Synthesis and antitumor activities of a series of novel quinoxalinhydrazides

Article abstract of DOI:10.1016/j.bmc.2006.09.073

Recently, we discovered a novel class of anticancer compounds with remarkable potency in a panel of cancer cell lines. A prototype compound, SC144, showed significant in vivo efficacy in mice xenograft models of human breast cancer cells. Herein, we report on a new synthetic route to SC144 and the synthesis of several of its analogues in order to understand required features for activity. A one-step coupling of 7-fluoro-4-chloropyrrolo[1,2-a]quinoxaline with pyrazin-2-carbohydrazide improved the yield significantly. Although several of the analogues showed significant activities, modification of the heteroacyl moiety had a dramatic effect on potency.

Full text of DOI:10.1016/j.bmc.2006.09.073

Bioorganic & Medicinal Chemistry 15 (2007) 288–294
Synthesis and antitumor activities of a series of novel
quinoxalinhydrazides
Fedora Grande,a,b Francesca Aiello,a,b Osvaldo De Grazia,a Antonella Brizzi,c
Antonio Garofaloa,* and Nouri Neamatib,*
a
`
Dipartimento di Scienze Farmaceutiche, Universita della Calabria, 87036 Arcavacata di Rende (Cs), Italy
bDepartment of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California,
1985 Zonal Avenue, Los Angeles, CA 90089, USA
c
`
Dipartimento Farmaco Chimico Tecnologico, Universita di Siena, Via A, Moro, 53100 Siena, Italy
Received 6 August 2006; revised 22 September 2006; accepted 26 September 2006
Available online 5 October 2006
Abstract—Recently, we discovered a novel class of anticancer compounds with remarkable potency in a panel of cancer cell lines. A
prototype compound, SC144, showed significant in vivo efficacy in mice xenograft models of human breast cancer cells. Herein, we
report on a new synthetic route to SC144 and the synthesis of several of its analogues in order to understand required features for
activity. A one-step coupling of 7-fluoro-4-chloropyrrolo[1,2-a]quinoxaline with pyrazin-2-carbohydrazide improved the yield sig-
nificantly. Although several of the analogues showed significant activities, modification of the heteroacyl moiety had a dramatic
effect on potency.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
interested in developing a coherent structure–activity
relationship amongst these novel compounds. There-
fore, in an effort to understand important features
for the remarkable antitumor activity of SC144, we
prepared a series of novel analogues to understand
the effect of substitution on the 2-carbohydrazide
moiety.
Recent advances in targeted therapeutics coupled with
new approaches in target identification have accelerat-
ed the need to design small-molecule compounds with
drug-like properties.1 Such molecules normally satisfy
the Lipinski’s rule-of-five and should preferably be
active orally.2,3 For targeted therapeutics against can-
cer, identification of lead compounds with novel
mechanisms of action, low toxicity, and enhanced
activity profiles is of paramount importance. Previ-
ously, we discovered that some of our small-molecule
HIV-1 integrase inhibitors exhibited remarkable cyto-
toxicity, which prompted us to seek to understand
their pharmacological properties.4 Although HIV-1
integrase has no cellular homologue, its inhibitors
may, however, inhibit other enzymes with similar ac-
tive site chemistry.5 Our extensive modifications of
some of those original leads4 resulted in the discovery
of a very promising analogue, SC144, with desirable
drug-like properties.6 Although the elucidation of
the mechanism of action of these compounds is un-
der active investigation in our laboratory, we were
2. Results and discussion
2.1. Chemistry
The synthesis of compounds designated as SC153–159
has been accomplished starting from the commercially
available 1-(2-aminophenyl)pyrrole 1. Compound 1
was treated with triphosgene in toluene under reflux to
give 2 in quantitative yield. The lactam obtained was
subsequently transformed into 4-chloro-1H-pyr-
rolo[1,2-a]quinoxaline 3 by treatment with phosphoryl
chloride. Reaction of 3 with hydrazine monohydrate in
DMF afforded 1-(H-pyrrolo[1,2-a]quinoxalin-4-yl)hy-
drazine 4. The hydrazine derivative was reacted with
the appropriate carboxylic acid in the presence of
EDC/DMAP to give SC155–159 (Scheme 1). The prep-
aration of SC153 and SC154 was accomplished by a
condensation step, using a procedure identical to that
*
Corresponding authors. Tel.: +1 323 442 2341; fax: +1 323 442 1390
(N.N.); e-mail addresses: garofalo@unical.it; neamati@usc.edu
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.09.073

F. Grande et al. / Bioorg. Med. Chem. 15 (2007) 288–294
289
Scheme 1.
