Anti-AIDS agents 90. novel C-28 modified bevirimat analogues as potent HIV maturation inhibitors

Article abstract of DOI:10.1021/jm301040s

In a continuing study of bevirimat (2), the anti-HIV-maturation clinical trials agent, 28 new betulinic acid (BA, 1) derivatives were designed and synthesized. Among these compounds, 17, with a C-28 MEM ester moiety, and 22, with a C-28 ethyl hexanoate, i

Full text of DOI:10.1021/jm301040s

Article
pubs.acs.org/jmc
Anti-AIDS Agents 90. Novel C‑28 Modified Bevirimat Analogues as
Potent HIV Maturation Inhibitors
Keduo Qian,*,†,∥ Ibrahim D. Bori,†,∥ Chin-Ho Chen,‡ Li Huang,‡ and Kuo-Hsiung Lee*,†,§
†Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North
Carolina 27599, United States
‡Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, United States
§Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 401, Taiwan
S
* Supporting Information
ABSTRACT: In a continuing study of bevirimat (2), the anti-
HIV-maturation clinical trials agent, 28 new betulinic acid (BA, 1)
derivatives were designed and synthesized. Among these
compounds, 17, with a C-28 MEM ester moiety, and 22, with a
C-28 ethyl hexanoate, increased the anti-HIV replication activity
compared with 2 by 2-fold while compounds 40, 41, 48, and 49,
with C-28 piperazine or piperidine amide substitutions, increased
the activity by 3- to 15-fold. The best new compound, 41,
exhibited an anti-HIV IC50 of 0.0059 μM compared with 0.087 μM for 2. All of the active compounds showed only
antimaturation effects, as confirmed by TZM-bl assay, in blocking the HIV replication. The results suggest that proper C-28
substitutions can further enhance the antimaturation activity of 2 without any antientry effects. Thus, 41 may serve as a
promising new lead for development of anti-AIDS clinical trial candidates.
INTRODUCTION
■
Although introduction of highly active antiretroviral therapy
(HAART) has significantly improved the treatment of HIV/
AIDS,1−4 HIV incidence is still increasing in many countries
and regions. Over 2.6 million new infections occurred in 2009
alone, contributing to the current global incidence of 33.3
million.5 New infections continue to outpace the number of
people placed on treatments, and the virus is suppressed rather
than eradicated.6−8 In addition, the efficacy of the treatments is
Figure 1.
hampered by the emergence of drug-resistant viral strains and
severe drug−drug interactions.9−11 Therefore, novel potent
Interestingly, it was discovered that certain C-28 side chains
could significantly enhance the anti-HIV maturation activity of
2 without introducing the second mechanism of antientry
effects. Their syntheses, anti-HIV evaluation, and structure−
activity relationship (SAR) correlations are reported in this
paper.
antiretroviral agents with different targets are still urgently
needed.
Triterpenes, such as betulinic acid (BA, 1),12 represent a
promising class of anti-HIV agents with novel mechanisms. In
our prior research on modified triterpenes, bevirimat [3-O-
(3′,3′-dimethylsuccinyl)betulinic acid, 2] was found to exhibit
Design. Prior C-28 modification of 1 focused only on the
remarkable anti-HIV-1 activity against primary and drug-
resistant HIV-1 isolates (Figure 1).13,14 Mechanism of action
promotion of anti-HIV entry activity. It was found that C-28
amide functionality, a second aminoalkanoic acid group near
the end of the C-28 side chain, and a 7−10 carbon alkane chain
study revealed that 2 blocks the last step of viral gag precursor
polyprotein processing from p25 (CA-SP1) to functional p24
between these two amide moieties are important to the
(CA), resulting in the production of noninfectious immature
enhanced antientry potency.22 In order to design antimatura-
tion promoting C-28 side chains without an antientry effect, we
first synthesized a group of BA derivatives (3, 4, 7−15, and 29)
with a C-28 ester moiety rather than the prior amide
functionality. Methoxymethyl (MOM), methoxyethoxymethyl
HIV-1 particles.15 Therefore, 2 represents a unique first in a
class of anti-HIV compounds termed maturation inhibitors
(MIs), and it succeeded in phase I and IIa clinical trials against
sensitive viruses during 2007−2009.14,16−20
Introduction of a C-28 side chain into a C-3 modified BA-
analog (e.g., 2) can generate a bifunctional HIV inhibitor with
both antimaturation and antientry activities.21 Consequently, in
the current study, additional C-28 side chains were investigated.
Received: July 16, 2012
Published: September 14, 2012
© 2012 American Chemical Society
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Figure 2.
Scheme 1
Scheme 2
(MEM), and tetraethylene glycol chains were incorporated to
improve the hydrophilicity of the compounds (7, 8, and 29)
and to investigate the influence of a polar heteroatom (e.g.,
oxygen) present within the C-28 side chain, which was
originally a pure carbon alkane chain. Bulky substitutions (9
and 12) and an unsaturated C-28 chain (11) were also
synthesized. Different lengths of the C-28 alkane chains with or
without a second ester group near the terminus were also
investigated (14 vs 10, 13, and 15). These 28-modified BA
derivatives were evaluated for anti-HIV replication activity to
determine if these compounds function as HIV-1 entry
inhibitors before the C-3 antimaturation pharmacophore, a
dimethylsuccinyl side chain, was incorporated to generate the
corresponding 28-substituted derivatives (16−24 and 30) of 2.
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Scheme 3
Scheme 4
In addition, a cyclic secondary amine from piperazinebutyric
acid or piperazinepentanoic acid, rather than a primary amine,
was used to form the amide bond with the C-28 carboxylic acid
of 1 to yield compounds 38 and 39. In this design, an additional
polar heteroatom nitrogen was present within the side chain,
and the substitution near the C-28 amide moiety was bulkier, in
order to reduce the potential antientry activity and promote the
antimaturation effect. The corresponding C-3 succinylated
compounds 40 and 41 were then prepared and evaluated in
comparison with 2. Previously synthesized compounds 42−49,
with C-28 piperidine side chains (Figure 2),23 were also tested
because of their structural similarity to 38−41. The SAR of the
antimaturation promoting activity of C-28 side chains was then
established.
55%. Compound 24 was synthesized in 75% yield by click
reaction of 20 with AZT in the presence of CuI and N,N-
diisopropylethylamine in DMF using a microwave synthesizer
at 120 °C for 30 min.
A tetraethyleneglycol chain was also introduced at the C-28
position of 2, and compounds 29 and 30 were prepared as
described in Scheme 3. Overall, 25 was first protected with
TBSCl to yield 26, which was then converted to bromo-
substituted 27 in 85% yield by reaction with CBr4 using PPh3
and pyridine. Reaction of 27 with 1 provided 28 in a yield of
72%, and deprotection of 28 using TBAF provided 29.
Compound 30 was prepared by a microwave reaction as
described above.
The syntheses of piperazine incorporated BA derivatives 38−
41 were achieved according to Scheme 4. The C-28 piperazine
side chains of 34 and 35 were prepared in toluene at 80 °C. C-3
acetyl protected BA (3-OAc-BA) was prepared previously23 and
was activated by oxalyl chloride before reacting with 34 and 35
to furnish 36 and 37 in 89% and 100% yields, respectively. After
hydrolysis, the resulting compounds 38 and 39 were reacted
with 2,2-dimethylsuccinic anhydride in a microwave synthesizer
to provide compounds 40 and 41 in approximately 40% yield.
CHEMISTRY
■
The syntheses of BA derivatives 3−6 were carried out
according to Scheme 1. Four equivalents of MOMCl or
MEMCl were reacted with 1 and DIEA in CH2Cl2 at room
temperature overnight or in THF at 65 °C for 6 h to generate
compounds 3 and 4, respectively, in 83% or 89% yield.
Hydrolysis of 3 and 4 using 4 N NaOH provided 5 and 6 in
over 90% yield.
RESULTS AND DISCUSSION
■
Scheme 2 depicts the synthetic pathway to compounds 7−
24. Compounds 7 and 8 were obtained by reducing the amount
of MOMCl or MEMCl to 1.1 equiv in the reaction mixture. N-
Hydroxysuccinimide and 1 were reacted in the presence of
EDCI and DMAP overnight to furnish 9 in 53% yield.
Compounds 10−15 (67−98% yield) were obtained by treating
1 with Cs2CO3, followed by adding different halogenated
alkanes or alkynes. Esterification of 7−11 and 13−15 with 2,2-
dimethylsuccinic anhydride was conducted using microwave
conditions rather than conventional oil-bath heating to yield
compounds 16−23. A catalytic amount of PTSA and THF as
solvent was used instead of pyridine, resulting in yields of 40−
The anti-HIV-1 replication activity of BA derivatives 3−24, 29,
30, and 38−49 was assessed in HIV-1NL4‑3 infected MT-4 cell
lines in parallel with AZT and 2, and the results are summarized
in Table 1. The results indicated that compounds with MOM
or MEM substitution at C-3 (5 and 6), C-28 (7 and 8), or both
positions (3 and 4) of 1 did not show anti-HIV activity.
