Synthesis and anti-HIV activity of bi-functional betulinic acid derivatives

Article abstract of DOI:10.1016/j.bmc.2005.11.016

Betulinic acid (BA) derivatives with a side chain at C-3 can inhibit HIV-1 maturation. On the other hand, BA derivatives with a side chain at C-28 can block HIV-1 entry. In order to combine the anti-maturation and anti-entry activities in a single molecul

Full text of DOI:10.1016/j.bmc.2005.11.016

Bioorganic & Medicinal Chemistry 14 (2006) 2279–2289
Synthesis and anti-HIV activity of bi-functional betulinic
acid derivatives
Li Huang,a Phong Ho,a Kuo-Hsiung Leeb and Chin-Ho Chena,*
aDepartment of Surgery, Duke University Medical Center, Durham, NC 27710, USA
bNatural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
Received 21 September 2005; revised 3 November 2005; accepted 4 November 2005
Available online 28 November 2005
Abstract—Betulinic acid (BA) derivatives with a side chain at C-3 can inhibit HIV-1 maturation. On the other hand, BA deriv-
atives with a side chain at C-28 can block HIV-1 entry. In order to combine the anti-maturation and anti-entry activities in a
single molecule, new bi-functional BA derivatives containing side chains at C-3 and C-28 have been synthesized. The most potent
compound ([[N-[3b-O-(30,30-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-7-aminoheptyl]-carbamoyl]methane) inhibited HIV-1 at an
EC50 of 0.0026 lM and was at least 20 times more potent than either the anti-maturation lead compound DSB or the anti-entry
lead compound IC9564. This bi-functional BA derivative was active against both HIV entry and maturation. These results sug-
gest that bi-functional BA derivatives with dual mechanisms of action have the potential to become clinically useful for AIDS
therapy.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
molecular mechanisms of action for both classes of
BA derivatives were quite unique in comparison with
currently known anti-HIV drugs that target HIV reverse
transcriptase or protease.5,9–11 For example, IC9564 was
reported to inhibit HIV-1 envelope-mediated membrane
fusion by targeting the HIV-1 envelope glycopro-
teins.9,12–14 On the other hand, DSB was found to inter-
fere with HIV-1 CA/SP1 processing. During HIV-1
maturation, the viral protease cleaves specific sites in
p55 Gag polyprotein to form the structural proteins
MA, CA (p24), NC, and p6 as well as the Gag spacer
peptides SP1 (p2) and SP2.15 DSB partially blocked
the cleavage of CA/SP1, which resulted in the produc-
tion of non-infectious HIV-1 particles.10,11,16 Thus,
DSB and IC9564 represent two distinct classes of BA
derivatives with different anti-HIV profiles.
Current clinical AIDS treatment relies on a multiple-
drug combination regime called highly active antiretro-
viral therapy (HAART), which uses drugs targeting at
least two viral proteins.1,2 However, this existing drug
therapy encounters problems such as the emergence of
drug resistant viruses and toxicities caused by long-term
drug usage.3,4 Therefore, it is important to continually
search for new drugs with novel mechanisms of action
that can be added to the current anti-HIV therapy.
Betulinic acid (BA) derivatives have been studied for
their potential as anti-HIV agents. Two classes of chem-
ically modified BA derivatives are reported to inhibit
HIV replication at nanomolar concentrations. Class I
compounds, such as DSB (also designated as PA-457),
possess side-chain modification at the C-3 position of
BA.5,6 Class II compounds, such as IC9564, possess
side-chain modification at the C-28 position.6–9
Although both classes of BA derivatives shared the same
betulinic acid core, they exhibit very different modes of
anti-HIV action. Previous studies suggested that the
Previous studies on anti-HIV BA derivatives suggested
that the C-3 side chain is the pharmacophore for anti-
maturation activity, while the C-28 side chain is the
pharmacophore for anti-entry activity.5–8 Structurally,
the C-3 and C-28 pharmacophores of these two com-
pound classes are located at the opposite ends of the
BA pentacyclic ring system. Therefore, a design that
incorporates both pharmacophores into one BA mole-
cule, such as 14, is chemically feasible. As proof of prin-
ciple, we previously synthesized two bi-functional BA
Keywords: HIV-1; Betulinic acid; Entry; Maturation.
*
Corresponding author. Tel.: +1 919 684 3819; fax: +1 919 684
3878; e-mail: chc@duke.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.016

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L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
H
H
O
O
28
H
O
H
OH
NH-(CH2)7-C-NH
COOH
H
O
3
H
OH
O
HO
H
H
HOOC
IC9564
DSB
anti-HIV fusion
H
anti-HIV maturation
O
O
H
NH(CH2)7-C-NH
O
COOH
H
OH
O
HOOC
H
Bifunctional compound (14)
derivatives, LH15 (4) and LH55 (8), which exhibited
improved anti-HIV-1 activity when compared to the
mono-functional BA derivatives with a side chain either
at the C-3 or the C-28 position.17 The goal of this study
was to develop new bi-functional BA derivatives that
can potently inhibit HIV-1 replication by targeting both
viral entry and maturation.
respectively, with n ranging from 5 to 10. Compound
11 has a bulky bicyclic aromatic moiety. All synthetic
bi-functional compounds contain two terminal carbox-
ylic acids, one in each of the C-3 and C-28 side chains,
except for compounds 11 and 16–20, which have a
terminal carboxylic acid in the C-3 but not the C-28
side chain.
As previously shown by Hashimoto et al., 30,30-di-
methylsuccinyl betulinic acid (DSB) is the most potent
class I anti-HIV maturation BA derivative.5 Other ana-
logs with different C-3 side chains, including an isomer
of DSB, 20,20-dimethylsuccinyl BA, showed a signifi-
cantly lower anti-HIV activity. In contrast, IC9564, a
class II anti-HIV entry BA derivative, has a much larger
side chain (–CONH–(CH2)7-statine) attached to the C-
28 position, which seems able to tolerate some changes
without a drastic reduction in the anti-entry activity.7,8
Therefore, we synthesized a series of new bi-functional
BA derivatives possessing a 30,30-dimethylsuccinyl (or
20,20-dimethylsuccinyl) at C-3 and a variety of amide
side chains at C-28. The range of length and bulkiness
in the C-28 side chain was designed to generate mole-
cules with optimal interactions with the putative viral
binding targets.
2. Chemistry
Schemes 1–4 show the four general routes for synthesiz-
ing bi-functional BA derivatives 4–13 and 15–20.
Synthesis of these compounds began with modification
at the C-28 position of either 3-OAc-BA or BA and
ended with modification at the C-3 position. For the
preparation of 4–10, 3-OAc-BA was treated with oxalyl
chloride, and the resulting acyl chloride was readily
coupled with the appropriate amine. Saponification with
4 N NaOH in THF/MeOH yielded the corresponding
C-28 derivatized BA intermediates 4a, 6a, 8a, and 10a.
These intermediates were esterified at C-3 with 2,2-di-
methylsuccinic anhydride, in the presence of dimethyl-
aminopyridine (DMAP) and pyridine under reflux, to
furnish the target bi-functional compounds 4–10
(Scheme 1).