described for the preceding compounds but using the
appropriate N-Boc-aminoacid, followed by a deprotec-
tion step by treatment with trifluoroacetic acid and ani-
sole (Scheme 1).
methyl carboxylates with hydrazine monohydrate in
refluxing ethanol.8
2.2. Antitumor activity
Compounds SC144 and SC160–166 were prepared by
reacting an appropriate chloro derivative of 5 with
hydrazine monohydrate to give pure fluoro-4-hydrazi-
nopyrrolo[1,2-a]quinoxalines 6. The subsequent N-acyl-
ation step with selected commercially available
carboxylic acids was performed using 2,20-dipyridyldi-
sulfide and triphenylphosphine as a condensing system.6
A more convenient approach for the synthesis of SC144
and SC160–166 (as hydrochloride salts) was discovered
by direct reaction of chloro derivatives of 5 with acy-
lhydrazides in ethanol under reflux (Scheme 2).7 Acy-
lhydrazides were prepared by the reaction of specific
All new compounds were tested in four human cancer
cell lines, a breast cancer cell, MDA-MB-435, and
three colon cancer cells. Compound SC153 was mod-
erately active against colon cancer lines and inactive
against the breast cancer line. All other indole ana-
logues (SC154–SC159) were inactive at 20 lM. In
the pyridino and pyrazino derivatives, the position
of the nitrogen atom on the ring appeared to be
important for activity. For example, SC160 and
SC162 were inactive, whereas SC161 was highly active
in all cell lines (Fig. 1). In all the cell lines we tested,
SC161, with IC50 values that range from 0.3 to 4 lM,
R1
N
N
NHNH
2
R2
.
R3COOH
NH2NH2 H2O
6
O
H
R1
R1
Cl
N
N
N
N
N
R3CONHNH2
N
R3
H
R2
R2
5
SC144; SC160; SC161;
SC162; SC163; SC164;
SC165; SC166
Scheme 2.

290
F. Grande et al. / Bioorg. Med. Chem. 15 (2007) 288–294
A
B
100
80
60
40
20
0
MDA-MB435
HCT116 p53+/+
HCT116 p53-/-
HT29
hLN-CaP
0.1
1
10
concentration (μM)
Figure 1. Compound SC161 shows remarkable activity in a panel of cell lines. (A) The IC50 values of SC161 range from 0.3 to 4 lM in representative
breast, colon, and prostate cell lines using MTT assay (B) SC161 completely block colony formation at doses P1 lM.
was more potent than the previously described
SC144.6 Interestingly, in colony formation assays at
doses above 1 lM, SC161 completely abolished cell
growth (Fig. 1). All compounds satisfy Lipinski’s
rule-of-five3 as calculated using ADMET PredictorTM
(Simulations Plus Inc.) (Table 1). For example,
SC161 has a molecular weight of 322.3 (MW < 500),
calculated log P of 1.82 (clog P < 5), two hydrogen
bond donors (HBD < 5), and six hydrogen bond
acceptors (HBA < 10). In addition, SC161, with only
three rotatable bonds, is very compact. The predicted
acidic and basic pKa values are also listed in Table 1.
Compound SC166 with IC50 values of 8–15 lM was
also active, but significantly less so than SC161. In
summary, while the substitution at the 2-carbohydraz-
ide moiety had a profound effect on activity, the posi-
tion of the fluorine atom on the benzo fused ring of
pyrroloquinoxaline did not seem to greatly influence
activity.
performed on a Perkin-Elmer 240C element analyzer,
and the results are within 0.4% of the theoretical
values. Yields refer to purified products and are
not optimized.
