New betulinic acid derivatives for bevirimat-resistant human immunodeficiency virus type-1

Article abstract of DOI:10.1021/jm3016969

Bevirimat (1, BVM) is an anti-HIV agent that blocks HIV-1 replication by interfering with HIV-1 Gag-SP1 processing at a late stage of viral maturation. However, clinical trials of 1 have revealed a high baseline drug resistance that is attributed to naturally occurring polymorphisms in HIV-1 Gag. To overcome the drug resistance, 28 new derivatives of 1 were synthesized and tested against compound 1-resistant (BVM-R) HIV-1 variants. Among them, compound 6 exhibited much improved activity against several HIV-1 strains carrying BVM-R polymorphisms. Compound 6 was at least 20-fold more potent than 1 against the replication of NL4-3/V370A, which carries the most prevalent clinical BVM-R polymorphism in HIV-1 Gag-SP1. Thus, compound 6 merits further development as a potential anti-AIDS clinical trial candidate.

Full text of DOI:10.1021/jm3016969

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Article
New Betulinic Acid Derivatives for Bevirimat-
Resistant Human Immunodeficiency Virus Type-1
Zhao Dang, Phong Ho, Lei Zhu, Keduo Qian, Kuo-Hsiung Lee, Li Huang, and Chin-Ho Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3016969 • Publication Date (Web): 04 Feb 2013
Downloaded from http://pubs.acs.org on February 18, 2013
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Title: New Betulinic Acid Derivatives for Bevirimat-Resistant Human Immunodeficiency
Virus Type-1
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Zhao Dang,† Phong Ho,† Lei Zhu,† Keduo Qian,‡ Kuo-Hsiung Lee,‡,§ Li Huang,*,† and Chin-Ho
Chen*,†
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†Surgical Science, Department of Surgery, Duke University Medical Center, Durham, North
Carolina 27710, USA
‡Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North
Carolina, Chapel Hill, North Carolina 27599, USA
§Chinese Medicine Research and Development Center, China Medical University and Hospital,
Taichung, Taiwan
KEYWORDS: Betulinic acid; Bevirimat; HIV-1; Maturation inhibitors; Bevirimat-resistance.
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ABSTRACT:
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Bevirimat (1, BVM) is an anti-HIV agent that blocks HIV-1 replication by interfering with HIV-
1 Gag-SP1 processing at a late stage of viral maturation. However, clinical trials of 1 have
revealed a high baseline drug resistance that is attributed to naturally-occurring polymorphisms
in HIV-1 Gag. To overcome the drug resistance, 28 new derivatives of 1 were synthesized and
tested against compound 1-resistant (BVM-R) HIV-1 variants. Among them, compound 6
exhibited much improved activity against several HIV-1 strains carrying BVM-R
polymorphisms. Compound 6 was at least 20-fold more potent than 1 against the replication of
NL4-3/V370A, which carries the most prevalent clinical BVM-R polymorphism in HIV-1 Gag-
SP1. Thus, compound 6 merits further development as a potential anti-AIDS clinical trial
candidate.
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INTRODUCTION
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After three decades of battle against Acquired Immunodeficiency Syndrome (AIDS) and its
causative agent human immunodeficiency virus type 1 (HIV-1), HIV-1 infection remains one of
the leading causes of infectious diseases worldwide with more than 30 million people currently
living with HIV/AIDS.1 Although current anti-retroviral therapy (ART) has drastically improved
the clinical outcome of AIDS, it fails to eradicate HIV-1 from patients.2-4 Thus, long-term
treatment is required to suppress HIV-1 replication in infected patients. The emergence of drug-
resistant HIV strains and the side effects/toxicities associated with long-term drug treatment
present additional obstacles for the clinical management of HIV-1 infection.5,6 Therefore,
identifying new drug candidates with novel mechanisms of action is vital to advancing AIDS
therapy.
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Bevirimat (1, BVM), also known as PA-457, DSB, and MPC-4326, is a first-in-class HIV-1
maturation inhibitor that interferes with the CA-SP1 cleavage step of Gag processing, resulting
in the accumulation of p25 and immature viral cores.7-10 Clinical trials indicated that 1 caused a
significant and clinically relevant reduction of the viral load in both ART-experienced and naive
patients.11,12 However, the clinical trials also revealed a high baseline drug resistance to 1. A
significant fraction (40–50%) of treated patients had pre-existing resistant viruses that had
significantly reduced sensitivity to 1.13-15 This BVM-resistance was linked to naturally-occurring
HIV polymorphisms in the SP1 region of HIV-1 Gag. Polymorphisms within three amino acids,
Gln-Val-Thr (Q-V-T), at Gag positions 369–371 of the SP1, such as 370A instead of V or
deleted V or T, were shown to be associated with the BVM-resistance (Table 1). A
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polymorphism of 362I in the CA region of Gag was also reported to confer resistance to
compound 1.16 Analysis of SP1 sequences indicated that 370A is the most prevalent
polymorphism among HIV-1 isolates from subtype B and C viruses.17 The high baseline drug
resistance due to the existing polymorphisms poses serious limitations to the clinical potential
and further development of compound 1.
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Table 1. Genotypes of NL4-3 Variants Carrying BVM-R Polymorphisms
Virus
(Gag)
CA
SP1
362
V L
369 371
NL4-3
A E A M S Q V T N S A
A E A M S Q ∆ T N S A
A E A M S Q A T N S A
A E A M S Q V ∆ N S A
A E A M S Q V T N S A
NL4-3/V370∆
NL4-3/V370A
NL4-3/T371∆
NL4-3/V362I
V L
V L
V L
V I
It has been shown that 1 does not bind to monomeric Gag, but rather binds to an immature Gag
to interfere with the CA-SP1 cleavage. It was suggested that 1 associated with an alpha helical
structure of SP1 of immature viral particles.18,19 SP1 with BVM-R polymorphisms could disrupt
the binding of 1, leading to a loss of sensitivity to the compound. The goal of this study was to
modify the structure of 1 to overcome the resistance. Our prior studies indicated that attachment
of a side chain at the C28 position of 1 resulted in improved activity. For example compound 30
(N-[3β-O-(3′,3′-dimethylsuccinyl)lup-20(29)-en-28-oyl]-5-piperazinepentanoic acid), with a C28
piperazinepentanoic acid side chain on the compound 1 molecule, exhibited a greater than 10-
fold increase in anti-HIV-1 maturation activity.20 In spite of its increased activity against wild
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type virus, 30 was not effective against BVM-R viruses. Nevertheless, its improved anti-HIV-1
activity against the NL4-3 strain suggested that C28 modifications could impact drug-target
interaction. Thus, the rationale of this study was that compound 1 analogs with optimized C28
side chains might improve drug-target interaction and overcome the resistance to compound 1.
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RESULTS
New analogs were synthesized with compound 1 as the scaffold and various spacer/R’ groups
at the C28 position (Figure 1). Compounds 2–18
H
contain an ω-aminoalkanoic acid spacer [H2N-
R
28
H
O
(CH2)n-COOH], where n ranged from 6–9.
Compounds 19–29 have a 1,ω-diaminoalkane
[H2N-(CH2)n-NH2] spacer, where n was 7–9.
O
3
H
HOOC
O
H
1 (Bevirimat): R = OH
New analogs:
2-18 R = -H2N-(CH2)n-COR', n = 6 - 9
19-29 R = -H2N-(CH2)n-NHR' n = 7 - 9
Figure 1. Structure of compound 1 analogs.
As shown in Scheme 1, compounds 2–18 were synthesized by first coupling the C28
carboxylic acid of 3-O-acetate-BA with benzyl ω-aminoalkanoates (Table 2). The coupling
reaction was then followed by alkaline hydrolysis to form intermediates 2a–4a and 18a, which
are known compounds that have been previously reported for their activities against HIV-1 and
HIV-2 viruses.21-23 Further modifications at the C28 terminal carboxylic acid of the above
intermediates with the corresponding amino acid ester in the presence of EDC, HOBt, and NMM
resulted in 2b–4b and 6b–18b. Treatment of 2b–4b and 6b–18b with 2,2-dimethylsuccinic
anhydride and DMAP in pyridine at 140 °C for 2 h using microwave radiation yielded the final
compounds 2–4 and 6–18. Compound 5 was obtained by alkaline hydrolysis of 4.
