Anti-AIDS agents 49. Synthesis, anti-HIV, and anti-fusion activities of IC9564 analogues based on betulinic acid

Article abstract of DOI:10.1021/jm020069c

The betulinic acid derivative IC9564 inhibits human immunodeficiency virus (HIV)-1 entry. Among a series of IC9564 derivatives, 5 and 20 were the most promising compounds against HIV infection with EC₅₀ values of 0.33 and 0.46 μM, respectively.

Full text of DOI:10.1021/jm020069c

J . Med. Chem. 2002, 45, 4271-4275
4271
An ti-AIDS Agen ts 49.1 Syn th esis, An ti-HIV, a n d An ti-F u sion Activities of IC9564
An a logu es Ba sed on Betu lin ic Acid
I-Chen Sun,† Chin-Ho Chen,‡ Yoshiki Kashiwada,§ J iu-Hong Wu,† Hui-Kang Wang,† and Kuo-Hsiung Lee*,†
Natural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360,
Department of Microbiology, Meharry Medical College, West Basic Science Building, Nashville, Tennessee 37208, and Niigata
College of Pharmacy, Kamishin’ei-cho, Niigata 950-21, J apan
Received February 11, 2002
The betulinic acid derivative IC9564 inhibits human immunodeficiency virus (HIV)-1 entry.
Among a series of IC9564 derivatives, 5 and 20 were the most promising compounds against
HIV infection with EC50 values of 0.33 and 0.46 µM, respectively. Both compounds inhibited
syncytium formation with EC50 values of 0.40 and 0.33 µM, respectively. The comparable EC50
values in the two assays suggested that these compounds are fusion inhibitors. The structure-
activity relationship data also indicated that a double bond in IC9564 can be eliminated and
the statine moiety can be replaced with L-leucine while retaining anti-HIV activity.
Ch a r t 1
In tr od u ction
Triterpenes constitute a promising class of anti-HIV
(human immunodeficiency virus) agents.2-7 Their modes
of action have been associated with several steps in the
virus life cycle, including fusion,8-11 reverse transcrip-
tion,12,13 and maturation.14-16 Current HIV therapy
lacks drugs targeted at the fusion step, although peptide
mimetics (T20 and T1249), recombinant protein (PRO-
542), and small molecular inhibitors (bicyclam AMD3100)
have shown promise in clinical trials.17
Mayaux et al. reported that the triterpene fusion
inhibitor RPR103611 (1, Chart 1) was active at a
submicromolar level.8 Subsequently, Labrosse et al.
determined that the drug sensitivity was linked to a
single amino acid change, I84S, on the loop region of
HIV-1 transmembrane glycoprotein gp419, and to the
stability of the gp120-gp41 complex.10 IC9564 (2, Chart
1) is a stereoisomer of and equipotent with RPR103611.
Results from a syncytium formation assay indicated
that IC9564 inhibits HIV-1 at the membrane fusion
step.11 Further analysis showed that HIV-1 gp120 was
the molecular target for IC9564. In an effort to discover
more potent anti-fusion triterpenes based on betulinic
acid, the present study has focused on modifications in
the isopropylene and C28 side chain.
acid chloride 6 with L-leucine methyl ester gave 7, which
was converted to 8 by the same conditions as described
for 5.
Conjugation of acid chloride 6 (Scheme 2) with various
amines (â-alanine, 8-aminocaprylic acid, and 11-ami-
noundecanoic acid methyl esters) in the presence of
DMAP, potassium carbonate, triethylamine, and pyri-
dine gave the corresponding amide derivatives 9-11.
Removal of the acetyl and methyl moieties using 4N
sodium hydroxide produced the acids 12-14. Com-
pounds 15-17 were obtained similarly to 7 using EDC/
HOBT in CH2Cl2 at room temperature in yields ranging
from 83% to 93%. Finally, saponification of the methyl
ester led to 18-20.