Among the synthetic bi-functional compounds, 4–7
and 12 have relatively small C-28 side chains, such as
leucine or glycinyl-leucine (Table 1). Conversely,
compounds 8–10, 13–15, and 16–20 have a long C-28
side chain of –CONH–(CH2)n–COOH, –CONH–
(CH2)n–COR (R = leucine, statine or 3-aminobenzoic
acid, respectively), and –CONH–(CH2)n–NH–COCH3,
Unprotected BA was used as the starting material for
the synthesis of 11 and 12 (Scheme 2). In this route, mild
conditions were used for the initial amide coupling, so
that protection of the C-3 hydroxyl group as the acetate
was not needed. After the initial coupling to assemble
the C-28 side-chain, 11 was obtained by subsequent
H
H
O
O
H
H
HN
NH-(CH2)10-COOH
COOH
O
O
H
H
O
O
HOOC
HOOC
H
H
8
4

L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
Table 1. The effect of BA derivatives against HIV-NL4-3 in MT4 cellsa
2281
H
O
28
H
R2
3
H
R1O
Betulinic acid: R1 = H and R2 = OH
H
Compound
R1
H
R2
EC50 (lM)b
IC50 (lM)b
>10
O
1 (IC9564)
0.053
NH(CH2)7-C-NH
COOH
OH
O
2 (DSB)
OH
0.075
0.45
7.5
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
HOOC
O
3
4
OH
8
10
12
8
O
L-Leucine
0.015
0.008
0.02
O
5
L-Leucine
O
6
D-Leucine
O
7
D-Leucine
0.01
9
O
8
–NH–(CH2)10–COOH
–NH–(CH2)10–COOH
–NH–(CH2)7–COOH
0.012
0.035
0.077
0.035
0.016
0.087
0.096
0.007
0.012
0.007
6
O
9
8
O
10
11
12
13
14
15
16
17
>10
N
N
O
O
2.5
N
O
O
O
O
O
O
O
>10
>10
>10
COOH
NHCH2-C-NH
O
NH(CH2)7-C-NH COOH
O
NH(CH2)7-C-NH
COOH
OH
O
9.5
NH(CH2)7-C-NH
COOH
–NH–(CH2)5–NH–COCH3
–NH–(CH2)6–NH–COCH3
9
7.5
(continued on next page)

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L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
Table 1 (continued)
Compound
R1
R2
EC50 (lM)b
IC50 (lM)b
18 (A12-2)
–NH–(CH2)7–NH–COCH3
0.0026
0.0036
0.012
8
O
O
HOOC
19
–NH–(CH2)8–NH–COCH3
–NH–(CH2)9–NH–COCH3
–NH–(CH2)7–NH–COCH3
7
7.4
HOOC
O
20
HOOC
18a (A43-D)
H
0.047
>10
Anti-HIV activity of Bi-functional BA derivatives.
a See anti-HIV assay in Section 5 for experimental procedures.
b EC50 is the concentration that inhibits HIV-1 replication by 50%; IC50 is the drug concentration that resulted in a 50% reduction of viable cells in a
4-day assay. Data represent an average of at least two experiments.
H
H
H
O
O
iv
O
i, ii
iii
H
H
H
R2
R'
OH
H
H
H
HO
R1O
AcO
H
H
H
O
3-O-Ac-BA
4a. R' = NH COOH
4. R1 =HOOC
5. R1 = HOOC
6. R1 = HOOC
R2 = NH
COOH
O
NH
6a. R' =
COOH
R2 =
COOH
COOH
NH
8a. R' = -NH(CH2)10-COOH
10a. R' = -NH(CH2)7COOH
O
O
NH
R2 =
7. R1 =HOOC
8. R1 = HOOC
R2 = NH
COOH
O
O
R2 = -NH(CH2)10-COOH
R2 = -NH(CH2)10COOH
R2 = -NH(CH2)7COOH
9. R1 = HOOC
O
10.R1 =
HOOC
Scheme 1. Reagents and conditions: (i) (CO)2Cl2/CH2Cl2, rt, 10 min; (ii) H2N–R0–COOMe/HOBT/TEA (or DIEA)/CH2Cl2, or H2N–R0–COOMe/
HOBT/CH2Cl2, rt, overnight; (iii) NaOH 4 N/THF/MeOH, rt, overnight; (iv) 2,2-dimethylsuccinic anhydride/Py/DMAP, reflux, overnight.
H
ii, iii
vi, iii
BA
v, iv
O
H
O
NH
NH COOH
H
HO
O
iv
H
12a
H
H
H
O
H
O
O
N
NH
H
N
COOH
NH
O
H
N
O
RO
O
H
HOOC
H
11. R =
12
HOOC
Scheme 2. Reagents and conditions: (ii) H2N–R0–COOMe/HOBT/TEA (or DIEA)/CH2Cl2, or H2N–R0–COOMe/HOBT/CH2Cl2, rt, overnight; (iii)
NaOH 4 N/THF/MeOH, rt, overnight; (iv) 2,2-dimethylsuccinic anhydride/Py/DMAP, reflux, overnight; (v) PyBop/HOBT/DIEA/NH4Cl/DMF, rt,
1 h; (vi) H2N–CH2–COOEt/DMAP/CH2Cl2, rt, overnight.
C-3 esterification with 2,2-dimethylsuccinic anhydride.
The complete assembly of the C-28 side chain of 12
required two steps of coupling, each followed by a step
of saponification to furnish the intermediate 12a. The
final C-3 esterification as described above completed
the synthesis of 12.
To synthesize 13 and 15 (Scheme 3), the intermediate
10a was coupled with leucine or p-aminobenzoic acid,
catalyzed by PyBop/HOBT/DIEA, to form the corre-
sponding amide. Subsequent saponification furnished
the intermediates 13a and 15a, which were finally
modified at C-3 to give 13 and 15, respectively.

L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
2283
H
H
H
O
iv
ii, iii
H
O
O
H
H
R
R
NH-(CH2)7-COOH
H
O
H
H
O
HO
HO
HOOC
H
O
H
H
O
C
13. R =
10a
1 3a. R =
NH(CH2
)
C
O
NH
NH
COOH
COOH
NH(CH2
)
NH
NH
COOH
COOH
7
7
O
15. R = NH(CH2)7
C
15a. R =
NH(CH2)7
C
Scheme 3. Reagents and conditions: (ii) H2N–R0–COOMe/HOBT/TEA (or DIEA)/CH2Cl2, or H2N–R0–COOMe/HOBT/CH2Cl2, rt, overnight;
(iii) NaOH 4 N/THF/MeOH, rt, overnight; (iv) 2,2-dimethylsuccinic anhydride/Py/DMAP, reflux, overnight.
H
H
H
viii, iii
i, vii
iv
O
O
O
H
O
H
O
3-O-Ac-BA
H
CH3
NH(CH2)n-NH
NH(CH2)n-NH CH3
NH(CH2 )nNH2
O
H
H
H
O
HO
AcO
HOOC
H
H
H
16a - 20a (n = 5 ~ 9)
16b - 20b (n = 5 ~ 9)
16 - 20 (n = 5 ~ 9)
Scheme 4. Reagents and conditions: (i) (CO)2Cl2/CH2Cl2, rt, 10 min; (iii) NaOH 4 N/THF/MeOH, rt, overnight; (iv) 2,2-dimethylsuccinic anhydride/
Py/DMAP, reflux, overnight; (vii) H2N–(CH2)n–NH2/CH2Cl2, n=5–9; (viii) Ac2O/DMAP/Py, rt, overnight.
