Anti-AIDS agents. 78. Design, synthesis, metabolic stability assessment, and antiviral evaluation of novel betulinic acid derivatives as potent anti-human immunodeficiency virus (HIV) agents

Article abstract of DOI:10.1021/jm900136j

In a continuing study of potent anti-HIV agents, seventeen 28,30-disubstituted betulinic acid (BA, 1) derivatives and seven novel 3,28-disubstituted BA analogues were designed, synthesized, and evaluated for in vitro antiviral activity. Among them, compound 21 showed an improved solubility and equal anti-HIV potency (EC₅₀ = 0.09 μM) when compared to HIV entry inhibitors 3b (IC9564, (3R,4S)-N′-[N-[3β-hydroxylup-20(29)-en- 28-oyl]-8-aminooctanoyl]-4-amino-3-hydroxy-6-methylheptanoic acid) and 4 (A43-D, [[N-[3β-O-(3′,3′-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-7- aminoheptyl]carbamoyl]methane). Using a cyclic secondary amine to form the C-28 amide bond increased the metabolic stability of the derivatives significantly in pooled human liver microsomes. The most potent compounds 47 and 48 displayed potent anti-HIV activity with EC₅₀ values of 0.007 and 0.006 μM, respectively. These results are slightly better than that of bevirimat (2, 3′,3′-dimethylsuccinylbetulinic acid), which is currently in phase IIb clinical trials. Compounds 47 and 48 should serve as attractive promising leads to develop next generation, metabolically stable, 3,28-disubstituted bifunctional HIV-1 inhibitors as clinical trials candidates.

Full text of DOI:10.1021/jm900136j

3248
J. Med. Chem. 2009, 52, 3248–3258
Anti-AIDS Agents. 78.† Design, Synthesis, Metabolic Stability Assessment, and Antiviral
Evaluation of Novel Betulinic Acid Derivatives as Potent Anti-Human Immunodeficiency Virus
(HIV) Agents
Keduo Qian,3 Donglei Yu,3 Chin-Ho Chen,‡ Li Huang,‡ Susan L. Morris-Natschke,3 Theodore J. Nitz,§ Karl Salzwedel,§
Mary Reddick,§ Graham P. Allaway,§ and Kuo-Hsiung Lee*,3
Natural Products Research Laboratories, Eshelman School of Pharmacy, UniVersity of North Carolina, Chapel Hill, North Carolina 27599,
Department of Surgery, Duke UniVersity Medical Center, Durham, North Carolina 27710, and Panacos Pharmaceuticals,
209 Perry Parkway, Gaithersburg, Maryland 20877
ReceiVed February 3, 2009
In a continuing study of potent anti-HIV agents, seventeen 28,30-disubstituted betulinic acid (BA, 1)
derivatives and seven novel 3,28-disubstituted BA analogues were designed, synthesized, and evaluated for
in vitro antiviral activity. Among them, compound 21 showed an improved solubility and equal anti-HIV
potency (EC50 ) 0.09 µM) when compared to HIV entry inhibitors 3b (IC9564, (3R,4S)-N′-[N-[3ꢀ-hydroxy-
lup-20(29)-en-28-oyl]-8-aminooctanoyl]-4-amino-3-hydroxy-6-methylheptanoic acid) and 4 (A43-D, [[N-[3ꢀ-
O-(3′,3′-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-7-aminoheptyl]carbamoyl]methane). Using a cyclic sec-
ondary amine to form the C-28 amide bond increased the metabolic stability of the derivatives significantly
in pooled human liver microsomes. The most potent compounds 47 and 48 displayed potent anti-HIV activity
with EC50 values of 0.007 and 0.006 µM, respectively. These results are slightly better than that of bevirimat
(2, 3′,3′-dimethylsuccinylbetulinic acid), which is currently in phase IIb clinical trials. Compounds 47 and
48 should serve as attractive promising leads to develop next generation, metabolically stable, 3,28-
disubstituted bifunctional HIV-1 inhibitors as clinical trials candidates.
Introduction
preferably with simplified treatment regimens (fewer pills and
less-frequent administration).
As the world enters the third decade of the AIDSa epidemic,
this pandemic has rapidly grown into the fourth leading cause
of mortality globally.1 Introduction of highly active antiretroviral
therapy (HAART), which employs a combination of nucleoside/
nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleo-
side reverse transcriptase inhibitors (NNRTIs), and/or protease
inhibitors (PIs), has significantly improved the treatment of
HIV/AIDS.2-5 However, the virus is suppressed rather than
eradicated by HAART.6-8 On HAART regimens, multiple drug
therapies can lead to increased adverse effects and toxicities
due to long-term use and drug-drug interactions.9,10 Moreover,
it inevitably leads to the emergence of multidrug-resistant viral
strains.11 In fact, a significant proportion of newly infected
individuals harbor HIV-1 isolates that are resistant to at least
one ART.12,13 Therefore, novel potent antiretroviral agents are
needed, with different targets than currently approved drugs and
Triterpenes, such as betulinic acid (BA, 1, Figure 1),14 represent
a promising class of anti-HIV agents with novel mechanisms. Two
types of BA derivatives have exhibited potent anti-HIV profiles.
C-3 esterification of BA led to the discovery of bevirimat (DSB,
PA-457, 2, 3′,3′-dimethylsuccinylbetulinic acid),15 which is a HIV-1
maturation inhibitor (MI) that blocks cleavage of p25 (CA-SP1)
to functional p24 (CA), resulting in the production of noninfectious
HIV-1 particles.16,17 Bevirimat (2) is currently in phase IIb clinical
trials launched by Panacos Pharmaceuticals, Inc.18,19 On the other
hand, the C-28 side chain was proven to be a necessary pharma-
cophore for anti-HIV entry activity, as seen with the equipotent
diastereomers 3a (RPR103611, (3S,4S)-N′-[N-[3ꢀ-hydroxy-lup-
20(29)-en-28-oyl]-8-aminooctanoyl]-4-amino-3-hydroxy-6-meth-
ylheptanoic acid)20,21 and 3b (IC9564, (3R,4S)-N′-[N-[3ꢀ-hydro-
xy-lup-20(29)-en-28-oyl]-8-aminooctanoyl]-4-amino-3-hydroxy-6-
methylheptanoic acid).22,23 Mechanism of action studies have
revealed that C-28 modified BA derivatives function at a post-
binding, envelope-dependent step involved in fusion of the virus
to the cell membrane.24 Recent studies further suggested that 3b
may also function by targeting the V3 loop of gp120, a domain
involved in chemokine receptor binding.25 Although 3a showed
potent antiviral activity in vitro, the clinical development of 3a by
Rhone-Poulenc (now Sanofi-Aventis) was stopped because of poor
“pharmacodynamic properties”.26 However, the high potency and
novel mechanism of 3a suggest that further modification of this
compound class as HIV entry inhibitors is warranted. Therefore,
in the current study, we mainly focused on the modification of
BA to maintain the anti-HIV activity while improving pharmaco-
kinetic properties.
†
For part 77, see the following. Xu, S.; Yan, X.; Chen, Y.; Xia, P.; Yu,
D.; Qian, K.; Xia, Y.; Yang, Z. Y.; Morris-Natschke, S. L.; Lee, K. H.
Anti-AIDS agents. 77. Synthesis and anti-HIV activity of 2′-monomethyl-
4-methyl DCK and 1′-thia-4-methyl DCK analogs. J. Med. Chem.,
submitted.
* To whom correspondence should be addressed. Phone: 919-962-0066.
Fax: 919-966-3893. E-mail: khlee@unc.edu.
3
University of North Carolina.
Duke University Medical Center.
Panacos Pharmaceuticals.
‡
§
a Abbreviations: AIDS, acquired immunodeficiency syndrome; HIV-1,
human immunodeficiency virus type 1; HAART, highly active antiretroviral
therapy; NRTIs, nucleoside/nucleotide reverse transcriptase inhibitors;
NNRTIs, non-nucleoside reverse transcriptase inhibitors; PIs, protease
inhibitors; MI, maturation inhibitor; BA, betulinic acid; P24 (CA), capsid;
CYPs, cytochrome P450; UGTs, UDP-glucuronosyltransferases; FMO,
flavin containing monooxygenase; NBS, N-bromosuccinimide; PDC, py-
ridinium dichromate; HOBt, hydroxybenzotriazole; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; DMAP, 4-(dimethylami-
no)pyridine; AZT, zidovudine.
Design
Structurally, the triterpenoid skeleton of BA contains three
functional groups: C-3 hydroxyl group, C-28 carboxylic acid
10.1021/jm900136j CCC: $40.75
2009 American Chemical Society
Published on Web 04/23/2009

Betulinic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3249
Figure 1
Scheme 1. General Synthetic Route for 28,30-Disubstituted BA Derivatives
group, and C-19 isopropenyl moiety. Because the C-19 isopro-
The fact that 2 has suitable pharmocokinetic properties for
penyl group has been less investigated, modification was first
carried out on this moiety. Previous research suggested that
saturation of the 20(29) double bond did not influence the
antiviral activity of the BA derivatives significantly.15 Therefore,
we focused on the modification at the C-30 allyl position in
order to better explore the SAR. Some early data suggested that
thioether-linked substitution on the C-30 position retained the
anti-HIV-1 potency slightly in the resulting BA analogues.27
In the current study, the bioisosteric oxygen ether linker was
chosen to replace the thioether group. Leucine and 8-aminooc-
tanoic acid were proved to improve entry inhibition in our
previous study; thus, they were incorporated into the C-28 side
chain of the diverse C-30 modified analogues to yield com-
pounds 10-30.
development, but 3a does not, led us to speculate that the C-28
side chain, which is critical for anti-HIV entry activity, may be
responsible for the poor performance of 3a in preclinical testing.
Indeed, in our study, we observed that C-28 modified BA
derivatives, including 4 (A43-D, [[N-[3ꢀ-O-(3′,3′-dimethylsucci-
nyl)-lup-20(29)-en-28-oyl]-7-aminoheptyl]carbamoyl]methane)28
which is the prior best antientry hit, are less water-soluble.
Meanwhile, although 2 has been demonstrated to be metabolized
primarily by UGT glucuronidation,29 there is no report regarding
to the metabolism of C-28 modified BA analogues. Therefore, in
the present study, in vitro metabolic stability assessment was first
carried out in pooled human liver microsomes (BD Biosciences),
which contain enzymes including cytochrome P450 (CYPs), UGTs,
and FMO, etc. The results revealed that C-28 modified BA

3250 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Qian et al.
