Solid-phase chemical synthesis and in vitro biological evaluation of novel 2β-piperazino-(20R)-5α-pregnane-3α,20-diol N-derivatives as anti-leukemic agents

Article abstract of DOI:10.1016/j.steroids.2012.07.012

The steroid nucleus is an interesting scaffold for the development of new therapeutic agents. Within the goal of identifying anticancer agents, new pregnane derivatives were prepared by using a sequence of liquid and solid-phase reactions. After we dehydrated epi-allopregnanolone in one step with diethylaminosulfur trifluoride and generated a 2,3α-epoxide, the regio- and stereo-selective aminolysis of this epoxide enabled us to obtain a 2β-piperazino-pregnane, whose secondary amine was protected as N-Fmoc-derivative. Using the difference in reactivity between OHs 3 and 20, we linked the pregnane nucleus-selectively on the polystyrene diethylbutylsilane resin via the OH in position 20. We next achieved in parallel the coupling of an amino acid (1st level of diversity) and the coupling of a carboxylic acid (2nd level of diversity) to generate two libraries of pregnane derivatives. The compounds inhibited the HL-60 leukemia cell growth and the most potent were three compounds (PD, LPC-37 and LPC-48) with a l-proline as first level of diversity and a cyclohexyl-carbonyl, a naphthalene-2-carbonyl or a 3-acetylbenzoyl as second level of diversity. LPC-48 efficiently inhibited HL-60 cell proliferation with IC₅₀ value of 1.9 μM and exhibited a low toxicity on normal peripheral blood lymphocytes (IC₅₀ = 31 μM). These results encouraged us to further evaluate the biological activity of these new aminosteroids by investigating their preliminary mechanism of action.

Full text of DOI:10.1016/j.steroids.2012.07.012

Steroids 77 (2012) 1403–1418
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Solid-phase chemical synthesis and in vitro biological evaluation of novel
2b-piperazino-(20R)-5
a
-pregnane-3
a,20-diol N-derivatives as anti-leukemic agents
1
1
⇑
Hajer Jegham , René Maltais , Philippe Dufour, Jenny Roy, Donald Poirier
Laboratory of Medicinal Chemistry, Oncology and Genomic Unit, CHUQ-CHUL Research Center and Laval University, Québec, Canada G1V 4G2
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 May 2012
Received in revised form 17 July 2012
Accepted 26 July 2012
Available online 14 August 2012
The steroid nucleus is an interesting scaffold for the development of new therapeutic agents. Within the
goal of identifying anticancer agents, new pregnane derivatives were prepared by using a sequence of
liquid and solid-phase reactions. After we dehydrated epi-allopregnanolone in one step with diethylami-
nosulfur trifluoride and generated a 2,3a-epoxide, the regio- and stereo-selective aminolysis of this
epoxide enabled us to obtain a 2b-piperazino-pregnane, whose secondary amine was protected as
N-Fmoc-derivative. Using the difference in reactivity between OHs 3 and 20, we linked the pregnane
nucleus-selectively on the polystyrene diethylbutylsilane resin via the OH in position 20. We next
achieved in parallel the coupling of an amino acid (1st level of diversity) and the coupling of a carboxylic
acid (2nd level of diversity) to generate two libraries of pregnane derivatives. The compounds inhibited
the HL-60 leukemia cell growth and the most potent were three compounds (PD, LPC-37 and LPC-48)
Keywords:
Aminosteroid
Pregnane
Leukemia
Cell viability
Lymphocytes
Apoptosis
with a
3-acetylbenzoyl as second level of diversity. LPC-48 efficiently inhibited HL-60 cell proliferation with
IC50 value of 1.9 M and exhibited a low toxicity on normal peripheral blood lymphocytes (IC50 = 31 M).
L-proline as first level of diversity and a cyclohexyl-carbonyl, a naphthalene-2-carbonyl or a
l
l
These results encouraged us to further evaluate the biological activity of these new aminosteroids by
investigating their preliminary mechanism of action.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
has incited interest owing to its ability to inhibit the proliferation
of breast cancer cells in vitro [7]; a few years later, a C19-steroid
Leukemia starts in blood-forming tissue such as the bone mar-
row and causes large numbers of blood cells to be produced and
enter the bloodstream [1]. Approximately 9000 deaths from acute
myeloid leukemia were predicted for 2009 in the United States, al-
most all in adults [2]. The therapeutic approach to acute leukemia
is usually chemotherapy by using anticancer agents; however, se-
vere side-effects and complications such as serious infection and
bleeding are major problems linked to this type of approach in
the clinical setting. In particular, the side effects of current chemo-
therapeutic treatments are sometimes fatal, especially among old-
er and immunocompromised patients. Further studies are still
required to explore novel and safer potent anticancer agents for
the treatment of leukemia [3].
derivative 2b-(4-methylpiperazinyl)-5a-androstane-3a,17b-diol
was reported to inhibit the proliferation of HL-60 cells (human
acute myeloid leukemia cells) and to promote cell differentiation
[8]. Taking advantage of our expertise in steroid chemistry, we
synthesized various 2b-derivatives of 5a-androstane-3a,17b-diol
and investigated their antiproliferative potential on HL-60 cells
[9–12]. From these compounds, the aminosteroid RM (Fig. 1)
was one of the most potent compounds as it exhibited an IC50 va-
lue of 0.6–2.1 lM [11,12]. Interestingly, RM showed cytotoxicity
on tumor cell lines but a very low cytotoxicity on normal cells
[12]. In order to extend our structure–activity relationship study
of this new family of cytotoxic agents, we generated pregnane
derivatives that were closely related to the androstane derivative
RM and bearing two levels of molecular diversity at position 2b
(Fig. 1).
In this report, we have optimized the use of diethylaminosulfur
trifluoride as dehydrating reagent, we described the chemical syn-
thesis of two libraries containing 40 new pregnane derivatives and
we presented data on their antiproliferative activity on HL-60 cells.
The toxicity of three members belonging to the two libraries (PD,
LPC-37 and LPC-48) was also evaluated on normal peripheral blood
lymphocytes and two compounds were selected for a preliminary
investigation of their mechanisms of action on HL-60 cells.
The steroid nucleus constitutes an interesting scaffold for the
development of new therapeutic agents [4–6]. However, until
now, few steroidal compounds have been investigated for poten-
tial antileukemic activity. In 1992, a novel class of aminosteroids
⇑
Corresponding author. Address: Laboratory of Medicinal Chemistry, Oncology
and Genomic Unit, CHUQ-CHUL Research Center (Bloc T), 2705 Laurier Boulevard,
Québec, Canada G1V 4G2. Tel.: +1 418 654 2296; fax: +1 418 654 2761.
E-mail address: donald.poirier@crchul.ulaval.ca (D. Poirier).
1
These authors contributed equally to this work.
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.07.012

1404
H. Jegham et al. / Steroids 77 (2012) 1403–1418
OH
R2 R1
O
O
OH
N
H
N
N
N
H
H
H
N
HO
H
H
H
HO
H
R1 = first level of diversity from
selected amino acids
R2 = second level of diversity from
RM (androstane nucleus)
selected carboxylic acids
Name
PD
R
O
O
O
N
N
N
OH
N
N
H
O
O
R
HO
H
H
LPC-37
LPC-48
N
N
H
Pregnane nucleus
(Libraries A and B)
O
N
N
O
Fig. 1. Chemical structures of the androstane derivative RM (compound B10 or 14 reported in reference 11 or 12, respectively), the general representation of pregnane library
members, and structures of three pregnane derivatives (PD, LPC-37 and LPC-48) selected for additional biological in vitro studies.
reflectance (ATR) were recorded on ABB model MB3000 FT-IR spec-
trophotometer (Québec, Qc, Canada) and the significant band re-
ported in cmꢀ1. Nuclear magnetic resonance (NMR) spectra were
recorded at 400 MHz for 1H and 100.6 MHz for 13C on a Bruker
Avance 400 digital spectrometer (Billerica, MA, USA). The chemical
shifts (d) were expressed in ppm and referenced to chloroform
(7.26 and 77.0 ppm), acetone (2.05 and 29.2 ppm) or methanol
(3.31 and 49.0 ppm), respectively, for 1H and 13C NMR. Low-resolu-
tion mass spectra (LRMS) were recorded on a PE Sciex API-150ex
apparatus (Foster City, CA, USA) equipped with a turbo ion-spray
source. High-resolution mass spectra (HRMS) were provided by
Pierre Audet at the Laval University Chemistry Department (Qué-
bec, QC, Canada). The purity of selected aminosteroids LPC-37
and LPC-48 was determined by high-performance liquid chroma-
tography (HPLC) (Waters Associates Milford, MA, USA) using a
Nova Pak C18 reversed-phase column (150 mm ꢁ 3.9 mm id,
2. Material and methods
2.1. Chemical synthesis
2.1.1. General
Epi-allopregnanolone was purchased from Steraloids (Wilton,
NH, USA). The polystyrene diethylbutylsilane (PS-DES) resin with
a loading of 1.58 mmol/g was supplied by Argonaut Technologies
(San Carlos, CA, USA). Chemical reagents were purchased from Sig-
ma–Aldrich Canada Ltd. (Oakville, ON, Canada) and Calbiochem-
Novabiochem Corp. (San Diego, CA, USA). The usual solvents were
obtained from Fisher Scientific (Montréal, QC, Canada) and were
used as received. Anhydrous dichloromethane (DCM) and dimeth-
ylformamide (DMF) were obtained from Sigma–Aldrich. The load-
ing of steroid 5 on resin was performed in peptide synthesis vessels
(25 mL) with frit equipped for vacuum filtration (ChemGlass Inc.;
Vineland, NJ, USA). The reaction vessel was shaken with a Burrell
wrist-action shaker model 75 (Pittsburgh, PA, USA). The libraries
were synthesized in parallel fashion using an automated synthe-
sizer (model ‘The solution’ from aapptec, Louisville, KY, USA) with
a reaction block (Ares block) of 96 wells (4 mL per well). The work-
up following final cleavage from the solid support was assisted
with a separator phase syringe (10 mL) from Biotage (Charlottes-
ville, VA, USA). Thin-layer chromatography (TLC) and flash-column
chromatography were performed on 0.20-mm silica gel 60 F254
plates and with 230–400 mesh ASTM silica gel 60, respectively
(E. Merck; Darmstadt, Germany). Melting points were recorded
on an Electrothermal IA9300 SERIES Digital Melting Point Appara-
tus and are uncorrected. Infrared spectra (IR) by attenuated total
4 lm, 60 Å). The names of steroid derivatives were obtained using
ACD/Labs (Chemist’s version) software (Toronto, ON, Canada).
2.1.2. Synthesis of solid-phase precursor 5
2.1.2.1. (5a)-pregn-2-en-20-one (1). To a solution of 5a-pregnan-
3b-ol-20-one (3.75 g, 11.8 mmol) in anhydrous DCM (200 mL) at
ꢀ78 °C under argon atmosphere was added diethylaminosulfur tri-
fluoride (DAST) (2.30 mL, 17.7 mmol) and the resulting solution
was stirred for 1 h at ꢀ78 °C. The solution was then directly evap-
orated in presence of silica gel and purified by flash chromatogra-
phy with EtOAc/hexanes (2:98) as eluant to give 1 (880 mg, 25%) as
a white solid (mp = 113–114 °C) with a ratio of C2AC3 vs. C3AC4 al-
kene isomer of 96:4. Data reported are for the major C2AC3 isomer.