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Compounds 19–29 were synthesized by coupling the diaminoalkane with the C28 carboxylic
acid of 3-O-acetate-BA to form amine intermediates 19a, 21a and 26a.21,23 Further coupling of
the above amine intermediate with a corresponding N-protected amino acid, followed by alkaline
hydrolysis of the esters at C3 resulted in compounds 19b–29b. Esterification of 19b–29b at the
C3 hydroxyl formed the end products 19–29 (Scheme 2; Table 3).
Activity of Compound 1 Derivatives against BVM-R HIV-1 Variant V370A. To evaluate the
effectiveness of the newly synthesized compound 1 analogs against BVM-R HIV-1 strains, a
panel of NL4-3 mutants carrying BVM-R polymorphisms was constructed.23 The NL4-3
sequences of the polymorphic site in SP1 and its flanking region are shown in Table 1 along with
the sequences of BVM-R mutants. The synthesized compounds were tested initially against both
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the wild type HIV-1 NL4-3 and NL4-3/V370A, a variant that carries the most prevalent BVM-R
polymorphism with an Ala at position 370 in the SP1 region.
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As expected, 1 potently inhibited wild type NL4-3 with an IC50 of 0.076 ꢀM, but was
ineffective at up to 6.8 ꢀM against the BVM-R variant NL4-3/V370A. Most of the newly
synthesized analogs (2–29) were more potent than 1 against wild type NL4-3 (Tables 2 and 3).
Twenty-four of the new compounds had IC50 values in the range of 0.01–0.05 ꢀM, six in the
range of 0.07–0.18 ꢀM, and three were over 0.2 ꢀM or inactive. In contrast to 1, most of the new
C28 modified analogs exhibited inhibitory activity against the BVM-R NL4-3/V370A variant
with IC50 values less than 3 ꢀM. Ten of the tested compounds exhibited significant potency
against this variant with IC50 values ranging from 0.16 to 0.47 ꢀM. Among them, compounds 5,
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6, 14, 15, 20, 21, 24, and 29 also had improved potency (IC50 ≤ 0.05 ꢀM) against wild type NL4-
3 compared with 1. Compound 6 with a C28 methyl nonanoyl-glutaminate side chain, exhibited
the best anti-HIV potency against both wild type virus and the BVM-R NL4-3/V370A variant
with IC50 values of 0.01 and 0.16 ꢀM, respectively. The improved activity of these compounds
over 1 was not accompanied with increased cytotoxicity, as their CC50 values were comparable
to that of 1 (Tables 2 and 3).
Table 2. Activities of Compounds 2–18 against HIV-1 NL4-3 and NL4-3/V370A
IC50a (ꢀM)
CC50b (ꢀM)
Compound
NL4-3
NL4-3/V370A
>5
n
6
R
0.076 +/- 0.018 (5)
0.05 +/- 0.014 (3)
>10
>10
1
2
2.46 ± 1.10 (3)
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0.05 +/- 0.017 (2)
>5
>10
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0.01 ± 0.0032 (2)
0.04 ± 0.016 (2)
1.51 ± 0.125 (2)
0.48 ± 0.32 (2)
>10
>10
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0.009 ± 0.002 (5)
0.01 ± 0.003 (3)
0.04 ± 0.013 (2)
0.16 ± 0.048 (5)
0.81 ± 0.28 (3)
2.54 ± 0.95 (2)
>10
>10
>10
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0.04 ± 0.017 (2)
0.10 ± 0.019 (2)
0.07 ± 0.042 (2)
2.78 ± 0.93 (2)
0.88 ± 0.31 (2)
1.29 ± 0.47 (2)
>10
>10
>10
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0.01 ± 0.005 (2)
0.18 ± 0.073 (2)
0.01 ± 0.0036 (3)
1.24 ± 0.052 (2)
2.16 ± 1.07 (2)
>10
>10
>10
0.42 ± 0.085 (3)
0.46 ± 0.22 (3)
0.33 ± 0.14 (2)
15
16
8
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8
0.03 ± 0.014 (2)
0.15 ± 0.062 (2)
6.49 ± 0.64 (2)
>10
17
18
30
0.04 ± 0.018 (2)
0.10 ± 0.045 (2)
0.006 ± 0.0016 (3)
2.08 ± 0.88 (2)
>10
>10
>10
9
0.70 ± 0.31 (2)
>3.3
aIC50: concentration that inhibited HIV-1 replication by 50%; presented as mean +/- standard deviation
(SD). bCC50: concentration that reduced cell viability by 50%. IC50 and CC50 were determined by using
CalcuSyn (Biosoft). The numbers in the parentheses represent the number of tests.
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Table 3. Activities of Compounds 19–29 against HIV-1 NL4-3 and NL4-3/V370A
5
6
IC50a (ꢀM)
CC50b (ꢀM)
Compound
7
8
9
n
7
R
NL4-3
NL4-3/V370A
2.6 ± 1.2 (2)
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21
0.01 ± 0.004 (2)
>10
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8
0.01 ± 0.003 (2)
0.03 ± 0.007 (3)
0.37 ± 0.15 (2)
0.36 ± 0.13 (3)
7.9 ± 0.65 (2)
>10
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22
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25
0.15 ± 0.05 (2)
0.02 ± 0.0072 (2)
0.03 ± 0.014 (2)
0.04 ± 0.018 (2)
0.33 ± 0.04 (2)
0.92 ± 0.21 (2)
0.33 ± 0.25 (2)
0.95 ± 0.34 (2)
>10
>10
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26
0.02 ± 0.008 (2)
1.20 ± 0.13 (2)
>10
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28
0.05 ± 0.016 (2)
0.02 ± 0.007 (2)
1.58 ± 0.33 (2)
1.04 ± 0.42 (2)
>10
>10
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9
29
0.04 ± 0.019 (2)
0.34 ± 0.22 (2)
8.3± 0.56 (2)
aIC50: concentration that inhibited HIV-1 replication by 50%; presented as mean +/- SD. bCC50:
concentration that reduced cell viability by 50%. IC50 and CC50 were determined by using CalcuSyn
(Biosoft). The numbers in the parentheses represent the number of tests.
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Compound 6 Inhibited Multiple NL4-3 Variants Carrying BVM-R Polymorphisms. Due to
its superior anti-HIV activity, compound 6 was tested against three more BVM-R variants that
carry BVM-R polymorphisms (Table 1). While these viruses were not sensitive to 1 (Table 4),
all four BVM-R variants with V370ꢀ, V370A, V362I, and T371ꢀ polymorphisms were sensitive
to 6. The IC50 values of 6 against these resistant variants ranged from 0.016 to 0.32 ꢀM
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compared with 0.008 ꢀM against wild type NL4-3 (Table 4). The rank order of sensitivity of the
BVM-R variants to 6 was NL4-3/V362I > NL4-3/T371ꢀ > NL4-3/V370A > NL4-3/V370ꢀ.
Table 4. Activities of Compound 6 against BVM-R HIV-1 Variants
.
IC50a (ꢀM)
Virus
Compound 1
0.076 ± 0.019
> 6.8
Compound 6
0.008 ± 0.0017
0.32 ± 0.077
NL4-3
NL4-3/V370Δ
NL4-3/V370A
NL4-3/T371Δ
NL4-3/V362I
> 6.8
0. 158 ± 0.048
0.067 ± 0.012
0.016±0.0042
> 6.8
> 6.8
aIC50: the numbers in the table are mean +/- SD from three independent experiments.
