Synthesis and evaluation of the antiproliferative activity of novel isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives

Article abstract of DOI:10.3109/14756366.2010.548326

A novel series of isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives was synthesized and evaluated in vitro against various human cancer cell lines for antiproliferative activity. These new compounds displayed activity against leukemia a

Full text of DOI:10.3109/14756366.2010.548326

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(5): 657–667
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.548326
OrIgInal arTIClE
Synthesis and evaluation of the antiproliferative activity
of novel isoindolo[2,1-a]quinoxaline and indolo[1,2-a]
quinoxaline derivatives
Vanessa Desplat1, Stéphane Moreau1, Solene Belisle-Fabre1, Denis iolat2, Juliette Uranga1,
Romain Lucas1, Laure de Moor1, Stéphane Massip1, Christian Jarry1, Djavad M. Mossalayi2, Pascal
Sonnet3, Gérard Déléris1, and Jean Guillon1
1Université Bordeaux Segalen, CNRS FRE 3396, Pharmacochimie, Bordeaux, 2Université Bordeaux Segalen, PPF
Médicaments-Parasitologie, Bordeaux, and 3Université de Picardie Jules Verne, UMR-CNRS 6219, Laboratoire des
Glucides, Faculté de Pharmacie, Amiens, France
abstract
A novel series of isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives was synthesized and evaluated
in vitro against various human cancer cell lines for antiproliferative activity. These new compounds displayed activity
against leukemia and breast cancer cell lines in the 3- to 18-µM concentration range.
Keywords: Isoindolo[2,1-a]quinoxaline, indolo[1,2-a]quinoxaline, antiproliferative agents
Introduction
structures 1, which could be defined as new bioisosteres
e pyrrolo[1,2-a]quinoxaline heterocyclic framework
constitutes the basis of an important class of compounds
possessing interesting biological activities. ese com-
pounds have been reported to serve as key intermedi-
ates for the assembly of several heterocycles including
antipsychotic agent1, anti-HIV agent2, adenosine A3
receptor modulator3, antiparasitic agents4–7, and antitu-
mor agents8,9. In this last field, the discovery and devel-
opment of novel therapeutic agents are one of the most
important goals in medicinal chemistry. Recently, we
have designed and developed a series of new interesting
antiproliferative-substituted pyrrolo[1,2-a]quinoxalines
I–II10,11 (Figure 1).
of the pyrrolo[1,2-a]quinoxaline skeleton (Figure 1).
Cyclizing open structures or creating an additional ring
system in a given structure represents one of the useful
methods in the search for biologically active conforma-
tions. e end result is a more constrained molecule,
with an imposed conformation12. Moreover, these new
isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxa-
line moieties 1 were substituted by a benzylpiperidinyl
benzimidazolone substituent or benzylpiperidinyl fluo-
robenzimidazole group in analogy to our previously bio-
active reference compounds I–II10,11. e antiproliferative
profile of the obtained derivatives 1 was then evaluated
in vitro against a panel of five leukemic cell lines: U937,
K562, Jurkat, U266, and HL60, and against one breast can-
cer cell line: MCF7. Moreover, to determine their respec-
tive cytotoxicity, the new isoindolo[2,1-a]quinoxaline
and indolo[1,2-a]quinoxaline derivatives were tested on
activated human peripheral blood mononuclear cells.
As an extension of our work on the development of
anticancer drugs of the pyrroloquinoxaline type10,11, we
synthesized new conformationally restricted analogues
of our previously synthesized compounds II such as
isoindolo[2,1-a]quinoxalineandindolo[1,2-a]quinoxaline
Address for Correspondence: Jean Guillon, Université Bordeaux Segalen, CNRS FRE 3396, Pharmacochimie, Bordeaux. E-mail: jean.
guillon@u-bordeaux2.fr
(Received 05 January 2010; revised 16 November 2010; accepted 12 December 2010)
657

658 V. Desplat et al.
R2
R3
N
N
N
N
R1
NH
N
R
R
N
N
O
N
I
II
1a-h
N
N
R1
−
R2
−
R3
−
R-
H-
H-
N
,
O
N
H
1a-h
N
F
H-
N
H
H-
Figure 1. Structure of compounds I, II and new synthesized substituted isoindolo- or indoloquinoxaline derivatives 1a-h.
washed with water (2×50mL), then brine (50mL) and
dried over sodium sulfate. e organic layer was con-
centrated under vacuum to give products 3a–c. Method
B: A suspension of methyl- or ethyl-substituted pyrrole-
2-carboxylate 2a–b (3.4 mmol), 1-fluoro-2-nitrobenzene
(5.1 mmol), and cesium carbonate (4.06 mmol) in 12mL
of DMF was irradiated for 10min. e irradiation was
programmed to maintain a constant temperature (200°C)
with a maximal output power of 850W. e reaction
mixture was then diluted in AcOEt (60mL), washed with
water (2×50mL) and brine (50mL) and then dried over
sodium sulfate. e organic layer was concentrated under
vacuum to give products 3a–b as oil.
Materials and methods
Chemistry
Instrumentation
Melting points were determined with an SM-LUX-POL Leitz
hot-stage microscope and reported uncorrected. IR spectra
were recorded on a BRUKER IFS-25 spectrophotometer.
NMR spectra were recorded on a BRUKER AVANCE 300
spectrometer (300MHz). Chemical shifts refer to tetram-
ethylsilane, which was used as an internal reference.
Analytical thin layer chromatography was carried out on
0.25 precoated silica gel plates (POLYGRAM SIL G/UV254
)
with visualisation by irradiation with a UV lamp. Silica gel
60 (70–230 mesh) was used for column chromatography.
Mass spectra were recorded on a Micromass-Waters Q-TOF
Ultima spectrometer. Elemental analyses were conducted
by CNRS, Vernaison, France. Compound LY-294002 was
purchased from Sigma-Aldrich. Microwave experiments
were carried out at atmospheric pressure using a micro-
wave reactor (MILESTONE Microwave Laboratory Systems
Start S). e instrument consists of a continuous focused
microwave power output from 0 to 850W.
