Betulinic acid derivatives: A new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action

Article abstract of DOI:10.1021/jm950670t

A series of ω-undecanoic amides of lup-20(29)-en-28-oic acid derivatives were synthesized and evaluated for activity in CEM 4 and MT-4 cell cultures against human immunodeficiency virus type 1 (HIV-1) strain III(B)/LAI. The potent HIV inhibitors which emerged, compounds 5a, 16a, and 17b, were all derivatives of betulinic acid (3β-hydroxylup-20(29)-en-28-oic acid). No activity was found against HIV-2 strain ROD. Compound 5a showed no inhibition of HIV-1 reverse transcriptase activity with poly(C)·oligo(dG) as template/primer, nor did it inhibit HIV-1 protease. Additional mechanistic studies revealed that this class of compounds interfere with HIV-1 entry in the cells at a postbinding step.

Full text of DOI:10.1021/jm950670t

1056
J . Med. Chem. 1996, 39, 1056-1068
Betu lin ic Acid Der iva tives: A New Cla ss of Hu m a n Im m u n od eficien cy Vir u s
Typ e 1 Sp ecific In h ibitor s w ith a New Mod e of Action
Michel Evers, Christe`le Poujade, Franc¸oise Soler, Yves Ribeill, Claude J ames, Yves Lelie`vre,
J ean-Christophe Gueguen, Daniel Reisdorf, Isabelle Morize, Rudi Pauwels,† Erik De Clercq,† Yvette He´nin,
Anne Bousseau, J ean-Franc¸ois Mayaux, J ean-Bernard Le Pecq, and Norbert Dereu*
Rhoˆne-Poulenc Rorer S.A., Centre de Recherche de Vitry-Alfortville, De´partements de Chimie Pharmaceutique, Biologie, et
Biotechnologie, 13, quai J ules Guesde, B.P. 14, F-94403 Vitry sur Seine, Cedex, France, and Rega Institute for Medical
Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received September 11, 1995X
A series of ω-undecanoic amides of lup-20(29)-en-28-oic acid derivatives were synthesized and
evaluated for activity in CEM 4 and MT-4 cell cultures against human immunodeficiency virus
type 1 (HIV-1) strain IIIB/LAI. The potent HIV inhibitors which emerged, compounds 5a , 16a ,
and 17b, were all derivatives of betulinic acid (3â-hydroxylup-20(29)-en-28-oic acid). No activity
was found against HIV-2 strain ROD. Compound 5a showed no inhibition of HIV-1 reverse
transcriptase activity with poly(C)‚oligo(dG) as template/primer, nor did it inhibit HIV-1
protease. Additional mechanistic studies revealed that this class of compounds interfere with
HIV-1 entry in the cells at a postbinding step.
The only approved drugs for the treatment of human
immunodeficiency virus (HIV) infections are the nucleo-
side analogs 3′-azido-3′-deoxythymidine (AZT),1 2′,3′-
dideoxyinosine (ddI),2 and 2′,3′-dideoxycytidine (ddC).3
They act through the inhibition of the HIV reverse
transcriptase (RT) activity and/or by a mechanism of
oligonucleotide chain termination. This noncurative
treatment suffers from important limitations such as
toxic side effects and the emergence of drug-resistant
strains. Other approaches currently under clinical
investigations include allosteric HIV-1 RT inhibitors
represented by TIBO,4a HEPT,4b nevirapine,4c and vari-
ous others (for a review, see ref 5) and HIV protease
inhibitors such as Ro 31-89596 and A 80987,7 as well
as the TAT inhibitor Ro 24-74298which was recently
dropped from development. The rapid appearance of
HIV-1 resistance to allosteric RT inhibitors9 (for a
commentary, see ref 9b) and the poor oral bioavailability
of the first-generation HIV protease inhibitors hamper
the development of these drugs. Drugs with a new
mode of action are urgently needed to overcome the
limitations of the current approaches.
Ch em istr y
The starting materials for the synthesis of 5a ,b are
the corresponding 3â-acetoxylup-20(29)-en-28-oic acid10
(2a ) and 3R-acetoxylup-20(29)-en-28-oic acid (2b) ob-
tained respectively from 3â-betulinic acid and 3R-
betulinic acid11 (Scheme 1). Treatment with oxalyl
chloride afforded acid chlorides 3a ,12b. Condensation
of methyl 11-aminoundecanoate13 with 3a ,b followed by
saponification of the resulting esters 4a ,b gave the two
stereoisomers 5a (3â-OH) and 5b (3R-OH). Compounds
5e,f were obtained in a similar way using methyl 11-
hydroxyundecanoate14 and methyl 11-(methylamino)-
undecanoate,15 respectively.
Our discovery that N-[3â-hydroxylup-20(29)-en-28-
oyl]glycine (betulinylglycine, 1b) displays a weak inhibi-
tion of the HIV-1 protease (data not shown) led us to
synthesize analogs of 1b in order to improve its activity
against the viral protease. No significant improvement
of the antiprotease activity was achieved. However,
N-[3â-hydroxylup-20(29)-en-28-oyl]-11-aminoundecano-
ic acid ((betulinylamino)undecanoic acid, 5a ), although
devoid of any activity against HIV-1 protease, was found
to inhibit the cytopathogenicity of HIV-1 in CEM 4 cells
at a concentration of 300 nM. From this lead, we
started a program to optimize the anti-HIV-1 activity
(in cell culture) by systematic alterations of the back-
bone of betulinic acid as well as the chain attached to
carbon 28. This report deals with variations in the
triterpene moiety.
Betulonic acid 2c16 was directly converted into its acid
chloride 3c using oxalyl chloride in CH2Cl2 at room
temperature. Compound 3c led to 5c by the same
pathway as described for 5a ,b. The synthesis of methyl
ether 5d was achieved using 2d as starting material.
The synthesis of 2d is described in the Experimental
Section. Saponification of 4c followed by Wolf-Kishner
reduction in a mixture of n-BuOH/ethylene glycol (1:5)
at reflux led to 5g in 16% yield. Reductive amination
of 4c (NH2OH‚HCl/NaBH3CN-TiCl3) followed by sa-
ponification gave the 3â amino derivative 5g.
The two-step tosylation-elimination reaction of betu-
linic acid 2a led to the 2,3-dehydro acid 6 (Scheme 2).
The latter was used as starting material for the syn-
thesis of 7b as described above for molecule 5c.
The synthesis of 9b and 10b is outlined in Scheme 3.
† Rega Institute for Medical Research.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1996.
The common starting acid 817 was first treated with
0022-2623/96/1839-1056$12.00/0 © 1996 American Chemical Society

Betulinic AcidssInhibitors with New Mode of Action
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1057
Sch em e 1
Sch em e 2
oxalyl chloride, and the crude acid chloride was reacted
with methyl 11-aminoundecanoate to yield the ester 9a .
Saponification of 9a gave 9b. Further reduction of the
ketone 9a by NaBH3CN led, after saponification, to 10b.
Scheme 4 describes the palladium-catalyzed hydro-
genation of the isopropylidene moiety in 4a . The ester
11a was further reacted with aqueous NaOH to afford
11b in 74% yield.
The methyl keto derivative 12 was prepared by
ozonolysis of 2a according to Vistrcil18 and transformed
as described for 2a into the undecylic acid derivative
13b via ester 13a . Haloform degradation (NaOH/Br2
at 5 °C) of the latter afforded the diacid 13c (Scheme
5).
The 30-bromo compound 14, a key intermediate for
the synthesis of isopropylidene-substituted compounds
depicted in Scheme 6, was obtained in quantitative yield
by vinylic bromination of 4a using N-bromosuccinimide
in CCl4. Various sulfur and nitrogen nucleophiles were
introduced via the Trost-Tsuji π-allylpalladium proce-
dure19 leading, after saponification of the intermediate
esters 15a -g, to 30-substituted acids 16a -g. Reaction
of silver acetate with bromo compound 14, in the
presence of a catalytic amount of Bu4N+Br3- in refluxing
toluene, gave diacetoxy ester 17a , which was trans-
formed into the hydroxy acid 17b on saponification
(Scheme 6).
The introduction of a free amino group and an
acetylamino group at position 30 was achieved via the
phthalimide intermediate 18 which could be obtained
from 14 and potassium phthalimide (Scheme 7). Treat-
ment of 18 with 1.5 equiv of hydrazine in methanol at
room temperature led to 30-amino diester 19a . Saponi-
fication of 19a gave 30-amino acid 19b in 36% yield,
whereas prior treatment with acetyl chloride followed
by alkaline cleavage of both ester functions led to 30-
acetylamino acid 19c.
Modifications of the carboxyl function are described
in Scheme 8. Acid chloride 3a was treated with NH3;
the crude amide obtained was then reacted with io-
dosobenzene diacetate leading to the intermediate iso-
cyanate 20.12 This isocyanate was the starting material
for the synthesis of molecules 21b and 22b. Acidic
hydrolysis of isocyanate 20 led to the corresponding
amino derivative which was then reacted with methyl
11-(chloroformyl)undecanoate20 to afford the correspond-
ing methyl ester 21a . Reaction of 20 with methyl 10-
aminodecanoate21 yielded the ureido compound 22a .