H. Jegham et al. / Steroids 77 (2012) 1403–1418
1405
IR (ATR)
0.78 (s, 19-CH3), 0.75–2.15 (residual CH and CH2), 2.07 (s, 21-
CH3), 2.60 (t, J = 9.1 Hz, 17
-CH), 5.57 (m, 2-CH and 3-CH). 13C
NMR (CDCl3) d: 11.7, 13.4, 20.9, 22.7, 24.4, 28.6, 30.2, 31.6, 31.7,
34.6, 35.6, 39.1, 39.7, 41.4, 44.2, 53.9, 56.7, 63.9, 125.8, 125.9,
209.8. LRMS for C21H32O [M+H]+: 301.1 m/z.
m
: 1704 (C@O). 1H NMR (acetone-d6) d: 0.60 (s, 18-CH3),
3445 (OH) and 1709 (OCON). 1H NMR (acetone-d6) d: 0.76 (s, 18-
CH3), 1.01 (s, 19-CH3), 1.07 (d, J = 6.1 Hz, 21-CH3), 0.70–1.75 (resid-
ual CH and CH2), 2.21 (m, CH), 2.40 and 2.52 (2 m, 2 ꢁ CH2N and
a
2a
-CH), 3.41 (broad s, 2 ꢁ CH2NCO), 3.65 (m, 20-CH), 4.04 (m,
3b-CH), 4.28 (t, J = 6.5 Hz, CH of Fmoc), 4.42 (d, J = 6.6 Hz, CH2O
of Fmoc), 7.34 (t, J = 7.4 Hz, 2 ꢁ CH of Fmoc), 7.42 (t, J = 7.4 Hz,
2 ꢁ CH of Fmoc), 7.67 (d, J = 7.3 Hz, 2 ꢁ CH of Fmoc), 7.87 (d,
J = 7.5 Hz, 2 ꢁ CH of Fmoc). 13C NMR (CDCl3) d: 12.6, 17.3, 21.2,
23.6, 24.4, 25.6, 28.3, 31.6, 32.8, 34.7, 35.4, 35.7, 38.4, 40.1, 42.6,
47.4, 48.0, 55.8, 56.0, 58.5, 63.7, 64.9, 67.2, 70.6, 120.0 (2ꢁ),
125.0 (2ꢁ), 127.0 (2ꢁ), 127.7 (2ꢁ), 141.3, (2ꢁ), 144.0 (2ꢁ),
155.1. LRMS for C40H54N2O4 [M+H]+: 627.4 m/z.
2.1.2.2. (5a,20R)-pregn-2-en-20-ol (2). To a solution of ketone 1
(860 mg, 2.86 mmol) in anhydrous DCM (120 mL) at ꢀ78 °C under
argon atmosphere was slowly added DIBAL-H (1.0 M in hexane)
(5.72 mL, 5.72 mmol). The solution was stirred at ꢀ78 °C for 3 h
and successively washed with an aqueous HCl solution (5%) and
a Rochelle salt solution. The organic phase was then filtered using
a phase separator syringe (Biotage) and evaporated. Purification by
flash chromatography (EtOAc/hexanes, 5:95) yielded 672 mg (78%)
2.1.3. Solid-phase synthesis of pregnane derivatives 9 (libraries A and
B)
2.1.3.1. 9H-fluoren-9-ylmethyl-4-[(2b,3a,5a,20R)-3,20-dihydroxy-
of 2 as a white solid (mp = 132–133 °C). IR (ATR)
m
: 3487 (OH). 1H
NMR (CDCl3) d: 0.76 (s, 18-CH3 and 19-CH3), 1.13 (d, J = 6.1 Hz, 21-
CH3), 0.70–2.10 (residual CH and CH2), 3.73 (m, 20-CH), 5.58 (m, 2-
CH and 3-CH). 13C NMR (CDCl3) d: 11.7, 12.5, 20.8, 23.6, 24.4, 25.6,
28.7, 30.3, 31.9, 34.6, 35.4, 39.8, 40.1, 41.4, 42.4, 54.0, 55.9, 58.6,
70.6, 125.8, 126.0. LRMS for C21H33 [MHꢀH2O]+: 285.2 m/z.
pregnan-2-yl]piperazine-1-carboxylate loaded resin 6. 1,3-Dichloro-
5,5-dimethylhydantoin (5.85 g, 29.7 mmol) in dry DCM (60 mL)
was added to PS-DES resin (5.44 g, 1.58 mmol/g theoretical load-
ing) that had been previously dried under vacuum over a period
of two days, placed into a 100 mL peptide flask under argon and al-
lowed to swell in dry DCM (25 mL). After a period of 1 h, the result-
ing chlorosilyl resin was washed under argon with dry DCM
(3 ꢁ 75 mL). The disappearance of the Si-H band at 2100 cmꢀ1
was confirmed by the IR spectrum. The resin was next used for
the loading step. The chlorosilyl resin was swollen in dry DCM
(20 mL) under argon atmosphere. A solution of imidazole (1.16 g,
17.0 mmol) and diol 5 (10.7 g, 16.8 mmol) in DCM (20 mL) was
subsequently added. The mixture was vortexed overnight at room
temperature using a Burrell wrist-action shaker. The loaded resin
was washed with DCM (3 ꢁ 75 mL) and dried overnight under vac-
2.1.2.3. (2a,3a,5a,20R)-2,3-epoxypregnan-20-ol (3). To a solution of
alkene 2 (660 mg, 2.18 mmol) in anhydrous DCM (20 mL) at 0 °C
under an atmosphere of argon was added in six portions m-chloro-
perbenzoic acid (m-CPBA) (733 mg, 3.27 mmol). The solution was
stirred for 1 h at 0 °C and then allowed to return to room temper-
ature overnight. The resulting solution was diluted with DCM
(30 mL) and successively washed with a Na2S2O3 aqueous solution
(10%) and a Na2CO3 aqueous solution (10%), dried with MgSO4, fil-
tered and evaporated to dryness. Purification by flash chromatog-
raphy (EtOAc/hexanes, 5:95) yielded 511 mg (73%) of 3 as a
uum to provide 7.3 g of resin 6 (loading of 0.40 mmol/g). IR (KBr):
m
white solid (mp = 154–156 °C). IR (ATR)
m
: 3475 (OH). 1H NMR
3442 (OH, alcohol), 1702 (C@O, carbamate) cmꢀ1. The free diol 5
(8.7 g) was easily recovered after flash chromatography using
EtOAc/hexanes (1:1) as eluent.
(acetone-d6) d: 0.62 (CH), 0.74 (s, 18-CH3), 0.79 (s, 19-CH3), 1.07
(d, J = 6.1 Hz, 21-CH3), 0.70–2.30 (residual CH and CH2), 3.06 (m,
2 ꢁ CH of epoxide), 3.62 (m, 20-CH). 13C NMR (CDCl3) d: 12.4,
12.9, 20.7, 23.6, 24.4, 25.6, 28.4, 29.0, 31.7, 33.6, 35.4, 36.2, 38.2,
39.9, 42.3, 51.1, 52.4, 53.7, 55.7, 58.5, 70.6. LRMS for C21H34O2
[M+H]+: 319.1 m/z.
2.1.3.2. Procedure for the preparation of resin-bound derivatives 7. A
solution of piperidine in DCM (20% v/v) (70 mL) was added to resin
6 (7.3 g, 0.4 mmol/g) and the suspension was vortexed using a Bur-
rell wrist-action shaker for 1 h at room temperature. The resin was
then filtered and washed successively with DCM (5 ꢁ 75 mL) and
MeOH (5 ꢁ 75 mL), and finally dried overnight to provide 6.5 g of
Fmoc deprotected resin. The resin was divided into portions
(1.80 g, 0.4 mmol/g in a 50 mL peptide flask). To each portion
2.1.2.4. (2b,3a,5a,20R)-2-(piperazin-1-yl)-pregnane-3,20-diol (4). To
the epoxide 3 (500 mg, 1.57 mmol) were added piperazine (3.5 g,
40.6 mmol) and water (1 mL). The suspension was heated over-
night at 150 °C and the resulting solution was cooled to room tem-
perature, diluted with DCM (50 mL) and washed three times with
water (100 mL). The organic layer was dried using a phase separa-
tor syringe (Biotage). Purification by flash chromatography (DCM/
MeOH/triethylamine, 95:4:1) yielded 529 mg (83%) of 4 as a white
was added a solution of the appropriate amino acid (Fmoc-
line-OH (2.5 g, 7.5 mmol), Fmoc- -proline-OH (2.5 g, 7.5 mmol),
Fmoc- -phenylalanine-OH (2.9 g, 7.5 mmol), Fmoc- -phenylala-
nine-OH (2.9 g, 7.5 mmol) or Fmoc- -tetrahydro-isoquinoline-3-
carboxylic acid (3.0 g, 7.5 mmol), benzotriazole-yl-oxy-tris-pyrr-
olidinophosphonium hexafluorophosphate (PyBOP) (3.9 g,
L-pro-
D
L
D
L
solid (mp = 114 °C). IR (ATR) m
: 3410 (OH and NH). 1H NMR (ace-
tone-d6) d: 0.75 (s, 18-CH3), 0.99 (s, 19-CH3), 1.07 (d, J = 6.1 Hz,
21-CH3), 0.70–2.25 (residual CH and CH2), 2.35, 2.50 and 2.77
7.5 mmol) and N-hydroxybenzotriazole (HOBt) (1.0 g, 7.5 mmol)
in DMF (25 mL) under argon atmosphere. Diisopropylethylamine
(DIPEA) (2.6 mL, 15 mmol) was added to the suspensions and the
peptide flasks were vortexed with a Burrell wrist-action shaker
for 5 h at room temperature. The resins were then filtered and
washed successively with DCM (5 ꢁ 25 mL) and MeOH
(5 ꢁ 25 mL), and finally dried overnight to give the resins 7. The
coupling reaction was repeated a second time in each case in order
to ensure complete coupling.
(3 m, 4 ꢁ CH2N and 2
a-CH), 3.62 (m, 20-CH), 4.01 (m, 3b-CH).
13C NMR (CD3OD) d: 12.8, 14.7, 22.0, 23.8, 25.5, 26.8, 29.2, 33.3,
34.3, 36.4, 37.2, 40.5, 41.1, 43.8, 46.7 (2ꢁ), 51.8 (2ꢁ), 57.0, 57.5,
59.4, 66.4, 66.9, 70.9. LRMS for C25H44N2O2 [M+H]+: 405.4 m/z.
2.1.2.5. 9H-fluoren-9-ylmethyl-4-[(2b,3a,5a,20R)-3,20-dihydroxy-
pregnan-2-yl]piperazine-1-carboxylate (5). To a solution of piperaz-
ino derivative 4 (520 mg, 1.27 mmol) in THF (12 mL) was added
water (3 mL), NaHCO3 (322 mg, 3.83 mmol) and Fmoc-O-succini-
mide (518 mg, 1.53 mmol). The solution was stirred overnight at
room temperature. The resulting solution was diluted with EtOAc
(50 mL), washed twice with water (70 mL), once with brine
(30 mL), dried with MgSO4, filtered and evaporated to dryness.
Purification by flash chromatography (DCM/MeOH, 98:2) yielded
2.1.3.3. Procedure for the preparation of resin-bound derivatives 8. To
each of the resin-bound derivatives 7 (2.5 g) was added 30 mL of a
20% (v/v) solution of piperidine in DCM (30 mL). The suspensions
were vortexed with a Burrell wrist-action shaker for 1 h at room
temperature. The resins were then filtered, washed successively
with DCM (5 ꢁ 30 mL) and MeOH (5 ꢁ 30 mL) and finally dried
530 mg (65%) of 5 as a white solid (mp = 84–87 °C). IR (ATR) m:

1406
H. Jegham et al. / Steroids 77 (2012) 1403–1418
overnight to provide Fmoc deprotected resins. For library A, por-
tions of 100 mg of the resins 7 were placed in tagged individual
1.13 (d, J = 6.0 Hz, 21-CH3), 0.69–2.25 (residual CH and CH2), 2.32–
-CH and CH2CO), 3.50 and 3.70 (2 m,
3 ꢁ CH2NCO and 20-CH), 3.85 (m, 3b-CH), 4.87 (dd, J1 = 3.3 Hz,
J2 = 8.3 Hz, NCHCO of Pro). LRMS for C38H64N3O4 [M+H]+:
626.5 m/z.
2.75 (broad m, 2 ꢁ CH2N, 2
a
syringe reactor (3 ꢁ 4 = 12 syringes; tagged
For library B, portions of 100 mg of the resin 7 of the appropriate
resin-bound amino acid derivatives ( -Pro, -Pro, -Phe, -Phe)
L
-Pro,
L-Phe,
L
-Tic).
L
D
L
D
were placed in reactor wells (4 mL) of the automated synthesizer
reaction block (4 ꢁ 7 = 28 wells). To each reactor syringe (library
A) or reactor well (library B) was successively added 0.7 mL of a
0.3 M solution of the appropriate carboxylic acid in DMF, 0.7 mL
of a 0.3 M solution of PyBOP and HOBt in DMF, and finally 0.7 mL
of 0.6 M solution of DIPEA in DMF. The resulting suspensions were
vortexed at 600 rpm for 3 h under argon atmosphere. The well and
syringe reactors were then filtered to remove the reaction solution
from the resin. The resins were subsequently washed successively
with DMF (2 ꢁ 3 mL), DCM (2 ꢁ 3 mL) and MeOH (2 ꢁ 3 mL). This
procedure was repeated a second time for each of the resin-bound
derivatives 8.
2.1.3.5.5. 3-cyclohexyl-N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxy-
pregnan-2-yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]propana-
mide (FC): 6.4 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3), 0.81 (s, 19-
CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.69–1.70 (residual CH and CH2),
2.20 (t, J = 7.8 Hz, CH2CO), 2.15 and 2.54 (2 m, 2 ꢁ CH2N and 2
a-
CH), 2.93 and 3.03 (2m, CH2Ph), 3.35 and 3.75 (2m, 2 ꢁ CH2NCO,
20-CH and 3b-CH), 5.17 (m, NHCHCO of Phe), 6.33 (d, J = 8.2 Hz,
NH) 7.20 (m, 3 ꢁ CH of CH2Ph), 7.28 (m, 2 ꢁ CH of CH2Ph). LRMS
for C42H66N3O4 [M+H]+: 676.5 m/z.
2.1.3.5.6. 3-cyclohexyl-1-[(3S)-3-({4-[(2b,3a,5a,20R)-3,20-dihydroxy-
pregnan-2-yl]piperazin-1-yl}carbonyl)-3,4-dihydroisoquinolin-2(1H)-
yl]propan-1-one (TC): 1.4 mg. 1H NMR (CDCl3) d: 0.75 (s, 18-CH3),
0.84 (s, 19-CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.72–1.85 (residual
2.1.3.4. Cleavage of the resin-bound derivatives 8 to obtain library
members of general formula 9. To each of the resin-bound deriva-
tives 8 was added 2 mL of an acid solution of 2 M methanolic
HCl (AcCl + MeOH) in DCM (20:80, v/v) and the resulting suspen-
sions were vortexed at 600 rpm for 1 h. DCM (1 mL) was added
and the suspensions were filtered and the recovered filtrate was
neutralized with 0.5 mL of 10% aqueous NaHCO3 (pH 8). The bipha-
sic solution was filtered using a phase separator syringe (Biotage)
and the resulting organic solution evaporated under reduced pres-
sure. The 12 (3 ꢁ 4) crude amide compounds of library A (Table 2)
were purified by filtration over a silica gel plug (10 mL) using
EtOAc/hexanes (1:1) (15 mL) and then EtOAc (20 mL). In another
experiment, the 28 (4 ꢁ 7) amide compounds of library B (Table 3)
were evaporated to dryness and judged sufficiently pure by TLC
and 1H NMR analyses for direct screening on HL-60 cells. All mem-
bers of libraries A and B were analyzed by TLC, 1H NMR and LRMS.
CH and CH2), 2.00–2.70 (broad m, 2 ꢁ CH2N, 2
a-CH and CH2CO),
3.05 (d, J = 5.3 Hz, CHCH2Ar), 3.30–3.78 (broad m, 2 ꢁ CH2NCO
and 20-CH), 3.86 (m, 3b-CH), 4.68 (q of AB system, J = 15.7 Hz,
NCOCH2Ar), 5.54 (t, J = 5.7 Hz, NHCHCO of Tic), 7.18 (m, 4 ꢁ CH
of Ar). LRMS for C43H66N3O4 [M+H]+: 688.3 m/z.
2.1.3.5.7. cyclohexyl[(2S)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypreg-
nan-2-yl]piperazin-1-yl}carbonyl) pyrrolidin-1-yl]methanone (PD):
8.0 mg. 1H NMR (CDCl3) d: 0.74 (s, 18-CH3), 0.84 (s, 19-CH3),
1.13 (d, J = 6.1 Hz, 21-CH3), 0.70–2.22 (residual CH and CH2),
2.34–2.70 (broad m, 2 ꢁ CH2N, 2
a-CH and CH2CHCO), 3.30–3.78
(broad m, 3 ꢁ CH2NCO and 20-CH), 3.85 (m, 3b-CH), 4.87 (dd,
J1 = 3.5 Hz, J2 = 8.2 Hz, NCHCO of Pro). 13C NMR (CDCl3) d: 12.6,
17.1(17.2), 23.6, 24.4, 24.8, 25.6, 25.8, 28.3, 28.7, 28.8, 29.0, 31.6,
32.9, 34.6, 35.3, 35.8, 38.4, 40.1, 42.5, 42.6, 42.7, 46.0, 46.9, 48.3,
48.4, 52.5, 55.7, 55.8, 56.0, 58.5, 61.1, 63.8, 64.9, 70.6, 170.4,
179.4. LRMS for C37H62N3O4 [M+H]+: 612.6 m/z.
2.1.3.5.8.
N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
2.1.3.5. Library A (12 aminosteroids; Table 2).
yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]cyclohexanecarboxa-
mide (FD): 5.1 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3), 0.81 (s, 19-
CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.70–1.87 (residual CH and CH2),
2.1.3.5.1.
1-[(2S)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]-3-methylbutan-1-one
(PA): 7.0 mg. 1H NMR (CDCl3) d: 0.74 (s, 18-CH3), 0.84 (s, 19-CH3),
0.96 and 0.98 (2 d, J = 6.2 Hz, (CH3)2CH), 1.13 (d, J = 6.1 Hz, 21-CH3),
0.69–2.28 (residual CH and CH2), 2.35–2.75 (broad m, 2 ꢁ CH2N
2.15 and 2.54 (2 m, 2 ꢁ CH2N, 2
a-CH and CH2CHCO), 2.93 and 3.02
(2 m, CH2Ph), 3.30 and 3.74 (2 m, 2 ꢁ CH2NCO, 20-CH and 3b-CH),
5.14 (m, NHCHCO of Phe), 6.35 (d, J = 8.0 Hz, NH), 7.20 (m, 3 ꢁ CH
of CH2Ph), 7.27 (m, 2 ꢁ CH of CH2Ph). LRMS for C41H64N3O4 [M+H]+:
662.5 m/z.
and 2
a
-CH), 3.48 and 3.70 (2 m, 3 ꢁ CH2NCO and 20-CH), 3.85
(m, 3b-CH), 4.89 (dd, J1 = 3.3 Hz, J2 = 8.1 Hz, NCHCO of Pro). LRMS
for C35H60N3O4 [M+H]+: 586.5 m/z.
2.1.3.5.9. cyclohexyl[(3S)-3-({4-[(2b,3a,5a,20R)-3,20-dihydroxypreg-
2.1.3.5.2.
N-[(2S)-1-{4-[(2b,3
a
,5
a
,20R)-3,20-dihydroxypregnan-2-
nan-2-yl]piperazin-1-yl}carbonyl)-3,4-dihydroisoquinolin-2(1H)-
yl]methanone (TD): 1.2 mg. 1H NMR (CDCl3) d: 0.75 (s, 18-CH3),
0.84 (s, 19-CH3), 1.14 (d, J = 5.9 Hz, 21-CH3), 0.70–1.90 (residual
yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]-3-methylbutanamide
(FA): 6.0 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3), 0.81 (s, 19-CH3),
0.93 (d, J = 6.0 Hz, (CH3)2CH), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.70–1.85
CH and CH2), 1.95–2.73 (broad m, 2 ꢁ CH2N, 2
a-CH and CH2CHCO),
(residual CH and CH2), 2.10 and 2.53 (2 m, 2 ꢁ CH2N, 2
a-CH and
3.05 (d, J = 5.9 Hz, CHCH2Ar), 3.30–3.78 (broad m, 2 ꢁ CH2NCO and
20-CH), 3.86 (m, 3b-CH), 4.67 (q of AB system, J = 15.6 Hz, NCOCH2-
Ar), 5.54 (m, NHCHCO of Tic), 7.18 (m, 4 ꢁ CH of Ar). LRMS for
CH2CO), 2.93 and 3.04 (2 m, CH2Ph), 3.30 and 3.74 (2 m, 2 ꢁ CH2-
NCO, 20-CH and 3b-CH), 5.17 (m, NHCHCO of Phe), 6.32 (d,
J = 8.2 Hz, NH) 7.20 (m, 3 ꢁ CH of CH2Ph), 7.28 (m, 2 ꢁ CH of CH2-
Ph). LRMS for C39H62N3O4 [M+H]+: 636.5 m/z.
C
42H64N3O4 [M+H]+: 674.3 m/z.
2.1.3.5.10. 1-[(2S)-2-({4-[(2b,3
a,5a
,20R)-3,20-dihydroxypregnan-2-
2.1.3.5.3.
1-[(3S)-3-({4-[(2b,3
a
,5
a
,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]nonan-1-one (PN):
yl]piperazin-1-yl}carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl]-3-
methylbutan-1-one (TA): 1.9 mg. 1H NMR (CDCl3) d: 0.75 (s, 18-
CH3), 0.84 (s, 19-CH3), 0.96 and 0.98 (2 d, J = 6.6 Hz, (CH3)2CH),
1.14 (d, J = 6.0 Hz, 21-CH3), 0.69–1.70 (residual CH and CH2),
8.5 mg. 1H NMR (CDCl3) d: 0.75 (s, 18-CH3), 0.84 (s, 19-CH3),
0.87 (t, J = 6.8 Hz, CH3CH2), 1.13 (d, J = 6.0 Hz, 21-CH3), 0.69–2.25
(residual CH and CH2), 2.30 (m, CH2CH2CON), 2.35–2.70 (broad
m, 2 ꢁ CH2N and 2 -CH), 3.52 and 3.70 (2 m, 3 ꢁ CH2NCO and
a
1.75–2.70 (broad m, 2 ꢁ CH2N,
2
a-CH and CH2CO), 3.05 (d,
20-CH), 3.86 (m, 3b-CH), 4.87 (dd, J1 = 3.3 Hz, J2 = 8.2 Hz, NCHCO
J = 6.1 Hz, CHCH2Ar), 3.30–3.78 (broad m, 2 ꢁ CH2NCO and 20-
CH), 3.85 (3b-CH), 4.67 (q of AB system, J = 15.8 Hz, NCOCH2Ar),
5.57 (t, J = 5.6 Hz, NHCHCO of Tic), 7.20 (m, 4 ꢁ CH of Ar). LRMS
for C40H62N3O4 [M+H]+: 648.4 m/z.
of Pro). LRMS for C39H68N3O4 [M+H]+: 642.5 m/z.
2.1.3.5.11. N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]nonanamide
(FN):
5.2 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3), 0.81 (s, 19-CH3),
0.88 (t, J = 6.7 Hz, CH3CH2), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.70–1.85
2.1.3.5.4. 3-cyclohexyl-1-[(2S)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxy-
pregnan-2-yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]propan-1-one
(residual CH and CH2), 2.16 and 2.53 (2 m, 2 ꢁ CH2N, 2
a-CH and
(PC): 7.9 mg. 1H NMR (CDCl3) d: 0.74 (s, 18-CH3), 0.83 (s, 19-CH3),
CH2CH2CON), 2.94 and 3.03 (2 m, CH2Ph), 3.31 and 3.75 (2 m,