Ethyl 2-(2-nitrophenyl)-4,5,6,7-tetrahydroisoindole-1
-carboxylate (3a). Yield: 95% (method A), 88% (method
1
B), yellow oil; IR vmax (KBr)/cm−1 1705 (CO); H NMR
(300 MHz, CDCl3) δ 8.04 (dd, 1H, J= 7.95 and 1.55 Hz,
H-3′), 7.66 (ddd, 1H, J= 7.95, 7.80 and 1.55 Hz, H-4′), 7.54
(ddd, 1H, J= 7.95, 7.80 and 1.55 Hz, H-5′), 7.38 (dd, 1H,
J= 7.95 and 1.55 Hz, H-6′), 6.63 (s, 1H, H-3), 4.10 (q, 2H,
J= 7.00 Hz, CH2), 2.91–2.86 (m, 2H, CH2), 2.61–2.58 (m,
2H, CH2), 1.83–1.78 (m, 4H, 2 CH2), 1.16 (t, 3H, J= 7.00
Hz, CH3). Anal. Calcd. for C17H18N2O4: C, 64.96; H, 5.77; N,
8.91. Found: C, 65.12; H, 5.72; N, 8.79.
Synthesis
of
ethyl
2-(2-nitrophenyl)-4,5,6,7-
tetrahydroisoindole-1-carboxylate, ethyl 2-(2-nitrophenyl)-
4,5-dihydrobenzo[e]isoindole-1-carboxylate and ethyl
1-(2-nitrophenyl)indole-2-carboxylate (3a-c). Method A:
To the solution of methyl or ethyl substituted pyrrole-2-
carboxylate 2a–c or (3.4 mmol) in 12mL of dimethylform-
amide (DMF), cesium carbonate (4.06 mmol) was added.
e mixture was stirred at room temperature for 10min,
then 1-fluoro-2-nitrobenzene (5.1 mmol) was added. e
reaction mixture was refluxed for 1h 30 min, then diluted
in AcOEt (60mL) after cooling. e reaction mixture was
Ethyl 2-(2-nitrophenyl)-4,5-dihydrobenzo[e]isoindole-
1-carboxylate (3b). Yield: 97% (method A), 86% (method
1
B), orange oil; IR ν (KBr)/cm−1 1710 (CO); H NMR
(300 MHz, CDCl3) δm8ax.19 (dd, 1H, J= 8.00 and 2.00 Hz,
H-9), 8.06 (dd, 1H, J= 8.10 and 1.50 Hz, H-3′), 7.69 (ddd,
1H, J= 8.10, 7.50 and 1.50 Hz, H-4′), 7.58 (ddd, 1H, J= 8.10,
7.50 and 1.50 Hz, H-5′), 7.44 (dd, 1H, J= 8.10 and 1.50 Hz,
H-6′), 7.31–7.18 (m, 3H, H-6, H-7 and H-8), 4.11 (q, 2H,
J= 7.15 Hz, CH2), 2.92–2.88 (m, 2H, CH2), 2.72-2.66 (m,
Journal of Enzyme Inhibition and Medicinal Chemistry

Antiproliferative Activity of Novel Isoindolo[2,1-a]quinoxaline and Indolo[1,2-a]quinoxaline Derivatives 659
2H, CH2), 1.05 (t, 3H, J= 7.15 Hz, CH3). Anal. Calcd. for
C21H18N2O4: C, 69.60; H, 5.01; N, 7.73. Found: C, 69.85; H,
4.92; N, 7.96.
(300 MHz, CDCl3) δ 7.79 (dd, 1H, J= 8.0 and 1.40 Hz, H-1),
7.66 (dd, 1H, J= 8.0 and 1.40 Hz, H-4), 7.63 (s, 1H, H-11),
7.45 (ddd, 1H, J= 8.0, 7.75 and 1.40 Hz, H-2), 7.36 (ddd,
1H, J= 8.0, 7.75 and 1.40 Hz, H-3), 3.17 (t, 2H, J= 5.80 Hz,
CH2), 2.78 (t, 2H, J= 5.80 Hz, CH2), 1.90–1.80 (m, 4H, 2
CH2). Anal. Calcd. for C15H13ClN2: C, 70.18; H, 5.10; N,
10.91. Found: C, 69.96; H, 5.38; N, 11.05.
Ethyl 1-(2-nitrophenyl)indole-2-carboxylate (3c). Yield:
75% (method A), yellow crystals, mp = 88°C13.
Synthesis
1-a]quinoxalin-6-one,
of
7,8,9,10-tetrahydro-5H-isoindolo[2,-
11,12-dihydro-5H-benzo[e]
isoindolo[2,1-a]quinoxalin-6-one and 5H-indolo[1,2-a]
quinoxalin-6-one (4a–c). Method A: A suspension of
3a–c (2.5 mmol) and iron powder (10 mmol) in 12 mL of
acetic acid was heated under reflux for 2 h. e reaction
mixture was cooled, suspended in 35 mL of a 1-M aque-
ous solution of HCl, agitated, then filtered off, washed
with 1 M (25 mL) HCl, followed by water, AcOEt, Et2O
and then dried to give 4a–c as a white solid. Method B:
A mixture of pyrrole-2-carboxylate ester 2a (0.25 mmol),
o-iodotrifluoroacetanilide (0.375 mmol), CuI (0.025
mmol), l-proline (0.05 mmol), and K2CO3 (0.75 mmol) in
5 mL of DMSO was stirred at 80°C for 24 h. To the cooled
solution, 1.5 mL of water was added. e mixture was then
heated at 60°C for 15 h. e mixture was cooled to room
temperature and diluted with 150 mL of ethyl acetate,
washed with water, dried over Na2SO4, and concentrated
in vacuum. e residue was purified by column chroma-
tography on silica gel (eluting with 1:1 petroleum ether/
ethyl acetate) to provide the desired product 4a.
6-Chloro-11,12-dihydro-benzo[e]isoindolo[2,1-a]qui-
noxaline (5b). Yield: 83%, beige crystals, mp = 173°C; H
1
NMR (300 MHz, CDCl3) δ 8.07 (dd, 1H, J= 8.20 and 1.40
Hz, H-7), 7.88 (dd, 1H, J= 7.80 and 1.20 Hz, H-1), 7.86 (s,
1H, H-13), 7.77 (dd, 1H, J= 7.80 and 1.20 Hz, H-4), 7.52
(ddd, 1H, J= 7.80, 7.20 and 1.20 Hz, H-2), 7.42 (ddd, 1H,
J= 7.80, 7.20 and 1.20 Hz, H-3), 7.40–7.32 (m, 2H, H-8
and H-10), 7.25 (ddd, 1H, J= 8.20, 7.55 and 1.40 Hz, H-9),
2.94–2.89 (m, 2H, CH2), 2.86–2.81 (m, 2H, CH2). Anal.
Calcd. for C19H13ClN2: C, 74.88; H, 4.30; N, 9.19. Found: C,
74.74; H, 4.19; N, 9.30.
6-Chloro-indolo[1,2-a]quinoxaline (5c). Yield: 84%,
pale-yellow crystals, mp = 129°C; 1H NMR (300 MHz,
CDCl3) δ 8.46 (dd, 1H, J= 8.20 and 1.30 Hz, H-11), 8.41
(dd, 1H, J= 8.40 and 1.10 Hz, H-1), 7.98 (dd, 1H, J= 8.40
and 1.10 Hz, H-4), 7.95 (dd, 1H, J= 8.20 and 1.30 Hz, H-8),
7.68–7.57 (m, 2H, H-2 and H-3), 7.51–7.43 (m, 2H, H-9
and H-10), 7.37 (s, 1H, H-7). Anal. Calcd. for C15H9ClN2:
C, 71.30; H, 3.59; N, 11.09. Found: C, 71.25; H, 3.82; N,
10.87.
