Anti-AIDS agents. Part 36: 17-carboxylated steroids as potential anti-HIV agents

Article abstract of DOI:10.1016/S0968-0896(99)00169-8

In our search for novel anti-HIV agents, seven 17-carboxylated steroid derivatives were synthesized and evaluated as potential anti-HIV agents. Compound 13 exhibited potent anti-HIV activity in acutely infected H9 lymphocytes with EC₅₀ and therapeutic index values of 0.8 μM and 300, respectively.

Full text of DOI:10.1016/S0968-0896(99)00169-8

Bioorganic & Medicinal Chemistry 7 (1999) 1907±1911
Anti-AIDS Agents. Part 36:1 17-Carboxylated Steroids as
Potential Anti-HIV Agents
Peng Xia, a,y Zheng-Yu Yang, b Yi Xia, b Yun-Qing Zheng, a L. Mark Cosentino c
and Kuo-Hsiung Lee b*
aDepartment of Organic Chemistry, School of Pharmacy, Shanghai Medical University, Shanghai 200032, People's Republic of China
bNatural Products Laboratory, Division of Medicinal Chemistry and Natural Products, School of Pharmacy,
University of North Carolina, Chapel Hill, NC 27599, USA
cBiotech Research Laboratories, 217 Perry Parkway, Gaithersburg, MD 20877, USA
Received 18 January 1999; accepted 22 March 1999
AbstractÐIn our search for novel anti-HIV agents, seven 17-carboxylated steroid derivatives were synthesized and evaluated as
potential anti-HIV agents. Compound 13 exhibited potent anti-HIV activity in acutely infected H9 lymphocytes with EC50 and
therapeutic index values of 0.8 mM and 300, respectively. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
replication activity in H9 lymphocyte cells with an EC50
value of 3 mg/mL. In addition, some sulfated sterols 2±58
obtained from marine invertebrates showed EC50 values
of 3±13 mM against HIV-1 and 2±8 mM against HIV-2.
Cosalane 6 and its analogue 79 displayed EC50 values of
HIV infection leads to the disease called acquired
immunode®ciency syndrome (AIDS), which has been a
life-threatening health problem since 1981.2 The magni-
tude of the AIDS pandemic and the need for more
e€ective therapy have prompted much exploration of
new potent anti-HIV agents. Many strategies, including
FDA approved anti-HIV reverse transcriptase (RT)
and protease agents, have been investigated to prevent
and/or control the spread of virus infection.3 Current
therapy can e€ectively control plasma viremia, but the
virus is suppressed rather than eradicated in HIV-infec-
ted individuals.4±6 To circumvent the existing thera-
peutic diculties, the discovery of new structures with
signi®cant bioactivity is meaningful, not only to obtain
more active agents, but also to ®nd new molecular tar-
gets for drug intervention.
5.1 and 0.55 mM, respectively, as inhibitors of HIV-1RF
.
More interestingly, a number of structurally more sim-
ple synthetic keto/epoxy steroids (8) have also been
reported to be e€ective HIV-1 Tat inhibitors, with IC50
values at micromolar concentrations and therapeutic
index values of 1.6±6.5.10
During routine anti-HIV screening, we recently dis-
covered that some 17-carboxylated steroid derivatives
from our compound library show anti-HIV activity. We
report herein on the synthesis and anti-HIV activity of
this structurally unique class of anti-HIV agents.
Chemistry
Steroids show a broad spectrum of biological activities
and numerous steroidal drugs are widely used in the
clinic. However, the anti-HIV activity of steroids is still
a new research area. Recent studies revealed that some
steroids (Fig. 1) are signi®cantly active in inhibiting
HIV infection; for example, suberosol (1),7 isolated
from Polyalthis suberosa, was found to show anti-HIV
The 17-carboxylated steroid derivatives 10, 11 and 12
were synthesized by literature methods11 as presented in
Scheme 1, but as shown in Experimental, the reported
tedious isolation of 11 was improved. After complet-
ing the Oppennauer oxidation of 10, if the excess
cyclohexanone and the cyclohexanol formed in the
reaction are removed exhaustively, the product 11 can
be easily isolated. In addition, we include spectral data
of 10±12 that are not given in the literature. The 17b-
carboxylic acid (12) was reacted either with oxalyl
chloride or thionyl chloride to give the corresponding
Key words: Anti-HIV; 17-carboxylated steroid.
