Synthesis of betulinic acid derivatives as entry inhibitors against HIV-1 and bevirimat-resistant HIV-1 variants

Article abstract of DOI:10.1016/j.bmcl.2012.06.080

Betulinic acid derivatives modified at the C28 position are HIV-1entry inhibitors such as compound A43D; however, modified at the C3 position instead of C28 give HIV-1 maturation inhibitor such as bevirimat. Bevirimat exhibited promising pharmacokinetic profiles in clinical trials, but its effectiveness was compromised by the high baseline drug resistance of HIV-1 variants with polymorphism in the putative drug binding site. In an effort to determine whether the viruses with bevirimat resistant polymorphism also altered their sensitivities to the betulinic acid derivatives that inhibit HIV-1 entry, a series of new betulinic acid entry inhibitors were synthesized and tested for their activities against HIV-1 NL4-3 and NL4-3 variants resistant to bevirimat. The results show that the bevirimat resistant viruses were approximately 5- to10-fold more sensitive to three new glutamine ester derivatives (13, 15 and 38) and A43D in an HIV-1 multi-cycle replication assay. In contrast, the wild type NL4-3 and the bevirimat resistant variants were equally sensitive to the HIV-1 RT inhibitor AZT. In addition, these three new compounds markedly improved microsomal stability compared to A43D.

Full text of DOI:10.1016/j.bmcl.2012.06.080

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5190–5194
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of betulinic acid derivatives as entry inhibitors against HIV-1
and bevirimat-resistant HIV-1 variants
Zhao Dang a, Keduo Qian b, Phong Ho a, Lei Zhu a, Kuo-Hsiung Lee b,c, Li Huang a, , Chin-Ho Chen a,
⇑
⇑
a Surgical Science, Department of Surgery, Box 2926, Duke University Medical Center, Durham, NC 27710-2926, United States
b Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, United States
c Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Betulinic acid derivatives modified at the C28 position are HIV-1entry inhibitors such as compound
A43D; however, modified at the C3 position instead of C28 give HIV-1 maturation inhibitor such as bevir-
imat. Bevirimat exhibited promising pharmacokinetic profiles in clinical trials, but its effectiveness was
compromised by the high baseline drug resistance of HIV-1 variants with polymorphism in the putative
drug binding site. In an effort to determine whether the viruses with bevirimat resistant polymorphism
also altered their sensitivities to the betulinic acid derivatives that inhibit HIV-1 entry, a series of new
betulinic acid entry inhibitors were synthesized and tested for their activities against HIV-1 NL4-3 and
NL4-3 variants resistant to bevirimat. The results show that the bevirimat resistant viruses were approx-
imately 5- to10-fold more sensitive to three new glutamine ester derivatives (13, 15 and 38) and A43D in
an HIV-1 multi-cycle replication assay. In contrast, the wild type NL4-3 and the bevirimat resistant vari-
ants were equally sensitive to the HIV-1 RT inhibitor AZT. In addition, these three new compounds mark-
edly improved microsomal stability compared to A43D.
Received 1 May 2012
Revised 18 June 2012
Accepted 25 June 2012
Available online 3 July 2012
Keywords:
HIV-1
Entry inhibitor
Maturation inhibitor
Betulinic acid
Berivimat
Berivimat-resistance
Ó 2012 Elsevier Ltd. All rights reserved.
The HIV-1 life cycle is initiated by a multi-step entry of HIV into
the target cell that involves interactions of HIV-1 gp120 to the cell
surface receptor CD4 and co-receptors CXCR4 or CCR5.1 Binding of
gp120 to the cellular receptors triggers conformational changes in
HIV-1 envelope glycoproteins that allow HIV-1 to enter the cells.2
Interference at the various steps of HIV entry has been proved to be
a successful strategy for drug development of anti-HIV therapy.