H. Jegham et al. / Steroids 77 (2012) 1403–1418
1407
2 ꢁ CH2NCO, 3b-CH and 20-CH), 5.16 (m, NHCHCO of Phe), 6.33 (d,
J = 8.1 Hz, NH), 7.20 (m, 3 ꢁ CH of CH2Ph), 7.28 (m, 2 ꢁ CH of CH2-
Ph). LRMS for C43H70N3O4 [M+H]+: 692.5 m/z.
NHCHCO of Phe), 7.16 (s, COCH@CAO), 7.25 (m, 3 ꢁ CH of CH2Ph),
7.31 (m, 2 ꢁ CH of CH2Ph), 7.47 (t, J = 7.1 Hz, CH of coumarin), 7.58
(d, J = 8.5 Hz, CH of coumarin), 7.75 (t, J = 8.6 Hz, CH of coumarin),
7.84 (d, J = 8.0 Hz, NH), 8.22 (dd, J1 = 1.6 Hz, J2 = 8.0 Hz, CH of cou-
marin). LRMS for C44H58N3O6 [M+H]+: 724.4 m/z.
2.1.3.5.12. 1-[(3S)-3-({4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl]nonan-
1-one (TD): 2.1 mg. 1H NMR (CDCl3) d: 0.75 (s, 18-CH3), 0.85 (s, 19-
CH3), 0.88 (t, J = 6.7 Hz, CH3CH2), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.71–
2.1.3.6.7. 2-{[(2R)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]carbonyl}-4H-chromen-4-
one (DPC-38): 9.6 mg. 1H NMR (CDCl3) d: 0.74 (s, 18-CH3), 0.85 (s,
19-CH3), 1.13 (d, J = 6.1 Hz, 21-CH3), 0.70–2.40 (residual CH and
1.85 (residual CH and CH2), 2.00–2.68 (broad m, 2 ꢁ CH2N, 2
a-CH
and CH2CH2CON), 3.05 (d, J = 4.9 Hz, CHCH2Ar), 3.32–3.74 (broad
m, 2 ꢁ CH2NCO and 20-CH), 3.86 (m, 3b-CH), 4.67 (q of AB system,
J = 15.7 Hz, NCOCH2Ar), 5.55 (m, NHCHCO of Tic), 7.18 (m, 4 ꢁ CH
of Ar). LRMS for C44H70N3O4 [M+H]+: 704.4 m/z.
CH2), 2.45–2.90 (broad m, 2 ꢁ CH2N and 2
a-CH), 3.30–4.10 (broad
m, 3 ꢁ CH2NCO, 20-CH and 3b-CH), 5.03 (m, NCHCO of Pro), 6.84 (s,
COCH@CAO), 7.45 (m, CH of coumarin), 7.51 (d, J = 8.2 Hz, CH of
coumarin), 7.72 (t, J = 8.6 Hz, CH of coumarin), 8.21 (dd,
J1 = 1.6 Hz, J2 = 8.0 Hz, CH of coumarin). LRMS for C40H56N3O6
[M+H]+: 674.4 m/z.
2.1.3.6. Library B (28 aminosteroids; Table 3).
2.1.3.6.1. {4-[(2b,3a,5a,20R)-3,17-dihydroxypregnan-2-yl]piperazin-
1-yl}[2S)-1(naphtalen-1-ylcarbonyl)pyrrolidin-2-yl]methanone (LPC-
37): 10 mg. 1H NMR (CDCl3) d: 0.73 (s, 18-CH3), 0.84 (s, 19-CH3),
1.12 (d, J = 6.1 Hz, 21-CH3), 0.70–2.30 (residual CH and CH2),
2.1.3.6.8.
N-[(2R)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]-4-oxo-4H-chromene-
2-carboxamide (DFC-38): 2.2 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-
CH3), 0.82 (s, 19-CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.68–2.20 (resid-
2.40–2.80 (broad m, 2 ꢁ CH2N and 2
a-CH), 3.40–3.95 (broad m,
3 ꢁ CH2NCO, 20-CH and 3b-CH), 5.14 (dd, J1 = 5.9 Hz, J2 = 8.0 Hz,
NCHCO of Pro), 7.53 (m, 2 ꢁ CH of Aryl), 7.67 (dd, J1 = 1.3 Hz,
J2 = 8.3 Hz, CH of Aryl), 7.86 (m, 3 ꢁ CH of Aryl), 8.10 (s, CH of Aryl).
LRMS for C41H58N3O4 [M+H]+: 656.4 m/z. See Section 2.1.4 for addi-
tional characterization of this compound.
ual CH and CH2), 2.08, 2.40 and 2.57 (3 m, 2 ꢁ CH2N and 2
a-CH),
2.95–3.80 (broad m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.33
(m, NHCHCO of Phe), 7.17 (s, COCH@CAO), 7.30 (m, CH2Ph), 7.47
(t, J = 7.6 Hz, CH of coumarin), 7.58 (d, J = 8.3 Hz, CH of coumarin),
7.75 (t, J = 8.6 Hz, CH of coumarin), 7.83 (d, J = 8.1 Hz, NH), 8.22 (dd,
J1 = 1.4 Hz, J2 = 8.0 Hz, CH of coumarin). LRMS for C44H58N3O6
[M+H]+: 724.4 m/z.
2.1.3.6.2.
N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]naphthalene-2-car-
boxamide (LFC-37): 7.0 mg. 1H NMR (CDCl3) d: 0.75 (s, 18-CH3),
0.82 (s, 19-CH3), 1.14 (d, J = 6.1 Hz, 21-CH3), 0.70–1.85 (residual
2.1.3.6.9. 6-{[(2S)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]carbonyl}-2,2-dimethyl-
2,3-dihydro-4H-pyran-4-one (LPC-39): 9.3 mg. 1H NMR (CDCl3) d:
0.74 (s, 18-CH3), 0.84 (s, 19-CH3), 1.13 (d, J = 6.0 Hz, 21-CH3),
1.50 (s, (CH3)2C), 0.70–2.25 (residual CH and CH2), 2.30–2.75
CH and CH2), 2.09, 2.21 and 2.57 (3 m, 2 ꢁ CH2N and 2
a-CH),
3.05–3.82 (4 m, 2 ꢁ CH2NCO, CH2Ph, 20-CH and 3b-CH), 5.43 (m,
NCHCO of Phe), 7.28 (m, CH2Ph), 7.56 (m, 2 ꢁ CH of Aryl), 7.90
(m, 4 ꢁ CH of Aryl), 8.31 (s, CH of Aryl). LRMS for C45H60N3O4
[M+H]+: 706.6 m/z.
(broad m, 2 ꢁ CH2N, 2
a-CH and CCH2CO), 3.30–3.90 (broad m,
3 ꢁ CH2NCO, 20-CH and 3b-CH), 4.90 (dd, J1 = 4.5 Hz, J2 = 7.4 Hz,
NCHCO of Pro), 5.91 (s, C@CHCO). LRMS for C38H60N3O6 [M+H]+:
654.4 m/z.
2.1.3.6.3. {4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-yl]piperazin-
1-yl}[(2R)-1-(naphthalen-2-ylcarbonyl)pyrrolidin-2-yl]methanone
(DPC-37): 8.5 mg. 1H NMR (CDCl3) d: 0.73 (s, 18-CH3), 0.84 (s, 19-
CH3), 1.13 (d, J = 6.0 Hz, 21-CH3), 0.70–2.30 (residual CH and CH2),
2.1.3.6.10. N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]-2,2-dimethyl-4-oxo-
3,4-dihydro-2H-pyran-6-carboxamide (LFC-39): 10 mg. 1H NMR
(CDCl3) d: 0.76 (s, 18-CH3), 0.81 (s, 19-CH3), 1.14 (d, J = 6.0 Hz,
21-CH3), 1.48 and 1.49 (2 s, (CH3)2C), 0.70–1.88 (residual CH and
2.45–3.10 (broad m, 2 ꢁ CH2N and 2
a-CH), 3.30–3.95 (broad m,
3 ꢁ CH2NCO, 20-CH and 3b-CH), 5.14 (dd, J1 = 5.9 Hz, J2 = 8.1 Hz,
NCHCO of Pro), 7.52 (m, 2 ꢁ CH of Aryl), 7.67 (dd, J1 = 1.3 Hz,
J2 = 8.5 Hz, CH of Aryl), 7.86 (m, 3 ꢁ CH of Aryl), 8.09 (s, CH of Aryl).
LRMS for C41H58N3O4 [M+H]+: 656.5 m/z.
CH2), 2.12 and 2.54 (2 m, 2 ꢁ CH2N, 2
a-CH and CCH2CO), 2.95–
3.80 (broad m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.20 (m,
NHCHCO of Phe), 6.29 (s, C@CHCO), 7.22 (m, 3 ꢁ CH of CH2Ph),
7.29 (m, 2 ꢁ CH of CH2Ph), 7.51 (d, J = 8.3 Hz, NH). LRMS for
2.1.3.6.4.
N-[(2R)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]naphthalene-2-car-
boxamide (DFC-37): 6.5 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3),
0.82 (s, 19-CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.70–1.85 (residual
C
42H62N3O6 [M+H]+: 704.4 m/z.
2.1.3.6.11. 6-{[(2R)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
CH and CH2), 2.07, 2.40 and 2.58 (3 m, 2 ꢁ CH2N and 2
a
-CH),
yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]carbonyl}-2,2-dimethyl-
2,3-dihydro-4H-pyran-4-one (DPC-39): 11 mg. 1H NMR (CDCl3) d:
0.74 (s, 18-CH3), 0.84 (s, 19-CH3), 1.13 (d, J = 6.0 Hz, 21-CH3),
1.49 and 1.50 (2s, (CH3)2C), 0.70–2.20 (residual CH and CH2),
3.05–3.80 (broad m, 2 ꢁ CH2NCO, CH2Ph, 20-CH and 3b-CH), 5.42
(m, NCHCO of Phe), 7.28 (m, CH2Ph), 7.56 (m, 2 ꢁ CH of Aryl),
7.90 (m, 4 ꢁ CH of Aryl), 8.31 (s, CH of Aryl). LRMS for
C
45H60N3O4 [M+H]+: 706.5 m/z.
2.30–2.84 (broad m, 2 ꢁ CH2N, 2
a-CH and CCH2CO), 3.25–3.90
(broad m, 3 ꢁ CH2NCO, 20-CH and 3b-CH), 4.90 (m, NCHCO of
2.1.3.6.5. 2-{[(2S)-2-({4-[(2b,3
a,5
a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]carbonyl}-4H-chromen-4-
one (LPC-38): 6.4 mg. 1H NMR (CDCl3) d: 0.74 (s, 18-CH3), 0.85 (s,
19-CH3), 1.13 (d, J = 6.1 Hz, 21-CH3), 0.70–2.35 (residual CH and
Pro), 5.91 (s, C@CHCO). LRMS for C38H60N3O6 [M+H]+: 654.5 m/z.
2.1.3.6.12. N-[(2R)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]-2,2-dimethyl-4-oxo-
3,4-dihydro-2H-pyran-6-carboxamide (DFC-39): 5.1 mg. 1H NMR
(CDCl3) d: 0.76 (s, 18-CH3), 0.81 (s, 19-CH3), 1.14 (d, J = 6.0 Hz,
21-CH3), 1.48 and 1.50 (2 s, (CH3)2C), 0.68–1.85 (residual CH and
CH2), 2.30–2.78 (broad m, 2 ꢁ CH2N and 2
a-CH), 3.30–4.10 (broad
m, 3 ꢁ CH2NCO, 20-CH and 3b-CH), 5.02 (m, NCHCO of Pro), 6.84 (s,
COCH@CAO), 7.44 (m, CH of coumarin), 7.51 (d, J = 8.5 Hz, CH of
coumarin), 7.72 (t, J = 8.5 Hz, CH of coumarin), 8.21 (d, J = 8.0 Hz,
CH of coumarin). LRMS for C40H56N3O6 [M+H]+: 674.4 m/z.
CH2), 2.10, 2.35 and 2.54 (3 m, 2 ꢁ CH2N, 2
a-CH and CCH2CO),
2.95–3.80 (broad m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.20
(m, NHCHCO of Phe), 6.29 (s, C@CHCO), 7.22 (m, 3 ꢁ CH of CH2Ph),
7.30 (m, 2 ꢁ CH of CH2Ph), 7.51 (d, J = 8.1 Hz, NH). LRMS for
2.1.3.6.6.
N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]-4-oxo-4H-chromene-
2-carboxamide (LFC-38): 6.0 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-
CH3), 0.82 (s, 19-CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.70–1.90 (resid-
C
42H62N3O6 [M+H]+: 704.4 m/z.
2.1.3.6.13. 4-{[(2S)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
ual CH and CH2), 2.00–2.60 (m, 2 ꢁ CH2N and 2
a
-CH) 2.98–3.80
yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]carbonyl}benzonitrile
(broad m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.33 (m,
(LPC-41): 12 mg. 1H NMR (CDCl3) d: 0.74 (s, 18-CH3), 0.84 (s, 19-

