Anti-AIDS agents 88. Anti-HIV conjugates of betulin and betulinic acid with AZT prepared via click chemistry

Article abstract of DOI:10.1016/j.tetlet.2012.02.022

In the present study, a new strategy to link AZT with betulin/betulinic acid (BA) by click chemistry was designed and achieved. This conjugation via a triazole linkage offers a new direction for the modification of anti-HIV triterpenes. Click chemistry provides an easy and productive way for linking two molecules, even when one of them is a large natural product. Among the newly synthesized conjugates, compounds 15 and 16 showed potent anti-HIV activity with EC₅₀ values of 0.067 and 0.10 μM, respectively, which are comparable to that of AZT (EC₅₀: 0.10 μM) in the same assay.

Full text of DOI:10.1016/j.tetlet.2012.02.022

Tetrahedron Letters 53 (2012) 1987–1989
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Anti-AIDS agents 88. Anti-HIV conjugates of betulin and betulinic acid
with AZT prepared via click chemistry
Ibrahim D. Bori a, Hsin-Yi Hung a, Keduo Qian a, , Chin-Ho Chen b, Susan L. Morris-Natschke a,
⇑
Kuo-Hsiung Lee a,c,
⇑
a Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
b Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 401, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, a new strategy to link AZT with betulin/betulinic acid (BA) by click chemistry was
designed and achieved. This conjugation via a triazole linkage offers a new direction for the modification
of anti-HIV triterpenes. Click chemistry provides an easy and productive way for linking two molecules,
even when one of them is a large natural product. Among the newly synthesized conjugates, compounds
Received 22 November 2011
Revised 31 January 2012
Accepted 6 February 2012
Available online 13 February 2012
15 and 16 showed potent anti-HIV activity with EC50 values of 0.067 and 0.10
comparable to that of AZT (EC50: 0.10 M) in the same assay.
lM, respectively, which are
l
Keywords:
Betulin
Ó 2012 Elsevier Ltd. All rights reserved.
Betulinic acid
AZT
Anti-HIV
Click chemistry
Due to the expanded and improved HIV programs, the number
of new global HIV infections declined by 19% over the past decade.
However, this progress is fragile and unevenly distributed. HIV
incidence is still increasing in some countries and regions, and
too many new infections are occurring, 2.6 million in 2009 alone,
contributing to the current global incidence of 33.3 million.1 New
infections continue to outpace the number of people placed on
treatment, and the efficacy of the treatments is hampered by the
emergence of drug-resistant viral strains and severe drug–drug
interactions. Therefore, novel potent antiretroviral agents with dif-
ferent targets and acceptable prices are still urgently needed.
Two lupine-type triterpenes betulin and betulinic acid (BA),
which are readily available from the birch tree in large quantity,
exhibit diverse pharmacological activities, including anti-HIV,
anti-cancer, and anti-inflammatory activities.2 Among the BA/bet-
ulin derivatives, bevirimat [3-O-(30,30-dimethylsuccinyl)betulinic
acid, 14] was found to exhibit remarkable anti-HIV-1 activity
against primary and drug-resistant HIV-1 isolates,3,4 representing
a unique first in a class of anti-HIV compounds termed maturation
inhibitors (MIs).4,5 Bevirimat has recently succeeded in Phase IIb
clinical trials.6–9
In our prior study, AZT (30-azido-30-deoxythymidine), a clini-
cally used nucleoside reverse transcriptase inhibitor (NRTI), was
conjugated with 3-O-(30,30-dimethylsuccinyl)betulin at its C-28 po-
sition using different linkers. The goal was to provide multi-target
therapeutics in one molecule in order to reduce the risk of drug–
drug interactions, which can occur from mixing monotherapies.10
The conjugates were formed via an ester bond, which was meant
to be subsequently hydrolyzed inside the cells and release two dif-
ferent chemical entities exerting two pharmacological functions,
anti-maturation and anti-reverse transcriptase. However, one po-
tential drawback of this design is that the hydroxyl group of AZT,
which needs to undergo phosphorylation inside the host cells to
become active, forms the linker bond with the betulin derivatives.
Consequently, the inhibitory ability of AZT is highly dependent on
the ability of the conjugate to dissociate. In order to improve this
issue, we investigated another strategy to link AZT with betulin
and BA via the azido group of AZT by using click chemistry.
Click chemistry as introduced by K. Barry Sharpless is a chemi-
cal philosophy to mimic nature’s ability to make carbon–hetero-
atom bonds rather than carbon–carbon bonds.11 Conjugation of
AZT with betulin and BA by this methodology has the following po-
tential benefits. (1) Click chemistry is designed to link the azido
group of AZT with an alkyne group on betulin/BA derivatives, leav-
ing the hydroxyl group of AZT free to be phosphorylated. (2) The
linking triazole group is physiologically stable in cells. Thus, the
new conjugated molecule will not be degraded inside the cells,
⇑
Corresponding authors. Tel.: +1 919 966 1394 (K.Q.); tel.: +1 919 962 0066;
fax: +1 919 966 3893 (K.-H.L.).
E-mail addresses: kdqian@unc.edu (K. Qian), khlee@unc.edu (K.-H. Lee).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2012.02.022