1058 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Evers et al.
Sch em e 3
Sch em e 4
Resu lts a n d Discu ssion
The anti-HIV-1 activity of reported triterpenes was
assessed in CEM 4 and MT-4 cells, and the results are
presented in Tables 1 and 2. The concentration that
produced 50% protection against the cytopathogenicity
(IC50) as stated was the mean for at least two experi-
ments. Structural variations in cycle A of the triterpene
highlight the importance of the 3â-hydroxy substituent.
Changing the hydroxy position from 3â (5a ) to 3R (5b)
led to a 10-fold drop in activity. The 3-keto derivative
5c was found of intermediate activity, whereas the
3-deoxy derivative 5g displayed no activity at all. This
points to a critical hydrogen bond interaction involving
the oxygen at the 3 position, which preferentially occurs
in the 3â position (Figure 1). The 3â-methoxy (5d ) and
the 3â-amino (5h ) derivatives were found inactive. The
inactivity of 5d might be explained by a steric hindrance
due to the presence of the methyl groups at the 4
position combined to the presence of the methyl sub-
stituent on the oxygen. The introduction of a second
hydroxyl at the 2 position (compound 10) led to a
complete loss of activity. Furthermore, the antiviral
activities of the diketone 9b and the 2,3-dehydro deriva-
tive 7b were rather low. Thus, almost all chemical
modifications in ring A led to considerable loss in
activity.
Saponification of esters 21a and 22a using aqueous
sodium hydroxide gave the acids 21b and 22b. As
outlined in Scheme 9, molecules 26b,c were obtained
from methyl betulinate (1c). Protection of the 3-hydroxy
function of the latter as its dimethyl tert-butylsilyl ether
followed by lithium aluminum hydride reduction gave
the betulinol derivative 24 in a quantitative yield.
Swern oxidation of 24 led to the aldehyde 25. Reductive
amination of the latter with methyl 11-aminounde-
canoate hydrochloride and sodium cyanoborohydride
afforded the dimethyl tert-butylsilyl-protected compound
26a . Deprotection of the 3-hydroxy group using stan-
dard ferric chloride in a 1:1 CH3CN-THF mixture
followed by saponification of the methyl ester led to 26c.
The isopropylidene could be substituted at position
Sch em e 5

Betulinic AcidssInhibitors with New Mode of Action
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1059
Sch em e 6
Sch em e 7
30 by different substituents. The antiviral activity of
the 30-(hydroxyethyl)thio (16a ), 30-[2-(diethylamino)-
ethyl]thio (16b), 30-(1-pyrrolidine) (16f), and 30-hydroxy
(17b) derivatives remained high but was not better than
that of the unsubstituted derivative 5a . This illustrates
a lack of steric requirements by the HIV-1 molecular
target for groups at position 30. However, an acidic
substituent such as 30-(carboxymethyl)thio (16c) was
clearly detrimental to potency. A similar drop of activity
was observed when a secondary nitrogen was directly
attached at position 30 (compounds 16e and 19c). The
combination of a free carboxyl and a secondary amine
as well as an unsubstituted amino moiety (compounds
16g and 19b) led to complete inactivation. An aromatic
substituent (16d ) also led to a significant drop in
activity. Hydrogenation of the double bond (compound
11b) reduced activity by 2-5-fold, and replacement of
the isopropylidene at position 19 by an acetyl or a
carboxyl group (compounds 13b,c) led to virtually
inactive molecules. In conclusion, the isopropylidene
seems important for optimal activity, probably due to
binding to a hydrophobic pocket. A certain lack of steric
hindrance accommodates a variety of substituents with-
out improvement of activity. However, unfavorable
interactions are observed for primary and secondary
amines as well as for the free carboxylic acid function.
Variations at the C28 carboxyl such as N-methylation
of the amide moiety in 5f, replacement of the amide by
an ester in 5e, and replacement of the carbonyl by a
methylene in 24b led to a complete loss of activity.
Figure 2 clearly indicates that the interactions of the
amide moiety with neighboring protons restrain the free
rotation of the carbonyl group, predetermining its
spatial orientation. The importance of a hydrogen-
donating NH group is higlighted by the fact that the
corresponding ester 5e is completly inactive. This was
further corroborated by the lack of biological activity of
the reversed amide 21b and the urea derivative 22b,
in which the NH group occupies a different spatial
position. Furthermore, 11-aminoundecanoic amides of

1060 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Evers et al.
Sch em e 8
Sch em e 9
other triterpenes, such as oleanic acid and hederagine,
proved to be devoid of any activity in the cellular screen
(data not shown). The dramatic loss of activity for most
of the modifications on the triterpene skeleton suggests
a stringent specificity for these compounds.
Exp er im en ta l Section
Melting points were recorded on a Kofler apparatus and are
uncorrected. Proton nuclear magnetic resonance spectra were
obtained on either a Brucker WM (250 MHz), a Brucker AC
(300 MHz), or a Brucker AM (400 MHz) spectrometer, and
proton chemical shifts are relative to tetramethylsilane as
internal standard. The following abbreviations are used to
denote signal patterns: s ) singlet, d ) doublet, t ) triplet, q
) quadruplet, br ) broad, m ) multiplet. The compounds
were analyzed by electronic impact (EI) on a Finnigan 3300
spectrometer at 70 eV, or by desorption chemical ionization
(DCI) on a Nermag R 10.10B spectrometer using NH3 as
reactant gas, or by liquid secondary ion mass spectrometry
(LISIMS) on a Kratos MS50 spectrometer. Where elementary
analyses are reported only by symbols of the elements in the
tables, results were within 0.4% of the theoretical values. All
reactions as well as column chromatography were monitored
routinely with the aid of thin layer chromatography with
precoated silica gel 60 F256 from Merck. The 3D structures
were built using the X-ray structure of 19R-lupeol methyl
ether23 as registered in the Cambridge Structural database.
No activity was found for the most potent inhibitors
of HIV-1, namely, 5a , 16a , and 17b, against the HIV-2
strain ROD. Furthermore, compound 5a showed no
inhibition in an in vitro HIV-1 RT assay using poly(C)‚-
olifo(dG) template/primer and did not inhibit HIV-1
protease22 at concentrations compatible with cellular
activity, suggesting a new mode of anti-HIV action for
these triterpenes. From their activity pattern following
addition at different times and their inhibitory effect
on HIV-1-induced syncytium formation (combined with
the lack of virus-cell binding inhibition), it was inferred
that these compounds interfere with virus entry in the
cells at a postbinding fusion step.22

Betulinic AcidssInhibitors with New Mode of Action
Ta ble 1. Anti-HIV-1 Activity in CEM and MT-4 Cells
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1061
anti-HIV-1 activity IC50 (µM)
no.
R1
R2
R3
X
mp, °C
formula
anal.a
CEM cells
MT-4 cells
5a
5b
5c
5d
5e
5f
5g
5h
16a
16b
16c
16d
16e
16f
16g
17b
19b
19c
21b
22b
26c
OH
H
H
OH
H
H
H
H
H
H
H
H
CONH
CONH
CONH
CONH
COO
115
182
90
100
122
154
85
145
180
145
194
146
214
146
220
174
220
160
130
225
182
C41H69NO4
C,H,N
C,H,N
C,bH,N
C,H,N
C,H,N
C,H,iN
C,H,N
C,cH,N
C,dH,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,jN
C,H,N
C,eH,N
C,H,kN
C,H,lN
C,fH,mN
C,gH,N
C,hH,nN
0.23
3.0
1.2
na
na
na
0.44
2.52
0.33
na
na
na
C
C
C
C
C
C
41H69NO4
41H67NO4
42H71NO4
41H68O5
42H71NO4
41H69NO3
dO
OMe
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CONMe
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
CONH
NHCO
NHCONH
CH2NH
na
na
nt
nt
NH2
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
C41H70N2O3
C43H73NO5S
C47H82N2O4S
C43H71NO6S
C47H72FNO4S
C43H74N2O5
SCH2CH2OH
SCH2CH2N(Et)2
SCH2COOH
SC6H4-p-F
NHCH2CH2OH
1-pyrrolidinyl
NHCH2COOH
OH
NH2
NHCOMe
H
H
H
0.29
1.0
na
2.0
5.75
1.0
na
0.20
na
1.25
na
0.27
0.63
1.61
1.37
2.0
0.42
nt
0.38
nt
0.26
na
C
45H76N2O4
C43H72N2O6
41H69NO5
C41H70N2O4
C
C
C
C
43H72N2O5
41H69NO4
40H68N2O4
na
na
na
nt
C41H71NO3
a
b
Elemental analyses were within 0.4% of theory unless otherwise noted. C: calcd, 77.19; found, 77.9. c C: calcd, 77.06; found, 73.4.
H: calcd, 11.04; found, 11.1. N: calcd, 4.38; found, 4.0. This sample was partially a sodium salt. C: calcd, 72.12; found, 71.1. e C:
d
calcd, 75.07; found, 73.4. f C: calcd, 76.95; found, 76.3. C: calcd, 74.95; found, 74.3. C: calcd, 78.66; found, variable results due to
g
h
incomplete combustion. H: calcd, 10.94; found, 11.3. j H: calcd, 10.80; found, 11.6. H: calcd, 10.77; found, 11.9. H: calcd, 10.41; found,
i
k
l
m
n
11.5. H: calcd, 10.87; found, 11.4. H: calcd, 11.43; found, 10.7. na: not active. nt: not tested.
Ta ble 2. Other Compounds
anti-HIV-1
activity IC50 (µM)
no.
mp,°C
formula
anal.a
CEM cells MT4 cells
7b
9b
82
90
C41H67NO3 C,H,N
1.8
3.0
na
1.5
12.0
50.0
2.6
nt
nt
0.58
nt
9.0
C
C
C
C
C
41H65NO5 C,H,N
41H69NO5 C,H,N
41H71NO4 C,H,cN
40H67NO5 C,bH,dN
39H65NO6 C,H,N
10b 102
11b 110-115
13b 150
13c 160
a
Elemental analyses were within 0.4% of theory unless other-
wise noted. C: calcd, 74.84; found, 74.3. c H: calcd, 11.5; found,
12.2. H: calcd, 10.52; found, 11.2.
b
d
After modifications of the structure to fit with the different
derivatives, energy minimization using Chem-X (Chemical
Design Ltd., Oxford) and/or Insight (Biosym Technologies Inc.,
San Diego) was performed.
Gen er a l P r oced u r e: Meth yl N-[3â-Hyd r oxylu p -20(29)-
en -28-oyl]-11-a m in ou n d eca n oa te (4a ). To a solution of 3â-
acetoxylup-20(29)-en-28-oyl chloride12 (3a ) (1.03 g, 2 mmol) in
CH2Cl2 (30 mL) at room temperature was added dropwise a
solution of methyl 11-aminoundecanoate hydrochloride (0.58
g, 2.3 mmol) in CH2Cl2 (30 mL), followed by triethylamine (0.62
mL, 4.45 mmol). The resulting solution was stirred for 16 h,
diluted with CH2Cl2 (50 mL), washed twice with water, dried
over magnesium sulfate, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
eluting with diisopropyl oxide to give 1.29 g (98.6%) of 4a as
a white meringue (Rf ) 0.31, eluent 15% ethyl acetate in
cyclohexane).
F igu r e 1. Three-dimensional structure of methyl 3â-hydroxy-
and methyl 3R-hydroxybetulinate and methyl betulonate.
(20 mL) was added aqueous NaOH (4 N, 4.5 mL). After
stirring for 24 h and concentrating the solution in vacuo, the
residue was suspended in distilled water (60 mL). The
suspension was treated with aqueous 5 N hydrochloric acid
(3.6 mL). After 20 min, the suspension was filtered, washed
with distilled water to pH 7, and dried to yield 1.2 g (98%) of
Gen er a l P r oced u r e: N-[3â-Hyd r oxylu p -20(29)-en -28-
oyl]-11-a m in ou n d eca n oic Acid (5a ). To a solution of meth-
yl N-[3â-hydroxylup-20(29)-en-28-oyl]-11-aminoundecanoate
(4a ) (1.28 g, 1.96 mmol) in a 1:1.5 mixture of CH3OH and THF
1
5a as a white solid: mp 115 °C; H-NMR (CDCl3, 400 MHz)
(ppm) 0.67 (br d, J ) 9 Hz, 1H, -H in 5), 0.76 (s, 3H, -CH3),