6-Chloro-isoindolo[2,1-a]quinoxaline (5d). Yield: 47%,
pale-yellow crystals, mp = 183°C; (Found M+: 253.0522,
C H9ClN2 requires 253.0533); 1H NMR (300 MHz, CDCl3)
δ185.65 (m, 1H, H-1), 8.54 (s, 1H, H-11), 8.12 (m, 1H, H-10),
8.03 (m, 1H, H-7), 7.92 (m, 1H, H-4), 7.65–7.61 (m, 2H, H-2
and H-3), 7.51–7.46 (m, 2H, H-8 and H-9). Anal. Calcd. for
C15H9ClN2: C, 71.30; H, 3.59; N, 11.09. Found: C, 71.56; H,
3.54; N, 10.93.
7,8,9,10-Tetrahydro-5H-isoindolo[2,1-a]quinoxalin-6-
one (4a). Yield: 81% (method A), 46% (method B), beige
1
crystals, mp = 273°C; IR νmax (KBr)/cm−1 1645 (CO); H
NMR (300 MHz, DMSO-d6) δ 10.96 (s, 1H, NH), 7.87 (dd,
1H, J= 7.90 and 1.45 Hz, H-1), 7.83 (s, 1H, H-11), 7.21–7.10
(m, 3H, H-2, H-3, and H-4), 2.95–2.91 (m, 2H, CH2), 2.66–
2.63 (m, 2H, CH2), 1.74–1.71 (m, 4H, 2 CH2). Anal. Calcd.
for C15H14N2O: C, 75.61; H, 5.92; N, 11.76. Found: C, 75.47;
H, 6.04; N, 11.71.
11,12-Dihydro-5H-benzo[e]isoindolo[2,1-a]quinox-
alin-6-one (4b). Yield: 79% (method A), beige crystals,
mp = 297°C; IR νmax (KBr)/cm−1 1650 (CO); 1H NMR
(300 MHz, DMSO-d6) δ 11.26 (s, 1H, NH), 8.85 (dd, 1H,
J= 8.10 and 1.90 Hz, H-7), 8.12 (s, 1H, H-13), 7.98 (dd, 1H,
J= 7.95 and 1.50 Hz, H-1), 7.27–7.17 (m, 6H, H-2, H-3, H-4,
H-8, H-9 and H-10), 2.85–2.82 (m, 2H, CH2), 2.76–2.73 (m,
2H, CH2). Anal. Calcd. for C19H14N2O: C, 79.70; H, 4.93; N,
9.78. Found: C, 79.93; H, 4.99; N, 9.67.
Synthesis of 4-(isoindolo[2,1-a]quinoxalin-6-yl)benz-
aldehydes and 4-(indolo[1,2-a]quinoxalin-6-yl)benzal-
dehyde (6a–d). To suspension of compound 5a–d (4.64
mmol) and Pd(PPh3)4 (0.232 mmol) in a mixture of tolu-
ene/EtOH (75/4.1 mL) under nitrogen were added K2CO3
(5.1 mmol) and 4-formylphenylboronic acid (5.1 mmol).
e reaction mixture was refluxed for 24 h, and the
cooled suspension was extracted with CH2Cl2 (3 × 80 mL).
e organic layer was washed with a saturated solution
of NaCl (95 mL), and the combined organic extracts were
dried over sodium sulfate, filtered, and evaporated under
reduced pressure. e crude residue was triturated in
ethanol. e resulting precipitate when was filtered,
washed with ethanol, and purified by column chroma-
tography on silica gel using dichloromethane as eluent
gave the pure product 6a–d.
4-(7,8,9,10-Tetrahydroisoindolo[2,1-a]quinoxalin-
6-yl)benzaldehyde (6a). Yield: 89%, yellow crystals,
mp = 172°C; IR νmax (KBr)/cm−1 1705 (CO); 1H NMR
(300 MHz, CDCl3) δ 10.14 (s, 1H, CHO), 8.05 (d, 2H,
J= 8.10 Hz, H-2 and H-6), 8.03 (dd, 1H, J= 8.05 and 1.20
Hz, H1′), 7.87 (s, 1H, H-11′), 7.82 (dd, 1H, J= 8.05 and 1.20
Hz, H-4′), 7.79 (d, 2H, J= 8.10, H-3 and H-5), 7.54 (ddd,
5H-Indolo[1,2-a]quinoxalin-6-one (4c). Yield: 94%
(method A), beige crystals, mp > 300°C13.
Synthesis of 6-chloro-isoindolo[2,1-a]quinoxalines and
6-chloro-indolo[1,2-a]quinoxaline (5a–d). A solution of
4a–d(30mmol)inPOCl3 (60 mL)wasrefluxedfor4 h. After
removing excess of reactive under vacuum, the residue
was carefully dissolved in water at 0°C and the resulting
solution was made basic with 32% aqueous ammonium
hydroxide solution. e precipitate was filtered, washed
with water, dried and extracted with dichloromethane.
e organic layer was washed with water, dried over mag-
nesium sulfate, and evaporated to dryness to give 5.
6-Chloro-7,8,9,10-tetrahydroisoindolo[2,1-a]quinoxa-
line (5a). Yield: 84%, beige crystals, mp = 127°C; 1H NMR
© 2011 Informa UK, Ltd.

660 V. Desplat et al.
1H, J= 8.05, 7.70 and 1.20 Hz, H-2′), 7.44 (ddd, 1H, J= 8.05,
7.70 and 1.20 Hz, H-3′), 2.84 (t, 2H, J= 6.20 Hz, CH2), 2.18
(t, 2H, J= 6.20 Hz, CH2), 1.85–1.75 (m, 2H, CH2), 1.69–1.60
(m, 2H, CH2). Anal. Calcd. for C22H18N2O: C, 80.96; H, 5.56;
N, 8.58. Found: C, 81.05; H, 5.70; N, 8.55.