Similarly, compounds 9−15 and 29 with a C-3 hydroxy group
and C-28 ester substitutions of different length or size, or with
heteroatoms within the side chains, did not show antiviral
activity, suggesting that 1 derivatives bearing ester modifica-
tions at the C-28 position do not exhibit anti-HIV-1 activity and
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potency (IC50 = 0.056 μM). For those compounds with one or
two polar oxygen atoms present within their C-28 side chain,
16 (MOM ester) had an anti-HIV IC50 of 0.17 μM, while 17
(MEM ester) had an IC50 of 0.046 μM and thus was 3.7-fold
more potent than 16 and 2-fold more potent than 2.
Compound 30, with a C-28 tetraethylene glycol side chain
terminating in a dimethyl succinyl ester, in addition to the
dimethyl succinyl ester at C-3, was equipotent (IC50 = 0.079
μM) with 2. Compounds with bulky C-28 substituents, such as
an N-succinimide ester (18), or conjugated to AZT through a
triazole linkage (24) displayed slightly reduced anti-HIV-1
activity (IC50 of 0.13 and 0.10 μM, respectively) relative to 2.
To further investigate the effect of C-28 side chains, cyclic
secondary amines from piperazinebutyric acid, piperazinepen-
tanoic acid, and piperidinebutyric acid, rather than a primary
amine, were used in amidation reactions with the C-28
carboxylic acid of 3-acetylated BA, resulting in 38, 39, and
42−45. None of these 1 analogues exhibited anti-HIV
replication activity in MT-4 lymphocytes, suggesting that
bulkier C-28 amide moieties compromised the antientry effect
of C-28 modified BAs.
Subsequently, the C-3 hydroxy group of 38, 39, and 42−45
was esterified with a dimethylsuccinyl moiety. Among the
resulting compounds, 40, 41, 48, and 49 showed significantly
increased antiviral activity (IC50 values ranging from 0.0059 to
0.035 μM). These results indicate that incorporating a cyclic
secondary amide group with an extended side chain at the C-28
position can remarkably enhance the activity of 2. Specifically, a
C-28 amide from piperidinebutyric acid with a second amide
group formed from ethylmorpholine (48) or propylmorpholine
(49) increased the antiviral activity of 2 by about 3-fold, with
IC50 values of 0.035 and 0.031 μM, respectively. The presence
of additional polar nitrogen heteroatom within the C-28 amide
side chain (from piperazinebutyric or piperazinepentanoic acid)
significantly increased the potency by 8- to 15-fold, as shown by
compound 40 (IC50 = 0.011 μM) and 41 (IC50 = 0.0059 μM),
respectively.
A TZM-bl assay was used to confirm the mechanism of
action of active compounds. This assay measures neutralization
in TZM-bl cells as a function of a reduction in Tat-induced
luciferase (Luc) reporter gene expression after a single round of
virus infection. The TZM-bl cell line is a HeLa cell clone that
was engineered to express CD4 and CCR5 and contains
integrated reporter gene for firefly luciferase and E.coli β-
galactosidase under control of an HIV-1 LTR. Expression of the
reporter genes is induced in trans by viral Tat protein soon after
infection, and the TZM-bl assay can detect HIV-entry
inhibitors sensitively. However, because maturation inhibitors
block HIV-1 replication only at the last step of the viral life
cycle and, thus, still permit the production of immature
noninfectious viral particles, the single round TZM-bl assay
cannot detect HIV-maturation inhibitors because the expres-
sion of reporter gene is not impaired in the first cycle.
Therefore, all of the novel active 3,28-modified BAs were
further screened in this assay system. As expected, none of the
compounds were active in the single round TZM-bl assay,
proving that they do not function as HIV entry inhibitors and,
thus, their antiviral replication activity should result from an
antimaturation mechanism of action.
Table 1. Anti-HIV-1 Replication Activities in HIV-1NL4‑3
Infected MT-4 Cell Lines
a
compd
AZT
IC50 (μM)
CC50 (μM)
TI
0.030
0.087
>37
14.2
1248
171
bevirimat (2)
b
c
3
NS
NT
b
c
4
NS
NT
b
c
5
NS
NT
b
c
6
NS
NT
b
c
7
NS
NT
b
c
8
NS
NT
b
c
9
NS
NT
b
c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
29
30
38
39
40
41
42
43
44
45
46
47
48
49
NS
NT
b
c
NS
NT
b
c
NS
NT
b
c
NS
NT
b
c
NS
NT
b
c
NS
NT
0.17
12.4
12.1
11.0
10.4
15.3
14.8
8.6
70.9
0.046
0.13
261.3
86.2
0.088
0.067
1.8
118.2
226.2
8.3
0.056
153.7
b
c
NS
NT
0.10
11.0
101.0
111.3
b
c
NS
NT
0.079
8.9
b
c
NS
NT
b
c
NS
NT
0.011
13.0
13.2
1181
2237
0.0059
b
c
NS
NT
b
c
NS
NT
b
c
NS
NT
b
c
NS
NT
0.24
>13.3
10.9
10.0
10.0
>55.5
155.8
286.1
321.2
0.07
0.035
0.031
a
All data presented are averages of at least three separate experiments.
IC50: concentration that inhibits HIV-1NL4‑3 replication by 50%. CC50:
concentration that inhibits mock-infected MT-4 cell growth by 50%.
b
TI = CC50/IC50. NS: no suppression at the testing concentration (10
c
μM). NT: not tested.
are not entry inhibitors. The results confirmed that C-3
dimethylsuccinyl substitution is essential to the antimaturation
activity of 2.
Diverse C-28 ester substituted 2 derivatives (16−24 and 30)
were then investigated to establish the SAR of the C-28 side
chain toward 2’s antimaturation potency. Compounds 19, 21,
and 23, with the C-28 alkane chain length increasing from
propyl to hexyl to octyl, respectively, showed a decreasing
activity trend. While 19 (IC50 = 0.088 μM) and 2 (IC50 = 0.087
μM) exhibited equivalent anti-HIV potency, compound 21 was
approximately 20-fold less potent (IC50 = 1.8 μM) and 23
showed no inhibition of HIV-1 replication. Compound 20, with
a C-28 prop-2-ynyl moiety, exhibited good antiviral activity
(IC50 = 0.067 μM), and 22, with a second ester substitution at
the terminus of a C-28 hexyl chain, showed increased antiviral
The following SAR conclusions summarize the effects of the
C-28 substitutions to promote the antimaturation activity of 2.
C-28 ester side chains with a three- to six-carbon alkyl chain
result in enhanced anti-HIV activity. The presence of a polar
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1
(C34H56O5 − H). H NMR (400 MHz, CDCl3): δ 5.28−5.22 (2H,
app d, J = 5.6 Hz, -COOCH2O-), 4.80, 4.67 (1H each, app d, J = 6.4
Hz, -OCH2O-), 4.73, 4.61 (1H each, s, H-29), 3.48, 3.38 (3H each, s,
-OCH3), 3.08−2.99 (2H, m, H-3, H-19), 1.69 (3H, s, H-30), 0.97,
0.95, 0.93, 0.83, 0.78 (3H each, s, 5 × CH3). 13C NMR (100 MHz,
CDCl3): δ 14.9, 16.2, 16.4, 16.5, 18.5, 19.6, 21.1, 24.4, 25.8, 28.2, 29.8,
30.8, 32.2, 34.6, 37.1, 37.3, 38.5, 38.9, 38.9, 41.0, 42.6, 47.1, 49.6, 50.8,
55.7, 56.0, 57.0, 57.9, 85.4, 90.1, 96.2, 109.9, 150.7, 175.8.
oxygen atom within the side chain and the introduction of a
second ester functionality at the terminus of the C-28 ester
chain can moderately enhance the antimaturation activity of 2,
as shown by 17 and 22. Meanwhile, while 1 analogues with C-
28 primary amide substitutions function as antientry agents, 1
analogues with C-28 secondary cyclic amide groups do not.
Instead, the latter compounds exhibit significant antimaturation
promoting activity. Thus far, compound 41, which has an
additional polar nitrogen heteroatom present in the C-28 amide
(piperazinepentanoic acid), showed the best antiviral repli-
cation activity acting through an antimaturation mechanism of
action.
To evaluate its potential as a drug candidate, 41 was further
assessed for its in vitro metabolic stability in pooled human
liver microsomes (HLMs). The results revealed that 86.15% of
41 remained intact after 60 min of incubation in HLMs.