1062 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Evers et al.
oxolup-20(29)-en-28-oyl]-11-aminoundecanoate (4c) (600 mg,
0.92 mmol) led to a gummy solid which was extracted twice
with ethyl acetate (25 mL). The organic solution was washed
with distilled water, dried over sodium sulfate, filtered, and
concentrated in vacuo to yield 340 mg (58%) of 5c as a white
powder: mp 90 °C; IR (KBr, cm-1) ν 3400, 3200-2250, 3075,
2930, 2860, 1710 (νCO acid), 1640, 1525, 1460, 885; MS(EI) 637,
609, 594, 409, 189, 69 (base).
3â-Meth oxylu p -20(29)-en -28-oic Acid (2d ). To a solution
of betulinic acid (1a ) (0.8 g, 1.75 mmol) in dry THF (30 mL)
was added portionwise a 60% slurry of NaH in mineral oil (0.3
g, 7.5 mmol). The resulting mixture was stirred at room
temperature for 1 h and then heated at reflux for 1 h. After
cooling to 40 °C, CH3I (1.2 mL, 19 mmol) was added, and the
resulting solution was heated at reflux for 16 h. After cooling
to room temperature, an additional portion of NaH in oil (0.3
g) and CH3I (3 mL) was added and the mixture heated at reflux
for an additional 24 h. At this point, the reaction was
complete. Distilled water (10 mL) was added dropwise, and
the solvent was removed. The residue was then dissolved in
CH2Cl2 (100 mL), and the solution was washed with distilled
water (5 × 40 mL), dried over Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by column chroma-
tography on silica gel eluting with CH2Cl2 to give 0.82 g (97%)
of methyl 3â-methoxylup-20(29)-en-28-oate as a white solid
melting at 198 °C.
F igu r e 2. Molecular models of the interactions between the
A solution of methyl 3â-methoxylup-20(29)-en-28-oate (1 g,
2.06 mmol) and dry LiI (4 g) in collidine (160 mL) was heated
at reflux under nitrogen during 7 h. The collidine was
concentrated in vacuo, and the residue was treated with
distilled water (150 mL). The pH was adjusted to 2 using
aqueous HCl (5 N), and the product was extracted with ethyl
acetate (3 × 100 mL). The organic phase was washed with
distilled water, dried over Na2SO4, filtered, and concentrated
in vacuo to give a semisolid orange residue which was purified
by column chromatography eluting with CH2Cl2 to yield 800
mg of 2d (82%) as a white meringue: mp 300 °C.
Met h yl N-[3â-Met h oxylu p -20(29)-en -28-oyl]-11-a m i-
n ou n d eca n oa te (4d ). 3â-Methoxylup-20(29)-en-28-oyl chlo-
ride (3d ), prepared from 3â-methoxylup-20(29)-en-28-oic acid
(2d ) (900 mg, 1.91 mmol) and thionyl chloride (0.7 mL) in CH2-
Cl2, was treated with methyl 11-aminoundecanoate hydro-
chloride (600 mg, 2 mmol) in the presence of triethylamine
(0.67 mL) as described for 4a . The residue was purified by
column chromatography on silica gel using CH2Cl2 as eluent
to yield 1.3 g (100%) of 4d as a resin (Rf ) 0.49, 20% ethyl
acetate in cyclohexane).
28-amide moiety and neighboring protons.
0.82 (s, 3H, -CH3), 0.93 (s, 3H, -CH3), 0.98 (s, 6H, -CH3), 1.56
(t, J ) 11.5 Hz, 1H, CH in 18), 1.70 (s, 3H, -CH3 in 29), 2.36
(t, J ) 7.5 Hz, 2H, -CH2-COO-), 2.44 (dt, J ) 11.5, 3 Hz, 1H,
CH in 13), 3.14 (dt, J ) 11.5, 4 Hz, 1H, CH in 19), 3.10-3.25
and 3.31 (2m, 1H each, -CONH-CH2-), 3.20 (dd, J ) 11, 5 Hz,
1H, CH in 3), 4.59 and 4.74 (2br s, 1H each, dCH2), 5.60 (t, J
) 5.5 Hz, 1H, -CONH-); IR (KBr, cm-1) ν 3390, 3250-2250,
3075, 2925, 2855, 1710, 1640, 1515, 1465, 1450, 1385, 1045,
1035, 808; MS(EI) 639, 611, 596, 203, 189, 81 (base).
Meth yl N-[3r-Acetoxylu p-20(29)-en -28-oyl]-11-am in ou n -
d eca n oa t e (4b). A solution of 3R-acetoxylup-20(29)-en-28-
oic acid (2b)11 (437 mg, 0.97 mmol) in CHCl3 (14 mL) and
oxalyl chloride (0.184 mL, 2.10 mmol) was stirred at room
temperature for 18 h. The mixture was concentrated in vacuo,
cyclohexane (20 mL) was added to the residue, and the mixture
was evaporated under reduced pressure. This was repeated
twice to yield 453 mg of crude 3R-acetoxylup-20(29)-en-28-oyl
chloride (3b) which was reacted with methyl 11-aminounde-
canoate hydrochloride as described for 4a . The residue was
purified by column chromatography on silica gel eluting with
15% ethyl acetate in cyclohexane to give 810 mg (98.5%) of 4b
as a white meringue (Rf ) 0.25, eluent 15% ethyl acetate in
cyclohexane).
N-[3r-Hydr oxylu p-20(29)-en -28-oyl]-11-am in ou n decan o-
ic Acid (5b). To a solution of methyl N-[3R-acetoxylup-20-
(29)-en-28-oyl]-11-aminoundecanoate (4b) (400 mg, 0.575 mmol)
in a 1:1.5 mixture of CH3OH and THF (10.5 mL) was added
aqueous NaOH (4 N, 4.8 mL). After the solution was stirred
for 24 h and concentrated in vacuo, the residue was suspended
in distilled water (35 mL). The suspension was treated with
aqueous hydrochloric acid (4 N, 6.8 mL), and after 20 min,
the suspension was filtered, washed with distilled water to
pH 7, and dried to yield 290 mg (78%) of 5b as a white solid:
mp 182 °C; IR (KBr, cm-1) ν 3400, 3200-2250, 3075, 2940,
2860, 1710 (νCO acid), 1525, 1455, 1065, 885; MS(EI) 639 (base),
624, 611, 596, 203, 189.
N-[3â-Meth oxylu p-20(29)-en -28-oyl]-11-am in ou n decan o-
ic Acid (5d ). Following the procedure described for 5a ,
methyl N-[3â-methoxylup-20(29)-en-28-oyl]-11-aminounde-
canoate (4d ) (1.1g, 1.64 mmol) led to 950 mg (88.2%) of 5d as
a white powder: mp 100 °C; IR (KBr, cm-1) ν 3400, 3125-
2250, 3075, 2935, 2860, 2825, 1710 (νCO acid), 1640 (νCO amide),
1525, 1465, 1455, 1390, 1375, 885; MS(DCI) 654, 121, 119, 69
(base).
Meth yl 11-[[3â-Acetoxylu p -20(29)-en -28-oyl]oxy]u n d e-
ca n oa te (4e). Crude acid chloride12 3a , prepared from 3â-
acetoxylup-20(29)-en-28-oic acid (2a ) (920 mg, 1.8 mmol) and
oxalyl chloride (0.2 mL, 2.2 mmol), was treated with methyl
11-hydroxyundecanoate14 as described for 4a . The residue was
purified by column chromatography on silica gel using 1%
ethyl acetate in CH2Cl2 as eluent to yield 550 mg (44%) of 4e
as a colorless oil (Rf ) 0.35, 1% ethyl acetate in CH2Cl2).
11-[3â-H yd r oxylu p -20(29)-en -28-oyl]oxy]u n d eca n oic
Acid (5e). Following the procedure described for 5a , methyl
11-[[3â-acetoxylup-20(29)-en-28-oyl]oxy]undecanoate (4e) (500
mg, 0.72 mmol) led to 420 mg (91%) of 5e as a white solid:
mp 122 °C; IR (KBr, cm-1) ν 3430, 3075, 2940, 2855, 2750,
1725 (νCO acid), 1640, 1465, 1455, 1390, 1375, 1045, 1035, 885;
MS(DCI) 658 (base), 641; MS(EI) 623, 411, 203, 189 (base).
Meth yl N-[3â-Acetoxylu p -20(29)-en -28-oyl]-N-m eth yl-
11-a m in ou n d eca n oa te (4f). Following the procedure de-
scribed for 4a , crude acid chloride12 3a (1 g, 2 mmol) was
reacted with methyl 11-(methylamino)undecanoate15 (0.54 g,
2 mmol) and triethylamine (0.8 mL) to yield an oil (1.2 g). The
crude oil was purified by two subsequent column chromatog-
Meth yl N-[3-Oxolu p -20(29)-en -28-oyl]-11-a m in ou n d e-
ca n oa te (4c). 3-Oxolup-20(29)-en-28-oyl chloride (3c), pre-
pared from 3-oxolup-20(29)-en-28-oic acid16 (2c) (1g, 2.2 mmol)
and oxalyl chloride (0.290 mL, 3.3 mmol), was treated with
methyl 11-aminoundecanoate hydrochloride (553 mg, 2.2
mmol) as described for 4a . The residue was purified by two
subsequent chromatographies on silica gel using respectively
2% methanol in CH2Cl2 and 10% ethyl acetate in CH2Cl2 as
eluents to yield 600 mg (60%) of 4c as a white meringue (Rf )
0.77, eluent 2% methanol in CH2Cl2).
N-[3-Oxolu p-20(29)-en -28-oyl]-11-am in ou n decan oic Acid
(5c). Following the procedure described for 5a , methyl N-[3-