Ch em istr y
The common intermediates acid chlorides 3 and 6
were obtained by reacting dihydrobetulinic and betulinic
acids with acetic anhydride followed by treating with
oxalyl chloride. Acid chloride 3 (Scheme 1) was coupled
with the readily prepared amine [4S-(8-aminooctanoy-
lamino)-3R-hydroxy-6-methylheptanoic acid benzyl es-
ter], synthesized as described in ref 11, in the presence
of EDC/HOBT to give 4. The protecting groups were
cleaved with 4N sodium hydroxide in MeOH/tetrahy-
drofuran (THF) to yield 5. Likewise, condensation of
Resu lts a n d Discu ssion
Compounds 4, 5, 7, and 9-20 were evaluated for anti-
HIV-1 activity in a MAGI assay against the virus NL4-
3. Compound 8 was also examined for inhibitory activity
in H9 lymphocytes.18 AZT was included during each
experiment as a positive drug control. The data are
presented in Table 1.
* To whom correspondence should be addressed. Tel: (919)962-0066.
Fax: (919)966-3893. E-mail: khlee@unc.edu.
† University of North Carolina.
‡ Meharry Medical College.
§ Niigata College of Pharmacy.
10.1021/jm020069c CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/05/2002

4272 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Sch em e 1. Synthesis of (R,S)Statine Derivatives
Sun et al.
Sch em e 2. Synthesis of l-Leucine Derivatives
The saturated analogue 5 was equipotent (EC50
)
µM). However, the methyl ester (16) of 19 showed
increased potency (EC50 ) 0.52 µM). When the meth-
ylene chain was extended to 10 carbons, the free acid
20 showed equivalent activity (EC50 ) 0.46 µM) to 16.
In contrast, the â-alanine derivative 18 totally lost anti-
HIV activity (EC50 > 39 µM). Furthermore, the methyl
esters (15 and 17) of 18 and 20 also lacked discernible
anti-HIV activity. Complete omission of the aminoal-
kanoic chain (8) dropped the inhibitory activity 8-fold.
Among the non-L-leucine intermediates, the esters 9-11
were active only at g10 µM. However, the deesterified
compound 14 was quite active (EC50 ) 0.7 µM), while
0.33 µM) with IC9564 (EC50 ) 0.4 µM). In contrast, the
EC50 value of its ester precursor 4 dropped to >28 µM.
These data suggested that a free hydroxy group at C3
and free carboxylic acid in the C28 side chain are
necessary for anti-HIV activity. This finding is consis-
tent with the results reported previously for IC9564 and
IC9563.11 Our results also suggest that the double bond
of the isopropylidene group might not play a key role
in the HIV inhibitory activity of this compound type.
Replacing the statine moiety of IC9564 with L-leucine
yielded the 4-fold less potent compound 19 (EC50 ) 1.38

Anti-AIDS Agent 49
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4273
Ta ble 1. Results of MAGI and Fusion Assays
continued overnight. The solution was diluted with CH2Cl2 (20
mL) and then washed three times with water (50 mL). The
CH2Cl2 layer was dried over anhydrous MgSO4 and concen-
trated under reduced pressure. The crude product was chro-
matographed on silica gel and crystallized from n-hexane and
EtOAc.
Gen er al P r ocedu r e for Syn th esizin g Com pou n ds 9-11.
A solution of amine ester (0.5 mmol) in dry CH2Cl2 (2 mL) was
treated with 4-(dimethylamino)pyridine (1.5 equiv mol), K2-
CO3 (1.5 equiv mol), dry triethylamine (0.2 mL), and anhy-
drous pyridine (3 mL). To the mixture was added acid chloride
6 (0.8 equiv mol), and stirring was continued overnight. The
solution was diluted with EtOAc (20 mL) and then washed
three times with 10% HCl solution (50 mL). The EtOAc layer
was dried over anhydrous MgSO4 and concentrated under
reduced pressure. The crude product was chromatographed
using a silica gel column and crystallized from n-hexane and
EtOAc.
