Anti-AIDS agents 69. Moronic acid and other triterpene derivatives as novel potent anti-HIV agents

Article abstract of DOI:10.1021/jm0601912

In a continuing structure-activity relationship study of potent anti-HIV agents, seven new triterpene derivatives were designed, synthesized, and evaluated for in vitro antiviral activity. Among them, moronic acid derivatives 19, 20, and 21 showed significant activity in HIV-1 infected H9 lymphocytes. Compounds 19 and 20 were also evaluated against HIV-1 NL4-3 and drug resistant strains in the MT-4 cell line. Compounds 19 and 20 showed better antiviral profiles than the betulinic acid analogue 8 (PA-457), which has successfully completed a Phase IIa clinical trial. Compound 20 showed potent anti-HIV activity with EC₅₀ values of 0.0085 μM against NL4-3, 0.021 μM against PI-R (a multiple protease inhibitor resistant strain), and 0.13 μM against FHR-2 (an HIV strain resistant to 8). Promising compound 20 has become a new lead for modification, and further development of 20-related compounds as clinical trial candidates is warranted.

Full text of DOI:10.1021/jm0601912

5462
J. Med. Chem. 2006, 49, 5462-5469
Anti-AIDS Agents 69. Moronic Acid and Other Triterpene Derivatives as
Novel Potent Anti-HIV Agents
Donglei Yu,† Yojiro Sakurai,† Chin-Ho Chen,‡ Fang-Rong Chang,§ Li Huang,‡ Yoshiki Kashiwada,| and Kuo-Hsiung Lee*,†
Natural Products Research Laboratories, School of Pharmacy, UniVersity of North Carolina, Chapel Hill, North Carolina 27599,
Medical Center, Box 2926, SORF, Duke UniVersity, Durham, North Carolina 27710, Graduate Institute of Natural Products,
Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, and Faculty of Pharmaceutical Sciences, Niigata UniVersity of Pharmacy and
Applied Life Sciences, Niigata 950-2081, Japan
ReceiVed February 17, 2006
In a continuing structure-activity relationship study of potent anti-HIV agents, seven new triterpene deriva-
tives were designed, synthesized, and evaluated for in vitro antiviral activity. Among them, moronic acid
derivatives 19, 20, and 21 showed significant activity in HIV-1 infected H9 lymphocytes. Compounds 19
and 20 were also evaluated against HIV-1 NL4-3 and drug resistant strains in the MT-4 cell line. Compounds
19 and 20 showed better antiviral profiles than the betulinic acid analogue 8 (PA-457), which has successfully
completed a Phase IIa clinical trial. Compound 20 showed potent anti-HIV activity with EC50 values of
0.0085 µM against NL4-3, 0.021 µM against PI-R (a multiple protease inhibitor resistant strain), and 0.13 µM
against FHR-2 (an HIV strain resistant to 8). Promising compound 20 has become a new lead for modification,
and further development of 20-related compounds as clinical trial candidates is warranted.
Introduction1
pene skeletons, including betulinic acid (BAa), glycyrrhetinic
acid (GLA), moronic acid (MA), oleanolic acid (OA), and ursolic
acid (UA). This article reports their design, synthesis, and SAR.
Design. From previous SAR studies of 8-type analogues, the
most potent derivatives contain either 3′,3′-dimethylsuccinyl or
3′,3′-dimethylglutaryl moieties at C-3.8,9,11 Thus, these side
chains were also used in the current study.
Acquired immunodeficiency syndrome (AIDS) remains an
exceptional crisis beccause of both its emergent and long-term
development. The epidemic remains extremely dynamic, grow-
ing and changing character as the human immunodeficiency
virus (HIV) exploits new opportunities for transmission. No
region of the world has been spared, and the AIDS pandemic
continues to outpace the global response to HIV treatment.2
Although current anti-AIDS drugs include inhibitors of reverse
transcriptase (RT), protease, and fusion,3 the increasing preva-
lence of drug resistant HIV strains is one of the major problems
for the treatment of HIV infection, driving the demand for the
development of novel drugs with new mechanisms of action.
Our first aim was to investigate the SAR of the triterpene core.
To optimize this core and gain more information about target
binding requirements, we designed and synthesized three sub-
stituted derivatives of triterpenes other than 1. Moreover, by
replacing the betulin core with other triterpenes, we could poten-
tially lower cytotoxicity, improve pharmacological profiles, and
obtain more SAR information. Lupeol (2) has the same skeleton
as 1 but lacks the C-28 carboxylic acid. OA (3) and UA (4)
have a six-membered E ring rather than the five-membered E
ring found in 1. MA (5) and 3 differ at the double bond position.
GLA (7) has a carboxylic acid at C-30 rather than at C-28.
Activity data with these latter compounds will provide informa-
tion on the contribution and positional requirement of the
carboxylic acid on antiviral activity. Previously synthesized
compounds 12-15 were also included in the current study.12
Among many C-28 amide BA derivatives, a relatively long
C-28 amide side chain is a common structural feature that is
essential for potent anti-HIV entry activity.8 These results
suggest that BA derivatives may interfere at more than one stage
of HIV infection, depending on the side chains on C-3 and C-28.