Compound 41’s long in vitro half-life (t1/2 of 327 min) and low
clearance (CLint of 0.0021 mL min−1 mg−1) proved that
incorporation of C-28 cyclic secondary amide chains would not
decrease the stability of 2, which has a long elimination half-life
(t1/2 of 56.3−69.5 h) in healthy volunteers.18
Overall, new BA derivatives were designed and synthesized in
order to investigate the modulating effects of C-28 substitution
toward 2’s antimaturation activity. It was discovered that certain
ester or cyclic secondary amide substitutions at C-28 will
enhance the antiviral activity of 2, acting only through an
antimaturation effect. The best new compound 41 showed
excellent anti-HIV activity with an IC50 of 0.0059 μM, 15-fold
better than that of 2. Compound 41 also exhibited good in vitro
metabolic stability in HLMs, and therefore, may serve as an
attractive new lead for the development of a next generation of
maturation inhibitors as anti-AIDS clinical trial candidates.
Methoxymethyl 3β-Hydroxylup-20(29)-en-28-oate (7). Yield,
190 mg (86% yield), starting from 200 mg of 1; white amorphous
powder. Mp 114−115 °C. HRMS (ESI−) m/z: 499.3745 (C32H52O4
− H). 1H NMR (400 MHz, CDCl3): δ 5.28−5.22 (2H, app q, J = 6.0,
5.6 Hz, -COOCH2O-), 4.73, 4.61 (1H each, s, H-29), 3.48 (3H, s,
-OCH3), 3.18 (1H, dd, J = 11.2, 5.2 Hz, H-3), 3.05−2.98 (1H, m, H-
19), 2.21−2.30 (2H, m, H-16), 1.69 (3H, s, H-30), 0.97, 0.96, 0.93,
0.82, 0.75 (3H each, s, 5 × CH3). 13C NMR (100 MHz, CDCl3): δ
14.9, 15.6, 16.2, 16.3, 18.5, 19.6, 21.1, 25.7, 27.6, 28.2, 29.8, 29.9, 30.7,
32.2, 34.6, 37.1, 37.4, 38.4, 38.9, 39.0, 40.9, 42.6, 47.1, 49.6, 50.8, 55.6,
56.9, 57.9, 79.1, 90.1, 109.9, 150.6, 175.8.
Syntheses of 4 and 8. Compound 1 (1 equiv), DIEA (4 equiv),
and MEMCl (4 equiv for 4 and 1.1 equiv for 8) were dissolved in THF
(5 mL) at room temperature. For 4, the mixture was stirred at 65 °C
under N2 for 6 h, and for 8, the mixture was stirred at 60 °C for 3 h.
The solution was then diluted with water, extracted with CH2Cl2, and
washed with brine. The organic layer was dried over anhydrous
MgSO4 and concentrated to dryness under reduced pressure. The
residue was chromatographed using a silica gel column to yield the
pure target compounds 4 and 8.
Methoxyethoxymethyl 3β-Methoxyethoxymethoxylup-
20(29)-en-28-oate (4). Yield, 311 mg (89% yield), starting from
252 mg of 1; white amorphous powder. Mp 102−105 °C. HRMS (ESI
+) m/z: 655.4579 (C38H64O7 + Na). 1H NMR (400 MHz, CDCl3): δ
5.37−5.30 (2H, app dd, J = 6.0, 5.8 Hz, -COOCH2O-), 4.81, 4.65 (1H
each, app d, J = 6.8 Hz, -OCH2O-), 4.71, 4.58 (1H each, s, H-29), 3.80,
3.71, 3.57, 3.50 (2H each, m, 2 × -OCH2CH2O-), 3.38 (6H, s, 2 ×
-OCH3), 3.09−3.05 (1H, dd, J = 11.4, 4.4, 4.0 Hz, H-3), 3.01−2.94
(1H, m, H-19), 1.68 (3H, s, H-30), 0.97, 0.93, 0.91, 0.83, 0.77 (3H
each, s, 5 × CH3). 13C NMR (100 MHz, CDCl3): δ 14.91, 16.25,
16.38, 16.49, 18.51, 19.59, 21.14, 24.30, 25.78, 28.27, 29.88, 30.76,
32.20, 34.60, 37.10, 37.27, 38.45, 38.86, 38.89, 41.98, 42.64, 47.11,
49.62, 50.79, 55.94, 56.93, 59.26, 59.31, 67.19, 69.70, 71.76, 72.07,
85.33, 89.08, 95.13, 109.88, 150.70, 175.79.
EXPERIMENTAL SECTION
■
Chemistry. The melting points were measured with a Fisher Johns
melting apparatus without correction. 1H NMR spectra were measured
on an Inova 400 MHz spectrometer using Me4Si (TMS) as internal
standard. The solvent used was CDCl3, unless otherwise indicated.
High resolution mass spectra were measured on a Shimadzu LCMS-
IT-TOF with ESI interface. HPLC for purity determinations was
conducted using a Shimadzu LCMS with a Grace Alltima 2.1 mm
×150 mm HP C18 5 μm column and a Shimadzu SPD-M20A detector
at 205 or 220 nm wavelength. The detailed solvent percentage
conditions for each tested compound are listed in the Supporting
Information. All target compounds were at least 95% pure. Optical
rotations were measured with a Jasco Dip-2000 digital polarimeter at
25 °C at the sodium D line. Thin-layer chromatography (TLC) was
performed on Merck precoated silica gel 60 F-254 plates. For
purification of all synthetic BA derivatives, a Grace Reveleris flash
system equipped with RevealX detection allowing for multisignal
(UV/ELSD) collection to low milligram quantities was used for
medium pressure column chromatography. Reveleris Navigator
method optimizer and Grace Reveleris flash silica cartridges were
employed for high quality separations. Commercial chemicals were
obtained from Sigma-Aldrich, Inc.
Methoxyethoxymethyl 3β-Hydroxylup-20(29)-en-28-oate
(8). Yield, 219 mg (92% yield), starting from 200 mg of 1; white
amorphous powder. Mp 119−121 °C. HRMS (ESI+) m/z: 567.4043
1
(C34H56O5 + Na). H NMR (400 MHz, CDCl3): δ 5.37−5.30 (2H,
app dd, J = 6.4, 6.0 Hz, -COOCH2O-), 4.73, 4.60 (1H each, s, H-29),
3.80, 3.56 (2H each, t, J = 5.2 Hz, -OCH2CH2O-), 3.39 (3H, s,
-OCH3), 3.18 (1H, m, H-3), 3.04−2.98 (1H, m, H-19), 1.68 (3H, s,
H-30), 1.00, 0.97, 0.92, 0.82, 0.75 (3H each, s, 5 × CH3). 13C NMR
(100 MHz, CDCl3): δ 14.9, 15.6, 16.2, 16.4, 18.5, 19.6, 21.1, 25.8,
27.6, 28.2, 29.9, 30.8, 32.2, 34.6, 37.1, 37.4, 38.4, 39.0, 39.1, 41.0, 42.7,
47.1, 49.6, 50.8, 55.6, 56.9, 59.3, 69.7, 71.8, 79.2, 89.1, 109.9, 150.7,
175.8.
Syntheses of 34 and 35. Piperazine 31 (2 equiv) was dissolved in
toluene, and the mixture was heated to 80 °C. Compound 32 or 33 (1
equiv) was added, and the reaction proceeded for 4 h. After the
mixture was cooled and filtered, the organic layer was concentrated
under reduced pressure. The residue was chromatographed using a
silica gel column to yield 500 mg of 34 (50%) and 498 mg of 35
(42%).
Syntheses of 36 and 37. Oxalyl chloride solution (2 M in
CH2Cl2, 10 mL) was added to 3-OAc-BA (1 equiv)22 in CH2Cl2 (10
mL), and the mixture was stirred for 2 h. After concentration under
vacuum, the residual mixture was treated with 34 or 35 (1.6 equiv) and
triethylamine (Et3N, 1.2 equiv) in CH2Cl2. The mixture was stirred at
room temperature overnight. The solution was then diluted with
CH2Cl2 (20 mL) and washed with brine. The organic layer was dried
over anhydrous MgSO4 and concentrated to dryness under reduced
Syntheses of 3 and 7. Compound 1 (1 equiv), DIEA (4 equiv),
and MOMCl (4 equiv for 3 and 1.1 equiv for 7) were dissolved in
CH2Cl2 (5 mL) at 0 °C. The mixture was stirred overnight under N2,
allowing a gradual warming to room temperature. The solution was
then diluted with CH2Cl2 (20 mL) and washed with brine. The
organic layer was dried over anhydrous MgSO4 and concentrated to
dryness under reduced pressure. The residue was chromatographed
using a silica gel column to yield the pure target compounds 3 and 7.
Methoxymethyl 3β-Methoxymethoxylup-20(29)-en-28-oate
(3). Yield, 149 mg (83% yield), starting from 179 mg of 1; white
amorphous powder. Mp 99−102 °C. HRMS (ESI−) m/z: 543.3991
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pressure. The residue was chromatographed using a silica gel column
to yield the pure target compounds 36 and 37.
using a silica gel column to yield 167 mg of 9 (53% yield), a white
amorphous powder. Mp 189−191 °C. HRMS (ESI+) m/z: 576.3656
(C34H51NO5 + Na). 1H NMR (400 MHz, CDCl3): δ 4.72, 4.61 (2H, s,
H-29), 3.18 (1H, dd, J = 11.2, 4.8 Hz, H-3), 2.95 (1H, m, H-19), 2.84,
2.82 (4H, d, J = 5.2 Hz, -COCH2CH2CO-), 2.47−2.42 (1H, m, H-13),
1.69 (3H, s, H-30), 0.98 (6H, s, 2 × CH3), 0.96, 0.82, 0.75 (3H each, s,
3 × CH3).