Examples of drugs that target HIV entry include the gp41 fusion
inhibitor enfuvirtide and the CCR5 receptor antagonist maravi-
roc.1,3,4 Enfuvirtide has high potency and broad spectrum in HIV
inhibition but as a peptide, has limited oral bioavailability. Marav-
iroc is very effective against HIV-1 R5 viruses that use the CCR5 co-
receptor for entry, but not X4 strains that use CXCR4 for entry. In
mid 90s, a betulinic acid (BA) derivative was reported with potent
anti-HIV-1 activity in the early stage of HIV life cycle.5 It has been
shown that BA derivatives with side chain modification at the C28
position inhibited viral entry by blocking the conformational
change of HIV gp120 during the process of HIV entry.6,7 Further
study suggested that these compounds targeted the V3 region of
HIV gp120, preventing the subsequent conformational changes in
HIV-1 gp41 in HIV entry.8 Among a series of C28 modified BA
derivatives, compound A43D exhibited the most potent anti-HIV-
1 entry activity.9
A43D was effective against a variety of HIV subtypes and dis-
played the strongest inhibition to the clade C HIV-1 strains.8 How-
ever, as shown in the results section, the microsomal stability of
A43D was poor when it was compared to bevirimat (BVM). BMV
is a BA derivative that targets HIV-1 maturation instead of entry
by blocking the processing of Gag precursor protein at a specific
step of CA-SP1 cleavage. In clinical trials, BVM displayed good bio-
availability and pharmacokinetic profiles.10 However, a high base-
line BVM resistance was uncovered during the clinical trials among
HIV-1 positive patients due to the polymorphism in the HIV-1 Gag
region.11,12 Therefore, it is important to study the possible drug
resistance issue in the synthesis of BA derivatives as potential
anti-HIV agent. A43D and BVM share the same BA scaffold
(Fig. 1). BVM possesses a dimethylsuccinic acid side chain at the
C3 position, while A43D has a long side chain modification at the
C28 position.13 The BA entry inhibitors, such as A43D, do not have
C3 side chain modifications but possess a relatively larger C28 side
chain with 7 to 9 carbons to be optimal for their anti-entry activity.
Thus, the objectives of this study are to synthesize new BA deriva-
tives with improved microsomal stability and to investigate the
Abbreviations: BA, betulinic acid; BVM, bevirimat; PEG, polyethylene glycol; Boc,
tert-butoxycarbonyl; t-Bu, tert-butyl; Bn, benzyl; Cpd, compound; AZT, 30-azido-30-
deoxythymidine; EDC, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride; THF, tetrahydrofuran; DCM, dichloromethane.
⇑
Corresponding authors. Tel.: +1 919 684 2952; fax: +1 919 684 3878 (L.H); tel.:
+1 919 684 3819; fax: +1 919 684 3878 (C.H.C).
E-mail addresses: lihuang@duke.edu (L. Huang), chc@duke.edu (C.-H. Chen).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.080

Z. Dang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5190–5194
5191
H
H
O
O
H
H
H
28
H
N
O
OH
HN
3
O
H
(CH2)7
O
HO
H
O
HO
H
Bevirimat
A43D
Figure 1. Chemical structures of the BA derivatives A43D and bevirimat.
effectiveness of the compounds not only to wild type HIV-1 but
also the BVM-resistant variants.
dihydrobetulinic acid scaffold with the same or similar C28 modi-
fication of 13.
It is clear that the HIV-1 Gag polymorphism greatly reduces the
effectiveness of BVM. In light of the structural similarity between
BVM and BA derivatives that inhibit HIV-1 entry, it is possible that
HIV-1 variants with BVM resistant polymorphism could have an al-
tered sensitivity to the BA entry inhibitors as well. Therefore, in
addition to identifying new HIV-1 entry inhibitors with improved
metabolic stability, their effectiveness against BVM resistant HIV-
1 strains was also determined. As a result, 38 new BA derivatives
with a variety of modifications at the C28 position were synthe-
sized. Among them, compounds 2–10 have a BA scaffold with
The C-28 modified BA or dihydro-BA derivatives were synthe-
sized using previously described methods.15 As shown in Scheme 1,
BA 3-O-acetate was treated with oxalyl chloride and subsequently
reacted with an alkyl di-amine or amino alkanoic acid ester in the
presence of triethylamine to form an intermediate of BA with a C28
amide linker (2a, 4a, 8a, 11a–13a, 24a). The intermediate was
hydrolyzed to remove the ester group(s) and then coupled with a
boc-amino acid, amino acid ester, or aminoalkyl cyano reagent in
the presence of N,N0-dicyclohexylcarbodimide/hydroxybenzotria-
zole/triethylamine to form the final products 2–28. Compounds
29–37, which contain some unusual linkers at their C28 position
such as ethylene glycol (PEG), fluoride substituted or unsaturated
hydrocarbons, were also synthesized using a protocol similar to
that described in Scheme 1. Compounds with a dihydro-BA scaf-
fold, such as 38 and 39, were synthesized by the same methods ap-
plied for compounds 13 and 31 except dihydro-BA 3-O-acetate was
used as the starting material.
C28 modified with a 1,
boc-masked amino acid. Compounds 11–28 are similar to the first
nine compounds except that the spacers are -aminoalkanoic acid
x-diamino alkane spacer and terminal
x
that terminate with an ester of amino acid or aminoalkyl cyano
moiety. The spacer length varied from 6 to 9 methylene groups
as suggested by previous structure–activity relationship stud-
ies.5,9,14,15 Compounds 29–37 contain new spacers such as a double
bond, fluoride substituent, or oxygen atoms in contrast to those
with methylene groups. Compounds 38 and 39 have the
Anti-HIV activity of BA derivatives in single cycle infectivity assay.