Hz, H-4”), 7.56–7.38 (m, 6H, H-2′, H-3′, H-5′, H-6′, H-2” and
H-3”), 7.26–7.23 (m, 1H, H benzimid.), 6.98–6.96 (m, 3H, H
benzimid.), 4.18–4.17 (m, 1H, CH pip.), 3.65 (s, 2H, CH2N),
3.02–2.99 (m, 2H, CH2 pip.), 2.75 (t, 2H, J=5.90 Hz, CH2),
2.43–2.38 (m, 2H, CH2 pip.), 2.18–2.09 (m, 4H, CH2 pip. and
CH2), 1.70–1.65 (m, 4H, CH2 and CH2 pip.), 1.57–1.54 (m,
2H, CH2). Anal. Calcd. for C34H33N5O: C, 77.39; H, 6.30; N,
13.27. Found: C, 77.33; H, 6.21; N, 13.45.
4-(11,12-Dihydrobenzo[e]isoindolo[2,1-a]quinoxalin-
6-yl)benzaldehyde (6b). Yield: 86%, yellow crystals,
mp = 174°C; IR νmax (KBr)/cm−1 1710 (CO); 1H NMR
(300 MHz, CDCl3) δ 10.07 (s, 1H, CHO), 8.09 (dd, 1H,
J= 8.10 and 1.35 Hz, H-7′), 7.98 (s, 1H, H-13′), 7.88 (dd,
1H, J= 8.10 and 1.20 Hz, H-1′), 7.86 (d, 2H, J= 8.10 Hz, H-2
and H-6), 7.83 (d, 2H, J= 8.10 Hz, H-3 and H-5), 7.58 (ddd,
1H, J= 8.00, 7.65 and 1.20 Hz, H-2′), 7.55 (ddd, 1H, J= 8.00,
7.65 and 1.20 Hz, H-3′), 7.26 (dd, 1H, J= 8.00 and 1.20 Hz,
H-4′), 6.98 (ddd, 1H, J= 8.10, 7.60, and 1.35 Hz, H-8′), 6.58
(ddd, 1H, J= 8.10, 7.60, and 1.35 Hz, H-9′), 6.15 (dd, 1H,
J= 8.10 and 1.35 Hz, H-10′), 3.00–2.95 (m, 2H, CH2), 2.92–
2.87 (m, 2H, CH2). Anal. Calcd. for C26H18N2O: C, 83.40; H,
4.85; N, 7.48. Found: C, 83.58; H, 4.97; N, 7.62.
1,3-Dihydro-1-{1-[4-(11,12-dihydrobenzo[e]
isoindolo[2,1-a]quinoxalin-6-yl)benzyl]piperidin-4-
yl}-2H-benzimidazol-2-one . 2 HCl (1b). Yield: 76%,
orange crystals, mp = 298°C; IR νmax (KBr)/cm−1 3400
1
(NH), 2650–2350 (NH+), 1695 (C=O); H NMR (300 MHz,
DMSO-d6) δ 11.93 (s, 1H, NH+), 11.04 (s, 1H, NH), 9.03
(s, 1H, H-13”), 8.49 (d, 1H, J = 7.50 Hz, H-7”), 8.18 (d, 1H,
J = 7.30 Hz, H-1”), 7.86–7.75 (m, 6H, H-2′, H-3′, H-5′, H-6′,
H-2” and H benzimid.), 7.62 (t, 1H, J = 7.30 Hz, H-3”), 7.27
(d, 1H, J = 7.30 Hz, H-4”), 7.03–6.98 (m, 4H, H-8” and 3H
benzimid.), 6.69 (t, 1H, J = 7.50 Hz, H-9”), 6.11 (d, 1H,
J = 7.50 Hz, H-10”), 4.61–4.59 (m, 1H, CH pip.), 4.45 (s, 2H,
CH2N), 4.40 (bs, 1H, NH+), 3.47–3.42 (m, 2H, CH2 pip.),
3.21–3.13 (m, 2H, CH2 pip.), 3.08–2.88 (m, 6H, CH2 pip.
and 2 CH2), 1.95–1.91 (m, 2H, CH2 pip.). Anal. Calcd. for
C38H35Cl2N5O: C, 70.37; H, 5.44; N, 10.80. Found: C, 70.46;
H, 5.57; N, 11.04.
4-(Indolo[1,2-a]quinoxalin-6-yl)benzaldehyde
(6c).
Yield: 83%, yellow crystals, mp = 196°C; IR νmax (KBr)/
cm−1 1690 (CO); 1H NMR (300 MHz, CDCl3) δ 10.18 (s, 1H,
CHO), 8.57 (d, 1H, J= 8.10 Hz, H-11′), 8.53 (d, 1H, J= 8.30
Hz, H-1′), 8.24 (d, 2H, J= 8.10 Hz, H-2 and H-6), 8.14–8.11
(m, 3H, H-3, H-5 and H-4′), 7.97 (d, 1H, J= 8.10, H-8′),
7.69 (t, 1H, J= 8.30 Hz, H-2′), 7.62 (t, 1H, J= 8.30 Hz, H-3′),
7.53–7.47 (m, 2H, H-9′ and H-10′), 7.29 (s, 1H, H-7′). Anal.
Calcd. for C22H14N2O: C, 81.97; H, 4.38; N, 8.69. Found: C,
82.17; H, 4.42; N, 8.89.
1,3-Dihydro-1-{1-[4-(indolo[1,2-a]quinoxalin-6-yl)
benzyl]piperidin-4-yl}-2H-benzimidazol-2-one(1c).Yield:
69%, yellow crystals, mp = 236°C; IR νmax (KBr)/cm−1 3350
(NH), 1685 (C=O); 1H NMR (300 MHz, DMSO-d6) δ 10.87
(s, 1H, NH), 8.77–8.73 (m, 2H, H-11” and H-1”), 8.05–8.00
(m, 4H, H-3′, H-5′, H-4” and H-8”), 7.71(t, 1H, J= 8.10 Hz,
H-2”), 7.61 (t, 1H, J= 8.10 Hz, H-3”), 7.59 (d, 2H, J= 8.10 Hz,
H-2′ and H-6′), 7.54-7.48 (m, 2H, H-9” and H-10”), 7.39
(s, 1H, H-7”), 7.28–7.25 (m, 1H, H benzimid.), 7.01–6.96
(m, 3H, H benzimid.), 4.23–4.14 (m, 1H, CH pip.), 3.66 (s,
2H, CH2N), 3.05–3.01 (m, 2H, CH2 pip.), 2.45–2.40 (m, 2H,
CH2 pip.), 2.21–2.13 (m, 2H, CH2 pip.), 1.70–1.64 (m, 2H,
CH2 pip.). Anal. Calcd. for C34H29N5O: C, 77.99; H, 5.58; N,
13.37. Found: C, 78.20; H, 5.62; N, 13.45.
4-(Isoindolo[2,1-a]quinoxalin-6-yl)benzaldehyde(6d).
Yield: 79%, yellow crystals, mp = 213°C; IR νmax (KBr)/
1
cm−1 1700 (CO); H NMR (300 MHz, CDCl3) δ 10.23 (s,
1H, CHO), 8.62 (s, 1H, H-11′), 8.24–8.20 (m, 1H, H-1′),
8.19–8.16 (m, 1H, H-10′), 8.15 (d, 2H, J= 8.40 Hz, H-2 and
H-6), 8.01 (d, 2H, J= 8.40 Hz, H-3 and H-5), 7.93 (d, 1H,
J= 8.70, H-4′), 7.69–7.65 (m, 2H, H-2′ and H-3′), 7.41–7.35
(m, 1H, H-9′), 7.31–7.28 (m, 1H, H-7′), 7.20–7.14 (m, 1H,
H-8′). Anal. Calcd. for C22H14N2O: C, 81.97; H, 4.38; N,
8.69. Found: C, 82.09; H, 4.34; N, 8.77.
Synthesis
( i s o i n d o l o [ 2 , 1 - a ] q u i n o x a l i n - 6 - y l ) b e n z y l ]
piperidin-4-yl}-2H-benzimidazol-2-ones and 1,3-
of
1,3-dihydro-1-{1-[4-
1,3-Dihydro-1-{1-[4-(isoindolo[2,1-a]quinoxalin-6-yl)
benzyl]piperidin-4-yl}-2H-benzimidazol-2-one
(1d).