Two compounds (10 and 11) with C-3 ester and C-28 amide
side chains exhibited both anti-fusion and maturation activity.13
These observations prompted us to hypothesize that the betulin
moiety serves as a molecular scaffold or docker, the C-28 amide
side chain is the pharmacophore for anti-HIV entry activity,
and the C-3 ester group has an important role in target interaction
during HIV-1 maturation. On the basis of this hypothesis, a
compound with both DMS on C-3 and a proper amide on C-28
will probably exhibit dual mechanisms of action, both anti-fusion
Triterpenes, including betulinic acid (1), constitute a promis-
ing class of anti-HIV agents. Two types of anti-HIV 1 deriva-
tives have exhibited potent anti-HIV profiles. In our prior
studies, we found that 3-O-(3′,3′-dimethylsuccinyl)-betulinic
acid (8) inhibits HIV-1 maturation by interfering with HIV-1
P24/P25 processing, which results in a noninfectious HIV-1
particle.4-7 The second type of anti-HIV 1 analogues contain
various C-28 amide modifications. IC9564 (9), a statine ana-
logue of 1, represents this compound type and acts at an early
stage of viral infection.8,9
Compound 8, designated as PA-457 by Panacos Pharma-
ceuticals, Inc.,10 is a maturation inhibitor directed against a novel
viral target. Panacos, Inc. recently concluded a successful Phase
IIa clinical trial with 8.10 Because 8 has a different target than
that of approved HIV drugs, it retains activity against virus
isolates resistant to currently available treatments, including RT
and protease inhibitors.
In the current study, we synthesized a series of new triterpene
derivatives with 3-, 28-, and both substitutions on different triter-
* To whom correspondence should be addressed. Phone: 919-962-0066.
Fax: 919-966-3893. E-mail: khlee@unc.edu.
† University of North Carolina.
‡ Duke University.
§ Kaohsiung Medical University.
a Abbreviations: BA, betulinic acid; MA, moronic acid: OA, oleanolic
acid; UA, ursolic acid; GLA, glycyrrhetinic acid.
| Niigata University of Pharmacy and Applied Life Sciences.
10.1021/jm0601912 CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/15/2006

Anti-AIDS Agents 69
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5463
Scheme 1a
a Conditions: (i) 2,2-dimethylsuccinyl anhydride, DMAP, pyridine, reflux 12 h; (ii) Ac2O, py, rt; (iii) oxalyl chloride, CH2Cl2, rt; (iv) leucine methyl
ester, Et3N, CH2Cl2, rt; (v) 2 N KOH, THF-MeOH (1:2), rt.
and anti-maturation, to increase potency and decrease HIV-
resistance profiles.
pound 33. Without isolation, 33 was treated with 2 N KOH in
MeOH and THF at 0 °C to afford target compound 19 in 70%
yield (Scheme 2).
Our previous article reported that 5, which is isolated from
Brazilian propolis, a herbal medicine widely used worldwide
today, exhibited significant anti-HIV activity (EC50 <0.1 µg/
mL; TI >186) in H9 lymphocytes.14 In addition, the observed
toxicity of MA derivatives is generally lower than that of BA
derivatives. This finding implies that changing the betulin core
to 5 may alter the cytotoxicity profile without impairing anti-
HIV potency. In our continuing development of novel triterpene
anti-HIV agents, compound 5 was dually functionalized at C-3
and C-28 on the basis of the SAR of BA derivatives.
The â-amyrin series triterpene glycyrrhizin or glycyrrhizic
acid (6) is used in Japanese clinics by intravenous administration
for the treatment of chronic viral hepatitis B. In some clinical
studies in Japan, the administration of 6 to AIDS patients
resulted in delayed progression of HIV infection symptoms.15
Therefore, we also modified 7, the aglycone of 6, with both
ester and amide side chains.
Chemistry. Compounds 2 and 7 were reacted separately with
2,2-dimethylsuccinic anhydride to yield target compounds 16
and 17, respectively. Compound 23, the acetate of 7, was then
treated with oxalyl chloride and, without isolation, further
reacted with leucine methyl ester hydrochloride to afford 24 in
a yield of 95% over two steps. Compound 24 was hydrolyzed
under basic conditions to 25 in a 95% yield. The 3â-hydroxy
group of 25 was reacted with 2,2-dimethylsuccinic anhydride
to yield final target product 18 in a 65% yield (Scheme 1).
The methanol extract (600 g) of propolis, collected by
Africanized Apis mellifera in southern Brazil, gave 5 in good
yield (1.5 g, 0.25%).14 Scheme 2 shows the synthetic steps to
preparing moronic acid derivatives 19-22. Using the same
method used for compound 24, compounds 26 and 27 were
synthesized from 5. To reduce the C-3 keto group, sodium boro-
hydride (NaBH4) was added to a solution of 26 or 27 in MeOH
and THF to produce the corresponding reduction products 28
and 29 in yields of 33% and 85%, respectively. At the same
time, 26 was also reduced to 32 in a 60% yield. Compounds
28 and 29 were hydrolyzed to 30 and 31 and yielded final target
products 20-22 in 30-40% yield. Compound 32 reacted with
2,2-dimethylsuccinyl anhydride to yield crude disuccinated com-
Results and Discussion
In our current study, newly synthesized triterpene derivatives
16-22 were evaluated for anti-HIV-1 replication activity against
two different viral strains in two different cell lines in parallel
with 8 and other previously synthesized compounds 10-15.
Two MA derivatives 19 and 20 were also evaluated for anti-
HIV-1 replication activity against three different viral strains
in MT-4 cells in parallel with 8 and compound 10, which has
the same C-3 and C-28 substituents as 20 but with a BA core.
The results are summarized in Tables 1 and 2, respectively.