1408
H. Jegham et al. / Steroids 77 (2012) 1403–1418
CH3), 1.13 (d, J = 6.0 Hz, 21-CH3), 0.68–2.30 (residual CH and CH2),
-CH), 3.35–3.90 (broad
m, 3 ꢁ CH2NCO, 20-CH and 3b-CH), 5.04 (dd, J1 = 5.5 Hz,
J2 = 8.2 Hz, NCHCO of Pro), 7.69 (q of AB system 4 ꢁ CH of 4-CN-
Ph-CO). LRMS for C38H55N4O4 [M+H]+: 631.4 m/z.
(m, 2 ꢁ CH of Aryl), 7.63 (d, J = 15.6 Hz, CH@CHCO). LRMS for
43H60N3O4 [M+H]+: 682.4 m/z.
2.1.3.6.21. {4-[(2b,3 ,5 ,20R)-3,20-dihydroxypregnan-2-yl]piperazin-
1-yl}[(2S)-1-{[(4S)-4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]car-
bonyl}pyrrolidin-2-yl]methanone (LPC-46): 12 mg. 1H NMR (CDCl3)
d: 0.74 (s, 18-CH3), 0.83 (s, 19-CH3), 1.13 (d, J = 6.0 Hz, 21-CH3),
1.74 (s, CH2@CCH3), 0.70–2.30 (residual CH and CH2), 2.32–2.75
2.45, 2.60 and 2.68 (3 m, 2 ꢁ CH2N and 2
a
C
a a
2.1.3.6.14.
4-cyano-N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxy-
pregnan-2-yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]benzamide
(LFC-41): 6.2 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3), 0.82 (s, 19-
CH3), 1.14 (d, J = 6.1 Hz, 21-CH3), 0.70–1.88 (residual CH and
(m, 2 ꢁ CH2N and 2
a
-CH), 3.30–3.90 (m, 3 ꢁ CH2NCO, 20-CH and
3b-CH), 4.73 (d, J = 13.4 Hz, CH2@CCH3), 4.91 (t, J = 6.9 Hz, NCHCO
CH2), 2.10, 2.22 and 2.56 (3 m, 2 ꢁ CH2N and 2
a
-CH), 3.00–3.84
of Pro), 6.14 (s, CH@CACO). LRMS for
650.5 m/z.
C
40H64N3O4 [M+H]+:
(broad m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.33 (m,
NHCHCO of Phe), 7.16 (d, J = 7.9 Hz, NH), 7.23 (m, 3 ꢁ CH of CH2Ph),
7.29 (m, 2 ꢁ CH of CH2Ph), 7.74 and 7.88 (2 d, J = 8.4 Hz, 4 ꢁ CH of
4-CN-Ph-CO). LRMS for C42H57N4O4 [M+H]+: 682.4 m/z.
2.1.3.6.22.
(4S)-N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypreg-
nan-2-yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]-4-(prop-1-en-
2-yl)cyclohex-1-ene-1-carboxamide (LFC-46): 7.5 mg. 1H NMR
(CDCl3) d: 0.76 (s, 18-CH3), 0.81 (s, 19-CH3), 1.14 (d, J = 6.0 Hz,
21-CH3), 1.74 (s, CH2@CCH3), 0.72–2.20 (residual CH and CH2),
2.1.3.6.15. 4-{[(2R)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-
yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]carbonyl}benzonitrile
(DPC-41): 14 mg. 1H NMR (CDCl3) d: 0.73 (s, 18-CH3), 0.84 (s, 19-
CH3), 1.13 (d, J = 6.0 Hz, 21-CH3), 0.68–2.32 (residual CH and
2.22–2.60 (broad m, 2 ꢁ CH2N and 2
a-CH), 2.90–3.82 (broad m,
2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 4.74 (d, J = 16.2 Hz, CH2-
@CCH3), 5.23 (m, NHCHCO of Phe), 6.60 (d, J = 8.1 Hz, NH), 6.70
(s, CH@CACO), 7.20 (m, 3 ꢁ CH of CH2Ph), 7.29 (m, 2 ꢁ CH of CH2-
Ph). LRMS for C44H66N3O4 [M+H]+: 700.5 m/z.
CH2), 2.40–2.90 (broad m, 2 ꢁ CH2N and 2
a-CH), 3.35–3.90 (broad
m, 3 ꢁ CH2NCO, 20-CH and 3b-CH), 5.05 (dd, J1 = 5.5 Hz, J2 = 8.2 Hz,
NCHCO of Pro), 7.69 (q of AB system, 4 ꢁ CH of 4-CN-Ph-CO). LRMS
for C38H55N4O4 [M+H]+: 631.4 m/z.
2.1.3.6.23. {4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-2-yl]piperazin-
2.1.3.6.16.
4-cyano-N-[(2R)-1-{4-[(2b,3
a
,5
a
)-3,20-dihydroxypreg-
1-yl}[(2R)-1-{[(4S)-4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]car-
bonyl}pyrrolidin-2-yl]methanone (DPC-46): 6.2 mg. 1H NMR (CDCl3)
d: 0.74 (s, 18-CH3), 0.84 (s, 19-CH3), 1.13 (d, J = 6.0 Hz, 21-CH3),
1.74 (s, CH2@CCH3), 0.70–2.30 (residual CH and CH2), 2.32–2.87
nan-2-yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]benzamide
(DFC-41): 5.4 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3), 0.82 (s, 19-
CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.72–1.85 (residual CH and CH2),
2.10, 2.39 and 2.60 (3 m, 2 ꢁ CH2N and 2
a
-CH), 3.00–3.80 (broad
(broad m, 2 ꢁ CH2N and 2 -CH), 3.35–3.90 (broad m, 3 ꢁ CH2NCO,
a
m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.30 (m, NHCHCO of
Phe), 7.26 (m, CH2Ph and NH), 7.74 and 7.88 (2 d, J = 8.4 Hz,
4 ꢁ CH of 4-CN-Ph-CO). LRMS for C42H57N4O4 [M+H]+: 681.5 m/z.
20-CH and 3b-CH), 4.73 (m, CH2@CCH3), 4.93 (d, J = 7.7 Hz, NCHCO
of Pro), 6.12 (s, CH@CACO). LRMS for C40H64N3O4 [M+H]+: 650.6 m/
z.
2.1.3.6.17. (2E)-1-[(2S)-2-({4-[(2b,3
a,5a
,20R)-3,20-dihydroxypreg-
2.1.3.6.24.
(4S)-N-[(2R)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypreg-
nan-2-yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]-3-phenylprop-2-
en-1-one (LPC-42): 11 mg. 1H NMR (CDCl3) d: 0.75 (s, 18-CH3), 0.85
(s, 19-CH3), 1.13 (d, J = 6.0 Hz, 21-CH3), 0.70–2.30 (residual CH and
nan-2-yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]-4-(prop-1-en-
2-yl)cyclohex-1-ene-1-carboxamide (DFC-46): 2.6 mg. 1H NMR
(CDCl3) d: 0.76 (s, 18-CH3), 0.81 (s, 19-CH3), 1.14 (d, J = 6.0 Hz,
21-CH3), 1.75 (s, CH2@CCH3), 0.70–2.20 (residual CH and CH2),
CH2), 2.43 and 2.69 (2 m ꢁ CH2N and 2
a-CH), 3.40–3.97 (m,
3 ꢁ CH2NCO, 20-CH and 3b-CH), 4.99 (dd, J1 = 3.7 Hz, J2 = 8.2 Hz,
NCHCO of Pro), 6.77 (d, J = 15.5 Hz, CH@CHCO), 7.36 (m, 3 ꢁ CH
of Aryl), 7.51 (m, 2 ꢁ CH of Aryl), 7.70 (d, J = 15.5 Hz, CH@CHCO).
LRMS for C39H58N3O4 [M+H]+: 632.4 m/z.
2.25–2.62 (broad m, 2 ꢁ CH2N and 2
a-CH), 2.90–3.80 (broad m,
2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 4.74 (d, J = 16.2 Hz, CH2-
@CCH3), 5.22 (m, NHCHCO of Phe), 6.60 (d, J = 8.0 Hz, NH), 6.69
(s, CH@CACO), 7.22 (m, 3 ꢁ CH of CH2Ph), 7.29 (m, 2 ꢁ CH of CH2-
Ph). LRMS for C44H66N3O4 [M+H]+: 700.6 m/z.
2.1.3.6.18.
(2E)-N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypreg-
nan-2-yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]-3-phenylprop-
2-enamide (LFC-42): 4.5 mg. 1H NMR (CDCl3) d: 0.75 (s, 18-CH3),
0.81 (s, 19-CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.70–1.88 (residual
2.1.3.6.25. 1-(3-{[(2S)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-
2-yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]carbonyl}phenyl)etha-
none (LPC-48): 11 mg. 1H NMR (CDCl3) d: 0.74 (s, 18-CH3), 0.84 (s,
19-CH3), 1.13 (d, J = 6.0 Hz, 21-CH3), 0.70–2.35 (residual CH and
CH and CH2), 2.15 and 2.55 (2 m, 2 ꢁ CH2N and 2
a-CH), 2.96–
3.80 (broad m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.30 (m,
NHCHCO of Phe), 6.42 (d, J = 15.6 Hz, CH@CHCO), 6.59 (m, NH),
7.25 (m, CH2Ph), 7.37 (m, 3 ꢁ CH of Aryl), 7.51 (m, 2 ꢁ CH of Aryl),
7.63 (d, J = 15.6 Hz, CH@CHCO). LRMS for C43H60N3O4 [M+H]+:
682.4 m/z.
CH2), 2.40–2.75 (broad m, 2 ꢁ CH2N and 2
a-CH), 2.62 (s, CH3CO),
3.40–3.90 (broad m, 3 ꢁ CH2NCO, 20-CH and 3b-CH), 5.07 (dd,
J1 = 5.7 Hz, J2 = 8.1 Hz, NCHCO of Pro), 7.51 (t, J = 7.7 Hz, CH of Aryl),
7.79 (d, J = 7.6 Hz, CH of Aryl), 8.02 (d, J = 7.8 Hz, CH of Aryl), 8.16
(s, CH of Aryl). LRMS for C39H58N3O5 [M+H]+: 648.5 m/z. See Sec-
tion 2.1.4 for additional characterization of this compound.
2.1.3.6.19. (2E)-1-[(2R)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypreg-
nan-2-yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]-3-phenylprop-2-
en-1-one (DPC-42): 6.4 mg. 1H NMR (CDCl3) d: 0.74 (s, 18-CH3),
0.84 (s, 19-CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.72–2.30 (residual
2.1.3.6.26.
3-acetyl-N-[(2S)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxy-
pregnan-2-yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]benzamide
(LFC-48): 4.0 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3), 0.82 (s, 19-
CH3), 1.14 (d, J = 6.1 Hz, 21-CH3), 0.70–1.85 (residual CH and
CH and CH2), 2.40–2.90 (broad m, 2 ꢁ CH2N and 2
a-CH), 3.35–
3.95 (broad m, 3 ꢁ CH2NCO, 20-CH and 3b-CH), 5.00 (m, NCHCO
of Pro), 6.77 (d, J = 15.5 Hz, CH@CHCO), 7.35 (m, 3 ꢁ CH of Aryl),
7.52 (m, 2 ꢁ CH of Aryl), 7.70 (d, J = 15.5 Hz, CH@CHCO). LRMS
for C39H58N3O4 [M+H]+: 632.5 m/z.
CH2), 2.10, 2.20 and 2.57 (m, 2 ꢁ CH2N and 2
a-CH), 2.65 (s, CH3CO),
3.02–3.82 (m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.37 (m,
NHCHCO of Phe), 7.16 (d, J = 8.0 Hz, NH), 7.28 (m, CH2Ph), 7.55 (t,
J = 7.8 Hz, CH of Aryl), 7.98 (d, J = 7.9 Hz, CH of Aryl), 8.11 (d,
J = 7.8 Hz, CH of Aryl), 8.36 (s, CH of Aryl). LRMS for C43H60N3O5
[M+H]+: 698.4 m/z.
2.1.3.6.20.
(2E)-N-[(2R)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxypreg-
nan-2-yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]-3-phenylprop-
2-enamide (DFC-42): 4.1 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3),
0.81 (s, 19-CH3), 1.14 (d, J = 6.0 Hz, 21-CH3), 0.71–1.88 (residual
2.1.3.6.27. 1-(3-{[(2R)-2-({4-[(2b,3a,5a,20R)-3,20-dihydroxypregnan-
2-yl]piperazin-1-yl}carbonyl)pyrrolidin-1-yl]carbonyl}phenyl)etha-
none (DPC-48): 11 mg. 1H NMR (CDCl3) d: 0.73 (s, 18-CH3), 0.84 (s,
19-CH3), 1.13 (d, J = 5.9 Hz, 21-CH3), 0.68–2.30 (residual CH and
CH and CH2), 2.08, 2.38 and 2.57 (3 m, 2 ꢁ CH2N and 2
a-CH),
2.98–3.80 (m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.30 (m,
NCHCO of Phe), 6.42 (d, J = 15.6 Hz, CH@CHCO), 6.58 (d,
J = 8.0 Hz, NH), 7.26 (m, CH2Ph), 7.38 (m, 3 ꢁ CH of Aryl), 7.51
CH2), 2.44–2.90 (broad m, 2 ꢁ CH2N and 2
a-CH), 2.62 (s, CH3CO),
3.40–3.92 (broad m, 3 ꢁ CH2NCO, 20-CH and 3b-CH), 5.07 (dd,