3.1. General procedure for the preparation of compounds
SC155–159
The preparation of 1H-indole-2-carboxylic acid N0-pyr-
rolo[1,2-a]quinoxalin-4-yl-hydrazide (SC155) is reported
as a representative example.
To a stirred solution of EDC (94 mg, 0.49 mmol)
and DMAP (cat.) in ethyl acetate (15 mL), 1-(H-pyr-
rolo-
[1,2-a]quinoxalin-4-yl)hydrazine
4
(77 mg,
0.39 mmol) and 2-indolecarboxylic acid (63 mg,
0.39 mmol) were added portionwise within 15 min.
The resulting mixture was stirred at room tempera-
ture for 24 h, then shaken with sodium bicarbonate
saturated solution and water. Evaporation of the
dried extract gave a residue, which was crystallized
to give SC155 as a white solid (82 mg, 62% yield);
3. Experimental
mp
IR (KBr) 3255, 1680 cmꢀ1
186 ꢁC
(dichloromethane/light
;
petroleum);
1H NMR (DMSO-d6)
All reactions were carried out under a nitrogen
atmosphere. Reaction progress was monitored by
TLC on silica gel plates (Merck 60, F254, 0.2 mm).
Organic solutions were dried over MgSO4. Evapora-
tion refers to removal of solvent on a rotary evapo-
rator under reduced pressure. Melting points were
measured using a Gallenkamp apparatus and are
uncorrected. IR spectra were recorded as thin films
on Perkin-Elmer 398 and FT 1600 spectrophotome-
6.75 (s, 1H), 7.05 (m, 1H), 7.20 (m, 4H), 7.40 (m,
3H), 7.65 (m, 1H), 8.10 (m, 1H), 8.35 (s, 1H),
9.55 (br s, 1H), 10.65 (br s, 1H), 11.80 (br s, 1H).
MS (CI) m/z 342 (MH+). Anal. (C20H15N5O) C,
H, N.
3.1.1. 1H-Indole-5-carboxylic acid N0-pyrrolo[1,2-a]qui-
noxalin-4-yl-hydrazide (SC156). Following the identical
procedure to that described for SC155, but using
2-indolecarboxylic acid (63 mg, 0.39 mmol), SC156
was obtained as a white solid (69 mg, 52% yield); mp
160 ꢁC (dichloromethane/light petroleum); IR (KBr)
ters. 1H NMR spectra were recorded on a Bruker
¨
300-MHz spectrometer with TMS as an internal
standard: chemical shifts are expressed in d values
(ppm) and coupling constants (J) in Hz. Mass spec-
tral data were determined by direct insertion at
70 eV with a VG70 spectrometer. Merck silica gel
(Kieselgel 60/230–400 mesh) was used for flash
chromatography columns. Elemental analyses were
3250, 1680 cmꢀ1 1H NMR (acetone-d6) 6.60 (d, 1H,
;
J = 3.6 Hz), 6.75 (t, 1H, J = 3.6 Hz), 7.23 (d, 1H,
J = 3.6 Hz), 7.29 (m, 2H), 7.51 (m, 3H), 7.85 (d, 1H,
J = 8.5 Hz), 8.03 (m, 1H), 8.20 (m, 1H), 8.39 (s, 1H),

F. Grande et al. / Bioorg. Med. Chem. 15 (2007) 288–294
291
9.60 (br s, 1H), 10.70 (br s, 1H), 11.45 (br s, 1H). MS
3.1.6. 3-Amino-propionic acid N0-pyrrolo[1,2-a]quinoxa-
lin-4-yl-hydrazide (SC154). Following a procedure iden-
tical to that described for SC153, but using N-Boc-R-
alanine (74 mg, 0.39 mmol), SC154 was obtained as a
white solid (92 mg, 88% yield based on compound 4).
Mp 164.5 ꢁC (dichloromethane/light petroleum); IR
(CI) m/z 342 (MH+). Anal. (C20H15N5O) C, H, N.
