Anti-AIDS agents 81. design, synthesis, and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors

Article abstract of DOI:10.1021/jm901782m

In our continuing study of triterpene derivatives as potent anti-HIV agents, different C-3 conformationally restricted betulinic acid (BA, 1) derivatives were designed and synthesized in order to explore the conformational space of the C-3 pharmacophore. 3-O-Monomethylsuccinyl-betulinic acid (MSB) analogues were also designed to better understand the contribution of the C-3′ dimethyl group of bevirimat (2), the first-in-class HIV maturation inhibitor, which is currently in phase IIb clinical trials. In addition, another triterpene skeleton, moronic acid (MA, 3), was also employed to study the influence of the backbone and the C-3 modification toward the anti-HIV activity of this compound class. This study enabled us to better understand the structure-activity relationships (SAR) of triterpene-derived anti-HIV agents and led to the design and synthesis of compound 12 (EC₅₀: 0.0006 μM), which displayed slightly better activity than 2 as a HIV-1 maturation inhibitor.

Full text of DOI:10.1021/jm901782m

J. Med. Chem. 2010, 53, 3133–3141 3133
DOI: 10.1021/jm901782m
Anti-AIDS Agents 81. Design, Synthesis, and Structure-Activity Relationship Study of Betulinic Acid
and Moronic Acid Derivatives as Potent HIV Maturation Inhibitors†
Keduo Qian,‡ Reen-Yun Kuo,‡ Chin-Ho Chen,§ Li Huang,§ Susan L. Morris-Natschke,‡ and Kuo-Hsiung Lee*,‡
‡Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, and
§Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710
Received December 1, 2009
In our continuing study of triterpene derivatives as potent anti-HIV agents, different C-3 conforma-
tionally restricted betulinic acid (BA, 1) derivatives were designed and synthesized in order to explore the
conformational space of the C-3 pharmacophore. 3-O-Monomethylsuccinyl-betulinic acid (MSB)
analogues were also designed to better understand the contribution of the C-30 dimethyl group of
bevirimat (2), the first-in-class HIV maturation inhibitor, which is currently in phase IIb clinical trials.
In addition, another triterpene skeleton, moronic acid (MA, 3), was also employed to study the influence
of the backbone and the C-3 modification toward the anti-HIV activity of this compound class. This
study enabled us to better understand the structure-activity relationships (SAR) of triterpene-derived
anti-HIV agents and led to the design and synthesis of compound 12 (EC50: 0.0006 μM), which displayed
slightly better activity than 2 as a HIV-1 maturation inhibitor.
Introduction
one ART.8 Therefore, novel potent antiretroviral agents with
different targets than currently approved drugs may hold
particular promise in addressing issues of current therapies.
Our prior modification study on betulinic acid (BA, 1)
resulted in the discovery of bevirimat [3-O-(30,30-dimethyl-
succinyl)-betulinic acid, 2].9 Bevirimat exhibits extremely potent
antiviral activity against HIV-1 primary isolates and several
drug-resistant viruses and represents a unique first in a class of
anti-HIV compounds termed maturation inhibitors (MIs).10,11
MIs block the last step of viral Gag precursor polyprotein
processing from p25 (CA-SP1) to functional p24 (CA), resulting
in the production of noninfectious immature HIV-1 particles.
Most importantly, MIs retain their high anti-HIV potency
against different viral strains that are resistant to current ARTs,
including AZT (NRTI), Nevirapine (NNRTI), and Indinavir
(PI). Compound 2 has recently succeeded in phase IIb clinical
trials and is in preparation for phase III clinical trials.12-14
To summarize our prior structure-activity relationship
(SAR) study of 2 and other 3-O-acyl-BA derivatives, we know
that C-3 ester substitution is important to the enhanced
antiviral activity.9,15 Within the C-3 side chain, the proper
length, a terminal carboxylic acid, and C-30 dimethyl substi-
tution contribute to antiviral potency. However, the C-3 ester
groups of prior BA analogues have mainly contained freely
rotatable alkyl chains. Therefore, inthe present study, fiveC-3
conformationally restricted BA analogues (4-8) were synthe-
sized in order to explore the conformational space of the C-3
pharmacophore. Two 3-O-monomethylsuccinyl betulinic
acid (MSB) derivatives (9-10) and compound 11 were further
designed to investigate how the methyl substituents on the C-3
side chain impact the antiviral potency.
It is estimated that at the end of 2008, approximately 33.2
million people were infected worldwide with human immuno-
deficiency virus (HIVa), the etiologic cause of acquired immu-
nodeficiency syndrome (AIDS). Each year, around 2.7 million
more people become infected with HIV and 2 million die of
AIDS.1 Considering that the development of a safe and effective
HIV vaccine is still in the future,2 the current research continues
to focus on the disease treatment by chemical anti-HIV agents.
Although significant progress has been made since the intro-
duction of highly active antiretroviral therapy (HAART),3,4
which employs a combinational use of nucleoside/nucleotide
reverse transcriptase inhibitors (NRTIs), non-nucleoside re-
verse transcriptase inhibitors (NNRTIs), and/or protease inhi-
bitors (PIs), it has also led to some serious problems, including
increased adverse effects and the emergence of multidrug-
resistant viral strains.5-7 Drug-resistant virus is then involved
in HIV transmission, and more than 25% of newly infected
individuals harbor HIV-1 isolates that are resistant to at least
†For part 80, see Lee, K. H. Discovery and Development of Natural
Product-derived Chemotherapeutic Agents Based on a Medicinal
Chemistry Approach J. Nat. Prod. 2010 DOI: 10.1021.np900821e.
*To whom correspondence should be addressed. Phone: 919-962-
0066. Fax: 919-966-3893. E-mail: khlee@unc.edu.
a Abbreviations: AIDS, acquired immunodeficiency syndrome; HIV-1,
human immunodeficiency virus type 1; BA, betulinic acid; MA, moronic
acid; HAART, highly active antiretroviral therapy; NRTI, nucleoside/
nucleotide reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse
transcriptase inhibitor; PI, protease inhibitor; MI, maturation inhibitor;
P24 (CA), capsid; P25 (CA-SP1), capsid precursor; Bevirimat, 3-O-(30,30-
dimethylsuccinyl)-betulinic acid; MSB, 3-O-monomethylsuccinyl betulinic
acid; EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlor-
ide; DMAP, 4-(dimethylamino)pyridine; TBDPS, tert-butyldimethylsilyl;
TBAF, tetra-n-butylammonium fluoride; THF, tetrahydrofuran; TEM-
PO, 2,2,6,6-tetramethyl-1-piperidinyloxy; PhI(OAc)2, (diacetoxyiodo)
benzene; NaBH4, sodium borohydride; Et3N, triethylamine; TFAA, tri-
fluoroacetic anhydride; AZT, zidovudine; PI, postinfection.