Scheme 2. Synthesis of 3,28-Disubstituted 3ꢀ-Amino BA Derivative
derivatives like 4 are quickly metabolized in liver microsomes.
Therefore, novel C-28 side chains were designed and synthesized.
A cyclic secondary amine (from 4-piperidinebutyric acid), rather
than a primary amine, was used to form the critical amide bond
with the C-28 carboxylic acid group, which yielded 38.
Moreover, although BA derivatives with both C-3 ester and
C-28 amide side chains exhibit both antientry and maturation
activity,30 they also display some metabolic problems, likely
due to the C-28 side chain. Thus, modifications were also carried
out on C-28 and C-3 side chains to enhance the stability as
well as increase the anti-HIV activity in the 3,28-disubstituted
BA analogues, which yielded compounds 36, 39, 40, and
45-48. This article reports the design, syntheses, SAR, and
metabolic stability assessment of these novel BA derivatives
as potent HIV-1 inhibitors.
intermediates 6 and 7. Reaction of silver acetate with 8 and 9
in the presence of a catalytic amount of the phase transfer
catalyst tetrabutylammonium bromide (Bu4NBr) in acetonitrile
gave diacetoxy esters 26 and 27, which were then converted to
30-hydroxyl BA derivatives 28 and 29. The 3,28,30-trisubsti-
tuted analogue 30 was acquired by reaction of 24 with 2,2-
dimethylsuccinic anhydride in the presence of DMAP in
pyridine.
The 3,28-disubstituted 3ꢀ-amino analogue 36 was successfully
prepared as described in Scheme 2. Oxidation of 1 with 2 equiv
of PDC produced the 3-keto-BA 31 (87% yield). L-Leucine methyl
ester was reacted with the C-28 carboxylic acid of 31 in the
presence of DMAP and EDCI to furnish 32. The keto moiety of
32 was then converted to an oxime by treatment with hydroxyl-
amine hydrochloride (NH2OH·HCl) in pyridine, which yielded 33
(90% yield).31 The 3ꢀ-amine 34 was readily prepared in 82% yield
from oxime 33 by enantioselective reduction of the Schiff base
with TiCl3 and NaCNBH3, as reported by Leeds and Kirst.32
Treatment of 34 with 2,2-dimethylsuccinic anhydride under DMAP
in pyridine yielded 35. Finally, hydrolysis of 35 with 2 N sodium
hydroxide furnished the desired 3,28-disubstituted 3ꢀ-amino BA
analogue 36.
The syntheses of 3,28-disubstituted 28-piperidine analogues
38-48 were carried out according to Scheme 3. The 3-O-Ac-BA
(5) was treated with oxalyl chloride in dichloromethane, followed
by reaction with the readily prepared 4-piperidinebutyric acid
methyl ester to provide 37 in a 94% yield. Saponification of 37
yielded the desired lead compound 38 quantitatively. Esterification
of 38 with 2,2-dimethylsuccinic anhydride and 2,2-dimethylglutaric
anhydride under DMAP in pyridine led to 39 and 40 in yields of
55% and 36%, respectively. The syntheses of 41-44 were carried
out by reacting 38 with different amines in the presence of HOBt
and EDCI. Reaction of the 3ꢀ-hydroxyl group of 41-44 with 2,2-
dimethylsuccinic anhydride provided the 3,28-disubstituted target
compounds 45-48 in yields of 58-81%.
Chemistry
Scheme 1 depicts the synthetic pathway of 28,30-disubstituted
BA derivatives 10-30. The C-3 ꢀ-hydroxyl group of BA was
protected as the acetate ester of 3-O-Ac-BA (5). Compound 5
was reacted with oxalyl chloride in dichloromethane to yield
an intermediate acid chloride, which was then treated with
leucine methyl ester or 8-aminooctanoic acid methyl ester to
furnish 6 and 7. Allylic bromination of 6 and 7 was carried out
using N-bromosuccinimide (NBS) in dilute acetonitrile at room
temperature to provide 30-bromo BA derivatives 8 and 9. The
bromide group of 8 and 9 was then substituted by a diverse set
of nucleophilic compounds to yield 10-20. In this step, the
desired nucleophilic compound was first treated with 10 equiv
of NaH in THF for 30 min. The 30-bromo 8 or 9 was then
reacted with the resulting suspension using a microwave
apparatus at 120 °C. After the mixture was cooled, 1 mL of
MeOH-H2O was added to convert the intermediate esters to
carboxylic acids by saponification, which furnished the target
compounds 10-20 in 59-77% yields. Reaction of 20 with
methylamine in the presence of HOBt/EDCI in dichloromethane
led to 21 in a 69% yield. Saponification of the 30-bromo 8 and
9 with 2 N sodium hydroxide in MeOH/THF yielded the
corresponding carboxylic acids 24 and 25. The previous reported
22 and 23 were also prepared by saponification of the ester
Results and Discussion
The anti-HIV-1 replication activities of the newly synthe-
sized BA derivatives 10-30, 36, 38-40, and 45-48 were
assessed in HIV-1IIIB infected MT-2 lymphocytes in parallel

Betulinic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3251
Scheme 3. General Synthetic Route for 3,28-Disubstituted 28-Piperidine BA Analogues
Table 1. Anti-HIV-1 Replication Activities in HIV-1IIIB Infected MT-2
with AZT and 2. Compounds 21 and 45-48 were further
evaluated against HIV-1NL4-3 in MT-4 cell lines and compared
with 3b and 4. Because these two antiviral screening systems
used slightly different protocols, the results may vary for
the same compounds. The bioassay data are summarized in
Tables 1 and 2, respectively, and the SAR conclusions are
summarized in Figure 2.
Within the 28,30-disubstituted BA derivatives, the C-28
leucine, C-30 substituted analogues 10-19, 24, and 28 did not
inhibit viral replication. Because the monosubstituted C-28
leucine derivative 22 did not exhibit antiviral replication activity
either, we postulated that the derivatives did not lose antiviral
potency due to the C-30 modification. However, because the
presence of different C-30 substitutions did not increase the anti-
HIV-1 activity, the C-19 isopropenyl moiety is unlikely to be
an activity pharmacophore. Nevertheless, considering that BA
derivatives with antientry necessary C-28 lipophilic side chains
are less water-soluble, C-30 allylic modification may still be
useful to influence pharmacokinetic properties, such as hydro-
philicity and solubility.
The introduction of a free hydroxyl group at C-30 in 29
reduced the antiviral activity relative to 23 by severalfold to an
EC50 value of 14.8 µM. This result suggested that a hydrogen
bond donor is not tolerated near the C-19 isopropenyl group, a
conclusion that is also supported by prior data that a primary
amine substituent at C-30 is unfavorable.27 In comparison,
analogues with 2-morpholinoethoxy (20) and bromide (25)
moieties at C-30 retained the antiviral activity of 23. Specifically,
the 28-aminooctanoic acid derivatives 23, 20, and 25 showed
Cell Linesa
compd
EC50 (µM)
CC50 (µM)
TI
AZT
2
0.056
0.011
NS
NS
NS
NS
NS
NS
NS
27.8
NS
26.6
1.8
0.09
NS
3.3
NS
2.3
NS
14.8
0.011
13.2
21.72
0.015
0.23
0.067
0.011
0.007
0.006
1870
40
33392
3636
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
28
29
30
36
38
39
40
45
46
47
48
40.7
25.3
24.9
21.7
16.7
24.4
20.8
34.6
27.5
35.8
24.8
22.3
29.8
33.4
26.8
36.9
30.7
40.7
33.2
35.9
41.0
20.8
33.2
33.3
19.2
17.3
18.5
1.2
1.4
13.8
250
10.1
16.1
2.8
3022
2.7
1.9
1389
145
497
1748
2473
3087
a All data presented are averages of at least three separate experiments
performed by Panacos Pharmaceuticals Inc., Gaithersburg, MD. EC50:
concentration that inhibits HIV-1IIIB replication by 50%. CC50: concentration
that inhibits mock-infected MT-2 cell growth by 50%. TI ) CC50/EC50.
NS: no suppression at concentrations below the CC50.
antiviral EC50 values of 3.3, 1.8, and 2.3 µM against HIV-1IIIB
,
respectively. These results demonstrated that the C-30 position
of BA can accommodate some diverse ethers without decreasing
the anti-HIV potency. Moreover, the introduction of the
morpholinoethoxy moiety in 20, which reduced the log P value
of 23 from 9.79 to 8.26 (calculated by ACD/LogP DB software),
resulted in an increase in the derivative’s solubility, confirming
that the C-30 position may serve as a good place to incorporate
water-solubilizing moieties.

3252 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Qian et al.
Table 3. In Vitro Metabolic Stability of Compounds 23 and 38a
Table 2. Anti-HIV-1 Replication Activities in HIV-1NL4-3 Infected MT-4
Cell Linesa
percentage of remaining parent compounds,
compd
EC50 (µM)
CC50 (µM)
TI
X ( SD (%)
3b
4
21
45
46
47
48
0.089
0.092
0.09
0.24
0.07
>10
>10
>10
>10
8.8
>112.4
>150
>138
>41.7
121.4
283.9
245.7
incubation time (min)
compd 23
100.0
93.4 ( 2.5
70.7 ( 1.5
61.9 ( 10.1
40.1 ( 1.6
7.9 ( 4.2
compd 38
0
5
15
30
60
120
100.0
101.7 ( 4.7
97.6 ( 6.2
81.4 ( 8.6
62.7 ( 4.3
53.8 ( 3.8
0.035
0.031
8.5
8.6
a All data presented are averages of at least two separate experiments
performed by Dr. Chin-Ho Chen, Duke University, NC. EC50: concentration
that inhibits HIV-1NL4-3 replication by 50%. CC50: concentration that inhibits
mock-infected MT-4 cell growth by 50%. TI ) CC50/EC50. NS: no
suppression at the testing concentration (10 µM).
a Data presented are averages of two separate experiments.
human liver microsomes. A similar result was found with
buspirone (t1/2 ) 31 min), an established fast-metabolized drug
used as reference in the same experiment, suggesting that 23
was degraded quite easily in the assay system. Comparatively,
it took about 125 min to lose 50% of the newly designed
analogue 38, indicating a much longer half-life (Table 3). This
result might be due to the increased steric hindrance at the C-28
pharmacophore of 38 so that the amide bond would be less
available to metabolic enzymes. Thus, 38 represents a more
stable lead for the development of C-28 modified BA derived
HIV-1 entry inhibitors and 3,28-disubstituted bifunctional
inhibitors.