Syntheses of 26 and 27. A mixture of tetraethylene glycol (25, 35
g, 5 equiv), TEA (1.5 equiv), and DMAP (0.1 equiv) was dissolved in
CH2Cl2 (200 mL) at 0 °C. TBSCl (5 g, 1 equiv) in CH2Cl2 (50 mL)
was added dropwise, and reaction continued overnight. The solution
was diluted with CH2Cl2 and washed with mildly acidic water and
brine. The organic layer was dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. The crude product was
purified by column chromatography to yield 50 g of TBS-protected
tetraethylene glycol 26 (90%).24 In the next step, a mixture of 26 (852
mg, 1 equiv) and CBr4 (2 equiv) was dissolved in CH2Cl2 (200 mL) at
0 °C. Pyridine (2.5 mL, 10 equiv) was added, followed by PPh3 (2
equiv). The mixture was stirred overnight with a gradual warming to
room temperature. The suspension was filtered through Celite, and
the organic layer was concentrated to dryness under reduced pressure.
The residue was chromatographed using a silica gel column to yield
872 mg of bromo-substituted TBS-protected tetraethylene glycol 27
(85% yield).25
Methyl N-[3β-Acetoxylup-20(29)-en-28-oyl]-4-piperazinebu-
tanoate (36). Yield, 177.5 mg (89% yield), starting from 150 mg of 3-
OAc-BA; white amorphous powder. Mp 203−205 °C. HRMS (ESI+)
1
m/z: 667.5055 (C41H66N2O5 + H). H NMR (400 MHz, CDCl3): δ
4.72, 4.60 (1H each, s, H-29), 4.47 (1H, dd, J = 12.4, 5.6 Hz, H-3),
3.68 (3H, s, -COOCH3), 3.61−3.52 (4H, m, 28-CON(CH2CH2)2N-),
3.01 (1H, m, H-19), 2.91−2.76 (4H, m, 28-CON(CH2CH2)2N-), 2.46
(2H, m, -NCH2-), 2.05 (3H, s, OCOCH3), 1.68 (3H, s, H-30), 1.02,
0.99, 0.96, 0.85, 0.81 (3H each, s, 5 × CH3).
Methyl N-[3β-Acetoxylup-20(29)-en-28-oyl]-5-piperazine-
pentanoate (37). Yield, 847 mg (100% yield), starting from 622.8
mg of 3-OAc-BA; white amorphous powder. Mp 217−218 °C. HRMS
(ESI+) m/z: 681.5219 (C42H68N2O5 + H). 1H NMR (400 MHz,
CDCl3): δ 4.73, 4.60 (1H each, s, H-29), 4.46 (1H, m, H-3), 3.67 (3H,
s, -COOCH3), 3.58−3.47 (4H, m, 28-CON(CH2CH2)2N-), 3.00 (1H,
m, H-19), 2.87−2.76 (4H, m, 28-CON(CH2CH2)2N-), 2.46 (2H, m,
-NCH2-), 2.06 (3H, s, OCOCH3), 1.69 (3H, s, H-30), 0.98, 0.97, 0.90,
0.86, 0.83 (3H each, s, 5 × CH3).
Syntheses of 5, 6, 38, and 39. To a solution of the appropriate
ester intermediates 3−4 and 36−37 (1 equiv) in MeOH (8 mL) and
THF (4 mL) was added 4 N NaOH (4 mL). The mixture was stirred
overnight and then neutralized with 20% HCl. The solution was dried
under vacuum and reconstituted with CH2Cl2. The organic layer was
washed with brine and dried over anhydrous MgSO4 and concentrated
to dryness under reduced pressure. The residue was chromatographed
using a silica gel column to yield the pure target compounds 5−6 and
38−39.
Syntheses of 10−15 and 28. Compound 1 (1 equiv) and
CS2CO3 (3 equiv) were dissolved in a 1:1 mixed solution of DMF and
THF (4 mL), and the mixture was stirred for 30 min at room
temperature. Bromo- or iodo-substituted compound (RX) or 27 (3
equiv) was then added, and the reaction was carried out overnight at
room temperature. Water was added to stop the reaction, and the
solution was extracted with EtOAc. The organic layer was washed
further with brine, dried over anhydrous MgSO4, and concentrated
under reduced pressure. The residue was chromatographed using a
silica gel column to yield the pure target compounds 10−15 and 28.
Propyl 3β-Hydroxylup-20(29)-en-28-oate (10). Yield, 56 mg
(86% yield), starting from 60 mg of 1; white amorphous powder. Mp
3β-Methoxymethoxybetulinic Acid (5). Yield, 85 mg (93%
yield), starting from 100 mg of 3; white amorphous powder. Mp 117−
1
119 °C. HRMS (ESI−) m/z: 499.3730 (C32H52O4 − H). H NMR
(400 MHz, CDCl3): δ 4.75, 4.62 (1H each, app d, J = 6.4 Hz,
-OCH2O-), 4.71, 4.59 (1H each, s, H-29), 3.36 (3H, s, -OCH3), 3.05−
2.96 (2H, m, H-3, H-19), 1.71 (3H, s, H-30), 0.97, 0.95, 0.90, 0.83,
0.77 (3H each, s, 5 × CH3). 13C NMR (100 MHz, CDCl3): δ 14.7,
16.1, 16.1, 16.3, 18.3, 19.4, 20.9, 24.2, 25.5, 28.0, 29.7, 30.6, 32.2, 34.3,
37.1, 38.4, 38.7, 40.7, 42.4, 47.0, 49.3, 50.5, 55.5, 55.7, 56.4, 77.3, 85.2,
96.0, 109.7, 150.4, 181.8.
1
106−107 °C. HRMS (ESI+) m/z: 499.4155 (C33H54O3 + H). H
NMR (400 MHz, CDCl3): δ 4.73, 4.60 (2H, s, H-29), 4.09 (2H, m,
28-COOCH2-), 3.18 (1H, dd, J = 11.2, 4.8 Hz, H-3), 3.01 (1H, m, H-
19), 1.68 (3H, s, H-30), 1.01 (6H, s, 2 × CH3), 0.94 (3H, s, CH3),
0.91 (3H, m, CH3), 0.82, 0.76 (3H each, s, 2 × CH3).
3β-Methoxyethoxymethoxybetulinic Acid (6). Yield, 118 mg
(92% yield), starting from 150 mg of 4; white amorphous powder. Mp
1
Prop-2′-ynyl 3β-Hydroxylup-20(29)-en-28-oate (11). Yield, 47
mg (72% yield), starting from 60 mg of 1; white amorphous powder.
Mp 131−133 °C. HRMS (ESI+) m/z: 495.3826 (C33H50O3 + H). 1H
NMR (400 MHz, CDCl3): δ 4.73, 4.61 (2H, s, H-29), 4.69, 4.65 (2H,
d, J = 2.2 Hz, 28-COOCH2-), 3.18 (1H, m, H-3), 3.01 (1H, m, H-19),
2.43 (1H, t, J = 2.2 Hz, -CCH), 1.69 (3H, s, H-30), 0.97 (6H, s, 2 ×
CH3), 0.93, 0.82, 0.75 (3H each, s, 3 × CH3).
121−124 °C. HRMS (ESI+) m/z: 567.4033 (C34H56O5 + Na). H
NMR (400 MHz, CDCl3): δ 4.80, 4.66 (1H each, app d, J = 6.8 Hz,
-OCH2O-), 4.70, 4.58 (1H each, s, H-29), 3.69, 3.52 (2H each, t, J =
4.4 Hz, -OCH2CH2O-), 3.36 (3H, s, -OCH3), 3.07 (1H, dd, J = 12.0,
4.4 Hz, H-3), 3.00 (1H, m, H-19), 1.67 (3H, s, H-30), 0.94, 0.91, 0.89,
0.80, 0.73 (3H each, s, 5 × CH3).
N-[3β-Hydroxylup-20(29)-en-28-oyl]-4-piperazinebutyric
Acid (38). Yield, 91 mg (79% yield), starting from 125 mg of 36;
white amorphous powder. Mp 153−154 °C. HRMS (ESI−) m/z:
3′-Phenylpropyl 3β-Hydroxylup-20(29)-en-28-oate (12).
Yield, 72 mg (96% yield), starting from 60 mg of 1; white amorphous
powder. Mp 169−172 °C. HRMS (ESI+) m/z: 575.4465 (C39H58O3 +
1
609.4646 (C38H62N2O4 − H). H NMR (400 MHz, CDCl3): δ 4.71,
1
4.60 (1H each, s, H-29), 3.65−3.49 (4H, m, 28-CON(CH2CH2)2N-),
3.17 (1H, m, H-3), 3.01 (1H, m, H-19), 2.82−2.68 (4H, m, 28-
CON(CH2CH2)2N-), 2.45 (2H, m, -NCH2-), 1.69 (3H, s, H-30), 1.01,
0.98, 0.89, 0.84, 0.78 (3H each, s, 5 × CH3).