Gen er a l P r oced u r e for Syn th esizin g Com p ou n d s 5, 8,
12-14, a n d 18-20. A solution of starting material (0.25
mmol), MeOH (1 mL), and THF (1 mL) was treated with 4 N
NaOH (0.5 mL). After the reaction was complete, the mixture
was acidified with hydrochloric acid to pH 4 and extracted
three times with CHCl3 (20 mL). Followed by the routine
workup, the residue was crystallized from n-hexane and
EtOAc.
EC50
compd
MAGI assay (µM)
fusion assay (µM)
4
5
7
8
>28
0.33
>40
3.22a
26.7
29.1
10.2
>47
>22
0.40
>32
5.48
21.4
>31
>29
>38
9
10
11
12
13
14
15
16
17
18
19
20
0.70
>38
0.31
7.63
0.86
>26
0.52
>32
>39
>31
1.38
0.46
1.10
0.33
IC9564
DP178
AZT
0.40
0.014
0.075
0.66
0.019
>75
a
Compound was tested in acutely infected H9 cells; see
Experimental Section for details.
(3R,4S)-Ben zyl N′-[N-[3â-Acet oxy-28-oyl]-8-a m in ooc-
ta n oyl]-4-a m in o-3-h yd r oxy-6-m eth ylh ep ta n oa te (4). Yield
both compounds (12 and 13) with shorter aminoalkanoic
acids were inactive. These results indicate that the
statine group in the C28 side chain of IC9564 can be
replaced by L-leucine and that the numbers of methyl-
enes in the aminoalkanoic chain are crucial to optimal
anti-HIV potency.
A known fusion inhibitor, DP178/T20, was used as a
positive control in the fusion and MAGI assays.19
Compounds 5, 8, 14, 16, 19, and 20 all had EC50 values
of e3.2 µM in the MAGI assay and were active against
membrane fusion with EC50 values of <5.5 µM. In
addition, the concentrations required to inhibit syncy-
tium formation were comparable to those required to
inhibit HIV-1 replication. Thus, HIV-1 envelope-induced
membrane fusion appears to be the primary target of
these compounds. Further mode of action studies will
be determined.
25
64%; an off-white amorphous powder; [R]D -16° (c ) 0.5,
1
CHCl3). H NMR (CDCl3): δ 0.74, 0.85 (3H each, both d, J )
7 Hz; 20-(CH3)2), 0.83, 0.84, 0.85, 0.92, 0.94 (3H each, all s;
4-(CH3)2, 8-CH3, 10-CH3, 14-CH3), 0.89, 0.92 (3H each, both d,
J ) 7 Hz; statine moiety -CH(CH3)2), 2.04 (3H, s; 3-OCOCH3),
2.18 (2H, t, J ) 7 Hz; -CH2CONH-), 2.52 (2H, d, J ) 6 Hz;
-CH2COO-), 3.16-3.25 (2H, m; -CONHCH2-), 3.64 (1H, d,
J ) 4 Hz; -CHOH), 3.98-4.12 (2H, m; -NHCH-, -CHOH-),
4.47 (1H, dd, J ) 5, 11 Hz; H-3), 5.15 (2H, s; -OCH2C6H5),
5.55-5.59 (2H, m; -NHCH2-, -NHCH-), 7.32-7.39 (5H, m;
-C6H5). Anal. (C55H88N2O7) C, H, N.
(3R,4S)-N′-[N-[3â-Hyd r oxy-28-oyl]-8-a m in oocta n oyl]-4-
a m in o-3-h yd r oxy-6-m eth ylh ep ta n oic Acid (5). Yield 88%;
a white powder; mp 114 °C (dec); [R]D25 -25° (c ) 2.34, CHCl3).