3.1.2. 1H-Indole-6-carboxylic acid N0-pyrrolo[1,2-a]qui-
noxalin-4-yl-hydrazide (SC157). Following a procedure
identical to that described for SC155, but using 6-indol-
ecarboxylic acid (63 mg, 0.39 mmol), SC157 was ob-
tained as a white solid (17 mg, 13% yield); mp
198.5 ꢁC (dichloromethane/light petroleum); IR (KBr)
1
(KBr) 3255, 1680 cmꢀ1; H NMR (DMSO-d6) 2.80 (m,
2H) 3.20 (m, 2H), 7.05 (m, 1H), 7.50 (m, 2H), 7.95 (m,
2H), 8.30 (m, 1H), 8.60 (m, 1H), 10.70 (br s, 1H),
11.25 (br s, 1H). MS (CI) m/z 270 (MH+). Anal.
(C14H15N5O) C, H, N.
1
3245, 1685 cmꢀ1; H NMR (acetone-d6) 6.55 (m, 1H),
6.85 (m, 1H), 7.28 (m, 1H), 7.28 (m, 3H), 7.45 (m,
1H), 7.60 (d, 1H, J = 8.1 Hz), 8.70 (m, 2H), 8.15 (s,
1H), 8.39 (m, 1H), 9.44 (br s, 1H), 10.55 (br s, 1H),
11.51 (br s, 1H). MS (CI) m/z 342 (MH+). Anal.
(C20H15N5O) C, H, N.
3.2. General procedure for the preparation of compounds
SC144 and SC160–166 (hydrochlorides)
The preparation of N0-(7-fluoropyrrolo[1,2-a]quinoxa-
lin-4-yl)pyrazine-2-carbohydrazidehydrochloride (SC144.HCl)
is reported as a representative example.
3.1.3. 1H-Indole-3-carboxylic acid N0-pyrrolo[1,2-a]qui-
noxalin-4-yl-hydrazide (SC158). Following a procedure
identical to that described for SC155, but using 3-indol-
ecarboxylic acid (63 mg, 0.39 mmol), SC158 was ob-
tained as a white solid (42 mg, 32% yield); mp
162.5 ꢁC (dichloromethane/light petroleum); IR (KBr)
A mixture of 7-fluoro-4-chloropyrrolo[1,2-a]quinoxaline
(200 mg, 0.90 mmol) and pyrazin-2-carbohydrazide
(125 mg, 0.90 mmol) in EtOH (2 mL) was refluxed for
5 h and then chilled overnight. The product was collect-
ed by filtration, washed with cold EtOH, and dried in
vacuo to give pure SC144.HCl (257 mg, 80 % yield).
mp 282 ꢁC (dec.) (methanol/ethyl acetate); IR (KBr)
1
3250, 1685 cmꢀ1; H NMR (CDCl3) 6.80 (m, 1H), 6.90
(t, 1H, J = 3.3 Hz), 7.08 (d, 1H, J = 3.2 Hz), 7.30–7.60
(m, 4H), 7.48 (m, 1H), 7.58 (m, 1H), 7.90 (m, 2H),
8.10 (m, 1H), 8.11 (s, 1H), 8.30 (m, 1H), 9.20 (br s,
1H), 10.25 (br s, 1H), 11.60 (br s, 1H). MS (CI) m/z
342 (MH+). Anal. (C20H15N5O) C, H, N.
3255, 1690 cmꢀ1 1H NMR (DMSO-d6) 3.75 (br s,
;
1H), 6.96 (m, 1H), 7.35 (t, 1H, J = 8.7 Hz), 7.68 (m,
2H), 8.30 (dd, 1H, J = 8.7, 4.8 Hz), 8.60 (s, 1H), 8.82
(m, 1H), 8.94 (d, 1H, J = 2.7 Hz), 9.22 (s, 1H), 11.67
(br s, 1H). MS (CI) m/z 323 (MH+). Anal.
(C16H12ClFN6O) C, H, N.