From the bioassay data, we discovered that compound 39
with the C-3 side chain of 2 incorporated into 38 showed very
potent anti-HIV-1 activity with an EC50 value of 0.015 µM and
TI of 1.4 × 103, proving that the presence of a bulky amide
moiety near C-28 does not reduce the antiviral potency of 2.
Compound 40, with a 4′,4′-dimethylglutaryl rather than 3′,3′-
dimethylsuccinyl C-3 ester side chain, had a higher antiviral
EC50 value (0.23 µM), indicating the importance of the
positioning of the dimethyl substitution in the C-3 modification
of BA. However, compound 38 itself showed decreased antiviral
activity compared with 23, likely due to the slightly reduced
length of the new C-28 side chain compared with the previous
8-aminooctanoic chain (23). Indeed, the BA derivative with
7-aminoheptanoic acid as the C-28 side chain showed 16-fold
decreased anti-HIV activity compared with 23,20 confirming the
importance of the length of the C-28 side chain.
Analogue 21 with methylamine linked to the terminal
carboxylic acid of 20 exhibited potent anti-HIV-1 activity with
an EC50 value of 0.09 µM and TI of 250 against both HIV-1IIIB
and HIV-1NL4-3 variants. These data are similar to those with
the prior best entry inhibitor 4 (EC50 ) 0.10 µM, TI > 100).
This result indicates that the amide moiety near the end of the
C-28 side chain is necessary for enhanced antiviral potency.
Interestingly, 21 differs from 4 in the direction of the terminal
amide linkage (-CONHCH3 in 21 and -NHCOCH3 in 4).
Compound 21 showed a significantly reduced log P value of
7.5 compared with the lead compound 23 and prior best hit 4.
Analogue 30 with a 3′,3′-dimethylsuccinyl side chain linked
to the C-3 ꢀ-hydroxyl group of 24 showed extremely potent
antiviral activity with an EC50 value of 0.011 µM and TI of 3.0
× 103, which are similar to those of 2 (EC50 ) 0.011 µM, TI
> 3.6 × 103). This result suggests that the presence of small
substitutions on C-30 of BA does not harm the high anti-HIV-1
potency of 2. Thus, incorporation of polar groups into the C-30
position may also help to improve the hydrophilicity of 3,28-
disubstituted BA derivatives.
Because an amide is generally more stable in vivo than an
ester moiety, we also synthesized 3,28-disubstituted 3ꢀ-amino
BA derived analogue 36. Its C-3 side chain is similar to that of
2 except for a C-3 amide rather than ester bond. However, the
antiviral activity of 36 against HIV-1IIIB decreased significantly
to an EC50 value of 13.2 µM, suggesting that bioisosteric
replacement of the C-3 ester bond with an amide moiety is not
tolerated.
Results from the metabolism study revealed that changing
the C-28 side chain from 8-aminooctanoic acid (23) to 4-pip-
eridinebutyric acid (38) could significantly increase the in vitro
metabolic stability. Specifically, approximately 50% of 23
disappeared after around 35 min of incubation with pooled
Compounds 47 and 48, which contain 3′,3′-dimethylsuccinyl
at C-3 and a morpholine ring at the end of C-28 side chain that
is separated from the terminal amide bond by a short alkyl spacer
(two or three methylenes), exhibited extremely potent anti-
HIV-1 replication activity against HIV-1IIIB with EC50 values
of 0.007 and 0.006 µM, respectively, which are almost 2-fold
better than that of 2. These two compounds were also 2- to
3-fold more potent than entry inhibitors 3b and 4 in the anti-
Figure 2. Summary of SAR of BA analogues.

Betulinic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3253
3-O-Acetylbetulinic Acid (5). A mixture of 1 (2.1 g), pyridine
(1.5 mL), and acetic anhydride (Ac2O, 20 mL) was stirred at room
temperature overnight until it became homogeneous. The mixture
was then poured into ice-cold water (30 mL) and stirred for 20
min. The crude product was filtered off and purified on a silica-gel
column to yield 1.98 g (87% yield) of pure 5, white amorphous
powder. Mp 289-291 °C. MS (ESI-) m/z: 497.38 (M- - H) for
HIV screening against HIV-1NL4-3. Compound 46, with mor-
pholine directly involved in the amide bond, and 2 had
equivalent anti-HIV-1IIIB potency (EC50 ) 0.011 µM). Com-
pound 45 with methylamine at the end of C-28 showed a slightly
decreased antiviral potency (EC50 ) 0.067 µM) against HIV-
1IIIB compared with 2. These results further confirm the
importance of the length of the C-28 side chain to the enhanced
antiviral potency in 3,28-disubstituted BA analogues. The better
potency of 47 and 48 indicates that the activity of 2 can be
increased with proper C-28 substitution. Moreover, because the
C-28 side chains in all prior potent HIV inhibitors terminate in
a free carboxylic acid or amide, the success of 47 and 48 also
demonstrates that other polar groups at the end of this side chain
can also increase antiviral potency.
1
C32H50O4. H NMR (300 MHz, CDCl3): δ 4.74, 4.61 (1H each, s,
H-29), 4.47 (1H, dd, J ) 9.9, 5.9 Hz, H-3), 3.01 (1H, m, H-19),
2.05 (3H, s, OCOCH3), 1.69 (3H, s, H-30), 0.97, 0.93, 0.86, 0.84,
0.83 (3H each, s, 5 × CH3).
Syntheses of BA Derivatives 6, 7, and 37. Oxalyl chloride
solution (2 M in CH2Cl2, 10 mL) was added to 5 (1 equiv) in
CH2Cl2 (10 mL) and stirred for 2 h. After concentration under
vacuum, the residual mixture was treated with leucine methyl ester
(1.6 equiv), 8-aminooctanoic acid methyl ester (1.6 equiv), or
4-piperidinebutyric acid methyl ester (1.6 equiv) and triethylamine
(Et3N, 1.2 equiv) in CH2Cl2. The mixture was stirred at room
temperature overnight until no starting material was observed by
TLC. The solution was then diluted with CH2Cl2 (20 mL) and
washed three times with brine and distilled water. The organic layer
was dried over anhydrous Na2SO4 and concentrated to dryness under
reduced pressure. The residue was chromatographed using a silica
gel column to yield the pure target compounds.
In conclusion, diverse 28,30-disubstituted BA analogues were
synthesized in our study. We discovered that a hydrogen bond
donor is not tolerated near the C-19 isopropenyl moiety.
Otherwise, C-30 substitution did not significantly influence the
anti-HIV-1 activity of BA derivatives. Therefore, the C-30
position serves as a good place to incorporate water-solubilizing
moieties to increase the hydrophilicity. The resulting analogue
21 showed a good solubility as well as equal potency against
HIV-1 compared with the previous best antientry hit 4. Using
a cyclic secondary amine moiety (piperidine) rather than a
primary amine to form the C-28 amide bond significantly
increased the metabolic stability of the derivatives in pooled
human liver microsome assessment. Subsequent introduction
of a second amide bond at the carboxylic terminus of this
metabolically stable C-28 side chain and introduction of the
3′,3′-dimethylsuccinyl side chain at the C-3 position resulted
in the discovery of 47 and 48, which showed extremely potent
antiviral activity slightly better than that of 2. They should serve
as attractive promising leads for the development of a next
generation of BA derived 3,28-disubstituted HIV-1 inhibitors,
as clinical trial candidates.
Methyl N-[3ꢀ-acetoxy-lup-20(29)-en-28-oyl]leucinate (6): 1.15 g
(80.5% yield) starting from 1 g of 5; white amorphous powder.
Mp 230-232 °C. MS (ESI+) m/z: 626.48 (M+ + H), 648.47 (M+
1
+ Na) for C39H63NO5. H NMR (300 MHz, CDCl3): δ 5.87 (1H,
d, J ) 8 Hz, -CONH-), 4.72, 4.59 (1H each, s, H-29), 4.64 (1H,
m, -NHCH-), 4.49 (1H, t, J ) 8 Hz, H-3), 3.73 (3H, s,
-COOCH3), 3.05 (1H, m, H-19), 2.10-2.20 (1H, m, H-13), 2.04
(3H, s, OCOCH3), 1.68 (3H, s, H-30), 1.01 (6H, s, leucine moiety
-(CH3)2), 0.97 (6H, s, 2 × CH3), 0.89, 0.84, 0.83 (3H each, s, 3
× CH3).
Methyl N-[3ꢀ-acetoxy-lup-20(29)-en-28-oyl]-8-aminooctano-
ate (7): 643 mg (98% yield) starting from 500 mg of 5; light-
yellow amorphous powder. Mp 104-105 °C. MS (ESI+) m/z: 654.5
1
(M+ + H) for C41H67NO5. H NMR (300 MHz, CDCl3): δ 5.57
(1H, t, J ) 6 Hz, -CONH-), 4.73, 4.60 (1H each, s, H-29), 4.45
(1H, m, H-3), 3.67 (3H, s, -COOCH3), 3.30-3.08 (3H, m, H-19,
-CONHCH2-), 2.50 (1H, m, H-13), 2.31 (2H, t, J ) 7 Hz,
-CH2COOCH3), 2.05 (3H, s, OCOCH3), 1.68 (3H, s, H-30), 0.97,
0.94 (3H each, s, 2 × CH3), 0.85, 0.84, 0.81 (3H each, s, 3 ×
CH3).
Experimental Section
Chemistry. The melting points were measured with a Fisher
Johns melting apparatus without correction. 1H NMR spectra were
measured on a 300 MHz Varian Gemini 2000 spectrometer using
Me4Si (TMS) as internal standard. The solvent used was CDCl3
unless otherwise indicated. Mass spectra were measured on
Shimadzu LCMS-2010 (ESI-MS). High resolution mass spectra
(HRMS) were measured on Shimadzu LCMS-IT-TOF with ESI
interface. Elemental analyses were performed by Atlantic Microlab,
Inc., Norcross, GA. Target compounds were analyzed for C, H and
gave values within (0.4% of the theoretical values. HPLC for purity
determinations were conducted using Shimadzu LCMS-2010 with
a Grace Alltima 2.1 mm × 100 mm HP C18 3 µm column and a
Shimadzu SPD-M20A detector at 200 nm wavelength. Two
different solvent systems for HPLC purity analyses were as follows:
(1) solvent B was acetonitrile, and solvent A was water with or
without 0.1% formic acid; (2) solvent B was methanol, and solvent
A was water with or without 0.1% formic acid. The flow rate was
0.3 mL/min. The isocratic HPLC mode was used, and the specific
solvent percentage conditions for each tested compound were listed
in the Supporting Information. All target compounds possessed a
purity of at least 95% as determined by elemental analysis or by
HPLC-UV-MS. Optical rotations were measured with a Jasco
Dip-2000 digital polarimeter at 20 °C at the sodium D line. Thin-
layer chromatography (TLC) and preparative thin-layer chroma-
tography (PTLC) were performed on Merck precoated silica gel
60 F-254 plates. Flash+ and CombiFlash systems (Teledyn-Isco)
were used as medium pressure column chromatography. Silica gel
(200-400 mesh) from Aldrich, Inc., was used for column chro-
matography. All other chemicals were obtained from Aldrich, Inc.