* Corresponding author. Tel.: +1-919-962-0066; fax: +1-919-966-
y3893; e-mail: khlee@unc.edu
Second corresponding author.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00169-8

1908
P. Xia et al. / Bioorg. Med. Chem. 7 (1999) 1907±1911
Figure 1. Anti-HIV steroids.
acid chloride, which was further treated in situ with tert-
butylamine to a€ord the 17b-caboxyamide (13). The
successful chlorination with thionyl chloride depends on
appropriate reaction conditions including reaction
medium, concentration of thionyl chloride and reaction
temperature (see Experimental). A reaction temperature
over 65ꢀC and a higher concentration of thionyl chlo-
ride will lead to destruction of the product.
cleavage of 3-keto-Á4-17b-carboxylic acid 12.12
Although the synthesis of enelactam 15 is known in the
literature,12 prior methods needed high temperature and
resulted in low yields. We found that the ring closure of
various A-nor-3,5-seco acids, including 14, could be
smoothly completed in high yield by treating of sub-
strate with ammonium acetate in acetic acid at re¯ux.
Compound 18 was synthesized previously by us.13 As
shown in Scheme 2, compound 12 was converted into
the ene-lactam (18) via chlorination of the 17b-carboxyl
Scheme 2 shows the synthetic route to 14±18. The A-
nor-3,5-seco acid 14 was easily prepared by oxidative
Scheme 1. (a) I2/Py; (b) CH3ONa/CH3OH; (c) Al(i-PrO)3, cyclohexanone/toluene; (d) KOH/CH3OH; (e) SOCl2/toluene; (f) (CH3)3CNH2/toluene.

P. Xia et al. / Bioorg. Med. Chem. 7 (1999) 1907±1911
1909
Scheme 2. (a) NaIO4, KMnO4, Na2CO3/t-BuOH; (b) CH3COONH4/CH3COOH; (c) SOCl2/toluene; (d) 3-tri¯uoromethyl-4-nitroaniline/toluene.
Table 1. Anti-HIV activity of 10±18 in acutely infected H9 lympho-
cytes
e€ects on the anti-HIV activity and 17-amide deriva-
tives seem to be bene®cial for increased anti-HIV activ-
ity and selectivity. Compound 10 was also examined, it
has the same 17-acid methyl ester group as compound
11, but di€erent structural features in the A-B rings (a
3b-hydroxy-5-ene moiety). Both compounds had similar
therapeutic index values, although compound 11
showed slightly greater anti-HIV activity with an EC50
value about twofold lower than that of 10. Opening the
A ring to give compound 14 abolished the anti-HIV
activity. Inserting a N atom at 4-position formed 3-oxo-
17b-carboxylated 4-azasteroids 15 and 18; these two 4-
aza compounds had similar therapeutic index values,
but were not comparable with compound 13. Thus,
modi®cation of the 17-position seemed to be more
meaningful than that of the A-B rings.
Compound
IC50 (mM)a
EC50 (mM)b
TIc
10
11
12
13
14
15
18
AZT
39.4
15.3
67.3
240
18.0
8.47
2.2
1.8
No suppression
0.8
No suppression
3.85
3.09
300
14.1
4.8
1875
3.67
1.55
41,667
0.045
a
Concentration that inhibits uninfected H9 cell growth by 50%.
Concentration that inhibits viral replication by 50%.
b
c
TI=therapeutic indec IC50/EC50
.
with thionyl chloride in toluene, amidation in situ with
3-tri¯uoromethyl-4-nitroaniline, oxidative cleavage of
ring A with NaIO4-KMnO4 and Na2CO3 in re¯uxing
tert-BuOH, and ring closure of the A-nor-3,5-seco acid
with ammonium acetate in acetic acid at high tempera-
ture. These improved procedures are easy to carry out
and produce high yields.
In summary, the discovery of the considerable anti-HIV
activity of steroid 3-ketone-4-ene 17b-amide (13) has
signi®cance in opening a new research area into a novel
structural class of anti-HIV agent. Therefore, we have
selected 13 as a promising, new lead compound for fur-
ther development of anti-HIV agents. The mechanism
and pharmacological action of this compound are cur-
rently under investigation.
