Anti-AIDS agents 73: Structure-activity relationship study and asymmetric synthesis of 3-O-monomethylsuccinyl-betulinic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.09.081

3-O-3′(or 2′)-Methylsuccinyl-betulinic acid (MSB) derivatives were separated by using recycle HPLC. The structures of four isomers were assigned by NMR and asymmetric synthesis. 3-O-3′S-Methylsuccinyl-betulinic acid (3′S-MSB, 4) exhibited potent anti-HIV

Full text of DOI:10.1016/j.bmcl.2007.09.081

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Bioorganic & Medicinal Chemistry Letters 17 (2007) 6553–6557
Anti-AIDS agents 73: Structure–activity relationship study
and asymmetric synthesis of 3-O-monomethylsuccinyl-betulinic
acid derivatives
Keduo Qian,a Kyoko Nakagawa-Goto,a Donglei Yu,a Susan L. Morris-Natschke,a
Theodore J. Nitz,b Nicole Kilgore,b Graham P. Allawayb and Kuo-Hsiung Leea,*
aNatural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
bPanacos Pharmaceuticals, 209 Perry Parkway, Gaithersburg, MD 20877, USA
Received 2 August 2007; revised 21 September 2007; accepted 24 September 2007
Abstract—3-O-30(or 20)-Methylsuccinyl-betulinic acid (MSB) derivatives were separated by using recycle HPLC. The structures of
four isomers were assigned by NMR and asymmetric synthesis. 3-O-30S-Methylsuccinyl-betulinic acid (30S-MSB, 4) exhibited
potent anti-HIV activity with an EC50 value of 0.0087 lM and a TI value of 6.3 · 103, which is comparable to the data for bevirimat
(DSB, PA-457), a current clinical trials drug that was also derived from betulinic acid. The anti-HIV potency of 4 was slightly better
than that of AZT.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Highly active antiretroviral therapy (HAART) com-
bines several anti-HIV drugs with different targets, and
its use has significantly improved AIDS treatment.1–3
However, it has also led to some serious problems, such
as increased toxicities and emergence of multi-drug
resistant HIV viral strains.4–7 Therefore, new classes of
compounds with novel modes of action against HIV
are still urgently needed.
has recently succeeded in Phase IIa clinical trials and
is currently in Phase IIb clinical trials.13,14
Our prior structure–activity relationship (SAR) study of
2 and other 3-O-acyl-betulinic acid derivatives suggested
that the proper length, a terminal carboxylic acid, and
C-30 dimethyl substitution of the C-3 side chain contrib-
ute to enhanced anti-HIV activity.9,15 However, it is still
unclear which, if either, C-30 methyl group within the
C-3 side chain of 2 contributes more toward activity.
We initially identified betulinic acid (BA, 1) as an anti-
HIV lead compound with an EC50 of 1.4 lM and ther-
apeutic index (TI) of 9.3 from the leaves of Syzygium
claviflorum.8 Our subsequent modification study re-
sulted in the discovery of 3-O-(30,30-dimethylsuccinyl)-
betulinic acid (bevirimat, DSB, PA-457, 2). Bevirimat
exhibits extremely potent anti-HIV activity with an
EC50 value of <0.00035 lM and a TI value of
>20,000.9,10 It was found to inhibit HIV-1 replication
by interfering with a previously unidentified viral target,
HIV-1 P24/P25 processing, resulting in the production
of a noninfectious HIV-1 particle.11,12 Therefore, bevir-
imat represents a unique first in a class of anti-HIV com-
pounds termed maturation inhibitors (MIs). Bevirimat
Therefore,
3-O-monomethylsuccinyl-betulinic
acid
(MSB) derivatives were designed to investigate the im-
pact of these methyl groups. This paper reports their
synthesis, isolation, structural assignment, and SAR
study.
Betulinic acid was treated with 2-methylsuccinic anhy-
dride and DMAP in pyridine at reflux to furnish the
30-MSB racemic isomers, as well as the undesired 20-
MSB isomers. The separation of the four compounds
(3–6)16 was challenging, since they differ only by the
presence of a 20 or 30 (R or S) methyl group, but was
successfully achieved by using recycle preparative HPLC
(Fig. 1).17 The purities of 3–6 were ascertained by
1
analytical HPLC (Fig. 2). By comparing the H NMR
spectra of the purified isomers with those of 2 and
3-O-20,20-dimethylsuccinyl BA, peaks 1 and 2 were
Keywords: Betulinic acid; Asymmetric synthesis; Anti-HIV activity.
*
Corresponding author. Tel.: +1 919 962 0066; fax: +1 919 966
3893; e-mail: khlee@unc.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.081