Methyl N-[3ꢀ-acetoxy-lup-20(29)-en-28-oyl]-4-piperidinebu-
tanoate (37): 1.02 g (94% yield) starting from 800 mg of 5; white
amorphous powder. Mp 195-197 °C. MS (ESI+) m/z: 666.5 (M+
1
+ H) for C42H67NO5. H NMR (300 MHz, CDCl3): δ 4.72, 4.57
(1H each, s, H-29), 4.47 (1H, dd, J ) 11.1, 5.7 Hz, H-3), 3.67
(3H, s, -COOCH3), 3.67-3.47 (4H, m, 28-CON(CH2CH2)2CH-),
2.99 (1H, m, H-19), 2.31 (2H, t, J ) 7 Hz, -CH2COOCH3), 2.05
(3H, s, OCOCH3), 1.68 (3H, s, H-30), 0.96 (6H, s, 2 × CH3), 0.94,
0.82, 0.75 (3H each, s, 3 × CH3).
Syntheses of BA Derivatives 8 and 9. A mixture of N-
bromosuccinimide (1.1 equiv) and 6 or 7 (1 equiv) in acetonitrile
(ACN, 30 mL) was stirred at room temperature until the starting
material was not observed by TLC. The mixture was concentrated
to dryness under reduced pressure and chromatographed over silica
gel to yield pure target compounds.
Methyl N-[3ꢀ-acetoxy-30-bromo-lup-20(29)-en-28-oyl]leucinate
(8): 297 mg (66% yield) starting from 400 mg of 6; light-yellow
amorphous powder. Mp 127-129 °C. MS (ESI+) m/z: 704.4 (M+
1
+ H), 706.4 (M+ + H) for C39H62BrNO5. H NMR (300 MHz,
CDCl3): δ 5.81 (1H, d, J ) 8 Hz, -CONH-), 5.11, 5.05 (1H each,
s, H-29), 4.65 (1H, m, -NHCH-), 4.45 (1H, t, J ) 8 Hz, H-3),
4.00 (2H, s, H2-30), 3.72 (3H, s, -COOCH3), 3.10 (1H, m, H-19),
2.50-2.32 (1H, m, H-13), 2.05 (3H, s, OCOCH3), 1.02 (6H, s,
leucine moiety -(CH3)2), 0.99 (6H, s, 2 × CH3), 0.89, 0.86, 0.85
(3H each, s, 3 × CH3).

3254 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Qian et al.
Methyl N-[3ꢀ-acetoxy-30-bromo-lup-20(29)-en-28-oyl]-8-ami-
nooctanoate (9): 402 mg (72.5% yield) starting from 360 mg of 7;
light-yellow amorphous powder. Mp 99-101 °C. MS (ESI+) m/z:
732.4 (M+ + H), 734.4 (M+ + H) for C41H66BrNO5. 1H NMR (300
MHz, CDCl3): δ 5.59 (1H, t, J ) 6 Hz, -CONH-), 5.13, 5.04
(1H each, s, H-29), 4.47 (1H, t, J ) 8.1 Hz, H-3), 4.00 (2H, s,
H2-30), 3.67 (3H, s, -COOCH3), 3.41-3.09 (3H, m, H-19,
-CONHCH2-), 2.46 (1H, m, H-13), 2.31 (2H, t, J)7.5 Hz,
-CH2COOCH3), 2.04 (3H, s, OCOCH3), 0.97, 0.93 (3H each, s, 2
× CH3), 0.89, 0.84, 0.83 (3H each, s, 3 × CH3).
2.85 (1H, t, J ) 7.5 Hz, H-19), 2.39 (3H, m, 30-OCH2CH2Ph(p-
OCH3), H-13), 0.95, 0.93, 0.90 (15H, s, leucine moiety -(CH3)2,
3 × CH3), 0.79, 0.75 (3H each, s, 2 × CH3). Anal. (C45H69O6N·
41/2H2O) C, H, O.
N-[3ꢀ-Hydroxy-30-(4′-fluorophenethoxy)-lup-20(29)-en-28-oyl]leu-
cine (15): 24 mg (40% yield) starting from 60 mg of 8; light-yellow
amorphous powder. Mp 102-104 °C. MS (ESI-) m/z: 706.4 (M-
- H). 1H NMR (300 MHz, CDCl3): δ 7.16-6.80 (5H, m, H ar-2′,
3′, 4′), 5.96 (1H, br s, -CONH-), 4.90, 4.89 (2H, br s, H-29),
4.48 (1H, m, -NHCH-), 3.92 (2H, s, H2-30), 3.61 (2H, t, J ) 7.2
Hz, 30-OCH2CH2Ph(p-F)), 3.17 (1H, m, H-3), 2.87 (1H, t, J ) 7.5
Hz, H-19), 2.36-2.10 (3H, m, 30-OCH2CH2Ph(p-F), H-13), 0.95,
0.88 (15H, s, leucine moiety -(CH3)2, 3 × CH3), 0.75, 0.73 (3H
each, s, 2 × CH3). Anal. (C44H66O5NF·31/2H2O) C, H, O.
N-[3ꢀ-Hydroxy-30-(4′-bromophenethoxy)-lup-20(29)-en-28-oyl]leu-
cine (16): 18 mg (41% yield) starting from 40 mg of 8; light-yellow
amorphous powder. Mp 127-129 °C. MS (ESI-) m/z: 706.4 (M-
- H). 1H NMR (300 MHz, CDCl3): δ 7.56-7.28 (5H, m, H ar-2′,
3′, 4′), 5.96 (1H, br s, -CONH-), 4.91, 4.90 (2H, br s, H-29),
4.48 (1H, m, -NHCH-), 3.91 (2H, s, H2-30), 3.60 (2H, t, J ) 7.0
Hz, 30-OCH2CH2Ph(p-Br)), 3.17 (1H, dd, J ) 11.0, 5.6 Hz, H-3),
2.89 (1H, t, J ) 7.5 Hz, H-19), 2.39 (1H, m, 30-OCH2CH2Ph(p-
Br)), 0.96 (12H, s, leucine moiety -(CH3)2, 2 × CH3), 0.82, 0.79,
0.75 (3H each, s, 3 × CH3). Anal. (C44H66O5NBr·2H2O) C, H, O.
N-[3ꢀ-Hydroxy-30-(4′-chlorophenethoxy)-lup-20(29)-en-28-oyl]leu-
cine (17): 16 mg (38% yield) starting from 40 mg of 8; light-yellow
amorphous powder. Mp 119-121 °C. MS (ESI-) m/z: 706.4 (M-
- H). 1H NMR (300 MHz, CDCl3): δ 7.18-6.87 (5H, m, H ar-2′,
3′, 4′), 5.96 (1H, br s, -CONH-), 4.91, 4.90 (2H, br s, H-29),
4.48 (1H, m, -NHCH-), 3.91 (2H, s, H2-30), 3.62 (2H, t, J ) 6.8
Hz, 30-OCH2CH2Ph(p-Cl)), 3.17 (1H, dd, J ) 11.0, 5.6 Hz, H-3),
2.87 (1H, t, J ) 7.5 Hz, H-19), 2.36-2.06 (3H, m, 30-
OCH2CH2Ph(p-Cl), H-13), 0.96 (15H, s, leucine moiety -(CH3)2,
3 × CH3), 0.81, 0.76 (3H each, s, 2 × CH3). Anal. (C44H66O5NCl·
H2O) C, H, O.
Syntheses of BA Derivatives 10-20. NaH (60% in mineral oil)
was washed three times with hexane. A solution of appropriate
nucleophilic compound (8 equiv) and NaH (10 equiv) in anhydrous
THF (1.5 mL) was stirred under dry nitrogen at room temperature
for 30 min. The 30-bromo BA derivative 8 or 9 (1 equiv) was then
added into the system. The mixture was heated using microwave
(Biotage) at 120 °C for 30 min. After the mixture was cooled to
room temperature, 1 mL of MeOH-H2O was added into the mixture
and stirred to transform the intermediate esters to carboxylic acids
by saponification. The mixture was neutralized with 10% HCl and
dried under vacuum and reconstituted with EtOAc. The organic
layer was washed with brine and dried over anhydrous Na2SO4 and
concentrated to dryness under reduced pressure. The residue was
chromatographed using a silica gel column to yield the pure target
compounds.
N-[3ꢀ-Hydroxy-30-ethoxy-lup-20(29)-en-28-oyl]leucine (10): 22
mg (59% yield) starting from 40 mg of 8; white amorphous powder.
Mp 128-130 °C. MS (ESI-) m/z: 612.4 (M- - H) for C38H63NO5.
1H NMR (300 MHz, CDCl3): δ 5.88 (1H, d, J ) 8 Hz, -CONH-),
4.93, 4.92 (2H, br s, H-29), 4.63-4.58 (1H, m, -NHCH-), 3.90
(2H, s, H2-30), 3.47 (2H, m, 30-OCH2CH3), 3.18 (1H, dd, J ) 11.1,
5.4 Hz, H-3), 2.99 (1H, m, H-19), 2.50-2.32 (1H, m, H-13), 1.00
(9H, br s, 30-OCH2CH3, leucine moiety -(CH3)2), 0.96 (6H, s, 2
× CH3), 0.89, 0.86, 0.85 (3H each, s, 3 × CH3).
N-[3ꢀ-Hydroxy-30-propoxy-lup-20(29)-en-28-oyl]leucine (11): 23
mg (60% yield) starting from 40 mg of 8; white amorphous powder.
Mp 116-117 °C. MS (ESI-) m/z: 626.5 (M- - H) for C39H65NO5.
1H NMR (300 MHz, CDCl3): δ 6.13 (1H, br s, -CONH-), 4.91,
4.90 (2H, br s, H-29), 4.52 (1H, m, -NHCH-), 3.90 (2H, s, H2-
30), 3.36 (2H, t, J ) 6.9 Hz, 30-OCH2CH2CH3), 3.18 (1H, dd, J )
11.1, 5.4 Hz, H-3), 2.99 (1H, m, H-19), 0.96, 0.94, 0.92, 0.89 (15H,
m, 30-O(CH2)2CH3, leucine moiety -(CH3)2, CH3-23, 24), 0.82,
0.81, 0.79 (3H each, s, 3 × CH3).
