Betulinic acid derivatives as human immunodeficiency virus type 2 (HIV-2) inhibitors

Article abstract of DOI:10.1021/jm9004253

Wepreviously reported that [[N-[3β-hydroxyllup-20(29)-en-28-oyl]-7- aminoheptyl]carbamoyl]methane (A43D, 4) was a potent HIV-1 entry inhibitor. However, 4 was inactive against HIV-2 virus, suggesting the structural requirements for targeting these two ret

Full text of DOI:10.1021/jm9004253

J. Med. Chem. 2009, 52, 7887–7891 7887
DOI: 10.1021/jm9004253
Betulinic Acid Derivatives as Human Immunodeficiency Virus Type 2 (HIV-2) Inhibitors
Zhao Dang,†,§ Weihong Lai,†,§ Keduo Qian,‡ Phong Ho,† Kuo-Hsiung Lee,‡ Chin-Ho Chen,*,† and Li Huang*,†
†Surgical Science, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, and ‡Natural Products Research
Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599. §These two authors contributed equally to
this manuscript.
Received April 2, 2009
We previously reported that [[N-[3β-hydroxyllup-20(29)-en-28-oyl]-7-aminoheptyl]carbamoyl]methane
(A43D, 4) was a potent HIV-1 entry inhibitor. However, 4 was inactive against HIV-2 virus, suggesting
the structural requirements for targeting these two retroviruses are different. In this study, a series of new
betulinic acid derivatives were synthesized, and some of them displayed selective anti-HIV-2 activity at
nanomolar concentrations. In comparison to compounds with anti-HIV-1 activity, a shorter C-28 side
chain is required for optimal anti-HIV-2 activity.
Introduction
Compounds 17 (IC9564)12 and 4 (A43D)10 are among the
most potent BA derivatives that inhibit HIV-1 entry by
targeting HIV-1 gp120.16,17 Chemical modifications of 1 at
C-3 resulted in compounds that inhibited HIV-1 maturation
as opposed to entry.13,18,19 The HIV-1 maturation inhibitor,
3-O-(30,30-dimethylsuccinyl)betulinic acid (bevirimat),20 is a
C-3 BA derivative currently under clinical trial for anti-HIV-1
therapy. Chemical modifications at C-3 and C-28 resulted in
bifunctional BA derivatives that inhibited HIV-1 entry and
maturation.9,10 [[N-[3β-O-30,30-Dimethylsuccinyllup-20(29)-
en-28-oyl]-7-aminoheptyl]carbamoyl]methane (A12-2) was
one of the most potent BA derivatives that inhibited HIV-1
replication at maturation and entry steps.10 Because of the
relatively low genetic homology between HIV-1 and HIV-2,
we hypothesized that the optimal pharmacophore of BA
derivatives required for inhibiting HIV-2 is different from
that for anti-HIV-1 activity. Since C-3 derivatives, such as 22
(bevirimat), did not inhibit HIV-2 replication in our assays
(Table 1), chemical synthesis in this study is focused on the
modification ofthe C-28 positionof 1. The results of thisstudy
support the hypothesis in that a shorter C-28 side chain of BA,
an equivalent of CONH(CH2)nR with n being 4 or 5, is needed
for optimal anti-HIV-2 activity.
HIV-2 is a retrovirus related to HIV-1, the causative agent
of AIDS. HIV-2 infections can also cause AIDS-like diseases,
but these usually progress more slowly than those caused by
HIV-1.1 While HIV-1 has spread to almost all of the countries
in the world, HIV-2 is mostly concentrated in countries of
West Africa.1,2 As the vaccine development against HIV-1
encounters tremendous challenges, progress in antiretroviral
therapy (ARTa) has been encouraging.3 However, almost all
drugs have been developed with a focus on their effectiveness
against HIV-1. Drug regimes for HIV-1 therapy have been
used for HIV-2 infections, but some of the drugs that were
potent against HIV-1 replication appeared to be less active or
inactive against HIV-2 virus. For example, HIV-2 is mostly
insensitive to non-nucleoside reverse transcriptase inhibitors
and the HIV-1 entry inhibitor Fuzeon.4,5 Some HIV-1 pro-
tease inhibitors have also beenshownto have reduced potency
against HIV-2.6 Given the fact that the genetic homology
between HIV-1 and HIV-2 is less than 50%,7 it is no surprise
that drugs currently used for HIV-2 are not as optimal as they
are for HIV-1.