Meanwhile, moronic acid (MA, 3), which was isolated from
Brazilian propolis, also exhibited promising anti-HIV activity
as a lead compound with an EC50 of 0.1 μg/mL.16 However,
insertion of the same 30,30-dimethylsuccinyl side chain found
r
2010 American Chemical Society
Published on Web 03/23/2010
pubs.acs.org/jmc

3134 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Qian et al.
Chemistry
Compounds 4 and 5 were synthesized by reaction of the
corresponding cycloalkanedicarboxylic acid with the C-3 β-
hydroxyl group of 1 in the presence of 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride (EDCI) and di-
methylaminopyridine (DMAP), resulting in yields of 26%
and 35%, respectively (Scheme 1). Compounds 6-8 and 11
were synthesized according to Scheme 2. Reaction of the
corresponding acid anhydride with 1 furnished the target
compounds in yields of 35-55%.
The synthesis of MSB analogues was reported before in
ref 17. As shown in Scheme 3, the hydroxyl of 3R-bromo-2-
methylpropanol (22a) was first protected by a tert-butyl-
dimethylsilyl (TBDPS) moiety to yield 23a quantitatively.
The bromide group was then replaced with a cyano moiety
(24a), which was reduced to an aldehyde moiety (25a)18 and
consequentially oxidized to a carboxylic acid (26a). Reaction
of 1 with 26a led to esterification of the 3β-hydroxy group of 1
to provide 27a. After cleavage of the TBDPS group with
TBAF in THF, the primary hydroxyl of 28 was oxidized to a
carboxylic acid in the presence of 2,2,6,6-tetramethyl-1-piper-
idinyloxy (TEMPO) and PhI(OAc)2 to give 3-O-(30R-
methylsuccinyl)-betulinic acid (30R-MSB, 9).19 The 30S-MSB
isomer (10) was synthesized using the same method starting
with 3S-bromo-2-methylpropanol (22b).
Scheme 4 depicts the synthesis of MA analogues. Com-
pound 3 was first treated with NaBH4 to yield 3β-hydroxyl
groupof 30. Differentacyl chloride was then reactedwith30in
the presence of Et3N to furnish 3-O-acyl-MA analogues
13-21.
The synthesis of 12 started from the commercially available
2-ethyl-2-methylsuccinic acid (31), which was stirred in tri-
fluoroacetic anhydride (TFAA) to form 2-ethyl-2-methylsuc-
cinic anhydride (32). The reaction of 32 with 1 furnished
3-O-(30-ethyl-30-methylsuccinyl) betulinic acid (12) in 55%
yield (Scheme 5).
Figure 1. Structures of MSB analogues and other 3-O-acyl BA
analogues.
in 2 at the 3β position of morolic acid (30), the C-3 ketone
reduced analogue of 3, unexpectedly did not increase the
antiviral activity.16 Because 2 and 30 share similar 3D con-
formational space of their triterpene skeletons, this un-
expected result led us to further investigate possible modi-
fications on analogues of 3 and 30.
Detailed SAR of triterpene-derived HIV-1 maturation inhi-
bitors has been established from the current study. This
analysis led to the design and synthesis of compound 12 with
slightly better activity than 2.
Results and Discussion
The newly synthesized conformationally restricted 3-O-
acyl-BA analogues (4-8) were first tested in acutely HIV-
1IIIB infected MT-2cell lines and compared to2 and AZT. The
data are listed in Table 1. Only the 2-methylmaleic acid
substituted BA derivative (8) showed moderate anti-HIV-1
activity, with a TI of 1.4 ꢀ 102 and an EC50 of 0.18 μM. A
pendant cyclopentyl or cyclohexyl ring within the C-3 side
chain reduced the antiviral potency of the derivatives (4-7)
significantly. One possible reason for the reduced or abolished
activity of these compounds is that the pendant ring moieties
are locked into a conformation that is not the bioactive one.
Alternatively, steric hindrance at the 20-position [as was seen
with 3-O-(20,20-dimethylsuccinyl) BA, which had an EC50 of
only 2.7 μM]9 may be a contributing factor to the loss in
potency. This hindrance may impart an unfavorable interac-
tion with either the binding site, resulting in reduced affinity,
or the triterpene template, leading to an unfavorable C-3 side
chain conformation.
In contrast, 3-O-(40,40-dimethylglutaryl)-betulinic acid (11)
had an antiviral EC50 of 0.048 μM and a TI of 8.7 ꢀ 102. This
result confirms that the dimethyl moiety is essential to the
anti-HIV-1 potency of 3-O-acyl-BA analogues. Although
moving the dimethyl substitution to the 20-position was highly
detrimental, moving the dimethyl group to the 40-position
of the C-3 side chain was well tolerated and significantly
Design. Because the presence of C-3 substitution is
critical to the anti-HIV-1 activity of BA derivatives, five
conformationally restricted 3-O-acyl-BA analogues (4-8)
(Figure 1) were first synthesized and evaluated in order to
further explore the conformational space of this pharma-
cophore. The C-30 dimethyl group was also moved toward
the 40-position in a C-3 glutaryl-substituted compound
(11) (Figure 1) to study the influence of different position-
ing of the methyl groups. In addition, although we know
that the presence of the C-30 dimethyl within the C-3 side
chain is vital to anti-HIV-1 activity, it is still unclear which,
if either, C-30 methyl group of 2 contributes more toward
activity. Therefore, 3-O-monomethylsuccinyl betulinic
acid (MSB) derivatives (9-10) (Figure 1) were designed
and synthesized.17 Their antiviral activities were then
evaluated in vitro against HIV-1IIIB replication in MT-2
cell lines. Diverse substitutions (13-21) (Scheme 4) were
also incorporated into 30 to study their influence on
the antiviral potency and the impact of the triterpene
skeleton itself on the antiviral activity. After reviewing
the initial promising bioassay results, compound 12
(Figure 1) was then designed to try to improve the antiviral
potency of 2.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3135
Scheme 1. Synthesis of Compounds 4 and 5
Scheme 2. Syntheses of Compounds 6-8 and 11
Scheme 3. Total Syntheses of Compounds 9 and 10
increased the antiviral activity compared with that of the
unesterified compound 1. However, the anti-HIV-1 activity
of 11 was still 20-fold less than that of 2, indicating that the
30-position, rather than 40-position, of the C-3 side chain
remains the optimal substitution position. Overall, results of
the conformationally restricted compounds (4-8) and 11
stimulated our interest in further study of the importance of
the C-30 dimethyl moiety and the exact contribution of both
groups to the anti-HIV-1 potency of 2.