1H NMR (CDCl3): δ 0.74, 0.85 (3H each, both d, J ) 7 Hz;
20-(CH3)2), 0.76, 0.83, 0.94, 0.96, 0.97 (3H each, all s; 4-(CH3)2,
8-CH3, 10-CH3, 14-CH3), 0.91, 0.94 (3H each, both d, J ) 7
Hz; statine moiety -CH(CH3)2), 2.22 (2H, dt, J ) 3, 7 Hz;
-CH2CONH-), 2.50 (2H, d, J ) 6 Hz; -CH2COOH), 3.13-
3.28 (3H, m; -NHCH2-, -CHOH-), 3.64 (1H, d, J ) 4 Hz;
-CHOH), 4.0-4.12 (2H, m; H-3, -NHCH-), 5.69 (1H, t, J )
6 Hz; -NHCH2-), 5.94 (1H, d, J ) 8 Hz; -NHCH-). Anal.
(C46H80N2O6) C, H, N.
Exp er im en ta l Section
The melting points were measured with a Fisher-J ohns
melting point apparatus and are uncorrected. The proton
nuclear magnetic resonance (1H NMR) spectra were obtained
using a Bruker AC-300 NMR spectrometer. All chemical shifts
are reported in parts per million from the internal standard
Me4Si (tetramethylsilane, TMS) with CDCl3 or C5D5N as
solvent. Elemental analyses were performed by Atlantic Mi-
crolab, Inc., Norcross, GA. Optical rotations were measured
with a J asco DIP-1000 polarimeter. Thin-layer chromatogra-
phy (TLC) was carried out on Merck precoated silica gel 60
F-254 plates. EM Kieselgel 60 (230-400 mesh ASTM) was
used for column chromatography. All new target compounds
were characterized by optical rotation, 1H NMR, and elemental
analysis.
Meth yl N-[3â-Acetoxy-20(29)-en -28-oyl]-L-leu cin a te (7).
Yield 75%; an off-white amorphous powder; mp 115-117 °C;
25
1
[R]D -2.4° (c ) 0.25, CHCl3). H NMR (CDCl3): δ 0.83, 0.84
(6H), 0.92, 0.95, 0.96 (6H) (3H each, except 0.84, 0.96, all s;
4-(CH3)2, 8-CH3, 10-CH3, 14-CH3, leucine moiety -(CH3)2), 1.68
(3H, s; H-30), 2.04 (3H, s; 3-OCOCH3), 2.45 (1H, dt, J ) 3, 12
Hz; H-13), 3.11 (1H, dt, J ) 4, 11 Hz; H-19), 3.73 (3H, s;
-COOCH3), 4.47 (1H, t, J ) 8 Hz; H-3), 4.58 (1H, s; H-29),
4.64 (1H, dt, J ) 5, 8 Hz; -NHCH-), 4.72 (1H, d, J ) 2 Hz;
H-29), 5.87 (1H, d, J ) 8 Hz; -CONH-). Anal. (C39H63NO5)
C, H, N.
N-[3â-Hydr oxy-20(29)-en -28-oyl]-L-leu cin e (8). Yield 40%;
P r ep a r a tion of Acid Ch lor id es 3 a n d 6. Oxalyl chloride
solution (2 M in CH2Cl2, 10 mL) was added to the 3-O-acetates
of dihydrobetulinic acid and betulinic acid (0.5 mmol) and
stirred for 2 h. Each mixture was concentrated to dryness
under reduced pressure. The residues were diluted with dry
CH2Cl2 (3 × 1 mL), concentrated to dryness under reduced
pressure, and used without further purification.
Gen er a l P r oced u r e for Syn th esizin g Com p ou n d s 4, 7,
a n d 15-17. A solution of amine ester (0.5 mmol) in dry CH2-
Cl2 (3 mL) was treated with EDC (1.5 equiv mol) and HOBT
(1.5 equiv mol). To the mixture was added acid chloride (0.8
equiv mol) or free acid (0.8 equiv mol), and stirring was
an off-white amorphous powder; mp 243-244 °C; [R]D25 -17.2°
1
(c ) 1.40, CHCl3). H NMR (CDCl3): δ 0.75, 0.82, 0.91, 0.96,
0.97 (9H) (3H each, except 0.97, all s; 4-(CH3)2, 8-CH3, 10-CH3,
14-CH3, leucine moiety -(CH3)2), 1.68 (3H, s; H-30), 2.42 (1H,
dt, J ) 3, 12 Hz; H-13), 3.10 (1H, dt, J ) 4, 11 Hz; H-19), 3.19
(1H, dd, J ) 5, 11 Hz; H-3), 4.59 (1H, s; H-29), 4.56-4.63 (1H,
m; -NHCH-), 4.73 (1H, d, J ) 2 Hz; H-29), 5.88 (1H, d, J )