Our previous work has been focused on the synthesis of
betulinic acid derivatives as potential anti-HIV-1 agents.8-10
Betulinic acid (BA, 1) is a pentacyclic triterpene found in
abundance in many plant species.11 Unmodified 1 was in-
active or exhibited very weak activity against HIV-1 replica-
tion. Chemical modification of 1 at C-28 and/or C-3 positions
resulted in potent anti-HIV-1 compounds. We and others
have synthesized C-28 modified BA or other pentacyclic
triterpene derivatives that inhibit HIV-1 entry.10,12-15
Results and Discussion
Chemistry. Compounds 4, 17, 7-11, and 1 are known
compounds that have been synthesized and reported for their
activities against HIV-1 virus.10,12 The C-28 modified BA
derivatives tested in this study were synthesized using meth-
ods previously described (Scheme 1). On the basis of their
C-28 side chain structures, four types of compounds were
synthesized. The first type of compounds included the ana-
logues 2-5. This type was synthesized by coupling diamino-
alkane [NH2(CH2)nNH2], where n was 5-8, with the C-28
carboxylic acid of 3-O-acetate-BA. The resultant C-28 termi-
nal amine intermediate was then acylated with acetic anhy-
dride following a step of alkaline hydrolysis of the C-3 ester
to form analogues 2-5. The amine intermediate 3a was
coupled with malonic acid in the presence of EDC and
The authors congratulate the Division of Medicinal Chemistry on its
100th anniversary.
*To whom correspondence should be addressed. For C.-H.C.: phone,
919-684-3819; fax, 919-684-3878; e-mail: chc@duke.edu. For L.H.:
phone, 919-684-2952; fax, 919-684-3878; e-mail: lihuang@duke.edu.
a Abbreviations: BA, betulinic acid; ART, antiretroviral therapy; EDC,
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride; SARs,
structure-activity-relationships; THF, tetrahydrofuran; DCM, dichloro-
methane; compds, compounds; AZT, 30-azido-30-deoxythymidine.
r
2009 American Chemical Society
Published on Web 06/15/2009
pubs.acs.org/jmc

7888 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Dang et al.
Table 1. Inhibitory Activity of BA and Its Derivatives against HIV-1 and HIV-2
IC50 (μM)a
TC50 (μM),a
cytotoxicity
compd
1 (BA)
X1
n
X2
R
HIV-1 NL4-3
HIV-2 KR.X3
OH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
inactive
inactive
inactive
inactive
>80
2
5
6
7
8
6
3
4
5
6
7
3
4
5
6
7
7
5
5
5
5
-NHCO-CH3
-NHCO-CH3
-NHCO-CH3
-NHCO-CH3
-NHCO-CH2COOH
-COOH
13.9 ( 3.3
48.1 ( 12.6
>66
3
4 (A43D)
0.027 ( 0.013
0.038 ( 0.011
0.14 ( 0.02
0.44 ( 0.02
inactive
inactive
inactive
5
inactive
inactive
>64
6
34.6 ( 13.8
31.2 ( 1.3
>72
7
2.8 ( 1.8
0.17 ( 0.14
0.15 ( 0.04
4.5 ( 4.3
inactive
8
-COOH
-COOH
32.8 ( 6.5
2.0 ( 0.6
11.1 ( 9.8
0.84 ( 0.52
inactive
9
10
>70
-COOH
-COOH
36.2 ( 1.2
34.2 ( 1.8
>67
11
12
-CO-Gly
-CO-Gly
3.7 ( 1.5
2.6 ( 2.8
0.35 ( 0.25
0.42 ( 0.33
0.64 ( 0.02
2.9 ( 1.3
0.37 ( 0.40
0.23 ( 0.10
0.63 ( 0.14
1.7 ( 0.9
inactive
13
14
inactive
34.7 ( 8.5
4.1 ( 2.1
0.12 ( 0.04
0.042 ( 0.013
48.9 ( 29.4
inactive
>65
>64
-CO-Gly
-CO-Gly
-CO-Gly
-CO-R0
-CO-Ala
15
16
>63
15.8 ( 4.1
33.2 ( 3.3
>63
17 (IC9564)
18
19
20
-CO-Tyr
-CO-Pro
-CO-Gly-Gly
54.6 ( 28.3
>60
>58
51.4 ( 29.7
>58
21
22 (bevirimat)
AZT
R = HOOC-C(CH3)2CH2CO-
0.13 ( 0.01
>30
>78
0.069 ( 0.01
a Data represent an average of three independent experiments. “Inactive” denotes compounds with TC50/IC50 < 2.
