Synthesis, Anti-HIV, and Cytotoxic Activity of New AZT Conjugates of Steroid Acids

Article abstract of DOI:10.1081/NCN-120026406

In an attempt to discover potent anti-HIV agents devoid of the serious toxicity of the current HIV-reverse transcriptase inhibitors, three steroid prodrugs of AZT have been synthesized and their anti-HIV profiles determined with CEM-SS cell line. Two of t

Full text of DOI:10.1081/NCN-120026406

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Synthesis, Anti-HIV, and Cytotoxic Activity of New AZT
Conjugates of Steroid Acids
a
b
b
a c
Zhengqing You , A. Moshin Mian , Ram P. Agarwal & Henry Joung Lee
a
College of Pharmacy and Pharmaceutical Sciences , Florida A & M University , Tallahassee,
Florida, USA
b
Department of Medicine , University of Miami School of Medicine , Miami, Florida, USA
c
College of Pharmacy and Pharmaceutical Sciences , Florida A & M University , Tallahassee,
FL, 32304, USA
Published online: 31 Aug 2006.
To cite this article: Zhengqing You , A. Moshin Mian , Ram P. Agarwal & Henry Joung Lee (2003) Synthesis, Anti-HIV, and
Cytotoxic Activity of New AZT Conjugates of Steroid Acids, Nucleosides, Nucleotides and Nucleic Acids, 22:11, 2049-2060,
DOI: 10.1081/NCN-120026406
To link to this article: http://dx.doi.org/10.1081/NCN-120026406
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 11, pp. 2049–2060, 2003
Synthesis, Anti-HIV, and Cytotoxic Activity of New AZT
Conjugates of Steroid Acids
1
Zhengqing You, A. Moshin Mian, Ram P. Agarwal,
2
2
1
,
*
and Henry Joung Lee
1
College of Pharmacy and Pharmaceutical Sciences, Florida A & M University,
Tallahassee, Florida, USA
2
Department of Medicine, University of Miami School of Medicine,
Miami, Florida, USA
ABSTRACT
In an attempt to discover potent anti-HIV agents devoid of the serious toxicity of
the current HIV-reverse transcriptase inhibitors, three steroid prodrugs of AZT
have been synthesized and their anti-HIV profiles determined with CEM-SS cell
line. Two of the prodrugs were active against HIV, though weaker than AZT.
The third agent was totally inactive against HIV. However, it demonstrated
remarkably high anti-growth activities. Further experiments established that
growth inhibition of the third agent was caused by induction of apoptosis rather
than general necrosis.
Key Words: AZT; Steroid; Prodrug; Anti-HIV; Cytotoxicity.
Azidothymidine (AZT), the first clinically approved agent for treatment of
acquired immunodeficiency syndrome (AIDS), acts by inhibiting the reverse tran-
[
1–3]
scriptase of human immunodeficiency virus (HIV).
Although AZT has attained
*
Correspondence: Henry Joung Lee, College of Pharmacy and Pharmaceutical Sciences,
Florida A & M University, Tallahassee, FL 32304, USA; Fax: (850) 599-3323; E-mail: henry.
lee@famu.edu.
2
049
DOI: 10.1081/NCN-120026406
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