The MSB analogues 9 and 10 were then synthesized and
evaluated in parallel with 2 and AZT against viral replication
in HIV-1IIIB infected MT-2 cell lines. A 1:1 mixture (33) of 9
and 10 was also tested. The anti-HIV-1 activity data of these
derivatives are also listed in Table 1. Among the MSB
derivatives, compound 10 with 30S-methyl showed very po-
tent antiviral activity with a TI of 3.8 ꢀ 103 and EC50 of 0.0087
μM, which is comparable to that of 2 (EC50: 0.0013 μM).
Compound 9 with 30R-methyl exhibited only moderate anti-
HIV-1 activity with a TI of 3.7 ꢀ 102 and EC50 of 0.12 μM.
The antiviral activity (EC50: 0.016 μM) of the mixture (33) of
the two stereoisomers fell in between those of 9 and 10. This
result indicates that the two C-30 methyl groups in the C-3
ester side chain contribute differently to the extremely potent
anti-HIV-1 activity of this compound class. We postulate that
interaction of the 30S-methyl group with the viral target might
be essential to the anti-HIV-1 activity of 2. The interaction of

3136 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Scheme 4. Synthesis of MA Analogues 13-21
Qian et al.
Scheme 5. Synthesis of Compound 12
the 30R-methyl group within the target is less significant but
still necessary because 2, with dimethyl substitution at C-30
position, is slightly more potent than 10.
Table 1. Anti-HIV-1 Replication Activities for BA and MA Derivatives
in Acutely Infected MT-2 Cell Linesa
compd
EC50 (μM)
IC50 (μM)
therapeutic index
To further elucidate the mechanism of action, the produc-
tion of virus from 10-treated HIV-1 wild-type (NL4-3) or
resistant variants transfected HeLa cells were subjected to
characterization. Radioimmunoprecipitation analyses re-
vealed that, like 2, 10 also functions as a maturation inhibitor.
In detail, 10 specifically inhibited the conversion of p25 (CA-
SP1) to p24 (CA) in both cell and virion lysates (Figure 3
showing the results from virion lysate), which led to defective
Gag processing and production of morphologically abnor-
mal, noninfectious virion particles.
The anti-HIV replication activity of newly synthesized MA
analogues 13-21 were tested against HIV-1IIIB and summarized
in Table 1 as well. These compounds have diverse substitutions
at the 3β position of analogue 30 to explore the conformational
space to enhance the antiviral potency. However, none of them
showed significant anti-HIV activity. Nevertheless, although the
insertion of the 30,30-dimethylsuccinyl side chain into the 3β of
30 did not follow the general trend of increasing the activity, the
current study indicates that a terminal polar moiety, such as a
carboxylic acid group, may still be necessary for the compounds
to exert their antiviral replication activity. It also proves that the
triterpene skeleton itself plays an important role in the enhanced
anti-HIV activity of BA derivatives.
AZT
2
0.034
0.0013
2.2
1870
42.78
41.89
40.93
41.06
40.27
42.51
43.83
32.78
41.75
36.41
>10
>10
>10
>10
>10
>10
>10
>10
>10
44.93
55000
32907
19
4
5
17.3
NS
2.4
6
7
NS
8
0.18
0.12
0.0087
0.048
0.0006
NS
236.2
365.3
3768
9
10
11
12
869.8
60683
13
14
NS
NS
15
16
NS
NS
17
18
NS
NS
19
20
NS
NS
21
33 (9 þ 10)
0.016
2808
a All data presented are averages of at least two separate experiments
performed by Panacos Pharmaceutical Inc. EC50: concentration that
inhibits HIV-1 replication by 50%. IC50: concentration that inhibits
mock-infected cell growth by 50%. TI = IC50/EC50. NS: no suppression
at the testing concentration.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3137
Figure 2. Isolation of the two isomers of compound 12 by JAI LC-918 recycling preparative HPLC. Mobile phase: 85% acetonitrile in water
(0.1% TFA). Flow rate: 4.0 mL/min. Detector: refractive index (RI). Column: Alltima 10 mm ꢀ 250 mm C18 5 μ. Peak 1 is 12a (30S isomer), and
peak 2 is 12b (30R isomer).
Figure 3. Effect of compound 10 on virus particle production and Gag processing in virion lysate. Note the accumulation of p25 in the presence
of 10. A1 V is a CA-SP1 mutant resistant to 2. L231 M is a PI-resistant viral mutant.
As stated above, our investigation of the C-30 chiral center of
the anti-HIV-1 clinical trials agent 2showed that the 30S-methyl
group is the major contributor to enhanced anti-HIV-1 activity,
while the 30R-methyl group is less important but still necessary.
Accordingly, we postulated that the latter effect may be due to
insufficient interaction or a slightly different positioning of the
30Sor 30Rmoiety. To confirm our hypothesis, one of the methyl
groups was further enlarged to an ethyl group to give the
30-ethyl-30-methyl substitution found in 12, which is a mixture
of 30R and 30S isomers. Compound 12 was evaluated and
compared with 2 and AZT in a HIV-1IIIB infected MT-2 cell
line. It showed very potent antiviral activity with an EC50 of
0.0006 μM and TI of 6.1 ꢀ 104, which was more active than 33,
the mixture of 9 and 10. Indeed, its potency was slightly better
than those of 10 and 2.
the high potency of 2. A C-3 terminal polar moiety, such as
carboxylic acid, is also very important to the antiviral activity,
as was proven by MA analogues 13-21. Further SAR study
of the C-30 chiral center of the newly designed MSB analogues
(9-10) revealed that the 30S-methyl group of 2 is the major
contributor to the compound’s enhanced anti-HIV-1 activity.
This result led us to design and synthesize compound 12,
which has an enlarged C-30 substituent (methylethyl instead of
dimethyl). As we anticipated, 12 showed extremely potent
antiviral activity and was slightly better than 2. Further
investigation confirmed that 12a, the 30S isomer, is the active
compound. This result provides us with better SAR informa-
tion tofurtherdesign the nextgeneration ofpotentBAderived
HIV-1 maturation inhibitors.