H). H NMR (400 MHz, CDCl3): δ 7.31−7.26, 7.22−7.18 (5H, m,
-C6H5), 4.73, 4.61 (2H, s, H-29), 4.09 (2H, m, 28-COOCH2-), 3.18
(1H, m, H-3), 3.02 (1H, m, H-19), 2.72 (1H, t, J = 6.4 Hz,
-CH2C6H5), 1.69 (3H, s, H-30), 0.97, 0.96, 0.91, 0.81, 0.75 (3H each,
s, 5 × CH3).
N-[3β-Hydroxylup-20(29)-en-28-oyl]-5-piperazinepentanoic
Acid (39). Yield, 624 mg (85% yield), starting from 800 mg of 37;
white amorphous powder. Mp 159−161 °C. HRMS (ESI−) m/z:
Hexyl 3β-Hydroxylup-20(29)-en-28-oate (13). Yield, 48 mg
(67% yield), starting from 60 mg of 1; white amorphous powder. Mp
1
1
623.4781 (C39H64N2O4 − H). H NMR (400 MHz, CDCl3): δ 4.71,
124−125 °C. HRMS (ESI+) m/z: 541.4629 (C36H60O3 + H). H
4.58 (1H each, s, H-29), 3.64−3.51 (4H, m, 28-CON(CH2CH2)2N-),
3.19 (1H, m, H-3), 2.99 (1H, m, H-19), 2.89−2.72 (4H, m, 28-
CON(CH2CH2)2N-), 2.46 (2H, m, -NCH2-), 1.68 (3H, s, H-30), 0.98,
0.95, 0.92, 0.82, 0.81 (3H each, s, 5 × CH3).
NMR (400 MHz, CDCl3): δ 4.73, 4.59 (2H, s, H-29), 4.07 (2H, m,
28-COOCH2-), 3.18 (1H, dd, J = 11.8, 4.4 Hz, H-3), 3.02 (1H, m, H-
19), 1.68 (3H, s, H-30), 0.97 (6H, s, 2 × CH3), 0.92 (3H, s, CH3),
0.90 (3H, m, CH3), 0.82, 0.75 (3H each, s, 2 × CH3).
N-Succinimide 3β-Hydroxylup-20(29)-en-28-oate (9). A mix-
ture of 1 (260 mg, 1 equiv), N-hydroxysuccinimide (1.5 equiv), EDCI
(1.5 equiv), and DMAP (0.1 equiv) was dissolved in CH2Cl2 (5 mL)
and stirred under N2 at room temperature overnight. The solution was
then diluted with CH2Cl2 (20 mL) and washed with brine. The
organic layer was dried over anhydrous MgSO4 and concentrated to
dryness under reduced pressure. The residue was chromatographed
Ethyl O-[3β-Hydroxylup-20(29)-en-28-oyl]-6-hexanoate (14).
Yield, 77 mg (98% yield), starting from 60 mg of 1; white amorphous
powder. Mp 191−194 °C. HRMS (ESI+) m/z: 599.4649 (C38H62O5 +
1
H). H NMR (400 MHz, CDCl3): δ 4.72, 4.59 (2H, s, H-29), 4.16−
4.02 (4H, m, 28-COOCH2-, -COOCH2CH3), 3.41 (1H, t, J = 6.8 Hz,
-OH), 3.17 (1H, dd, J = 11.8, 4.4 Hz, H-3), 3.00 (1H, m, H-19), 2.31
(2H, m, -CH2COO-), 1.68 (3H, s, H-30), 1.26 (3H, t, J = 7.2 Hz,
8133
dx.doi.org/10.1021/jm301040s | J. Med. Chem. 2012, 55, 8128−8136

Journal of Medicinal Chemistry
Article
-COOCH2CH3), 0.97 (6H, s, 2 × CH3), 0.91, 0.82, 0.75 (3H each, s, 3
× CH3).
z: 680.4161 (C40H59NO8 − H). 1H NMR (400 MHz, CDCl3): δ 4.72,
4.60 (2H, s, H-29), 4.47 (1H, dd, J = 11.2, 5.2 Hz, H-3), 2.99 (1H, m,
H-19), 2.84, 2.82 (4H, d, J = 5.2 Hz, -COCH2CH2CO-), 2.64−2.41
(2H, m, H-2′), 1.68 (3H, s, H-30), 1.28 (6H, s, 2 × CH3-3′), 0.98
(6H, s, 2 × CH3), 0.91, 0.82, 0.75 (3H each, s, 3 × CH3).
Octyl 3β-Hydroxylup-20(29)-en-28-oate (15). Yield, 70 mg
(94% yield), starting from 60 mg of 1; white amorphous powder. Mp
146−147 °C. HRMS (ESI+) m/z: 569.4936 (C38H64O3 + H). 1H
NMR (400 MHz, CDCl3): δ 4.73, 4.60 (2H, s, H-29), 4.07 (2H, m,
28-COOCH2-), 3.17 (1H, dd, J = 12.0, 4.0 Hz, H-3), 3.02 (1H, m, H-
19), 1.68 (3H, s, H-30), 0.96 (6H, s, 2 × CH3), 0.92 (3H, s, CH3),
0.89 (3H, t, J = 6.4 Hz, CH3), 0.82, 0.75 (3H each, s, 2 × CH3).
2-(2-(2-(2-O-tert-Butyldimethylsilylethoxy)ethoxy)ethoxy)-
ethyl 3β-hydroxylup-20(29)-en-28-oate (28). Yield, 142 mg (72%
yield), starting from 120 mg of 1; white amorphous powder. Mp 203−
Propyl 3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-28-oate
(19). Yield, 23 mg (46% yield), starting from 40 mg of 10; white
amorphous powder. Mp 109−111 °C. HRMS (ESI−) m/z: 625.4465
1
(C39H62O6 − H). H NMR (400 MHz, CDCl3): δ 4.73, 4.60 (2H, s,
H-29), 4.48 (1H, dd, J = 11.0, 4.8 Hz, H-3), 4.09 (2H, m, 28-
COOCH2-), 3.00 (1H, m, H-19), 2.58−2.39 (2H, m, H-2′), 1.68 (3H,
s, H-30), 1.28, 1.27 (3H each, s, 2 × CH3-3′), 1.00, 0.98, 0.93 (3H
each, s, 3 × CH3), 0.89 (3H, t, J = 6.4 Hz, CH3), 0.82, 0.75 (3H each,
s, 2 × CH3).
1
206 °C. HRMS (ESI+) m/z: 747.5603 (C44H78O7Si + H). H NMR
(400 MHz, CDCl3): δ 4.73, 4.62 (2H, s, H-29), 4.25 (2H, m,
-COOCH2CH2O-), 3.96−3.54 (2H each, m, -COOCH2CH2O-, 3 ×
-OCH2CH2O-), 3.17 (1H, dd, J = 11.2, 4.8 Hz, H-3), 3.01 (1H, m, H-
19), 1.68 (3H, s, H-30), 0.98 (12H, s, CH3, -Si(CH3)2C(CH3)3), 0.91,
0.89, 0.82, 0.75 (3H each, s, 4 × CH3), 0.21 (6H, s, -Si(CH3)2C-
(CH3)3).
Prop-2′-ynyl 3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-28-
oate (20). Yield, 28 mg (55% yield), starting from 40 mg of 11; light
yellow amorphous powder. Mp 127−129 °C. HRMS (ESI−) m/z:
621.4175 (C39H58O6 − H). 1H NMR (400 MHz, CDCl3): δ 4.73, 4.60
(2H, s, H-29), 4.69, 4.66 (2H, d, J = 2.4 Hz, 28-COOCH2-), 4.47 (1H,
dd, J = 11.8, 5.6 Hz, H-3), 3.00 (1H, m, H-19), 2.62−2.40 (3H, m, H-
2′, -CCH), 1.69 (3H, s, H-30), 1.30, 1.28 (3H each, s, 2 × CH3-3′),
0.97 (6H, s, 2 × CH3), 0.93, 0.81, 0.75 (3H each, s, 3 × CH3).