2050
You et al.
a mainstay status in the treatment of AIDS, its serious dose-related side effects have
[
4]
necessitated ever-growing research to find safer and more effective analogs. One
promising approach to improve the therapeutic index has been the prodrug strategy.
This approach couples AZT with an acid to form an ester which is designed to gra-
dually release AZT by metabolism. Since AZT has a short plasma half-life due to
0
[5]
[6]
glucuronidation at the 5 -OH, and poor cellular penetrating capability, the ester-
ification is expected to increase drug stability against metabolic destruction and
improve its cellular bio-availability, thus reducing dose-related toxicity. The most
studied AZT prodrugs have been carboxylic ester and phosphoester. The former
[
7]
[8]
includes use of bicyclams attached to an acid group, retinoic acids, 1,4-dihydro-
[9]
nicotinic acids, and steroid acids. Some of the latter are derived from salicyl alco-
lipophilic glycosides.
with AZT, four rationales were employed. 1. Biologically inactive steroid acids
obtained based on the antedrug concept
acids are designed to improve lipophilicity and bio-availability of the resulting pro-
[
8]
[10]
[11]
[12]
hols,
ether lipids,
In design of new steroid conjugates
[
13]
are devoid of systemic toxicity. 2. The
0
drugs. 3. Protection of the 5 -OH could increase drug half-life in vivo. 4. Binding of
[
14]
steroid conjugates to transcortin could further improve metabolic stability. In this
study, two biologically inactive steroid acids (2,3) synthesized in our lab and one
commercially available, non-toxic acid (1, cholenic acid) were conjugated with
AZT. Preliminary anti-HIV bioassays showed that the two esters (5,6) were active
against HIV, while the third (4) was not. However, the intriguing activity=toxicity
profile of the third conjugate led to an in-depth evaluation of its anti-proliferation
activities. Presented in this paper are both anti-HIV results and anti-growth profile
of these three compounds.
RESULTS AND DISCUSSIONS
Chemistry
AZT conjugates with steroids were synthesized through ester formation of
steroid acids with AZT alcohol as shown in Sch. 1. Either 1,3-dicyclohexylcarbo-
diimide (DCC) or carbonyldiimidazole (CDI) was used as the coupling agent.
Cholenic acid (1) underwent coupling with AZT only under the DCC conditions,
while acid 3 yielded the desired product 6 only under the CDI conditions.
Although acid 2 could form some desired product 5 under the CDI conditions, the
DCC conditions gave much higher yield of 5. Acid 1 was purchased from
[
15,16]
Steraloids Inc. (Wilton, N.H.). The two biologically inactive acids (2 and 3)
were prepared as shown in Sch. 2. Acid 2 was synthesized by a 4-step procedure
starting from enone 9. Nitrile 8 was formed cleanly through a one-step conversion
[
17]
of enone 9 with metal fulminate.
yielded methyl ester 7
Mattox rearrangement and methanolysis then
which eventually gave target acid 2 by alkaline hydro-
lysis. Acid 3 was synthesized from diol 11 which in turn was derived from pred-
[
18]
[
19]
nisolone using a known literature procedure.
Acetonide 10 was obtained from
diol 11 with 2,2-dimethoxypropane and p-toluenesulfonic acid. Alkaline hydro-
lysis then gave acid 3.

AZT Conjugates of Steroid Acids
2051
Scheme 1.
Biology
The ester conjugates were subjected to in-vitro anti-HIV bioassays with CD4
expressing lymphocytes (CEM-SS cell line). Drug efficacy (EC ) was obtained by
5
0
adding HIV and AZT conjugate to the cell to measure protection of the drug against
HIV-induced cell death. The cytotoxicity (IC ) data was obtained from adding only
5
0
the conjugate to the cell to measure cell death caused by the agent. Therapeutic index
TI) is the ratio IC =EC . As summarized in Table 1, conjugates 5 and 6 showed
TIs of 370 and 184 as compared to >150 for AZT. The two active agents have
(
5
0
50
ꢀ
ꢀ
Scheme 2. a) Hg(CNO)
5%; c) NaOH=MeOH=H
MeOH=H O, 75%.
2
=LiBr=Et
3
N=HOAc=DMF, 60 C, 70%; b) MeOH=HCl, ꢁ20 C,
ꢀ
6
2
O, 87%; d) Me
2
C(MeO)
2
=TsOH=DMF, 50 C, 54%; e) NaOH=
2