To identify which isomer is the active component, the two
C-30 diastereoisomers (12a and 12b) of 12 were separated by
using recycling preparative HPLC (JAI LC-918). After four
cycles of separation, the two isomers were obtained success-
fully (Figure 2). Their purities were ascertained by analytical
Experimental Section
Chemistry. The melting points were measured with a Fisher
Johns melting apparatus without correction. H NMR spectra
1
were measured on a 300 MHz Varian Gemini 2000 spectrometer or
500 MHz Inova spectrometer using Me4Si (TMS) as internal
standard. The solvent used was CDCl3 unless otherwise indicated.
Mass spectra were measured on Shimadzu LCMS-2010 and
LCMS-IT/TOF (ESI-MS). HPLC for purity determinations were
conducted using Shimadzu LCMS-2010 with a Grace Alltima
2.1 mm ꢀ 150 mm HP C18 5 μ column or a 2.1 mm ꢀ 100 mm
HP C18 3 μ column and a Shimadzu SPD-M20A detector at
205 nm wavelength. Two different solvent systems for HPLC
purity analyses were as follows: (1) acetonitrile:water=80:20, (2)
MeOH:water=90:10. The isocratic HPLC mode was used, and the
flow rate was 0.3 mL/min. All target compounds were at least 95%
pure, as determined 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) was performed
on Merck precoated silica gel 60 F-254 plates. Flashþ and Combi-
Flash systems were used as medium pressure column chromato-
graphy. All other chemicals were obtained from Aldrich, Inc.
1
HPLC. By comparing the H NMR spectra and the optical
rotation data of 2, 9, 10, 12a, and 12b, we assigned 12a as the
30S isomerand 12b asthe 30R isomer. The antiviral activities of
12a and 12b were determined in HIV-1NL4-3 infected MT-4
lymphocytes, where 12a was active and 12b showed no
antiviral activity. This result indicates that within the C-3
pharmacophore of BA-derived maturation inhibitors, the 30S
substitution is critical to the anti-HIV activity. Modification
on this site may further increase the antiviral potency of the
current maturation inhibitors.
To conclude our SAR investigation of the C-3 modification
on triterpene-derived anti-HIV agents, we found that no
conformationally restricted 3-O-acyl BA analogue (4-8)
showed significant antiviral activity, indicating that C-30
dimethyl substitution of the succinyl side chain is crucial to

3138 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Qian et al.
Synthesis of BA Derivatives 4 and 5. A solution of 1 (1 equiv),
EDCI (8 equiv), DMAP (2 equiv), and the proper cyclo-
alkanedicarboxylic acid (5 equiv) in anhydrous CH2Cl2 (8 mL)
was stirred at rt 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 the pure
target compounds.
s, CH3-23, 24), 0.86, 0.85, 0.83 (3H each, s, CH3-25, 26, 27).
[R]20D -0.77° (c=0.13, MeOH).
Synthesis of 23a and 23b. A solution of 22a or 22b (1 equiv),
imidazole (1.1 equiv), and TBDPSCl (1.1 equiv) in dry DMF
was stirred at rt until the starting material was not observed by
TLC. The reaction mixture was diluted with EtOAc and washed
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 the pure target compounds.
(3R-Bromo-2-methylpropoxy)(tert-butyl)diphenylsilane (23a).
Yield 2.03 g (100%) from 22a; colorless oil. MS (ESIþ) m/z:
391.2 (Mþ þ H), 413.2 (Mþ þ Na) for C20H27BrOSi.
(3S-Bromo-2-methylpropoxy)(tert-butyl)diphenylsilane (23b).
Yield 1.44 g (100%) from 22b; colorless oil. MS (ESIþ) m/z:
391.1 (Mþ þ H), 413.1 (Mþ þ Na) for C20H27BrOSi.
Synthesis of 24a and 24b. To a solution of 23a or 23b (1 equiv)
in dry DMSO (10 mL) was added sodium cyanide (3 equiv). The
mixture was stirred at 120 °C for 2 h until the starting material
was not observed. After cooling to rt, the reaction was diluted
with diethyl ether (30 mL) and washed with distilled water. The
organic layer was dried over anhydrous Na2SO4 and concen-
trated to dryness under reduced pressure. The residue was
chromatographed using a silica gel column to yield the pure
target compounds.
3β-O-[(20R, 30R)-30-Carboxycyclopentanecarbonyl]-betulinic
Acid (4). Yield 26% (starting with 150 mg of 1); white amor-
phous powder; mp 228-230 °C. MS (ESI-) m/z: 595.4
(M- - H) for C37H56O6. 1H NMR (300 MHz, CDCl3): δ
4.70, 4.57 (1H each, s, H-29), 4.45 (1H, dd, J = 7.8, 5.7 Hz,
H-3), 3.20 (1H, m, H-19), 3.01, 2.88 (1H each, m, H-30, H-20),
1.85-2.06 (6H, m, 3 ꢀ CH2, H-40, 50, 60), 1.65 (3H, s, H-30), 0.94
(6H, s, 2 ꢀ CH3), 0.85, 0.81, 0.75 (3H each, s, 3 ꢀ CH3). [R]20
D
-15.29° (c=0.17, MeOH).
3β-O-[(20R, 30R)-30-Carboxycyclohexanecarbonyl]-betulinic
Acid (5). Yield 35% (starting with 150 mg of 1); white amor-
phous powder; mp 233-235 °C. MS (ESI-) m/z: 609.4
(M- - H) for C38H58O6. 1H NMR (300 MHz, CDCl3): δ
4.71, 4.59 (1H each, s, H-29), 4.46 (1H, dd, J=10.2, 5.1 Hz,
H-3), 2.98 (1H, m, H-19), 2.89, 2.71 (1H each, m, H-30, H-20),
1.84-2.16 (8H, m, 4 ꢀ CH2, H-40, 50, 60, 70), 1.66 (3H, s, H-30),
4-(tert-Butyldiphenylsilyloxy)-3R-methylbutanenitrile (24a).
Yield 1.70 g (93%) starting with 23a; colorless oil. MS (ESIþ)
m/z: 338.2 (Mþ þ H) for C21H27NOSi.
0.94 (9H, s, 3 ꢀ CH3), 0.81, 0.79 (3H each, s, 2 ꢀ CH3). [R]20
D
-27.00° (c=0.20, MeOH).
4-(tert-Butyldiphenylsilyloxy)-3S-methylbutanenitrile (24b).