3.1.4.
yl)benzohydrazide (SC159). Following
2-Methoxy-N0-(H-pyrrolo[1,2-a]quinoxalin-4-
procedure
a
identical to that described for SC155, but using 2-
methoxybenzoic acid (60 mg, 0.39 mmol), SC159
was obtained as a white solid (98 mg, 75% yield);
mp 204.5 ꢁC (dichloromethane/light petroleum); IR
3.2.1. N0-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)pyridine-
2-carbohydrazide hydrochloride (SC160.HCl). Following
the same procedure described for compound
SC144.HCl, but using picolinohydrazide (124 mg,
0.90 mmol), SC160.HCl was obtained as a yellow solid
(263 mg; 82% yield). mp > 280 ꢁC (dec.) (ethanol/ethyl
acetate); IR (KBr) 3245, 1750 cmꢀ1; 1H NMR (CD3OD)
(ppm): 8.80 (dd, 2H, J = 6.2, 1.4 Hz); 8.45 (m, 1H); 8.18
(dd, 1H, J = 9.2, 4.9 Hz); 8.03 (d, 2H, J = 6.0); 7.60 (m,
1H); 7.47 (dd, 1H, J = 9.2, 2.7 Hz); 7.28 (m, 1H); 7.0
(dd, 1H J = 4.2, 2.8 Hz). MS (CI) m/z 322 (MH+). Anal.
(C17H13ClFN5O) C, H, N.
(KBr) 3200, 1675 cmꢀ1 1H NMR (DMSO-d6) 3.90
;
(s, 3H), 6.70 (m, 1H), 7.15 (m, 5H), 7.45 (m, 2H),
7.75 (m, 1H), 8.00 (m, 1H), 8.25 (br s, 1H), 9.75
(br s, 1H), 10.30 (br s, 1H). MS (CI) m/z 333
(MH+). Anal. (C19H16N4O2) C, H, N.
3.1.5. Thiazolidine-4-carboxylic acid N0-pyrrolo[1,2-
a]quinoxalin-4-yl-hydrazide (SC153). Starting from N-
Boc-thiazolidine-4-carboxylic acid (90 mg, 0.39 mmol),
tert-butyl-4-[(2-pyrrolo[1,2-a]quinoxalin-4-ylhydrazi-
no)carbonyl]-1,3- thiazolidine-3-carboxylate was ob-
tained as
a
solid, after crystallization (hexanes).
3.2.2.
N0-(9-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)pyra-
The solid obtained was added to a stirred mixture
of TFA (2 mL) and anisole (2 mL) at 0 ꢁC. The
reaction mixture was allowed to reach to room tem-
perature and stirred for a further 50 min. Evapora-
tion of the volatiles by azeotropization with
toluene (3 · 3 mL) gave SC153 as a pale yellow solid
(66 mg, 55% yield based on compound 4). mp
162 ꢁC (ethyl acetate/hexanes); IR (KBr) 3255,
zine-2- carbohydrazide hydrochloride (SC161.HCl). Fol-
lowing the same procedure described for compound
SC144.HCl, but using 4-chloro-9-fluoropyrrolo[1,2-
a]quinoxaline (200 mg, 0.90 mmol), SC161.HCl was
obtained as a yellow solid (303 mg; 94% yield). mp
248 ꢁC (dec.) (ethanol/ethyl acetate); IR (KBr) 3250,
1
1680 cmꢀ1; H NMR (DMSO-d6) (ppm): 10.90 (br s,
1H); 9.80 (br s, 1H); 9.20 (s, 1H); 8.90 (s, 1H); 8.80
(s, 1H); 8.10 (m, 1H); 7.20 (m, 5H); 6.80 (s, 1H).
MS (CI) m/z 323 (MH+). Anal. (C16H12ClFN6O) C,
H, N.