2052
You et al.
a
Table 1. Anti-HIV activity and cytotoxicity in CEM-SS cell line.
b
EC50 (M)
c
IC50 (M)
Drug
TI(IC =EC )
50 50
ꢁ
6
4
Inactive
4.7 ꢂ 10
1.3 ꢂ 10
1.2 ꢂ 10
> 1 ꢂ 10
ꢁ8
ꢁ8
ꢁ9
ꢁ5
ꢁ5
ꢁ6
5
3.5 ꢂ 10
6.5 ꢂ 10
7.0 ꢂ 10
370
184
> 150
6
AZT
a
Numbers derived by averaging two repeat experiments.
5
b
c
5
0% Effective concentration against HIV cytopathic effects.
0% Inhibitory concentration for cell growth.
weaker anti-HIV potency than AZT, but appear to possess relatively low cytotoxi-
city (relatively high IC50 value). The National Cancer Institute, which performed
the anti-HIV screen test with CEM-SS cells, uses AZT as a reference compound with
ꢁ
6
its concentration up to 1 ꢂ 10 M which is considered quite high. As shown in Table
ꢁ
5
ꢁ5
1
, compounds 5 and 6 gave IC values of 1.3 ꢂ 10 and 1.2 ꢂ 10 M respectively.
5
0
Conjugate 4 provided no protection against HIV-induced death, but higher cytotoxi-
city than 5 or 6. The intriguing profile of conjugate 4 prompted further evaluations
of its growth inhibition in comparison to 5 and 6.
Cell growth inhibitory activity against human lymphoblasts (H9 cells) for com-
pounds 4–6 and AZT is depicted in Fig. 1. Compound 4 was the most potent with an
IC50 value of 2.5 mg=mL, while the values for the compounds 6 and 5 were estimated
4
Figure 1. Cell growth inhibition by compounds 4–6. H9 cells (5 ꢂ 10 cells=mL), in triplicate,
ꢀ
were incubated with various concentrations of compounds 4–6. After 72 h incubation at 37 C,
trypan blue excluding cells were counted and the % cell growth inhibition was calculated from
the untreated controls.

AZT Conjugates of Steroid Acids
2053
to be 10 and 12 mg=mL, respectively. AZT was apparently even weaker than 5 and 6.
Conjugate 4 was further tested against ddC resistant H9 cells (H9=ddC cells that
have reduced thymidine and deoxycytidine kinase activities); HL60, HL60=araC
(
human promyelocytic leukemic cell lines, parental and araC resistant), H29,
H29=FU (human colon adenocarcinomas, parental and FU resistant), MCF7
human breast carcinoma) and BL5 (Burkitt lymphoma cell line). Preliminary results
(
show that, at 10 mg=mL, it produced 85–100% growth inhibition in all of these
human tumor cell lines.
The question of whether the alcohol and acid components within conjugate 4
were responsible for the observed cytotoxicity was addressed by exposure of H9 cells
to AZT, cholenic acid, AZT plus cholenic acid, and 4. As shown in Fig. 2, compound
4
gave not only far greater cytotoxicity than either AZT or cholenic acid, but also
significantly higher activity than a combination of AZT plus cholenic acid, even
though the mixture produced more than the additive effect of the two components.
At 5 mg=mL, conjugate 4 prevented growth of H9 cells almost completely.
The effect of drug exposure time on cell growth inhibition is shown in
Fig. 3. Treatment with conjugate 4 at 1 or 5 mg=mL for 4 h did not produce any inhi-
bition, while 8 h of incubation resulted in 20–25% inhibition. Substantially more
cytotoxicity was observed after 24 h of exposure at 5 mg=mL, although much longer
(72 h) exposure only gave a plateau. The data appear to suggest that at least one cell
cycle drug exposure be required to cause a significant growth inhibition.
Figure 2. Cytotoxicity of compound 4 and its components (AZT and cholenic acid). H9
4
cells (5 ꢂ 10 cells=mL), in triplicate, were incubated with compound 4 (5 mg=mL), AZT
(
10 mg=mL), CA [cholenic acid (10 mg=mL)] and a combination of AZT þ CA (10 mg=mL each)
ꢀ
for 72 h at 37 C. Trypan blue excluding cells were counted and the % cell growth inhibition
was calculated from the untreated controls.