H. Jegham et al. / Steroids 77 (2012) 1403–1418
1409
J1 = 5.7 Hz, J2 = 8.1 Hz, NCHCO of Pro), 7.51 (t, J = 7.7 Hz, CH of Aryl),
7.79 (d, J = 7.6 Hz, CH of Aryl), 8.02 (d, J = 7.8 Hz, CH of Aryl), 8.16
(s, CH of Aryl). LRMS for C39H58N3O5 [M+H]+: 648.4 m/z.
streptomycin/mL) and stimulated by leucoagglutinin (PHA-L)
(Sigma–Aldrich, Oakville, ON, Canada) in a 5% CO2 humidified
atmosphere at 37 °C [13,14].
2.1.3.6.28.
3-acetyl-N-[(2R)-1-{4-[(2b,3a,5a,20R)-3,20-dihydroxy-
pregnan-2-yl]piperazin-1-yl}-1-oxo-3-phenylpropan-2-yl]benzamide
(DFC-48): 3.0 mg. 1H NMR (CDCl3) d: 0.76 (s, 18-CH3), 0.82 (s, 19-
CH3), 1.14 (d, J = 6.1 Hz, 21-CH3), 0.68–1.85 (residual CH and CH2),
2.2.2. Cell proliferation and viability assay
2.2.2.1. Inhibition of HL-60 cells proliferation. Triplicate cultures of
1 ꢁ 104 cells in a total of 100
lL medium in 96-well culture plates
2.08, 2.40 and 2.60 (3 m, 2 ꢁ CH2N and 2a-CH), 2.65 (s, CH3CO),
were incubated at 37 °C, 5% CO2. Doxorubicin (Novapharm,
Toronto, ON, Canada) was used as positive control. Compounds were
dissolved in ethanol to prepare the stock solution of 1 ꢁ 10ꢀ2 M.
These compounds and doxorubicin were diluted at multiple con-
centrations in culture medium, added to each well, and incubated
3.02–3.80 (m, 2 ꢁ CH2NCO, 20-CH, 3b-CH and CH2Ph), 5.37 (m,
NHCHCO of Phe), 7.16 (d, J = 7.9 Hz, NH), 7.29 (m, CH2Ph), 7.55 (t,
J = 7.7 Hz, CH of Aryl), 7.98 (d, J = 7.8 Hz, CH of Aryl), 8.11 (d,
J = 7.8 Hz, CH of Aryl), 8.36 (s, CH of Aryl). LRMS for C43H60N3O5
[M+H]+: 698.4 m/z.
for 72 h. Following each treatment, 20 lL of 3-(4,5-dimethylthia-
zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-H-tet-
razolium (MTS) reagent (Promega, Madison, WI, USA) were added
to each well and the reaction was stopped after 4 h. MTS is con-
verted to water-soluble colored formazan by dehydrogenase en-
zymes present in metabolically active cells. Subsequently, the
plates were read at 490 nm using a microplate reader (Molecular
Devices, Sunnyvale, CA, USA). Data were reported as the percent-
age of cell proliferation inhibition compared to the control (basal
2.1.4. Purification and additional characterization of LPC-37 and LPC-
48
The two aminosteroids selected for a preliminary investigation
of their mechanism of action on HL-60 cells, LPC-37 and LPC-48,
were resynthesized using 2 ꢁ 375 mg of resin 7 (0.56 mmol/g) fol-
lowing the general procedure described above for 8 and 9. The two
compounds released from the resin were purified by flash chroma-
tography using DCM/MeOH (95:5) as eluent to give LPC-37 and
LPC-48.
cell proliferation) for two concentrations of 1 lM and 10 lM. For
IC50 determination, the percentage of cell proliferation was plotted
against the drug concentration ranging from 10ꢀ8 to 10ꢀ4 M and
the value resulting in 50% cell growth inhibition was determined.
2.1.4.1. LPC-37: 49 mg. IR (ATR) m
: 3433 (OH) and 1636 (CON). 1H
NMR and LRMS data are reported in previous section. 13C NMR
(CDCl3) d: 12.2, 17.2, 21.2, 23.6, 24.4, 25.6, 28.3, 29.4, 29.7, 31.6,
32.9, 34.6, 35.3, 35.8, 38.4, 40.1, 42.6, 46.3, 48.2, 48.8, 50.3, 55.8,
56.0, 56.2, 58.5, 63.8, 64.9, 70.6, 124.5, 126.5, 127.1, 127.4, 127.7,
128.0, 128.6, 132.5, 133.7, 133.9, 169.5, 170.4. HRMS for
2.2.2.2. Inhibition of peripheral blood lymphocytes proliferation. The
antiproliferative activity of tested compounds and doxorubicin in
PBL was assessed by culturing cells (1 ꢁ 105 cells/100
lL culture
medium) in 96-well culture plates in presence of leucoagglutinin
C
41H58N3O4 [M+H]+: 656.4422, found 656.4425. HPLC purity:
(PHA-L) (2
were then added and treated cells were incubated for 72 h. After
each incubation period, MTS (20 L) was added to each well. The
lg/mL culture medium) for 48 h, compounds to test
93.4%.
l
2.1.4.2. LPC-48: 52 mg. IR (ATR)
m
: 3425 (OH), 1690 (CO) and 1632
reaction was stopped after 4 h. Absorbance was plotted against
(CON). 1H NMR and LRMS data are reported in previous section.
13C NMR (CDCl3) d: 12.6, 17.2, 21.2, 23.6, 24.4, 25.5, 25.6, 26.7,
28.3, 29.4, 31.6, 33.1, 34.7, 35.3, 35.8, 38.4, 40.1, 42.6, 46.1, 48.0,
48.7, 50.2, 55.8, 55.9, 56.0, 56.4, 58.5, 63.9, 65.0, 70.5, 127.4,
128.7, 129.7, 131.8, 136.8, 137.1, 168.4, 170.1, 197.5. HRMS: calcu-
lated for C39H58N3O5 [M+H]+: 648.4371, found 648.4376. HPLC
purity: 92.4%.
the drug concentration and IC50 values were determined [13,15].
2.2.2.3. Assay for cell viability. Cell viability was determined by the
trypan blue dye exclusion test. HL-60 cells were plated into a 6-
well plate at 2.5 ꢁ 105 cells/mL in a total volume of 2 mL. Cells
were then exposed or not (control) for 24, 48 or 72 h at 37 °C to
LPC-37 or LPC-48. After each incubation period, the number of cells
that did not take up trypan blue was counted using a haemocytom-
eter [16].
2.2. Biological evaluations
2.2.1. Cellular material
2.2.1.1. Human promyelocytic leukemia cells. HL-60 (ATCC,
Rockville, MD, USA) were routinely grown in suspension in 90%
RPMI 1640 (Sigma, Saint Louis, MO, USA) containing
(2 nM), and antibiotics (100 IU penicillin/mL, 100 g streptomy-
2.2.3. Flow cytometric cell cycle analysis
HL-60 cells were plated at a density of 1 ꢁ 106 cells per
100 mm ꢁ 15 mm culture dish in a total volume of 10 mL and ex-
posed or not (control) for 24, 48 or 72 h at 37 °C to LPC-37 or LPC-
48. After treatment, cells were collected by centrifugation for 5 min
L-glutamine
l
cin/mL) and supplemented with 10% (v/v) fetal bovine serum
(FBS), in a 5% CO2 humidified atmosphere at 37 °C. Cultures were
maintained two times per week by addition of fresh medium or
replacement of medium.
at 400g, resuspended in 300 lL of ice-cold PBS, fixed in ice-cold
95% ethanol and stored at ꢀ20 °C for at least 30 min. Fixed cells
were collected by centrifugation and pellets were resuspended in
PBS containing RNase A (500 UI/mL). Samples were kept at room
temperature for 30 min. To determine cellular DNA content, cells
2.2.1.2. Peripheral blood lymphocytes isolation. Normal human
peripheral blood lymphocytes (PBL) were isolated from buffy coats
kindly provided by Dr. Jean Sévigny (CHUQ-CHUL Research Cen-
ter). In summary, mononuclear cells were isolated on Histopaque
(Sigma–Aldrich, Oakville, ON, Canada) density gradient
(d = 1.077) by centrifugation (300g, 30 min). They were then
washed in 0.02 M phosphate-buffered saline (PBS) at pH 7.2. Fur-
ther purification of lymphocytes from peripheral blood mononu-
clear cells was performed by adhesion of monocytes to plastic
plates for 4 h at 37 °C. Lymphocytes were grown in RPMI 1640
were stained with propidium iodide (50 lg/mL), incubated for
20 min on ice then analyzed by flow cytometry using a Coulter
EPICS XL (Beckman-Coulter, Miami, FL, USA). The percentages of
cells in G0/G1, S and G2/M phases and the percentage of cells in
sub-G0/G1 peak were calculated using MultiCycle AV Software
(Phoenix Flow Systems, San Diego, CA, USA) that eliminated the
debris effect [17].
2.2.4. Morphological analysis with fluorescence microscopy
To evaluate the apoptotic activity of LPC-48, we performed nu-
clear staining with the DNA-binding dye Hoechst 33342 (Invitro-
gen, Burlington, ON, Canada). HL-60 cells were plated into 6-well
medium (1 ꢁ 106 cells/mL) supplemented with 10% FBS,
L-gluta-
mine (2 nM) and antibiotics (100 IU penicillin/mL, 100 lg

1410
H. Jegham et al. / Steroids 77 (2012) 1403–1418
plates at 106 cells/well and exposed or not (control) to increasing
concentrations of LPC-48 for 72 h. Cells were collected by centrifu-
gation at 200g for 5 min, washed with ice-cold PBS and then fixed
with 2% paraformaldehyde in PBS for 10 min at 4 °C. Fixed cells
found too much costly to use allopregnanolone (3a-OH) to gener-
ate 1. In fact, considering the cost of this commercially available
natural product, we found advantageous to use epi-allopregnano-
lone as starting material rather than allopregnanolone (1200-fold
more costly), even with an expected twice lowest yield of 1. Thus,
the reaction on epi-allopregnanolone with 1.2 eq of DAST at ꢀ78 °C
in DCM gave the pregnene 1 with a high C-2,3 ratio (98:2) but a
moderate yield of 25% (entry 18). The DAST procedure was found
more appropriate than the classical methods for alcohol dehydra-
tation previously used [10], which always gave a higher amount
(>10%) of undesired steroidal C-3,4 androstene. We also favored
the one-step DAST procedure over the five-step classical procedure
that we previously used starting from dihydrotestosterone, a 3-
keto androstane derivative [11].
were washed with PBS, incubated with Hoechst 33342 (10
mL) for 15 min in the dark, then placed on slides and observed un-
der fluorescence microscope (excitation 352 nm, emission
lg/
a
461 nm; NIKON TE2000-E). Apoptotic cells were identified by con-
densation of chromatin and fragmentation of nuclei. Pictures were
obtained at 40ꢁ, using a video camera Q-imaging (Burnaby, BC,
Canada) [18].
3. Results and discussion
The carbonyl of 1 was stereoselectively reduced with DIBAL-H
to give the corresponding alcohol 2 in 78% yield (Fig. 2). Following
Cram’s rule [21], and in accordance with NMR data [22], the 20R
isomer (or 20b-OH) was isolated. The subsequent epoxidation of
3.1. Synthesis of new pregnane derivatives
3.1.1. Synthesis of solid phase precursor 5
alkene 2 with m-CPBA stereoselectively afforded the 2,3a-epoxide
Compound 5, the pregnane to be linked on a polymeric support
for the solid-phase synthesis, was obtained following a 5-step
sequence of reactions (Fig. 2). In the first step, the 3b-OH of epi-
allopregnanolone was dehydrated regioselectively using diethyla-
minosulfur trifluoride (DAST) as dehydrating reagent. The proce-
dure was adapted from a previously published result showing
that a C-2,3 alkene was obtained as a minor side-product when
androsterone (ADT) was fluorinated with DAST [19,20]. In fact,
we optimized the reaction conditions using the steroid scaffold
ADT and epi-ADT (Fig. 3) in order to maximize the C-2,3 vs. C-3,4
alkene ratio (inseparable mixture) as well as the reaction yield
(Table 1). The use of 1.2 equivalent of DAST at ꢀ78 °C in dichloro-
methane (DCM) gave interesting ratio for ADT (95:5) and epi-ADT
(96:4) with a higher yield of alkene for ADT (57%) than for epi-ADT
(30%) (entries 3 and 12). Changing DCM for THF or toluene did not
improve the quantity of C-2,3 alkene (entries 3, 8 and 9). The use of
dimethylaminosulfur trifluoride (Me-DAST) instead of DAST gave
the same yield and alkene ratio (entries 15–17). Although the best
3 (83% yield), which was next regio- and stereo-selectively opened
with piperazine to give 4. N-Fmoc protection of the NH of piperaz-
ino derivative 4 completed the synthesis of the solid-phase precur-
sor 5.
3.1.2. Solid-phase synthesis of libraries A and B
N-Fmoc-2b-piperazino-5a-pregnan-3a,20b-diol (5) was cou-
pled to chlorosilyl resin, which was previously generated in situ
from butyldiethylsilane polystyrene (PS-DES) resin. In the coupling
reaction, the resin was swelled in dry DCM and treated with imid-
azole and the steroid 5 (Fig. 4) to give the coupled steroid 6. Sim-
ilarly as previously observed for the synthesis of androstane
derivatives [11], the low reactivity of the 3a-OH group at proxim-
ity of a bulky N-Fmoc-piperazine group allowed a regioselective
coupling at the more reactive 20-OH. The Fmoc protecting group
of 6 was easily removed with a solution of 20% piperidine in
DMF and the compound 7 was obtained by coupling a N-Fmoc ami-
no acid thus introducing the first level of diversity. In the next step,
the N-Fmoc group of 7 was hydrolyzed and the steroid-bound resin
yield of alkene was obtained when dehydrating a 3a-OH group,
such as ADT, instead of a 3b-OH group, such as epi-ADT, it was
epi-allopregnanolone
a
O
OH
OH
c
b
O
3
1
2
d
OH
OH
HN
FmocN
N
N
e
HO
HO
4
5
Fig. 2. Synthesis of 5 from epi-allopregnanolone. Reagents and conditions: (a) DAST, DCM, ꢀ78 °C, 1 h, 25% yield; (b) DIBAL-H, DCM, ꢀ78 °C, 78% yield; (c) m-CPBA, DCM, 0 °C,
73% yield; (d) piperazine, H2O, 160 °C, 24 h, 83% yield; (e) Fmoc-OSu, NaHCO3 (1 M), THF/H2O (5:1, v/v), rt, 65% yield.