Betulinic AcidssInhibitors with New Mode of Action
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1063
raphies on silica gel eluting with 10% ethyl acetate in
cyclohexane followed by 1% ethyl acetate in CH2Cl2 for the
second column yielding 250 mg (18%) of 4f as a colorless oil
(Rf ) 0.5, eluent 10% ethyl acetate in cyclohexane).
N-[3â-Hyd r oxylu p -20(29)-en -28-oyl]-N-m eth yl-11-a m i-
n ou n d eca n oic Acid (5f). Following the procedure described
for 5a , methyl N-[3â-acetoxylup-20(29)-en-28-oyl]-N-methyl-
11-aminoundecanoate (4f) (230 mg, 0.32 mmol) led to 200 mg
(94.3%) of 5f as a white powder: mp 154 °C; IR (KBr, cm-1) ν
3430, 3070, 2930, 2855, 2750-2250, 1710 (νCO acid), 1630, 1610
(νCO amide), 1465, 1455, 1390, 1375, 1040, 880; MS(EI) 653
(base), 638, 625, 411, 189.
Meth yl N-[3â-Am in olu p -20(29)-en -28-oyl]-11-a m in ou n -
d eca n oa te (4g). A solution of methyl N-[3-oxolup-20(29)-en-
28-oyl]-11-aminoundecanoate (4c) (500 mg, 0.77 mmol) in
pyridine (10 mL) and hydroxylamine hydrochloride (110 mg,
1,54 mmol) was heated for 45 min at 112 °C. After cooling to
room temperature, distilled water (125 mL) was added and
the residue extracted with ethyl acetate (4 × 35 mL). The
combined organic layers were washed with distilled water (3
× 35 mL), dried over magnesium sulfate, filtered, and evapo-
rated under reduced pressure. The residue was purified by
column chromatography on silica gel eluting with 20% ethyl
acetate in cyclohexane to yield to 200 mg (39%) of the
corresponding oxime.
A solution of the crude oxime (190 mg, 0.28 mmol) and
ammonium acetate (216 mg, 4 mmol) in CH3OH (25 mL) was
treated with solid sodium cyanoborohydride (250 mg, 4 mmol).
The medium was cooled to 10 °C, 15% aqueous titanium
trichloride (0.86 mL, 0.84 mmol) was added dropwise over 5
min, and the mixture was left at room temprature for 12 h.
Aqueous sodium hydroxide was added until pH ) 10, followed
by addition of CH2Cl2 (180 mL). The organic layer was washed
to neutrality with distilled water, dried over magnesium
sulfate, filtered, and evaporated in vacuo to give 160 mg (88%)
of 4g as a white meringue which was used in the next step
without further purification.
N-[3â-Am in olu p -20(29)-en -28-oyl]-11-a m in ou n d eca n o-
ic Acid (5g). A solution of methyl N-[3â-aminolup-20(29)-
en-28-oyl]-N-methyl-11-aminoundecanoate (4g) (150 mg, 0.23
mmol) and aqueous sodium hydroxide (5 N, 0.05 mL) in C2H5-
OH (11 mL) was stirred at room temperature for 15 h. The
solution was evaporated under reduced pressure and the
residue triturated with distilled water (3 mL). The solid was
filtered, washed with distilled water (5 × 2 mL), dried, and
washed with ethyl acetate (2 × 2 mL) to yield 110 mg (73%)
of 5g as a white powder: mp 145 °C; IR (KBr, cm-1) ν 3400,
3100 to 2250, 3075, 2930, 2865, 1710 (νCO acid), 1640, 1525,
1465, 1455, 1385, 1375, 885; MS (LSIMS/glycerol) 639 (base),
622.
N-[Lu p-20(29)-en -28-oyl]-11-am in ou n decan oic Acid (5h ).
To a solution of N-[3-oxolup-20(29)-en-28-oyl]-11-aminounde-
canoic acid (5c) (480 mg, 0.75 mmol) in a mixture of n-butanol
(10 mL) and ethylene glycol (50 mL) was added hydrazine
hydrate (0.36 mL, 0.75 mmol). The mixture was heated at
130 °C for 3h and allowed to cool to room temperature.
Potassium hydroxide (3.4 g, 61 mmol) was added. n-Butanol
was distilled off; then the medium was heated at 210 °C for
3h. The medium was allowed to cool to room temperature;
distilled water (20 mL) was added under stirring followed by
acidification to pH ) 1 using aqueous hydrochloric acid (4 N,
10 mL). The solid obtained was filtered, dried, and purified
by column chromatography on silica gel using 35% ethyl
acetate in cyclohexane to yield crude 5g which was triturated
in a mixture of C2H5OH and distilled water to yield 123 mg of
5h (27%) as a white powder: mp 85 °C; IR (KBr, cm-1) ν 3390,
3200-2250, 3070, 2930, 2855, 1640 (νCO acid), 1540, 1470,
1455, 1390, 1375, 885; MS(DCI) 623 (base).
that precipitated was collected by filtration and used in the
next step without further purification.
Sodium acetate was added to a solution of this white solid
in DMF (360 mL), and the mixture was heated at 120 °C for
6 h. The solvent was evaporated in vacuo, and the creamy
residue was treated with water (200 mL) and extracted with
CH2Cl2 (3 × 100 mL). The organic layer was dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
eluting with 10% ethyl acetate in cyclohexane to yield 3.45 g
(71%) of 6: mp 225 °C.
Meth yl N-[Lu p a -2(3),20(29)-d ien -28-oyl]-11-a m in ou n -
d eca n oa te (7a ). Lupa-2(3),20(29)-dien-28-oyl chloride, pre-
pared from lupa-2(3),20(29)-dien-28-oic acid (6) (500 mg, 1.14
mmol) and oxalyl chloride (0.2 mL), was treated with 11-
aminoundecanoate hydrochloride (0.33 g, 1.31 mmol) and
triethylamine (0.38 mL) as described for 4a . The residue was
purified by column chromatography on silica gel using 2%
ethyl acetate in CH2Cl2 as eluent to yield 0.58 g (80%) of 7a
as a white gum (Rf ) 0.46, eluent 2% ethyl acetate in CH2-
Cl2).
N-[Lu p a -2(3),20(29)-d ien -28-oyl]-11-a m in ou n d eca n o-
ic Acid (7b). Following the procedure described for 5a ,
methyl N-[lupa-2(3),20(29)-dien-28-oyl]-11-aminoundecanoate
(7a ) (470 mg, 0.74 mmol) led to 460 mg of crude 7b which was
purified by column chromatography on silica gel eluting with
35% ethyl acetate in cyclohexane yielding 430 mg (89%) of 7b
as a white solid: mp 82 °C; IR (KBr, cm-1) ν 3420, 3125-2500,
3070, 2940, 2875, 1750 (νCO acid), 1650 (νCO amide), 1530, 1465,
1450, 1390, 1375, 890; MS(DCI) 622(base); MS(EI) 607, 594,
579, 393, 189 (base).
2,3-Dioxolu p -20(29)-en -28-oic Acid (8). Oxygen was
passed through a solution of 3-oxolup-20(29)-en-28-oic acid (2c)
(5.27 g, 11.6 mmol) and potassium tert-butylate (3.48 g, 31
mmol) in tert-butyl alcohol (248 mL) at room temperature
during 1 h. Then aqueous HCl (0.1 N, 31 mL) was added. After
stirring for 15 min, the insoluble material was filtered off and
the filtrate was concentrated in vacuo. The residue was
treated three times with cyclohexane (50 mL) and concentrated
in vacuo to dryness. The residue was purified by column
chromatography on silica gel eluting with 20% ethyl acetate
in cyclohexane to yield 3.3 g (61%) of 8 as a yellow meringue
(Rf ) 0.35, eluent 20% ethyl acetate in cyclohexane).
Meth yl N-[2,3-Dioxolu p -20(29)-en -28-oyl]-11-a m in ou n -
d eca n oa te (9a ). 2,3-Dioxolup-20(29)-en-28-oyl chloride, pre-
pared from 2,3-dioxolup-20(29)-en-28-oic acid (8) (2 g, 4.26
mmol) and oxalyl chloride (0.72 mL, 8.4 mmol), was treated
with 11-aminoundecanoate hydrochloride (1.16 g, 4.6 mmol)
and triethylamine (1.3 mL) as described for 4a . The residue
was purified by column chromatography on silica gel using
3% ethyl acetate in CH2Cl2 as eluent to yield 0.94 g (33.1%) of
9a as a white gum (Rf ) 0.74, eluent 10% ethyl acetate in CH2-
Cl2).
N-[2,3-Dioxolu p a -20(29)-en -28-oyl]-11-a m in ou n d eca n o-
ic Acid (9b). Following the procedure described for 5a ,
methyl N-[2,3-dioxolup-20(29)-en-28-oyl]-11-aminoundecanoate
(9a ) (470 mg, 0.7 mmol) led to 420 mg of crude 9b which was
purified by column chromatography on silica gel using 5%
methanol in chloroform yielding 234 mg (51%) of 9b as a white
meringue: mp 90 °C; IR (KBr, cm-1) ν 3455, 3325-2375, 3070,
2930, 2850, 1710 (νCO acid), 1650, 1500, 1455, 1380, 1235, 890;
MS(EI) 651, 636, 624, 189, 121 (base).
Met h yl N-[2â,3â-Dih yd r oxylu p -20(29)-en -28-oyl]-11-
a m in ou n d eca n oa te (10a ). To a solution of methyl N-[2,3-
dioxolup-20(29)-en-28-oyl]-11-aminoundecanoate (9a ) (400 mg,
0.6 mmol) in methanol (50 mL) were added NaBH3CN (280
mg, 4.45 mmol) and TiCl4 (3.8 mL, 4.47 mmol). The resulting
dark blue solution was stirred at room temperature for 48 h.
Water (100 mL) and ethyl acetate (150 mL) were added, and
the organic phase was washed with water, dried over magne-
sium sulfate, filtered, and concentrated in vacuo. The residue
was purified by column chromatography on silica gel eluting
with 33% ethyl acetate in CH2Cl2 yieding 130 mg (32.3%) of
10a as a white meringue (Rf ) 0.13, eluent cyclohexane-ethyl
acetate-dichloromethane, 7:2:1).
Lu p a -2(3),20(29)-d ien -28-oic Acid (6). To a solution of
betulinic acid1a (5 g, 10.9 mmol) in pyridine (50 mL) was added
p-toluenesulfonyl chloride (4.6 g, 24 mmol), and the yellow
solution was stirred for 24 h at room temperature. An
additional amount of p-toluensulfonyl chloride (2.3 g, 12 mmol)
was added. After additional stirring for 24 h at room tem-
perature, water (150 mL) was added. The white solid (7.33 g)