Scheme 4 shows the synthetic steps for the preparation
of 16–20. The protected 3-OAc-BA was subjected to
two successive coupling steps for the C-28 side chain
assembly. The acyl chloride of 3-OAc-BA was first
treated with excess alkyldiamine to form 16a–20a, which
were further coupled with acetic anhydride in the pres-
ence of DMAP and pyridine. Subsequent saponification
resulted in 16b–20b, which were finally esterified at C-3
to form the corresponding bi-functional compounds
16–20, respectively.
and 2,2-dimethylsuccinic anhydride in the presence of
DMAP. This reaction produced two isomers: 20,20-
dimethyl and 30,30-dimethylsuccinyl BA derivatives. Un-
der our synthetic conditions, the 30,30-dimethylsuccinyl
isomer is the dominant product, especially for com-
pounds with long C-28 side chains. Accordingly, with
12–20, only the 30,30-dimethylsuccinyl BA derivatives
was obtained. However, for 4a and 6a, which have rela-
tively short C-28 side chains, approximately equal
amount of both isomers (4/5 and 6/7, respectively) were
obtained. Compound 8a with C-28 undecanoic acid side
chain also afforded two isomers (8 and 9) upon esterifi-
cation, with 8 as the major and 9 as the minor products.
Compound 11, which has a C-28 aromatic bicyclic ring
system, was the only compound that formed predomi-
nantly the 20,20-dimethylsuccinyl BA derivative. These
results suggest that the C-3 acylation of BA derivatives
was sterically influenced by its C-28 moiety. The two
isomers formed after esterification were separated by re-
verse-phase HPLC. Extensive NMR experiments includ-
ing HMQC and HMBC were performed on compound 7
to establish its structure as the 20,20-dimethyl isomer.
The distinct proton signal patterns of the methylene
To synthesize compound 14, extra protection/de-protec-
tion steps were needed to preserve the statine hydroxyl
group (Scheme 5). The MEM ether was chosen as it
was stable during the following steps of saponification
and C-3 acylation. However, the cleavage of the MEM
ether did not occur as readily as anticipated. No reac-
tion occurred after ZnBr2 treatment in DCM for over
2 days; however, cleavage of the MEM ether was finally
successful by using B-chlorocatecholborane.18
The final esterification of the C-3 hydroxyl involves
reflux of a pyridine mixture of the BA intermediate
1. MEM chloride/DIEA/CH2Cl2, rt, overnight
2. NaOH 4N/THF/MeOH, rt, overnight
H
H
O
O
H
H
O
COOH
OMEM
NH(CH2)7CONH
NH(CH2)7CONH
H
H
O
OH
H
AcO
HO
H
H
14b MEM = CH2OCH2CH2OCH3
14a
1. 2,2-dimethyl succinic anhydride/Py/DMAP, reflux, overnight
2. B-chlorocatecholborane, CH2Cl2, rt, overnight
O
H
NH(CH2)7CONH
COOH
O
H
OH
O
HOOC
H
14
Scheme 5.

2284
L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
group, derived from 20,20-dimethyl (in 7) or 30,30-dim-
ethylsuccinyl (in 6), were used as the basis to determine
the structures of all remaining isomers.5
13–15 in the order and identity of the terminal amide
group (–CONHR0–COOH for 13–15; –NHCOCH3 for
16–20). By using a variety of diaminoalkanes with n
ranging from 5 to 9, BA derivatives with various chain
lengths attached to C-28 were obtained. Compounds
18 and 19 with n equal to 7 and 8, respectively, are the
most active compounds obtained thus far in our study
(EC50 = 0.0026 and 0.0036 lM, respectively).
3. Results and discussion
3.1. Anti-HIV activity of bi-functional BA derivatives
As shown in Table 1, all new bi-functional compounds
exhibited anti-HIV activity. Compounds 4–7, which
contain a C-28 leucine, showed EC50 values of around
0.02 lM. The leucine stereochemistry, either D or L,
did not affect the anti-HIV potency. Bi-functional BA
3.2. Anti-fusion activity of bi-functional BA derivatives
To determine if the bi-functional BA derivatives indeed
inhibit both HIV-1 entry and maturation activities, the
most potent bi-functional BA derivative (18) was chosen
for mechanistic studies. A luciferase-based fusion assay
was used to determine the anti-HIV-1 entry activity of
18. TZM cells that express luciferase upon fusion with
envelope-expressing COS cells were used as fusion part-
ners. A Promega luciferase assay kit was used to quan-
tify luciferase activity in the fused cells using a Biotek
luminometer. In the absence of inhibitors, HIV-1 enve-
lope-mediated fusion of a sample in 96-well plates yield-
ed an average of 70,000 relative luminescence units
(control RLU). The drug concentration that reduced
the control RLU by 50% is defined as 50% effective con-
centration (EC50). The mono-functional class I BA
derivative, DSB, did not exhibit significant anti-fusion
activity (Fig. 1). On the other hand, the mono-function-
al class II BA derivative 18a (A43-D in Fig. 1) is a
potent anti-fusion inhibitor (EC50 0.080 lM). The bi-
functional analog 18 (A12-2 in Fig. 1) was also active
(EC50 0.22 lM) in the anti-fusion assay, although not
as potent as 18a (Fig. 1). Since 18a and 18 share the
same anti-fusion C-28 functional side chain, the results
suggest that the side chain at C-3 has a negative impact
on the anti-fusion activity of 18.
derivatives
5
and 7, which have 3-O-20,20-dim-
ethylsuccinyl substitution, were approximately twice
as potent as their 3-O-30,30-dimethylsuccinyl isomers,
4 and 6. However, this rank order of activity was not
uniform among all derivatives. For example, with the
two C-28 aminoundecanoic acid isomers, compound 8
with a 3-O-30,30-dimethylsuccinyl side chain was 3-fold
more potent than its 3-O-20,20-dimethylsuccinyl isomer,
9. This latter result corresponds to the prior and cur-
rent results with the mono-functional BA derivatives.
For example, DSB (2) contains a 3-O-30,30-dim-
ethylsuccinyl side chain, is clearly much more potent
than its 3-O-20,20-dimethylsuccinyl isomer (3). These
data suggested that the C-28 side chain might affect
the interaction between the C-3 pharmacophore and
its target.
Compound 12 with C-3 30,30-dimethylsuccinyl and C-28
glycinyl-leucine side chains showed similar potency to
the leucine compounds 4–7. Compound 11, which con-
tains a C-3 20,20-dimethylsuccinyl and a fused ring sys-
tem at C-28, was less active and more toxic to the cells
when compared to the bi-functional BA derivatives with
short side chains at C-28 such as 4–7.
3.3. The anti-maturation activity of bi-functional BA
derivatives
Bi-functional BA derivatives 13–15 all have a 30,30-di-
methylsuccinyl moiety at C-3 and a large C-28 amido
side chain containing an aminooctanoyl moiety
terminated with an amino acid [–CONH–(CH2)7–
CONH–R, R = leucine, statine, or 3-aminobenzoic acid,
respectively]. Although DSB and IC9564 are among the
most potent mono-functional class I and II BA deriva-
tives, respectively, bi-functional compound 14, which
has an identical side chain to that of IC9564 at C-28
and to that of DSB at C-3, exhibited a weaker anti-
HIV activity than either mono-functional compound.
The anti-HIV potency of 13 was about the same as 14.
On the other hand, 15, the benzoic acid bi-functional
derivative, was significantly more potent than the statine
analog 14, the leucine analog 13, and both DSB and
IC9564. These results suggested that the structure-activ-
ity relationships of the bi-functional BA derivatives are
different from those reported for mono-functional BA
derivatives.