Scheme 1. Synthesis of Types 1-4 BA Derivativesa
a Reaction conditions: (i) oxalyl chloride; (ii) NH2(CH2)nNH2; (iii) acetic anhydride, Py; (iv) NaOH, THF, MeOH; (v) CH2(COOH)2, EDC, Et3N; (vi)
NH2(CH2)nCOOBn, Et3N; (vii) R-ester, EDC, Et3N.

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7889
Et3N to form the second type of compound (6) after the
saponification mediated by sodium hydroxide. The third
type of compound was synthesized by coupling the ω-
aminoalkanoates of varying lengths to the C-28 carboxylic
acid of 3-O-acetate-BA. Compounds 7-11 were obtained
after alkaline hydrolysis of esters at C-3 and C-28 side chains.
Further coupling of the C-28 terminal carboxylic acid of
7-11 with mono- or diamino acids furnished the fourth type
of compound, which included 12-16 and 18-21.
Results. These new BA derivatives, along with 1 and some
known BA derivatives, were tested for their anti-HIV-1 and
HIV-2 activity (Table 1). The unmodified 1 did not exhibit
anti-HIV-1 or anti-HIV-2 activity. For anti-HIV-1 activity,
4, 17, and the newly synthesized 3 were three of the most
potent inhibitors against HIV-1 NL4-3 replication. Com-
pound 3 was the most potent compound with its IC50 at
27 nM. Compounds with side chains less than six methylene
groups exhibited a significantly weaker anti-HIV-1 activity
as demonstrated by 2. Compound 5, with eight methylenes in
its C-28 side chain, was less potent than 4 against HIV-1
replication. In contrast to type 1 analogues, which have
acetamide termini at their C-28 side chains, 17 and type
2-4 compounds possessed carboxylic acid at the terminus of
their C-28 side chain. Among them, derivatives with C-28
methylene groups equal to 7 exhibited the most potent
anti-HIV-1 activity when compared to analogues with a
shorter linker in their respective compound types (Table 1).
Although 4 and 17 were two of the most potent anti-HIV-1
BA derivatives, 17 exhibited weak anti-HIV-2 activity (about
70 times less potent than its anti-HIV-1 activity) and 4 was
inactive against HIV-2 KR.X3 infection. The striking differ-
ence in their potencies against HIV-1 and HIV-2 suggested
that the optimal pharmacophore against HIV-2 is different
from that required for anti-HIV-1 activity. To obtain potent
anti-HIV-2 compounds, BA derivatives with a variety of C-
28 side chains were synthesized and tested for their anti-HIV-
2 activity. Similar to 4, the newly synthesized analogues 2, 3,
and 5 with acetamide termini were inactive against HIV-2
replication. This result suggested that a free terminal car-
boxylic acid at the C-28 side chain of BA might be needed for
HIV-2 inhibition as shown by 17. Therefore, type 1 com-
pounds were further modified by substituting the C-28
acetamide terminus with malonic acid. Except for compound
6, the resultant type 2 compounds were mostly unstable
because of decomposition of the terminal malonamide
R-NHCO-CH2-COOH into acetamide R-NHCO-CH3. How-
ever, 6 was less potent than 3 against HIV-1 and was inactive
against HIV-2 (Table 1). Thus, a free terminal carboxylic
acid at C-28 alone was not sufficient for anti-HIV-2 activity.
To systematically investigate the optimal length of the C-
28 side chain required for anti-HIV-2 activity, a series of BA
derivatives with C-28 side chains in the form of -NH
(CH2)nCOOH, where n ranged from 3 to 7, were synthesized.
As shown in Table 1, type 3 compounds 7-11 were either
inactive or displayed weak anti-HIV-1 activity with IC50 at
low micromolar concentrations. Most of these compounds
were more potent against HIV-2 than HIV-1 except 11,
which was inactive against HIV-2 replication. Within this
type, 8 and 9 exhibited the most potent anti-HIV-2 activity
with IC50 at 0.17 and 0.15 μM, respectively. These results
suggested that the optimal anti-HIV-2 activity was achieved
when n = 4 or 5 for the length of the C-28 side chain. In
contrast, the optimal anti-HIV-1 activity was achieved when
n=7 or 8 for the length of the C-28 side chain.10,12
Figure 1. Differential sensitivity of HIV-2 KR.X3 and HIV-2 KR.