First, our new results confirm prior reports that a 3′,3′-
dimethylsuccinyl side chain is superior to 3′,3′-dimethylglutaryl
at the C-3 position. Accordingly, OA derivative 13 with the
latter substituent was inactive, whereas OA analogue 12 with
the former substituent had an EC50 value of 0.32 µM. Similarly,
among comparable UA analogues, 15 was 10-fold weaker than
14. (Due to changes in the screening system after 2000, our
prior publications reported different activities for 12-15 in H9
lymphocytes.12,16
)
Compounds 16 and 8 have identical structures, except at C-28,
which is a methyl group in the former and a carboxylic acid in
the latter compound. Compound 16 showed dramatically
decreased activity in both HIV-1 infected H9 lymphocytes and
MT-4 cells with EC50 values of 2.6 and 21.6 µM, respectively.
OA analogue 12 and UA analogue 14, which also have a
carboxylic acid at C-28, were 10-50-fold more potent than 16
in the H9 lymphocyte assay; however, they were still less active
than 8. GLA analogue 17, which has a C-28 methyl and a C-30
carboxylic acid, was inactive in both assays. These results
indicate that the carboxylic acid at C-28 might be required for
high anti-HIV activity in this compound type.
From the 3D structures of 1, 3â-hydroxymoronic acid, and 7
shown in Figure 4, the C-28 carboxylic acid is oriented in the
same direction in 1 and 3â-hydroxymoronic acid, even though
they have different E rings. However, the C-30 carboxylic acid
of 7 is oriented in a different direction. Accordingly, a

5464 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Yu et al.
Scheme 2a
a Conditions: (i) oxalyl chloride, CH2Cl2, rt; (ii) amino acid methyl esters, Et3N, CH2Cl2, rt; (iii) NaBH4, MeOH/THF, rt; (iv) 2 N KOH, THF-MeOH
(1:2), rt; (v) 2,2-dimethylsuccinyl anhydride, DMAP, pyridine, reflux 12 hr.
substituent on this position of 7 will create a totally different
molecular shape compared to that of analogous BA and MA
derivatives. Therefore, like 17, disubstituted derivative 18 was
inactive in both assays.
Similar to disubstituted BA derivatives (10 and 11), di-
substituted MA derivatives (19-21) also exhibited high potency.
Compound 21 with 3-(3′,3′-dimethylsuccinyl) and 28-undeca-
exhibited weaker activity in this system. Interestingly, unlike
in H9 lymphocytes, compound 20, which showed the best
activity (EC50 ) 0.0085 µM) in MT-4 cells, was more potent
than 8 in this assay. Compound 19 also exhibited slightly better
activity than 8 with an EC50 value of 0.045 µM. However, the
most active compound in H9 lymphocytes, 21, showed an EC50
value of only 0.11 µM. The discrepancy might result from
differences in the assay protocols, especially the viral infecting
dose.
Because 19 and 20 were active in both assays, these two
compounds, together with 10, were also screened against other
viral strains. The data are shown in Table 2. PI-R is HIV-1
M46I/L63P/V82T/I84V, an HIV-1 strain resistant to multiple
protease inhibitors,17 and FHR-2 is an HIV-1 strain resistant to
8.7 Both 19 and 20 showed better activity (EC50 ) 0.088 and
0.021 µM) than 8 against the multi-PI resistant strain but were
noic amide substituents exhibited the highest potency (EC50
)
0.007 µM; TI ) 3400, in H9 lymphocytes), with results similar
to those of 8 in the same assay. Compounds 19 and 20 also
demonstrated high potencies with EC50 values of 0.017 and
0.016 µM, respectively. Compound 22, the 3-(2′,2′-dimethyl-
succinyl) isomer of 21, almost lost anti-HIV activity.
These compounds were also tested in an HIV-1 replication
assay using MT-4 cells infected with the NL4-3 strain, which
is a T-cell adapted X4 wild-type HIV-1 virus. Most compounds

Anti-AIDS Agents 69
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5465
Table 1. Anti-HIV Activities of Triterpene Derivatives Against HIV-1
Strains in H9 Lymphocytes and MT-4 Cells
The key structural feature of disubstituted compounds 10,
11, 19, 20, and 21 is the presence of both side chains at positions
3 and 28. This feature enables our target compounds to be active
against viruses that are resistant to 8, which has a side chain
only at C-3. Therefore, this modification study indicated that
the BA scaffold can be replaced by an analogous triterpene,
MA, without a loss of activity. Disubstituted MA derivatives
with side chains on both C-3 and C-28 are likely to have dual
functions and lower cytotoxicity as well as better drug resistance
profiles than their parent compound 8. However, the detailed
molecular mechanisms of action of these new disubstituted MA
derivatives are yet to be determined. Their mechanisms of action
might be different from that of 8. Additional modification and
SAR studies are in progress with an aim to continually improve
potency, particularly against drug-resistant viral strains. Further
development of compounds related 20 as next generation clinical
trial candidates is warranted.
HIV-1IIIB in H9
lymphocytes
NL4-3 in MT-4 cells
IC50
EC50
IC50
EC50
compd
(µM)
(µM)
TI
(µM)
(µM)
TI
8
>42.8 0.007
NT
>6250
>5a
12.2
11
0.096
0.010
0.007
8.3
NS
8.3
>52
>1220
1570
10
11
12
13
14
15
16
17
18
19
20
21
22
NT
>42.7 0.32
>133
20.2
2.4
NS
28.5 0.079
28.5 0.68
>45.1 2.6
NS
360
42
21
2.5
NS
>17.2 >36.1 21.6
>1.7
NS
NS
0.045
0.0085
0.11
NS
>42.7 0.017
>42.7 0.0156
24.0 0.007
>32.6 9.3
2500
2735
3400
23.5
22.5
19.5
522
2650
177.3
>3.5
NS
a The highest concentration tested for 8 was 5 µM; there was no observ-
able cytotoxicity at this concentration. NT, not tested in this assay; NS, no
suppression.