8 Hz; -CONH-). Anal. (C36H59NO4) C, H, N.
Met h yl N-[3â-Acet oxy-20(29)-en -28-oyl]-3-a m in op r o-
p a n oa te (9). Yield 76%; a white powder; mp 112-114 °C;
[R]D25 +20.3° (c ) 1.43, CHCl3). 1H NMR (CDCl3): δ 0.83, 0.84

4274 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Sun et al.
(6H), 0.91, 0.95 (3H each, except 0.84, all s; 4-(CH3)2, 8-CH3,
10-CH3, 14-CH3), 1.68 (3H, s; H-30), 2.04 (3H, s; 3-OCOCH3),
2.41 (1H, dt, J ) 3, 12 Hz; H-13), 2.54-2.58 (2H, m; -CH2-
COOCH3), 3.10 (1H, dt, J ) 4, 11 Hz; H-19), 3.42-3.61 (2H,
m; -CONHCH2-), 3.70 (3H, s; -COOCH3), 4.47 (1H, dd, J )
5, 11 Hz; H-3), 4.59 (1H, s; H-29), 4.74 (1H, d, J ) 2 Hz; H-29),
6.20 (1H, t, J ) 6 Hz; -CONH-). Anal. (C36H57NO5) C, H, N.
Meth yl N-[3â-Acetoxy-20(29)-en -28-oyl]-8-a m in oocta n -
4.74 (1H, d, J ) 2 Hz; H-29), 5.60 (1H, t, J ) 6 Hz;
-CONHCH2-), 5.81 (1H, d, J ) 8 Hz; -CONHCH(CH2-)-
CO-). Anal. (C45H76N2O5) C, H, N.
Met h yl N′-[N-[3â-H yd r oxy-20(29)-en -28-oyl]-11-a m i-
n ou n d eca n oyl]-L-leu cin a te (17). Yield 87%; a yellow gum;
mp 92-94 °C; [R]D25 +3.4° (c ) 0.93, CHCl3). 1H NMR (CDCl3)
δ 0.75, 0.82, 0.94 (6H), 0.96 (9H) (3H each, except 0.94, 0.96,
all s; 4-(CH3)2, 8-CH3, 10-CH3, 14-CH3, leucine moiety -(CH3)2),
1.68 (3H, s; H-30), 2.18-2.24 (2H, m; -CH2CONH-), 2.45 (1H,
dt, J ) 3, 12 Hz; H-13), 3.11-3.34 (4H, m; H-3, H-19,
-CONHCH2-), 3.73 (3H, s; -COOCH3), 4.59 (1H, s; H-29),
4.66 (1H, dt, J ) 5, 8 Hz; -NHCH(CH2-)CO-), 4.73 (1H, d,
J ) 2 Hz; H-29), 5.58 (1H, t, J ) 6 Hz; -CONHCH2-), 5.81
(1H, d, J ) 8 Hz; -CONHCH(CH2-)CO-). Anal. (C48H82N2O5)
C, H, N.
N′-[N-[3â-Hydr oxy-20(29)-en -28-oyl]-3-am in opr opan oyl]-
L-leu cin e (18). Yield 77%; a white powder; mp 180-182 °C;
[R]D25 +3.6° (c ) 0.28, C5H5N). 1H NMR (CDCl3): δ 0.75, 0.81,
0.91, 0.96 (12H) (3H each, except 0.96, all s; 4-(CH3)2, 8-CH3,
10-CH3, 14-CH3, leucine moiety -(CH3)2), 1.68 (3H, s; H-30),
2.35-2.52 (3H, m; H-13, -CH2CONH-), 3.08 (1H, dt, J ) 4,
11 Hz; H-19), 3.18 (1H, dd, J ) 5, 11 Hz; H-3), 3.45-3.57 (2H,
m; -CONHCH2-), 4.53-4.59 (2H, m; -NHCH(CH2-)CO-,
H-29), 4.73 (1H, br s; H-29), 6.32 (1H, d, J ) 8 Hz; -CON-
HCH2-), 6.40 (1H, t, J ) 6 Hz; -CONHCH(CH2-)CO-). Anal.