X3-YU-2/V3 to 14. A dose-dependent inhibition of the two viruses
was determined in the presence of various concentrations of 14 as
indicated. Each data point represents the mean ( SD of four
independent experiments.
To explore the effect of additional terminal moieties at the
C-28 side chain on anti-HIV-2 activity, type 4 compounds
12-16 were synthesized by coupling a glycine to the car-
boxylic acid of 7-11. The addition of a glycine to C-28 side
chain of 7 did not significantly change the anti-HIV-2
activity as shown by 12. However, the same glycine addition
resulted in compounds 13 and 14 with reduced anti-HIV-2
activity (approximately 15- and 2-fold reduction) when
compared to their precursors 8 and 9, respectively. On the
other hand, 10-fold or more increases in anti-HIV-2 activities
were observed for compounds 15 and 16 when compared to
their precursors without the glycine addition. Overall, 14 and
15 were the two most potent anti-HIV-2 compounds with a
glycine terminus. However, 14 and 15 were 2- to 3-fold less
potent compared to the type 3 compounds 8 and 9. Among
the above potent anti-HIV-2 derivatives, 8 and 14 preferen-
tially inhibited HIV-2 with an IC50 of approximately 2 log10
lower than that against HIV-1 (Table 1).
Since both hexanoic-moiety-containing 9 and 14 exhibited
the most potent anti-HIV-2 activity from types 3 and 4
compounds, the hexanoic acid was again modified in an
attempt to further diversify the C-28 terminus. As a result,
additional type 4 compounds 18-21 were synthesized
with their C-28 side chains in the form of -NH(CH2)5CO-
aa, where aa was a mono- or diamino acid. Among the single
amino acid coupled hexanoic derivatives, 19, with a tyrosine
terminus, displayed the most potent anti-HIV-2 activity
with IC50 at 0.23 μM. Although all the monoamino acid
derivatives remained quite active against HIV-2 (IC50 ran-
ging from 0.23 to 0.63 μM), these compounds were less
potent than 9 (IC50=0.15 μM). For the BA derivatives with
a hexanoic moiety, their anti-HIV-2 activity based on their
terminus, was in the order of none > tyrosine > glycine =
alanine>proline. The dipeptide derivative 21 was approxi-
mately 10-fold less potent than 9.
We have previously shown that the BA derivatives 4 and
17 inhibit HIV-1 entry by targeting the V3 loop of HIV-1
gp120.17 To determine whether the V3 loop of gp120 is
responsible for HIV-2 inhibition, the sensitivities of HIV-2
KR.X3 and HIV-2 KR.X3-YU-2/V3 were determined in the
presence of 14. HIV-2 KR.X3-YU-2/V3 is a chimeric virus
containing the V3 loop of HIV-1 YU-2 instead of the original
V3 of HIV-2 KR.X3. Compound 14 was used for this study
because it was the most potent anti-HIV-2 BA derivative
available before 9 was synthesized. Compound 14 inhibited
HIV-2 KR.X3 in a dose-dependent manner with an IC50 of
0.35 μM (Figure 1). The HIV-2 chimeric virus with YU-2 V3

7890 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Dang et al.
loop, HIV-2 KR.X3-YU-2/V3, was approximately 5-fold
less sensitive to 14 when compared to HIV-2 KR.X3. Since
the only difference between the two viruses was the V3 loop,
the results suggested that the V3 loop of HIV-2 was a possible
target of 14.
Procedure for Synthesizing Type 1 Compounds 2-5. To a
stirring solution of 3-O-Ac-BA (0.3-0.7 mmol) in dichloro-
methane (DCM, 2 mL) was added oxalyl chloride (7-12 equiv).
After the mixture was stirred for 10 min, the organic solvent
was removed under vacuum. The residue was dissolved in DCM
and to it was added the corresponding 1,ω-diaminoalkane
(4-5 equiv) in DCM. The mixture was stirred overnight and
then concentrated. The residue was washed with water and
dissolved in ethanol. After filtration, the ethanolic solution
was concentrated and the residue was chromatographed on
Si gel to yield the corresponding amine intermediates 2a-5a.