1064 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Evers et al.
N-[2â,3â-Dih yd r oxylu p -20(29)-en -28-oyl]-11-a m in ou n -
d eca n oic Acid (10b). Following the procedure described for
5a , methyl N-[2â,3â-dihydroxylup-20(29)-en-28-oyl]-11-ami-
noundecanoate (10a ) (120 mg, 0.179 mmol) led to 87 mg (74%)
of 10b as a white powder: mp 102 °C; IR(KBr, cm-1) ν 3415,
3150, 2250, 3075, 2930, 2860, 1710 (νCO acid), 1635 (νCO amide),
1520, 1460, 1390, 1375, 1055, 1045, 885; MS(DCI) 656 (base);
MS(EI) 641, 627 (base), 612, 427, 203, 121, 119.
2250, 2930, 2855, 1710 (νCO acid), 1640 (νCO amide), 1525, 1465,
1455, 1390, 1375, 1045, 1030; MS(EI) 643, 328, 610, 189 (base),
81, 69.
Meth yl N-[3â-Acetoxy-30-br om olu p -20(29)-en -28-oyl]-
11-a m in ou n d eca n oa te (14). A suspension of methyl N-[3â-
acetoxylup-20(29)-en-28-oyl]-11-aminoundecanoate (4a ) (10.4
g, 15 mmol) and N-bromosuccinimide (5.3 g, 30 mmol) in CCl4
(300 mL) was stirred for 72 h at room temperature. The solid
was filtered, and the filtrate was concentrated in vacuo to yield
11.7 g (100%) of 14 as a white meringue contaminated with
5-10% of the corresponding bromovinylic compounds (Rf )
0.32, eluent 20% ethyl acetate in cyclohexane). This was used
without further purification.
Meth yl N-(3â-Acetoxylu p a n -28-oyl)-11-a m in ou n d eca -
n oa te (11a ). A solution of methyl N-[3â-hydroxylup-20(29)-
en-28-oyl]-11-aminoundecanoate (4a ) (1 g, 1.44 mmol) in
methanol (50 mL) was hydrogenated at 1 atm and room
temperature over 0.1 g of Pd/C. After 2 h, the catalyst was
filtered off and the solution concentrated in vacuo. The residue
was purified by column chromatography on silica gel eluting
with 10% CH2Cl2 and 10% ethyl acetate in cyclohexane to yield
600 mg (59.7%) of 11a as a colorless syrup (Rf ) 0.51, eluent
20% ethyl acetate in cyclohexane).
Gen er a l P r oced u r e: Meth yl N-[3â-Acetoxy-30-[(2′-h y-
d r oxyeth yl)th io]lu p -20(29)-en -28-oyl]-11-a m in ou n d eca -
n oa te (15a ). To a solution of palladium acetate (180 mg, 0.8
mmol) and triphenylphosphine (840 mg, 3.2 mmol) in THF (25
mL) was added triethylamine (0.112 mL, 0.8 mmol). After
stirring at room temperature for 30 min, the yellow-orange
resulting suspension was added to a solution of methyl N-[3â-
a cet oxy-30-br om olu p-20(29)-en -28-oyl]-11-a m in ou n deca -
noate (14) (3.2 g, 4 mmol) and 2-mercaptoethanol (0.56 mL, 8
mmol) in THF (100 mL). After heating at reflux for 144 h,
the mixture was allowed to cool to room temperature, and CH2-
Cl2 (200 mL) was added. The organic phase was washed with
distilled water (3 × 50 mL), dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel using 30% ethyl acetate in
cyclohexane as eluent to yield 1.85 g (60%) of 15a as a white
meringue (Rf ) 0.68, eluent 40% ethyl acetate in cyclohexane).
N-[3â-H yd r oxy-30-[(2′-h yd r oxyet h yl)t h io]lu p -20(29)-
en -28-oyl]-11-am in ou n decan oic Acid (16a). Aqueous NaOH
(5 N, 23 mL) was added to a solution of methyl N-[3âb-acetoxy-
30-[(2′-hydroxyethyl)thio]lup-20(29)-en-28-oyl]-11-aminounde-
canoate (15a ) (1.80 g, 2.33 mmol) in a 1:1 mixture of CH3OH
and THF (140 mL). After stirring for 24 h, the mixture was
acidified to pH ) 2 by aqueous hydrochloric acid (4 N).
Distilled water (500 mL) was added. After stirring at room
temperature for 2 h, the solid was filtered, washed with
distilled water (6 × 30 mL), and recrystallized from a 3:7
mixture of distilled water and CH3OH (50 mL) to yield 1.30 g
(78%) of 16a as white crystals: mp 180 °C; IR (KBr, cm-1) ν
3390, 3125-2250, 3075, 2930, 2860, 1710 (νCO acid), 1630 (νCO
amide), 1535, 1470, 1450, 1390, 1375, 1040, 1030, 900; MS-
(DCI) 716 (base), 698, 640; MS(EI) 638, 203, 189, 44 (base).
Met h yl N-[3â-Acet oxy-30-[[(2,2-d iet h yla m in o)et h yl]-
t h io]lu p -20(29)-en -28-oyl]-11-a m in ou n d eca n oa t e (15b ).
Following the procedure described for 15a , methyl N-[3â-
a cet oxy-30-br om olu p-20(29)-en -28-oyl]-11-a m in ou n de-
canoate (14) (1.0 g, 1.3 mmol) and (2,2-diethylamino)ethane-
thiol (0.39 mL, 2.6 mmol) led to a residue which was purified
by column chromatography on silica gel using 5% CH3OH in
CH2Cl2 as eluent to yield 600 mg (60%) of 15b as a white
meringue (Rf ) 0.21, eluent 5% MeOH in CH2Cl2).
N-[3â-Hyd r oxy-30-[[(2,2-d ieth yla m in o)eth yl]th io]lu p -
20(29)-en -28-oyl]-11-a m in ou n d eca n oic Acid (16b). Aque-
ous NaOH (5 N, 3.6 mL) was added to a solution of methyl
N-[3â-acetoxy-30-[[(2,2-diethylamino)ethyl]thio]lup-20(29)-en-
28-oyl]-11-aminoundecanoate (15b) (600 mg, 0.72 mmol) in a
1:1.5 mixture of CH3OH and THF (30 mL). After stirring for
72 h at room temperature, the solution was concentrated in
vacuo. Distilled water (25 mL) was added to the residue; the
suspension was acidified to pH ) 2 by aqueous hydrochloric
acid (4 N) and extracted with CH2Cl2 (4 × 20 mL). The organic
layer was washed with distilled water (5 × 10 mL), dried over
magnesium sulfate, filtered, and concentrated under reduced
pressure. The residue was triturated with diisopropyl ether
(15 mL) to yield 350 mg of 16b (63%) as a white solid: mp
145 °C; IR (KBr, cm-1) ν 3415, 3125- 2250, 3075, 2930, 2860,
1720 (νCO acid), 1635 (νCO amide), 1525, 1465, 1455, 1390, 1375,
1045, 890; MS(FAB, p-nitrobenzyl alchohol) 771 (base), 638,
542.
N -(3â-H yd r oxylu p a n -28-oyl)-11-a m in ou n d e ca n oa t e
(11b). Following the procedure described for 5a , methyl N-(3â-
acetoxylupan-28-oyl)-11-aminoundecanoate (4a) (600 mg, 0.859
mmol) led to 410 mg (74%) of 11b as a white solid: mp 110-
115 °C; IR (KBr, cm-1) ν 3400, 3150-2125, 2930, 2860, 1710
(νCO acid), 1640 (νCO amide), 1520, 1465-1450, 1380, 1370,
1045; MS(EI) 641, 626 (base), 420.
3â-Acetoxy-30-n or -20-oxolu p a n -28-oic Acid (12).18 Ozo-
nized oxygen was passed in a solution of 3â-acetoxylup-20-
(29)-en-28-oic acid (2a ) (5 g, 10 mmol) and methanol (0.6 mL,
15 mmol) in CH2Cl2 (200 mL) and cooled to -60 °C until the
characteristic blue color of Ozone appeared. After stirring for
30 min at -60 °C, dimethyl sulfide (3.0 mL, 41 mmol) was
added and the resulting solution was allowed to slowly warm
to room temperature. After stirring for 12 h at room temper-
ature, the solution was concentrated in vacuo. The residue
was purified by column chromatography on silica gel eluting
with 25% ethyl acetate in cyclohexane to give 3.9 g of crude
12. Triturating with diisopropyl ether (25 mL) yielded 3.3 g
(66%) of 12 as a white solid: mp >250 °C.
Meth yl N-(3â-Acetoxy-30-n or -20-oxolu p a n -28-oyl)-11-
a m in ou n d eca n oa te (13a ). 3â-Acetoxy-30-nor-20-oxolupan-
28-oyl chloride, prepared from 3â-acetoxy-30-nor-20-oxolupan-
28-oic acid (12)18 (3.5 g, 6.96 mmol) and oxalyl chloride (1.5
mL), was treated with methyl 11-aminoundecanoate hydro-
chloride (1.94 g, 7.7 mmol) and triethylamine (2.2 mL) as
described for 4a . The residue was purified by column chro-
matography on silica gel using 30% ethyl acetate in cyclohex-
ane as eluent to yield 4.75 g (98%) of 13a as a white meringue
(Rf ) 0.28, eluent 5% MeOH in CH2Cl2).
N-(3â-Hydr oxy-30-n or -20-oxolu pan -28-oyl)-11-am in ou n -
d eca n oic Acid (13b). Following the procedure described for
5a , methyl N-(3â-acetoxy-30-nor-20-oxolupan-28-oyl)-11-ami-
noundecanoate (13a ) (700 mg, 1 mmol) led to 650 mg of crude
13b which was purified by column chromatography on silica
gel eluting with 50% ethyl acetate in cyclohexane to give an
oily residue. Triturating with a mixture of THF (3 mL) and
isopropyl oxide (16.5 mL) led to 500 mg (78%) of 13b as a white
solid: mp 150 °C; IR (KBr, cm-1) ν 3400, 3125, 2250, 2930,
2855, 1710 (νCO acid), 1630, 1640 (νCO amide), 1530, 1465, 1455,
1390, 1375, 1045; MS(EI) 641, 599, 582, 189, 81, 43 (base).
N-(19-Car boxy-3â-h ydr oxy-20,29,30-tr in or lu pan -28-oyl)-
11-a m in ou n d eca n oic Acid (13c). To a solution of methyl
N-(3â-a cet oxy-30-n or -20-oxolu pa n -28-oyl)-11-a m in ou n -
decanoate (13a ) (2.3 g, 3.3 mmol) in dioxane (50 mL) cooled
to 10 °C was added dropwise in 10 min a solution of sodium
hypobromide obtained by mixing sodium hydroxide (2.6 g, 64
mmol), bromine (0.7 mL, 13 mmol), dioxane (12 mL), and
distilled water (18 mL) at 5 °C. After stiring for 12 h at room
temperature, sodium sulfite (500 mg, 4.85 mmol) in distilled
water (5 mL) was added. The mixture was acidified to pH )
1 with aqueous hydrochloric acid (4 N) and diluted with
distilled water (50 mL). The precipitate was filtered, washed
successively with distilled water (50 mL), dioxane (2 mL), and
diethyl ether (10 mL), and then dried to yield 800 mg (38%) of
13c as a white solid: mp 160 °C; IR(KBr, cm-1) ν 3400, 3250-
Meth yl N-[3â-Acetoxy-30-[[(eth oxyca r bon yl)m eth yl]-
t h io]lu p -20(29)-en -28-oyl]-11-a m in ou n d eca n oa t e (15c).
Following the procedure described for 15a , methyl N-[3â-
a cet oxy-30-br om olu p-20(29)-en -28-oyl]-11-a m in ou n deca -