Yield: 72%, yellow crystals, mp = 255°C; IR νmax (KBr)/cm−1
1
dihydro-1-{1-[4-(indolo[1,2-a]quinoxalin-6-yl)benzyl]
piperidin-4-yl}-2H-benzimidazol-2-one (1a–d). e pH
of a solution of the aldehyde 6a–d (2.5 mmol) and 4-(2-
ketobenzimidazol-1-yl)piperidine (3.0 mmol) in 40 mL
methanol was adjusted to 6 by the dropwise addition
of acetic acid. Powered sodium cyanoborohydride (6.9
mmol) was then added, and the resultant mixture was
refluxed for 5 h. e hot precipitate formed was filtered,
washed with methanol then with diethyl ether and dried
under reduced pressure to give the compounds 1a–d.
1,3-Dihydro-1-{1-[4-(7,8,9,10-tetrahydroisoindolo[2,1-a]
quinoxalin-6-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2
-one (1a). Yield: 43%, pale-yellow crystals, mp=289°C; IR
3345 (NH), 1690 (C=O); H NMR (300 MHz, DMSO-d6) δ
10.83 (s, 1H, NH), 9.24 (s, 1H, H-11”), 8.64 (dd, 1H, J= 8.50
and 1.80 Hz, H-10”), 8.02–7.99 (m, 1H, H-1”), 7.95 (d, 1H,
J= 8.50 Hz, H-4”), 7.75 (d, 2H, J= 7.95 Hz, H-3′ and H-5′),
7.73–7.67 (m, 2H, H-2” and H-3”), 7.62 (d, 2H, J= 7.95 Hz,
H-2′ and H-6′), 7.36–7.27 (m, 1H, H-9”), 7.26–7.22 (m, 2H,
H-7” and H benzimid.), 7.17–7.11 (m, 1H, H-8”), 7.01–6.96
(m, 3H, H benzimid.), 4.26–4.16 (m, 1H, CH pip.), 3.73 (s,
2H, CH2N), 3.09–3.05 (m, 2H, CH2 pip.), 2.50–2.37 (m, 2H,
CH2 pip.), 2.25–2.17 (m, 2H, CH2 pip.), 1.73–1.68 (m, 2H,
CH2 pip.). Anal. Calcd. for C34H29N5O: C, 77.99; H, 5.58; N,
13.37. Found: C, 78.17; H, 5.43; N, 13.30.
Synthesis of 5-fluoro-2-{1-[4-(isoindolo[2,1-a]qui-
noxalin-6-yl)benzyl]piperidin-4-yl}-1H-benzimidazoles
ν
max (KBr)/cm−1 3350 (NH), 1685 (C=O); 1H NMR (300MHz,
CDCl3) δ 10.86 (s, 1H, NH), 8.29 (s, 1H, H-11”), 8.18 (dd, 1H,
J=8.00 and 1.25 Hz, H-1”), 7.80 (dd, 1H, J=8.00 and 1.25
and
5-fluoro-2-{1-[4-(indolo[1,2-a]quinoxalin-6-yl)
benzyl]piperidin-4-yl}-1H-benzimidazole (1e–h). e
Journal of Enzyme Inhibition and Medicinal Chemistry

Antiproliferative Activity of Novel Isoindolo[2,1-a]quinoxaline and Indolo[1,2-a]quinoxaline Derivatives 661
pH of a solution of the aldehyde 6a–d (2.5 mmol) and
pip.), 1.93–1.82 (m, 2H, CH2 pip.). Calcd. for C34H28FN5:
C, 77.69; H, 5.37; N, 13.32. Found: C, 77.56; H, 5.48; N,
13.11.
4-(5-fluorobenzimidazol-2-yl)piperidine (3.0 mmol) in
40 mL methanol was adjusted to 6 by the dropwise addi-
tion of acetic acid. Powered sodium cyanoborohydride
(6.9 mmol) was then added, and the resultant mixture
was refluxed for 5 h. After removal of the methanol by
rotary evaporation, the residue was triturated in water
and extracted with dichloromethane. e organic layer
was washed with water, dried over magnesium sulfate
and evaporated to dryness. Column chromatography of
the residue on silica gel using ethyl acetate–cyclohexane
(1/1) then methanol–chloroform (1/9) as eluents gave the
crude product. is solid was then triturated with diethyl
ether, filtered, washed with diethyl ether and dried under
reduced pressure to give the compounds 1e–h.
5-Fluoro-2-{1-[4-(7,8,9,10-tetrahydroisoindolo[2,-
1-a]quinoxalin-6-yl)benzyl]piperidin-4-yl}-1H-
benzimidazole (1e). Yield: 63%, pale-yellow crystals,
mp = 152°C; IR νmax (KBr)/cm−1 3340 (NH), 1685 (C=O);
1H NMR (300 MHz, DMSO-d6) δ 12.41 (s, 1H, NH), 8.30
(s, 1H, H-11”), 8.19 (d, 1H, J= 8.10 Hz, H-1”), 7.80 (d,
1H, J= 8.10 Hz, H-4”), 7.57–7.31 (m, 7H, H-2′, H-3′, H-5′,
H-6′, H-2”, H-3” and H benzimid.), 7.23–7.20 (m, 1H, H
benzimid.), 6.99–6.96 (m, 1H, H benzimid.), 3.63 (s, 2H,
CH2N), 2.98–2.93 (m, 2H, CH2 pip.), 2.87–2.85 (m, 1H,
CH pip.), 2.76 (t, 2H, J= 5.85 Hz, CH2), 2.17–2.12 (m, 4H,
CH2 and CH2 pip.), 2.00–1.98 (m, 2H, CH2 pip.), 1.90–1.84
(m, 2H, CH2 pip.), 1.71–1.68 (m, 2H, CH2), 1.56–1.54 (m,
2H, CH2). Anal. Calcd. for C34H32FN5: C, 77.10; H, 6.09; N,
13.22. Found: C, 77.24; H, 6.32; N, 13.15.
5 - F l u o r o - 2 - { 1 - [ 4 - ( 1 1 , 1 2 - d i h y d r o b e n z o [ e ]
isoindolo[2,1-a]quinoxalin-6-yl)benzyl]piperidin-4-
yl}-1H-benzimidazole (1f). Yield: 40%, yellow crystals,
mp = 179°C; IR νmax (KBr)/cm−1 3340 (NH), 1690 (C=O);
1H NMR (300 MHz, DMSO-d6) δ 12.33 (s, 1H, NH), 8.54
(s, 1H, H-13”), 8.29 (d, 1H, J= 7.80 Hz, H-7”), 7.91 (dd,
1H, J= 8.10 and 1.20 Hz, H-1”), 7.61 (ddd, 1H, J= 8.10,
7.90 and 1.20 Hz, H-2”), 7.51 (d, 2H, J= 8.10 Hz, H-3′ and
H-5′), 7.50–7.48 (m, 2H, H-3” and H benzimid.), 7.23 (d,
2H, J= 8.10 Hz, H-2′ and H-6′),7.22–7.20 (m, 2H, H-4” and
H benzimid.), 7.02–6.91 (m, 2H, H-8” and H benzimid.),
6.52 (t, 1H, J= 7.80 Hz, H-9”), 6.11 (d, 1H, J= 7.80 Hz,
H-10”), 3.57 (s, 2H, CH2N), 2.97–2.81 (m, 7H, CH2 pip., CH
pip. and 2 CH2), 2.15–2.04 (m, 4H, 2 CH2 pip.), 1.91–1.83
(m, 2H, CH2 pip.). Calcd. for C38H32FN5: C, 79.01; H, 5.58;
N, 12.12. Found: C, 78.95; H, 5.57; N, 12.04.