Inhibition of CA-SP1 Processing of BVM-R HIV-1 Variant by Compound 6. As a
maturation inhibitor, compound 1 causes the accumulation of p25 by interfering with the HIV-1
Gag CA-SP1 cleavage.7-10 To determine whether anti-HIV-1 maturation was indeed responsible
for the observed anti-HIV activity, 6 was tested in a standard anti-maturation assay that measures
p25 accumulation.10,18 NL4-3 and the BVM-R variant NL4-3/V370A were produced by 293T
cells in the presence of 1 or 6. The Gag CA-SP1 (p25) protein and its processed product p24
were detected with Western blot analysis. As expected, 1 interfered with the cleavage of Gag
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CA-SP1 in wild type NL4-3 as shown by p25 accumulation, but did not inhibit the cleavage of
Gag CA-SP1 in the drug resistant variant NL4-3/V370A under the same experimental conditions
(Figure 2a). These results agree with the anti-viral assay results of 1. On the other hand,
treatment of the BVM-R NL4-3/V370A virus with 6 resulted in accumulation of CA-SP1 (p25),
the signature event of inhibition of HIV-1 maturation. The accumulation of p25 was even more
pronounced when the BVM-R NL4-3/V362I virus was produced in the presence of 6 (Figure 2b).
This was consistent with the anti-viral results that NL4-3/V362I was most sensitive to 6 among
the BVM-R mutants. These data indicated that 6 is a potent maturation inhibitor effective against
both wild type and BVM-R mutants.
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Figure 2. Inhibition of CA-SP1 processing. The anti-maturation activity of 1 and 6 was determined by
transfecting 293T cells with the following plasmids: (a) pNL4-3 or pNL4-3/V370A; (b) pNL4-3/V362I.
The viruses produced in the presence of the compounds were collected two days after transfection.
Compound 6 was Inactive against HIV-1 Entry. We and others have previously shown that
betulinic acid (BA) derivatives with C28 modified side chains can block HIV-1 replication at the
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viral entry step.25,26 To determine whether 6 also inhibits HIV-1 entry, 6 was tested in a fusion
assay that was previously established for studying HIV-1 entry inhibitors.23 A43D (31), a BA
derivative with a C28 modified side chain but without the C3 side chain of 1, was used as a
positive control for inhibition of HIV-1 entry.24 The results indicated that 1 and 6 do not inhibit
HIV-1 entry, while 31 strongly inhibited the HIV-1 NL4-3 Env-mediated cell-cell fusion with an
IC50 of 0.042 ꢀM (Figure 3). These results suggested that 6 inhibited the maturation of BVM-R
viruses without affecting viral entry.
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100
75
Compound 31
50
Compound 1
Compound 6
25
0
1
10
100
1000
10000
Compound (nM)
Figure 3. Compound 6 did not inhibit HIV-1 Env–mediated cell-cell fusion. COS cells (1 × 106 cells/mL)
were transfected by electroporation with HIV-1 NL4-3 Env-expressing vector (2 ꢀg) for one day before
mixing with TZM-bl cells for fusion. The luciferase expression measured as relative luciferase units
(RLU) in the fusion cells was quantified 24 hours after cell mixing.24 HIV-1 Env-mediated cell-cell fusion
in the absence of compounds is defined as 100% control. Each data point in the figure represents mean +/-
SD of three tests.
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DISCUSSION and CONCLUSIONS
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This study demonstrates that structural optimization can result in analogs of 1 that are effective
against BVM-R viruses. Compound 6 was at least 20-fold more potent than 1 against four BVM-
R viruses. The improved potency against BVM-R viruses is due to the ability of the modified
compounds to inhibit maturation of the viruses. Most of the derivatives of 1 that had increased
potency against the resistant variants were also more potent against BVM-R viruses. However,
improved potency against the sensitive virus alone was not sufficient to overcome resistance to
compound 1. For example, compound 30 was more potent than other tested compounds against
the compound 1-sensitive virus NL4-3, but it was ineffective against BVM-R variants. It appears
that the improved activity against BVM-R viruses is associated with the BA scaffold modified at
the C28 position with a long aliphatic side chain containing a spacer with 7–9 methylenes and
terminated with a suitable amide group. This structural feature is shared by 5, 6, 14, 15, 20, 21,
24, and 29, which displayed significant improvements in potency against BVM-R viruses.
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The BVM-R variants were not equally sensitive to 6. The differential sensitivity is probably
due to the variation in the amino acid residues at the drug resistant polymorphism site. In
summary, it is possible to overcome resistance to 1, since 6 showed markedly improved activity
against the variant with the most prevalent BVM-R polymorphism, 370A in SP1 of HIV-1 Gag.
However, optimal structural requirement(s) for inhibition of each BVM-R variant may vary,
since the drug resistant polymorphisms involve multiple amino acid residues.
EXPERIMENTAL SECTION
General. Chemistry. All solvents and reagents were used as received from Sigma-Aldrich or
other commercial sources. Positive or negative HR-FABMS were recorded on a Shimadzu
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LCMS-IT-TOF or a Joel SX-102 mass spectrometer. 1H and 13C NMR spectra were measured on
a Varian Mercury 300 MHz spectrometer. Samples were dissolved in CDCl3 with TMS as an
internal standard unless specified. Silica gel chromatography was carried out on an ISCO
CombiFlash Rf flash chromatograph system with a pre-packed Redi Sep Rf Si gel column
(Teledyne ISCO), and MeOH/DCM (0–10% gradient) as mobile phase. Compounds were
analyzed and purified (as needed) by using a Varian ProStar HPLC system with a PDA detector
and Agilent Zorbax C18 columns (5 ꢀM particle size, 4.6 × 250 mm and 9.4 × 250 mm for
analytical and semi preparative scales, respectively). The mobile phase was composed of
solution A (5% acetonitrile in water with 0.045% trifluoroacetic acid) and solution B
(water/methanol/acetonitrile = 5:10:85 with 0.045% trifluoroacetic acid). A linear gradient of
80% to 100% of solution B with a flow rate at 1 mL/min or 4 mL/min was used in HPLC
experiments. The compounds were analyzed with the UV absorption displayed at 220 nm and
recorded at a range from 200 to 250 nm. All tested compounds have purity of 95% or above
determined by HPLC analysis.
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General Procedure for Preparation of Compounds 2–4, 6–18 (Scheme 1). The
intermediates 2b–4b and 6b–18b were synthesized using previously described methods.21-23
Under enclosed conditions flushed with N2, oxalyl chloride (2 mL, 20 mmol) was added to a
stirring solution of betulinic acid-3-O-acetate (500 mg, 1 mmol) in DCM (anhydrous, 6 mL).
After being stirred for 1 h at rt, the reaction mixture was concentrated and benzyl 9-amino
nonanoate (315 mg, 1.2 mmol) in DCM (anhydrous, 6 mL) and Et3N (0.85 mL, 6 mmol) were
added. After being stirred overnight at rt, the reaction mixture was concentrated and
chromatographed on Si-gel to yield 472 mg (63% yield) of intermediate. To a solution of this
intermediate in THF (4 mL) and MeOH (4 mL), NaOH (2 mL, 4N) was added drop-wise. After
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being stirred overnight at rt, the reaction mixture was neutralized with HCl (1N). The precipitate
was collected, washed with water, and dried in vacuum to give hydrolyzed intermediate 18a (303
mg, 80% yield).
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A solution of 18a (45 mg, 0.07 mmol) in DCM (3 mL) was added to EDC-HCl (17 mg, 0.09
mmol), HOBt (11 mg, 0.09 mmol), NMM (17 mL, 0.15 mmol), and methyl glutaminate (33 mg,
0.21 mmol). After being stirred overnight at rt, the reaction mixture was diluted with DCM (40
mL), and then washed with water and brine. The organic layer was dried with Na2SO4 and
concentrated in vacuum. The residue was chromatographed on Si-gel and reverse phase HPLC to
yield compound 18b (28 mg, 51% yield).