Partial inhibition of p25 processing is believed to be
responsible for the anti-maturation activity of
DSB.10,11 Thus, the anti-maturation activity of BA
derivatives can be estimated by detecting p25 using Wes-
tern blot analysis. Unlike the viral replication assay,
detection of p25 accumulation allowed us to specifically
120
100
80
60
A43-D
A12-2
DSB
40
20
0
0.001
0.01
0.1
1
10
[Compounds] µM
Figure 1. BA derivatives inhibited HIV-1 envelope-mediated mem-
brane fusion. The fusion assay performed in the absence of drugs is
defined as 100% control fusion. A43-D is compound 18a and A12-2 is
compound 18 described in the text. Each data point represents the
mean SD of two duplicated experiments.
Compounds 16–20 were also obtained as 3-O-30,30-di-
methylsuccinyl BA derivatives. The C-28 side chain of
16–20 is N-acetyl diamino alkane [–CONH–(CH2)n–
NHCOCH3]. Thus, their structures vary from those of

L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
2285
chain can negatively affect the anti-fusion activity exert-
ed by the C-28 pharmacophore. However, even though
these two pharmacophores might interfere with each
other, the overall potency of the bi-functional BA deriv-
atives 18 was at least a log higher than the mono-func-
tional 18a or DSB in the HIV-1 replication assay
(Table 1). Given the evidence that 18 was active in both
HIV fusion and maturation assays, this improved anti-
viral potency is likely due to synergism between these
two modes of action.
p25
p24
Figure 2. BA derivatives interfere with HIV-1 p25 processing. The
partially processed p25 and matured p24 were detected with a rabbit
anti-p24 antiserum on a Western blot. A12-2 is compound 18 described
in the text.
5. Experimental
5.1. General experiment procedures
All melting points were determined with a Fisher–Johns
melting point apparatus without correction. Positive
and negative HR-FABMS were recorded on a Joel
evaluate the anti-maturation activity of BA derivatives.
DSB at 5 lM was used as a marker for 100% inhibition,
since DSB completely inhibited HIV NL4-3 replication
at this concentration. BA was used as a negative control,
since it did not inhibit NL4-3 replication. The results
indicated that the bi-functional BA derivative 18 (A12-
2 in Fig. 2) is comparable to DSB in inhibiting the pro-
cessing of p25 (Fig. 2).
1
SX-102 spectrometer. H and other NMR spectra were
measured on a Varian Mercury 300 or Varian Inova
500 or Bruker DRX-400 spectrometer. Other than as
noted, all samples were dissolved in CDCl3 with TMS
as internal standard. Si gel chromatography was carried
out on a Biotage Horizon Flash chromatograph system
with prepacked Si gel column. HPLC was performed on
a Varian ProStar solvent delivery and PDA detector
with Agilent Zorbax ODS or C-8 columns
(4.6 mm · 25 cm and 9.4 mm · 25 cm for analytical
and semipreparative scales, respectively).
4. Conclusions
In this paper, we described a straightforward approach
to obtain bi-functional BA derivatives by combining
two pharmacophores from two classes of anti-HIV BA
agents, each with its own unique mode of action, into
one molecule. Two derivatives, 18 and 19, were found
to be at least 10- to 20-fold more potent than the leading
anti-maturation mono-functional BA derivative DSB or
the anti-entry mono-functional BA derivative IC9564.
Further mechanistic study also proved that 18 was
active in both anti-HIV entry and anti-HIV maturation
assays. Both 18 and 19 contain the C-3 pharmacophore
of DSB and a new C-28 pharmacophore –CONH–
(CH2)nNH–COCH3. Based on the anti-HIV potency
of 16–20, n is optimally equal to 7. Prior reports suggest-
ed that a terminal carboxylic acid in the C-28 amido side
chain was important for anti-HIV entry activity.5–8
However, while both bi-functional 18 and its C-28
mono-functional analogs 18a do not have a terminal
carboxylic acid, they are quite potent against HIV-1 in-
duced fusion (Fig. 1). Therefore, we propose that the
terminal carboxylic acid at C-28 is not a requirement
for anti-HIV entry activity. Instead, a carbonyl moiety
near the terminal of C-28 side chain, such as the one
in –CONH-(CH2)7NH–COCH3, may be critical for po-
tent anti-entry activity. In addition, the bi-functional
analog 14, which possesses the C-3 pharmacophore of
DSB and the C-28 pharmacophore of IC9564, exhibited
unexpectedly low anti-HIV potency. This result suggests
that although the two pharmacophores at the C-3 and
C-28 positions of BA are located far apart, they may
interfere with each other in target binding. Indeed, the
fact that the anti-fusion activity of bi-functional com-
pound 18 was weaker than that of its mono-functional
analog 18a, also suggested that the presence of C-3 side
5.2. Procedure for preparation of compounds 4–10
5.2.1. General procedure for coupling at C-28 position. A
mixture of 3-O-Ac-BA (250–350 mg, 0.5–0.8 mmol) and
oxalyl chloride (6 mmol, 2 M in CH2Cl2) was stirred
for 10 min. After concentration under vacuum, the
residual mixture was treated with L- or D-leucine meth-
yl ester, 11-aminodecanoic acid methyl ester, or 7-
aminoheptanoic acid methyl ester (1–2 mmol), HOBT
(1 equiv) and triethylamine (0.3 mL) in dichlorometh-
ane. The mixture was then stirred at room temperature
overnight, then diluted with CH2Cl2, washed with
water and brine, dried over Na2SO4, and finally con-
centrated under vacuum. The residue was chromato-
graphed on Si gel to yield the corresponding C-28
amide intermediates.
5.2.2. General procedure for saponification. To the solu-
tion of above intermediates in THF/MeOH (50%,
4 mL) was added aqueous NaOH (1 mL, 4 M). After
stirring for 4–10 h at rt, the mixture was neutralized with
1 N HCl. The resulting precipitate was collected, washed
with water, and dried under vacuum to yield the corre-
sponding C-3 unprotected and C-28 amide intermediates
4a, 6a, 8a, and 10a with yields of 60%, 52%, 57%, and
80%, respectively, for the above two steps.
5.2.3. General procedure for coupling at C-3 position. The
mixture of above intermediate (4a, 6a, 8a, or 10a),
2,2-dimethylsuccinic anhydride (5–10 equiv), and
DMAP (1 equiv) in pyridine (anhydrous, 4 mL) was re-
fluxed overnight. The mixture was then concentrated

2286
L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
under vacuum, re-dissolved in MeOH, and purified with
reverse-phase HPLC. Intermediates 4a, 6a, and 8a each
gave two isomers: 4 and 5, 6 and 7, and 8 and 9, respec-
tively. Compound 10 was the only isomer isolated after
acylation of 10a.
5.2.3.5. N-[3b-O-(30,30-Dimethylsuccinyl)-lup-20(29)-
en-28-oyl]-11-aminoundecanoic acid (8). Yield 16%. Mp
115–120 ꢁC. Positive FABMS m/z 768 (M+H)+; HR-
FABMS calcd for C47H78NO7 768.5778, found
768.5752. 1H NMR (400 MHz)
d 5.76 (1H, t,
J = 5.5 Hz, NH), 4.74, 4.53 (each 1H, each s, @CH2),
4.51 (1H, dd, J = 5.0 Hz, J = 11.0 Hz, H-3), 3.30–3.41,
3.08–3.15 (2H, m, –CH2NH), 3.05–3.08 (1H, m, H-19),
2.78, 2.47 (each 1H, each d, J = 15.0 Hz, –CH2–COO–
CH–), 2.23–2.37 (2H, m, –CH2–COOH), 2.17 (1H, dt,
J = 13.0 Hz, J = 3.4 Hz, H-13), 1.69 (3H, s, CH3-30),
1.30, 1.24 (each 3H, each s, –C(CH3)2–COOH), 0.75,
0.82, 0.83, 0.94, 0.97 (each 3H, each s, 5· CH3).