(C39H64N2O5) C, H, N.
25
oa te (10). Yield 69%; a yellow gum; mp 83-85 °C; [R]D
1
+17.4° (c ) 0.3, CHCl3). H NMR (CDCl3): δ 0.83, 0.84 (6H),
0.93, 0.96 (3H each, except 0.84, all s; 4-(CH3)2, 8-CH3, 10-
CH3, 14-CH3), 1.68 (3H, s; H-30), 2.04 (3H, s; 3-OCOCH3), 2.31
(2H, t, J ) 7 Hz; -CH2COOCH3), 2.46 (1H, dt, J ) 3, 12 Hz;
H-13), 3.10-3.32 (3H, m; H-19, -CONHCH2-), 3.67 (3H, s;
-COOCH3), 4.47 (1H, t, J ) 8 Hz; H-3), 4.59 (1H, s; H-29),
4.73 (1H, d, J ) 2 Hz; H-29), 5.57 (1H, t, J ) 6 Hz; -CON-
H-). Anal. (C41H67NO5) C, H, N.
Met h yl N-[3â-Acet oxy-20(29)-en -28-oyl]-11-a m in ou n -
d eca n oa te (11). Yield 65%; an off-white amorphous powder;
mp 75-77 °C; [R]D +15.1° (c ) 0.49, CHCl3). 1H NMR
25
(CDCl3): δ 0.83, 0.84 (6H), 0.93, 0.96 (3H each, except 0.84,
all s; 4-(CH3)2, 8-CH3, 10-CH3, 14-CH3), 1.68 (3H, s; H-30), 2.04
(3H, s; 3-OCOCH3), 2.30 (2H, t, J ) 8 Hz; -CH2COOCH3),
2.45 (1H, dt, J ) 3, 12 Hz; H-13), 3.10-3.36 (3H, m; H-19,
-CONHCH2-), 3.67 (3H, s; -COOCH3), 4.46 (1H, t, J ) 8 Hz;
H-3), 4.59 (1H, s; H-29), 4.73 (1H, d, J ) 2 Hz; H-29), 5.57
(1H, t, J ) 6 Hz; -CONH-). Anal. (C44H73NO5) C, H, N.
N-[3â-Hydr oxy-20(29)-en -28-oyl]-3-am in opr opan oic Acid
N′-[N-[3â-Hyd r oxy-20(29)-en -28-oyl]-8-a m in oocta n oyl]-
L-leu cin e (19). Yield 64%; a white powder; mp 147-149 °C;
25
25
1
(12). Yield 87%; a white powder; [R]D +25.0° (c ) 0.58,
[R]D -2.9° (c ) 1.37, CHCl3). H NMR (CDCl3): δ 0.75, 0.82,
0.92, 0.96 (12H) (3H each, except 0.96, all s; 4-(CH3)2, 8-CH3,
10-CH3, 14-CH3, leucine moiety -(CH3)2), 1.68 (3H, s; H-30),
2.18-2.29 (2H, m; -CH2CONH-), 2.41 (1H, dt, J ) 3, 12 Hz;
H-13), 3.07-3.31 (4H, m; H-3, H-19, -CONHCH2-), 4.59-
4.66 (2H, m; -NHCH(CH2-)CO-, H-29), 4.73 (1H, br s; H-29),
5.77 (1H, t, J ) 6 Hz; -CONHCH2-), 6.25 (1H, d, J ) 8 Hz;
-CONHCH(CH2-)CO-). Anal. (C44H74N2O5) C, H, N.