A mixture of intermediate 18b (15 mg, 0.02 mmol), 2,2-dimethylsuccinic anhydride (35 mg,
0.27 mmol), and DMAP (3 mg, 0.025 mmol) in pyridine (anhydrous, 0.5 mL) was heated to 140
°C for 2 h in a microwave oven (Biotage initiator). The mixture was then concentrated under
vacuum. The residue was chromatographed on Si-gel and reverse phase HPLC to yield
compound 18 (6 mg, 34% yield).
The above method was used to synthesize compounds 2–4 and 6–17 using different reagents
such as other alkanoates and/or amino acid derivatives instead of benzyl 9-amino nonanoate and
methyl glutaminate, respectively.
Procedure for Preparation of Compound 5. NaOH (32 mg, 0.8 mmol) was added to a
solution of 4 (4 mg, 0.005 mmol) in THF (1.4 mL), H2O (0.7 mL) and MeOH (0.7 mL). After
being stirred overnight at rt, the reaction mixture was neutralized with HCl (1N). The mixture
was diluted with DCM (30 mL), and then washed with water and brine. The organic layer was
dried with Na2SO4 and concentrated in vacuum. The residue was chromatographed on reverse
phase HPLC to yield compound 5 (3 mg, yield 76%).
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General Procedure for Preparation of Compounds 19–29 (Scheme 2). Under enclosed
conditions flushed with N2, oxalyl chloride (12 mmol) was added to a stirring solution of
betulinic acid-3-O-acetate (300 mg, 0.6 mmol) in DCM (anhydrous, 3 mL). After being stirred
for 1 h at rt, the reaction was concentrated to dryness and was re-dissolved in DCM (anhydrous,
3 mL). The latter solution was added drop-wise to a stirring solution of 1,7-diaminoheptane (470
mg, 3.6 mmol) in DCM (10 mL). After being stirred overnight at rt, the reaction mixture was
concentrated, re-dissolved in MeOH, and filtered. The filtrate was concentrated and
chromatographed on Si-gel to yield 19a (256 mg, yield 69%).
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To a stirring solution of 19a (43 mg, 0.07 mmol) in DCM (2 mL), EDC-HCl (53 mg, 0.27
mmol), HOBt (53 mg, 0.4 mmol), TEA (0.2 mmol), and Boc-glutamine (50 mg, 0.2 mmol) were
added. After being stirred overnight at rt, the reaction mixture was diluted with DCM (40 mL),
and then washed with water and brine. The organic layer was dried with Na2SO4 and
concentrated in vacuum. The residue was chromatographed on Si-gel and reverse phase HPLC to
yield intermediate (46 mg, 78% yield). To the mixture of this intermediate in THF (1 mL) and
MeOH (1 mL), NaOH (0.5 mL, 4N) was added drop-wise. After being stirred for 5 h at rt, the
reaction mixture was neutralized with HCl (1N), and then extracted with DCM. After removal of
the organic solvent under vacuum, the residue was chromatographed on Si-gel to give 19b (26
mg, 65% yield).
Using the same procedure described above to obtain 18 from 18b, 19b was further modified at
the C3 position to furnish compound 19 (9 mg, 32%).
The above method (for obtaining 19 from betulinic acid-3-O-acetate) was used to synthesize
compounds 20–29 using different reagents such as other 1,ω-diaminoalkane and/or amino acid
derivatives instead of 1,7-diaminoheptane and Boc-glutamine, respectively.
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Methyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-7-aminoheptanoyl]-L-
7
8
9
glutaminate (2): Yield 7 mg (22%). 1H NMR (CDCl3/Pyridine-d5) δ 7.03 (bs, 1H, -CONH-), 6.81
and 5.68 (2bs, 1H each, -CONH2), 5.83 (bs, 1H, -CONH-), 4.68 (s, 1H, =CH), 4.51–4.56 (m, 2H,
-COCH-, =CH), 4.43 (dd, J = 4.7 Hz, J = 11.1 Hz, -CH- in 3), 3.67 (s, 3H, -OCH3), 3.04–3.24
(m, 3H, -CONH-CH2-, -CH- in 19), 2.66 and 2.55 (2d, J = 15.3 Hz, 1H each, -C(CH3)2-CH2-),
1.62 (s, 3H, -CH3 in 30), 1.29 and 1.28 (2s, 3H each, -CO-C(CH3)2), 0.89 (s, 3H, -CH3), 0.86 (s,
3H, -CH3), 0.78 (s, 3H, -CH3), 0.73 (s, 6H, 2 × -CH3). Calcd for C49H79N3O9Na (M+Na)+:
876.5714. Found: 876.5714.
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Methyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-8-aminooctanoyl]-L-
glutaminate (3): Yield 10 mg (33%). 1H NMR δ 6.95 and 6.88 (2bs, 1H each, -CONH2), 6.64 (d,
J = 7.7 Hz, 1H, -CONH-), 5.75 (t, J = 5.4 Hz, 1H, -CONH-), 4.72 (s, 1H, =CH), 4.55–4.62 (m,
2H, -COCH-, =CH), 4.47 (dd, J = 6.5 Hz, J = 9.1 Hz, 1H, -CH- in 3), 3.77 (s, 3H, -OCH3), 3.04–
3.30 (m, 3H, -CONH-CH2-, -CH- in 19), 2.69 and 2.54 (2d, J = 15.5 Hz, 1H each, -C(CH3)2-
CH2-), 1.68 (s, 3H, -CH3 in 30), 1.29 and 1.27 (2s, 3H each, -CO-C(CH3)2), 0.96 (s, 3H, -CH3),
0.91 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.78 (s, 3H, -CH3). Calcd for C50H82N3O9 (M+H)+:
868.6046. Found: 868.6043.
Methyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-L-
1
glycinate (4): Yield 4 mg (34%). H NMR δ 6.07 (bs, 1H, -CONH-), 5.60 (t, J = 6.0 Hz, 1H, -
CONH-), 4.73 and 4.58 (2s, 1H each, =CH2), 4.48 (m, 1H, -CH- in 3), 4.05 (d, J = 5.2 Hz, 2H, -
CO-CH2-NH-), 3.76 (s, 3H, -OCH3), 3.10–3.30 (m, 3H, -CONH-CH2-, -CH- in 19), 2.42–2.69
(m, 5H, -C(CH3)2-CH2-, -CO-CH2-, -CH- in 13), 1.67 (s, 3H, -CH3 in 30), 1.29 (m, 6H, -CO-
C(CH3)2), 0.95 (s, 3H, -CH3), 0.92 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.80 (s, 3H, -CH3).
Calcd for C48H79N2O8 (M+H)+: 811.5725. Found: 811.5729.
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N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic acid-28-oyl]-9-aminononanoyl]-L-glycine (5):
5
6
1
Yield 3 mg (76%). H NMR (CDCl3/Pyridine-d5) δ 7.10 and 6.94 (2bs, 1H each, 2 × -CONH-),
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8
9
4.80 and 4.63 (2s, 1H each, =CH2), 4.54 (m, 1H, -CH- in 3), 4.15 (t, J = 4.3 Hz, 2H, -CO-CH2-
NH-), 3.14–3.36 (m, 3H, -CONH-CH2-, -CH- in 19), 2.64–2.79 (m, 5H, -C(CH3)2-CH2-, -CO-
CH2-, -CH- in 13), 1.70 (s, 3H, -CH3 in 30), 1.30 (m, 6H, -CO-C(CH3)2), 0.97 (s, 6H, 2 × -CH3),
0.87 (s, 3H, -CH3), 0.82 (s, 3H, -CH3), 0.79 (s, 3H, -CH3). Calcd for C47H76N2O8Na (M+Na)+:
819.5499. Found: 819.5470.
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Methyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-L-
1
glutaminate (6): Yield 15 mg (40%). H NMR (CDCl3/Pyridine-d5) δ 7.03 (d, J = 7.5 Hz, 1H, -
CONH-), 6.86 (bs, 1H, 1H in -CONH2), 5.85–5.89 (m, 2H, -CONH- and 1H in -CONH2), 4.66
(s, 1H, =CH), 4.48–4.55 (m, 2H, -COCH-, =CH), 4.42 (dd, J = 4.5 Hz, J = 11.1 Hz, 1H, -CH- in
3), 3.64 (s, 3H, -OCH3), 3.05–3.24 (m, 3H, -CONH-CH2-, -CH- in 19), 2.65 and 2.53 (2d, J =
15.1 Hz, 1H each, -C(CH3)2-CH2-), 1.61 (s, 3H, -CH3 in 30), 1.26 and 1.21 (2s, 3H each, -CO-
C(CH3)2), 0.88 (s, 3H, -CH3), 0.85 (s, 3H, -CH3), 0.76 (s, 3H, -CH3), 0.71 (s, 6H, 2 × -CH3). 13C
NMR (CDCl3/Pyridine-d5, 75 MHz) δ 179.9, 176.3, 175.1,173.8, 172.7, 171.6, 151.2, 109.5,
81.3, 55.7, 55.6, 52.5, 52.1, 50.7, 50.3, 47.0, 45.3, 42.6, 40.9, 40.7, 39.3, 38.6 × 2, 37.9, 37.2,
36.5, 34.5, 33.9, 32.0, 31.1, 30.0, 29.9, 29.6, 29.3, 29.2 × 2, 28.0, 27.1, 26.1, 25.7 × 2, 25.6, 23.8,
21.1, 19.6, 18.3, 16.7, 16.3 × 2, 14.8. Calcd for C51H84N3O9 (M+H)+: 882.6202. Found:
882.6203.
t-Butyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-L-
1
glutaminate (7): Yield 2 mg (17%). H NMR δ 7.52, 6.91, and 5.64 (3bs, 1H each, -CONH-, -
CONH2), 6.23 (d, J = 7.8 Hz, 1H, -CONH-), 4.73 and 4.60 (2s, 1H each, =CH2), 4.40–4.50 (m,
2H, -CO-CH-NH-, -CH- in 3), 2.94–3.16, 3.42–3.70 (m, 3H, -CONH-CH2-, -CH- in 19), 2.79
and 2.47 (2d, J = 15.0 Hz, 1H each, -C(CH3)2-CH2-), 1.68 (s, 3H, -CH3 in 30), 1.47 (s, 9H, -
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C(CH3)3), 1.28 (m, 6H, -CO-C(CH3)2), 0.96 (s, 3H, -CH3), 0.92 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -
CH3), 0.78 (s, 3H, -CH3), 0.77 (s, 3H, -CH3). Calcd for C54H89N3O9Na (M+Na)+: 946.6496.
Found: 946.6498.
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Benzyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-L-
glutaminate (8): Yield 5 mg (28%). 1H NMR δ 7.36 (bs, 5H, ArH), 7.22 (bs, 2H, -CONH2-), 6.83
(bs, 1H, -CONH-), 6.59 (d, J = 6.9 Hz, 1H, -CONH-), 5.19 (s, 2H, -OCH2-), 4.73 (s, 1H, =CH),
4.60 (m, 2H, -CO-CH-NH-, =CH), 4.71 (m, 1H, -CH- in 3), 3.30–3.42, 3.00–3.17 (m, 3H, -
CONH-CH2-, -CH- in 19), 2.71 and 2.54 (2d, J = 15.9 Hz, 1H each, -C(CH3)2-CH2-), 1.68 (s,
3H, -CH3 in 30), 1.28 (m, 6H, -CO-C(CH3)2), 0.96 (s, 3H, -CH3), 0.90 (s, 3H, -CH3), 0.82 (s, 3H,
-CH3), 0.78 (s, 6H, 2 × -CH3). Calcd for C57H87N3O9Na (M+Na)+: 980.6340. Found: 980.6355.
Methyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-L-
isoleucinate (9): Yield 14 mg (48%). 1H NMR δ 6.18 (d, J = 8.7 Hz, 1H, -CONH-), 5.71 (bs, 1H,
-CONH-), 4.72 (s, 1H, =CH), 4.59–4.67 (m, 2H, -CONH-CH-, =CH), 4.48 (t, J = 6.0 Hz, -CH-
in 3), 3.74 (s, 3H, -OCH3), 3.05–3.26 (m, 3H, -CONH-CH2-, -CH- in 19), 2.67 and 2.56 (2d, J =
15.9 Hz, 2H, -C(CH3)2-CH2-), 2.39 (t, J = 10.2 Hz, 1H, -CH- in 13), 2.63 (t, J = 7.2 Hz, 2H, -
CO-CH2-), 1.67 (s, 3H, -CH3 in 30), 1.29 (m, 6H, -CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.91 (s, 6H,
2 × -CH3), 0.89 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.79 (s, 3H, -CH3). Calcd for
C52H86N2O8Na (M+Na)+: 889.6282. Found: 889.6294.
Methyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-L-
1
prolinate (10): Yield 17 mg (59%). H NMR δ 5.75 (bs, 1H, -CONH-), 4.72 and 4.59 (2s, 1H
each, =CH2), 4.45–4.54 (m, 2H, -CON-CH-, CH- in 3), 3.51-3.77 (m, 5H, -OCH3, -CONCH2-),
3.04–3.25 (m, 3H, -CONH-CH2-, -CH- in 19), 2.67 and 2.56 (2d, J = 16.2 Hz, 2H, -C(CH3)2-
CH2-), 1.68 (s, 3H, -CH3 in 30), 1.29 (m, 6H, -CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.91 (s, 3H, -
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CH3), 0.82 (s, 6H, 2 × -CH3), 0.79 (s, 3H, -CH3). Calcd for C51H82N2O8Na (M+Na)+: 873.5969.
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5
6
Found: 873.5989.
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9
Methyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-L-
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phenylalaninate (11): Yield 18 mg (67%). H NMR δ 7.26–7.28 (m, 3H, ArH), 7.08 (d, 2H, J =
6.6 Hz, ArH), 5.92 (d, J = 7.2 Hz, 1H, -CONH-), 5.59 (bs, 1H, -CONH-), 4.90 (dd, J = 6.0 Hz, J
= 12.9 Hz, 1H, -CO-CH-NH-), 4.72 and 4.58 (2s, 1H each, =CH2), 4.47 (t, J = 6.3 Hz, 1H, -CH-
in 3), 3.72 (s, 3H, -OCH3), 3.67 (d, 2H, J = 6.9 Hz, -CH2-C6H6), 3.05–3.30 (m, 3H, -CONH-
CH2-, -CH- in 19), 2.52–2.69 (m, 2H, -C(CH3)2-CH2-), 2.44 (t, J = 9.9 Hz, 1H, -CH- in 13), 1.67
(s, 3H, -CH3 in 30), 1.28 (m, 6H, -CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.92 (s, 3H, -CH3), 0.82 (s,
6H, 2 × -CH3), 0.79 (s, 3H, -CH3). Calcd for C55H84N2O8Na (M+Na)+: 923.6125. Found:
923.6142.
Methyl N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic acid-28-oyl]-9-aminononanoyl]-L-valinate
1
(12): Yield 8 mg (45%). H NMR δ 6.09 (d, J = 8.4 Hz, -CONH-), 5.67 (m, 1H, -CONH-), 4.72
(s, 1H, =CH), 4.56–4.60 (m, 2H, =CH, -CO-CH-NH-), 4.48 (t, J = 6.9 Hz, 1H, -CH- in 3), 3.75
(s, 3H, -OCH3), 3.07–3.28 (m, 3H, -CONH-CH2-, -CH- in 19), 2.67 and 2.56 (2d, J = 15.9 Hz,
1H each, -C(CH3)2-CH2-), 2.41 (t, J = 11.7 Hz, -CH- in 13), 2.27 (t, J = 7.5 Hz, 2H, -COCH2-),
1.68 (s, 3H, -CH3 in 30), 1.28 (m, 6H, -CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.92 (m, 6H, 2 × -
CH3), 0.89 (s, 3H, 2 × -CH3), 0.82 (s, 6H, 2 × -CH3), 0.79 (s, 3H, -CH3). Calcd for
C51H84N2O8Na (M+Na)+: 875.6125. Found: 875.6118.
Dimethyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-L-
1
glutamate (13): Yield 12 mg (64%). H NMR δ 6.57 (d, J = 7.8 Hz, -CONH-), 5.78 (bs, 1H, -
CONH-), 4.72 and 4.59 (2s, 1H each, =CH2), 4.61–4.66 (m, 1H, -CO-CH-NH-), 4.61 (t, J = 6.6
Hz, 1H, -CH- in 3), 3.76 and 3.69 (2s, 3H each, 2 × -OCH3), 3.12–3.32 (m, 2H, -CONH-CH2-),
3.03–3.09 (m, 1H, -CH- in 19), 2.67 and 2.57 (2d, J = 15.9 Hz, 1H each, -C(CH3)2-CH2-), 2.15–
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2.48 (m, 7H, 2 × –COCH2-, -NHCH-CH2-, -CH- in 13), 1.68 (s, 3H, -CH3 in 30), 1.28 (m, 6H, -
CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.91 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.79 (s, 3H, -CH3).
Calcd for C52H84N2O10Na (M+Na)+: 919.6024. Found: 919.6052.
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Methyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-L-
alaninate (14): Yield 7 mg (42%). 1H NMR δ 6.33 (d, J = 6.3 Hz, -CONH-CH-), 5.76 (bs, 1H, -
CONH-), 4.73 (s, 1H, =CH), 4.53–4.63 (m, 2H, =CH, -CO-CH-NH-), 4.48 (t, J = 8.4 Hz, 1H, -
CH- in 3), 3.77 (s, 3H, -OCH3), 3.15–3.30 (m, 2H, -CONH-CH2-), 3.07 (t, 1H, J = 8.4 Hz, 1H, -
CH- in 19), 2.68 and 2.57 (2d, J = 15.6 Hz, 1H each, -C(CH3)2-CH2-), 2.38 (t, J = 10.8 Hz, 1H, -
CH- in 13), 2.27 (t, J = 7.2 Hz, 2H, -COCH2-) 1.68 (s, 3H, -CH3 in 30), 1.43 and 1.41 (d, J = 7.5
Hz, 3H, -CH-CH3), 1.30 (m, 6H, -CO-C(CH3)2), 0.96 (s, 3H, -CH3), 0.91 (s, 3H, -CH3), 0.82 (s,
6H, 2 × -CH3), 0.80 (s, 3H, -CH3). Calcd for C49H80N2O8Na (M+Na)+: 847.5812. Found:
847.5813.
N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-
aminoacetonitrile (15): Yield 12 mg (46%). 1H NMR δ 6.57 and 5.66 (2bs, 1H each, 2 × -CONH-
), 4.72 and 4.59 (2s, 1H each, =CH2), 4.48 (t, 1H, J = 6.0 Hz, -CH- in 3), 4.18 (d, J = 5.7 Hz, 2H,
-CH2-CN), 3.08–3.34 (m, 3H, -CONH-CH2-, -CH- in 19), 2.67 and 2.55 (2d, J = 15.9 Hz, 2H, -
C(CH3)2-CH2-), 2.42 (t, J = 11.4 Hz, 1H, -CH- in 13), 2.25 (t, J = 7.5 Hz, 2H, -CO-CH2-), 1.68
(s, 3H, -CH3 in 30), 1.29 (m, 6H, -CO-C(CH3)2), 0.96 (s, 3H, -CH3), 0.92 (s, 3H, -CH3), 0.82 (s,
6H, 2 × -CH3), 0.80 (s, 3H, -CH3). Calcd for C47H75N3O6Na (M+Na)+: 800.5554. Found:
800.5535.
N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-
1
aminopropionitrile (16): Yield 11 mg (46%). H NMR δ 6.40 (bs, 1H, -CONH-), 5.73 (t, J = 5.1
Hz, 1H, -CONH-), 4.71 and 4.59 (2s, 1H each, =CH2), 4.48 (t, 1H, J = 6.9 Hz, -CH- in 3), 3.52
(dd, J = 6.0 Hz, J = 15.6 Hz, 2H, -CONH-CH2-), 3.14–3.30 (m, 2H, -CONH-CH2-), 3.07 (dt, J =
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3.6 Hz, J = 14.7 Hz, 1H, -CH- in 19), 2.53–2.70 (m, 4H, -C(CH3)2-CH2-, -CH2-CN), 2.39 (t, J =
11.7 Hz, 1H, -CH- in 13), 2.24 (t, J = 8.1 Hz, 2H, -CO-CH2-), 1.68 (s, 3H, -CH3 in 30), 1.29 (m,
6H, -CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.91 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.79 (s, 3H, -
CH3). Calcd for C48H77N3O6Na (M+Na)+: 814.5710. Found: 814.5719.
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N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononanoyl]-N-methyl-
aminoacetonitrile (17): Yield 7 mg (39%). 1H NMR δ 5.75 (bs, 1H, -CONH-), 4.72 and 4.59 (2s,
1H each, =CH2), 4.46 (t, 1H, J = 6.6 Hz, -CH- in 3), 4.37 (s, 2H, -CH2-CN), 3.04–3.28 (m, 6H, -
CONH-CH2-, -N-CH3, -CH- in 19), 2.67 and 2.55 (2d, J = 15.9 Hz, 2H each, -C(CH3)2-CH2-),
2.34–2.39 (m, 3H, -NCO-CH2-, -CH- in 13), 1.67 (s, 3H, -CH3 in 30), 1.28 (m, 6H, -CO-
C(CH3)2), 0.95 (s, 3H, -CH3), 0.91 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.79 (s, 3H, -CH3).
Calcd for C48H78N3O6 (M+H)+: 792.5891. Found: 792.5923.
Methyl
N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-10-aminodecanoyl]-L-
glutaminate (18): Yield 6 mg (34%). 1H NMR δ 7.16 and 6.91 (2bs, 1H each, -CONH2-) 6.56 (d,
J = 6.9 Hz, 1H, -CONH-), 5.71 (bs, 1H, -CONH-), 4.72 (1s, 1H each, =CH), 4.59 (m, 2H, -
CONH-CH-, =CH), 4.48 (m, 1H, -CH- in 3), 3.77 (s, 3H, -OCH3-), 3.33–3.42 and 3.02–3.34 (m,
3H, -CO-CH2-, -CH- in 19), 2.71 and 2.55 (2d, J = 15.6 Hz, 1H each, -C(CH3)2-CH2-), 1.68 (s,
3H, -CH3 in 30), 1.29 (m, 6H, -CO-C(CH3)2), 0.96 (s, 3H, -CH3), 0.91 (s, 3H, -CH3), 0.82 (s, 3H,
-CH3), 0.80 (s, 3H, -CH3), 0.79 (s, 3H, -CH3). Calcd for C52H85N3O9Na (M+Na)+: 918.6183.
Found: 918.6173.
N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-7-aminoheptyl]-Na-boc-L-
glutaminamide (19): Yield 9 mg (32%). 1H NMR δ 6.96 (d, J = 15.3 Hz, 1H, -CONH-), 6.73 (bs,
1H, -CONH-), 6.06 (bs, 2H, -CONH2), 5.71–5.81 (m, 1H, -CONH-), 4.72 and 4.59 (2s, 1H each,
=CH2), 4.45 (t, 1H, J = 8.4 Hz, -CH- in 3), 4.10–4.23 (m, 1H, -COCH-), 3.03–3.35 (m, 5H, 2 × -
CONH-CH2-, -CH- in 19), 2.69 and 2.54 (2d, J = 15.9 Hz, 1H each, -C(CH3)2-CH2-), 2.32–2.39
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(m, 3H, -CH2-CONH2, -CH- in 13), 1.67 (s, 3H, -CH3 in 30), 1.43 (s, 9H, -C(CH3)3), 1.29 (m,
6H, -CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.91 (s, 3H, -CH3), 0.81 (s, 6H, 2 × -CH3), 0.78 (s, 3H, -
CH3). Calcd for C53H89N4O9 (M+H)+: 925.6630. Found: 925.6591.