N-[3ꢀ-Hydroxy-30-morpholino-lup-20(29)-en-28-oyl]leucine (18):
22 mg (41% yield) starting from 60 mg of 8; white amorphous
1
powder. Mp 98-100 °C. MS (ESI-) m/z: 653.5 (M- - H). H
NMR (300 MHz, CDCl3): δ 5.61 (1H, d, J ) 6 Hz, -CONH-),
4.92, 4.90 (H each, s, H-29), 4.63-4.58 (1H, m, -NHCH-), 3.72
(4H, m, 30-N(CH2CH2)2O), 3.17 (1H, dd, J ) 11.1, 5.4 Hz, H-3),
3.00 (3H, m, H-19, H2-30), 2.53 (4H, m, 30-N(CH2CH2)2O), 2.42
(1H, m, H-13), 0.96 (6H, s, leucine moiety -(CH3)2), 0.92 (6H, s,
2 × CH3), 0.86, 0.81, 0.75 (3H each, s, 3 × CH3). Anal.
(C40H66O5N2 ·2H2O) C, H, O.
N-[3ꢀ-Hydroxy-30-butoxy-lup-20(29)-en-28-oyl]leucine (12): 10
mg (37% yield) starting from 30 mg of 8; yellow amorphous
powder. Mp 104-105 °C. MS (ESI-) m/z: 640.2 (M- - H) for
C40H67NO5. 1H NMR (300 MHz, CDCl3): δ 6.01 (1H, br s,
-CONH-), 4.90, 4.88 (2H, br s, H-29), 4.58 (1H, m, -NHCH-),
3.89 (2H, s, H2-30), 3.37 (2H, m, 30-OCH2(CH2)2CH3), 3.17 (1H,
m, H-3), 3.01 (1H, m, H-19), 0.99 (9H, br s, 30-O(CH2)3CH3,
leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3), 0.86, 0.84, 0.81
(3H each, s, 3 × CH3).
N-[3ꢀ-Hydroxy-30-(2′-morpholinoethoxy)-lup-20(29)-en-28-oyl]leu-
cine (19): 26 mg (56% yield) starting from 50 mg of 8; white
amorphous powder. Mp 89-91 °C. MS (ESI-) m/z: 697.4 (M- -
H). 1H NMR (300 MHz, CDCl3): δ 5.61 (1H, d, J ) 8 Hz,
-CONH-), 4.92, 4.90 (H each, s, H-29), 4.59 (1H, m, -NHCH-),
3.94 (2H, s, H2-30), 3.72 (4H, m, -N(CH2CH2)2O), 3.58 (2H, t, J
) 5.7 Hz, 30-OCH2CH2-morpholine), 3.18 (1H, dd, J ) 11.4, 4.6
Hz, H-3), 3.01 (1H, m, H-19), 2.60 (2H, t, J ) 5.4 Hz,
30-OCH2CH2-morpholine), 2.53 (4H, m, -N(CH2CH2)2O), 1.00
(6H, s, leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3), 0.89, 0.85,
0.80 (3H each, s, 3 × CH3). Anal. (C42H70O6N2 ·H2O) C, H, O.
N-[3ꢀ-Hydroxy-30-(2′-morpholinoethoxy)-lup-20(29)-en-28-oyl]-
8-aminooctanoic acid (20): 51 mg (64% yield) starting from 80
mg of 9; white amorphous powder. Mp 111-112 °C. MS (ESI-)
m/z: 725.5 (M- - H) for C44H74N2O6. 1H NMR (300 MHz, CDCl3):
δ 5.61 (1H, d, J ) 8 Hz, -CONH-), 4.91, 4.90 (H each, s, H-29),
3.94 (2H, s, H2-30), 3.72 (4H, m, -N(CH2CH2)2O), 3.58 (2H, t, J
) 5.7 Hz, 30-OCH2CH2-morpholine), 3.18 (3H, m, -CONHCH2-,
H-3), 3.01 (1H, m, H-19), 2.60 (2H, t, J ) 5.4 Hz, 30-OCH2CH2-
morpholine), 2.53 (4H, m, -N(CH2CH2)2O), 2.28 (2H, t, J ) 7.5
Hz, -CH2COOH), 0.96 (6H, s, 2 × CH3), 0.92, 0.81, 0.75 (3H each,
s, 3 × CH3).
N-[3ꢀ-Hydroxy-30-phenethoxy-lup-20(29)-en-28-oyl]leucine (13):
37 mg (77% yield) starting from 50 mg of 8; light-yellow
amorphous powder. Mp 155-157 °C. MS (ESI-) m/z: 688.4 (M-
- H). 1H NMR (300 MHz, CDCl3): δ 7.68-7.62 (2H, m, H ar-3′),
7.28-7.20 (3H, m, H ar-2′, 4′), 5.97 (1H, br s, -CONH-), 4.91,
4.90 (2H, br s, H-29), 4.44 (1H, m, -NHCH-), 3.93 (2H, s, H2-
30), 3.64 (2H, t, J ) 7.2 Hz, 30-OCH2CH2Ph), 3.17 (1H, dd, J )
11.1, 5.4 Hz, H-3), 2.91 (1H, m, H-19), 2.57 (2H, m, 30-
OCH2CH2Ph), 0.95 (12H, s, leucine moiety -(CH3)2, CH3-23, 24),
0.89, 0.78, 0.74 (3H each, s, 3 × CH3). Anal. (C44H67O5N·2H2O)
C, H, O.
N-[3ꢀ-Hydroxy-30-(4′-methoxyphenethoxy)-lup-20(29)-en-28-
oyl]leucine (14): 46 mg (64% yield) starting from 70 mg of 8; light-
yellow amorphous powder. Mp 128-129 °C. MS (ESI-) m/z: 718.5
(M- - H). 1H NMR (300 MHz, CDCl3): δ 7.27, 7.16-7.13,
6.85-6.82 (5H, m, H ar-2′, 3′, 4′), 5.97 (1H, br s, -CONH-),
4.91, 4.89 (H each, br s, H-29), 4.48 (1H, m, -NHCH-), 3.93
(2H, s, H2-30), 3.79 (3H, s, ar-OCH3), 3.60 (2H, t, J ) 7.2 Hz,
30-OCH2CH2Ph(p-OCH3)), 3.17 (1H, dd, J ) 11.1, 5.4 Hz, H-3),
Syntheses of BA Derivatives 26 and 27. A solution of 30-bromo
BA derivative 8 or 9 (1 equiv), silver acetate (AgOAc, 2 equiv)
and tetrabutylammonium bromide (Bu4NBr, 0.2 equiv) in aceto-
nitrile (1.5 mL) was heated using microwave at 100 °C for 25 min.

Betulinic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3255
The precipitant was filtered, and the solution was concentrated to
dryness under vacuum. The residue was chromatographed over a
silica gel column to yield the pure diacetoxy intermediates 26 and
27.
H2-30), 3.17 (1H, dd, J ) 11.2, 5.6 Hz, H-3), 3.01 (1H, m, H-19),
2.34 (1H, m, H-13), 1.10 (6H, s, leucine moiety -(CH3)2), 0.99
(6H, s, 2 × CH3), 0.86, 0.83, 0.80 (3H each, s, 3 × CH3). [R]D25
-39.3 ° (c 0.35, MeOH).
N-[3ꢀ,30-Dihydroxy-lup-20(29)-en-28-oyl]-8-aminooctanoic acid
(29): 50 mg (80% yield) starting from 58 mg of 27; white
amorphous powder. Mp 135-137 °C. MS (ESI-) m/z: 612.5 (M-
- H) for C38H63NO5. 1H NMR (300 MHz, CDCl3): δ 5.61 (1H, br
s, -CONH-), 4.94, 4.90 (1H each, s, H-29), 4.12 (2H, s, H2-30),
3.25-3.15 (3H, m, H-3, -CONHCH2-), 3.01 (1H, m, H-19), 2.34
(2H, t, J ) 7.6 Hz, -CH2COOH), 2.06 (1H, m, H-13), 0.97, 0.96
(3H each, s, 2 × CH3), 0.92, 0.82, 0.75 (3H each, s, 3 × CH3).
[R]2D5 -143.3 ° (c 0.10, MeOH).
N′-[3ꢀ-N-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]leucine (36):
24 mg (98%) starting from 25 mg of 35; white amorphous powder.
Mp 248-250 °C. MS (ESI-) m/z: 695.5 (M- - H) for C42H68N2O6.
1H NMR (300 MHz, CDCl3): δ 5.87 (1H, d, J ) 7.6 Hz,
-CONH-), 4.72, 4.58 (1H each, s, H-29), 4.64 (1H, m,
-NHCH-), 3.59 (1H, m, H-3), 2.99 (1H, m, H-19), 2.64-2.42
(2H, m, H-2′), 1.68 (3H, s, H-30), 1.30, 1.26 (3H each, s, 2 ×
CH3-3′), 1.00 (6H, s, leucine moiety -(CH3)2), 0.96 (6H, s, 2 ×
CH3), 0.89, 0.86, 0.85 (3H each, s, 3 × CH3). [R]2D5 -16.1 ° (c
0.28, MeOH).
Methyl N-[3ꢀ,30-diacetoxy-lup-20(29)-en-28-oyl]leucinate (26):
80 mg (68% yield) starting from 120 mg of 117; off-white
amorphous powder. Mp 201-203 °C. MS (ESI+) m/z: 684.5 (M+
+ H) for C41H65NO7. 1H NMR (300 MHz, CDCl3): δ 5.69 (1H, br
s, -CONH-), 4.97, 4.94 (2H, d, J ) 9, H-29), 4.58-4.52 (3H, m,
-NHCH-, H2-30), 4.45 (1H, t, J ) 8 Hz, H-3), 3.72 (3H, s,
-COOCH3), 3.10 (1H, m, H-19), 2.50-2.32 (1H, m, H-13), 2.08
(6H, s, 2 × OCOCH3), 1.05 (6H, s, leucine moiety -(CH3)2), 0.96
(6H, s, 2 × CH3), 0.89, 0.82, 0.81(3H each, s, 3 × CH3).