1
C5H5N). H NMR (C5D5N): δ 0.83, 0.97, 1.02, 1.08, 1.17 (3H
each, all s; 4-(CH3)2, 8-CH3, 10-CH3, 14-CH3), 1.75 (3H, s;
H-30), 2.85-2.93 (3H, m; H-13, -CH2COOH), 3.39 (1H, t, J )
8 Hz; H-3), 3.51 (1H, dt, J ) 4, 11 Hz; H-19), 3.68-3.84 (2H,
m; -NHCH2-), 4.71 (1H, s; H-29), 4.89 (1H, d, J ) 2 Hz; H-29),
8.05 (1H, br s; -CONH-). Anal. (C33H53NO4) C, H, N.
N-[3â-Hyd r oxy-20(29)-en -28-oyl]-8-a m in oocta n oic Acid
(13). Yield 82%; an off-white amorphous powder; mp 114-
N ′-[N -[3â-H yd r oxy-20(29)-e n -28-oyl]-11-a m in ou n d e -
ca n oyl]-L-leu cin e (20). Yield 91%; an off-white amorphous
25
116 °C; [R]D +5.1° (c ) 0.65, CHCl3). 1H NMR (CDCl3): δ
powder; [R]D -0.9° (c ) 2.87, CHCl3). 1H NMR (CDCl3): δ
25
0.75, 0.81, 0.93, 0.96 (6H) (3H each, except 0.96, all s; 4-(CH3)2,
8-CH3, 10-CH3, 14-CH3), 1.68 (3H, s; H-30), 2.35 (2H, t, J ) 8
Hz; -CH2COOH), 2.45 (1H, dt, J ) 3, 12 Hz; H-13), 3.09-
3.25 (4H, m; H-3, H-19, -CONHCH2-), 4.59 (1H, s; H-29), 4.73
(1H, d, J ) 2 Hz; H-29), 5.58 (1H, t, J ) 6 Hz; -CONH-).
Anal. (C38H63NO4) C, H, N.
0.75, 0.82, 0.92, 0.95, 0.96 (6H), 0.97 (3H each, except 0.96,
all s; 4-(CH3)2, 8-CH3, 10-CH3, 14-CH3, leucine moiety -(CH3)2),
1.68 (3H, s; H-30), 2.16-2.32 (2H, m; -CH2CONH-), 2.40 (1H,
dt, J ) 3, 12 Hz; H-13), 3.10 (1H, dt, J ) 4, 11 Hz; H-19),
3.16-3.30 (3H, m; H-19, -CONHCH2-), 4.59-4.66 (2H, m;
-NHCH(CH2-)CO-, H-29), 4.72 (1H, br s; H-29), 5.71 (1H, t,
J ) 6 Hz; -CONHCH2-), 6.02 (1H, d, J ) 8 Hz; -CONHCH-
(CH2-)CO-). Anal. (C47H80N2O5) C, H, N.
N-[3â-H yd r oxy-20(29)-en -28-oyl]-11-a m in ou n d eca n o-
ic Acid (14). Yield 81%; an off-white amorphous powder; mp
25
1
102-104 °C; [R]D +8.5° (c ) 0.33, CHCl3). H NMR (CDCl3):
δ 0.75, 0.82, 0.93, 0.96 (6H) (3H each, except 0.96, all s;
4-(CH3)2, 8-CH3, 10-CH3, 14-CH3), 1.68 (3H, s; H-30), 2.35 (2H,
t, J ) 7 Hz; -CH2COOH), 2.45 (1H, dt, J ) 3, 12 Hz; H-13),
3.10-3.36 (4H, m; H-3, H-19, -CONHCH2-), 4.59 (1H, s;
H-29), 4.74 (1H, d, J ) 2 Hz; H-29), 5.59 (1H, t, J ) 6 Hz;
-CONH-). Anal. (C41H69NO4) C, H, N.