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N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-7-aminoheptyl]-Na-boc-L-
alaninamide (20): Yield 6 mg (23%). 1H NMR δ 6.53 (t, J = 4.8 Hz, 1H, -CONH-), 5.77 (t, J =
5.4 Hz, 1H, -CONH-), 5.30 (bs, 1H, -CONH-), 4.72 and 4.59 (2s, 1H each, =CH2), 4.68 (t, 1H, J
= 6.9 Hz, -CH- in 3), 4.16–4.23 (m, 1H, -COCH-), 3.03–3.28 (m, 5H, 2 × -CONH-CH2-, -CH- in
19), 2.67 and 2.56 (2d, J = 16.2 Hz, 1H each, -C(CH3)2-CH2-), 2.38 (t, J = 12.0 Hz, 1H, -CH- in
13), 1.67 (s, 3H, -CH3 in 30), 1.44 (s, 9H, -C(CH3)3), 1.35 (d, J = 6.9 Hz, 3H, -CH-CH3), 1.28
(m, 6H, -CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.90 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.79 (s,
3H, -CH3). Calcd for C51H86N3O8 (M+H)+: 868.6415. Found: 868.6462.
N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-8-aminooctyl]-Na-boc-L-
glutaminamide (21): Yield 8 mg (35%). 1H NMR δ 7.17, 6.86, 6.60, 5.73 and (4bs, 1H each, 3 ×
-CONH-, 1 H in -CONH2-), 4.72 and 4.59 (2s, 1H each, =CH2), 4.47 (t, 1H, J = 6.6 Hz, -CH- in
3), 4.13 (m, 1H, -COCH-), 3.34–3.42 (m, 1H, 1H in -CONH-CH2-), 3.23 (dd, J = 6.3 Hz, J =
13.8 Hz, 2H, -CONH-CH2-), 3.02–3.12 (m, 2H, 1H in -CONH-CH2-, -CH- in 19), 2.71 and 2.54
(2d, J = 15.6 Hz, 1H each, -C(CH3)2-CH2-), 1.68 (s, 3H, -CH3 in 30), 1.43 (s, 9H, -C(CH3)3),
1.28 (m, 6H, -CO-C(CH3)2), 0.96 (s, 3H, -CH3), 0.91 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.79
(s, 3H, -CH3). Calcd for C54H90N4O9Na (M+Na)+: 961.6605. Found: 961.6623.
N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic acid-28-oyl]-8-aminooctyl]-Na-boc-L-valinamide
(22): Yield 11 mg (41%). 1H NMR δ 6.45 (bs, 1H, -CONH-), 5.74 (t, J = 5.7 Hz, 1H, -CONH-),
5.40 (bs, 1H, -CONH-), 4.72 and 4.58 (2s, 1H each, =CH2), 4.47 (t, 1H, J = 7.2 Hz, -CH- in 3),
3.84 (t, J = 7.8 Hz, 1H, -COCH-), 3.06–3.29 (m, 6H, 2 × -CONH-CH2-, -CH-(CH3)2, -CH- in
19), 2.67 and 2.56 (2d, J = 15.9 Hz, 1H each, -C(CH3)2-CH2-), 2.38 (t, J = 9.9 Hz, 1H, -CH- in
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13), 1.67 (s, 3H, -CH3 in 30), 1.43 (s, 9H, -C(CH3)3), 1.35 (d, J = 6.9 Hz, 3H, -CH-CH3), 1.28
(m, 12H, -CH-(CH3)2, -CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.91 (s, 3H, -CH3), 0.81 (s, 6H, 2 × -
CH3), 0.79 (s, 3H, -CH3). Calcd for C54H91N3O8Na (M+Na)+: 932.6704. Found: 932.6708.
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N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-8-aminooctyl]-Na-boc-L-
alaninamide (23): Yield 3 mg (17%). 1H NMR δ 6.36 (bs, 1H, -CONH-), 5.71 (t, J = 5.4 Hz, 1H,
-CONH-), 5.18 (bs, 1H, -CONH-), 4.72 and 4.59 (2s, 1H each, =CH2), 4.48 (t, 1H, J = 6.3 Hz, -
CH- in 3), 4.16–4.25 (m, 1H, -COCH-), 3.06–3.30 (m, 5H, 2 × -CONH-CH2-, -CH- in 19), 2.67
and 2.56 (2d, J = 15.9 Hz, 1H each, -C(CH3)2-CH2-), 1.68 (s, 3H, -CH3 in 30), 1.44 (s, 9H, -
C(CH3)3), 1.35 (d, J = 7.2 Hz, 3H, -CH-CH3), 1.29 (m, 6H, -CO-C(CH3)2), 0.95 (s, 3H, -CH3),
0.89 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.80 (s, 3H, -CH3). Calcd for C52H86N3O8 (M-H)¯ :
880.6415. Found: 880.6389.
2-(Boc-amino)-4-cyano-N-[N’-[3β-O-(3’,3’-dimethylsuccinyl)-betulinic
acid-28-oyl]-8-
1
aminooctyl]butanamide (24): Yield 2 mg (22%). H NMR δ 6.32 (bs, 1H, -CONH-), 5.68 (bs,
1H, -CONH-), 5.30 (bs, 1H, -CONH-), 4.72 and 4.59 (2s, 1H each, =CH2), 4.46 (m, 1H, -CH- in
3), 4.16–4.27 (m, 5H, -CH2-CH2-CN, -COCH-), 3.10–3.30 (m, 5H, 2 × -CONH-CH2-, -CH- in
19), 2.68 and 2.56 (2d, J = 15.0 Hz, 1H each, -C(CH3)2-CH2-), 1.68 (s, 3H, -CH3 in 30), 1.45 (s,
9H, -C(CH3)3), 1.30 (m, 6H, -CO-C(CH3)2), 0.92 (s, 6H, 2 × -CH3), 0.89 (s, 3H, -CH3), 0.83 (s,
6H, 2 × -CH3), 0.80 (s, 3H, -CH3). Calcd for C54H87N4O8 (M-H)¯ : 919.6524. Found: 919.6481.
N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic acid-28-oyl]-8-aminooctyl]-Na-boc-L-prolinamide
1
(25): Yield 9 mg (34%). H NMR δ 6.90 and 6.48 (2bs, 1H each, 2 × -CONH-), 4.72 and 4.59
(2s, 1H each, =CH2), 4.75 (t, 1H, J = 9.6 Hz, -CH- in 3), 4.22 (t, J = 4.5 Hz, 1H, -COCH-), 3.06–
3.50 (m, 7H, 2 × -CONH-CH2-, -CON-CH2-, -CH- in 19), 2.66 and 2.56 (2d, J = 16.2 Hz, 1H
each, -C(CH3)2-CH2-), 2.39 (t, J = 10.8 Hz, 1H, -CH- in 13), 1.68 (s, 3H, -CH3 in 30), 1.45 (s,
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× -CH3), 0.80 (s, 3H, -CH3). Calcd for C54H88N3O8 (M-H)¯ : 906.6571. Found: 906.6523.
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acid-28-oyl]-9-aminononactyl]-Na-boc-L-
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glutaminamide (26): Yield 6 mg (21%). H NMR δ 7.10, 6.93 and 6.63 (3s, 1H each, 2 × -
CONH-, 1H in -CONH2), 5.75 (t, J = 5.4 Hz, 1H, -CONH-), 5.71 (bs, 1H, 1H in -CONH2), 4.72
and 4.59 (2s, 1H each, =CH2), 4.48 (dd, 1H, J = 6.0 Hz, J = 9.6 Hz, -CH- in 3), 4.21 (dd, J = 4.2
Hz, J = 6.0 Hz, 1H, -COCH-), 3.05–3.40 (m, 5H, 2 × -CONH-CH2-, -CH- in 19), 2.75 and 2.53
(2d, J = 15.9 Hz, 1H each, -C(CH3)2-CH2-), 1.68 (s, 3H, -CH3 in 30), 1.43 (s, 9H, -C(CH3)3),
1.29 and 1.27 (2s, 3H each, -CO-C(CH3)2), 1.11 (s, 3H, -CH3), 0.96 (s, 3H, -CH3), 0.91 (s, 3H, -
CH3), 0.82 (s, 6H, 2 × -CH3), 0.79 (s, 3H, -CH3). Calcd for C55H91N4O9 (M-H)¯ : 951.6786.