Hexyl 3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-28-oate
(21). Yield, 26 mg (52% yield), starting from 40 mg of 13; white
amorphous powder. Mp 118−120 °C. HRMS (ESI−) m/z: 667.4931
2-(2-(2-(2-Hydroxyethoxy)ethoxy)ethoxy)ethyl 3β-hydroxyl-
up-20(29)-en-28-oate (29). To a solution of 28 (170 mg, 1 equiv)
in THF (5 mL) was added TBAF (1.5 equiv). The mixture was stirred
overnight at room temperature. The organic layer was concentrated to
dryness under reduced pressure and the residue was chromatographed
using a silica gel column to yield 166 mg of 29 (98% yield), a white
amorphous powder. Mp 178−180 °C. HRMS (ESI+) m/z: 633.4743
1
(C42H68O6 − H). H NMR (400 MHz, CDCl3): δ 4.73, 4.60 (2H, s,
1
H-29), 4.47 (1H, dd, J = 11.2, 4.8 Hz, H-3), 4.07 (2H, m, 28-
COOCH2-), 3.01 (1H, m, H-19), 2.54−2.41 (2H, m, H-2′), 1.68 (3H,
s, H-30), 1.31, 1.28 (3H each, s, 2 × CH3-3′), 0.97 (6H, s, 2 × CH3),
0.92 (3H, s, CH3), 0.89 (3H, m, CH3), 0.81, 0.75 (3H each, s, 2 ×
CH3).
(C38H64O7 + H). H NMR (400 MHz, CDCl3): δ 4.73, 4.61 (2H, s,
H-29), 4.25 (2H, m, -COOCH2CH2O-), 3.65−3.44 (2H each, m,
-COOCH2CH2O-, 3 × -OCH2CH2O-), 3.17 (1H, dd, J = 11.6, 4.8 Hz,
H-3), 3.01 (1H, m, H-19), 1.68 (3H, s, H-30), 0.98, 0.97, 0.89, 0.82,
0.76 (3H each, s, 5 × CH3).
Ethyl O-[3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-28-oyl]-
6-hexanoate (22). Yield, 28 mg (46% yield), starting from 50 mg of
14; light yellow amorphous powder. Mp 179−181 °C. HRMS (ESI−)
Syntheses of 16−23, 30, 40, and 41. The appropriate BA
derivative (1 equiv), 2,2-dimethylsuccinic anhydride (5 equiv), DMAP
(1.6 equiv), and PTSA (catalytic amount) was dissolved in THF (1.5
mL), and the mixture was stirred at 130 °C for 2 h using a microwave
synthesizer (Biotage). Water was added, and the mixture was then
extracted with CH2Cl2 (15 mL). The organic layer was dried over
anhydrous MgSO4 and concentrated to dryness under reduced
pressure. The residue was chromatographed using a silica gel column
to yield pure target compounds 16−23, 30, 40, and 41.
1
m/z: 725.4989 (C44H70O8 − H). H NMR (400 MHz, CDCl3): δ
4.72, 4.59 (2H, s, H-29), 4.48 (1H, t, J = 7.8 Hz, H-3), 4.18−4.02 (4H,
m, 28-COOCH2-, -COOCH2CH3), 3.01 (1H, m, H-19), 2.62−2.43
(2H, m, H-2′), 2.31 (2H, m, -CH2COO-), 1.68 (3H, s, H-30), 1.31,
1.28 (3H each, s, 2 × CH3-3′), 1.26 (3H, t, J = 7.2 Hz,
-COOCH2CH3), 0.98 (6H, s, 2 × CH3), 0.91, 0.82, 0.75 (3H each,
25
s, 3 × CH3). [α]D −16.6° (c 0.22, MeOH).
Methoxymethyl 3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-
28-oate (16). Yield, 25 mg (40% yield), starting from 52 mg of 7;
white amorphous powder. Mp 107−109 °C. HRMS (ESI−) m/z:
Octyl 3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-28-oate
(23). Yield, 31 mg (51% yield), starting from 50 mg of 15; white
amorphous powder. Mp 136−137 °C. HRMS (ESI−) m/z: 695.5248
(C44H72O6 - H). 1H NMR (400 MHz, CDCl3): δ 4.73, 4.60 (2H, s, H-
29), 4.47 (1H, t, J = 7.6 Hz, H-3), 4.07 (2H, m, 28-COOCH2-), 3.01
(1H, m, H-19), 2.61−2.40 (2H, m, H-2′), 1.68 (3H, s, H-30), 1.28
(6H, s, 2 × CH3-3′), 0.97 (6H, s, 2 × CH3), 0.92 (3H, s, CH3), 0.89
(3H, t, J = 6.4 Hz, CH3), 0.82, 0.75 (3H each, s, 2 × CH3).
1
627.4247 (C38H60O7 − H). H NMR (400 MHz, CDCl3): δ 11.11
(1H, br s, -COOH), 5.28−5.22 (2H, app d, J = 5.8 Hz, -COOCH2O-),
4.72, 4.60 (1H each, s, H-29), 4.47 (1H, dd, J = 11.2, 5.6 Hz, H-3),
3.48 (3H, s, -OCH3), 3.00 (1H, m, H-19), 2.64−2.42 (2H, m, H-2′),
1.68 (3H, s, H-30), 1.30, 1.28 (3H each, s, 2 × CH3-3′), 0.98, 0.97,
0.92, 0.81, 0.75 (3H each, s, 5 × CH3). 13C NMR (100 MHz, CDCl3):
δ 14.9, 16.2, 16.4, 16.7, 18.4, 19.6, 21.1, 23.8, 25.3, 25.7, 25.8, 28.1,
29.9, 30.8, 32.2, 34.5, 37.1, 37.3, 38.0, 38.4, 38.6, 40.7, 41.0, 42.6, 44.9,
47.1, 49.6, 50.7, 55.7, 57.0, 57.9, 81.8, 90.1, 109.9, 150.6, 171.2, 175.8,
182.6.
2-[2-[2-[2-O-(3′,3′-Dimethylsuccinyl)ethoxy]ethoxy]ethoxy]-
ethyl 3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-28-oate (30).
Yield, 50 mg (41% yield), starting from 100 mg of 29; light yellow
amorphous powder. Mp 165−167 °C. HRMS (ESI−) m/z: 759.5013
1
(C44H72O10 − H). H NMR (400 MHz, CDCl3): δ 4.73, 4.60 (2H, s,
Methoxyethoxymethyl 3β-O-(3′,3′-Dimethylsuccinyl)lup-
20(29)-en-28-oate (17). Yield, 30.5 mg (41% yield), starting from
60 mg of 8; white amorphous powder. Mp 115−118 °C. HRMS
(ESI−) m/z: 671.4511 (C40H64O8 − H). 1H NMR (400 MHz,
CDCl3): δ 11.16 (1H, br s, -COOH), 5.37−5.30 (2H, app d, J = 6.0
Hz, -COOCH2O-), 4.72, 4.59 (1H each, s, H-29), 4.47 (1H, t, J = 7.6
Hz, H-3), 3.80, 3.56 (2H each, t, J = 5.2 Hz, -OCH2CH2O-), 3.42 (3H,
s, -OCH3), 3.01 (1H, m, H-19), 2.67−2.46 (2H, m, H-2′), 1.68 (3H, s,
H-30), 1.29, 1.28 (3H each, s, 2 × CH3-3′), 1.00, 0.97, 0.92, 0.82, 0.76
(3H each, s, 5 × CH3). 13C NMR (100 MHz, CDCl3): δ 14.9, 16.2,
16.4, 16.7, 18.4, 19.6, 21.1, 23.8, 25.3, 25.7, 25.8, 28.1, 29.9, 30.7, 32.2,
34.5, 37.1, 37.3, 37.9, 38.4, 38.6, 40.7, 41.0, 42.6, 45.0, 47.1, 49.6, 50.7,
55.7, 56.9, 59.3, 69.7, 71.7, 81.8, 89.1, 109.9, 150.6, 171.2, 175.8, 183.0.
H-29), 4.48 (1H, dd, J = 11.4, 4.8 Hz, H-3), 4.26 (2H, m,
-COOCH2CH2O-), 3.68−3.41 (2H each, m, -COOCH2CH2O-, 3 ×
-OCH2CH2O-), 3.01 (1H, m, H-19), 2.64−2.42 (2H, m, H-2′), 1.68
(3H, s, H-30), 1.30, 1.28 (3H each, s, 2 × CH3-3′), 0.98, 0.97, 0.91,
0.82, 0.76 (3H each, s, 5 × CH3).
N-[3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-28-oyl]-4-pi-
perazinebutyric Acid (40). Yield, 45 mg (53% yield), starting from
70 mg of 38; light yellow amorphous powder. Mp 127−129 °C.
1
HRMS (ESI−) m/z: 737.5109 (C44H70N2O7 − H). H NMR (400
MHz, CDCl3): δ 4.71, 4.60 (1H each, s, H-29), 4.47 (1H, dd, J = 11.6,
4.8 Hz, H-3), 3.65−3.49 (4H, m, 28-CON(CH2CH2)2N-), 3.01 (1H,
m, H-19), 2.82−2.68 (4H, m, 28-CON(CH2CH2)2N-), 2.64−2.40
(3H, m, H-2′, -NCH2-), 1.68 (3H, s, H-30), 1.31, 1.28 (3H each, s, 2 ×
25
25
[α]D −13.8° (c 0.28, MeOH).