Yield 1.163 g (93%) starting with 23b; colorless oil. MS
(ESIþ) m/z: 338.2 (Mþ þ H) for C21H27NOSi.
Synthesis of BA Derivatives 6-8 and 11. A solution of 1
(1 equiv), DMAP (2 equiv), and the proper acid anhydride
(5 equiv) in anhydrous pyridine (1.5 mL) was stirred at 160 °C
for 2 h using microwave. The reaction mixture was diluted with
EtOAc (20 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 the pure target compounds.
Synthesis of 25a and 25b. To a solution of 24a or 24b (1 equiv)
in anhydrous toluene (10 mL) was added diisobutylaluminium
hydride (DIBALH, 1.1 equiv) dropwise at 0 °C under argon.
After stirring for 30 min, ice water was added slowly followed by
10% HCl and aqueous saturated potassium tartrate. Stirring
was continued for 15 min at 0 °C, and then the aqueous layer was
extracted three times with CH2Cl2. 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.
4-(tert-Butyldiphenylsilyloxy)-3R-methylbutanal (25a). Yield
1.02 g (95%) starting with 24a; colorless oil. MS (ESIþ) m/z:
341.2 (Mþ þ H) for C21H28O2Si.
3β-O-(30-Carboxycyclohex-50-enecarbonyl)-betulinic Acid (6).
Yield 40% (starting with 100 mg of 1); white amorphous
powder; mp 178-180 °C. MS (ESI-) m/z: 607.4 (M- - H) for
1
C38H56O6. H NMR (300 MHz, CDCl3): δ 5.70 (2H, m, H-50,
60), 4.72, 4.60 (1H each, s, H-29), 4.48 (1H, m, H-3), 3.02 (1H, m,
H-19), 2.83, 2.72 (1H each, m, H-30, H-20), 2.32-2.27 (4H, m,
2 ꢀ CH2, H-40, 70), 1.68 (3H, s, H-30), 1.01, 0.97 (3H each, s, 2 ꢀ
CH3), 0.89, 0.83, 0.81 (3H each, s, 3 ꢀ CH3). [R]20D þ14.17° (c=
0.18, MeOH).
4-(tert-Butyldiphenylsilyloxy)-3S-methylbutanal (25b). Yield
1.047 g (90%) starting with 24a; colorless oil. MS (ESIþ) m/z:
341.2 (Mþ þ H) for C21H28O2Si.
3β-O-[30-Carboxybicyclo[2.2.1]hept-50-enecarbonyl]-betulinic
Acid (7). Yield 35% (starting with 100 mg of 1); white amor-
Synthesis of 26a and 26b. To a solution of 25a or 25b (1 equiv)
phous powder; mp 165-167 °C. MS (ESI-) m/z: 619.4 (M-
-
in dry DMSO (1.0 mL) was added NaH2PO4 H2O (0.8 equiv in
3
H) for C39H56O6. 1H NMR (300 MHz, CDCl3): δ 5.73 (2H, m,
H-50, 60) 4.71, 4.59 (1H each, s, H-29), 4.45 (1H, m, H-3), 3.01
(1H, m, H-19), 2.95, 2.76 (1H each, m, H-30, H-20), 2.52-2.39
(4H, m, 2 ꢀ CH2, H-40, 70), 1.67 (3H, s, H-30), 0.96 (6H, s, 2 ꢀ
CH3), 0.91, 0.85, 0.82 (3H each, s, 3 ꢀ CH3). [R]20D þ24.00° (c=
0.10, MeOH).
2.0 mL H2O) and 80% NaClO2 (1.5 equiv in 2.0 mL of H2O)
dropwise over 5 min. The mixture was stirred overnight. The
reaction was quenched with saturated NH4Cl solution and
extracted with EtOAc. The organic layer was washed with brine,
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.
4-(tert-Butyldiphenylsilyloxy)-3R-methylbutanoic Acid (26a).
Yield 700 mg (100%) starting with 25a; colorless oil. MS (ESI-)
m/z: 355.2 (M- - H) for C21H28O3Si. 1H NMR (300 MHz,
CDCl3): δ 7.68-7.62 (4H, m, H ar-20), 7.45-7.33 (6H, m, H ar-
30, 40), 3.59, 3.43 (2H, dd, J = 7.6, 1.5 Hz, H-4), 2.61-2.30
(3H, m, H-2, H-3), 1.05 (9H, s, SiC(CH3)3), 0.95 (3H, d, J=10
Hz, H-5).
3β-O-[(Z)-30-Carboxybut-2-enoyl]-betulinic Acid (8). Yield
55% (starting with 150 mg of 1); red amorphous powder; mp
169-171 °C. MS (ESI-) m/z: 567.4 (M- - H) for C35H52O6. 1H
NMR (300 MHz, CDCl3): δ 5.89 (1H, s, H-20), 4.70, 4.58 (1H
each, s, H-29), 4.46 (1H, m, H-3), 2.96 (1H, m, H-19), 1.93 (3H, s,
CH3-30), 1.66 (3H, s, H-30), 0.94, 0.93, 0.90 (3H each, s, 3 ꢀ
CH3), 0.80, 0.79 (3H each, s, 2 ꢀ CH3). [R]20D þ9.41° (c=0.17,
MeOH).
3β-O-(40,40-Dimethylglutaryl)-betulinic Acid (11). Yield 53%
(starting with 100 mg of 1); white amorphous powder; mp
228-230 °C. MS (ESI-) m/z: 597.4 (M- - H) for C37H58O6.
1H NMR (300 MHz, CDCl3): δ 4.73, 4.61 (1H each, s, H-29),
4.45 (1H, dd, J=9.0, 5.2 Hz, H-3), 3.00 (1H, m, H-19), 2.32 (2H,
dd, J=6.6, 5.4 Hz, H-20), 2.16 (1H, m, H-13), 1.69 (3H, s, H-30),
1.22, 1.21 (3H each, d, J=6 Hz, 2 ꢀ CH3-40), 0.97, 0.91 (3H each,
4-(tert-Butyldiphenylsilyloxy)-3S-methylbutanoic Acid (26b).