Methyl N-[3ꢀ,30-diacetoxy-lup-20(29)-en-28-oyl]-8-aminooc-
tanoate (27): 77.8 mg (69.5% yield) starting from 80 mg of 118;
white amorphous powder. Mp 167-169 °C. MS (ESI+) m/z: 712.5
1
(M+ + H) for C43H69NO7. H NMR (300 MHz, CDCl3): δ 5.60
(1H, t, J ) 4.6 Hz, -CONH-), 4.94, 4.90 (1H each, s, H-29),
4.56 (2H, s, H2-30), 4.45 (1H, t, J ) 7 Hz, H-3), 3.66 (3H, s,
-COOCH3), 3.41-3.09 (3H, m, H-19, -CONHCH2-), 2.46 (1H,
m, H-13), 2.31 (2H, t, J ) 7.5 Hz, -CH2COOCH3), 2.05 (6H, s,
2 × OCOCH3), 0.97, 0.96, 0.85, 0.81, 0.80 (3H each, s, 5 × CH3).
Syntheses of BA Derivatives 22-25, 28, 29, 36, and 38. To a
solution of the appropriate ester intermediates 6-9, 26, 27, 35, and
37 (1 equiv) in MeOH (8 mL) and THF (4 mL) was added 2 N
NaOH (4 mL). The mixture was stirred overnight and then
neutralized with 20% HCl. The solution was dried under vacuum
and reconstituted with EtOAc. The organic layer was washed with
brine and dried over anhydrous Na2SO4 and concentrated to dryness
under reduced pressure. The residue was chromatographed using a
silica gel column to yield the pure target compounds.
N-[3ꢀ-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidinebutyric acid
(38): 190 mg (100%) starting from 200 mg of 37; white amorphous
powder. Mp 145-146 °C. MS (ESI-) m/z: 608.4 (M- - H) for
1
C39H63NO4. H NMR (300 MHz, CDCl3): δ 4.72, 4.57 (1H each,
s, H-29), 3.67-3.47 (4H, m, 28-CON(CH2CH2)2CH-), 3.19 (1H,
m, H-3), 2.99 (1H, m, H-19), 2.31 (2H, t, J ) 8.4 Hz,
-CH2COOH), 1.68 (3H, s, H-30), 0.96 (6H, s, 2 × CH3), 0.94,
0.82, 0.81 (3H each, s, 3 × CH3). [R]2D5 -22.7 ° (c 0.33, MeOH).
Synthesis of BA Derivatives 21 and 41-44. A solution of 20 or
38 (1 equiv), EDCI (2 equiv), N-hydroxybenzotriazole (HOBt, 1
equiv), Et3N (0.05 mL), and the appropriate amine (2 equiv) in
anhydrous CH2Cl2 (8 mL) was stirred at room temperature overnight
until the starting material was not observed by TLC. The solution
was diluted with CH2Cl2 (20 mL) and washed three times with
brine and distilled water. The organic layer was dried over
anhydrous Na2SO4 and concentrated to dryness under reduced
pressure. The residue was chromatographed using a silica gel
column to yield pure target compounds.
N-[3ꢀ-Hydroxy-lup-20(29)-en-28-oyl]leucine (22): 27 mg (100%
yield) starting from 30 mg of 6; white amorphous powder. Mp
243-244 °C. MS (ESI-) m/z: 568.42 (M- - H) for C36H59NO4.
1H NMR (300 MHz, CDCl3): δ 5.86 (1H, d, J ) 8 Hz, -CONH-),
5.11, 5.02 (1H each, s, H-29), 4.65 (1H, m, -NHCH-), 3.17 (1H,
dd, J ) 9.7, 5.4 Hz, H-3), 3.10-3.03 (1H, m, H-19), 1.68 (3H, s,
H-30), 1.00 (6H, s, leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3),
0.83, 0.80, 0.79 (3H each, s, 3 × CH3). [R]2D5 -17.2 ° (c 1.40,
CHCl3).
N-[3ꢀ-Hydroxy-lup-20(29)-en-28-oyl]-8-aminooctanoic acid (23):
37 mg (100% yield) starting from 40 mg of 7; white amorphous
powder. Mp 110-112 °C. MS (ESI-) m/z: 596.5 (M- - H) for
C38H63NO4. 1H NMR (300 MHz, CDCl3): δ 5.60 (1H, br s,
-CONH-), 4.73, 4.60 (1H each, s, H-29), 3.21-3.09 (4H, m, H-3,
H-19, -CONHCH2-), 2.31 (2H, t, J ) 6.9 Hz, -CH2COOH),
2.10-2.20 (1H, m, H-13), 1.68 (3H, s, H-30), 0.97 (6H, s, 2 ×
CH3), 0.85, 0.79, 0.75 (3H each, s, 3 × CH3). [R]2D5 -3.6 ° (c 0.19,
CHCl3). [R]2D5 -8.48 ° (c 0.20, MeOH).
ꢀ-Hydroxy-30-(2′-morpholinoethoxy)-lup-20(29)-en-28-oyl]-8-
aminooctanoyl]aminomethane (21): 21 mg (69% yield) starting
from 30 mg of 20; white amorphous powder. Mp 106-107 °C.
1
MS (ESI+) m/z: 740.5 (M+ + H) for C45H77N3O5. H NMR (300
MHz, CDCl3): δ 5.61 (2H, m, 2 × -CONH-), 4.92, 4.90 (H each,
s, H-29), 3.94 (2H, s, H2-30), 3.72 (4H, m, -N(CH2CH2)2O), 3.58
(2H, t, J ) 5.7 Hz, 30-OCH2CH2-morpholine), 3.28-3.14 (3H, m,
-CONHCH2-, H-3), 3.01 (1H, m, H-19), 2.81 (3H, d, J ) 4.8
Hz, -CONHCH3), 2.60 (2H, t, J ) 5.4 Hz, 30-OCH2CH2-
morpholine), 2.53 (4H, m, -N(CH2CH2)2O), 2.16 (2H, t, J ) 7.5
Hz, -CH2CONHCH3), 0.96 (6H, s, 2 × CH3), 0.92, 0.81, 0.75
(3H each, s, 3 × CH3).
N-[3ꢀ-Hydroxy-30-bromo-lup-20(29)-en-28-oyl]leucine (24): 102
mg (100% yield) starting from 110 mg of 8; white amorphous
powder. Mp 102-104 °C. MS (ESI-) m/z: 646.41, 648.39 (M- -
H) for C36H58BrNO4. 1H NMR (300 MHz, CDCl3): δ 5.86 (1H, d,
J ) 8 Hz, -CONH-), 5.11, 5.02 (1H each, s, H-29), 4.65 (1H, m,
-NHCH-), 3.90 (2H, s, H2-30), 3.17 (1H, dd, J ) 9.7, 5.4 Hz,
H-3), 3.10-3.03 (1H, m, H-19), 1.00 (6H, s, leucine moiety
-(CH3)2), 0.96 (6H, s, 2 × CH3), 0.83, 0.80, 0.79 (3H each, s, 3
× CH3). [R]2D5 -10.5 ° (c 0.15, MeOH).
N′-[N-[3ꢀ-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidinebutanoyl]-
aminomethane (41): 46 mg (100% yield) starting from 50 mg of
38, off-white amorphous powder. Mp 202-204 °C. MS (ESI+)
m/z: 623.5 (M+ + H) for C40H66N2O3. 1H NMR (300 MHz, CDCl3):
δ 5.44 (1H, br s, -CONHCH3), 4.69, 4.54 (1H each, s, H-29),
3.58-3.54 (4H, m, 28-CON(CH2CH2)2CH-), 3.16 (1H, m, H-3),
2.95 (1H, m, H-19), 2.78 (3H, d, J ) 4.8 Hz, -CONHCH3), 2.16
(2H, t, J ) 7.5 Hz, -CH2CONHCH3), 1.66 (3H, s, H-30), 0.93
(6H, s, 2 × CH3), 0.92, 0.90, 0.79 (3H each, s, 3 × CH3). [R]D25
-10.7° (c 0.19, MeOH).
N-[3ꢀ-Hydroxy-30-bromo-lup-20(29)-en-28-oyl]-8-aminooctano-
ic acid (25): 44 mg (95% yield) starting from 50 mg of 9; white
amorphous powder. Mp 119-122 °C. MS (ESI-) m/z: 674.4 (M-
- H) for C38H62BrNO4. 1H NMR (300 MHz, CDCl3): δ 5.60 (1H,
br s, -CONH-), 5.13, 5.04 (1H each, s, H-29), 4.00 (2H, s, H2-
30), 3.21-3.09 (4H, m, H-3, H-19, -CONHCH2-), 2.34 (2H, m,
-CH2COOH), 0.96 (6H, s, 2 × CH3), 0.92, 0.82, 0.81 (3H each,
s, 3 × CH3). [R]2D5 -16.5 ° (c 0.22, MeOH).
ꢀ-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]morpho-
line (42): 53 mg (87% yield) starting from 50 mg of 38, white
amorphous powder. Mp 132-133 °C. MS (ESI+) m/z: 679.5 (M+
1
N-[3ꢀ,30-Dihydroxy-lup-20(29)-en-28-oyl]leucine (28): 24 mg
(98% yield) starting from 30 mg of 26; white amorphous powder.
Mp 145-148 °C. MS (ESI-) m/z: 584.5 (M- - H) for C36H59NO5.
1H NMR (300 MHz, CDCl3): δ 5.89 (1H, br s, -CONH-), 4.91,
4.90 (1H each, s, H-29), 4.68 (1H, m, -NHCH-), 4.12 (2H, s,
+ H) for C43H70N2O4. H NMR (300 MHz, CDCl3): δ 4.69, 4.54
(1H each, s, H-29), 3.66-3.60 (8H, m, 28-CON(CH2CH2)2CH-,
-CON(CH2CH2)2O), 3.42 (4H, m, -CON(CH2CH2)2O), 3.14 (1H,
m, H-3), 2.95-2.60 (1H, m, H-19), 2.27 (2H, t, J ) 9.2 Hz,
-CH2CON(CH2CH2)2O), 1.65 (3H, s, H-30), 0.93 (3H each, s, 2

3256 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Qian et al.
× CH3), 0.91, 0.83, 0.79 (3H each, s, 3 × CH3). [R]2D5 -20.0° (c
0.31, MeOH).
dropwise over 45 min. The mixture was stirred at room temperature
overnight and then treated with 2 N NaOH until pH 10. The solution
was dried under vacuum, and the residual aqueous solution was
extracted with CH2Cl2 and washed with distilled water until pH 7
was attained. The organic layer was then dried over anhydrous
Na2SO4 and concentrated to dryness under vacuum. The residue
was chromatographed using a silica gel column to yield 80 mg
(82%) of pure 34, white amorphous powder. Mp 135-137 °C. MS
(ESI+) m/z: 582.5 (M+ + H) for C37H59NO4. 1H NMR (300 MHz,
CDCl3): δ 5.86 (1H, d, J ) 7 Hz, -CONH-), 4.72, 4.58 (1H each,
s, H-29), 4.64 (1H, m, -NHCH-), 3.73 (3H, s, -COOCH3), 3.05
(1H, m, H-19), 2.44 (1H, m, H-3), 2.10-1.90 (1H, m, H-13), 1.68
(3H, s, H-30), 0.97 (9H, s, CH3-23, leucine moiety-(CH3)2), 0.96,
0.94, 0.93, 0.92 (3H each, s, 4 × CH3).