Experimental Section
Chemistry. Melting points were measured with a Fisher-Johns
melting apparatus without correction. The proton nuclear magnetic
resonance (1H NMR) spectra were measured on 300 MHz Varian
Gemini 2000 and 500 MHz Varian Inova spectrometers using TMS
as internal standard. The solvent used was CDCl3 unless indicated.
Mass spectra were obtained on an Agilent 1100 MSD ion trap mass
spectrometer or a PE-SCIEX API-3000 with a turbo ion spray
source. Elemental analyses were performed by Atlantic Microlab,
Inc., Norcross, GA. All target compounds were analyzed for C, H,
and N and gave values within (0.4% of the theoretical values.
Thin-layer chromatography (TLC) was performed on PTLC silica
gel 60 F254 plates (0.5 mm, Merck). Teledyn-Isco companion sys-
tems were used as medium-pressure column chromatography.
Compound 7 was provided by Tokiwa Pharmaceuticals, Inc. All
other chemicals were obtained from Aldrich, Inc.
Table 2. Comparison of Anti-HIV Activities of Selected Derivatives
Against Several HIV-1 Strains in MT-4 Cellsa
EC50 (µM) in different
viral strains
IC50
compd
NL4-3
PI-R
FHR-2
(µM)
8
0.096
0.010
0.045
0.0085
0.013
0.43
NS
0.05
2.78
0.13
0.019
>5b
12.2
23.5
21.0
>37.5
10
19
20
AZT
0.006
0.088
0.021
0.019
a NL4-3 is a T-cell adapted HIV-1 strain X4 wild-type virus; PI-R is an
HIV-1 strain HIV-1 M46I/L63P/V82T/I84V, resistant to multiple protease
inhibitors;17 and FHR-2 is an HIV-1 strain resistant to 8.7 EC50 is the
concentration that inhibits HIV-1 replication by 50%, and IC50 is the concen-
tration that decreases 50% of the viable cell number. b The highest concen-
tration tested for DSB was 5 µM; there was no observable cytotoxicity at
this concentration. NS, no suppression at testing concentration, 5 µM.
3â-O-(3′,3′-Dimethylsuccinyl)-lupeol (16). Compound 2 (21.3
mg, 0.05 mmol) was heated with 2,2-dimethylsuccinic anhydride
(25.6 mg, 0.2 mmol) and 4-dimethylamino pyridine (DMAP, 12.2
mg, 0.1 mmol) in pyridine (2 mL) to reflux overnight. After the
addition of EtOAc (50 mL), the mixture was worked up with 2 N
HCl and H2O. The evaporation of EtOAc gave a mixture of the
starting material and succinate, which was separated by SiO2 column
chromatography (CH2Cl2/MeOH 100:2). Yield 63%; white amor-
phous powder; mp 193-194 °C; MS (ESI-) m/z: 553.5 (M- - 1)
Table 3. Inhibition of HIV-1 Envelope-Mediated Membrane Fusiona
EC50
IC50
compd
(µM)
(µM)
1
for C36H58O4. H NMR (500 MHz) δ 0.79, 0.81, 0.83, 0.84, 0.94,
8
9
19
20
21
>17.1
>17.1
>13.2
>14.6
>14.3
>13.0
1.03 (3H each, s, CH3-23, 24, 25, 26, 27 and 28), 1.29, 1.31 (3H
each, s, CH3-3′×2), 1.68 (3H, s, CH3-30), 1.91 (2H, m, H-2), 2.37
(1H, m, H-19), 2.57, 2.67 (1H each, d, J ) 15.5 Hz, H-2′), 4.49
(1H, dd, J ) 11.0, 5.5 Hz, H-3), 5.30 (1H, s, COOH-4′), 4.68,
4.56 (1H each, d, J ) 2 Hz, H-29). Anal. (C36H58O4), C 77.77, H
10.50.
0.0086
5.85
3.58
5.21
a EC50 is the drug concentration that inhibits HIV envelope-mediated
membrane fusion by 50%; IC50 values (cytotoxicity) for both cell types
were determined in a one-day assay because the fusion assay is also a one-
day assay. The highest concentration tested: 10 µg/mL.
3â-O-(3′,3′-Dimethylsuccinyl)-glycyrrhetinic Acid (17). Fol-
lowing the procedure described for 16, compound 17 was obtained
from 7 as a white amorphous powder (235 mg, 0.5 mmol) in a
yield of 60%; mp >300 °C; MS (ESI-) m/z: 597.4 (M- - 1) for
less active than 10 (EC50 ) 0.006 µM). As for the FHR-2 HIV-1
strain, which is resistant to 8, compound 20 retained some
potency with an EC50 of 0.13 µM but was less active than 10
(EC50 ) 0.05 µM), whereas 19 showed only weak activity (EC50
) 2.78 µM). With a reduced carboxylic acid in the 28-side
chain, 19 showed less activity than 20 against all tested viral
strains; thus, a carboxylic acid within the leucine side chain is
more favorable for target interaction. Our results indicate that
MA derivatives 19, 20, and 21 exhibited weak anti-HIV fusion
activity (Table 3). BA derivatives 10 and 11 also exhibited anti-
fusion activity in a previous study.13 The anti-fusion activity of
these compounds are weaker than that of 9 (EC50 0.0086 µM).