5-Fluoro-2-{1-[4-(indolo[1,2-a]quinoxalin-6-yl)-
benzyl]piperidin-4-yl}-1H-benzimidazole (1g). Yield:
86%, yellow crystals, mp = 139°C; IR νmax (KBr)/cm−1 3345
(NH), 1690 (C=O); 1H NMR (300 MHz, DMSO-d6) δ 12.27
(s, 1H, NH), 8.76-8.73 (m, 2H, H-11” and H-1”), 8.04–7.98
(m, 4H, H-3′, H-5′, H-4” and H-8”), 7.70 (ddd, 1H, J= 8.40,
7.20 and 1.20 Hz, H-2”), 7.62–7.54 (m, 3H, H-2′, H-6′ and
H-3”), 7.51–7.45 (m, 2H, H-9” and H-10”), 7.39 (s, 1H,
H-7”), 7.36-7.31 (m, 1H, H benzimid.), 7.22–7.18 (m, 1H,
H benzimid.), 7.02–6.94 (m, 1H, H benzimid.), 3.64 (s, 2H,
CH2N), 2.99–2.90 (m, 2H, CH2 pip.), 2.87–2.83 (m, 1H, CH
pip.), 2.21–2.13 (m, 2H, CH2 pip.), 2.04–2.00 (m, 2H, CH2
5-Fluoro-2-{1-[4-(isoindolo[2,1-a]quinoxalin-6-yl)
benzyl]piperidin-4-yl}-1H-benzimidazole (1h). Yield:
66%, yellow crystals, mp = 196°C; IR νmax (KBr)/cm−1 3350
(NH), 1690 (C=O); 1H NMR (300 MHz, DMSO-d6) δ 12.48
(bs, 1H, NH), 9.24 (s, 1H, H-11”), 8.64 (dd, 1H, J= 8.10
and 2.10 Hz, H-10”), 8.03–7.99 (m, 1H, H-1”), 7.95 (d, 1H,
J= 8.40 Hz, H-4”), 7.72 (d, 2H, J= 7.85 Hz, H-3′ and H-5′),
7.70–7.66 (m, 2H, H-2” and H-3”), 7.58 (d, 2H, J= 7.85 Hz,
H-2′ and H-6′), 7.48–7.44 (m, 1H, H benzimid.), 7.35–7.21
(m, 3H, H-7”, H-9” and H benzimid.), 7.16–7.10 (m, 1H,
H-8”), 6.99–6.92 (m, 1H, H benzimid.), 3.68 (s, 2H, CH2N),
3.02–2.97 (m, 2H, CH2 pip.), 2.92–2.83 (m, 1H, CH pip.),
2.23-2.15 (m, 2H, CH2 pip.), 2.08–2.01 (m, 2H, CH2 pip.),
1.92–1.78 (m, 2H, CH2 pip.). Anal. Calcd. for C34H28FN5:
C, 77.69; H, 5.37; N, 13.32. Found: C, 77.42; H, 5.21; N,
13.57.
2-(2-Aminophenyl)-2H-isoindole-1-carboxylic acid
(8). A solution of 1-cyano-2-(2-aminophenyl)isoindole 7
(3 mmol) in acetic acid (10 mL) was refluxed for 30 min.
After cooling, the reaction mixture was poured into ice
water (150 mL) and stirred for 15 min. e resulting solu-
tion was evaporated under reduced pressure. e residue
was cooled and triturated in diethyl ether. e precipitate
was collected by filtration and dried to give 8. Yield: 98%,
orange crystals, mp > 300°C; IR νmax (KBr)/cm−1 3360–2500
1
(NH2 and OH), 1660 (C=O); H NMR (300 MHz, DMSO-
d6) δ 12.03 (bs, 1H, COOH), 8.92 (s, 1H, H-3), 8.44 (dd, 1H,
J = 8.10 and 0.90 Hz, H-7), 8.37 (dd, 1H, J = 8.10 and 0.90
Hz, H-4), 7.89–7.86 (m, 1H, H-6′), 7.57 (dd, 1H, J = 8.10
and 1.20 Hz, H-3′), 7.46 (ddd, 1H, J = 8.10, 7.50 and 1.20
Hz, H-4′), 7.34–7.28 (m, 3H, H-5, H-6 and H-5′), 6.91 (s,
2H, NH2). Anal. Calcd. for C15H12N2O2: C, 71.42; H, 4.79; N,
11.10. Found: C, 71.37; H, 4.96; N, 11.39.
2-(2-Fluorophenyl)-2H-isoindole-1-carbonitrile (9).
To a solution of sodium hydrogen sulfite (0.015 mol) in
water (38 mL) was added phthalaldehyde (0.015 mol).
e mixture was stirred until the solid was dissolved,
and 2-fluoroaniline (0.015 mol) was added. e reaction
was heated at 70°C for 30min, cooled, KCN (0.052 mol)
in water (8.0 mL) was added, and the mixture was heated
at 70°C for an additional 90 min. e solid formed upon
cooling was filtered, washed with water and extracted
with dichloromethane. e organic layer was dried over
magnesium sulfate and evaporated to dryness. Column
chromatography of the residue on silica gel using dichlo-
romethane as eluent gave 9. Yield: 32%, pale-yellow crys-
tals, mp = 120°C; IR ν (KBr)/cm−1 2200 (C≡N); 1H NMR
(300 MHz, CDCl3) δ m7a.x73 (dd, 1H, J= 8.40 and 0.90 Hz,
H-4), 7.69 (dd, 1H, J= 8.40 and 0.90 Hz, H-7), 7.59–7.48
(m, 3H, H-4′, H-5′ and H-6′), 7.38–7.28 (m, 3H, H-3′, H-3
and H-5), 7.17 (ddd, 1H, J= 8.40, 7.80 and 0.90 Hz, H-6).
Anal. Calcd. for C15H9FN2: C, 76.26; H, 3.84; N, 11.86.
Found: C, 76.36; H, 3.77; N, 11.65.
5H-Isoindolo[2,1-a]quinoxalin-6-one (4d). A suspen-
sion of 2-(2-fluorophenyl)-2H-isoindole-1-carbonitrile
© 2011 Informa UK, Ltd.