Betulinic AcidssInhibitors with New Mode of Action
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1065
noate (14) (1 g, 1.3 mmol) and ethyl 2-mercaptoacetate (0.23
mL, 2 mmol) led to a crude solid which was purified by column
chromatography on silica gel using 20% ethyl acetate in
cyclohexane as eluent to yield 580 mg (55%) of 15c as a
colorless gum (Rf ) 0.52, eluent 30% ethyl acetate in cyclo-
hexane).
N-[3â-Hyd r oxy-30-[(ca r boxym eth yl)th io]lu p -20(29)-en -
28-oyl]-11-a m in ou n d eca n oic Acid (16c). Following the
procedure described for 16a , methyl N-[3â-acetoxy-30-[[(ethyl-
oxycarbonyl)methyl]thio]lup-20(29)-en-28-oyl]-11-aminounde-
canoate (15c) (540 mg, 0.67 mmol) led to a solid which was
first triturated with CH3CN (10 mL) and recrystallized from
a 3:7 mixture of water and CH3OH (4 mL) to yield 350 mg
(88%) of 16c as white crystals: mp 194 °C; IR (KBr, cm-1) ν
3405, 3125-2250, 3075, 2930, 2860, 1710 (νCO acid), 1635 (νCO
amide), 1535, 1470, 1450, 1390, 1375, 1040, 1030, 900; MS-
(LSIMS/glycerol) 730 (base), 712, 638.
to yield 650 mg (65%) of 15f as a white solid (Rf ) 0.23, eluent
5% MeOH in CH2Cl2).
N-[3â-Hyd r oxy-30-p yr r olid in olu p -20(29)-en -28-oyl]-11-
a m in ou n d eca n oic Acid (16f). To a solution of methyl N-[3â-
acetoxy-30-pyrrolidinolup-20(29)-en-28-oyl]-11-aminoun-
decanoate (15f) (650 mg, 0.85 mmol) in a 1:1.5 mixture of CH3-
OH and THF (15 mL) was added aqueous NaOH (5 N, 3.4 mL).
After stirring for 12 h at room temperature, the medium was
concentrated under reduced pressure, the residue was sus-
pended in distilled water (10 mL), and the solid was filtered.
The residue was dissolved in a mixture of dioxane (20 mL)
and distilled water (5 mL), acidified to pH ) 4 with solid citric
acid, and extracted with CHCl3 (5 × 40 mL). The organic
layers were collected, washed with distilled water (3 x 20 mL),
dried over magnesium sulfate, filtered, and concentrated under
reduced pressure. The residue was purified by column chro-
matography on silica gel using CHCl3-CH3OH-20% ammonia
(24:6:1) as eluent. The resulting solid was triturated with CH3-
CN (10 mL) to yield 240 mg (40%) of 16f as a white solid: mp
146 °C; IR (KBr, cm-1) ν 3425, 3125-2250, 3075, 2930, 2860,
2795, 1715 (νCO acid), 1640 (νCO amide), 1520, 1465, 1455, 1390,
1375, 1045, 1035, 900; MS(DCI) 709 (base); MS(EI) 648, 410,
84 (base).
Meth yl N-[3â-Acetoxy-30-[[(m eth oxyca r bon yl)m eth yl]-
am in o]-20(29)-en -28-oyl]-11-am in ou n decan oate (15g). Fol-
lowing the procedure described for 15a , methyl N-[3â-acetoxy-
30-bromolup-20(29)-en-28-oyl]-11-aminoundecanoate (14) (1 g,
1.3 mmol) and methyl glycinate hydrochloride (490 mg, 3.9
mmol) yielded a residue which was purified by column chro-
matography on silica gel using 50% ethyl acetate in cyclohex-
ane as eluent to yield 180 mg (18%) of 15g as a white solid (Rf
) 0.41, eluent 5% MeOH in CH2Cl2).
N-[3â-Hyd r oxy-30-[[(h yd r oxyca r bon yl)m eth yl]a m in o]-
20(29)-en -28-oyl]-11-a m in ou n d eca n ic Acid (16g). To a
solution of methyl N-[3â-acetoxy-30-[[(methoxycarbonyl)-
methyl]amino]-20(29)-en-28-oyl]-11-aminoundecanoate (15g)
(650 mg, 0.85 mmol) in a 1:2 mixture of CH3OH and THF (11
mL) was added aqueous NaOH (1 N, 1.14 mL). After stirring
for 48 h at room temperature, the medium was diluted with
distilled water (15 mL) and then acidified to pH ) 4 with solid
citric acid. After stirring for 30 min at room temperature, the
solid was filtered and dissolved in dioxane (5 mL). To the
solution was added distilled water (45 mL). The solid obtained
was filtered to yield 100 mg (74%) of 16g as a white solid: mp
220 °C; IR (KBr, cm-1) ν 3415, 3125-2250, 3075, 2930, 2860,
1715 (νCO acid), 1630 (νCO amide), 1530, 1465, 1455, 1390, 1375,
1045, 910; MS(LSIMS/glycerol) 713 (base).
Meth yl N-[3â,30-Diacetoxylu p-20(29)-en -28-oyl]-11-am i-
n ou n d eca n oa te (17a ). A suspension of methyl N-[3â-ac-
etoxy-30-bromolup-20(29)-en-28-oyl]-11-aminoundecanoate (14)
(5.1 g, 6.6 mmol), silver acetate (1.65 g, 9.9 mmol), and
tetrabutylammonium bromide (250 mg) in toluene (130 mL)
was refluxed for 2 h. The solids were filtered and washed with
toluene (25 mL). The filtrate was concentrated under reduced
pressure to give 5.5 g (100%) of 17a as a glaze (Rf ) 0.15,
eluent 20% ethyl acetate in cyclohexane).
Meth yl N-[3â-Acetoxy-30-[(4′-flu or op h en yl)th io]lu p -20-
(29)-en -28-oyl]-11-a m in ou n d eca n oa te (15d ). Following the
procedure described for 15a , methyl N-[3â-acetoxy-30-bromo-
lup-20(29)-en-28-oyl]-11-aminoundecanoate (14) (1 g, 1.3 mmol)
and 4-fluorothiophenol (0.28 mL, 2.6 mmol) led to a residue
which was purified by column chromatography on silica gel
using CH2Cl2 as eluent to yield 700 mg (66%) of 15d as a white
glass (Rf ) 0.15, eluent 10% ethyl acetate in cyclohexane).
N-[3â-Hyd r oxy-30-[(4′-flu or op h en yl)th io]lu p -20(29)-en -
28-oyl]-11-a m in ou n d eca n ic Acid (16d ). Following the
procedure described for 16b, methyl N-[3â-acetoxy-30-[(4′-
fluorophenyl)thio]lup-20(29)-en-28-oyl]-11-aminoundeca-
noate (15d ) (700 mg, 0.85 mmol) led to a residue which was
recrystallized from CH3CN (10 mL) to yield 250 mg (38%) of
16d as a white solid: mp 146 °C; IR (KBr, cm-1) ν 3415, 3125-
2250, 3075, 2930, 2860, 1710 (νCO acid), 1635 (νCO amide), 1465,
1450, 1390, 1375, 1045, 1030, 890; MS(DCI) 766 (base).
Meth yl N-[3â-Acetoxy-30-[(2′-h yd r oxyeth yl)a m in o]lu p -
20(29)-en -28-oyl]-11-a m in ou n d eca n oa te (15e). Following
the procedure described for 15a , methyl N-[3â-acetoxy-30-
bromolup-20(29)-en-28-oyl]-11-aminoundecanoate (14) (1 g, 1.3
mmol) and ethanolamine (0.24 mL, 3.9 mmol) led to a residue
which was purified by column chromatography on silica gel
using 10% CH3OH in CH2Cl2 as eluent to yield 450 mg (45%)
of 15e as a white glass (Rf ) 0.4, eluent 10% MeOH in CH2-
Cl2).
N-[3â-Hyd r oxy-30-[(2′-h yd r oxyeth yl)a m in o]lu p -20(29)-
en -28-oyl]-11-a m in ou n d eca n oic Acid (16e). To a solution
of methyl N-[3â-acetoxy-30-[(2′-hydroxyethyl)amino]lup-20-
(29)-en-28-oyl]-11-aminoundecanoate (15d) (700 mg, 0.85 mmol)
in a 1:1.5 mixture of CH3OH and THF (15 mL) was added
aqueous NaOH (5 N, 2.4 mL). After stirring for 12 h at room
temperature, the medium was acidified to pH ) 4 with solid
citric acid, diluted with distilled water (100 mL), and extracted
with CHCl3 (4 × 30 mL). The organic layers were collected,
washed with distilled water (2 × 20 mL), dried over magne-
sium sulfate, filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatography
on silica gel using CHCl3-CH3OH-NH3 (24:6:1) as eluent.
Trituration of the solid obtained with CH3CN (10 mL) yielded
230 mg (56%) of 16e as a white solid: mp 214 C; 1H-NMR
(DMSO-d6, 400 MHz) (ppm) 0.62 (m, 1H, -H in 5), 0.64 (s, 3H,
-CH3), 0.76 (s, 3H, -CH3), 0.83 (s, 3H, -CH3), 0.86 (s, 3H, -CH3),
0.92 (s, 3H, -CH3), 1.53 (m, 1H, -H in 18), 2.13 (t, J ) 7.5 Hz,
2H, -CH2-COO-), 2.52 (m, 1H, -H in 13), 2.56 (t, J ) 6 Hz, 2H,
-NH-CH2-CH2-), 2.80-3.00 (m, 3H, 1H of -CONH-CH2-H in 3
and -H in 19), 3.08 (limit ab, J ) 16 Hz, 12, -CH2-NH-), 3.12
(m, 1H, the other H of -CONH-CH2-), 3.45 (t, J ) 6 Hz, 2H,
-CH2O-), 4.75 and 4.80 (2 br s, 1H each, dCH2), 7.52 (t, J )
5.5 Hz, 1H, -CONH-); IR (KBr, cm-1) ν 3410, 3125-2250, 3080,
2930, 2860, 1710 (νCO acid), 1630 (νCO amide), 1530, 1465, 1455,
1385, 1045, 895; MS(DCI) 699 (base); MS(EI) 668, 471, 410.
Meth yl N-[3â-Acetoxy-30-p yr r olid in olu p -20(29)-en -28-
oyl]-11-a m in ou n d eca n oa te (15f). Following the procedure
described for 15a , methyl N-[3â-acetoxy-30-bromolup-20(29)-
en-28-oyl]-11-aminoundecanoate (14) (1 g, 1.3 mmol) and
pyrrolidine (0.33 mL, 3.9 mmol) were stirred for 12 h at room
temperature. The residue was purified by column chroma-
tography on silica gel using 5% CH3OH in CH2Cl2 as eluent
N-[3â,30-Dih yd r oxylu p -20(29)-en -28-oyl]-11-a m in ou n -
d eca n oic Acid (17b). Following the procedure described for
5a , methyl N-[3â,30-diacetoxylup-20(29)-en-28-oyl]-11-ami-
noundecanoate (17a ) (650 mg, 0.86 mmol) led to 17b as a white
1
solid: mp 174 °C; H-NMR (CDCl3, 400 MHz) (ppm) 0.69 (br
d, J ) 9 Hz, 1H, -H in 5), 0.78 (s, 3H, -CH3), 0.83 (s, 3H, -CH3),
0.93 (s, 3H, -CH3), 0.96 (s, 3H, -CH3), 0.97 (s, 3H, -CH3), 1.67
(t, J ) 11.5 Hz, 1H, -H in 18), 2.37 (t, J ) 7.5 Hz, 2H, -CH2-
COO-), 2.43 (dt, J ) 11.5, 3 Hz, 1H, -H in 13), 3.03 (dt, J )
11.5, 4 Hz, 1H, -H in 19), 3.18 and 3.28 (2m, 1H each, -CONH-
CH2-), 3.20 (dd, J ) 11, 5 Hz, 1H, -H in 3), 4.13 (limit ab, 2H,
-CH2O-), 4.91 and 4.96 (2br s, 1H each, dCH2), 5.62 (t, J )
5.5 Hz, 1H, -CONH-); IR (KBr, cm-1) ν 3400, 3100-2250, 3075,
2925, 2850, 1710 (νCO acid), 1635 (νCO amide), 1520, 1465, 1450,
1385, 1370, 1045, 1025, 880; MS(EI) 655, 640, 637, 409, 187
(base).
Meth yl N-[3â-Acetoxy-30-p h th a lim id olu p -20(29)-en -28-
oyl]-11-a m in ou n d eca n oa te (18). To a solution of methyl
N-[3â-acetoxy-30-bromolup-20(29)-en-28-oyl]-11-aminounde-
canoate (14) (2 g, 2.58 mmol) in toluene (70 mL) at room