MAGI Assa y.20 Hela-CD4/CCR5/â-gal cells were plated on
a 48 well plates at 10 000 cells/well and cultured in DMEM
medium containing 500 µg/mL of G418 and 250 µg/mL of
hygromycin for 1 day. The cells were infected with virus
dilutions in the presence of various concentrations of anti-HIV
agents and incubated for 2 days at 37 °C. The infected cells
were stained blue by adding X-gal at 0.4 mg/mL to the culture.
The cells were fixed with a solution containing 1% formalde-
hyde and 0.2% glutaraldehyde before staining. The numbers
of infected cells were counted by using an AlphaImager (Alpha
Innotech). An anti-HIV compound that inhibits 50% of virus
infection is defined by its ability to reduce the number of
infected cells by 50%, for example, from 400 blue cells to 200
blue cells. The MAGI assay detects the presence of the HIV-1
transactivating protein, Tat, during the viral replication. It is
a fast and accurate assay for HIV-1 entry inhibitors such as
IC9564 and its derivatives.
Cell F u sion Assa y.11 The fusion assays were performed
by transfecting the monkey kidney cells (COS) with the
expression vector pSRHS that contains the HIV-1 NL4-3
envelope gene. The original HXB envelope in pSRHS was
replaced with the NL4-3 envelope. Electroporation was per-
formed to express the HIV-1 envelope on COS cells. Briefly,
COS cells (106) in culture medium were incubated with 2 µg
of the envelope expression vector on ice for 10 min. The
electroporation was performed using a gene pulser (BioRad,
Hercules, CA) with capacitance set at 950 µF and a voltage at
Meth yl N′-[N-[3â-Hyd r oxy-20(29)-en -28-oyl]-3-a m in o-
p r op a n oyl]-L-leu cin a te (15). Yield 83%; a white powder; mp
25
1
134-136 °C; [R]D +3.1° (c ) 0.45, CHCl3). H NMR (CDCl3):
δ 0.75, 0.82, 0.93 (6H), 0.96 (9H) (3H each, except 0.93, 0.96,
all s; 4-(CH3)2, 8-CH3, 10-CH3, 14-CH3, leucine moiety -(CH3)2),
1.68 (3H, s; H-30), 2.35-2.53 (3H, m; H-13, -CH2CONH-),
3.11 (1H, dt, J ) 4, 11 Hz; H-19), 3.17 (1H, dd, J ) 5, 11 Hz;
H-3), 3.49-3.57 (2H, m;-CONHCH2-), 3.74 (3H, s;-COOCH3),
4.59 (1H, s; H-29), 4.57-4.64 (1H, m; -NHCH(CH2-)CO-),
4.73 (1H, d, J ) 2 Hz; H-29), 5.91 (1H, d, J ) 8 Hz;
-CONHCH(CH2-)CO-), 6.42 (1H, t, J ) 6 Hz; -CONHC-
H2-). Anal. (C40H66N2O5) C, H, N.
Meth yl N′-[N-[3â-Hyd r oxy-20(29)-en -28-oyl]-8-a m in ooc-
ta n oyl]-L-leu cin a te (16). Yield 93%; an off-white amorphous
powder; mp 107-108 °C; [R]D25 +3.7° (c ) 1.0, CHCl3). 1H NMR
(CDCl3): δ 0.75, 0.82, 0.93 (6H), 0.96 (9H) (3H each, except
0.93, 0.96, all s; 4-(CH3)2, 8-CH3, 10-CH3, 14-CH3, leucine
moiety -(CH3)2), 1.68 (3H, s; H-30), 2.18-2.24 (2H, m; -CH2-
CONH-), 2.45 (1H, dt, J ) 3, 12 Hz; H-13), 3.11-3.33 (4H,
m; H-3, H-19, -CONHCH2-), 3.73 (3H, s; -COOCH3), 4.59
(1H, s; H-29), 4.65 (1H, dt, J ) 5, 8 Hz; -NHCH(CH2-)CO-),

Anti-AIDS Agent 49
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150 mA. The transfected COS cells were cultured for 1 day
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Ack n ow led gm en t . This investigation was sup-
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National Institute of Allergy and Infectious Diseases
awarded to K.-H.L. and C.-H.C., respectively.
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