1690 cmꢀ1
;
1H NMR (methanol-d4) 3.15 (dd, 1H,
J = 10.9, 4.9) 3.30 (dd, 1H, J = 10.9, 7.1 Hz), 4.11
(0.5 of ABq, 1H, J = 9.7 Hz), 4.25 (0.5 of ABq,
1H, J = 9.7 Hz), 4.45 (dd, 1H, J = 7.1, 4.9 Hz), 6.92
(m, 1H), 7.41 (m, 3H), 7.71 (d, 1H, J = 7.4 Hz),
8.09 (d, 1H, J = 9.3 Hz), 8.38 (m, 1H), 10.40 (br s,
1H), 11.20 (br s, 1H). MS (CI) m/z 314 (MH+).
Anal. (C15H15N5OS) C, H, N.
3.2.3. N0-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)nicotinyl
hydrazide hydrochloride (SC162.HCl). Following the
same procedure described for compound SC144.HCl,
but using nicotinohydrazide (124 mg, 0.90 mmol),

Table 1. Cytotoxicity and physicochemical properties of quinoxalinhydrazides
Selected physicochemical propertiesb
S+ log P N_FrRotB HDB HBA
a
Compound
R
R1
IC50 (lM)
MDA-MB- 435 HCT 116 p53+/+ HCT 116 p53ꢀ/ꢀ HT 29 MW Acid-pred pKa Basic-pred pKa
H
N
SC153
SC154
SC155
>20
>20
>20
15
3
10
2
14
1
313.4 10.31
6.17, 3.71, ꢀ1.06, ꢀ3.85 1.21
8.54, 4.14, ꢀ0.81, ꢀ3.78 0.71
4.15, 1.54, ꢀ1.19, ꢀ3.94 3.21
4
5
3
3
3
3
5
5
4
S
>20
>20
>20
>20
>20
>20
269.3 10.4
NH2
H
N
341.4 9.48; 11.93
SC156
>20
>20
>20
>20
341.4 9.86; 14.18
4.13, 1.59, ꢀ0.69, ꢀ3.77 3.20
3
3
4
HN
SC157
SC158
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
341.4 9.87; 14.50
341.4 9.75; 12.75
332.3 9.69
4.17, 2.05, ꢀ0.59, ꢀ3.64 3.18
4.21, 2.11, ꢀ0.90, ꢀ3.80 3.19
3
3
4
3
3
2
4
4
5
N
H
NH
OCH3
SC159
3.92, ꢀ1.10, ꢀ4.17
3.18
Compound
R1
R2
R3
IC50 (lM)
Selected physicochemical properties
MDA-
MB-435
HCT 116
p53+/+
HCT 116
p53ꢀ/ꢀ
HT 29
hLN-
CaP
MW
Acid-
pred pKa
Basic-pred
pKa
S+log P
N_Fr RotB
HDB
HBA
5
N
N
SC160
SC161
F
H
F
>20
>20
>20
17
>20
0.4
321.3
9.3
3.69, 0.66, ꢀ1.32, ꢀ3.58
2.50
1.82
3
2
2
H
3
2
0.4 0.01
0.3 0.07
0.3 0.06
322.3
9.08
3.31, 0.91, ꢀ0.97, ꢀ2.79, ꢀ5.20
3
6
N

F. Grande et al. / Bioorg. Med. Chem. 15 (2007) 288–294
293
SC162.HCl was obtained as a white solid (231 mg; 72%
yield). Mp > 250 ꢁC (dec.) (ethanol/ethyl acetate); IR
(KBr) 3200, 1750 cmꢀ1 1H NMR(DMSO-d6) (ppm):
;
10.90 (br s, 1H); 9.80 (br s, 1H); 9.12 (s, 1H); 8.77 (s,
1H); 8.32 (m, 2H); 8.15 (m, 1H); 7.59 (m, 1H); 7.15
(m, 2H); 6.77 (s, 1H); 4.10 (m, 2H). m/z 322 (MH+).
Anal. (C17H13ClFN5O) C, H, N.