CH3-3′), 1.01, 0.98, 0.91, 0.84, 0.76 (3H each, s, 5 × CH3). [α]D
N-Succinimide 3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-
28-oate (18). Yield, 17 mg (39% yield), starting from 35 mg of 9;
light yellow amorphous powder. Mp 164−166 °C. HRMS (ESI−) m/
−17.5° (c 0.28, MeOH).
N-[3β-O-(3′,3′-Dimethylsuccinyl)lup-20(29)-en-28-oyl]-5-pi-
perazinepentanoic Acid (41). Yield, 199 mg (55% yield), starting
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from 300 mg of 39; white amorphous powder. Mp 135−137 °C.
injected directly onto a Shimadzu LC−MS 2010 system by
autosampler. The HPLC−MS analysis was carried out with an
electrospray ionization source (ESI). An Alltima C18 column (5 μm,
150 mm × 2.1 mm) was used for HPLC with a gradient elution at a
flow rate of 0.3 mL/min. The elution conditions for 41 were ACN (B)
in water (A) at 30% for 0−3 min, 95% for 3−9 min, and 30% for 9−12
min. The MS conditions were optimized to detector voltage of +1.7
kV, positive acquisition mode with selected ion monitoring (SIM) of
the appropriate molecular weights of the testing compounds. The
CDL temperature was 250 °C, and heat block temperature was 200
°C. The neutralizing gas flow was 1.5 L/min. The peak heights of test
compounds at different time points were converted to the percentage
remaining, and the peak height values at initial time (0 min) served as
100%. The slope of the linear regression from log of the percentage
remaining versus incubation time relationships (−k) was used to
calculate in vitro half-life (t1/2) by the formula of in vitro t1/2 = 0.693/
k, regarded as first-order kinetics. Conversion to in vitro clearance
(CLint in units of (mL/min)/mg protein) was calculated by the
formula:28 CLint = (0.693/in vitro t1/2)[(mL incubation)/(mg of
microsomes)]. Fast and moderate metabolizing reference compounds
terfenadine and propranolol, respectively, have t1/2 of 21.14 and 40.82
min, respectively, in these assay conditions.
1
HRMS (ESI−) m/z: 751.5251 (C45H72N2O7 − H). H NMR (400
MHz, CDCl3): δ 4.73, 4.60 (1H each, s, H-29), 4.47 (1H, dd, J = 11.2,
4.8 Hz, H-3), 3.64−3.51 (4H, m, 28-CON(CH2CH2)2N-), 3.00 (1H,
m, H-19), 2.89−2.72 (4H, m, 28-CON(CH2CH2)2N-), 2.61−2.40
(3H, m, H-2′, -NCH2-), 1.69 (3H, s, H-30), 1.30, 1.28 (3H each, s, 2 ×
25
CH3-3′), 0.98, 0.97, 0.92, 0.82, 0.75 (3H each, s, 5 × CH3). [α]D
−18.4° (c 0.30, MeOH).
[1-(3′-Deoxythymidine)-1H-1,2,3-triazol-4-yl]methyl 3β-O-
(3′,3′-Dimethylsuccinyl)lup-20(29)-en-28-oate (24). Compound
20 (25 mg, 1.3 equiv), AZT (1 equiv), CuI (0.2 equiv), and i-Pr2EtN
(2 equiv) were dissolved in DMF (1 mL), and the mixture was stirred
at 120 °C for 30 min using a microwave synthesizer (Biotage). The
solution was diluted with EtOAc and then washed with water,
saturated NaHCO3, and brine. The organic layer was dried over
anhydrous MgSO4 and concentrated to dryness under reduced
pressure. The residue was chromatographed using a silica gel column
to yield 20 mg of 24 (75%), a white amorphous powder. Mp 186−189
°C. HRMS (ESI+) m/z: 890.5227 (C49H71N5O10 + H). 1H NMR (400
MHz, CDCl3): δ 9.31 (1H, br, s, -NH), 7.79 (1H, s, triazol-H), 7.48,
7.44 (1H, d, J = 16 Hz, Thy-H), 6.21 (1H, t, J = 5.6 Hz, ribose-CH-2′),
5.42 (1H, m, ribose-CH-4′), 5.28−5.20 (2H, m, 28-COOCH2-), 4.72,
4.60 (2H, s, H-29), 4.46 (1H, m, H-3), 4.04, 3.80−3.73 (4H, m,
ribose-CH-5′-CH2-OH), 3.06−2.96 (3H, m, H-19, ribose-CH2-3′),
2.70−2.56 (2H, m, H-2′), 1.94 (3H, s, Thy-CH3), 1.67 (3H, s, H-30),
1.30, 1.28 (3H each, s, 2 × CH3-3′), 1.01 (6H, s, 2 × CH3), 0.93, 0.81,
0.75 (3H each, s, 3 × CH3).
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional information on compound purity, high-resolution
mass spectroscopic data, and HPLC analysis results of the
target compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
HIV-1NL4‑3 Replication Inhibition Assay in MT-4 Lympho-
cytes. A previously described HIV-1 infectivity assay was used.26,27 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). The 50% inhibition
concentration (IC50) was defined as the concentration that inhibits
HIV-1NL4‑3 replication by 50%.
Cytotoxicity Assay. A CytoTox-Glo cytotoxicity assay (Promega)
was used to determine the cytotoxicity of the synthesized BA
derivatives. Mock-infected MT-4 cells were cultured in the presence of
various concentrations of the compounds for 2 days. Percent of viable
cells was determined by following the protocol provided by the
manufacturer. The 50% cytotoxic concentration (CC50) was defined as
the concentration that caused a 50% reduction of cell viability.
TZM-bl Assay. Anti-HIV-1 activity was measured as reduction in
Luc reporter gene expression after a single round of virus infection of
TZM-bl cells. HIV-1 at 200 TCID50 and test samples were mixed in a
total volume of 100 mL of growth medium in 96-well black solid plates
(Corning-Costar). After 48 h of incubation, culture medium was
removed from each well and 100 mL of Bright Glo luciferase reagent
was added to each culture well. The luciferase activity in the assay wells
was measured using a Victor 2 luminometer. The 50% inhibitory dose
(IC50) was defined as the sample concentration that caused a 50%
reduction in relative luminescence units (RLU) compared to virus
control wells after subtraction of background RLU.
AUTHOR INFORMATION
■
Corresponding Author
*For K.Q.: phone, 919-883-5306; fax, 919-966-3893; e-mail,
kdqian@unc.edu. For K.-H.L.: phone, 919-962-0066; fax, 919-
966-3893; e-mail, khlee@unc.edu.
Author Contributions
∥These authors contributed equally to the paper.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This investigation was supported by Grant AI-077417 from the
National Institute of Allergy and Infectious Diseases (NIAID)
awarded to K.-H.L. I.D.B. thanks the NIAID for a Minority
Research Supplement grant. Thanks are also due to the support
of Taiwan Department of Health Clinical Trial and Research
Center of Excellence (Grant DOH100-TD-B-111-004).
Microsomal Stability Assay. Stock solutions of 41, as well as
reference compounds propranolol and terfenadine (1 mg/mL), were
prepared by dissolving the pure compound in DMSO and were stored
at 4 °C. Before the assay, the stock solution was diluted with ACN to
0.1 mM. For measurement of metabolic stability, all compounds were
brought to a final concentration of 1 μM with 0.1 M potassium
phosphate buffer at pH 7.4, which contained 0.1 mg/mL human liver
microsomes and 5 mM MgCl2. The incubation volumes were 300 μL,
and reaction temperature was 37 °C. Reactions were started by adding
60 μL of NADPH (final concentration of 1.0 mM) and quenched by
adding 600 μL of ice-cold ACN to stop the reaction at 5, 15, 30, and
60 min time points. Samples at 0 min time point were prepared by
adding 600 μL of ice-cold ACN first, followed by 60 μL of NADPH.
Incubations of all samples were conducted in duplicate. After
quenching, all samples were centrifuged at 12 000 rpm for 5 min at
0 °C. The supernatant was collected, and 30 μL of the supernatant was
ABBREVIATIONS USED
■
ACN, acetonitrile; AIDS, acquired immunodeficiency syn-
drome; HIV-1, human immunodeficiency virus type 1; BA,
betulinic acid; HAART, highly active antiretroviral therapy; MI,
maturation inhibitor; P24 (CA), capsid; P25 (CA-SP1), capsid
precursor; bevirimat, 3-O-(3′,3′-dimethylsuccinyl)betulinic
acid; AZT, zidovudine; MOMCl, methoxymethyl chloride;
MEMCl, methoxyethoxymethyl chloride; DIEA, diisopropyle-
thylamine; THF, tetrahydrofuran; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; DMAP, 4-
(dimethylamino)pyridine; TBS, tert-butyldimethylsilyl; TEA,
triethylaluminum; PTSA, p-toluenesulfonic acid; TBAF, tetra-n-
butylammonium fluoride; HLM, human liver microsome; PI,
postinfection
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Sciences Series; Royal Society of Chemistry: Cambridge, U.K., 2009;
pp 374−391.