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K. Qian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6553–6557
H
H
H
COOH
H
COOH
O
H
H
3
HO
O
HO
3'
H
H
O
2 (Bevirimat, PA-457, DSB)
1 (Betulinic Acid)
O
O
3'
(R)
H
OH
OH
OH
OH
3 R =
4 R =
5 R =
6 R =
(R)
O
2'
H
O
O
O
OH
H
H
O
O
(S)
RO
(S)
O
3- -Monomethylsuccinyl
O
Betulinic Acid (MSB)
Figure 1. JAI LC-918 recycling preparative HPLC. Mobile phase: 85% ACN 0.1% TFA; flowrate: 5.0 mL/min; detector: refractive index (RI);
column: Alltima C18 5 lm 250 mm · 22 mm. Peak 1: 3-O-(30 S-methylsuccinyl)betulinic acid (4); peak 2: 3-O-(30 R-methylsuccinyl)-betulinic acid (3);
peak 3: 3-O-(20 S-methylsuccinyl)-betulinic acid (6); peak 4: 3-O-(20 R-methylsuccinyl)-betulinic acid (5).
assigned as the 30-MSBs, and peaks 3 and 4 as the 20-
MSBs. However, we still needed to determine the stereo-
chemistry of the C-30 and C-20 chiral centers. Therefore,
the 30 R and 30 S isomers were also prepared through
asymmetric synthesis as described below.
succinyl)-betulinic acid (30R-MSB, 3). The 30S-MSB
isomer (4) was synthesized using the same method start-
ing with 3S-bromo-2-methylpropanol. By comparing
the HPLC retention times and 1H NMR spectra of these
compounds, we determined that peak 1 is the 30S diaste-
reoisomer and peak 2 is the 30R diastereoisomer.
As shown in Scheme 1, the hydroxy group of 3R-bromo-
2-methylpropanol was first protected by reaction with
TBDPSCl in DMF. The bromide group was then re-
placed with an aldehyde group by reaction first with so-
dium cyanide in DMSO, followed by reduction using
DIBALH.18 The aldehyde was oxidized to a carboxylic
acid in the presence of NaClO2 and NaH2PO4 in DMSO
to yield 9a. Reaction of 1 with 9a in the presence of
EDCI and DMAP led to esterification of the 3b-hydro-
xy group of 1 with the carboxylic acid of 9a to provide
10a. After cleavage of the TBDPS group with TBAF in
THF, the primary hydroxy group of 11 was oxidized to
a carboxylic acid in the presence of TEMPO, PhI(OAc)2
in a solution of H2O–CH2Cl2 to give 3-O-(30R-methyl
Furthermore, the reaction of 2R-methylsuccinic anhy-
dride with betulinic acid yielded the 30 R-MSB (3) and
20 R-MSB (5) isomers. The HPLC retention time indi-
cated that peak 4 is the 20 R isomer (5). Similarly, peak
3 was confirmed as the 20 S isomer (6). Thus, the stereo-
chemistry of the 30 or 20 chiral center in 3–6 was fully
determined.
Anti-HIV activities of all four synthetic compounds (3–
6) and 2 were evaluated according to the procedure
described in Refs. 19 and 20. The synthetic intermediate
12 and a 1:1 mixture (13) of the 30 R- and 30 S-MSB iso-
mers (3 and 4) were also tested.

K. Qian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6553–6557
6555
mAU
mAU
400
300
200
100
0
Extract-194nm,4nm (1.00)
Extract-194nm,4nm (1.00)
400
300
200
100
0
-100
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5 min
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5 min
Peak 3: Cmpd 6 (2'S-MSB), 7.581 min
Peak 1: Cmpd 4 (3'S-MSB), 7.437 min
mAU
mAU
600
500
400
300
200
100
0
Extract-194nm,4nm (1.00)
Extract-194nm,4nm (1.00)
300
200
100
0
-100
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5 min
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5 min
Peak 2: Cmpd 3 (3'R-MSB), 7.521 min
Peak 4: Cmpd 5 (2'R-MSB), 7.681 min
Figure 2. Purity confirmation of the four MSB (3–6) isomers by Shimadzu LC-20AT. Column: Alltima C18 5 lm 150 mm · 2.1 mm.
O
a, b
c, d
e
HO
Br
TBDPSO
CN
TBDPSO
OH
*
*
*
8a ( )
7a ( )
R
9a ( )
R
R
8b ( )
7b ( )
S
9b ( )
S
S
H
H
H
O
O
f
H
OH
OH
O
O
H
H
3'
*
TBDPSO
HO
O
O
H
H
*
11 (3'R)
12 (3'S)
10a ( )
R
10b ( )
S
H
O
H
g
OH
O
H
H
3'
HO
O
*
O
3 (3'R)
4
(3'S)
Scheme 1. Reagents and conditions: (a) TBDPSCl, imidazole, quant; (b) NaCN, DMSO, 93%; (c) DIBALH, 95%; (d) NaClO2, NaH2PO4, DMSO,
quant; (e) EDCI, DMAP, BA, 25-48%; (f) TBAF, THF, quant; (g) TEMPO, PhI(OAc)2, H2O, CH2Cl2, 75–88%.