5.2.3.1. 3b-O-(30,30-Dimethylsuccinyl)-28-N-L-leuci-
nyl-betulinic acid (4). Yield 32%. Mp 230–233 ꢁC. Posi-
tive FABMS m/z 698 (M+H)+; HR-FABMS calcd for
C42H68NO7 698.4996, found 698.4998. 1H NMR
(500 MHz) d 6.03 (1H, d, J = 7.8 Hz, –NH–), 4.74,
4.55 (each 1H, each s, @CH2), 4.65 (1H, dd,
J = 7.6 Hz, J = 13.0 Hz, –NH–CH–), 4.54 (1H, dd,
J = 6.2 Hz, J = 10.0 Hz, H-3), 3.04 (1H, dt, J = 4.0 Hz,
J = 11.0 Hz, H-19), 2.89, 2.37 (each d, each 1H,
J = 14.5 Hz, –CH2–COO–CH), 2.52–2.77 (m, 1H, H-
13), 1.68 (3H, s, CH3-30), 1.31, 1.21 (each 3H, each s,
–C(CH3)2–COOH), 0.94–0.97 (6H, m, 2 · CH3), 0.79
(6H, s, 2 · CH3), 0.67 (3H, s, CH3). A complete chemi-
cal shift assignment of carbon and proton of 4 derived
from 13C and HMQC NMR was provided as supple-
mentary data (Table 2).
5.2.3.6. N-[3b-O-(20,20-Dimethylsuccinyl)-lup-20(29)-
en-28-oyl]-11-aminoundecanoic acid (9). Yield 3.2%. Mp
97–100 ꢁC. Positive FABMS m/z 768 (M+H)+; HR-FAB-
1
MS calcd for C47H78NO7 768.5778, found 768.5752. H
NMR (400 MHz) d 5.63 (1H, br s, –NH–), 4.73, 4.59
(each 1H, each s, @CH2), 4.65–4.68 (1H, m, H-3), 3.27–
3.33, 3.16–3.22 (2H, m, CH2NH), 3.11 (1H, dt,
J = 4 Hz, J = 11 Hz, H-19), 2.73 (2H, br s,
–C(CH3)2–CH2–COOH), 2.23–2.44 (1H, m, H-13), 2.35
(2H, t, J = 7.5 Hz, –CH2–COOH), 1.68 (3H, s, CH3-30),
1.28 (6H, s, –C(CH3)2–CH2), 0.96 (6H, s, 2· CH3), 0.93
(3H, s, CH3), 0.87 (3H, d, J = 5.5 Hz, CH3), 0.78 (3H,
d, J = 24.0 Hz, CH3).
5.2.3.2. 3b-O-(20,20-Dimethylsuccinyl)-28-N-L-leuci-
nyl-betulinic acid (5). Yield 26%. Mp 250–252 ꢁC. Posi-
tive FABMS m/z 698 (M+H)+; HR-FABMS calcd for
C42H68NO7 698.4996, found 698.4996. 1H NMR
(400 MHz) d 5.82 (1H, d, J = 7.6 Hz, –NH–), 4.70,
4.58 (each 1H, each s, @CH2), 4.44–4.55 (2H, m,
–NH–CH– and H-3), 3.05–3.10 (1H, dt, J = 4.0 Hz,
J = 11 Hz, H-19), 2.66, 2.59 (each 1H, each d,
J = 16.0 Hz, –CH2–COOH), 2.44–2.50 (1H, dt,
J = 3 Hz, J = 11 Hz, H-13), 1.67 (3H, s, CH3-30), 1.30,
1.28 (each 3H, each s, –C(CH3)2–CH2), 0.91–0.98 (9H,
m, 3· CH3), 0.83 (3H, d, J = 13 Hz, CH3), 0.81 (3H,
d, J = 11.0 Hz, CH3).
5.2.3.7. N-[3b-O-(30,30-Dimethylsuccinyl)-lup-20(29)-
en-28-oyl]-8-aminooctanoic acid (10). Yield 70%. Mp
116–120 ꢁC. Positive FABMS m/z 726 (M+H)+; HR-
FABMS calcd for C44H72NO7 726.5309, found
726.5309. 1H NMR (300 MHz)
d 5.65 (1H, t,
J = 5.8 Hz, –NH–), 4.73, 4.59 (each 1H, each s,
@CH2), 4.48 (1H, t, J = 7.8 Hz, H-3), 3.28–3.68, 3.12–
3.18 (2H, m, –CH2NH), 3.08–3.10 (1H, m, H-19),
2.67, 2.56 (each 1H, each d, J = 15.8 Hz, –CH2–COO–
CH–), 2.38–2.47 (1H, m, H-13), 2.34 (2H, t,
J = 7.5 Hz, –CH2–COOH), 1.68 (3H, s, CH3-30), 1.30,
1.28 (each 3H, each s, –C(CH3)2–COOH), 0.79, 0.92,
0.95 (each 3H, each s, 3· CH3), 0.82 (6H, s, 2· CH3).
5.2.3.3. 3b-O-(30,30-Dimethylsuccinyl)-28-N-D-leuci-
nyl-betulinic acid (6). Yield 45%. Mp 205–207 ꢁC. Posi-
tive FABMS m/z 698 (M+H)+; HR-FABMS calcd
for C42H68NO7 698.4996, found 698.4996. 1H NMR
(300 MHz) d 6.04 (1H, d, J = 7.8 Hz, –NH–), 4.74,
4.59 (each 1H, each s, @CH2), 4.65 (1H, dd,
J = 7.6 Hz, J = 13.0 Hz, –NH–CH–), 4.53 (1H, dd,
J = 6.1 Hz, J = 9.7 Hz, H-3), 3.03 (1H, dt, J = 4.
3 Hz, J = 10.0 Hz, H-19), 2.90, 2.35 (each 1H, each
d, J = 14.5 Hz, –CH2–COO–CH), 2.67 (1H, t,
J = 13.8 Hz, H-13), 1.68 (3H, s, CH3-30), 1.31, 1.21 (each
3H, each s, –C(CH3)2–COOH), 0.94–0.98 (6H, m, 2·
CH3), 0.79 (s, 6H, 2· CH3), 0.67 (3H, s, CH3).
5.2.4. Preparation of 11. A mixture of betulinic acid
(200 mg, 0.44 mmol), PyBop (343 mg, 0.66 mmol),
HOBT
(88 mg,
0.66 mmol),
DIEA
(0.26 mL,
1.76 mmol), and NH4Cl (5 mg, 0.88 mmol) in DMF
(4 mL) was stirred for 1 h at room temperature. The
mixture was then diluted with EtOAc (50 mL), washed
with diluted HCl (1 N) and brine, and then dried over
Na2SO4. The organic layer was concentrated under vac-
uum and chromatographed on Si gel to yield the HOBT
derivative of betulinic acid. The resulting intermediate
was further modified following the general procedure
of C-3 coupling described above to yield 11.