REFERENCES
■
(1) Barbaro, G.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T.
Highly active antiretroviral therapy: current state of the art, new agents
and their pharmacological interactions useful for improving
therapeutic outcome. Curr. Pharm. Des. 2005, 11, 1805−1843.
(2) Ho, D. D.; Neumann, A. U.; Perelson, A. S.; Chen, W.; Leonard,
J. M.; Markowitz, M. Rapid turnover of plasma virions and CD4
lymphocytes in HIV-1 infection. Nature 1995, 373, 123−126.
(3) Hammer, S. M.; Katzenstein, D. A.; Hughes, M. D.; Gundacker,
H.; Schooley, R. T.; Haubrich, R. H.; Henry, W. K.; Lederman, M. M.;
Phair, J. P.; Niu, M.; Hirsch, M. S.; Merigan, T. C. A trial comparing
nucleoside monotherapy with combination therapy in HIV-infected
adults with CD4 cell counts from 200 to 500 per cubic millimeter.
AIDS Clinical Trials Group Study 175 Study Team. N. Engl. J. Med.
1996, 335, 1081−1090.
(17) Smith, P. F.; Ogundele, A.; Forrest, A.; Wilton, J.; Salzwedel, K.;
Doto, J.; Allaway, G. P.; Martin, D. E. Phase I and II study of the
safety, virologic effect, and pharmacokinetics/pharmacodynamics of
single-dose 3-O-(3′,3′-dimethylsuccinyl)betulinic acid (bevirimat)
against human immunodeficiency virus infection. Antimicrob. Agents
Chemother. 2007, 51, 3574−3581.
(18) Martin, D. E.; Blum, R.; Doto, J.; Galbraith, H.; Ballow, C.
Multiple-dose pharmacokinetics and safety of bevirimat, a novel
inhibitor of HIV maturation, in healthy volunteers. Clin. Pharmaco-
kinet. 2007, 46, 589−598.
(19) Martin, D. E.; Blum, R.; Wilton, J.; Doto, J.; Galbraith, H.;
Burgess, G. L.; Smith, P. C.; Ballow, C. Safety and pharmacokinetics of
bevirimat (PA-457), a novel inhibitor of human immunodeficiency
virus maturation, in healthy volunteers. Antimicrob. Agents Chemother.
2007, 51, 3063−3066.
(4) Gulick, R. M.; Meibohm, A.; Havlir, D.; Eron, J. J.; Mosley, A.;
Chodakewitz, J. A.; Isaacs, R.; Gonzalez, C.; McMahon, D.; Richman,
D. D.; Robertson, M.; Mellors, J. W. Six-year follow-up of HIV-1-
infected adults in a clinical trial of antiretroviral therapy with indinavir,
zidovudine, and lamivudine. AIDS 2003, 17, 2345−2349.
(20) http://investor.myriad.com/releasedetail.cfm?ReleaseID=
360258.
(21) Huang, L.; Ho, P.; Lee, K. H.; Chen, C. H. Synthesis and anti-
HIV activity of bi-functional betulinic acid derivatives. Bioorg. Med.
Chem. 2006, 14, 2279−2289.
(22) Qian, K.; Morris-Natschke, S. L.; Lee, K. H. HIV entry
inhibitors and their potential in HIV therapy. Med. Res. Rev. 2009, 29,
369−393.
(23) Qian, K.; Yu, D.; Chen, C. H.; Huang, L.; Morris-Natschke, S.
L.; Nitz, T. J.; Salzwedel, K.; Reddick, M.; Allaway, G. P.; Lee, K. H.
Anti-AIDS agents. 78. Design, synthesis, metabolic stability assess-
ment, and antiviral evaluation of novel betulinic acid derivatives as
potent anti-human immunodeficiency virus (HIV) agents. J. Med.
Chem. 2009, 52, 3248−3258.
(24) Jiang, Z. X.; Yu, Y. B. The design and synthesis of highly
branched and spherically symmetric fluorinated oils and amphiles.
Tetrahedron 2007, 63, 3982−3988.
(25) Zhang, W.; Oya, S.; Kung, M. P.; Hou, C.; Maier, D. L.; Kung,
H. F. F-18 polyethyleneglycol stilbenes as PET imaging agents
targeting Aβ aggregates in the brain. Nucl. Med. Biol. 2005, 32, 799−
809.
(26) Zhu, C. B.; Zhu, L.; Holz-Smith, S.; Matthews, T. J.; Chen, C.
H. The role of the third beta strand in gp120 conformation and
neutralization sensitivity of the HIV-1 primary isolate DH012. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 15227−15232.
(27) Qian, K.; Kuo, R. Y.; Chen, C. H.; Huang, L.; Morris-Natschke,
S. L.; Lee, K. H. Anti-AIDS agents 81. Design, synthesis, and
structure−activity relationship study of betulinic acid and moronic acid
derivatives as potent HIV maturation inhibitors. J. Med. Chem. 2010,
53, 3133−3141.
(28) Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones, B.
C.; MacIntyre, F.; Rance, D. J.; Wastall, P. The prediction of human
pharmacokinetic parameters from preclinical and in vitro metabolism
data. J. Pharmacol. Exp. Ther. 1997, 283, 46−58.
(5) World Health Organization. Health Topics: HIV/AIDS. http://
www.who.int/topics/hiv_aids/en/ (accessed 2011).
(6) Chun, T. W.; Carruth, L.; Finzi, D.; Shen, X.; DiGiuseppe, J. A.;
Taylor, H.; Hermankova, M.; Chadwick, K.; Margolick, J.; Quinn, T.
C.; Kuo, Y. H.; Brookmeyer, R.; Zeiger, M. A.; Barditch-Crovo, P.;
Siliciano, R. F. Quantification of latent tissue reservoirs and total body
viral load in HIV-1 infection. Nature 1997, 387, 183−188.
(7) Finzi, D.; Hermankova, M.; Pierson, T.; Carruth, L. M.; Buck, C.;
Chaisson, R. E.; Quinn, T. C.; Chadwick, K.; Margolick, J.;
Brookmeyer, R.; Gallant, J.; Markowitz, M.; Ho, D. D.; Richman, D.
D.; Siliciano, R. F. Identification of a reservoir for HIV-1 in patients on
highly active antiretroviral therapy. Science 1997, 278, 1295−1300.
(8) Wong, J. K.; Hezareh, M.; Gunthard, H. F.; Havlir, D. V.; Ignacio,
C. C.; Spina, C. A.; Richman, D. D. Recovery of replication-competent
HIV despite prolonged suppression of plasma viremia. Science 1997,
278, 1291−1295.
(9) Piscitelli, S. C.; Flexner, C.; Minor, J. R.; Polis, M. A.; Masur, H.
Drug interactions in patients infected with human immunodeficiency
virus. Clin. Infect. Dis. 1996, 23, 685−693.
(10) Louie, M.; Markowitz, M. Goals and milestones during
treatment of HIV-1 infection with antiretroviral therapy: a patho-
genesis-based perspective. Antiviral Res. 2002, 55, 15−25.
(11) Boden, D.; Hurley, A.; Zhang, L.; Cao, Y.; Guo, Y.; Jones, E.;
Tsay, J.; Ip, J.; Farthing, C.; Limoli, K.; Parkin, N.; Markowitz, M.
HIV-1 drug resistance in newly infected individuals. JAMA, J. Am. Med.
Assoc. 1999, 282, 1135−1141.
(12) 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. Anti-
AIDS agents, 11. Betulinic acid and platanic acid as anti-HIV principles
from Syzigium claviflorum, and the anti-HIV activity of structurally
related triterpenoids. J. Nat. Prod. 1994, 57, 243−247.
(13) Kashiwada, Y.; Hashimoto, F.; Cosentino, L. M.; Chen, C. H.;
Garrett, P. E.; Lee, K. H. Betulinic acid and dihydrobetulinic acid
derivatives as potent anti-HIV agents. J. Med. Chem. 1996, 39, 1016−
1017.
(14) Kanamoto, T.; Kashiwada, Y.; Kanbara, K.; Gotoh, K.;
Yoshimori, M.; Goto, T.; Sano, K.; Nakashima, H. Anti-human
immunodeficiency virus activity of YK-FH312 (a betulinic acid
derivative), a novel compound blocking viral maturation. Antimicrob.
Agents Chemother. 2001, 45, 1225−1230.
(15) 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. PA-457: a potent HIV
inhibitor that disrupts core condensation by targeting a late step in
Gag processing. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13555−13560.
(16) Qian, K.; Nitz, T. J.; Yu, D.; Allaway, G. P.; Morris-Natschke, S.
L.; Lee, K. H. Anti-AIDS Agents 76. From Natural Product to Clinical
Trials: Bevirimat, a Plant-Derived Anti-AIDS Drug. In Natural Product
Chemistry for Drug Discoverys; Buss, T., Butler, M., Eds.; Biomolecular
8136
dx.doi.org/10.1021/jm301040s | J. Med. Chem. 2012, 55, 8128−8136