1066 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Evers et al.
temperature were added potassium phthalimide (0.56 g, 3
mmol) and 18-crown-6 ether (0.68 g, 2.58 mmol). The resulting
suspension was heated for 8 h at 85 °C and then allowed to
cool to room temperature. The insoluble material was filtered
off, and the filtrate was concentrated under reduced pressure.
The pink oil obtained was purified by column chromatography
on silica gel using 30% ethyl acetate in cyclohexane as eluent
to yield 1.60 g (73%) of 18 as a white meringue (Rf ) 0.8, eluent
5% MeOH in CH2Cl2).
Meth yl N-[3â-Acetoxy-30-a m in olu p -20(29)-en -28-oyl]-
11-a m in ou n d eca n oa te (19a ). A solution of methyl N-[3â-
acetoxy-30-phthalimidolup-20(29)-en-28-oyl]-11-aminounde-
canoate (18) (1.6 g, 2.06 mmol) and hydrazine hydrate (98%,
0.12 mL, 2.5 mmol) in methanol (50 mL) was stirred for 48 h
at room temperature, acidified under stirring to pH ) 2 with
etheral hydrochloric acid (13 N), and then evaporated under
reduced pressure. The residue was purified by column chro-
matography on silica gel eluting with 10% methanol in CH2-
Cl2 to give 700 mg (47%) of 19a as a white solid directly used
without further purification in the next step.
N-[3â-Hyd r oxy-30-a m in olu p -20(29)-en -28-oyl]-11-a m i-
n ou n d eca n oic Acid (19b). Aqueous NaOH (1 N, 1.7 mL)
was added to a solution of methyl N-[3â-acetoxy-30-aminolup-
20(29)-en-28-oyl]-11-aminoundecanoate (19a ) (300 mg, 0.42
mmol) in a 1:1.5 mixture of CH3OH and THF (20 mL). After
stirring for 12 h at room temperature and concentrating the
solution in vacuo, the residue was suspended in distilled water
(30 mL). The medium was acidified to pH ) 4 with solid citric
acid, and the solid obtained was filtered, washed with distilled
water (2 × 2 mL), dried, and then purified by column
chromatography on silica gel eluting with a mixture of CHCl3-
CH3OH-20% ammonia (24:6:1) to yield 100 mg (36%) of 19b
as a white solid: mp 220 °C; IR (KBr, cm-1) ν 3420, 3125-
2250, 3080, 2930, 2860, 1710 (νCO acid), 1640 (νCO amide), 1525,
1465, 1455, 1390, 1375, 1045, 1035, 880; MS(DCI) 655 (base);
MS(EI) 426, 409, 189, 187, 121, 119, 69 (base).
under reduced pressure. The residue was purified by column
chromatography on silica gel using 10% CH2Cl2 in diisopropyl
ether as eluent to yield 330 mg (36%) of 21a as colorless gum
(Rf ) 0.76, eluent 10% CH2Cl2 in diisopropyl ether).
11-[[3â-Acetoxy-28-n or lu p -20(29)-en -17-yl]ca r ba m oyl]-
u n d eca n oic Acid (21b). Aqueous NaOH (4 N, 3.3 mL) was
added to a solution of methyl 11-[[3â-acetoxy-28-norlup-20-
(29)-en-17-yl]carbamoyl]undecanoate (21a) (330 mg, 0.47 mmol)
in a 1:1.5 mixture of CH3OH and THF (5 mL). After stiring
for 12 h at room temperature, distilled water was added (22
mL), and the medium was acidified to pH ) 2 with aqueous
hydrochloric acid (4 N, 4.5 mL). The suspension was stirred
for 1 h at room temperature and extracted with CH2Cl2 (3 ×
15 mL). The organic layers were collected, washed with
distilled water (3 × 15 mL), dried over sodium sulfate, and
concentrated in vacuo. The solid was purified by column
chromatography on silica gel using 10% CH3OH in CH2Cl2 as
eluent to yield 130 mg (43%) of 21b as a white solid: mp 130
°C; IR (KBr, cm-1) ν 3450, 3075, 2930, 2855, 2700-2250, 1710
(νCO acid), 1645 (νCO amide), 1510, 1470, 1455, 1390, 1375,
1045, 885; MS(DCI) 640 (base); MS(EI) 410, 230, 212 (base).
Meth yl N-[3â-Acetoxy-28-n or lu p -20(29)-en -28-oyl]-11-
a m in ou n d eca n oa te (22a ). A solution of 3â-acetoxy-17â-
isocyanato-28-norlup-20(29)-ene (20)12 (500 mg, 1 mmol),
methyl 10-aminodecanoate hydrochloride21 (276 mg, 1.1 mmol),
and triethylamine (0.18 mL, 1.28 mmol) in CHCl3 (15 mL) was
stirred for 16 h at room temperature. Then CHCl3 (25 mL)
and aqueous hydrochloric acid (0.1 N, 25 mL) were added. The
mixture was stirred for an additional 30 min, and the aqueous
phase was extracted with CHCl3 (25 mL). The combined
organic phase was mixed, washed with distilled water (3 ×
25 mL), dried over magnesium sulfate, and evaporated under
reduced pressure to give a solid which was purified by column
chromatography eluting with 10% ethyl acetate in CH2Cl2 to
yield 430 mg (61%) of 22a as a white meringue (Rf ) 0.26,
eluent 10% ethyl acetate in CH2Cl2).
N-[3â-Hyd r oxy-30-(a cetyla m in o)lu p -20(29)-en -28-oyl]-
11-a m in ou n d eca n oic Acid (19c). Acetyl chloride (0.036 mL,
0.5 mmol) and triethylamine (0.07 mL, 0.5 mmol) in CH2Cl2
(1 mL) was added at room temperature to a solution of methyl
N-[3â-acetoxy-30-aminolup-20(29)-en-28-oyl]-11-aminounde-
canoate (19a ) (300 mg, 0.42 mmol) in CH2Cl2 (15 mL). The
resulting solution was stirred for 12 h , washed with distilled
water (3 × 10 mL), dried over magnesium sulfate, and
concentrated in vacuo. The resulting solid (300 mg, 95%) was
used without further purification in the next step.
Following the procedure described for 5a , methyl N-[3â-
acetoxy-30-(acetylamino)lup-20(29)-en-28-oyl]-11-aminounde-
canoate (300 mg, 0.43 mmol) led to 150 mg (53%) of 19c as a
white solid: mp 160 °C; IR (KBr, cm-1) ν 3400, 3135-2250,
3080, 2930, 2860, 1715 (νCO acid), 1630 (νCO amide), 1530, 1465,
1450, 1390, 1375, 1045, 1030, 880; MS(DCI) 697 (base).
Meth yl 11-[[3â-Acetoxy-28-n or lu p -20(29)-en -17-yl]ca r -
ba m oyl]u n d eca n oa te (21a ). Distilled water (10 mL) and
hydrochloric acid in diethyl ether solution (5 N, 40 mL) were
added to a solution of 3â-acetoxy-17â-isocyanato-28-norlup-
20(29)-ene (20)12 (1.89 g, 3.8 mmol) in CH2Cl2 (47 mL). The
biphasic mixture was stirred for 24 h at room temperature
and then concentrated under reduced pressure. The resulting
solid (2.1 g) was purified by two subsequent column chro-
matographies on silica gel using CH2Cl2 and 50% methanol
in CH2Cl2 as eluent to yield 1.60 g (85%) of crude 3â-acetoxy-
17â-amino-28-norlup-20(29)-ene as a white solid (Rf ) 0.53,
eluent 10% methanol in CH2Cl2) which was used without
further purification in the next step.
N-[3â-Hydr oxy-28-n or lu p-20(29)-en -28-oyl]-11-am in ou n -
d eca n oic Acid (22b). Following the procedure described for
5a , methyl N-[3â-acetoxy-28-norlup-20(29)-en-28-oyl]-11-ami-
noundecanoate (22a ) (420 mg, 0.6 mmol) led to 345 mg (89%)
of 22b as a white powder: mp 225 °C; IR (KBr, cm-1) ν 3410,
3075, 2925, 2855, 2750-2250, 1715 (νCO acid), 1640, 1625 (νCO
urea), 1555, 1465, 1455, 1390, 1375, 1040, 885; MS(DCI) 641
(base); MS(EI) 410, 189, 187, 81, 69 (base).
Meth yl 3â-[(Dim eth yl-ter t-bu tylsilyl)oxy]lu p -20(29)-en -
28-oa te (23). To a solution of methyl betulinate (1c) (6.5 g,
13.8 mmol) in DMF (165 mL) were added imidazole (2.82 g,
41.4 mmol) and tert-butyldimethylsilyl chloride (4.25 g, 27.6
mmol). After 35 min at room temperature, the suspension
turned to a yellowish solution. After an additional 25 min of
stirring, a white solid appeared. The resulting suspension was
stirred for 40 h at room temperature, and distilled water (1500
mL) was added. The white solid was filtered, washed with
distilled water (5 × 250 mL), and dried over KOH in vacuo to
yield 8.1 g (100%) of methyl 3â-[(dimethyl-tert-butylsilyl)oxy]-
lup-20(29)-en-28-oate (23) as a white solid: mp 190 °C.
3â-[(Dim eth yl-ter t-bu tylsilyl)oxy]lu p-20(29)-en -28-ol (24).
To a solution of methyl 3â-[(dimethyl-tert-butylsilyl)oxy]lup-
20(29)-en-28-oate (23) (8.4 g, 14.5 mmol) in THF (46 mL) was
added lithium aluminum hydride (1.65 g, 42.9 mmol) portion-
wise in 45 min. The resulting mixture was stirred at room
temperature for 16 h. At this point, the reaction was complete
and water (1.9 mL) was added under stirring. Then aqueous
sodium hydroxide (5 N 1.4 mL) and distilled water (6.4 mL)
were added. The resulting suspension was stirred at room
temperature for 1 h and filtered. The solid was washed with
CH2Cl2 (6 × 50 mL). The organic layers were collected, dried
over magnesium sulfate, and concentrated in vacuo to give 8.0
g (100%) of 24 as a white meringue (Rf ) 0.43, eluent 20%
ethyl acetate in cyclohexane).
A solution of this 3â-acetoxy-17â-amino-28-norlup-20(29)-
ene in THF (10 mL) and a solution of methyl (chloroformyl)-
undecanoate,20 prepared from 11-(methoxycarbonyl)undecanoic
acid (526 mg, 2 mmol) and thionyl chloride in CHCl3 (20 mL),
were stirred at room temperature, and triethylamine (0.56 mL,
4 mmol) was added dropwise within 10 min. After stirring
for 12 h at room temperature, CH3OH (5 mL) was added,
stirring was maintained for 1 h, and distilled water (40 mL)
was added. The mixture was extracted with CH2Cl2 (2 × 50
mL). The organic layers were collected, washed with distilled
water (3 × 25 mL), dried over sodium sulfate, and evaporated
3â-[(Dim eth yl-ter t-bu tylsilyl)oxy]lu p-20(29)-en -28-al (25).
A solution of DMSO (0.34 mL, 4.4 mmol) in CH2Cl2 (5 mL)
was added dropwise to a -60 °C cooled solution of oxalyl
chloride (0.2 mL, 2.2 mmol) in CH2Cl2 (5 mL). After 5 min of
stirring at -60 °C, a solution of 3â-[(dimethyl-tert-terbutyl-
silyl)oxy]lup-20(29)-en-28-ol (24) (1.1 g, 2 mmol) in CH2Cl2 (10