Anti-HIV activity
Table 1 shows the anti-HIV activities of 10±15 and 18
with AZT included in the same experiment for compar-
ison. The results of the HIV growth inhibition assay
indicated that 3-ketone-4-ene 17-(N-tert-butyl) amide
(13) displayed potent anti-HIV activity in acutely infected
H9 lymphocytes with an EC50 value of 0.8 mM and a
good therapeutic index (TI) of 300. However, replacing
the 17-amide with a 17-carboxylic acid methyl ester gave
compound 11, which was 10-fold less active (EC50=
8.47 mM) than 13. Furthermore, compound 11 displayed
a greater than 160-fold lower TI value. In particular, a
hydrophilic carboxylic group at 17-position (12) abol-
ished the anti-HIV activity. Thus, in the 3-ketone 4-ene
system, the substituents at the 17-position have great
Experimental
Melting points were obtained in capillary tubes and are
uncorrected. The IR spectra were obtained in potassium
bromide discs on
a Perkin±Elmer 783 spectro-
1
photometer. The H NMR spectra were determined on
a JNM-FX-400 spectrometer with TMS as internal
reference. The MS spectra were recorded on an
HP5989A instrument. Elemental analyses (C, H, and N)
were performed by Shanghai Institute of Organic
Chemistry. Speci®c rotations were measured on a WZZ-
2 instrument. All reactions were monitored by TLC on
silica gel plate.

1910
P. Xia et al. / Bioorg. Med. Chem. 7 (1999) 1907±1911
Methyl 3ꢀ-Hydroxy-5-androstene-17ꢀ-carboxylate (10).
Iodine (43.33 g, 0.17 mol) was added portionwise over
30 min to a solution of pregnenolone 9 (50.00 g,
0.16 mol) in 134 mL of pyridine at 90ꢀC under stirring.
After re¯uxing 1.5 h, the reaction mixture was cooled to
room temperature. Filtration, washing with a pyridine
and petroleum ether, and drying in air a€orded 82.40 g
of crude pregnonolone 21-pyridinium iodide as a yel-
low-tan powder, mp 224±226ꢀC. (lit.11 mp 228±230ꢀC).
The crude pyridinium iodide (66.96 g, 0.13 mol) was
added portionwise over 30 min to a re¯uxing solution of
sodium methoxide (9.18 g, 0.17 mol) in 200 mL of
methanol. The dark solution was re¯uxed for another
1 h. After cooling to 45ꢀC, 200 mL of water was added
slowly under stirring followed by neutralization with
13 mL of 6 N HCl solution. Filtration, washing with 1:1
methanol:water, and drying gave 41.67 g of crude
methyl 3b-hydroxy-5-androstene-17b-carboxylate (10),
mp 170±174ꢀC (97.25% from pregnenolone 9). Crystal-
lization from EtOAc o€ered the analytical sample, mp
174±176ꢀC. MS m/z: 332 (M+), 313; 1H NMR (CDCl3):
d0.67 (s, 3H, C13-CH3), 1.01 (s, 3H, C10-CH3), 3.67 (s,
3H, -OCH3), 5.34±5.35 (m, 1H, 6-H)
temperature for 0.5 h. The reaction mixture was diluted
with EtOAc and washed with water. The organic layer
was dried over anhydrous Na2SO4 and the solvent was
evaporated to give N-tert-Butyl-3-oxoandrost-4-ene-
17b-carboxamide (13), 1.67 g; mp 198±202ꢀC. Recrys-
tallization from acetone gave an analytical sample 1.50 g
(89.34%); mp 218±219ꢀC. Anal. calcd for C24H37NO2:
C, 77.58; H, 10.04; N, 3.77. Found: C, 77.37; H, 10.26;
N, 3.61; MS: m/z 371 (M+), 271; IR (KBr): 3420, 2920,
1
1725, 1500 cm 1; H NMR (CDCl3): d 0.72 (s, 3H, C13-
CH3), 1.20 (s, 3H, C10-CH3), 5.10 (s, 1H, N-H), 5.70 (s,
1H, 4-H).
17ꢀ-Carboxy-5-oxo-A-nor-3,5-secoandrostan-3-oic acid
(14). 12 mL of sat aq sodium carbonate was added to a
solution of 3-oxoandrost-4-ene-17b-carboxylic acid (12)
(2.00 g, 6.36 mmol) in 40 mL of tert-butyl alcohol.
Under re¯uxing,
a solution of NaIO4 (11.52 g,
53.86 mmol) and KMnO4 (64.00 mg, 0.40 mmol) in
30 mL of water was added dropwise. The reaction mix-
ture was re¯uxed for 3 h then cooled to room tempera-
ture. After ®ltration and washing with water, the
combined ®ltrates were concentrated under reduced
pressure to remove most of tert-butyl alcohol. The aq
residue was cooled and acidi®ed with 6 N HCl to pH 2.
The product was extracted with CH2Cl2, washed with
water and dried over anhydrous Na2SO4. Filtration and
removal of solvent gave the crude product. Crystal-
lization from EtOAc gave 17b-carboxy-5-oxo-A-nor-
3,5-secoandrostan-3-oic acid (14) 1.50 g (70.42%), mp
190±193ꢀC (lit.12 mp 189±192ꢀC).