These data suggest that the disubstituted compounds 10, 11,
19, 20, and 21 can block HIV-1 entry.
1
C36H54O7. H NMR (500 MHz, CD3OD) δ 0.83, 0.89, 0.90, 0.92,
1.14, 1.16 (3H each, s, CH3-23, 24, 25, 26, 27 and 28), 1.25, 1.26
(3H each, s, CH3-3′×2), 1.43 (3H, s, CH3-29), 2.28 (1H, m, H-18),
2.51 (2H, m, H-7), 4.48 (1H, m, H-3), 5.58 (1H, s, H-12). Anal.
(C36H54NO7), C 72.30, H 8.90.
3â-O-Acetylglycyrrhetinic Acid (23). Compound 7 (935 mg,
2 mmol) was reacted with Ac2O (0.5 mL) in pyridine (5 mL) and
dichloromethane (10 mL) at room temperature for 1 day. The
mixture was washed with 20% HCl and H2O. The evaporation of
CH2Cl2 followed by silica gel column chromatography (CH2Cl2/
MeOH, 100:2) gave 23. Yield 54%; white powder; mp 175-177
°C; MS (ESI-) m/z: 511.5 (M- - 1) for C32H48O5. 1H NMR (500
MHz, DMSO-d6) δ 0.76, 0.83, 1.04, 1.07, 1.10, 1.15 (3H each, s,
CH3-23, 24, 25, 26, 27 and 28), 1.37 (3H, s, CH3-29), 2.0 (3H, s,

5466 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Yu et al.
Figure 1. Related triterpene natural products.
Figure 2. Previously modified anti-HIV triterpene derivatives.
CH3CO-3), 2.07 (1H, m, H-18), 2.63 (1H, m, H-7), 4.42 (1H, dd,
J ) 12.0, 4.5 Hz, H-3), 5.41 (1H, s, H-12), 12.18 (1H, COOH-30).
N-(Olean-3â-O-acetyl-11-oxo-12-en-30-oyl)-L-leucine Methyl
Ester (24). Following the procedure described for 26, compound
24 was obtained in 99% yield from compound 23; white amorphous
powder; mp 103-105 °C; MS (ESI+) m/z: 662.7 (M+ + Na),
640.7 (M+ + 1) for C39H61NO6. 1H NMR (500 MHz) δ 0.81, 0.88,
1.14, 1.15, 1.17, 1.38 (3H each, s, CH3-23, 24, 25, 26, 27 and 28),
0.94, 0.95 (3H each, d, J ) 6.5 Hz, -CH(CH3)2-30 side chain),
1.57 (3H, s, CH3-29), 2.05 (3H, s, CH3CO-3), 2.28 (1H, m, H-18),

Anti-AIDS Agents 69
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5467
Figure 3. Newly synthesized triterpene derivatives.
145-147 °C; MS (ESI+) m/z: 734.5 (M+ + Na) for C42H65NO8.
1H NMR (300 MHz) δ 0.78, 0.84, 0.86, 1.08, 1.12, 1.14 (3H each,
s, CH3-23, 24, 25, 26, 27, and 28), 0.91, 0.93 (3H each, d, J ) 7.2
Hz, -CH(CH3)2-30 side chain), 1.29 (6H, s, 2 × CH3-3′), 1.30 (3H,
s, CH3-29), 4.51 (1H, m, H-3), 4.69 (1H, m, -NHCH-), 5.68 (1H,
s, H-12), 6.49 (1H, br.s, -CONH-). Anal. (C42H65NO8), C 71.14, H
9.18, N 2.22.
N-(Olean-3-oxo-18-en-28-oyl)-leucine Methyl Ester (26). MA
(5, 454 mg, 1 mmol) was dissolved in CH2Cl2 (5 mL) and ice-
cooled. To this solution, oxalyl chloride (20 mL of 2.0 M solution
in CH2Cl2) was added dropwise with stirring. The mixture was
stirred at room temperature for 2 h, then CH2Cl2 was evaporated.
To the residue, CH2Cl2 (5 mL, ×3) was added and evaporated to
give a yellow solid. This residue was dissolved in CH2Cl2 (5 mL),
and a CH2Cl2 solution (10 mL) of leucine methyl ester hydro-
chloride (543 mg, 3 mmol) and Et3N (303 mg, 3 mmol) was added.
The reaction mixture was left overnight at room temperature. After
washing with water three times, CH2Cl2 was removed to give a
viscous oil, which was then subjected to silica gel column chroma-
tography (CH2Cl2/MeOH, 100:1) to give 26. 88% yield; mp 86-
88 °C; MS (ESI+) m/z: 582.6 (M+ + 1) for C37H59NO4. 1H NMR
(300 MHz) δ 0.78, 0.96, 0.99, 1.01, 1.01, 1.03, 1.08 (3H each, s,
CH3-23, 24, 25, 26, 27, 29 and 30), 0.93, 0.94 (3H each, d, J ) 6
Hz, -CH(CH3)2-28 side chain), 3.73 (3H, s, COOCH3), 4.61 (1H,
m, H-3), 5.37 (1H, s, H-19), 6.13 (1H, d, J ) 7.8 Hz, -CONH-).