To the corresponding amine intermediates 2a-5a in pyridine
(anhydrous, 1 mL), acetic anhydride (0.5 mL) was added and
stirred at room temperature overnight. The mixture was con-
centrated and redissolved in DCM. After the mixture was
washed with 1 N HCl, water, and brine, the organic layer was
concentrated and chromatographed on Si gel to yield 2b-5b.
Procedure for Saponification of Ester at C-3 or/and C-28.
Esters were hydrolyzed in a mixture of MeOH/THF/4 N NaOH
aq (2:2:1). The intermediates 2b-5b were dissolved in organic
solution (1-2 mL), and aqueous NaOH solution (0.5-1 mL)
was added. After being stirred overnight, the reaction mixture
was neutralized with aqueous 1 N HCl. The resulting precipitate
was washed with water and dried in vacuum to yield the final
compounds 2-5.
Conclusion
In summary, several important SARs for anti-HIV-2 acti-
vity of BA derivatives were observed in this study. First, for
anti-HIV-2 activity, the optimal length of the C-28 side chain
is achieved when n=5 in the form of -NH(CH2)nCOR, as
seen for 9 and 14 (Table 1). A gradual drop of anti-HIV-2
activity was observed when n is greater or smaller than 5.
Second, in contrast to the anti-HIV-1 BA derivatives, a
carboxylic acid terminus of C-28 side chain is important for
anti-HIV-2 activity. The potent anti-HIV-1derivative3, along
with analogues with various lengths of C-28 side chains that
did not possess a carboxylic acid terminus, was completely
inactive against HIV-2. Third, the addition of one amino acid
to the carboxylic terminus of C-28 side chain moderately
compromised the anti-HIV-2 activity of the BA derivatives,
especially when the C-28 side chain was at the optimal length
with n=5. On the other hand, the addition of one amino acid
increased anti-HIV-2 activity when n was 6 or 7. These results
provide clues for further characterization of ligand-target
interaction and synthesis of a next generation of anti-HIV-2
BA derivatives. In addition to the novel anti-HIV-2 BA
derivatives, this study also synthesized a potent HIV-1 entry
inhibitor, 3, with improved anti-HIV-1 activity (IC50=0.027 μM)
when compared to the previously synthesized most potent BA
derivative 4 with an IC50 of 0.038 μM.
Procedure for Synthesizing Type 2 Compound 6. To the
mixture of the above amine intermediate 3a (0.2 mmol) in
DCM (2 mL) were added malonic acid (10 equiv), EDC (2 equiv),
and Et3N (10 equiv). After being stirred at room temperature
overnight, the mixture was concentrated, redissolved in DCM,
washed with water, brine, and dried over Na2SO4. The organic
layer was concentrated and chromatographed on Si gel to yield
the intermediate 6a. After the saponification procedure as de-
scribed above, 6 was obtained as a solid.
Procedure for Synthesizing Type 3 Compounds 7-11. To a
stirring solution of 3-O-Ac-BA (0.1-0.6 mmol) in DCM (4 mL)
was added oxalyl chloride (10 equiv). After the mixture was
stirred for 10 min, the organic solvent was removed under
vacuum. The residue was dissolved in DCM, then reacted with
the corresponding ω-aminoalkanoate (1.2-1.5 equiv) in DCM
and Et3N (6 equiv) overnight. The reaction mixture was diluted
with DCM before being washed with water, brine and then dried
over Na2SO4. After concentration in vacuum, the residue was
chromatographed on Si gel to yield the corresponding 7a-11a,
which yielded the corresponding 7-11 after saponification.
Procedure for Synthesizing Type 4 Compounds 12-16. To a
solution of the corresponding intermediates 7a-11a (0.2mmol)in
DCM (2 mL) were added glycine methyl ester hydrochloride
(2.7 equiv), Et3N (7.5 equiv), and EDC (3.0 equiv). After being
stirred overnight at room temperature, the mixture was diluted
with DCM, washed with water, brine, and dried over Na2SO4.
The organic layer was concentrated under vacuum, and the
residue was chromatographed on Si gel. The pure intermediate
was collected and subjected to saponification as described
above to furnish the corresponding 12-16.