Yield 830 mg (76%) starting with 25b; colorless oil. MS (ESI-)
m/z: 355.2 (M- - H) for C21H28O3Si. 1H NMR (300 MHz,
CDCl3): δ 7.68-7.62 (4H, m, H ar-20), 7.45-7.33 (6H, m, H
ar-30, 40), 3.58, 3.42 (2H, dd, J=7.6, 1.5 Hz, H-4), 2.60-2.30
(3H, m, H-2, H-3), 1.05 (9H, s, SiC(CH3)3), 0.95 (3H, d, J=
10 Hz, H-5).

Article
Synthesis of 27a and 27b. A solution of 1 (1 equiv), EDCI
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3139
2.84, 2.42 (1H each, dd, J=15.9, 8.3, 5.6, H-20), 2.10-2.20 (1H,
m, H-13), 1.69 (3H, s, H-30), 1.28, 1.26 (3H, d, J=6 Hz, CH3-30),
0.97 (6H, s, 2 ꢀ CH3), 0.86, 0.83, 0.80 (3H each, s, 3 ꢀ CH3).
[R]20D þ13.00° (c=0.05, MeOH).
(8 equiv), DMAP (2 equiv), and 26a or 26b (5 equiv) in
anhydrous CH2Cl2 (5 mL) was stirred at rt overnight until the
starting material was not observed by TLC. The solution was
diluted with CH2Cl2 (15 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.
3β-O-(30S-Methylsuccinyl)-betulinic Acid (10). Yield 88%
from 29, white amorphous powder; mp 279-281 °C. MS
1
(ESI-) m/z: 569.38 (M- - H) for C35H54O6. H NMR (300
MHz, CDCl3): δ 4.73, 4.60 (1H each, s, H-29), 4.51 (1H, dd, J=
11.1, 4.8 Hz, H-3), 2.96-3.03 (1H, m, H-19), 2.86-2.93 (1H, m,
H-30), 2.70, 2.61 (1H each, dd, J = 16.2, 6.3, 4.8 Hz, H-20),
2.06-2.16 (1H, m, H-13), 1.69 (3H, s, H-30), 1.27, 1.25 (3H, d,
J=6 Hz, CH3-30), 0.97, 0.94, 0.86, 0.85, 0.81 (3H each, s, CH3-
23, 24, 25, 26, 27). [R]20D -12.88° (c=0.08, MeOH).
3β-O-[40-(tert-Butyldiphenylsilyloxy)-30R-methylbutanoyl]-
betulinic Acid (27a). Yield 32% starting with 340 mg BA,
white powder; mp 179-181 °C. MS (ESI-) m/z: 793.5 (M-
-
H) for C51H74O5Si. 1H NMR (300 MHz, CDCl3): δ 7.68-7.62
(4H, m, H ar-20), 7.45-7.33 (6H, m, H ar-30, 40), 4.72, 4.60 (1H
each, s, H-29), 4.45 (1H, m, H-3), 3.58, 3.42 (2H, m, H-40),
2.87-2.95 (2H, m, H-19, H-30), 2.64-2.42 (2H, m, H-20), 1.69
(3H, s, H-30), 1.28, 1.26 (3H, d, J=6 Hz, CH3-30), 1.01 (9H, s,
SiC(CH3)3), 0.96 (6H, s, 2 ꢀ CH3), 0.89, 0.85, 0.82 (3H each, s,
3 ꢀ CH3).
Synthesis of 12. 2-Ethyl-2-methylsuccinic acid (50 mg, 4
equiv) was stirred in TFAA at rt for 3 h until the reaction
mixture became homogeneous. The solution was then concen-
trated to dryness under reduced pressure to yield 2-ethyl-2-
methylsuccinic anhydride (32). Compound 32 was reacted with-
out further purification with 1 (36 mg, 1 equiv) and DMAP
(19 mg, 2 equiv) in anhydrous pyridine (1.5 mL), stirring at
160 °C for 2 h in microwave. The reaction mixture was diluted
with EtOAc (10 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 30 mg (55%) of 12; white amorphous powder.
3β-O-(30S-Ethylmethylsuccinyl)-betulinic Acid (12a). mp
186-187 °C. MS (ESI-) m/z: 597.4 (M- - H) for C37H58O6.
1H NMR (500 MHz, CDCl3): δ 4.71, 4.59 (1H each, s, H-29),
4.48 (1H, m, H-3), 3.00 (1H, m, H-19), 2.75, 2.52 (1H each, d, J=
15.5, H-20), 1.66 (3H, s, H-30), 1.34-1.32 (2H, m, CH2-30), 1.23
(3H, s, CH3-30), 1.18 (2H, m, CH3-300), 0.94 (6H, s, 2 ꢀ CH3),
0.85, 0.84, 0.78 (3H each, s, 3 ꢀ CH3). [R]20D -9.00° (c=0.10,
MeOH).
3β-O-(30R-Ethylmethylsuccinyl)-betulinic Acid (12b). mp
179-181 °C. MS (ESI-) m/z: 597.4 (M- - H) for C37H58O6.
1H NMR (500 MHz, CDCl3): δ 4.71, 4.58 (1H each, s, H-29),
4.51 (1H, dd, J=10.2, 6.4 Hz, H-3), 2.99 (1H, m, H-19), 2.98,
2.26 (1H each, d, J=15.5, H-20), 1.67 (3H, s, H-30), 1.35 (2H, m,
CH2-30), 1.25 (3H, s, CH3-30), 1.19 (3H, m, CH3-300), 0.98, 0.95
(3H each, s, 2 ꢀ CH3), 0.86, 0.82, 0.79 (3H each, s, 3 ꢀ CH3).
[R]20D þ9.57° (c=0.07, MeOH).
3β-O-[40-(tert-Butyldiphenylsilyloxy)-30S-methylbutanoyl]-
betulinic Acid (27b). Yield 48% starting with 170 mg BA, white
powder; mp 186-187 °C. MS (ESI-) m/z: 793.5 (M- - H) for
C51H74O5Si. 1H NMR (300 MHz, CDCl3): δ 7.68-7.62 (4H, m,
H ar-20), 7.45-7.33 (6H, m, H ar-30, 40), 4.73, 4.60 (1H each, s,
H-29), 4.51 (1H, dd, J=11.1, 4.8 Hz, H-3), 3.60-3.42 (2H, m,
H-40), 2.96-3.03 (1H, m, H-19), 2.86-2.93 (1H, m, H-30), 2.73,
2.69 (1H each, dd, J=11.2, 5.7, 4.8 Hz, H-20), 1.69 (3H, s, H-30),
1.27, 1.25 (3H, d, J=6 Hz, CH3-30), 1.02 (9H, s, SiC(CH3)3),
0.97, 0.94, 0.86, 0.85, 0.81 (3H each, s, CH3-23, 24, 25, 26, 27).