2054
You et al.
Figure 3. Effect of compound 4 on growth as a consequence of drug exposure time. H9 cells
were incubated with compound 4 (1 mg or 5 mg=mL) for 4, 8, or 24 h when the cells were
washed and reincubated in fresh drug free medium for 68, 64, and 48 h respectively (final incu-
bation time is 72 h). Another set of cells were exposed to compound 4 (1 mg or 5 mg=mL)
continuously for 72 h. Cells were counted and then % growth was determined from the
appropriate controls.
The role of apoptosis in the cellular cytotoxicity of compound 4 was studied and
the results are summarized in Fig. 4. In the experiments, fractions of cells in various
stages of apoptotic death were assessed by simultaneous Annexin V and propidium
iodide staining using flow cytometry. As indicated, major fractions of H9 tumor cells
treated with 4 were in early (Fig. 4C lower right quadrant) or late (Fig. 4C upper
right quadrant) stages of apoptotic cell death. In contrast, such effect was signifi-
cantly less on cells treated with either AZT (Fig. 4B) or cholenic acid (Fig. 4D).
Compound 4 at 10 mg=mL appeared to have induced close to 90% of the cells to
apoptosis, indicating that the growth inhibition was due to induction of apoptosis
and not general necrosis.
EXPERIMENTAL
Chemistry
Prednisolone was purchased from The Upjohn Company, Kalamazoo, MI.
3,24-Bisnor-5-cholenic acid-3b-ol acetate was obtained from Steraloids, Inc., New-
2
port, RI. All the other chemical reagents were ordered from Aldrich chemical
company, Milwaukee, WI. Preparative flash column chromatography was per-
formed on silica gel (200–425 mesh, Davisil G633), and the solvents used were
laboratory grade (Fisher Scientific, Fair Lawn, NJ). NMR spectra were obtained

AZT Conjugates of Steroid Acids
2055
Figure 4. Effect of compounds on the induction of apoptosis as measured by Annexin V
staining. H9 cells treated with (A) PBS (controls); (B) AZT (10 mg=mL); (C) compound 4
(
10 mg=mL); and (D) cholenic acid (10 mg=mL) for 48 h, were stained with Annexin V-FITC
and propidium iodide. Percentages of apoptotic cells are shown in the right corners. Annexin
V-FITC signal is shown on the X axis; propidium iodide (PI) signal is shown on the Y axis.
Ten thousand events were collected for each sample.
with a Brucker HX-300 spectrometer and the chemical shifts reported in parts per
million (ppm) down field from tetramethylsilane as an internal standard. Elemental
analysis was carried out by Galbraith Laboratories, Knoxville, TN. Melting points
were determined on a Thomas Hoover Capillary Melting Point Apparatus and
uncorrected.
0
0
0
3
-Azido-3 -deoxythymidine-5 -[3b-acetyloxy-22,23-bisnor-5-cholenate] (4). A mix-
ture of acid 1 (200 mg, 0.51 mmol), AZT (160 mg, 0.59 mmol), DCC (140 mg,
.68 mmol) and DMAP (14 mg, 0.11 mmol) in dry DMF (2 mL) was stirred under
0
a nitrogen atmosphere for 3 days and then poured in water (100 mL). The resulting
aqueous suspension was extracted with ethyl acetate=hexanes (2=1, 300 mL). The
organic solution was condensed via rotary evaporation and the resulting residue puri-
fied by flash chromatography (3=1 benzene=acetone) to yield, after evaporation of
solvent, 4 as a white solid: 66 mg (20%). Proton NMR (DMSO-d6): 11.355 (1H, s,
0
0
0
NH), 7.459 (1H, d, J ¼ 1Hz, H-6 ), 6.105 (1H, t, J ¼ 6.8 Hz, H-1 ), 5.326 (1H, m, H-6),