The anti-HIV activities of the BA derivatives were initially
H
H
H
O
O
O
H
1.(CO)2Cl2
H
R1
1. NaOH/THF/MeOH
H
HN
R1 R2
(CH2)n
HN
2. NH2(CH )nR1
H
OH
H
(CH2)n
2. R'COOH or
NH2R', EDC, Et3N,
DCM
Et3N,DCM2
H
AcO
H
HO
H
AcO
H
2 - 9 n = 7 - 9 R1-R2 = -NH-AA-Boc
2a, 4a, 8a
R
1 = -NH2
n = 7 - 9
11a - 13a, 24a
Betulinic acid-3-O-acetate
10 n = 8 R1-R2 = -NH-C(=O)-CH(NHBoc)-
R
1 = -C(=O)OBn
n = 6 - 9
C2H4CN
11-25 n = 6 - 9 R1-R2 = -C(=O)-AA esters
26 n = 8 R1-R2 = -C(=O)-NHCH2CN
27 n = 8 R1-R2 = -C(=O)-NHC2H4CN
28 n = 8 R1-R2 = -C(=O)-N(Me)-CH2CN
1.(CO)2Cl2
2. N H2-R1-COOMe
H
H
H
O
O
O
H
H
NH2R'COOMe
EDC,Et3N,DCM
H
HN
HN R1 OH
O
NaOH/THF/MeOH
OMe
R1 R2
H
HN
H
H
H
R1
HO
H
HO
AcO
H
O
31b R1 = -(CH2)5CH=CH-C(=O)-Gly-OMe
29 R1 = -(CH2)5CH=CH
30 R1 = -(CH2)5CH=CF
33b R1 = -PEG2CH2
NaOH
NaOH
29a R1 = -(CH2)5CH=CH
30a R1 = -(CH2)5CH=CF
33a R1 = -PEG2CH2
31
R
1 = -(CH2)5CH=CH-C(=O)-Gly
32b R1 = -(CH2)5CH=CF-C(=O)-Gly-OMe
32 R1 = -(CH2)5CH=CF-C(=O)-Gly
33c R1 = -PEG2CH2C(=O)-Gly-OMe
35b R1 = -PEG2C2H4NH
35a R1 = -PEG2C2H4NH
NaOH
NaOH
33
R
1 = -PEG2CH2C(=O)-Gly
34c R1 = -PEG2CH2C(=O)-Ala-OMe
34 R1 = -PEG2CH2C(=O)-Ala
35
36
R
R
1 =-PEG2C2H4NHC(=O)-Me
1 =-PEG2C2H4NHC(=O)-CH(Me)2
37 R1
=
N
N
N
H
PEG2
NH
O
N
Scheme 1. Synthesis of betulinic acid derivatives.

5192
Z. Dang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5190–5194
evaluated in a single cycle HIV-1 infection assay with NL4-3 virus
(Table 1).15 This assay detects HIV-1 tat-mediated luciferase pro-
duction in TZM-bl cells after HIV-1 entry. Therefore, it is a conve-
nient and effective assay for HIV-1 entry, but not maturation. The
purpose of using this assay was to evaluate and identify new BA
derivatives with potent anti-HIV entry activity. The results indi-
cated that most of these new compounds displayed anti-HIV activ-
ities except for those compounds with PEG linkers (33–37). The
results indicated that saturated hydrocarbon linkers are better
than less saturated side chains (29–32) for anti-HIV-1 activity of
these compounds. Previous studies have suggested that the opti-
mal linker length of the C28 side chain is 8 methylene groups for
BA derivatives to exhibit maximal activity.5,15 The data of this
study indicate that the optimal linker length to be 7 or 8 methylene
groups for compounds with a C28 –(CH2)n-NH-COR side chain (1–
10), and 8 or 9 methylene groups for compounds with a C28 –
(CH2)n-CO-NHR side chain (11–28). A terminal glutamine group
(2, 5, 13, 15, 24, 38 and 39) is favored among a variety of amino
acid residues (aa) investigated for the most effective anti-HIV-1
activity. None of the tested compounds were toxic to TZM-bl cells
in the single-cycle assay at 4 lM.