3.2.4. N0-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)20-fluoro-
benzoylhydrazide hydrochloride (SC163.HCl). Following
the same procedure described for compound
SC144.HCl,
but
using
2-fluorobenzohydrazide
(139 mg; 0.90 mmol), SC163.HCl was obtained as a
white solid (222 mg; 66% yield). Mp 247 ꢁC (dec.) (etha-
1
nol/ethyl acetate); IR (KBr) 3250, 1675 cmꢀ1; H NMR
(DMSO-d6) (ppm): 10.45 (br s, 1H); 9.75 (br s, 1H); 8.30
(s, 1H); 8.15 (m, 1H), 7.80 (m, 1H); 7.60 (m, 1H); 7.35
(m, 2H); 7.20 (m, 2H); 6.78 (m, 1H); 4.09 (m, 2H). m/z
339 (MH+). Anal. (C18H13ClFN4O) C, H, N.
3.2.5. N0-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)thiophene-
2-carbohydrazide hydrochloride (SC164.HCl). Following
the same procedure described for compound
SC144.HCl, but using thiophene-2-carbohydrazide
(128 mg; 0.90 mmol), SC164.HCl was obtained as a
white solid (251 mg; 77% yield). Mp 261 ꢁC (dec.) (etha-
1
nol/ethyl acetate); IR (KBr) 3245, 1680 cmꢀ1; H NMR
(CD3OD) (ppm): 8.41 (m, 1H); 8.18 (dd, 1H, J = 9.3,
5.0 Hz); 7.89 (dd, 1H, J = 3.8, 1.1 Hz); 7.82 (d, 1H,
J = 4.9 ); 7.57 (m, 1H); 7.49 (dd, 1H, J = 9.3, 2.8) 7.28
(m, 1H); 7.21 (dd, 1H, J = 4.9, 3.8) 6.98 (dd, 1H,
J = 4.2,
(C16H12ClFN4SO) C, H, N.
2.8 Hz).
m/z
327
(MH+).
Anal.
3.2.6. N0-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)furan-2-
carbohydrazide hydrochloride (SC165.HCl). Following
the same procedure described for compound
SC144.HCl,
but
using
furan-2-carbohydrazide
(113 mg; 0.90 mmol), SC165.HCl was obtained as a pale
yellow solid (243 mg; 78% yield). Mp 256 ꢁC (dec.) (eth-
anol/ethyl acetate); IR (KBr) 3240, 1700 cmꢀ1; 1H NMR
(CD3OD) (ppm): 8.48 (m, 1H); 8.25 (m, 1H); 7.85 (m,
1H); 7.65 (dd, 1H J = 4.3, 1.2 Hz); 7.55 (dd, 1H
J = 9.2, 2.7 Hz); 7.35 (m, 2H); 7.05 (m, 1H); 6.73 (m,
1H). m/z 311 (MH+). Anal. (C16H12ClFN4O2) C, H, N.
3.2.7. N0-(7-Fluoropyrrolo[1,2-a]quinoxalin-4- yl)quinox-
aline-2-carbohydrazide hydrochloride (SC166.HCl). Fol-
lowing the same procedure described for compound
SC144.HCl, but using quinoxaline-2-carbohydrazide
(170 mg; 0.90 mmol), SC166.HCl was obtained as a pale
yellow solid (327 mg; 89% yield). Mp 280 ꢁC (dec.) (eth-
anol/ethyl acetate); IR (KBr) 3255, 1750 cmꢀ1; 1H NMR
(CD3OD) (ppm): 9.55 (s, 1H); 8.47 (m, 1H); 8.28 (m,
1H); 8.20 (m, 2H); 7.97 (m, 2H); 7.68 (dd, 1H, J = 4.5,
1.2); 7.44 (dd, 1H, J = 9.3, 2.5); 7.32 (m, 1H); 7.04
(dd, 1H, J = 4.2, 2.5). m/z 373 (MH+). Anal.
(C20H14ClFN6O) C, H, N.