Procedure for Synthesizing Type 4 Compounds 18-21. The
synthesis of these compounds was achieved by the same proce-
dure as described above for 12-16, using intermediate 9
(for synthesis of 18-20) or 14 (for synthesis of 21) and the
corresponding amino acid methyl ester hydrochloride.
HIV-1 and HIV-2 Virus Infection Assay. Inhibition of
HIV-1NL4-3, HIV-2 KR.X3, or HIV-2 KR.X3-YU2/V3 infec-
tion was measured through reduction in luciferase gene expres-
sion after a single round of virus infection of TZM-bl cells, as
previously described.17 HIV-2 KR.X3 and HIV-2 KR.X3-YU2/V3
were generously provided by Dr. George Shaw, University of
Alabama. For the antiviral assays, 200 TCID50 of virus was used
to infect TZM-bl cells in the presence of various concentrations
of compounds. Two days after infection, the culture medium
was removed from each well and 100 μL of Bright Glo reagent
When comparing anti-HIV-1 and anti-HIV-2 activities,
most of the very potent HIV-1 inhibitors, such as 4 and its
analogues, appeared to have poor or no activity against HIV-
2, and vice versa, for mostactiveanti-HIV-2 compounds, such
as 8, 14, 18-20. These observations support our hypothesis
that the optimal pharmacophores required to target HIV-1
and HIV-2 are different. In terms of mechanism of action, 14
appeared to block HIV-2 replication by targeting the V3 loop
of gp120. The homology between the V3 loops of HIV-1
(YU-2) and HIV-2 (KR) is approximately 30%.21 It is likely
that the drug binding pocket in HIV-1 V3 loop is structurally
and conformationally different from that in HIV-2 V3 loop.
Experimental Section
General Experimental Procedures. Positive or negative HR-
FABMS data were recorded on a Shimadzu LCMS-IT-TOF or a
Joel SX-102 mass spectrometer. 1H and other NMR spectra were
measured on a Varian Mercury 300 or 500 spectrometer. Samples
were dissolved in CDCl3 or pyridine-d5 with TMS as an internal
standard. Silica gel chromatography was carried out on a Biotage
Horizon flash chromatograph with prepacked Si gel column. The
purity of BA derivatives was analyzed by using a Varian ProStar
HPLC system with a PDA detector and Agilent Zorbax ODS or
C-8 column. The mobile phase was composed of solution A (5%
acetonitrile in water with 0.045% trifluoroacetic acid) and solu-
tion B (water/methanol/acetonitrile = 5:10:85 with 0.045% tri-
fluoroacetic acid). A linear gradient of 80% to 100% of solution B
with a flow rate at 1 mL/min or with flow rate at 4 mL/min was
used to elute the compounds. The compounds were analyzed with
the UV absorption displayed at 220 nm and recorded at a range
from 200 to 250 nm. All the tested compounds have purity of 95%
or above except for the inactive unmodified 1 (BA), which was
purchased from Sigma-Aldrich with 90% purity.

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7891
(Promega, Luis Obispo, CA) was added to the cells for measure-
ment of luminescence using a Victor 3 luminometer. The 50%
inhibitory concentration (IC50) was defined as the concentration
that caused a 50% reduction of luciferase activity (relative light
units) compared to virus control wells.
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Cytotoxicity Assay. A CytoTox-Glo cytotoxicity assay (Pro-
mega) was used to determine the cytotoxicity of the synthesized
BA derivatives. TZM-bl cells were cultured in the presence of
various concentrations of the compounds for 2 days. Percent of
viable cells was determined by following the protocol provided
by the manufacturer. The 50% cytotoxic concentration (TC50)
was defined as the concentration that caused a 50% reduction of
cell viability.
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Acknowledgment. This work was supported by the Na-
tional Institute of Allergy and Infectious Diseases (NIAID)
Grant AI-65310 awarded to C.-H.C., National Institutes of
Drug Abuse (NIDA)Grant DA-024589 awarded to L.H., and
in part by Grant AI-077417 from NIAID awarded to K.-H.L.
The authors are grateful to Dr. George Dubay of the Depart-
ment of Chemistry and Dr. Anthony Ribeiro of the NMR
Spectroscopy Center of Duke University for their assistance
on mass and NMR spectroscopy data collection. We also
thank Dominique Soroka for her help on preparation of this
manuscript.
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Supporting Information Available: Spectroscopic and HPLC
analytical data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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