N′-[N-[3ꢀ-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-
2-aminoethylmorpholine (43): 54 mg (91% yield) starting from 55
mg of 38, white amorphous powder. Mp 114-116 °C. MS (ESI+)
m/z: 722.6 (M+ + H), 744.5 (M+ + Na) for C45H75N3O4. 1H NMR
(300 MHz, CDCl3): δ 6.79 (1H, br, s, -CONHCH2-), 4.68, 4.53
(1H each, s, H-29), 3.69-3.67 (8H, m, 28-CON(CH2CH2)2CH-,
-CH2N(CH2CH2)2O), 3.31 (2H, m, -CONHCH2-), 3.17 (1H, m,
H-3), 2.95-2.60 (1H, m, H-19), 2.43 (6H, m, -CH2N(CH2CH2)2O),
2.17 (2H, t, J ) 7.5 Hz, -CH2CONHCH2-), 1.63 (3H, s, H-30),
0.93 (3H each, s, 2 × CH3), 0.90, 0.79, 0.72 (3H each, s, 3 ×
CH3). [R]2D5 -10.6° (c 0.15, MeOH).
N′-[N-[3ꢀ-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-
3-aminopropylmorpholine (44): 54 mg (90% yield) starting from
50 mg of 38, white amorphous powder. Mp 122-124 °C. MS
(ESI+) m/z: 736.6 (M+ + H) for C46H77N3O4. 1H NMR (300 MHz,
CDCl3): δ 5.93 (1H, br, s, -CONHCH2-), 3.15 (1H, m, H-3),
2.95-2.60 (1H, m, H-19), 2.47-2.41 (6H, m, -CH2N(CH2CH2)2O),
2.16 (2H, t, J ) 7.5 Hz, -CH2CONHCH2-), 1.65 (3H, s, H-30),
0.93 (3H each, s, 2 × CH3), 0.91, 0.79, 0.72 (3H each, s, 3 ×
CH3). [R]2D5 -7.5° (c 0.18, MeOH).
Synthesis of BA Derivatives 30, 35, 39, 40, 45-48. A solution
of the appropriate BA analogue (1 equiv), DMAP (1.5 equiv), and
the appropriate acid anhydride (5 equiv) in anhydrous pyridine (1.5
mL) was stirred at 160 °C for 2 h using microwave (Biotage). The
reaction mixture was diluted with EtOAc (15 mL) and washed three
times with 20% HCl solution and distilled water. The organic layer
was dried over anhydrous Na2SO4 and concentrated to dryness under
reduced pressure. The residue was chromatographed using a silica
gel column to yield pure target compounds.
3-Deoxybetulinic Acid (31). To a solution of 1 (2 g, 1 equiv) in
DMF was added pyridium dichromate (PDC, 2 equiv). The mixture
was stirred at room temperature for 2 h. The reaction mixture was
diluted with EtOAc (30 mL), and the precipitate was filtered through
a short pack of Florisil. The solution was washed with 20% HCl
and distilled water. The organic layer was dried over anhydrous
Na2SO4 and concentrated to dryness under vacuum. The residue
was chromatographed using a silica gel column to yield 1.68 g
(87%) of pure 31, white powder. Mp 246-248 °C. MS (ESI-)
N-[3ꢀ-O-(3′,3′-Dimethylsuccinyl)-30-bromo-lup-20(29)-en-28-
oyl]leucine (30): 20 mg (32% yield) starting from 50 mg of 24;
light-yellow amorphous powder. Mp 105-107 °C. MS (ESI-) m/z:
1
774.5 (M- - H) for C42H66BrNO7. H NMR (300 MHz, CDCl3):
δ 5.86 (1H, d, J ) 8 Hz, -CONH-), 5.11, 5.02 (1H each, s, H-29),
4.65 (1H, m, -NHCH-), 4.54 (1H, dd, J ) 11.2, 5.7 Hz, H-3),
3.90 (2H, s, H2-30), 2.99 (1H, m, H-19), 2.64-2.42 (2H, m, H-2′),
1.30, 1.26 (3H each, s, 2 × CH3-3′), 1.00 (6H, s, leucine moiety
-(CH3)2), 0.96 (6H, s, 2 × CH3), 0.87, 0.86, 0.81 (3H each, s, 3
× CH3).
1
m/z: 453.3 (M- - H) for C30H46O3. H NMR (300 MHz, CDCl3):
δ 4.72, 4.58 (1H each, s, H-29), 3.09 (1H, m, H-19), 2.41-2.26
(2H, m, H-2), 1.69 (3H, s, H-30), 0.98, 0.97, 0.96, 0.92, 0.89 (3H
each, s, 5 × CH3).
MethylN′-[3ꢀ-N-(3′,3′-dimethylsuccinyl)-lup-20(29)-en-28-oyl]leuci-
nate (35): 37 mg (38% yield) starting from 80 mg of 34; white
amorphous powder. Mp 187-189 °C. MS (ESI+) m/z: 711.5 (M+
Methyl N-[3-Deoxy-lup-20(29)-en-28-oyl]leucinate (32). A solu-
tion of 31 (500 mg, 1 equiv), DMAP (0.6 equiv), and EDCI (1.6
equiv) in CH2Cl2 was stirred at 0 °C for 30 min. Leucine methyl
ester (1.6 equiv) and Et3N (1 equiv) was then added into the system
and stirred at room temperature overnight. The mixture was diluted
with CH2Cl2 (20 mL) and washed with brine. The organic layer
was then dried over anhydrous Na2SO4 and concentrated to dryness
under vacuum. The residue was chromatographed using a silica
gel column to yield 372 mg (58%) of pure 32, white amorphous
powder. Mp 191-193 °C. MS (ESI+) m/z: 582.5 (M+ + H) for
1
+ H), 733.4 (M+ + Na) for C43H70N2O6. H NMR (300 MHz,
CDCl3): δ 5.69 (1H, br s, -CONH-), 4.70, 4.58 (1H each, s, H-29),
4.62 (1H, m, -NHCH-), 3.73 (3H, s, -COOCH3), 3.59 (1H, m,
H-3), 2.99 (1H, m, H-19), 2.64-2.42 (2H, m, H-2′), 1.68 (3H, s,
H-30), 1.30, 1.26 (3H each, s, 2 × CH3-3′), 1.09 (6H, s, leucine
moiety -(CH3)2), 0.97 (6H, s, 2 × CH3), 0.92, 0.82, 0.80 (3H each,
s, 3 × CH3).
N-[3ꢀ-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-pipe-
ridinebutyric acid (39): 20 mg (41% yield) starting from 50 mg of
38, white amorphous powder. Mp 116-118 °C. MS (ESI+) m/z:
738.6 (M+ + H). MS (ESI-) m/z: 736.5 (M- - H) for C45H71NO7.
1H NMR (300 MHz, CDCl3): δ 4.72, 4.57 (1H each, s, H-29), 4.47
(1H, t, J ) 7.5, H-3), 3.65-3.50 (4H, m, 28-CON(CH2CH2)2CH-),
2.98 (1H, m, H-19), 2.64-2.42 (2H, m, H-2′), 2.30 (2H, t, J ) 7.2
Hz, -CH2COOH), 1.68 (3H, s, H-30), 1.27, 1.25 (3H each, s, 2 ×
CH3-3′), 0.95, 0.93, 0.84, 0.83, 0.82 (3H each, s, 5 × CH3). [R]D25
-27.7° (c 0.30, MeOH).
1
C37H59NO4. H NMR (300 MHz, CDCl3): δ 5.87 (1H, d, J ) 8.4
Hz, -CONH-), 4.70, 4.59 (1H each, s, H-29), 4.64 (1H, m,
-NHCH-), 3.73 (3H, s, -COOCH3), 3.05 (1H, m, H-19),
2.41-2.26 (2H, m, H-2), 1.68 (3H, s, H-30), 1.06, 1.02 (3H each,
s, leucine moiety -(CH3)2), 0.98 (3H, s, CH3), 0.96 (6H, s, 2 ×
CH3), 0.92, 0.89 (3H each, s, 2 × CH3).
Methyl N-[3-Oxime-lup-20(29)-en-28-oyl]leucinate (33). A solu-
tion of 32 (230 mg, 1 equiv) and hydroxylamine hydrochloride (4
equiv) in pyridine (10 mL) was heated at 50 °C for 2 h. After
cooling to room temperature, the reaction mixture was diluted with
CH2Cl2 and washed three times by 20% HCl and brine. The organic
layer was then dried over anhydrous Na2SO4 and concentrated to
dryness under vacuum. The residue was chromatographed using a
silica gel column to yield 235 mg (90%) of pure 33, white
amorphous powder. Mp 213-215 °C. MS (ESI+) m/z: 582.5 (M+
+ H) for C37H59NO4. 1H NMR (300 MHz, CDCl3): δ 5.87 (1H, d,
J ) 8.4 Hz, -CONH-), 4.70, 4.59 (1H each, s, H-29), 4.64 (1H,
m, -NHCH-), 3.73 (3H, s, -COOCH3), 3.05 (1H, m, H-19),
2.20-2.15 (2H, m, H-2), 1.67 (3H, s, H-30), 1.05, 1.02 (3H each,
s, leucine moiety -(CH3)2), 0.98 (3H, s, CH3), 0.96 (6H, s, 2 ×
CH3), 0.94, 0.92 (3H each, s, 2 × CH3).
N-[3ꢀ-O-(4′,4′-Dimethylglutaryl)-lup-20(29)-en-28-oyl]-4-pipe-
ridinebutyric acid (40): 12 mg (38% yield) starting from 30 mg of
38, white amorphous powder. Mp 143-145 °C. MS (ESI+) m/z:
752.4 (M+ + H), (ESI-) m/z: 750.4 (M- - H) for C46H73NO7. 1H
NMR (300 MHz, CDCl3): δ 4.72, 4.57 (1H each, s, H-29), 4.47
(1H, t, J ) 7.5, H-3), 3.65-3.50 (4H, m, 28-CON(CH2CH2)2CH-),
3.00 (1H, m, H-19), 2.35-2.30 (4H, m, H-2′, -CH2COOH), 1.68
(3H, s, H-30), 1.27, 1.25 (3H each, s, 2 × CH3-3′), 0.96 (6H, s, 2
× CH3), 0.89, 0.86, 0.82 (3H each, s, 3 × CH3). [R]2D5 -23.1° (c
0.20, MeOH).