2056
You et al.
0
0
4
(
(
.43 (2H, m), 4.213 (2H, m, H-5 ), 3.958 (1H, m, H-3 ), 1.969 (3H, s, Ac), 1.786
00
3H, d, J ¼ 1Hz, Me-5 ), 1.121 (3H, d, J ¼ 6.8 Hz, H-22), 0.963 (3H, s, Me), 0.648
3H, s, Me). Carbon-13 NMR (75.47 MHz, DMSO-d6): 175.4, 169.71, 163.61,
1
5
2
50.35, 139.46, 136.01, 121.99, 109.77, 83.58, 80.48, 79.16, 73.16, 62.96, 60.15,
5.58, 52.42, 49.34, 41.92, 41.85, 37.65, 36.47, 36.06, 35.54, 31.33, 31.20, 27.32,
6.71, 23.88, 21.04, 20.47, 18.93, 16.83, 12.12, 11.70. Analysis Calculated for
C H N O : C64.03%, H7.43%, N10.98%. Found: C63.82%, H7.61%, N10.80%.
7
3
4
47
5
0
0 0
-Azido-3 -deoxythymidine-5 -[21,21-dimethoxy-3,20-dioxo-11b-hydroxy-1,4-preg-
nadiene-16a-carboxylate] (5). Methyl ester 7 was obtained through one-step con-
3
[
17,18]
version of enone 9 to hydroxynitrile 8 followed by methanolysis.
800 mg, 1.8 mmol) was dissolved in methanol (10 mL) and aqueous NaOH (4%,
0 mL) added. The mixture was stirred for 20 min and then treated with aqueous
Ester 7
(
1
HCl (5%) to reach a pH 3. The methanol was removed by rotary evaporation,
and the remaining aqueous mixture extracted with ethyl acetate (100 mL). The
organic solution was washed with water (20 mL) and then dried over sodium sulfate.
The dried solution was condensed and the residue chromatographed (9=1
EtOAc=hexanes) to give acid 2 as a white powder, after evaporation of solvent:
7
00 mg (86%). Proton NMR (DMSO-d6): 12.1 (1H, br s, COOH), 7.31 (1H, d,
J ¼ 9.8 Hz, H-1), 6.15 (1H, dd, J ¼ 9.8, 2.0 Hz, H-2), 5.90 (1H, br s, H-4), 4.63
1H, m), 4.53 (1H, s, H-21), 4.21 (1H, m), 3.29 (3H, s, MeO), 3.26 (3H, s, MeO),
.09 (1H, d, J ¼ 9.3 Hz, H-17), 1.37 (3H, s, H-19), 0.81 (3H, s, H-18). Acid 2
200 mg, 0.46 mmol) was mixed with AZT (165 mg, 0.62 mmol), DCC (140 mg,
.68 mmol) and DMAP (10 mg, 0.08 mmol) in dry DMF (2 mL). The mixture was
stirred under a nitrogen atmosphere for 4 days and then poured in water
100 mL). The aqueous mixture was extracted with EtOAc=hexanes (3=1, 300 mL),
(
3
(
0
(
and the organic layer washed with water (3 ꢂ 80 mL). The organic solution was then
condensed and the resulting residue allowed to pass flash column (EtOAc) to yield
conjugate 5 as a white solid after evaporation of solvent: 150 mg (47%). Proton
NMR (CDCl3): 9.382 (1H, s, NH), 7.160 (1H, d, J ¼ 10.1 Hz, H-1), 7.077 (1H, br
0
0
d, J ¼ 1 Hz, H-6 ), 6.149 (1H, dd, J ¼ 10.1, 1.8 Hz, H-2), 5.959 (1H, t, J ¼ 6.4 Hz,
0
H-1 ), 5.899 (1H, br s, H-4), 4.364 (1H, s, H-21), 4.324 (1H, m, H-11), 4.235 (1H,
0
0
dd, J ¼ 12.1, 5.1 Hz, H-5 ), 4.104 (1H, dd, J ¼ 12.1, 4.0 Hz, H-5 ), 4.05 (1H, m, H-
0
0
4
), 3.90 (1H, m, H-3 ), 3.485 (1H, m, H-16), 3.267 (3H, s, MeO), 3.256 (3H, s,
0
0
MeO), 3.157 (1H, d, J ¼ 9.0 Hz, H-17), 1.861 (3H, d, J ¼ 1 Hz, Me-5 ), 1.329 (3H,
s, H-19), 0.827 (3H, s, H-18). Carbon-13 NMR (75.47 MHz, CDCl3): 203.91,
1
1
4
86.54, 175.06, 169.83, 163.82, 156.14, 150.11, 135.47, 127.80, 122.40, 111.44,
03.54, 85.50, 81.47, 69.63, 63.62, 61.17, 60.57, 56.01, 55.19, 54.66, 54.36, 47.31,
5.31, 43.94, 41.46, 37.18, 33.54, 31.74, 30.95, 29.17, 20.94, 16.63, 12.48. Analysis
Calculated for C H N O =0.5 H O: C59.12%, H6.42%, N10.14%. Found:
2
3
4
43
5
10
C58.85%, H6.57%, N10.23%.
0
0 0
-Azido-3 -deoxythymidine-5 -[11b-hydroxy-17,20a-isopropylidenedioxy-3-oxo-1,4-
3
pregnadiene-21-oate] (6). Diol 11 was obtained by literature procedure.
[
19]
It
200 mg, 0.5 mmol) was mixed with 2,2-dimethoxypropane (3 g, 28 mmol) and
(
TsOH.H O (20 mg, 0.1 mmol) in anhydrous DMF (3 mL), and stirred under a nitro-
2
ꢀ
gen atmosphere at 40 C for 2 days. The resulting mixture was then diluted with