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K. Qian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6553–6557
Table 1. Anti-HIV activities for 3-O-monomethylsuccinyl-betulinic
acid derivatives in HIV-1 infected MT-2 cell line18
References and notes
a
b
1. Palella, F. J., Jr.; Delaney, K. M.; Moorman, A. C.;
Loveless, M. O.; Fuhrer, J.; Satten, G. A.; Aschman, D. J.;
Holmberg, S. D. N. Eng. J. Med. 1998, 338, 853.
2. Mohri, H.; Bonhoeffer, S.; Monard, S.; Perelson, A. S.;
Ho, D. D. Science 1998, 279, 1223.
3. Klein, M. B.; Willemot, P.; Murphy, T.; Lalonde, R. G.
AIDS 2004, 18, 1895.
4. Piscitelli, S. C.; Flexner, C.; Minor, J. R.; Polis, M. A.;
Masur, H. Clin. Infect. Dis. 1996, 23, 685.
5. Louie, M.; Markowitz, M. Antiviral Res. 2002, 55,
15.
6. Boden, D.; Hurley, A.; Zhang, L.; Cao, Y.; Guo, Y.;
Jones, E.; Tsay, J.; Ip, J.; Farthing, C.; Limoli, K.; Parkin,
N.; Markowitz, M. J. Am. Med. Assoc. 1999, 282, 1135.
7. Condra, J. H. Haemophilia 1998, 4, 610.
Compound
EC50 (lM)
IC50 (lM)
TI (IC50/EC50)
AZT
2
0.034
0.0013
1870
55,330
30,555
961
>42.78
>43.83
32.78
3
0.12
0.0087
4
6274
—
5
No suppression
0.44
>43.83
>43.83
>44.93
>44.93
6
100
—
12
13
No suppression
0.016
5323
a Concentration that inhibits HIV-1 replication by 50%.
b Concentration that is toxic to 50% of mock-infected MT-2 cells.
The anti-HIV activities of the synthesized compounds
are summarized in Table 1. Among these derivatives,
the 30S-MSB isomer (4) showed very potent anti-HIV
activity with a TI of 6.3 · 103 and EC50 of 0.0087 lM,
which is comparable to that of 2 (EC50: 0.0013 lM)
and slightly better than that of AZT (EC50: 0.034 lM)
in this assay system. The 30R-MSB isomer (3) exhibited
only moderate anti-HIV activity with a TI of 9.6 · 102
and EC50 of 0.12 lM. The antiviral activity (EC50:
0.016 lM) of the mixture (13) of the two stereoisomers
falls in between those of 3 and 4. This result indicates
that the two C-30 methyl groups in the DSB side chain
contribute differently to the extremely potent anti-HIV
activity of this compound class. We postulate that an
interaction of the 30 S-methyl group with the viral target
might be essential to the anti-HIV activity of 2. The
interaction of the 30 R-methyl group within the target
is less significant, but still necessary, since 2, with di-
methyl substitution at C-30, is slightly more potent than
the 30 S-MSB isomer (4). In comparison with 3 and 4,
both 20-MSBs (5 and 6) exhibited no or little activity
in the same assay. These results agree with our previous
SAR study, indicating that methyl substitution at the C-
30 position improves the antiviral activity much more
significantly than at the C-20 position.
8. Fujioka, T.; Kashiwada, Y.; Kilkuskie, R. E.; Cosentino,
L. M.; Ballas, L. M.; Jiang, J. B.; Janzen, W. P.; Chen, I.
S.; Lee, K. H. J. Nat. Prod. 1994, 57, 243.
9. Kashiwada, Y.; Hashimoto, F.; Cosentino, L. M.; Chen, C.
H.; Garrett, P. E.; Lee, K. H. J. Med. Chem. 1996, 39, 1016.
10. Hashimoto, F.; Kashiwada, Y.; Cosentino, L. M.; Chen,
C. H.; Garrett, P. E.; Lee, K. H. Bioorg. Med. Chem. 1997,
5, 2133.
11. Li, F.; Goila-Gaur, R.; Salzwedel, K.; Kilgore, N. R.;
Reddick, M.; Matallana, C.; Castillo, A.; Zoumplis, D.;
Martin, D. E.; Orenstein, J. M.; Allaway, G. P.; Freed, E.
O.; Wild, C. T. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
13555.
12. Zhou, J.; Chen, C. H.; Aiken, C. Retrovirology 2004, 1, 15.
13. Beatty, G. L. J.; Eron, J.; Pollard, R.; Saag, M.; Doto, J.,
Salzwedel, K. Wild, C.; Allaway, G.; Jacobson, J.; Martin,
D. Presented at the 45th Conference on Antimicrobial
Agents and Chemotherapy, Washington, DC, December
2005.
14. Panacos. http://www.panacos.com/ne_press_releases.htm.
03/12/2007.
15. Yu, D.; Wild, C. T.; Martin, D. E.; Morris-Natschke, S.
L.; Chen, C. H.; Allaway, G. P.; Lee, K. H. Expert Opin.
Investig. Drugs 2005, 6, 681.
16. Spectroscopic data for 3-O-(30R-methylsuccinyl)-betulinic
acid (3): white amorphous powder. Mp 268–271 ꢁC. MS
(ESI-) m/z: 569.38 (MꢀꢀH) for C35H54O6. 1H NMR
(300 MHz, CDCl3): d 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), 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). 3-O-(30S-meth-
ylsuccinyl)-betulinic acid (4): white amorphous powder.
Mp 279–281 ꢁC. MS (ESI-) m/z: 569.38 (MꢀꢀH) for
In addition, compound 12, which differs structurally
from 3 only in the replacement of a primary hydroxyl
group for a terminal carboxylic acid in the 3-O-acyl side
chain, had no activity. This result is also consistent with
our findings in prior studies that the terminal carboxylic
acid is essential for enhanced antiviral activity.15
1
C35H54O6. H NMR (300 MHz, CDCl3): d 4.73, 4.60 (1H
In conclusion, 30-MSBs and 20-MSBs were prepared in
our study to investigate the SAR of the C-30 chiral cen-
ter of the anti-HIV clinical trials agent bevirimat (2).
Our results suggest that the 30 S-methyl group of 2 is
the major contributor to the compound’s extremely high
anti-HIV potency, since 2 and 30 S-MSB (4) exhibit
comparable anti-HIV activity, while the 30 R isomer
shows much lower activity.
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). 3-O-(20R-methylsuccinyl)-betulinic acid (5):
white amorphous powder. Mp 250–252 ꢁC. MS (ESI-) m/z:
569.38 (MꢀꢀH) for C35H54O6. 1H NMR (300 MHz,
CDCl3): d 4.73, 4.60 (1H each, s, H-29), 4.52 (1H, t,
J = 7.8 Hz, H-3), 2.90–3.10 (2H, m, H-19, H-20), 2.67, 2.42
(1H each, dd, J = 16.8, 9.6, 4.2, H-30), 2.13–2.20 (1H, m,
H-13), 1.68 (3H, s, H-30), 1.26, 1.23 (3H, d, J = 7.2 Hz,
CH3-20), 0.97 (6H, s, 2· CH3), 0.86, 0.83, 0.81 (3H each, s,
3· CH3). 3-O-(20S-methylsuccinyl)-betulinic acid (6):
white amorphous powder. Mp 256–258 ꢁC. MS (ESI-) m/
z: 569.38 (MꢀꢀH) for C35H54O6. 1H NMR (300 MHz,
Acknowledgment
This investigation was supported by Grant AI-33066
from the National Institute of Allergy and Infectious
Diseases (NIAID) awarded to K.H. Lee.