N-(Olean-3-oxo-18-en-28-oyl)-aminoundecanoic Acid Methyl
Ester (27). Following the procedure described for 26, compound
27 was obtained in 51% yield; white amorphous powder; mp 90-
92 °C; MS (ESI+) m/z: 652.9 (M+ + 1) for C42H69NO4. 1H NMR
(500 MHz) δ 0.77, 0.95, 0.98, 0.98, 1.01, 1.03, 1.08 (3H each, s,
CH3-23, 24, 25, 26, 27, 29 and 30), 3.30 (2H, m, -NHCH2-), 3.67
(3H, s, COOCH3), 5.32 (1H, s, H-19), 5.82 (1H, t, J ) 5.8 Hz,
-CONH-).
N-(Olean-3â-hydroxy-18-en-28-oyl)-leucine Methyl Ester (28).
Compound 26 (920 mg, 1.58 mmol) was dissolved in a mixture of
MeOH and THF (50 mL, 3 mL) and stirred in an ice bath. To the
solution, solid NaBH4 (597 mg, 15.8 mmol) was slowly added,
and the reaction mixture was kept overnight at room temperature.
Water and CH2Cl2 (50 mL, 100 mL) were added to the warmed
reaction mixture, and insoluble solid was filtered off. The aqueous
and organic layers were separated, and evaporation of the organic
solvent gave an oily residue, which was subjected to silica gel
Figure 4. 3D structures of 1, 3â-hydroxymoronic acid, and 7.
2.81 (1H, m, H-7), 3.75 (3H, s, COOCH3), 4.52 (1H, dd, J ) 12.0,
5.2 Hz, H-3), 4.67 (1H, m, -NHCH-), 5.77 (1H, s, H-12), 5.93 (1H,
d, J ) 8.5 Hz, -CONH-).
N-(Olean-3â-hydroxy-11-oxo-12-en-30-oyl)-L-leucine (25). Fol-
lowing the procedure described for 30, compound 25 was obtained
in 98% yield from 24; white amorphous powder; mp 184-186 °C;
1
MS (ESI-) m/z: 582.7 (M- - 1) for C36H57NO5. H NMR (500
MHz, CD3OD) δ 0.79, 0.80, 0.96, 1.13, 1.14, 1.25 (3H each, s,
CH3-23, 24, 25, 26, 27 and 28), 0.93, 0.95 (3H each, d, J ) 6.0
Hz, -CH(CH3)2-30 side chain), 1.42 (3H, s, CH3-29), 3.16 (1H,
dd, J ) 12.0, 5.0 Hz, H-3), 4.52 (1H, m, -NHCH-), 5.74 (1H, s,
H-12).
N-[3â-O-(3′,3′-Dimethylsuccinyl)-olean-11-oxo-12-en-30-oyl]-
L-leucine (18). Yield 65% from 25; white amorphous powder; mp

5468 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18
Yu et al.
column chromatography (CH2Cl2/MeOH, 200:1). Compounds 32
(300 mg, 60%) and 28 (560 mg, 33%) were isolated and crystallized
from a mixture of CH2Cl2 and MeOH; mp 145-146 °C; MS (ESI-)
m/z: 582.7 (M- - 1) for C37H61NO4; 1H NMR (300 MHz) δ 0.76,
0.76, 0.87, 0.97, 0.98, 0.99, 1.00 (3H each, s, CH3-23, 24, 25, 26,
27, 29 and 30), 0.93, 0.94 (3H each, d, J ) 6.3 Hz, -CH(CH3)2-28
side chain), 3.20 (1H, dd, J ) 10.6, 5.6 Hz, H-3), 3.72 (3H,
s, -COOCH3-28 side chain), 4.61 (1H, td, J ) 8.4, 4.5 Hz,
-CH(CH3)2-28 side chain), 5.36 (1H, s, H-19), 6.12 (1H, d, J )
8.4 Hz, -CONH-).
N-(Olean-3-hydroxy-18-en-28-oyl)-aminoundecanoic Acid
Methyl Ester (29). Following the procedure described for 28,
compound 29 was obtained in yield 85% from 27; white amorphous
powder; mp 152-154 °C; MS (ESI+) m/z: 676.8 (M+ + Na),
654.7 (M+ + 1) for C42H71NO4. 1H NMR (300 MHz) δ 0.74, 0.75,
0.84, 0.94, 0.95, 0.96, 0.99 (3H each, s, CH3-23, 24, 25, 26, 27, 29
and 30), 3.18 (2H, m, -NHCH2-), 3.29 (1H, m, H-3), 3.65 (3H, s,
COOCH3), 5.30 (1H, s, H-19), 5.80 (1H, t, J ) 6.0 Hz, -CONH-).
N-(Olean-3â-hydroxy-18-en-28-oyl)-leucine (30). Compound
28 (25 mg) was dissolved in a mixture of MeOH (2 mL) and THF
(1 mL), and 2 N KOH (0.5 mL) was added to this mixture at 0 °C
with stirring. The reaction mixture was acidified with 2 N HCl
and extracted with CH2Cl2 to yield compound 30 in a 96% yield
after the evaporation of solvent; mp 245-247 °C; MS (ESI-)
m/z: 568.6 (M- - 1) for C36H59NO4. 1H NMR (300 MHz, in CD3-
OD and CDCl3 mixture) δ 0.70, 0.76, 0.84, 0.90 (6H, br. s), 0.95
(br. s), 0.98 (3H each, s, CH3-23, 24, 25, 26, 27, 29 and 30), 0.90,
0.95 (3H each, br.s, -CH(CH3)2-28 side chain), 3.09 (1H, dd, J )
10.8, 5.2 Hz, H-3), 4.46 (1H, m, -CH(CH3)2-28 side chain), 5.34
(1H, s, H-19), 6.54 (1H, d, J ) 8.7 Hz, -CONH-).