H. Jegham et al. / Steroids 77 (2012) 1403–1418
1411
O
O
R2
R1
HO
ADT (R1 = OH, R2 = H)
epi-ADT (R1 = H, R2 = OH)
epi-allopregnanolone
a
Me
F
Et
H
Me
b
H
F
H
H
N
S
c
F
F
H
Et
HO
H
H
O
H
Et2NHSF2
DAST
Alcohol
Intermediate obtained
from ADT
a
b
c
19
Me
Me
Me
2
1
F
4
3
H
H
H
2,3-Alkene (Δ2)
3,4-Alkene (Δ3)
Fluoride
Fig. 3. Synthesis of C-2,3 and C-3,4 alkenes from the corresponding steroidal alcohol using DAST as dehydrating agent.
Table 1
Optimisation of dehydration reaction (synthesis of steroidal alkenes from the corresponding C-3 alcohol).a
Entry
Steroidal alcoholb
Reagentc
Eq.d
T (°C)
Solvent
Yield of alkene (%)e
Steroidal alkene D2 D3 ratiof
:
1
2
3
4
5
6
7
8
ADT (3
ADT (3
ADT (3
ADT (3
ADT (3
ADT (3
ADT (3
ADT (3
ADT (3
Epi-ADT (3b-OH)
Epi-ADT (3b-OH)
Epi-ADT (3b-OH)
Epi-ADT (3b-OH)
Epi-ADT (3b-OH)
Epi-ADT (3b-OH)
a
a
a
a
a
a
a
a
a
-OH)
-OH)
-OH)
-OH)
-OH)
-OH)
-OH)
-OH)
-OH)
DAST
DAST
DAST
DAST
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
1.2
2.4
1.2
1.2
1.2
1.2
0
ꢀ40
ꢀ78
ꢀ90
ꢀ78
ꢀ78
ꢀ78
ꢀ78
ꢀ78
0
ꢀ40
ꢀ78
ꢀ90
ꢀ78
ꢀ78
ꢀ78
ꢀ78
ꢀ78
DCM
DCM
DCM
DCM
DCM
DCM
DCM
THF
Toluene
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
55
ND
57
56
60
ND
21
ND
ND
31
ND
30
26
40
29
78
28
25
91:9
93:7
95:5
94:6
93:7
94:6
87:13
88:12
92:8
94:6
95:5
96:4
97:3
95:5
97:3
94:6
97:3
98:2
DASTg
DASTh
DASTi
DAST
9
DAST
DAST
DAST
DAST
DAST
DAST
Me-DAST
Me-DAST
Me-DAST
DAST
10
11
12
13
14
15
16
17
18
ADT (3a-OH)
Epi-alloPREG (3b-OH)
Epi-alloPREG (3b-OH)
a
The reaction was performed in anhydrous conditions (argon atm.). After 1 h, the mixture was evaporated in presence of silica gel and the solid added on the top of a silica
gel column to perform a flash chromatography.
b
Androsterone (ADT) is an androstane C-19 steroid; epi-allopregnanolone is a pregnane C-21 steroid.
DAST, diethylaminosulfur trifluoride; Me-DAST, dimethylaminosulfur trifluoride.
Equivalent (Eq.) of reagent vs. the steroidal alcohol.
c
d
e
Yield of 2,3- and 3,4-alkenes isolated after a silica gel flash-column chromatography. ND: yield not determined.
f
Ratio of 2,3-alkene (
Concentrated.
D D
2) and 3,4-alkene ( 3) determined by 1H NMR spectroscopy.
g
h
i
Diluted (5ꢁ).
Triethylamine was added to the reaction mixture.

1412
H. Jegham et al. / Steroids 77 (2012) 1403–1418
Table 2
Table 3
Library A compounds with their elements of molecular diversity (R1 and R2) and % of
Library B compounds with their elements of molecular diversity (R1 and R2) and % of
HL-60 cell growth inhibition reported at two concentrations (1
l
M/10
l
M).a
HL-60 cell growth inhibition reported at two concentrations (1
l
M/10
l
M).a
OH
OH
R2 R1
R2 R1
N
H
N
N
H
N
H
H
H
H
HO
HO
H
H
R2
R1
R2
R1
O
O
O
O
O
O
O
H
H
N
H
N
N
N
N
N
N
L-Pro
L-Phe
D-Pro
D-Phe
L-Phe (F)
L-Tic (T)
L-Pro (P)
LPC-37
LFC-37
DPC-37
0/0
DFC-37
O
O
6/86a
(IC50 = 1.9 lM)
0/0
0/0
PA
FA
TA
O
0/75a
0/81
0/62
C-37
Isovaleric acid (A)
LPC-38
0/89
LFC-38
0/32
DPC-38
0/24
DFC-38
0/0
PC
0/93
FC
0/19
TC
O
O
0/66
CO-38
Cyclopentylpro-
pionic acid (C)
LPC-39
0/0
LFC-39
0/94
DPC-39
0/0
DFC-39
0/75
O
PD
13/88
FD
5/46
TD
0/42
O
O
O
C-39
Cyclohexyl-
carboxylic acid (D)
LPC-41
0/85
LFC-41
0/10
DPC-41
0/0
DFC-41
0/46
O
O
PN
FN
TN
2/76
0/0
0/45
NC
O
Nonanoic acid (N)
C-41
C-42
a
Human promyelocytic leukemia (HL-60) cells were incubated for three (3) days
with tested compounds and the cell growth inhibition reported in % at two con-
centrations (1 and 10 M). The number of metabolically active cells was measured
using the MTS methodology. The reference compound doxorubicin inhibited 90%
and 93% of cell concentration at 1 and 10 M, whereas the aminosteroid RM
LPC-42
7/83
LFC-42
0/0
DPC-42
0/72
DFC-42
0/0
l
l
inhibited 28% and 85% of cell growth at the same concentrations.
LPC-46
0/87
LFC-46
0/0
DPC-46
0/78
DFC-46
0/0
O
submitted to an acylation with a carboxylic acid (second level of
diversity) providing 8. The final compound 9 was released with
an acid treatment and neutralized with a 10% aqueous bicarbonate
solution using an automated work-up procedure. The pregnane
derivatives were prepared in parallel and are represented by the
H
C-46
O
LPC-48
7/82
(IC50 = 1.9 lM)
LFC-48
0/55
DPC-48
0/0
DFC-48
0/23
model libraries
A
(3 ꢁ 4 = 12 compounds, Table 2) and
B
(4 ꢁ 7 = 28 compounds, Table 3). All members of the libraries were
analyzed by TLC, and the results confirmed the good reactivity of
all building blocks used in the elaboration of the libraries, except
O C-48
for library A where the acylation with the bulky N-Fmoc-L-tetrahy-
a
Human promyelocytic leukemia (HL-60) cells were incubated for three (3) days
with tested compounds and the cell growth inhibition reported in % at two con-
centrations (1 and 10 M). The number of metabolically active cells was measured
using the MTS methodology. The reference compound doxorubicin inhibited 78%
and 84% of cell growth at concentrations of 1 and 10 M, respectively.
dro-isoquinoline-3-carboxylic acid did not reach completion. For
those incomplete reactions, we thus proceeded to a fast purifica-
tion on silica gel to remove the unreacted piperazino-steroid. All li-
brary members showed the expected M+H signal by LRMS analyses
whereas TLC analyses confirmed the library uniformity and al-
lowed us to perform a random sampling. These selected library
members were then characterized by 1H NMR. Furthermore, the
aminosteroids LPC-37 and LPC-48, showing the best antiprolifera-
l
l
tive activities on HL-60 cells, were resynthesized, purified by chro-
matography and fully characterized by IR, 1H NMR, 13C NMR,
HRMS, and their purity determined by HPLC. Additional NMR

H. Jegham et al. / Steroids 77 (2012) 1403–1418
1413
experiments (HSQC, HMBC, COSY and NOESY) of the representative
compound PD allowed the assignment of all carbon signals and key
pears that eleven compounds out of twelve inhibited the cell
growth at 10 M, but only the pregnane derivative PD, with
-proline as building block, inhibited the cell growth at 1 lM
l
proton signals (Fig. 5) as well as to confirm the 2b-piperazino-3
a-
L
OH substitution (Fig. 6).
(13%). For comparison, the androstane derivative RM, which
has the same side-chain at position 2b than PD but a different
steroid nucleus, inhibited 28% and 85% of HL-60 cell prolifera-
3.2. Biological evaluation
3.2.1. Primary screening
The cytotoxic activity of newly synthesized aminosteroids
from libraries A and B was evaluated on the human promyelo-
cytic leukemia HL-60 cell line. We chose this cell line because
the first steroid derivative reported to exert an inhibitory activ-
ity on leukemic cells was tested on HL-60 cells [11]. In addi-
tion, the latter cells are well characterized and required
simple maintenance in vitro. The cell proliferation assay was
tion at 1 and 10 lM, respectively.
The data from the first library indicated that the antiprolifera-
tive effect is modulated by the nature of the amino acid. We then
extended our structure–activity relationship study by testing com-
pounds from library B. The molecular diversity was enriched by
adding
D
-proline,
D-phenylalanine and new carboxylic acids as
building blocks (Table 3). Interestingly, the combination of
L
-pro-
line with a naphthalene-2 carbonyl, a 3-phenylacryloyl or a 3-acet-
ylbenzoyl resulted in three hits (LPC-37, LPC-42 and LPC-48).
When inverting the stereochemistry of the L-proline, the activity
of the related compounds drastically dropped. In fact, compounds
performed at concentrations of 1 and 10 lM for each compound
of libraries A and B, and the results were expressed as the per-
centage of cell growth inhibition (Tables 2 and 3). Our results
show that the 40 compounds (libraries A and B) were not sim-
ilarly effective against HL-60 cells. The aminosteroids from both
libraries have two levels of diversity. The first level is provided
LPC-37, LPC-42 and LPC-48 inhibited >81% of cell growth at 10
whereas their analogues bearing a -proline as first level of diver-
sity had no antiproliferative effect except DPC-42 which inhibited
72% of cell growth at 10 M. Only a weak potency was exhibited by
lM
D
l
compounds bearing a phenylalanine as amino acid. However, com-
pounds LFC-39 and DFC-39 having a 6,6-dimethyl-4-oxo-5,6-dihy-
dro-4,4-pyran-2-carbonyl as second level of molecular diversity
by an amino acid (L-proline, L-phenylalanine or L-tetrahydro-
zisoquinoline-3-carboxylic acid) whereas the second level origi-
nated from a carboxylic acid (isovaleric acid, cyclopentylprop-
ionic acid, cyclohexyl-carboxylic acid or nonanoic acid). From
the % of HL-60 cell growth inhibition reported in Table 2, it ap-
inhibited >75% of cell growth at 10
M.
lM, but they had no effect at
1
l
OH
Si
H
Si Cl
FmocN
a
N
+
HO
5
PS-DES-Resin-Cl
PS-DES Resin
b
Si
O
Si
O
R
O
FmocN
FmocN
N
N
R1
N
c, d
HO
HO
6
7
c, e
Si
O
O
O
R
N
R
N
OH
R2
R2
N
N
O
R1
N
O
R1
N
f
HO
HO
8
9
Fig. 4. Synthesis of libraries A and B. Reagents and conditions: (a) 1,3-dichloro-5,5-dimethylhydantoin, DCM, rt; (b) imidazole, DCM, rt; (c) piperidine 20% (v/v), DCM, rt; (d) N-
Fmoc-aminoacid, PyBOP, HOBt, DIPEA, DMF, rt; (e) carboxylic acid, PyBOP, HOBt, DIPEA, DMF, rt; (f) (i) HCl (2 M)/MeOH (AcCl + MeOH) in DCM (20:80, v/v); (ii) 10% NaHCO3.