AZT Conjugates of Steroid Acids
2057
EtOAc=hexanes (1=1, 250 mL) and washed with saturated aqueous NaHCO3
(2 ꢂ 10 mL) and water (15 mL). The organic solution was condensed and the result-
ing residue chromatographed (3=2 EtOAc=hexanes) to give acetonide 10 as a white
solid, after evaporation of solvent: 120 mg (54%). Proton NMR (CDCl ): 7.268 (1H,
3
d, J ¼ 10.1 Hz, H-1), 6.261 (1H, dd, J ¼ 10.1, 1.9 Hz, H-2), 6.006 (1H, br s, H-4),
4
1
.567 (1H, s, H-20), 4.445 (1H, m, H-11), 3.774 (3H, s, MeO), 1.495 (3H, s, Me),
.440 (3H, s, Me), 1.368 (3H, s, Me), 0.945 (3H, s, H-18). Acid 3 was obtained, as
a white solid, from acetonide 10 with the procedure for preparation of acid 2, and
a yield of 75% achieved. This acid (3, 120 mg, 0.28 mmol) was mixed with CDI
(
100 mg, 0.61 mmol) in anhydrous DMF (1.5 mL) and stirred under a nitrogen atmo-
sphere for 20 min. AZT (130 mg, 0.49 mmol) was then added. Stirring continued (N2)
for 3 days and the mixture was poured in water (70 mL). The aqueous mixture was
extracted with EtOAc=hexanes (2=1, 150 mL). The organic solution was condensed
and the residue chromatographed (EtOAc) to yield conjugate 6 as a white solid, after
evaporation of solvent: 95 mg (52%). Proton NMR (CDCl3): 9.456 (1H, s, NH),
0
0
7
.187 (1H, d, J ¼ 10.1 Hz, H-1), 7.084 (1H, br d, J ¼ 1 Hz, H-6 ), 6.153 (1H, dd,
0
J ¼ 10.1, 1.7 Hz, H-2), 5.925 (1H, t, J ¼ 6.4 Hz, H-1 ), 5.902 (1H, br s, H-4), 4.464
0
0
(
1H, s, H-20), 4.40 (1H, H-11), 4.31 (2H, m, H-5 ), 4.103 (1H, m, H-4 ), 3.98 (1H,
0
00
m, H-3 ), 1.827 (3H, s, Me-5 ), 1.384 (3H, s, Me), 1.344 (3H, s, Me), 1.212 (3H, s,
Me), 0.954 (3H, s, H-18). Carbon-13 NMR (75.47 MHz, CDCl3): 186.64, 170.82,
1
8
3
70.35, 163.94, 156.42, 150.08, 135.69, 128.28, 122.27, 111.54, 111.23, 94.79, 86.16,
1.48, 77.54, 69.98, 63.83, 60.54, 55.31, 50.80, 46.22, 44.10, 39.25, 37.16, 33.57,
1.95, 31.45, 27.79, 27.36, 23.71, 20.97, 17.84, 12.53. Analysis Calculated for
C H N O =0.5H O: C60.52%, H6.57%, N10.38%. Found: C60.82%, H6.77%,
2
3
4
43
5
9
N9.94%.
Biology
Anti-HIV assay was carried out with CD4 expressing lymphocytes (CEM-SS cell
line) through the following standard procedure. Agent is dissolved in dimethyl sulf-
oxide and diluted 1:100 in cell culture medium before preparing serial half-log10
dilutions. CD4 expressing lymphocytes are added and after a brief interval HIV-1
is added, resulting in a 1:200 final dilution of the compound. Uninfected cells with
the compound serve as a toxicity control, and infected and uninfected cells without
ꢀ
the compound serve basic controls. Cultures are incubated at 37 C in a 5% carbon
dioxide atmosphere for 6 days. The tetrazolium salt, XTT, is added to all wells, and
cultures are incubated to allow formazan color development be viable cells. Indivi-
dual wells are analyzed spectrophotometrically to quantitate formazan production,
and in addition are viewed microscopically for detection of viable cells and confirma-
tion of protective activity. Drug-treated virus-infected cells are compared with drug-
treated non-infected cells and with other appropriate controls (untreated infected
and untreated non-infected cells, drug-containing wells without cells, etc.) on the
same plate. Data are reviewed in comparison with other tests done at the same time
and a determination about activity is made.
Growth inhibition assays were performed in triplicate by seeding
4
ꢂ 10 cells=mL in 24-well plates (Costar). Test compounds were added, and the
5