5.2.3.4. 3b-O-(20,20-Dimethylsuccinyl)-28-D-leucinyl-
betulinic acid (7). Yield 37%. Mp 235–237 ꢁC. Positive
FABMS m/z 698 (M+H)+; HR-FABMS calcd for
C42H68NO7 698.4996, found 698.4996. 1H NMR
(300 MHz) d 5.81 (1H, d, J = 7.5 Hz, –NH–), 4.70,
4.58 (each 1H, each s, @CH2), 4.48–4.56 (2H, m,
–NH–CH– and H-3), 3.05 (1H, dt, J = 4.0 Hz,
J = 11.0 Hz, H-19), 2.65, 2.58 (each 1H, each d,
J = 16.0 Hz, –CH2–COOH), 2.46 (1H, t, J = 12.0 Hz,
H-13), 1.67 (3H, s, CH3-30), 1.30, 1.28 (each 3H, each
s, –C(CH3)2–CH2), 0.91–0.98 (9H, m, 3· CH3),
0.82–0.86 (6H, m, 2· CH3).
5.2.4.1. O-[3b-O-(20,20-Dimethylsuccinyl)-lup-20(29)-
en-28-oyl]-1-hydroxybenzotriazole (11). Yield 64%. Mp
151–154 ꢁC. Negative FABMS m/z 700 (MꢀH)ꢀ; HR-
FABMS calcd for C42H58N3O6 700.4326, found
700.4326. 1H NMR (300 MHz, Pyridine-d5) d 8.20
(1H, d, J = 8.5 Hz, Ar-H), 7.67 (s, 1H, Ar-H), 7.64
(1H, dd, J = 8.5 Hz, J = 15.0 Hz, Ar-H), 7.42 (1H, dt,
J = 1.2 Hz, J = 6.6 Hz, Ar-H), 4.88, 4.75 (each 1H, each

L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
2287
s, @CH2), 4.71 (1H, d, J = 4.7 Hz, H-3), 3.13 (1H, dd,
J = 4.7 Hz, J = 11.0 Hz, H-19), 2.91 (2H, d, J = 6.0 Hz,
–CH2–COOH), 2.65 (1H, d, J = 2.8 Hz, CH), 2.38–
2.47 (1H, m, CH), 2.31 (1H, t, J = 12.0 Hz, H-13),
2.06–2.20 (1H, m, CH), 1.72 (3H, s, CH3-30), 1.53
(6H, s, –C(CH3)2–CH2–COOH), 0.71, 0.92, 0.95, 0.96,
1.02, (each 3H, s, 5· CH3).
furnished the corresponding bi-functional compounds
13 and 15.
5.2.6.1.
N0-[N-[3b-O-(30,30-Dimethylsuccinyl)-lup-
20(29)-en-28-oyl]-8-aminooctanoyl]-L-leucine (13). Yield
37%. Mp 131–134 ꢁC. Positive FABMS m/z 839
(M+H)+; HR-FABMS calcd for C50H83N2O8
1
839.6149, found 839.6194. H NMR (300 MHz, Pyri-
5.2.5. Preparation of 12. A mixture of betulinic acid
(300 mg, 0.66 mmol), DCC (1 mL, 1 M in dichlorometh-
ane), glycine ethylester (180 mg, 1.3 mmol), and DMAP
(96 mg, 0.8 mmol) in dichloromethane (20 mL) was stir-
red overnight at room temperature. The mixture was
then diluted with dichloromethane (50 mL), washed
with water and brine, dried over Na2SO4, and then con-
centrated under vacuum. The residue was chromato-
graphed on Si gel to yield the glycine ester derivative
of betulinic acid, which was then subjected to hydrolysis
(see Section 5.2.2) to yield the glycine BA. The resulting
glycine BA (150 mg, 0.28 mmol) was treated with leucine
methyl ester (100 mg, 0.7 mmol), PyBop (580 mg,
1.1 mmol), and HOBT (45 mg, 0.33 mmol) in CH2Cl2
(20 mL) at room temperature and stirred overnight.
The reaction mixture was then diluted with methylene
chloride (20 mL), washed with water and brine, dried
over Na2SO4, and then concentrated under vacuum.
Subsequent saponification (see Section 5.2.2) on the
above glycinyl-leucine intermediate yielded 12a,
which was further modified at C-3 as described above
to afford 12.
dine-d5) d 5.91 (1H, d, J = 7.0 Hz, NH), 5.21 (1H, br
s, NH), 4.91, 4.74 (each 1H, each s, @CH2), 4.73–4.77
(1H, m, H-3), 3.62 (1H, t, J = 11.0 Hz, CH–NH–),
3.45–3.55, 3.30–3.40 (each 1H, m, NH–CH2), 3.07
(1H, t, J = 11.4 Hz, H-19), 2.95, 2.87 (2H, each d,
J = 15.6 Hz, CH2–COO–CH), 2.40–2.50 (2H, m,
–CH2–COO–NH), 1.77 (3H, s, CH3-30), 1.52 (6H, s,
–C(CH3)2–COOH), 0.75, 0.95, 1.03, 1.09 (each 3H, s,
4· CH3), 0.99 (3H, d, J = 5.0 Hz, CH3), 0.92 (6H, s,
2· CH3).
5.2.6.2.
N0-[N-[3b-O-(30,30-Dimethylsuccinyl)-lup-
20(29)-en-28-oyl]-8-aminooctanoyl]-3-aminobenzoic acid
(15). Yield 63%. Mp 168–170 ꢁC. Positive FABMS
m/z
845
(M+H)+;
HR-FABMS
calcd
for
C51H77N2O8 845.5680, found 845.5680. 1H NMR
(300 MHz) d 8.02 (2H, d, J = 8.5 Hz, Ar-H), 7.12
(1H, br s, CO–NH–Ar), 7.64 (2H, d, J = 7.5 Hz, Ar-
H), 5.65 (1H, t, J = 5.8 Hz, –NH–), 4.71, 4.58 (each
1H, each s, @CH2), 4.47 (1H, dd, J = 5.0 Hz,
J = 11.0 Hz, H-3), 3.46–3.52, 2.93–2.99 (2H, m,
–CH2NH), 3.08–3.18 (1H, m, H-19), 2.78, 2.47 (each
1H, each d, J = 14.5 Hz, –CH2–COO–CH–), 2.36–
2.47 (1H, m, –CH), 2.16 (2H, s, CH2CO–NH–), 1.67
(3H, s, CH3-30), 1.32, 1.26 (each 3H, each s,
–C(CH3)2–COOH), 0.41, 0.57, 0.76, 0.77, 0.92 (each
3H, each s, 5· CH3).
5.2.5.1.
N0-[N-[3b-O-(30,30-Dimethylsuccinyl)-lup-
20(29)-en-28-oyl]-L-glycinyl]-L-leucine (12). Yield 31%
overall from betulinic acid. Mp 204–207 ꢁC. Positive
FABMS m/z 755 (M+H)+; HR-FABMS calcd for
C44H71N2O8 755.5210, found 755.5210. 1H NMR
(300 MHz) d 6.87 (1H, d, J = 8.0 Hz, NH), 6.63 (1H,
br s, NH), 4.70, 4.55 (each 1H, each s, @CH2),
4.60–4.66 (1H, m, –NH–CH–), 4.44 (1H, dd,
J = 5.4 Hz, J = 11.0 Hz, H-3), 3.94 (2H, d, J = 4.8 Hz,
–CH2–NH–), 3.08 (1H, dt, J = 4.0 Hz, J = 11.0 Hz,
H-19), 2.67, 2.56 (each d, each 1H, J = 15.3 Hz,
–CH2–COO–CH), 2.43 (t, 1H, J = 9.5 Hz, H-13), 1.64
(3H, s, CH3-30), 1.30, 1.29 (each 3H, each s,
–C(CH3)2–COOH), 0.91–0.95 (9H, m, 3· CH3), 0.72,
0.73, 0.78, 0.86 (each 3H, each s, 4· CH3).