N′-[N-[3ꢀ-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-pi-
peridinebutanoyl]aminomethane (45): 17 mg (48% yield) starting
from 30 mg of 41, white amorphous powder. Mp 166-169 °C.
1
Methyl N-[3ꢀ-Amino-lup-20(29)-en-28-oyl]leucinate (34). To a
solution of 33 (100 mg, 1 equiv) and ammonium acetate (15 equiv)
in MeOH was added sodium cyanoborohydride (NaCNBH3, 20
equiv) under nitrogen atmosphere. The mixture was cooled to 0
°C, and 15% aqueous titanium trichloride (TiCl3, 3 equiv) was added
MS (ESI+) m/z: 751.6 (M+ + H) for C46H74N2O6. H NMR (300
MHz, CDCl3): δ 5.43 (1H, br s, -CONHCH3), 4.69, 4.54 (1H each,
s, H-29), 4.47 (1H, t, J ) 7.5, H-3), 3.68-3.60 (4H, m, 28-
CON(CH2CH2)2CH-), 2.98 (1H, m, H-19), 2.79 (3H, d, J ) 3.4
Hz, -CONHCH3), 2.67-2.62 (2H, m, H-2′), 2.14 (2H, t, J ) 6.8

Betulinic Acid DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3257
Hz, -CH2CONHCH3), 1.65 (3H, s, H-30), 1.30, 1.25 (3H each, s,
2 × CH3-3′), 0.93, 0.91 (3H each, s, 2 × CH3), 0.90, 0.80, 0.79
(3H each, s, 3 × CH3). [R]2D5 -25.0 ° (c 0.12, MeOH).
was acetonitrile (B) in water (A, with 0.1% formic acid and 5 mM
ammonium acetate) at 55%. After staying at initial condition for 3
min, the concentration of B increased linearly to 90% at 15 min
and stayed at 90% for 2 min. The mobile phase was then returned
to the initial condition and re-equilibrated for 3 min. The MS
conditions were optimized to detector voltage: +1.35 kV; acquisi-
tion mode, SIM of the appropriate molecular weights of the testing
compounds. The CDL temperature is 200 °C, heat block is 230
°C, and neutralizing gas flow is 1.5 L/min. Samples were injected
by autosampler. Electrospray ionization was operated in the positive
ion mode. Full-scan spectra were also monitored over the range of
180-1000 m/z.
HIV-1IIIB Replication Inhibition Assay in MT-2 Lymphocytes.
The evaluation of HIV-1 inhibition was carried out as follows. The
human T-cell line, MT-2, was maintained in continuous culture
with complete medium (RPMI 1640 with 10% fetal calf serum
supplemented with L-glutamine at 5% CO2 and 37 °C. Test samples
were first dissolved in dimethyl sulfoxide (DMSO) at a concentra-
tion of 10 mg/mL to generate master stocks with dilutions made
into tissue culture media to generate working stocks. The following
drug concentrations were routinely used for screening: 100, 20, 4,
and 0.8 µg/mL. For agents found to be active, additional dilutions
were prepared for subsequent testing so that an accurate EC50 value
could be determined. Test samples were prepared, and to each
sample well, was added 90 µL of media containing MT-2 cells at
3 × 105 cells/mL and 45 µL of virus inoculum (HIV-1IIIB isolate)
containing 125 TCID50. Control wells containing virus and cells
only (no drug) and cells only (no virus or drug) were also prepared.
A second identical set of samples were added to cells under the
same conditions without virus (mock infection) for cytotoxicity
determinations (CC50 defined below). In addition, AZT and beviri-
mat were also assayed during each experiment as positive drug
controls. On day 4 postinfection, the assay was terminated and
culture supernatants were harvested for p24 antigen ELISA analysis.
The compound cytotoxicity was determined by XTT using the
mock-infected sample wells. The detailed procedure was described
previously.33,34 If a test sample inhibited virus replication and was
not cytotoxic, its effects were reported in the following terms: EC50,
the concentration of the test sample that was able to suppress HIV
replication by 50%; CC50, the concentration of test sample that was
toxic to 50% of the mock-infected cells; therapeutic index (TI),
the ratio of the CC50 to EC50.
N′-[N-[3ꢀ-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-pi-
peridinebutanoyl]morpholine (46): 23 mg (55% yield) starting from
35 mg of 42, off-white amorphous powder. Mp 122-124 °C. MS
(ESI+) m/z: 807.6 (M+ + H) for C49H78N2O7. 1H NMR (300 MHz,
CDCl3): δ 4.69, 4.54 (1H each, s, H-29), 4.45 (1H, t, J ) 6.9,
H-3), 3.66-3.61 (8H, m, 28-CON(CH2CH2)2CH-, -CON
(CH2CH2)2O), 3.45-3.42 (4H, m, -CON(CH2CH2)2O), 2.99-2.82
(1H, m, H-19), 2.67-2.52 (2H, m, H-2′), 2.28 (2H, t, J ) 7.8 Hz,
-CH2CON(CH2CH2)2O), 1.65 (3H, s, H-30), 1.26 (6H, s, 2 × CH3-
3′), 0.92, 0.90 (3H each, s, 2 × CH3), 0.79 (6H, s, 2 × CH3), 0.77
(3H, s, CH3). [R]D25 -19.1° (c 0.41, MeOH).
N′-[N-[3ꢀ-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-pi-
peridinebutanoyl]-2-aminoethylmorpholine (47): 14 mg (41%
yield) starting from 30 mg of 43, white amorphous powder. Mp
121-123 °C. MS (ESI+) m/z: 850.4 (M+ + H) for C51H83N3O7.
1H NMR (300 MHz, CDCl3): δ 7.01 (1H, br, s, -CONHCH2-),
4.70, 4.54 (1H each, s, H-29), 4.45 (1H, t, J ) 10.2, H-3),
3.81-3.78 (8H, m, 28-CON(CH2CH2)2CH-, -CH2N(CH2CH2)2O),
3.48 (2H, m, -CONHCH2-), 2.85-2.70 (7H, m, -CH2N
(CH2CH2)2O, H-19), 2.60-2.52 (2H, m, H-2′), 2.14 (2H, t, J )
7.5 Hz, -CH2CONHCH2-), 1.25 (6H, s, 2 × CH3-3′), 1.63 (3H,
s, H-30), 0.93 (3H each, s, 2 × CH3), 0.90, 0.79, 0.72 (3H each, s,
3 × CH3). [R]2D5 -18.0° (c 0.16, MeOH).
N′-[N-[3ꢀ-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-pi-
peridinebutanoyl]-3-aminopropylmorpholine (48): 17 mg (47%
yield) starting from 30 mg of 44, white amorphous powder. Mp
125-127 °C. MS (ESI+) m/z: 864.6 (M+ + H) for C52H85N3O7.
1H NMR (300 MHz, CDCl3): δ 6.76 (1H, br, s, -CONHCH2-),
4.69, 4.54 (1H each, s, H-29), 4.30 (1H, m, H-3), 3.81-3.67 (8H,
m, 28-CON(CH2CH2)2CH-, -CH2N(CH2CH2)2O), 3.28 (2H, m,
-CONHCH2-), 2.96 (1H, m, H-19), 2.80-2.73 (6H, m,
-CH2N(CH2CH2)2O), 2.60-2.52 (2H, m, H-2′), 2.16 (2H, t, J )
7.5 Hz, -CH2CONHCH2-), 1.65 (3H, s, H-30), 1.25, 1.24 (6H, s,
2 × CH3-3′), 0.92, 0.90 (3H each, s, 2 × CH3), 0.80, 0.79, 0.78
(3H each, s, 3 × CH3). [R]2D5 -14.1° (c 0.24, MeOH).
In Vitro Metabolic Stability Assessment. Materials. BA deriva-
tives 23 and 38 were synthesized and characterized in our study.
NADPH, MgCl2, KH2PO4, formic acid, and ammonium acetate were
purchased from Sigma-Aldrich. Reference compounds (fast-
metabolized, buspirone, propranolol; moderate-metabolized, atenolol;
and slow-metabolized, imipramine) were also purchased from
Sigma-Aldrich. HPLC-grade acetonitrile and water were purchased
from VWR. Pooled human liver microsomes (lot no. 70196) were
purchased from BD biosciences (Woburn, MA).
Sample Preparation. Stock solutions of 23 and 38 (1 mg/mL)
were prepared by dissolving the pure compound in methanol and
stored at 4 °C. For measurement of metabolic stability, four
reference compounds as well as test compounds 23 and 38 were
brought to a final concentration of 3 µM with 0.1 M potassium
phosphate buffer at pH 7.4, which contained 0.2 mg/mL human
liver microsome and 5 mM MgCl2. The incubation volumes were
800 µL. Reactions were started by adding 80 µL of NADPH (final
concentration of 1.0 mM) and stopped by taking the aliquots over
time, then adding to 1.5 volumes of ice-cold acetonitrile. Incubations
of all samples were conducted in duplicate. For each sample, 100
µL aliquots were taken out at 0, 5, 15, 30, 60, 120 min time points.
After addition of 150 µL of ice-cold acetonitrile, the mixture was
centrifuged at 12 000 rpm for 5 min at 0 °C. The supernatant was
collected, and 20 µL of the supernatant was directly injected to
LCMS. The following controls were also conducted: (1) positive
control incubations that contain liver microsomes, NADPH, and
the fast-metabolized substrate propranolol; (2) negative control
incubations that omit NADPH; (3) baseline control that only contain
liver microsomes and NADPH.
HIV-1NL4-3 Replication Inhibition Assay in MT-4 Lymphocytes.
A previously described HIV-1 infectivity assay was used.30,35
A
96-well microtiter plate was used to set up the HIV-1NL4-3 replication
screening assay. NL4-3 variants at a multiplicity of infection (MOI)
of 0.01 were used to infect MT4 cells. Culture supernatants were
collected on day 4 postinfection for the p24 antigen capture using
an ELISA kit from ZeptoMetrix Corporation (Buffalo, NY).
Acknowledgment. This investigation was supported by
Grant AI-077417 from the National Institute of Allergy and
Infectious Diseases (NIAID) awarded to K.-H.L.
Supporting Information Available: Additional information on
compound purity, high-resolution mass spectral data, HPLC analysis
results, and elemental analysis data of the target compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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