1414
H. Jegham et al. / Steroids 77 (2012) 1403–1418
Fig. 5. NMR (HSQC) experiment of the representative aminosteroid PD showing all carbon (13C) signals and key proton (1H) signals.
Fig. 6. NMR (NOESY) experiment with PD showing the key interactions between 19-CH3 and 3b-CH (3a
-OH) and between 3b-CH and CH2-10 (2b-piperazine).
The whole results obtained from this primary screening as-
sessed the key role of the amino acid -proline (first level of molec-
quently synthesized in larger quantities, purified by chromatogra-
phy and their purity assessed by HPLC.
L
ular diversity) in the cytotoxic activity. The role of the carboxylic
acid introduced in the second level of molecular diversity is how-
ever less important. We then selected the two representative ami-
3.2.2. IC50 values determination and selective-cytotoxicity of LPC-37
and LPC-48
nosteroids LPC-37 and LPC-48 for
mechanistic investigations. These two aminosteroids were conse-
a
series of preliminary
As a complementary part for the primary screening, it was
thought worthwhile to evaluate the differential growth inhibitory

H. Jegham et al. / Steroids 77 (2012) 1403–1418
1415
Fig. 9. Number of viable HL-60 cells after a treatment with LPC-37 or LPC-48. The
cells (5 ꢁ 105) were exposed to 10
lM of each compound for 24, 48 and 72 h. The
viable cells were measured by the trypan blue exclusion method using
a
haemocytometer. Each data point represents the mean of a triplicate (mean SD).
The results are significantly different from the control. ⁄⁄p < 0.01 vs. control,
##p < 0.01 vs. 0 h.
Fig. 7. Selective cytotoxicity of compounds PD, LPC-37 and LPC-48 towards HL-60
cells (A) and normal PBL (B). Cells were exposed to the aminosteroid at
concentrations ranging from 10ꢀ8 M to 10ꢀ4 M, for 72 h. Single points are average
of triplicate.
Fig. 8. Inhibition of HL-60 cell growth by compounds LPC-37, LPC-48 and
doxorubicin (Doxo). The cells (5 ꢁ 104) were seeded in a total volume of 100
lL
and exposed to 10 lM of each compound for 24, 48 and 72 h. The cell proliferation
was measured by MTS method. Each data point represents the mean of a triplicate
(mean SD). The results are significantly different from the control. ⁄⁄p < 0.01 vs.
control.
Fig. 10. Cell cycle analysis of HL-60 cells treated with LPC-37 (5 lM) or LPC-48
(5 lM). After 24 h, 48 h and 72 h of treatment, cells were labelled with propidium
iodide and analyzed by flow cytometry. ⁄⁄p < 0.01 vs. control, ⁄p < 0.05 vs. control.
effect of aminosteroids LPC-37 and LPC-48 on both cancerous and
normal cell lines. We first determined their IC50 values (the con-
centration required to inhibit cell growth by 50%) on cancerous
(HL-60) cells as well as on normal PBL. Both LPC-37 and LPC-48
after 72 h of incubation (Fig. 7A). Since chemotherapy very often
induces severe side effects, which are in part a consequence of
destruction of normal cells, we also tested the aminosteroids on
gave the same cell growth inhibition (IC50 = 1.9 lM) on HL-60 cells

1416
H. Jegham et al. / Steroids 77 (2012) 1403–1418
normal cells. Our findings point to a selective effect between tumor
and normal cells of the aforementioned compounds. Indeed, as
illustrated in Fig. 7B, the normal PBL were about 16-fold less sen-
cells, and there were roughly, no viable LPC-48 treated cells after
48 h of treatment. Thus, the cytotoxicity of the two compounds
was confirmed.
sitive to the inhibitory activity of LPC-48, with an IC50 of 31.3
LPC-37 was slightly less selective as it displayed IC50 value of
9.5 M.
After assessing that the growth inhibitory effect of LPC-37 and
LPC-48 on HL-60 cells was dose-dependent, we examined whether
it was also time-dependent. As shown in Fig. 8, a treatment with
lM.
3.2.4. LPC-37 and LPC-48 induce a G0/G1 cell cycle arrest
l
Having assessed the cytotoxicity of the two pregnane deriva-
tives LPC-37 and LPC-48, we next investigated their preliminary
mechanism of action. Particular interest was focused on their effect
on the cell cycle and on apoptosis induction. We performed cell-
10 lM of either LPC-37 or LPC-48 led to almost 80% cell growth
cycle analysis of HL-60 cells treated with 5 lM of either of the
inhibition upon the first 24 h of treatment. Longer incubation re-
sulted in a weak increase in growth inhibition. Indeed, after 72 h
of treatment, LPC-37 and LPC-48 inhibited 85% and 90% of cell
growth, respectively. Thus, the maximum of cell growth inhibition
is reached in the first 24 h of treatment. In addition, the two tested
compounds proved to be nearly equipotent to doxorubicin at
two compounds at different time periods. The percentages of cells
in Sub-G0/G1, G0/G1, S and G2/M were calculated and presented in
Fig. 10. The two aminosteroids significantly increased the amount
of G0/G1-phase cell population (59% in control vs. 70% and 68% in
cells treated with LPC-37 and LPC-48, respectively). Concomitant
with this increase in the amount of G0/G1 cells, we observed a de-
crease in the amount of S cells (33% in control vs. 19% and 23% in
cells treated with LPC-37 and LPC-48, respectively). Moreover, a
significant accumulation of sub-G0/G1 cells was detected (20%
and 32% in cells treated with LPC-37 and LPC-48, respectively, vs.
8% in control). Cells in sub-G0/G1 are hypodiploid cells and their
accumulation suggests that they have undergone apoptosis. The
pattern of cell cycle distribution changes was the same after 48 h
and 72 h of treatment. Interestingly, the percentage of Sub-G0/G1
cells at the different time periods was more important in LPC-48
treated cells than in LPC-37 treated cells, which points to an en-
hanced and faster occurring toxicity for LPC-48. These results sug-
gest that the two pregnane derivatives LPC-37 and LPC-48 induce a
strong G0/G1 cell cycle block followed by apoptosis in HL-60 cells.
10 lM concentration.
3.2.3. LPC-37 and LPC-48 induce cell death in HL-60 cells
To determine whether LPC-37 and LPC-48 growth inhibition of
HL-60 cells was related to a cytotoxic or a cytostatic effect, we
evaluated the cell viability of HL-60 after treatment with 10 lM
of either compound for 24, 48 and 72 h. Apart from the MTS meth-
od, which evaluates cell growth but cannot distinguish between
growth arrest and apoptosis or necrosis; cell viability can be as-
sessed using the trypan blue exclusion test, which is based on
the principle that living cells possess intact cell membranes that
exclude this dye, whereas, non-viable cells (late apoptotic and ne-
crotic cells) with damaged plasma membrane take it up [23]. The
trypan blue staining revealed that the two compounds had a sim-
ilar cytotoxic effect on HL-60 cells (Fig. 9). It is noteworthy that cell
viability underwent a drastic decline in the early hours of treat-
ment, as LPC-37 and LPC-48 reduced the viability to 35% and
14%, respectively, after 24 h. An increasing effect was observed
for the two compounds with an increase in exposure time. In fact,
the number of viable cells treated with LPC-37 decreased to 95,000
3.2.5. Effects of LPC-48 on cell nuclear morphology and DNA
fragmentation
Apoptotic cells were first identified by a series of typical mor-
phological changes, and morphology still constitutes important
experimental proof of the underlying process [24]. To further
investigate the mechanism of this novel family of pregnane
Fig. 11. Nuclear morphology of HL-60 cells treated with 0 (A, control), 30 (B), 50 (C) and 80 (D) lM of LPC-48 for 72 h and stained with Hoechst 33342 dye. Fluorescent
pictures were taken at 40ꢁ magnification using a NIKON TE2000-E microscope. Nuclear condensation is evident in cells with bright, condensed and rounded nuclei.
Membrane blebbing and apoptotic bodies are also evident.

H. Jegham et al. / Steroids 77 (2012) 1403–1418
1417
derivatives inducing cytotoxicity, we selected compound LPC-48,
and used fluorescence microscopy to determine whether it in-
duced apoptosis or necrosis of HL-60 cells at the single cell level.
LPC-48 treated and untreated HL-60 cells were stained with the
synthetic fluorescent bisbenzimidazole dye Hoechst 33342. This
molecule is a cell membrane permeant, A–T base pair selective,
minor groove binding, double stranded DNA-selective binding
and live-cell stain [25]. Fig. 11 shows the representative morphol-
this new family of pregnane derivatives induces a G0/G1 cell cycle
arrest of HL-60 cells leading to apoptosis. Additional studies will be
necessary to elucidate their exact mechanism of action. Identifica-
tion of this mechanism would help us to rationalize our results and
design other active compounds more efficiently.
Acknowledgments
ogy of HL-60 cells when exposed to 0, 30, 50 and 80 lM of LPC-48
We thank the Canadian Institutes of Health Research (CIHR) and
the Cancer Research Society (CRS) for their financial support. We
also thank Dr. Charles Doillon (CHUQ-CHUL Research Center) for
providing the Hoechst 33342 dye, the use of the inverted micro-
scope and for helpful discussions. We greatly appreciated the gift
of buffy coats from Dr. Jean Sévigny (CHUQ-CHUL Research Cen-
ter). Thanks to Marie-Claude Trottier and Micheline Harvey for
NMR analyses and for careful reading of the manuscript,
respectively.
for 72 h. Numerous morphological changes typical to apoptosis
were detected: plasma membrane blebbing, cellular shrinkage,
mitochondria depolarization, chromatin condensation and DNA
fragmentation. The control HL-60 cells appeared normal with
round and homogeneous nuclei (Fig. 11A). After treatment with
30 lM of LPC-48, alterations in the structure, size and shape of
the cell nucleus were detected. Chromatins of condensed appear-
ance, cell shrinkage and nuclear fragmentation as well as a forma-
tion of apoptotic bodies were thus observed (Fig. 11B). In addition
to these morphological changes, the cells treated with 50 and
References
80 lM of LPC-48 showed an impairment of the plasma membrane
and cell disintegration (Fig. 11C and 11D).
[1] National Cancer Institute. Available at: www.cancer.gov/cancertopics/types/
leukemia.
The sub-G0/G1 peak on flow cytometry and the nuclear mor-
phology of LPC-48 treated cells strongly suggest that the new syn-
thesized pregnane derivatives trigger apoptosis in HL-60 cells [26].
The process by which LPC-37 and LPC-48 block cell cycle progres-
sion is still to be clarified. A Western-blot analysis would deter-
mine whether the two compounds regulate the expression of
G1-phase associated proteins. In addition, in order to assess that
the two aminosteroids induce apoptosis in treated HL-60 cells,
the translocation of phosphatidylserine at the cell surface can be
examined by an annexine binding assay.
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when using alternative known sequences of reaction. The solid-
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amino acids (first level of diversity) and carboxylic acids (second
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showing good activity in previous screenings on HL-60 cells
2,3
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a
a-androstane-
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5a
a
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[11,12]. We selected three amino acids (
level of diversity and four different carboxylic acids as capping
groups for library A and four amino acids ( -Pro, -Pro, -Phe,
-Phe) and seven carboxylic acids capping groups for library B.
L-Pro, L-Phe, L-Tic) as first
L
D
L
D
These two libraries are small in size, but their preparation opens
the door to the synthesis of bigger libraries of pregnane derivatives.
When we compared the pregnane derivative PD (Library A) to
RM, a first generation aminosteroid with an androstane nucleus
[11,27] in the same protocol, they gave similar antiproliferative
activity on HL-60 cells showing the potential of both pregnane
and androstane nuclei as molecular scaffold. When tested on nor-
mal lymphocytes, the pregnane derivatives LPC-37 and LPC-48 (Li-
brary B) were found to preferentially inhibit the proliferation of
cancerous HL-60 cells. Our data presented in this study show that
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