X. J. Med. Chem. 1995, 38, 4929.
15. Physicochemical characteristics of compounds 10, [d4U]-
butyne-[HI-236], (see Scheme 2 for numbering): mp: 121–
20
122 °C; ½aꢀD þ 22:4ꢁ (c 1.10, CHCl3); IR (CHCl3) mmax
cmꢂ1: 3934 (s) (N–H), 3688 (m) (N–H), 2304 (s) (CꢃC),
1696 (m) (CO), 1606 (m) (C@C), 1425 (s) (C@S); 1H NMR
(400 MHz, CDCl3) d: 11.05 (1H, t, J = 4.2 Hz, NH), 8.70
(1H, br s, NH), 7.99 (1H, d, J = 2.6 Hz, H-600000), 7.97 (1H, s,
H-6), 7.56 (1H, dd, J = 8.8, 2.6 Hz, H-400000), 6.97 (1H, m, H-
10), 6.80 (1H, m, H-3000), 6.77 (2H, m, H-6000, H-5000), 6.66
(1H, d, J = 8.8 Hz, H-300000), 6.37 (1H, dt, J = 5.8, 1.5 Hz, H-
30), 5.86 (1H, m, H-20), 4.93 (1H, m, H-40), 4.05 (2H, t,
J = 5.9 Hz, H-400), 3.95 (2H, m, H-20000), 3.80 (2H, m, H-50),
3.78 (3H, s, OMe), 2.98 (2H, m, H-300), 2.84 (2H, t,
J = 5.8 Hz, H-10000); 13C NMR (100 MHz, CDCl3) d: 178.9
C@S), 162.2 (C-4), 153.4 (C-4000), 151.5 (C-200000), 150.9 (C-
1000), 149.1 (C-2), 146.6 (C-600000), 143.6 (C-6), 140.8 (C-400000),
135.6 (C-30), 129.3 (C-2000), 128.9 (C-20), 118.0 (C-3000), 113.4
(C-300000), 112.8 (C-6000), 111.4 (C-5000), 110.8(C-500000), 100.3 (C-
200), 91.3 (C-100), 90.4 (C-10), 87.7 (C-40), 72.5 (C-5), 66.8 (C-
400), 62.9 (C-50), 55.6 (O–Me), 45.7 (C-20000), 30.2 (C-10000),
21.0 (C-300); HRMS (ES) m/z Found: [M+H]+, 642.1008.
Calcd for C28H29BrN5O6S (M+H), 642.1022.
´
´
4. (a) Velazquez, S.; Alvarez, R.; San-Felix, A.; Jimeno, M.
L.; De Clercq, E.; Balzarini, J.; Camarasa, M. J. J. Med.
´
´
Chem. 1995, 38, 1641; (b) Velazquez, S.; Tun˜on, V.;
Jimeno, M. L.; Chamorro, C.; De Clercq, E.; Balzarini, J.;
Camarasa, M. J. J. Med. Chem. 1999, 42, 5188; (c)
´
´
Velazquez, S.; Lobaton, E.; De Clercq, E.; Koontz, D. L.;
Mellors, J. W.; Balzarini, J.; Camarasa, M. J. J. Med.
Chem. 2004, 47, 3418.
5. (a) Renoud-Grappin, M.; Fossey, C.; Fontaine, G.;
´
Laduree, D.; Aubertin, A. M.; Kirn, A. Antiviral Chem.
Chemother. 1998, 9, 205; (b) Gavriliu, D.; Fossey, C.;
´
Ciurea, A.; Delbederi, Z.; Sugeac, E.; Laduree, D.;
Schmidt, S.; Laumond, G.; Aubertin, A. M. Nucleos.
Nucleot. 2002, 21, 505; (c) Sugeac, E.; Fossey, C.; Laduree,
D.; Schmidt, S.; Laumond, G.; Aubertin, A. M. J. Enzym.
Inhib. 2003, 18, 175.
16. Mosmann, T. J. Immunol. 1983, 65, 55.
17. Ciurea, A.; Fossey, C.; Gavriliu, D.; Delbederi, Z.;
6. Pedersen, L.; Jørgensen, P. T.; Nielsen, S.; Hansen, T. H.;
Nielsen, J.; Pedersen, E. B. J. Med. Chem. 2005, 48, 1211.
7. Mao, C.; Sudbeck, E. A.; Venkatachalam, T. K.; Uckun,
F. M. Bioorg. Med. Chem. Lett. 1999, 9, 1593.
´
Sugeac, E.; Laduree, D.; Schmidt, S.; Laumond, G.;
Aubertin, A. M. J. Enzyme Inhib. 2004, 19, 511.
18. Cahard, D.; McGuigan, C.; Balzarini, J. Mini-Rev. Med.
Chem. 2004, 4, 371.
8. Ruth, J. L.; Cheng, Y. C. J. Biol. Chem. 1982, 257, 10261.