Betulinic AcidssInhibitors with New Mode of Action
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1067
mL) was added dropwise. After additional stirring (15 min)
at -60 °C, triethylamine (1.4 mL, 10 mmol) was added. The
mixture was allowed to warm to room temperature and stirred
for 72 h; then, it was diluted with CH2Cl2 (50 mL). The organic
layer was washed with distilled water (4 × 50 mL), dried over
magnesium sulfate, filtered, and concentrated in vacuo to give
1.1 g (100%) of 25 as an oil which was used without further
purification (Rf ) 0.36, eluent 10% ethyl acetate in cyclohex-
ane).
Meth yl 11-[[[3â-[(Dim eth yl-ter t-bu tylsilyl)oxy]lu p -20-
(29)-en -28-yl]m eth yl]a m in o]u n d eca n oa te (26a ). A solu-
tion of methyl 11-aminoundecanoate hydrochloride (5.43 g,
21.6 mmol) in MeOH (150 mL) was added dropwise to a
solution of 3â-[(dimethyl-tert-butylsilyl)oxy)lup-20(29)-en-28-
al (25) (2.0 g, 3.6 mmol) in CH2Cl2 (40 mL). The resulting
solution was heated for 60 h at 55 °C. The suspension was
cooled at room temperature, and solid sodium cyanoboro-
hydride (330 mg, 5.25 mmol) was added. After additional
stirring for 48 h at room temperature, distilled water was
added (100 mL) and the medium was extracted with CH2Cl2
(3 × 100 mL). The organic layers were collected, washed with
distilled water (3 × 50 mL), dried over magnesium sulfate,
and concentrated under reduced pressure. The residue (1.92
g) was purified by column chromatography on silica gel using
30% ethyl acetate in cyclohexane as eluent to yield 530 mg
(23%) of 26a as a white meringue (Rf ) 0.84, eluent 15% ethyl
acetate in cyclohexane).
CEM 4 cell suspension (125 µL of 5 × 104 cells/mL) was then
added, and cultures were incubated for 1 h at 37 °C (5% CO2).
Cells were infected with 100 µL of virus suspension (100-200
CCID50) and cultured for at least 5 days. Mock-infected
cultures were carried out in parallel to determine the cyto-
toxicity of the compounds. To assess cell viability, a solution
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (10 µL at 7 mg/mL in PBS) was added to 100 µL of cell
suspension. After 3-h incubation at 37 °C, most of the
supernatant was removed and the formazan precipitate was
dissolved in 100 µL of 0.04 M HCl in 2-propanol. The
absorbance at 540 nm was measured with a Biotek EL-311
microplate reader. The concentration that produced 50%
inhibition (IC50) was estimated as the mean for at least two
experiments.
An tivir a l Assa y in MT-4 Cells. Compounds were tested
according to the MTT method, as described by Pauwels et al.24
Ack n ow led gm en t. We thank Marc Vuilhorgne for
assistance in obtaining analytical data. Financial sup-
port was obtained from the Agence National de Recher-
ches sur le SIDA (ANRS).
Su p p or tin g In for m a tion Ava ila ble: NMR data of com-
pounds 5b-h , 7b, 9b, 10b, 11b, 13b,c, 16a -g, 19b,c, 21b,
22b, and 26c (7 pages). Ordering information can be found
on any current masthead page.
Meth yl 11-[[[3â-Hyd r oxylu p -20(29)-en -28-yl]m eth yl]-
a m in o]u n d eca n oa te (26b). Solid anhydrous ferric chloride
(215 mg, 1.32 mmol) was added to a solution of methyl 11-
[[[3â-[(dimethyl-tert-butylsilyl)oxy]lup-20(29)-en-28-ylmethyl]-
amino]undecanoate (26a ) (500 mg, 0.66 mmol) in a 1:1 mixture
of CH3CN and THF (20 mL), and the reaction mixture was
stirred at room temperature for 12 h. Distilled water (15 mL)
and CH2Cl2 (15 mL) were added. The aqueous layer was
extracted with CH2Cl2 (3 × 15 mL); the organic phase was
collected, washed with distilled water (3 × 50 mL), dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
oily residue was triturated with 15% ethyl acetate in cyclo-
hexane (5 mL), yielding 160 mg (38%) of 26b as a beige solid
(Rf ) 0.42, eluent 10% methanol in CH2Cl2).
11-[[[3â-Hyd r oxylu p -20(29)-en -28-yl]m eth yl]a m in o]u n -
d eca n oic Acid (26c). To a solution of methyl N-[[3â-
hydroxylup-20(29)-en-28-yl]methyl]-11-aminoundecanoate (26b)
(150 mg, 0.2 mmol) in a 1:1.5 mixture of CH3OH and THF
(3.30 mL) was added aqueous NaOH (4 N, 2.15 mL). After
stirring for 60 h at room temperature, distilled water (10 mL)
was added and the medium acidified to pH ) 4.3 using 4 N
aqueous hydrochloric acid. After stirring for 2 h at room
temperature, the medium was extracted using CH2Cl2 (3 ×
50 mL). The organic layers were collected, washed with
distilled water (3 × 50 mL), dried over magnesium sulfate,
filtered, and concentrated in vacuo. The residual solid was
triturated with CH2Cl2 (3 mL) to yield 56 mg (45%) of 26c as
beige crystals: mp 182 °C; IR (KBr, cm-1) ν 3400, 3075, 2935,
2860, 2750-2250, 1710 (νCO acid), 1640 (νCO amide), 1525,
1455, 1390, 1375, 1065, 880; MS(EI) 624, 551, 523, 214 (base).
Biologica l Meth od s. An tivir a l Assa y in CEM 4 Cells.
Cells: The CEM 4, a subclone enriched in CD4 receptors, was
obtained from the CEM T-lymphoblastoid tumor cell line. This
was originally isolated from a child with acute lymphoblastic
leukemia. Cells were grown at 37 °C in a CO2 incubator (5%)
in RPMI 1640 medium, supplemented with 10% heat-inacti-
vated fetal calf serum, penicillin (100 IU/mL), glutamine (100
µg/mL), streptomycin (100 µg/mL) and polybrene (Sigma; 2 µg/
mL).
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