1988
I. D. Bori et al. / Tetrahedron Letters 53 (2012) 1987–1989
which should reduce the potential drug–drug interactions. (3) In
addition, the triazole linkage formed by click chemistry may also
offer extra interaction with virus proteins. Based on this rationale,
the present study reports the synthesis and anti-HIV activity of the
newly designed AZT–betulin and AZT–BA conjugates.
the target compounds. In addition, the reaction time was short-
ened significantly to 30 min by using microwave conditions at
120 °C (followed by dilution with EtOAc, washing with NaHCO3
and brine, and chromatography), rather than the prior overnight
esterification reaction. Therefore, the click reaction provides a
much more productive and rapid approach to obtain the final prod-
ucts of AZT–betulin/BA conjugates.
The anti-HIV activity data of all new compounds are summa-
rized in Table 1. Among the tested compounds, conjugates 8 and
13 with AZT linked through the C-3 side chain of betulin/BA
showed moderate antiviral activity with EC50 values of 0.19 and
The synthetic route to compounds conjugated at the C-3 posi-
tion of betulin is outlined in Scheme 1. The C-28 hydroxyl of betu-
lin was first protected by the reaction with tert-butyldimethylsilyl
chloride (TBSCl) to yield the silyl ether 1. Prop-2-ynyl groups were
then introduced at the C-3 position as either an ether (2) or carbon-
ate ester (3). Compounds 2 and 3 were then reacted with the azido
group of AZT in the presence of Cu and CuSO4ꢀ5H2O to furnish final
compounds 8 and 9 in quantitative yields. Analogous final com-
pounds 10 and 11 were obtained by the same click reaction of
AZT with the C-28 de-protected betulin derivatives 4 and 5. Oxida-
tion of the C-28 hydroxyl of 4 with Jones reagent yielded 6, which
was also reacted with AZT to yield 12, an AZT–BA conjugate. Final-
ly, a 30,30-dimethylsuccinyl ester was introduced at the C-28 posi-
tion of 4 to yield compound 7, which was converted by click
chemistry to the conjugate 13. Scheme 2 depicts the synthesis of
AZT–bevirimat conjugates. A prop-2-ynyl ester moiety was added
to the C-28 position of bevirimat (14) to furnish 15, followed by
the click reaction of 15 with AZT to yield the final compound 16.
Compared with the previous yields (42.7–87.3%) for conjugating
AZT with betulin derivatives via an ester bond,10 the click reactions
of AZT with betulin/BA derivatives were achieved quantitatively,
which is a significant advantage in the last step of synthesizing
0.35 lM, respectively. The C-3 ether bond (8) was favored for activ-
ity compared with the C-3 carbonate ester bond (corresponding
conjugate 9 was inactive). Conjugates with a free C-28 hydroxyl
(10 and 11) or carboxylic acid group (12) showed no inhibitory
activity, compared with conjugates with a C-28 TBS ether (8) or
dimethylsuccinyl ester (13). Between the two active conjugates,
the smaller C-28 TBS side chain in 8 was favored compared with
the longer dimethylsuccinyl side chain in 13. Overall, in this lim-
ited data set, when AZT and betulin/BA are conjugated at the C-3
position, a shorter distance between AZT–betulin/BA and the pres-
ence of a small extra side chain at C-28 are important for the con-
jugates’ antiviral activity.
The non-conjugated 15 with a 3-O-30,30-dimethylsuccinyl side
chain at C-3 and propynyl ester moiety at C-28 showed potent
anti-HIV activity with an EC50 of 0.067 lM and a selectivity index
(SI, ratio of cytotoxic/anti-viral activity) of 226. The AZT–bevirimat
H
O
OH
H
CH3
OH
H
H
HN
OTBS
g
H
OTBS
b for 2
H
H
OTBS
a
H
O
N
H
O
HO
H
H
c for 3
H
H
HO
R
2
3
R=O
R=OCOO
H
H
R=O
R=OCOO
8
9
1
R
betulin
N
H
N
N
d
O
CH3
OH
HN
H
H
OH
N
O
OH
H
O
H
g
H
H
R
N
4 R=O
H
10 R=O
R=OCOO
R
N
H
N
R=OCOO
5
11
e
O
N
H
CH3
HN
f
H
H
OH
O
O
OH
H
O
H
O
g
O
H
H
6
O
N
N
H
12
N
O
H
O
N
O
OH
CH3
OH
H
HN
O
O
H
H
O
g
O
OH
H
O
O
H
O
7
H
O
N
N
H
N
13
Scheme 1. AZT conjugation at C-3 position. Reagents and conditions: (a) TBSCl, DMF/THF (1:1), DMAP, DIPEA, 0 °C; (b) propargyl bromide, NaH, THF; (c) triphosgene,
pyridine, THF,12 and then propargyl alcohol, pyridine, THF; (d) TBAF, THF; (e) Jones reagent, acetone; (f) 2,2-dimethylsuccinic anhydride, DMAP, DMF, 70 °C; (g) AZT, Cu,
CuSO4ꢀ5H2O, H2O, t-BuOH, under N2.13,14