K. Qian et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6553–6557
6557
CDCl3): d 4.74, 4.61 (1H each, s, H-29), 4.48 (1H, dd,
J = 10.8, 4.8 Hz, H-3), 2.80–3.01 (2H, m, H19, H-20), 2.76,
2.46 (1H each, dd, J = 16.9, 9.5, 4.7, H-30), 2.10–2.18 (1H,
m, H-13), 1.69 (3H, s, H-30), 1.25, 1.23 (3H, d, J = 6 Hz,
CH3-20), 0.97, 0.92 (3H each, s, 2· CH3), 0.84-0.86 (9H, m,
3· CH3).
cycle 4, and subjected again to recycle preparative HPLC,
which yielded pure compounds at cycle 7.
18. Keyling-Bilger, F.; Schmitt, G.; Beck, A.; Luu, B. Tetra-
hedron 1996, 47, 14891.
19. Yu, D.; Sakurai, Y.; Chen, C. H.; Chang, F. R.; Huang,
L.; Kashiwada, Y.; Lee, K. H. J. Med. Chem. 2006, 49,
5462.
20. Roehm, N. W.; Rodgers, G. H.; Hatfield, S. M.; Glase-
brook, A. L. J. Immunol. Methods 1991, 142, 257.
17. Peak 4 (compound 5) was collected during cycle 3, and
peak 1 (compound 4) during cycle 4. Peak 2 (compound 3)
and peak 3 (compound 6) were collected together during