Methyl 3-Oxo-4-androstene-17ꢀ-carboxylate (11). A
solution of crude 10 from above reaction (5.00 g,
15.04 mmol) and 6.25 mL of cyclohexanone in 50 mL of
toluene was heated until 20 mL of distillate was
removed. After cooling to 70ꢀC, Al(i-PrO)3 (1.44 g,
7.05 mmol) was added and the mixture was re¯uxed for
4 h followed by reduced distillation in vacuum (20 mm
Hg, 170ꢀC) to exhaustively remove the solvent, the
excess cyclohexanone, and the cyclohexanol formed
from the reaction. After cooling to room temperature,
the residue solidi®ed when a little methanol was added.
Filtration and washing with acetone gave 3.51 g of
methyl 3-oxo-4-androstene-17b-carboxylate (11) as
white crystals (70.62%), mp 126±128ꢀC. MS m/z: 330
3-Oxo-4-aza-5-androstene-17ꢀ-carboxylic acid (15). To
a solution of 17b-carboxy-5-oxo-A-nor-3,5-secoandro-
stan-3-oic acid (14) (0.50 g, 1.50 mmol) in glacial acetic
acid (20 mL) was added ammonium acetate (1.15 g,
14.95 mmol). The reaction mixture was re¯uxed for 10 h
and cooled to room temperature. Distillation under
reduced pressure removed most of acetic acid. The resi-
due as poured into 100 mL of ice-water. Filtration,
washing with water and drying gave 3-oxo-4-aza-5-
androstene-17b-carboxylic acid (15) 0.385 g (81.6%),
mp >310ꢀC; Elemental analysis for C19H27NO3 was
1
(M+); H NMR (CDCl3): d 0.69 (s, 3H, C13-CH3), 1.17
(s, 3H, C10-CH3), 3.66 (s, 3H, -OCH3), 5.72 (s, 1H, 4-H).
3-Oxoandrost-4-ene-17ꢀ-carboxylic acid (12). 18 mL of
10% aq KOH was added to a solution of methyl 3-oxo-
4-androstene-17b-carboxylate (11) (6.91 g, 0.02 mol) in
56 mL of MeOH. The reaction mixture was re¯uxed for
5 h and poured into 300 mL of ice-water. Filtration and
washing with water gave, on drying, 6.63 g of 3-oxoan-
drost-4-ene-17b-carboxylic acid (12) (100%), mp 244±
250ꢀC. MS (e/z): 316 (M+); 1H NMR (CDCl3): d
0.77 (s, 3H, C13-CH3), 1.28 (s, 3H, C10-CH3), 5.73 (s,
1H, 4-H).
veri®ed; MS: m/z 317 (M+), 318 (M++1), 302, 274,
1
256; IR (KBr): 3210, 2918, 1730, 1680, 1650, 1445 cm
;
1H NMR (pyridine-d5): d 1.0 (s, 3H, C13-CH3), 1.12 (s,
3H, C10-CH3), 5.21 (m, 1H, 6-H).
N-(30-Tri¯uoromethyl-40-nitrophenyl)-3-oxoandrost-4-ene-
17ꢀ-carboxamide (16). Prepared as for 13 with the sub-
stitution of 3-tri¯uoromethyl-4-nitroaniline for tert-
butyl and stirred overnight at room temperature. After
dilution with EtOAc and washing with water, the
organic layer was dried over anhydrous Na2SO4.
Removal of the solvent and crystallization from acetone
N-tert-Butyl-3-oxoandrost-4-ene-17ꢀ-carboxamide (13).
3-Oxoandrost-4-ene-17b-carboxylic acid (12) (1.43 g,
4.52 mmol) was dissolved in 100 mL of dry toluene;
25 mL of toluene was distilled and pyridine (2.43 mL)
was added under cooling with an ice-water bath. After
gave
N-(30-tri¯uoromethyl-40-nitrophenyl)-3-oxoand-
rost-4-ene-17b-carboxamide (16) 0.57 g (71.50%), mp
226±228ꢀC; Elemental analysis for C27H31N2O4F3 was
veri®ed; MS: m/z 504 (M+); IR (KBr): 3300, 2940,
stirring
a few min, thionyl chloride (0.794 mL,
10.88 mmol) was added dropwise at 5ꢀC. The mixture
was stirred at the same temperature for 3 h. After
removing excess thionyl chloride by distillation under
reduced pressure at 60ꢀC; tert-butylamine (4.15 mL,
39.49 mmol) was added at 5ꢀC, then stirred at the same
1
1700, 1660, 1520 cm 1; H NMR (CDC13): d 0.80 (s,
3H, C13-CH3), 1.25 (s, 3H, C10-CH3), 5.75 (s, 1H, 4-H),
7.97 (d, J=8.76 Hz, 1H, Ar-H), 8.07 (s, 1H, Ar-H), 8.10
(s, 1H, N-H), 8.14 (d, J=8.76 Hz, 1H, Ar-H).