Synthesis of 28 and 29. A solution of 27a or 27b (1 equiv) and
TBAF (1.0 M in THF, 3 equiv) in anhydrous THF was stirred at
0 °C for 1.5 h. The mixture was then allowed to warm to rt and
stirred until there was no starting material detected by TLC. The
reaction was diluted with 15 mL of CH2Cl2 and washed with
saturated NH4Cl solution and brine. 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.
3β-O-(40-Hydroxy-30R-methylbutanoyl)-betulinic Acid (28).
Yield 100% from 27a, white powder; mp 201-203 °C. MS
(ESI-) m/z: 555.4 (M- - H) for C35H56O5. 1H NMR (300 MHz,
CDCl3): δ 4.73, 4.60 (1H each, s, H-29), 4.54 (1H, dd, J=9.9, 5.7
Hz, H-3), 3.55, 3.42 (2H, m, H-40), 3.01 (1H, m, H-19),
2.87-2.95 (1H, m, H-30), 2.70-2.42 (2H, m, H-20), 1.68 (3H, s,
H-30), 1.27, 1.25 (3H, d, J=6 Hz, CH3-30), 0.93 (6H, s, 2 ꢀ CH3),
0.85, 0.84, 0.81 (3H each, s, 3 ꢀ CH3). [R]20D þ15.50° (c=0.12,
MeOH).
Synthesis of 30. To a solution of 3 (1 g, 1eq) in anhydrous
THF was added sodium borohydride (209 mg, 2.5 equiv). The
reaction was neutralized with 10% HCl after stirring in rt for 4 h
and extracted with EtOAc. 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 905 mg (90%) of 30; white amorphous powder;
mp 197-199 °C. MS (ESI-) m/z: 455.5 (M- - H) for C30H47O3.
1H NMR (300 MHz, CDCl3): δ 5.15 (1H, s, H-19), 3.19 (1H, dd,
J=10.8, 5.6 Hz, H-3), 0.97, 0.96 (3H each, s, H-29, H-30), 0.95
(6H, s, H-24, H-26), 0.84 (3H, s, H-25), 0.75, 0.74 (3H each, s,
H-23, H-27).
Synthesis of MA Derivatives 13-21. To a solution of 30 in dry
CH2Cl2 were added triethylamine (50 μL) and corresponding
acyl chloride (50 μL). The reaction was stirred at rt for 30 min.
The mixture was diluted with CH2Cl2 and washed with 10%
HCl. 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.
3β-O-(20-Methylbutyryl)-moronic Acid (13). Yield 73%
(starting with 20 mg of 1); white amorphous powder; mp
237-239 °C. MS (ESI-) m/z: 539.4 (M- - H) for C35H56O4.
1H NMR (300 MHz, CDCl3): δ 5.17 (1H, s, H-19), 4.47 (1H, dd,
J=10.0, 5.6 Hz, H-3), 2.35 (1H, dd, J=10.0, 6.0 Hz, H-20), 1.96
(2H, m, H-30), 1.17, 1.15 (3H, d, J=6 Hz, CH3-20), 0.98, 0.97 (3H
each, s, H-29, H-30), 0.91 (3H, m, H-40), 0.88, 0.84 (6H), 0.82
(3H each, s, H-26, H-25, H-24, H-23), 0.77 (3H, s, H-27).
3β-O-(40-Hydroxy-30S-methylbutanoyl)-betulinic Acid (29).
Yield 100% from 27b, white powder; mp 229-231 °C. MS
(ESI-) m/z: 555.4 (M- - H) for C35H56O5. 1H NMR (300 MHz,
CDCl3): δ 4.73, 4.60 (1H each, s, H-29), 4.51 (1H, dd, J=12.5,
5.6 Hz, H-3), 3.58, 3.43 (2H, m, H-40), 2.97-3.01 (1H, m, H-19),
2.83-2.92 (1H, m, H-30), 2.70, 2.62 (1H each, dd, J=16.2, 6.3,
4.8 Hz, H-20), 1.69 (3H, s, H-30), 1.27, 1.25 (3H, d, J=6 Hz,
CH3-30), 1.01, 0.96, 0.86, 0.85, 0.81 (3H each, s, CH3-23, 24, 25,
26, 27). [R]20D -16.10° (c=0.11, MeOH).
Synthesis of 9 and 10. To a solution of 28 or 29 (1 equiv) in
CH2Cl2 (5 mL) was added TEMPO (0.1 equiv) and PhI(OAc)2
(1.5 equiv). The mixture was stirred at rt until the starting
material was not observed by TLC. The reaction was filtered
through thin silica gel pad and eluted with CH2Cl2 and washed
with saturated NH4Cl solution and brine. 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.
3β-O-(30R-Methylsuccinyl)-betulinic Acid (9). Yield 75%
from 28, white amorphous powder; mp 268-271 °C. MS
1
(ESI-) m/z: 569.38 (M- - H) for C35H54O6. H NMR (300
MHz, CDCl3): δ 4.73, 4.60 (1H each, s, H-29), 4.54 (1H, dd, J=
9.9, 5.7 Hz, H-3), 3.01 (1H, m, H-19), 2.87-2.95 (1H, m, H-30),

3140 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Qian et al.
3β-O-Propionyl-moronic Acid (14). Yield 57% (starting with
11.2 mg of 1); white amorphous powder; mp 210-211 °C. MS
(ESI-) m/z: 511.5 (M- - H) for C33H52O4. 1H NMR (300 MHz,
CDCl3): δ 5.175 (1H, s, H-19), 4.48 (1H, dd, J=9.9, 6.6 Hz, H-3),
2.32 (2H, q, J=7.2 Hz, H-20), 1.14 (3H, t, J=7.8 Hz, H-30), 0.99,
0.98 (3H each, s, H-29, H-30), 0.88 (6H), 0.84 (6H), 0.77, (3H
each, s, H-26, H-25, H-24, H-23, H-27).
3β-O-Cyclobutancarbnyl-moronic Acid (15). Yield 58%
(starting with 12.2 mg of 1); white amorphous powder; mp
231-233 °C. MS (ESI-) m/z: 537.4 (M- - H) for C35H54O4. 1H
NMR (300 MHz, CDCl3): δ 5.17 (1H, s, H-19), 4.48 (1H, dd, J=
11.7, 4.8 Hz, H-3), 3.12 (1H, quintet, J = 8.4 Hz, H-20),
2.34-2.13, 2.04-1.86 (4H, m, H-30, H-50),1.62-1.27 (2H, m,
H-40), 0.99, 0.98, 0.97, 0.88, 0.84 (6H), 0.77 (3H each, s, H-29,
H-30, H-25, H-26, H24, H-23, H-27).