5.2.7. Preparation of 14. A mixture of 14a12,19 (130 mg,
0.146 mmol), DIEA (0.2 mL, 1.1 mmol), and MEMCl
(0.4 mL, 3.2 mmol) in anhydrous dichloromethane
(5 mL) was stirred overnight at room temperature. The
mixture was then diluted with EtOAc (50 mL), washed
with saturated NaHCO3 (2 · 25 mL) and brine, dried
over Na2SO4, and concentrated under vacuum to give
the MEM ether (160 mg, gum) without further
purification.
To a solution of MEM ether (160 mg) in THF/MeOH
(50%, 4 mL) was added NaOH (4 N, 1 mL). The mix-
ture was stirred overnight, then neutralized with HCl
(1 N), and extracted with EtOAc (2 · 25 mL). The
organic layer was washed with water and brine, dried
over Na2SO4, and then concentrated under vacuum to
give 14b (130 mg) without purification.
5.2.6. General procedure for preparation of 13 and 15. A
mixture of 10a (260 ꢁ370 mg, 0.4–0.6 mmol) in dichlo-
romethane (5 mL) was treated with leucine methyl ester
or methyl-4-aminobenzoate (0.6 mmol), PyBop (312 mg,
0.6 mmol), HOBT (81 mg, 0.6 mmol), and DIEA
(0.5 mL, 5.2 mmol) in dichloromethane (50 mL) and
stirred overnight at room temperature. The mixture
was diluted with 20 mL dichloromethane and washed
with diluted HCl, water, and brine, and then dried over
Na2SO4. After removal of organic solvent, the residue
was saponified following the procedure described above.
The collected precipitate was purified on Si gel to yield
the corresponding C-28 mono-substituted betulinic acid
derivatives 13a and 15a in 33% and 16% yields, respec-
tively. Further modification using the general procedure
for coupling at C-3, as described for compounds 4–10,
A mixture of 14b (130 mg), DMAP (20 mg, 0.16 mmol),
and 2,2-dimethylsuccinic anhydride (200 mg, 1.6 mmol)
in anhydrous pyridine (5 mL) was refluxed overnight.
After being concentrated in vacuo, the residue was dilut-
ed with dichloromethane, washed with water and brine,
dried over Na2SO4, and then concentrated under vacu-
um. The residue was purified with Si gel chromatogra-
phy to yield the 3,28-bi-functional BA derivative
(40 mg, 28% yield in three steps).

2288
L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
A mixture of above bi-functional derivative MEM ether
(40 mg, 0.04 mmol) and B-chlorocatecholborane
(100 mg, 0.65 mmol) in dry dichloromethane (2 mL)
was stirred overnight, after which water (1 mL) was add-
ed and the reaction mixture was stirred for 20 min. Then
the mixture was diluted with dichloromethane (25 mL),
washed with water and brine, dried over Na2SO4, and
concentrated under vacuum. The residue was then puri-
fied with reverse-phase HPLC to yield 14 (10 mg).
19), 2.56, 2.67 (each 1H, d, J = 16.0 Hz, –CH2–COO–
CH), 2.43 (1H, dt, J = 2.5 Hz, J = 12.0 Hz, H-13), 2.01
(3H, s, COCH3), 1.68 (3H, s, CH3-30), 1.28, 1.30 (each
3H, s, –C(CH3)2–COOH), 0.80, 0.91, 0.96 (each 3H, s,
3· CH3) 0.83 (6H, s, 2· CH3). A complete chemical shift
assignment of carbon and proton of 16 derived from 13C
and HMQC NMR was provided as Supplementary data
(Table 2).
5.2.8.2. [[N-[3b-O-(30,30-Dimethylsuccinyl)-lup-20(29)-
en-28-oyl]-6-aminohexyl]-carbamoyl]methane (17). Yield
17%. Mp 137 ꢁC (dec). Positive FABMS m/z 725
(M+H)+; HR-FABMS calcd for C44H73N2O6
5.2.7.1. (3R,4S)-N0-[N-[3b-O-(30,30-Dimethylsuccinyl)-
lup-20(29)-en-28-oyl]-8-aminooctanoyl]-4-amino-3-hydr-
oxyl-6-methylheptanoic acid (14). Yield 28%. Mp 128–
130 ꢁC. Positive FABMS m/z 883 (M+H)+; HR-FAB-
MS calcd for C52H87N2O9 883.6411, found 883.6412.
1H NMR (300 MHz) d 5.91 (1H, d, J = 7.0 Hz, NH),
5.82 (1H, br s, NH), 4.72, 4.60 (each 1H, each s,
@CH2), 4.89 (1H, dd, J = 5.2 Hz, J = 11.0 Hz, H-3),
4.39–4.10 (2H, m, CH–NH and CH–OH), 3.47–3.53,
2.91–2.98 (each 1H, m, NH–CH2), 2.77, 2.47 (each
1H, d, J = 15.0 Hz, –CH2–COO–CH), 1.68 (3H, s,
CH3-30), 1.29, 1.25 (each 3H, s, –C(CH3)2–COOH),
0.89–0.96 (12H, m, 4· CH3), 0.73, 0.97, 0.81 (each 3H,
s, 3· CH3).
1
725.5474, found 725.5469. H NMR (300 MHz) d 5.89
(1H, br s, NH), 5.70 (1H, t, J = 6.0 Hz, NH), 4.70,
4.58 (each 1H, each s, @CH2), 4.47 (1H, dd,
J = 6.5 Hz, J = 10.5 Hz, H-3), 3.17–3.29 (4H, m, 2·
CH2–NH), 3.10 (1H, dt, J = 3.5 Hz, J = 14.0 Hz, H-
19), 2.56, 2.66 (each 1H, d, J = 16.0 Hz, –CH2–COO–
CH), 2.43 (1H, t, J = 12.0 Hz, H-13), 1.99 (3H, s,
COCH3), 1.67 (3H, s, CH3-30), 1.27, 1.29 (each 3H, s,
–C(CH3)2–COOH), 0.79, 0.91, 0.94 (each 3H, s, 3·
CH3) 0.81 (6H, s, 2· CH3).
5.2.8.3. [[N-[3b-O-(30,30-Dimethylsuccinyl)-lup-20(29)-
en-28-oyl]-7-aminoheptyl]-carbamoyl]methane (18). Yield
29%. Mp 117–120 ꢁC. Positive FABMS m/z 739
(M+H)+; HR-FABMS calcd for C45H75N2O6
5.2.8. General procedure for preparation of 16–20. The
synthesis of C-28 mono-modified BA was accomplished
by following the procedures described previously by De-
reu.7,8 In brief, a mixture of 3-O-acyl betulinic acid
(300 mg, 0.6 mmol) and oxalyl chloride (6 mmol, 2 M
in CH2Cl2) was stirred for 10 min and then dried under
vacuum. The residue was redissolved in dichlorometh-
ane and added slowly to a solution of the appropriate
diaminoalkane (400 mg, 2.5–3.9 mmol) in dichlorometh-
ane (4 mL). After stirring overnight, the reaction mix-
ture was concentrated, washed with water, redissolved
in EtOH, and filtered to remove insoluble solid. The
EtOH solution was concentrated under vacuum, and
the residue was purified with Si gel chromatography to
yield the corresponding amine intermediates 16a–20a.