662 V. Desplat et al.
9 (3 mmol) and 85% potassium hydroxide (12 mmol)
in tert-butanol (21 mL) was heated at 80°C for 4 h, then
cooled, poured into crushed ice, and stirred for 10min.
e resulting precipitate was filtered, washed with water
then with diethyl ether, and dried under reduced pressure
to give 4d. Yield: 80%, white crystals, mp > 260°C; (Found
MNa+: 257.0699, C15H10N2O23Na requires 257.0691); IR
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
afforded the corresponding fused pyrroles 2a–b using
a Barton-Zard reaction14–16. e not commercially avail-
able, starting material 2-nitro-3,4-dihydronaphthalene
was readily prepared by the nitration of the correspond-
ing alkene17. e preparation of N-aryl pyrroles 3a-c
was then obtained by nucleophilic substitution of the
various alkyl pyrrole-2-carboxylates 2a–c with 1-fluoro-
2-nitrobenzene using cesium carbonate as the base in
refluxing DMF solution3,10. e preparation of 3a and 3b
was also performed under microwave irradiation.
Reduction of the nitro moiety of 3a–c with iron in
hot glacial acetic acid produced the spontaneous ring
closure onto the ester to afford the desired isoindolo- or
indoloquinoxalinones 4a–cthrough a one-pot reduction–
cyclization step3,13. CuI/L-proline-catalyzed coupling of
2-iodotrifluoroacetanilide with pyrrole-2-carboxylate
ester 2ain DMSO at 80−90°C followed by in situ hydrolysis
at 60°C also afforded the isoindolo[2,1-a]quinoxalin-6-
one 4a18. e lactams 4a–d were subsequently chlorode-
hydroxylated with phosphorous oxychloride, leading to
the 6-chloroquinoxalines 5a–d. Coupling chloro deriva-
tives 5a–d with 4-formylphenylboronic acid in the pres-
ence of Pd(PPh3)4 as a catalyst under Suzuki-Miyaura
cross-coupling conditions proceeded to afford the sub-
stituted benzaldehydes 6a–d19,10. Reductive amination of
benzaldehydes 6a–d with 4-(2-ketobenzimidazolin-1-yl)
piperidine or 4-(5-fluorobenzimidazol-2-yl)piperidine
using NaBH3CN provided the final products 1a–h. e
3D spatial determinations of 1a and 1c were established
by X-ray crystallography20 and confirmed the structures
in the solid state as anticipated on the basis of IR and 1H
NMR data (Figure 2).
ν
(KBr)/cm−1 1645 (CO); 1H NMR (300 MHz, DMSO-d6)
δm1a1x.40 (s, 1H, NH), 8.78 (s, 1H, H-11), 8.35 (dd, 1H, J= 8.10
and 1.00 Hz, H-1), 8.31 (dd, 1H, J= 7.90 and 0.90 Hz, H-10),
7.83 (dd, 1H, J= 7.90 and 0.90 Hz, H-7), 7.45–7.39 (m, 2H,
H-2 and H-4), 7.33–7.22 (m, 3H, H-3, H-8 and H-9). Anal.
Calcd. for C15H10N2O: C, 76.91; H, 4.30; N, 11.96. Found: C,
77.09; H, 4.10; N, 12.07.
Biology
Cell culture. e human leukemic cell lines U937, K562,
and HL60, and the breast cancer cell-line MCF7 were
grown in RPMI 1640 medium (Life Technology, France)
supplemented with 10% fetal calf serum, antibiotics
(100 U/mL penicillin, 100 µg/mL streptomycin), and
l-glutamin, at 37°C, 5% CO2 in air. e toxicity of various
molecules was also evaluated on non-activated, freshly
isolated normal human peripheral blood mononuclear
cells (PBMNC) and phytohemagglutinin (lymphop-
roliferative agent) (PHA)-induced cells. PBMNC from
blood of healthy volunteers were obtained following
centrifugation on Ficoll gradient. Cells were then incu-
bated in medium alone or induced to enter cell cycle by
the addition of PHA (5 µg/mL, Murex Biotech Limited,
Dartford, UK).
Cytotoxicity Test. e 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetra-
zolium (MTS) cell proliferation assay (Promega, France)
is a colorimetric assay system, which measures the
reduction of a tetrazolium component (MTS) into forma-
zan produced by the mitochondria of viable cells. Cells
were washed twice in phosphate buffer saline and plated
in quadruplicate into microtiter-plate wells in 100-µL
culture media without or with our various compounds
at increasing concentrations (0, 1, 5, 10, 20, and 50 µM).
After 3 h of incubation at 37°C with 20 µL MTS/well,
the plates were read using an ELISA microplate reader
(ermo, Electrocorporation) at 490 nm wavelength. e
amount of colour produced was directly proportional to
the number of viable cells. e results are expressed as
the concentrations inhibiting cell growth by 50% after a
3 days incubation period. e 50% inhibitory concentra-
tions (IC50) were determined by linear regression analy-
sis, expressed in μM SD (Microsoft Excel).
e synthesis of the isoindoloquinoxalinone deriva-
tive 4d was first supported by the work of the Diana
et al.21,22 using 2-(2′-aminophenyl)-1-cyano-isoindole 7
as a key intermediate (Scheme 2). e isolation of deriva-
tive 7 was possible by a Strecker-type synthesis between
substituted 1,2-phenylendiamine and phthalaldehyde
in water and in the presence of potassium cyanide and
sodium hydrogensulfite21,22. Refluxing compound 7 in
acetic acid followed by an in situ hydrolysis failed in
the synthesis of lactam 4d, and only furnished the 2-(2-
aminophenyl)-2H-isoindole-1-carboxylic acid 8. e
structural identification of compound 8 was unambigu-
1
ously established on the basis of IR and H NMR data.
e zwitterionic structure of 8 has been confirmed by IR
spectrometry. e IR spectrum indicated the presence
of a carboxylate COO− band and a sharp ammonium
NH3+ band, respectively, observed at 1660 and 1910 cm−1.
Moreover, the 1H NMR spectrum showed a broad singlet
at 12.03 ppm (1H), highly characteristic for a carboxylic
acid proton, and a singlet at 6.91 ppm (2H), attributed to
the amine group.