N-(Olean-3â-hydroxy-18-en-28-oyl)-aminoundecanoic Acid
(31). Following the procedure described for 30, compound 31 was
obtained in 85% yield from 29; white amorphous powder; mp 168-
170 °C; MS (ESI+) m/z: 640.7 (M+ + 1) for C41H69NO4. 1H NMR
(300 MHz) δ 0.74, 0.75, 0.85, 0.94, 0.95, 0.96, 0.97 (3H each,
s, CH3-23, 24, 25, 26, 27, 29 and 30), 3.17 (2H, m, -NHCH2-),
3.30 (1H, m, H-3), 5.30 (1H, s, H-19), 5.83 (1H, t, J ) 5.7 Hz,
-CONH-).
N-(Olean-3â-hydroxy-18-en-28-oyl)-(1-hydroxymethyl-3-
methyl)-butylamide (32). Compound 32 was obtained in 60% yield
from compound 26; mp 253-254 °C; MS (ESI-) m/z: 554.7 (M-
- 1) for C36H61NO3; 1H NMR (300 MHz) δ 0.76, 0.77, 0.86, 0.97,
0.98, 0.99, 1.00 (3H each, s, CH3-23, 24, 25, 26, 27, 29 and 30),
0.91, 0.93 (3H each, d, J ) 6.6 Hz, -CH(CH3)2-28 side chain),
3.20 (1H, dd, J ) 10.8, 5.1 Hz, H-3), 3.51 (1H, dd, J ) 10.5, 6.3
Hz, -CH2OH-28 side chain), 3.66 (1H, dd, J ) 10.5, 3.5 Hz, -CH2-
OH-28 side chain), 4.02 (1H, m, -CH(CH3)2-28 side chain), 5.33
(1H, s, H-19), 5.87 (1H, d, J ) 8.1 Hz, -CONH-).
N-[3â-O-(3′,3′-Dimethylsuccinyl)-olean-18-en-28-oyl]-(1-
hydroxymethyl-3-methyl)-butylamide (19). Compound 32 (130
mg, 0.23 mmol) was heated with 2,2-dimethylsuccinic anhydride
(180 mg, 1.38 mmol) and DMAP (56 mg, 0.46 mmol) in pyridine
(6 mL) to reflux overnight. After the addition of EtOAc (50 mL),
the mixture was washed twice with 2 N HCl (5 mL) and H2O. The
evaporation of EtOAc gave crude disuccinates 33 as an oil. The
oil was treated with 2 N KOH (7 mL) in a mixture of MeOH (15
mL) and THF (2 mL) under ice-cooling and then kept at room
temperature overnight. The reaction mixture was acidified with
2 N HCl, and the organic solvent was evaporated. The residue was
extracted by CH2Cl2 to give an oil, which was subjected to silica
gel column chromatography. Using a mixture of CH2Cl2 and MeOH
(100:1), compound 19 was obtained as an oil and was crystallized
from EtOAc to give 105 mg of 19 as a white amorphous powder
in a yield of 70.0%; mp 237-238 °C; MS (ESI-) m/z: 682.8
(M- - 1) for C42H69NO6. 1H NMR (300 MHz) δ 0.76, 0.80, 0.83,
0.87, 0.97, 0.99 (6H) (3H each, s, CH3-23, 24, 25, 26, 27, 29 and
30), 0.90, 0.92 (3H each, d, J ) 4.4 Hz, -CH(CH3)2-28 side chain),
1.27, 1.29 (3H each, s, 2 × CH3-3′), 3.49 (1H, dd, J ) 10.8, 3.9
Hz, -CH2OH-28 side chain), 3.64 (1H, dd, J ) 10.8, 6.3 Hz, -CH2-
OH-28 side chain), 4.03 (1H, m, -NHCH-), 4.49 (1H, dd, J )
9.9, 5.4 Hz, H-3), 5.34 (1H, s, H-19), 5.88 (1H, d, J ) 8.1 Hz,
-CONH-). Anal. (C42H69NO6), C 73.59, H 10.20, N 1.99.
N-[3â-O-(3′,3′-Dimethylsuccinyl)-olean-18-en-28-oyl]-L-
leucine (20). Compound 20 was obtained in 30% yield 30; white
amorphous powder; mp 223-224 °C; MS (ESI-) m/z: 696.8
1
(M- - 1) for C42H67NO7. H NMR (300 MHz, CDCl3) δ 0.76,
0.77, 0.83, 0.85, 0.93 (6H), 1.03 (3H each, s, CH3-23, 24, 25, 26,
27, 29 and 30), 0.99 (6H, d, J ) 7.2 Hz, -CH(CH3)2-28 side chain),
1.28, 1.31 (3H each, s, 2 × CH3-3′), 4.52 (1H, dd, J ) 8.1, 4.8 Hz,
H-3), 4.68 (1H, m, -NHCH-), 5.37 (1H, s, H-19), 6.32 (1H, d, J
) 8.1 Hz, -CONH-). Anal. (C42H67NO7), C 71.98, H 9.72, N 2.01.
N-[3â-O-(3′,3′-Dimethylsuccinyl) olean-18-en-28-oyl]-amino-
undecanoic Acid (21). Compound 21 was obtained in 48% yield
from compound 31; white amorphous powder; mp 131-132 °C;
MS (ESI+) m/z: 790.6 (M+ + Na) for C47H77NO7. 1H NMR (500
MHz) δ 0.76, 0.80, 0.85, 0.86, 0.96, 0.98, 1.00 (3H each, s, CH3-
23, 24, 25, 26, 27, 29 and 30), 1.26, 1.29 (3H each, s, 2 × CH3-
3′), 4.54 (1H, dd, J ) 11.5, 5.0 Hz, H-3), 5.32 (1H, s, H-19), 5.87
(1H, dd, J ) 8.5, 3.5 Hz, -CONH-). Anal. (C47H77NO7), C 73.12,
H 9.95, N 1.76.