I. D. Bori et al. / Tetrahedron Letters 53 (2012) 1987–1989
1989
H
H
H
a
b
OH
H
OH
O
H
H
O
O
O
O
O
H
H
H
HO
HO
HO
O
O
H
H
H
O
O
betulinic acid
15
bevirimat (14)
O
N
CH3
HN
O
OH
O
c
H
N
N
O
H
N
O
O
H
HO
O
H
16
O
Scheme 2. AZT conjugation at C-28 position. Reagents and conditions: (a) 2,2-dimethylsuccinic anhydride, DMAP, DMF, 70 °C; (b) propargyl bromide, Cs2CO3, DMF/THF (1:1),
room temperature; (c) AZT, Cu, CuSO4ꢀ5H2O, H2O, t-BuOH, under N2.
Table 1
activity comparable to bevirimat and AZT. Mechanistic studies
are currently ongoing.
Anti-HIV data against HIV-1NL4-3 infected MT-4 cells
a
Compound
EC50
(lM)
CC50
(l
M)
SI
Acknowledgments
b
2
3
4
5
6
7
8
9
10
11
12
13
15
16
—
—
—
—
—
—
—
—
—
—
—
—
>4.6
—
—
—
—
—
—
—
—
>24
—
—
This investigation was supported by Grant AI-077417 from the
National Institute of Allergy and Infectious Diseases (NIAID)
awarded to K.-H.L. This study was also supported in part by the
Taiwan Department of Health Clinical Trial and Research Center
of Excellence (DOH100-TD-B-111-004). Efficient purification of
all the synthetic BA analogs was performed with a Grace RevelerisÒ
flash chromatography system equipped with RevealX(TM) detec-
tion allowing for multisignal (UV/ELSD) collection to low mg quan-
tities. RevelerisÒ Navigator method optimizer and Grace RevelerisÒ
flash silica cartridges were employed for high quality separations.
0.19
—
—
—
—
—
—
—
—
0.35
0.067
0.10
0.077
0.10
>4.6
15.3
11.2
13.2
140
>13
226
101
171
1385
Bevirimat (14)
AZT
References and notes
a
Selectivity index = CC50/EC50 (CC50, the concentration of test sample that was
1. HIV/AIDS strategy 2011, http://www.who.int/topics/hiv_aids/en/.
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H. J. Med. Chem. 1996, 39, 1016–1017.
cytotoxic to 50% of the mock-infected cells; EC50, the concentration of the test
sample that was effective to suppress HIV replication by 50%).
b
No activity.
4. Kanamoto, T.; Kashiwada, Y.; Kanbara, K.; Gotoh, K.; Yoshimori, M.; Goto, T.;
Sano, K.; Nakashima, H. Antimicrob. Agents Chemother. 2001, 45, 1225–1230.
5. Li, F.; Goila-Gaur, R.; Salzwedel, K.; Kilgore, N. R.; Reddick, M.; Matallana, C.;
Castillo, A.; Zoumplis, D.; Martin, D. E.; Orenstein, J. M.; Allaway, G. P.; Freed, E.
O.; Wild, C. T. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13555–13560.
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G. P.; Martin, D. E. Antimicrob. Agents Chemother. 2007, 51, 3574–3581.
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2007, 46, 589–598.
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C.; Ballow, C. Antimicrob. Agents Chemother. 2007, 51, 3063–3066.
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Natural Product Chemistry for Drug Discovery, for the Royal Society of Chemistry
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374–391.
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Takaishi, Y. Bioorg. Med. Chem. 2010, 18, 6451–6646.
11. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004–
2021.
12. Fioravanti, S.; Pellacani, L.; Tardella, P. A. Tetrahedron 2009, 65, 5747–5751.
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conjugate 16 also exhibited anti-HIV activity with an EC50 of
0.10
lM and a SI of 101. Thus, these two compounds and AZT
(EC50: 0.10
lM) showed comparable activity in our assay system.
As a pioneer compound, conjugate 16 demonstrates that a click
reaction can be used easily with a large natural product (e.g. BA),
which contains a terminal alkyne moiety, to form conjugates. The
potent anti-HIV activity of compounds 15 and 16 proves that the
incorporation of a C-28 side chain in bevirimat may result in
retained or even increased antiviral activity. We are currently
investigating the synthesis and antiviral evaluation of additional
AZT–bevirimat conjugates with different sizes/lengths of linkers
between the C-28 carboxylic acid and the triazole moiety formed
by click reaction with AZT.
In conclusion, conjugation via a triazole linkage offers a new
direction of modification of anti-HIV triterpenes. Click chemistry
provides an easy and productive way for linking two molecules,
even when one of them is a large natural product. Preliminary re-
sults indicated that AZT–betulin/BA conjugates with proper substi-
tutions at the C-3 and C-28 positions showed potent antiviral