2058
You et al.
ꢀ
trypan blue-excluding cells were counted after 72 h of incubation at 37 C. IC
[
5
20,21]
0
values were calculated from growth inhibition curves as described previously.
6
Apoptosis experiments were carried out by treating H9 cells (1 ꢂ 10 ) with
compound 4 (10 mg=mL), AZT (10 mg=mL) or cholenic acid (10 mg=mL) for 48 h. Cells
were collected by centrifugation, washed with growth medium, and stained with
Annexin V-FITC and propidium iodide (apoptosis dilution kit; Sigma), according to
[
22]
the manufacturer’s protocol, and the fluorescence was analyzed via flow cytometry.
SUMMARY
Three new steroidal conjugates of AZT have been synthesized and their anti-
HIV profiles in CEM-SS cell line evaluated. Two of them (5 and 6) were active
against HIV-1, though weaker than AZT. The weaker activity does not necessarily
mean that this prodrug approach has failed, since the test was only conducted in
an in vitro system which may be lacking in esterase enzymes that are abundant in
the in vivo system, thus the prodrugs may be insufficiently cleaved to yield the active
drug AZT. The third conjugate (4) exhibited higher cytotoxicity but no anti-HIV
activity. Further experiments established that compound 4 is a highly potent anti-
growth agent which inhibited growth by induction of apoptosis rather than general
necrosis.
ACKNOWLEDGMENTS
We would like to thank the National Cancer Institute for performing the anti-
HIV screening assays. Special thanks are also extended to Drs. Tieran Han and
Yolong Yang for technical assistance on growth inhibition experiments. Financial
support for this project has been provided by NIH grants AM 21627, RR 08111,
RR 02660 and AI 20360.
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Received February 19, 2003
Accepted August 20, 2003