Found: 951.6762.
N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononactyl]-Na-boc-L-
1
alaninamide (27): Yield 12 mg (45%). H NMR δ 6.42 and 5.50 (2bs, 1H each, 2 × -CONH-),
5.70 (t, J = 5.4 Hz, 1H, -CONH-), 4.72 and 4.59 (2s, 1H each, =CH2), 4.47 (dd, 1H, J = 6.0 Hz, J
= 9.6 Hz, -CH- in 3), 4.21 (dd, J = 3.3 Hz, J = 6.3 Hz, 1H, -COCH-), 3.02–3.30 (m, 5H, 2 × -
CONH-CH2-, -CH- in 19), 2.67 and 2.55 (2d, J = 15.6 Hz, 1H each, -C(CH3)2-CH2-), 2.38 (t, J =
6.0 Hz, 1H, -CH- in 13), 1.67 (s, 3H, -CH3 in 30), 1.43 (s, 9H, -C(CH3)3), 1.35 (d, J = 7.2 Hz,
3H, -CHCH3), 1.28 and 1.27 (2s, 3H each, -CO-C(CH3)2), 0.95 (s, 3H, -CH3), 0.91 (s, 3H, -CH3),
0.82 (s, 6H, 2 × -CH3), 0.79 (s, 3H, -CH3). Calcd for C53H90N3O8 (M+H)+: 896.6728. Found:
896.6773.
N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononactyl]-Na-boc-L-
glycinamide (28): Yield 6 mg (34%). 1H NMR δ 6.41, 5.72 and 5.34 (3bs, 1H each, 3 × -CONH-
), 4.73 and 4.59 (2s, 1H each, =CH2), 4.46–4.51 (m, 1H, -CH- in 3), 3.80 (d, J = 3.6 Hz, 2H, -
COCH2-NH-), 3.02–3.36 (m, 5H, 2 × -CONH-CH2-, -CH- in 19), 2.70 and 2.56 (2d, J = 15.9 Hz,
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1H each, -C(CH3)2-CH2-), 1.68 (s, 3H, -CH3 in 30), 1.45 (s, 9H, -C(CH3)3), 1.25 (m, 6H, -CO-
C(CH3)2), 0.96 (s, 3H, -CH3), 0.92 (s, 3H, -CH3), 0.82 (s, 6H, 2 × -CH3), 0.80 (s, 3H, -CH3).
Calcd for C52H88N3O8 (M+H)+: 882.6571. Found: 882.6553.
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N-[N’-[3β-O-(3’,3’-Dimethylsuccinyl)-betulinic
acid-28-oyl]-9-aminononactyl]-Na-boc-L-
asparaginamide (29): Yield 8 mg (41%). 1H NMR δ 6.72, 6.21, 6.13 and 5.69 (4bs, 1H each, 3 ×
-CONH-, 1H in -CO-NH2), 4.73 and 4.59 (2s, 1H each, =CH2), 4.42–4.50 (m, 2H, -COCH-, -
CH- in 3), 3.64–3.24 and 2.84–3.20 (m, 7H, 2 × -CONH-CH2-,-COCH2-, -CH- in 19), 2.78 and
2.47 (2d, J = 15.0 Hz, 1H each, -C(CH3)2-CH2-), 1.68 (s, 3H, -CH3 in 30), 1.46 (s, 9H, -
C(CH3)3), 1.25 (m, 6H, -CO-C(CH3)2), 0.96 (s, 3H, -CH3), 0.92 (s, 3H, -CH3), 0.82 (s, 3H, -
CH3), 0.81 (s, 3H, -CH3), 0.75 (s, 3H, -CH3). Calcd for C54H89N4O9 (M-H)¯ : 937.6630. Found:
937.6595.
Construction of BVM-R Variants. Construction of these mutants was achieved by using a
QuickChange site-directed mutagenesis kit purchased from Stratagene as previously described.23
The plasmid, pNL4-3, was used as a template to create all the mutants listed in Table1. Each
mutation was introduced into pNL4-3 following the protocol provided by Stratagene.
Multi-Cycle Viral Replication in MT4 Cell Assay. HIV-1 NL4-3 or the resistant variants
with a multiplicity of infection (MOI) of 0.001 TCID50/cell was used to infect MT4 cells in the
presence of compounds at various concentrations. On day 4 post-infection, supernatant samples
were harvested and assayed for p24 using an ELISA kit from Perkin Elmer. The antiviral
potency is defined as the drug concentration that reduces the amount of p24 by 50% (IC50).
Cytotoxicity Assay. A CytoTox-Glo™ cytotoxicity assay (Promega) was used to determine
the cytotoxicity of the synthesized BA derivatives. MT4 cells were cultured in the presence of
various concentrations of the compounds for 4 days. Percent of viable cells was determined by
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following the protocol provided by the manufacturer. The 50% cytotoxic concentration (CC50)
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Determination of Anti-HIV-1 Maturation Activity. Analysis of anti-maturation activity was
performed as described previously.10,18 Briefly, 293T cells were transfected with plasmids with
corresponding proviruses, such as pNL4-3. The culture supernatant was harvested for Western
blot analysis two days after transfection. The signature event of anti-maturation activity of
compound 1 is a partial inhibition of CA-SP1 (p25) cleavage that occurs without interfering with
other Gag cleavage sites. Inhibition of CA-SP1 cleavage resulted in an accumulation of p25.
Accumulation of CA-SP1 (p25) was analyzed with a Western blot analysis using a monoclonal
antibody produced by the HIV-1 p24 hybridoma, 183-H12-5C (NIH AIDS Reagent Program).
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Fusion Assay. The fusion assay used in this study was described previously.24 The fusion
assay was performed by transfecting monkey kidney cells (COS) with the expression vector pars
that contained HIV-1 Env and tat genes. COS cells (1 × 106 cells/mL) were mixed with 5 µg of
the Env-expressing vector and incubated on ice for 10 minutes. Electroporation was performed
using a gene pulsar (Bio-Rad, Hercules, CA) with capacitance set at 950 ꢀF and voltage at 150
V. The transfected COS cells were cultured for one day and then mixed with TZM-bl cells.
TZM-bl cells were incubated with the Env-expressing COS cells in the presence of inhibitors in
96-well flat-bottom plates (Costar) overnight. Fusion was measured by quantifying luciferase
activity in the fused cells using a Bright-Glow luciferase assay kit (Promega, Luis Obispo, CA).
Inhibition of the Env-mediated membrane fusion was expressed as a percentage of the control
(Env-mediated membrane fusion in the absence of inhibitors).
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ASSOCIATED CONTENT
AUTHOR INFORMATION
Corresponding Author:
9
*For C. H. C.: phone, 919-684-3819; e-mail, chc@duke.edu. For L.H.: phone, 919-684-2952; e-
mail, lihuang@duke.edu.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID)
Grant AI-65310 (C.H.C.), in part by NIAID Grants AI-077417 and AI-033066 (K.H.L.), and by
the D. P. Bolognesi Award (L.H.), Duke University Medical Center.
ABBREVIATIONS USED
AIDS, acquired immunodeficiency syndrome; ART, antiretroviral therapy; BA, betulinic acid;
BVM-R, bevirimat-resistant; DCM, dichloromethane; DMAP, 4-Dimethylaminopyridine; DSB,
3-O-(3’,3’-dimethylsuccinyl)-betulinic
acid;
EDC,
N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimide hydrochloride; HIV-1, human immunodeficiency virus type 1; HOBt,
hydroxybenzotriazole; NMM, N-methylmorpholine; THF, tetrahydrofuran.
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