Anti-HIV activity of BA derivatives against BVM-resistant variant
(V370A). In contrast to the discussed C28 modified BA derivatives
with anti-HIV entry activities, it is known that the C3 modified
BA derivative BVM [3-O-(30,30-dimethylsuccinyl)-betulinic acid]
does not inhibit HIV-1 entry. However, it is a potent HIV-1 matura-
tion inhibitor. Phase II clinical trials revealed a high baseline drug
resistance due to pre-existing polymorphisms at the QVT-motif
Table 1
Inhibition of HIV-1 NL4-3 by BA entry inhibitors
H
H
H
O
O
O
H
O
H
H
H
HN
R
R'
R'
H
H
(CH2)8
HO
HO
HO
H
H
H
H
N
38, 39
H
N
29 - 37
(CH2)n
(CH2)n
1 - 10 R =
11 - 28 R =
R'
R'
H
O
N
a
a
Compound
n
R0
EC50
(lM)
Compound
n
R0
EC50 (lM)
1 (A43D)
7
7
7
8
8
8
8
9
–COCH3
–Gln-NHBoc
0.08
0.04
0.11
0.09
0.06
21
22
23
24
25
26
27
28
8
8
8
9
9
8
8
8
–Phe-OMe
–Pro-OMe
–(O-tBu)-Thr-OMe
–Gln-OMe
–Asn-OMe
–NHCH2CN
0.72b
0.16b
3.49b
0.06
2
3
4
5
6
7
8
–Ala-NHBoc
–Ala-NHBoc
–Gln-NHBoc
–Val-NHBoc
–Pro-NH-Boc
–Gln-NHBoc
0.08
2.43b
0.16
0.12
0.44b
0.34b
0.81b
–NHC2H4CN
–N(Me)CH2CN
O
H
9
9
8
–Ala-NHBoc
>4b
29
30
—
—
>4b
N
(CH2)5
OH
OH
O
O
H
NHBoc
N
10
0.08
>4b
CN
–Gln-OMe
–Gln-OMe
(CH2)5
F
O
H
N
OH
>4b
31
32
—
—
0.15
(CH2)5
11
12
6
7
N
H
O
O
O
H
N
OH
0.14
0.41b
(CH2)5
N
H
F
O
H
N
>4b
>4b
>4b
>4b
O
OH
13
14
15
16
8
8
8
8
–Gln-OMe
–Gln-O-tBu
–Gln-OBn
–Asn-OMe
0.09
0.14
0.12
0.11
33
34
35
36
—
—
—
—
O
N
H
O
O
H
N
O
OH
O
O
N
H
O
H
N
O
O
N
H
O
H
N
O
O
O
N
H
N
O
N
H
N
N
17
8
–Glu-Di-OMe
0.06
37
—
>4b
N
H
O
N
H
18
19
20
8
8
8
–Ala-OMe
–Ile-OMe
–Leu-OMe
0.07
0.14
0.16
38
39
—
—
–Gln-OMe
–Gln-OH
0.05
0.04
a
Data is average of two independent experiments.
Data from one experiment.
b

Z. Dang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5190–5194
5193
were 2- to 3-fold more potent against the resistant variant, while
AZT was 2 times less potent against the resistant variant in a single
cycle infection assay (Fig. 3).
Inhibition of HIV-1 and BVM-resistant variants by BA entry inhib-
itors in multiple-cycle replication assay. To further evaluate the BA
entry inhibitors on BVM-resistant HIV variants, the new BA deriv-
atives were tested against a panel of HIV-1 variants with the
V370A,
DV370, or DT371 genotype using a multi-cycle viral repli-
Figure 2. Genotype of three BVM-resistant variants. BVM-resistant variants
(V370 , V370A, and T371 ) were constructed from HIV-1 NL4-3 with mutations
D
D
cation assay in MT4 cells. These resistant strains showed markedly
decreased sensitivity to BVM when compared with the wild type
NL4-3 (Table 2). Five synthesized compounds (2, 13, 15, 38 and
39) were tested against HIV-1 NL4-3 and the resistant variants in
the multi-cycle viral replication assay. The majority of the tested
compounds were found to be at least 4-fold more potent against
BVM-resistant variants than the wild type NL4-3 (Table 2). The
in the QVT motif of HIV-1 Gag (17). The symbol
acid.