N-[3â-O-(2′,2′-Dimethylsuccinyl)-olean-18-en-28-oyl]-amino-
undecanoic Acid (22). Compound 22 was obtained in 10% yield
from compound 31 as a dia-isosteric isomer of 21, white amorphous
powder; mp 109-111 °C; MS (ESI+) m/z: 790.6 (M+ + Na) for
1
C47H77NO7. H NMR (500 MHz) δ 0.76, 0.76, 0.86, 0.95, 0.96,
0.98, 1.00 (3H each, s, CH3-23, 24, 25, 26, 27, 29 and 30), 1.27,
1.31 (3H each, s, 2 × CH3-2′), 4.40 (1H, dd, J ) 12.0, 4.5 Hz,
H-3), 5.32 (1H, s, H-19), 5.85 (1H, t, J ) 7.5 Hz, -CONH-). Anal.
(C47H77NO7), C 73.36, H 10.10, N 1.78.
HIV Growth Inhibition Assay in H9 Lymphocytes. The evalu-
ation of HIV-1 inhibition was carried out as follows using H9
lymphocytes. The human T-cell line, H9, was maintained in contin-
uous culture with complete medium (RPMI 1640 with 10% fetal
calf serum supplemented with L-glutamine at 5% CO2 and 37 °C.
Test samples were prepared as described previously,6 and to each
sample well was added 90 µL of media containing H9 cells at 3 ×
105 cells/mL and 45 µL of virus inoculum (HIV-1 IIIIB 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 set of samples were prepared identical to the first and
were added to cells under identical conditions without virus (mock
infection) for toxicity determinations (IC50 defined below). In
addition, AZT was also assayed during each experiment as a posi-
tive drug control. On days 1 and 4 postinfection (PI), spent media
were removed from each well and replaced with fresh media. On
day 6 PI, the assay was terminated, and culture supernatants were
harvested for analysis of virus replication by p24 antigen capture.
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:
IC50, the concentration of test sample that was toxic to 50% of the
mock-infected cells; EC50, the concentration of the test sample that
was able to suppress HIV replication by 50%; and the Therapeutic
index (TI), the ratio of the IC50 to EC50.
Anti-HIV Assay in MT4 Cells. A previously described HIV-1
infectivity assay was used in the experiments.13 A 96-well microtiter
plate was used to set up the HIV-1 replication assay. HIV-1 at a
multiplicity of infection (MOI) of 0.01 was used to infect MT4
cells. Culture supernatants were collected on day 4 postinfection
for the p24 assay using an ELISA kit from ZeptoMetrix Corporation
(Buffalo, New York).
Cell Fusion Assay. A protocol modified from a previously
described fusion assay was used in this study.9 TZM cells that
expressed luciferase upon fusion with envelope-expressing COS
cells were used as fusion partners. The fusion assays were performed
by transfecting monkey kidney cells (COS) with an expression
vector pSRHS that contained the HIV-1 NL4-3 envelope genes.
Electroporation was performed to express the HIV-1 envelope on
COS cells. Briefly, COS cells (106) in culture medium were incu-
bated with 2 µg of the envelope expression vector on ice for 10
min. Electroporation was performed using a gene pulser (BioRad,

Anti-AIDS Agents 69
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 18 5469
targeting of the final step of virion maturation. J. Virol. 2004, 78,
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Hercules, CA) with capacitance set at 950 µF and voltage at 150 V.
The transfected COS cells were cultured for 1 day before mixing
with TZM cells. TZM cells (10 × 104) were incubated with COS
cells (104) in 96-well flat-bottomed plates (Costar) in a 100 µL
culture medium. Compounds to be tested at various concentrations
in 10 µL of culture medium were incubated with the cell mixtures
at 37 °C for 24 h. Luciferase activity was quantified using a Biotek
luminometer.
(9) Holz-Smith, S. L.; Sun, I. C.; Jin, L.; Matthews, T. J.; Lee, K. H.;
Chen, C. H. Role of human immunodeficiency virus (HIV) type 1
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Salzwedel, K.; Wild, C.; Allaway, G.; Jacobson, J.; Martin, D. Safety
and antiviral activity of PA-457, the first-in-class maturation inhibitor,
in a 10-day monotherapy study in HIV-1 infected patients. Presented
at the 45th Conference on Antimicrobial Agents and Chemotherapy,
Washington, DC, Dec16-19, 2005.
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M.; Chen, C. H.; Yang, L. M.; Lee, K. H. Anti-AIDS agents. 34.
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Fujioka, T.; Yamagishi, T.; Cosentino, L. M.; Kozuka, M.; Okabe,
H.; Ikeshiro, Y.; Hu, C. Q.; Yeh, E.; Lee, K. H. Anti-AIDS agents
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Acknowledgment. This investigation was supported by
Grant AI-33066 from the National Institute of Allergy and
Infectious Diseases (NIAID) awarded to K.H.L. Thanks are also
due to Dr. Takashi Tatsuzaki, President of Tokiwa Phytochemi-
cal Co., Ltd., Chiba, Japan, for supplying 7.
Supporting Information Available: Elemental analysis data
for compounds 16-22. This material is available free of charge
via the Internet at http://pubs.acs.org.
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