3β-O-Valeroryl-moronic Acid (16). Yield 84% (starting with
13.5 mg of 1); white amorphous powder; mp 197-199 °C. MS
(ESI-) m/z: 539.4 (M- - H) for C35H56O4. 1H NMR (300 MHz,
CDCl3): δ 5.17 (1H, s, H-19), 4.48 (1H, dd, J=10.6, 6.0 Hz, H-3),
2.30 (2H, t, J=6.0 Hz, H-20), 1.76 (2H, m, H-30), 0.99, 0.98, 0.97,
0.88, 0.84 (6H), 0.77, (3H each, s, H-29, H-30, H-26, H-25, H-24,
H-23, H-27).
infection) for toxicity determinations (IC50 defined below). In
addition, AZT and 2 were also assayed during each experiment as
positive drug controls. On day 1 PI (postinfection), 140 μL of fresh
cell specific media was added to each cell. On day 4 PI, the assay was
terminated and culture supernatants were harvested for p24 antigen
ELISA analysis. The compound toxicity was determined by XTT
using the mock-infected sample wells. If a test sample inhibited virus
replication and was not toxic, its effects were reported in the
following terms: EC50, the concentration of the test sample that
was able to suppress HIV replication by 50%, IC50, the concentra-
tion of test sample that was toxic to 50% of the mock-infected cells,
and therapeutic index (TI), the ratio of the IC50 to EC50.
HIV-1 Maturation Inhibition Assay.10 HeLa cells were trans-
fected with pNL4-3 or mutated HIV-1 virus (A1 V or L231M)
and cultured in the absence or presence of 10. Two days post-
transfection, cells were metabolically labeled for 2 h with
35S-Met/Cys. Cell lysates were prepared, and virions were
pelleted by ultracentrifugation. Cell and viral lysates were
immunoprecipitated with HIV-Ig. Western blotting was then
performed,22,23 and p25 and p24 are indicated.
HIV-1NL4-3 Replication Inhibition Assay in MT-4 Lympho-
cytes.21 A previously described HIV-1 infectivity assay was used.
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 PI for the p24 antigen
capture using an ELISA kit from ZeptoMetrix Corporation
(Buffalo, NY).
3β-O-Isovaleroryl-moronic Acid (17). Yield 54% (starting
with 12.2 mg of 1); white amorphous powder; mp 134-
1
136 °C. MS (ESI-) m/z: 539.4 (M- - H) for C35H56O4. H
NMR (300 MHz, CDCl3): δ 5.17 (1H, s, H-19), 4.48 (1H, dd, J=
11.6, 5.6 Hz, H-3), 2.17 (2H, d, J=10.0, H-20), 1.63 (1H, m,
H-30), 0.99 (6H), 0.97 (6H), 0.95, 0.88, 0.84, 0.835, 0.77, (3H
each, s, 2 ꢀ H-40, H-29, H-30, H-26, H-25, H-24, H-23, H-27).
3β-O-Cyclopetanecarbonyl-moronic Acid (18). Yield 75%
(starting with 20 mg of 1); white amorphous powder; mp
209-210 °C. MS (ESI-) m/z: 551.4 (M- - H) for C36H56O4.
1H NMR (300 MHz, CDCl3): δ 5.17 (1H, s, H-19), 4.48 (1H, dd,
J = 10.6, 5.6 Hz, H-3), 3.13 (1H, quintet, J = 6.0 Hz, H-20),
2.36-2.13, 2.06-1.89 (4H, m, H-30, H-60),1.78-1.19 (4H, m, H-
40, H-50), 1.00, 0.98, 0.97, 0.88, 0.84 (6H), 0.77 (3H each, s, H-29,
H-30, H-25, H-26, H24, H-23, H-27).
Acknowledgment. We thank Panacos Pharmaceutical Inc.
for the anti-HIV replication assay in MT-2 lymphocytes. 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, and
HPLC analysis results of the target compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
3β-O-Butyryl-moronic Acid (19). Yield 71% (starting with
20 mg of 1); white amorphous powder; mp 242-244 °C. MS
(ESI-) m/z: 525.5 (M- - H) for C34H54O4. 1H NMR (300 MHz,
CDCl3): δ 5.18 (1H, s, H-19), 4.48 (1H, dd, J=10.0, 6.0 Hz, H-3),
2.28 (2H, t, J=6.0 Hz, H-20), 1.76 (2H, m, H-30), 1.00, 0.98 (3H
each, s, H-29, H-30), 0.97 (3H, t, J=6.0 Hz, H-40), 0.92, 0.88,
0.84 (6H), 0.77, (3H each, s, H-26, H-25, H-24, H-23, H-27).
3β-O-Isobutyryl-moronic Acid (20). Yield 66% (starting with
20 mg of 1); white amorphous powder; mp 206-207 °C. MS
(ESI-) m/z: 525.4 (M- - H) for C34H54O4. 1H NMR (300 MHz,
CDCl3): δ 5.18 (1H, s, H-19), 4.46 (1H, dd, J=10.0, 6.0 Hz, H-3),
2.54 (1H, septet, J=6.0 Hz, H-20), 1.19, 1.17, 1.16, 1.15 (6H, d,
J=3.0 Hz, H-30, CH3-20), 0.98 (9H), 0.88, 0.85, 0.84, 0.77 (3H
each, s, H-29, H-30, H-25, H-26, H24, H-23, H-27).
3β-O-Trimethylacetyl-moronic Acid (21). Yield 34% (starting
with 25 mg of 1); white amorphous powder; mp 103-105 °C. MS
(ESI-) m/z: 539.5 (M- - H) for C35H56O4. 1H NMR (300 MHz,
CDCl3): δ 5.17 (1H, s, H-19), 4.46 (1H, dd, J=10.0, 5.4 Hz, H-3),
1.15 (9H, s, 3 ꢀ CH3-20), 0.98, 0.97, 0.96, 0.88, 0.84 (6H), 0.77
(3H each, s, H-29, H-30, H-25, H-26, H24, H-23, H-27).
HIV-1IIIB Replication Inhibition Assay in MT-2 Cell Lines.17,20,21
The evaluation of HIV-1 inhibition was carried out as follows using
MT-2 lymphocytes. Test samples were first dissolved in dimethyl
sulfoxide (DMSO). The following drug concentrations were routi-
nely 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 μLof
media containing MT-2 cells at 3 ꢀ 105 cells/mL and 45 μL of virus
inoculum (HIV-1 IIIB 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
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