3.3. Cell culture
Human breast cancer cells MDA-MB-435 and colon
cancer HT29, p53 mutant were purchased from the

294
F. Grande et al. / Bioorg. Med. Chem. 15 (2007) 288–294
American Type Cell Culture (Manassas, VA). The
HCT116 P53+/+ and HCT116 P53ꢀ/ꢀ cells were
kindly provided by Dr. Bert Vogelstein (Johns Hop-
kins Medical Institutions, Baltimore, MD). Human
prostate cancer cells, LNCaP, were kindly provided
by Richard Cote (University of Southern California
Keck School of Medicine). Cells were maintained
as monolayer cultures in RPMI 1640 media supple-
mented with 10% fetal bovine serum (Gemini-
Bioproducts, Woodland, CA) and 2 mmol/L L-gluta-
mine at 37 ꢁC in a humidified atmosphere of 5%
CO2. To remove the adherent cells from the flask
for passaging and counting, cells were washed with
PBS without calcium or magnesium, incubated with
a small volume of 0.25% trypsin–EDTA solution
(Sigma–Aldrich, St. Louis, MO) for 5–10 min, and
washed with culture medium and centrifuged. All
experiments were performed using cells at exponen-
tial growth stage. Cells were routinely checked for
mycoplasma contamination using a PCR-based assay
(Stratagene, Cambridge, UK).
3.6. Colony formation assay
Colony formation assays were also performed to con-
firm the activity of these compounds as described.10
Briefly, cells were plated in 6-well plates at a density of
100 cells/well and allowed to attach. The next day, serial
dilutions of the corresponding compounds were added
and allowed to incubate for 24 h. After exposure, cells
were washed in PBS and cultured in free media until col-
onies were formed (8–10 days). Cells were subsequently
washed, fixed with a 1% glutaraldehyde solution for
30 min, and stained with a solution of crystal violet
(2%) for 30 min. After staining, cells were thoroughly
washed with water. Colonies were imaged on the Versa-
Doc Imaging System (Bio-Rad) and counted using the
Quantity One quantitation software package (Bio-
Rad). The data reported represent means of at least
three independent experiments.
References and notes
1. Neamati, N.; Barchi, J. J., Jr. Curr. Top. Med. Chem.
2002, 2, 211–227.
3.4. Drugs
2. Lipinski, C. A. J. Pharmacol. Toxicol. Methods 2000, 44,
235–249.
3. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.
J. Adv. Drug Deliv. Rev. 1997, 23, 3–25.
Stock solutions (10 mM) of all compounds were pre-
pared in DMSO and stored at ꢀ20 ꢁC. Further dilutions
were made fresh in PBS or cell-culture media.
4. Plasencia, C.; Dayam, R.; Wang, Q.; Pinski, J.; Burke, T.
R., Jr.; Quinn, D. I.; Neamati, N. Mol. Cancer Ther. 2005,
4, 1105–1113.
3.5. Cytotoxicity assays
Cytotoxicity was assessed by a 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay as
previously described.9 Briefly, cells were seeded in 96-
well microtiter plates and allowed to attach. Cells were
subsequently treated with continuous exposure to the
corresponding drug for 72 h. An MTT solution (at a fi-
nal concentration of 0.5 mg/mL) was added to each well,
and cells were incubated for 4 h at 37 ꢁC. After removal
of the medium, DMSO was added and the absorbance
was read at 570 nm. All assays were done in triplicate.
The IC50 was then determined for each drug from a plot
of log (drug concentration) versus percentage of cells
killed.
5. Melek, M.; Jones, J. M.; O’Dea, M. H.; Pais, G.; Burke, T.
R., Jr.; Pommier, Y.; Neamati, N.; Gellert, M. Proc. Natl.
Acad. Sci. U.S.A 2002, 99, 134–137.
6. Plasencia, C.; Grande, F.; Oshima, T.; Sanchez, T.; Aiello,
F.; Garofalo, A.; Neamati, N. J. Med. Chem. 2006, under
review.
7. Reich, M. F.; Fabio, P. F.; Lee, V. J.; Kuck, N. A.; Testa,
R. T. J. Med. Chem. 1989, 32, 2474–2478.
8. Fand, T. I.; Spoerri, P. E. J. Am. Chem. Soc. 1952, 74,
1345.
9. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J.
D.; Mitchell, J. B. Cancer Res. 1987, 47, 936–942.
10. Munshi, A.; Hobbs, M.; Meyn, R. E. Methods Mol. Med.
2005, 110, 21–28.