D denotes deletion of an amino
D
V370 and DT371 variants were particularly sensitive to these en-
try inhibitors when compared to the V370A variant and were at
least 5- to 10-fold more sensitive to the tested BA entry inhibitors
when compared with the wild type NL4-3 virus. NL4-3 and the
V370A variant were equally sensitive to the non-nucleoside HIV-
1 RT inhibitor, TMC-278.
In vitro metabolic stability of BA derivatives. The five selected best
entry inhibitors derived from BA (2, 13, 15, 38 and 39) were inves-
tigated for their microsomal stability using human liver micro-
somal preparations under oxidative conditions with a reference
compound terfenadine. Terfenadine has
a moderate-to-fast
half-life in vivo of around 3.5 h. The results in Table 3 show that
glutamine residue of the C28 side chain with boc protected amine
terminus (2) exhibited the fastest metabolism with t1/2 of
18.93 min. Leaving the glutamine terminus unprotected (39) did
not significantly change its microsomal stability (t1/2: 24.75 min)
when compared to A43D. The Benzyl ester protection of the gluta-
mine terminus significantly improved microsomal stability of 15
Figure 3. BVM-resistant HIV-1 variants were more sensitive to BA entry inhibitors.
Inhibition of HIV-1 infection was measured as the reduction in luciferase gene
expression in TZM-bl cells after a single round of virus infection as described
previously (15). The bevirimat sensitive virus HIV-1 NL4-3 (solid lines) and
resistant virus NL4-3 V370A (dashed lines) were used in this assay. Control: HIV-1
NL4-3 or HIV-1 V370A infection, expressed as relative fluorescence units, in the
absence of the compounds.
with a t1/2 of 41.01 min when compared to that of A43D (t1/2
:
25.48 min) and terfenadine (t1/2: 29.87 min). In addition to the im-
proved microsomal stability, compound 15 exhibited similar anti-
viral activity against NL4-3 and better activity against the BVM-
resistant V370A variant when compared with A43D.
In summary, we have designed and synthesized 38 new BA
derivatives with different C28 side chains. Among them, com-
pounds 2, 13, 15, 38 and 39, carrying a glutamine terminus,
showed potent anti-HIV activity with EC50 ranging from 0.04 to
(amino acid residues 369–371) within HIV-1 Gag SP1.12,16,17 Vari-
ation such as V370A, V370M, or V370 deletion resulted in high
resistance to BVM, while deletion of T371 resulted in medium
resistance to BVM.18,19 The V370A polymorphism was found to
be the most prevalent among HIV-1 positive patients.18,19 We have
constructed a panel of NL4-3 variants with BVM resistant geno-
types (Fig. 2).
The V370A variant was tested extensively in our study to eval-
uate anti-HIV activity of new BA derivatives against the BVM-resis-
tant strain. We observed that BA derivatives 1 (A43D), 13 and 15
0.12 lM in a single cycle HIV-1 NL4-3 infection assay (Table 1).
These BA entry inhibitors were in general more potent against
the BVM-resistant variants than the wild type NL4-3 (Fig. 2, Ta-
ble 2). In addition, compounds 13, 15 and 38 showed improved
metabolic stability in vitro when compared to the lead compound
A43D. These results suggest that the new BA derivatives may serve
as promising leads for further drug development against HIV-1.
Table 2
Effect of BA derivatives on HIV BVM-resistant viruses
Compound
EC50
(
lM)
Toxicityb TD50
(lM)
NL4-3a
V370Aa
D
T371b,c
D
V370b,c
BVM
1 (A43D)
2
13
15
38
39
TMC-278
0.076
>4
>4
>4
0.006
ND
ND
0.023
ND
>10, <20
12.6
7.4
4.8
15.0
4.6
0.12 0.08
0.36 0.09
1.72 0.83
0.19 0.14
0.58 0.74
0.83 0.37
0.00055 0.00014
0.19 0.35
0.09 0.11
0.02 0.02
0.05 0.03
0.02 0.01
0.24 0.28
0.027
NDc
ND
0.026
ND
ND
ND
ND
ND
18.6
>0.1
0.00052 0.00011
a
b
c
Data is the mean of two or more independent experiments and standard deviation.
Data is the average of two independent experiments.
ND = not determined.

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Z. Dang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5190–5194
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Microsomal stability of BA derivatives
Compound
Terfenadine A43D
2
13
15
38
39
in vitro t1/2 (min)a 29.87
25.48 18.93 30.13 41.01 31.64 24.75
0.272 0.366 0.230 0.169 0.219 0.280
CLint (ml/min/mg)a 0.232
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Acknowledgment
This work was supported by the National Institute of Allergy
and Infectious Diseases (NIAID) Grant AI-65310 awarded to C.H.
Chen, The D.P. Bolognesi Award (Surgical Science, Surgery Depart-
ment, Duke University) to L. Huang, and in part by Grant
AI-077417 from NIAID awarded to K.H. Lee.
14. Huang, L.; Yuan, X.; Aiken, C.; Chen, C. H. Antimicrob. Agents Chemother. 2004,
48, 663.
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2009, 52, 7887.
Supplementary data
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