P. Xia et al. / Bioorg. Med. Chem. 7 (1999) 1907±1911
1911
17ꢀ-[(N-30-Tri¯uoromethyl-40-nitrophenyl)aminocarbonyl]-
5-oxo-A-nor-3,5-secoandrostan-3-oic acid (17). Prepared
from 16 in same manner as 14 from 12. Removal of the
solvent gave crude product (17) 0.52 g (94.70%); mp
198±220ꢀC. Recrystallization from EtOAc a€orded an
analytical sample, 0.33 g, mp 240±242ꢀC; Elemental
analysis for C26H31N2O6F3 was veri®ed; MS: m/z
524(M+), 506, 319, 273 (base peak); IR (KBr): 1725,
1700, 1685, 1545, 1525, 1320 cm 1; 1H NMR (CDCl3): d
0.82 (s, 3H, C13-CH3), 1.14 (s, 3H, C10-CH3), 7.62 (s,
1H, 17-NH), 7.94±8.00 (m, 3H, Ar-H).
4 days. Cell-free supernatants were collected on day 4
for use in our in-house p24 antigen ELISA assay. P24
antigen is a core protein of HIV and therefore is an
indirect measure of virus present in the supernatants.
Toxicity was determined by performing cell counts by a
Coulter Counter on the mock-infected H9 cells which
had either received culture medium (no toxicity) or test
sample or AZT.
Acknowledgements
N-(30-Tri¯uoromethyl-40-nitrophenyl)-30-oxo-4-azaandrost-
5-ene-17ꢀ-carboxamide (18). Prepared from 17 in same
procedure as 15 from 14. N-(30-Tri¯uoromethyl-40-
nitrophenyl)-30-oxo-4-azaandrost-5-ene-17b-carboxamide
(18) 0.95 g (65.5%); mp 267±269ꢀC; Elemental analysis
for C26H30N3O4F3 was veri®ed; MS: m/z 506 (M++1),
505 (M+); 490, 475, 300, 247; IR (KBr): 1670, 1650,
This investigation was supported by grants from the
Science Foundation of National Education Ministry of
P. R. China awarded to Peng Xia and by grant AI-
33066 from the National Institute of Allergies and
Infectious Diseases awarded to K. H. Lee.
References
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1540, 1520, 1340 cm 1; H NMR (pyridine-d5): d 1.08
(s, 3H, C13-CH3), 1.15 (s, 3H, C10-CH3), 2.75 (m, lH,
17-H), 5.20 (m, 1H, 6-H), 8.20±8.65 (m, 3H, Ar-H),
10.36 (s, lH, 4-NH), 11.20 (s, 1H, 17-CONH-).
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continuous culture with complete medium (RPMI 1640
with 10% fetal calf serum (FCS) supplemented with l-
glutamine at 5% CO2 and 37ꢀC. Aliquots of this cell
line were only used in experiments when in log-phase of
growth. Test samples were ®rst dissolved in dimethyl
sulfoxide (DMSO). The following were the ®nal drug
concentrations routinely used for screening: 100, 20, 4
and 0.8 m/mL, but for active agents, additional dilutions
were prepared for subsequent testing so that an accurate
EC50 value could be achieved. As the test samples were
being prepared, an aliquot of the T cell line, H9, was
infected with HIV-1 (IIIB isolate) while another aliquot
was mock-infected with complete medium. The mock-
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stock virus used for these studies typically had a TCID50
value of 104 Infectious Units/mL. The appropriate
amount of virus for a multiplicity of infection (m.o.i.)
between 0.1 and 0.01 Infectious Units/cell was added to
the ®rst aliquot of H9 cells. The other aliquot of H9
cells only received culture medium and then was incu-
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cells. After a 4 h incubation at 37ꢀC and 5% CO2, both
cell populations were washed three times with fresh
medium and then added to the appropriate wells of a
24-well-plate containing the various concentrations of
the test drug or culture medium (positive infected con-
trol/negative drug control). In addition, AZT was also
assayed during each experiment as a positive drug con-
trol. The plates were incubated at 37ꢀC and 5% CO2 for
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