1
739.5625, found 739.5615. H NMR (300 MHz) d 5.62
(2H, t, J = 5.0 Hz, 2· NH), 4.72, 4.59 (each 1H, each
s, @CH2), 4.49 (1H, dd, J = 6.5 Hz, J = 10.0 Hz, H-3),
3.09–3.30 (5H, m, 2· CH2–NH and H-19), 2.55, 2.67
(each 1H, d, J = 16.0 Hz, –CH2–COO–CH), 2.46 (1H,
t, J = 11.0 Hz, H-13), 1.99 (3H, s, COCH3), 1.68 (3H,
s, CH3-30), 1.29, 1.30 (each 3H, s, –C(CH3)2–COOH),
0.80, 0.93, 0.96 (each 3H, s, 3· CH3) 0.83 (6H, s, 2·
CH3).
5.2.8.4. [[N-[3b-O-(30,30-Dimethylsuccinyl)-lup-20(29)-
en-28-oyl]-8-aminooctyl]-carbamoyl]methane (19). Yield
59%. Mp 113–115 ꢁC. Positive FABMS m/z 753
(M+H)+; HR-FABMS calcd for C46H77N2O6
To a solution of the above amine intermediate (16a–20a)
in anhydrous pyridine (4 mL) were added acetic anhy-
dride (2 equiv) and DMAP (1 equiv). After stirring
overnight at room temperature, the mixture was concen-
trated under vacuum. The residue was chromatographed
on Si gel to yield the corresponding C-28 mono-substi-
tuted betulinic acid derivative, which was subjected to
saponification as described above to yield the corre-
sponding C-28 mono-modified intermediates 16b–20b.
Further modification of 16b–20b using the general pro-
cedure for coupling at C-3 yielded the corresponding
bi-functional compounds 16–20.
1
753.5781, found 753.5779. H NMR (300 MHz) d 5.83
(1H, br s, NH), 5.67 (1H, t, J = 5.5 Hz, NH), 4.72,
4.58 (each 1H, each s, @CH2), 4.47 (1H, t, J = 7.0 Hz,
H-3), 3.06–3.29 (5H, m, 2· CH2–NH and H-19), 2.55,
2.66 (each 1H, d, J = 16.0 Hz, –CH2–COO–CH), 2.43
(1H, t, J = 10.0 Hz, H-13), 2.00 (3H, s, COCH3), 1.67
(3H, s, CH3-30), 1.27, 1.29 (each 3H, s, –C(CH3)2–
COOH), 0.79, 0.91, 0.95 (each 3H, s, 3· CH3) 0.82
(6H, s, 2· CH3).
5.2.8.5. [[N-[3b-O-(30,30-Dimethylsuccinyl)-lup-20(29)-
en-28-oyl]-9-aminonoyl]-carbamoyl]methane (20). Yield
40%. Mp 120–122 ꢁC. Positive FABMS m/z 698
(M+H)+; HR-FABMS calcd for C47H79N2O6
5.2.8.1. [[N-[3b-O-(30,30-Dimethylsuccinyl)-lup-20(29)-
en-28-oyl]-5-aminopentyl]-carbamoyl]methane (16). Yield
27%. Mp 137 ꢁC (dec). Positive FABMS m/z 711
(M+H)+; HR-FABMS calcd for C43H71N2O6
1
767.5938, found 767.5944. H NMR (300 MHz) d 5.72
(1H, br s, NH), 5.64 (1H, t, J = 5.5 Hz, NH), 4.72,
4.59 (each 1H, each s, @CH2), 4.48 (1H, t, J = 7.0 Hz,
H-3), 3.08–3.31 (5H, m, 2· CH2–NH and H-19), 2.55,
2.67 (each 1H, d, J = 16.0 Hz, –CH2–COO–CH), 2.42
(1H, t, J = 12.0 Hz, H-13), 2.00 (3H, s, COCH3),
1.68 (3H, s, CH3-30), 1.28, 1.29 (each 3H, s,
1
711.5312, found 711.5313. H NMR (500 MHz) d 6.02
(1H, br s, NH), 5.67 (1H, t, J = 6.0 Hz, NH), 4.72,
4.59 (each 1H, each s, @CH2), 4.48 (1H, dd,
J = 6.0 Hz, J = 10.0 Hz, H-3), 3.16–3.32 (4H, m, 2·
CH2–NH), 3.12 (1H, dt, J = 4.0 Hz, J = 11.0 Hz, H-

L. Huang et al. / Bioorg. Med. Chem. 14 (2006) 2279–2289
2289
–C(CH3)2–COOH), 0.80, 0.92, 0.95 (each 3H, s, 3· CH3)
0.82 (6H, s, 2· CH3).
collection; Dr. Susan Morris-Natschke in the School
of Pharmacy, University of North Carolina at Chapel
Hill, for critical reading of the manuscript.
5.3. Anti-HIV assay
An HIV-1 infectivity assay previously described was
used in the experiments.20 A 96-well microtiter plate
was used to set up the HIV-1 NL4-3 replication assay.
HIV-1 NL4-3 at a multiplicity of infection (MOI) of
0.01 was used to infect MT4 cells. Culture supernatants
were collected on day 4 post infection for the p24 assay
using an ELISA kit from ZeptoMetrix Corporation
(Buffalo, New York).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2005.11.016.
References and notes
5.4. Cell fusion assay
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TZM cells that express luciferase upon fusion with enve-
lope-expressing COS cells were used as fusion partners.
The fusion assays were performed by transfecting mon-
key kidney cells (COS) with the expression vector
pSRHS that contains the HIV-1 NL4-3 envelope genes.
Electroporation was performed to express the HIV-1
envelope on COS cells. Briefly, COS cells (106) in culture
medium were incubated with 2 lg of the envelope
expression vector on ice for 10 min. Electroporation
was performed using a gene pulser (BioRad, Hercules,
CA) with capacitance set at 950 lF and voltage at
150 V. The transfected COS cells were cultured for one
day before mixing with TZM cells. TZM cells
(10 · 104) were incubated with COS cells (104) in 96-well
flat-bottomed plates (Costar) in 100 lL culture medium.
Compounds to be tested at various concentrations in
10 lL culture medium were incubated with the cell mix-
tures at 37 ꢁC for 24 hours. A Promega luciferase assay
kit was used to quantify luciferase activity in the fused
cells using a Biotek luminometer. The drug concentra-
tion that reduced the control HIV-1 envelope-mediated
fusion by 50% is defined as 50% effective concentration
(EC50).
5.5. HIV-1 maturation assay
10. Zhou, J.; Yuan, X.; Dismuke, D.; Forshey, B. M.;
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Acknowledgments
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This work was supported by the National Institutes of
Health (Grants AI50504) awarded to C. H. Chen and
in part by Grant AI-33066 from the National Institute
of Allergy and Infectious Diseases (NIAID) awarded
to K. H. Lee. The authors thank Dr. George Dubay
of the Department of Chemistry and Dr. Anthony Ribe-
iro of the NMR Spectroscopy Center of Duke Universi-
ty for their help on mass and NMR spectroscopy data