results and discussion
Chemistry
e synthesis of the new isoindolo- or indoloquinoxa-
line derivatives 1a–h has been accomplished according
to the sequence depicted in Scheme 1. Condensation
of nitrocyclohexenes with ethyl isocyanoacetate in the
e preparation of 4d was achieved by using the fol-
lowing strategy via the 2-(2-fluorophenyl)-2H-isoindole-
1-carbonitrile 9 (Scheme 2). is cyano-substituted
isoindole 9 was also preliminary prepared by
a
Journal of Enzyme Inhibition and Medicinal Chemistry

Antiproliferative Activity of Novel Isoindolo[2,1-a]quinoxaline and Indolo[1,2-a]quinoxaline Derivatives 663
H
NO2
N
NO2
R3
COOC2H5
R1
ii
i
N
R3
COOC2H5
R2
+
R2
R1
CN
COOC2H5
2a-c
R2
R1
3a-c
iv
iii
R2
R2
R3
R3
N
N
R1
R1
v
N
vi
Cl
N
H
O
4a-d
5a-d
R2
R2
R3
R3
N
N
R1
N
N
R1
vii
R
N
1a-h
6a-d
CHO
R1−
R2−
R3−
R-
R1−
R2−
R3−
R-
H -
H -
H -
N
N
N
F
1a
1e
O
O
N
H
N
H
H -
N
N
N
F
F
1b
1c
1d
1f
1g
1h
N
H
N
H
H -
H -
N
N
N
H
O
O
N
H
H -
H -
F
N
H
N
H
Scheme 1. Reagents and conditions: (i) DBU, THF/t-BuOH, 50°C; (ii) 1-fluoro-2-nitrobenzene, Cs2CO3, DMF, Δ; (iii) Fe, CH3COOH, Δ; (iv)
1) o-iodotrifluoroacetanilide, K2CO3, CuI, L-proline, DMSO, 80°C; 2) H2O, 60°C (v) POCl3, Δ; (vi) OHC-C6H4-B(OH)2, Pd[P(C6H5)3]4, K2CO3,
toluene, EtOH, Δ; (vii) 4-(2-ketobenzimidazolin-1-yl)piperidine or 4-(5-fluorobenzimidazolin-2-yl)piperidine, NaBH3CN, MeOH, Δ.
© 2011 Informa UK, Ltd.

664 V. Desplat et al.
C17
C16
C2
C12
C15
C14
C3
C4
C11
C1
C6
C2
C4
N10
C13
C8
C5
N9
C1
C9
C7
C3
C14
C16
C23
C22
C18
C19
C6
C8
C10
C19
C20
N7
C5
C15
C21
C13
C20
C11
N12
C23
C22
C21
C24
C18
C17
C24
N25
N25
C30
C26
C30
C29
C26
C27
C28
C29
N31
C27
C28
036
C32
C37
N31
C35
C35
C38
C39
C40
C32
C33
N33
C39
C34
C37
036
N34
C40
C38
1a
1c
Figure 2. e ORTEP drawing of 1,3-dihydro-1-{1-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-ones 1a
and 1c with thermal ellipsoids at 30% level.
Strecker-type synthesis between 2-fluoroaniline and
o-phthalodicarboxaldehyde in water and in the presence
of potassium cyanide and sodium hydrogensulfite23.
Treatment of 9 with potassium hydroxide in tert-butanol
at 80°C led directly to the tetracyclic lactam 4d involving
selective hydrolysis to amide, which was able to carry
out intramolecular displacement of aromatic fluorine
through an anionic ring closure24–27. Structure elucida-
tion of 4d was performed using high-resolution mass
spectrometry analysis (experimental mass 257.0699,
theoretical mass for C15H10N2O23Na 257.0691). Structural
confirmation of compound 4d was also achieved by IR
and 1H NMR spectroscopy on the basis of previous results
for 5H-pyrrolo[1,2-a]quinoxalin-4-one series published
by our group4–7,10,11. In the 1H NMR spectrum, we observed
a D2O exchangeable singlet at 11.40 ppm characteristic of
the NH lactam function. Moreover, the data also showed
a singlet at 8.78 ppm attributed to the proton in position
11 of the isoindolo[2,1-a]quinoxaline skeleton. In the IR
spectrum, lactam 4d showed a strong stretching carbonyl
band at 1645 cm−1.
cell line (MCF7). Compound LY-294002 (Figure 3), which
showed antiproliferative activity against the HL60, U937
and K562 cell lines28–31, was applied as a referential cyto-
toxic agent. e results of the antiproliferative activity
studies are summarized in Table 1.
Among compounds 1a–h, the benzylpiperidinyl
fluorobenzimidazole derivatives 1e–g exhibited the best
antiproliferative activity on the growth of human myeloid
U937 cell line (IC50 from 3.5 to 4 µM) in comparison with
their benzylpiperidinyl benzimidazolone analogues 1a–c
that were found inactive (IC50 > 50 µM). Surprisingly, the
isoindoloquinoxaline 1h, the structural aromatic ana-
logue of the tetrahydroisoindoloquinoxaline 1e, showed
moderate antiproliferative activity with an IC50 value of
42 µM. Moreover, in terms of structure–activity relation-
ships discussion, it could be also noticed that the IC50
of compound 1g bearing an indole moiety (IC50 = 4 µM)
was found 10 times lower than those of its isoindolo ana-
logue 1h (IC50 = 42 µM). Introduction of a fused benzene
nucleus (compound 1f) onto the tetrahydroisoindole ring
(compound 1e) did not lead to significant modification
in the antiproliferative activity (IC50 = 4 µM for 1f versus
3.5 µM for 1e).
Biology
Antiproliferative Effect. e eight new compounds 1a–h
were tested in MTS assay for their in vitro antiprolifera-
tive activity against five human leukemic cell lines (U937,
K562, Jurkat, U266, and HL60), and one breast cancer
Similarprofileofantiproliferativeactivitywasobserved
against the human myeloma cell line U266. Hence, the
three quinoxalines 1e–g, bearing the benzylpiperidinyl
fluorobenzimidazole moiety in their 4-position, were
Journal of Enzyme Inhibition and Medicinal Chemistry

Antiproliferative Activity of Novel Isoindolo[2,1-a]quinoxaline and Indolo[1,2-a]quinoxaline Derivatives 665
NH2
CHO
N
COOH
NH2
CN
N
CHO
NH2
i
+
ii
8
NH2
7
N
N
H
O
CHO
4d
F
F
CHO
F
N
i
CN
+
N
iii
CONH2
NH2
9
Scheme 2. Reagents and conditions: (i) NaHSO3, KCN, H2O, 70°C; (ii) CH3COOH, Δ; (iii) KOH, tert-BuOH, 80°C.
nature of the substitution at position 4 in the isoindolo-
quinoxaline derivatives 1d and 1h was less detrimental
O
for the activity (IC50 of 11 and 13 µM, respectively). e
benzylpiperidinyl fluorobenzimidazole compounds pos-
sessing a fused tetrahydroisoindole, dihydrobenzo[e]
O
N
isoindole, or indole nucleus (compounds 1e–g) exhib-
ited better antiproliferative activities than their isoindolo
analogue 1h. In a general way, this kind of structure–
activity profile was also observed against the HL60 and
Jurkat cell lines.
O
Against the HL60 human acute promyeloid leuke-
mia cell line, most of the tested compounds showed
antiproliferative activity with IC50 values from 3 to 18 µM,
except 1b and 1c that were found inactive (IC50 > 50 µM).
As in a general way, isoindolo- and indoloquinoxalines
having a benzylpiperidinyl fluorobenzimidazole moi-
ety at position 4 exhibited better activities than their
benzylpiperidinyl benzimidazolone homologues (i.e.,
IC50 = 4 µM for 1e versus 13.5 µM for 1a, 3 µM for 1f com-
pared >50 µM for 1b, and 4 µM for 1g versus >50 µM
for 1c). Surprisingly, this kind of observation could not
be applied to compounds 1d and 1h. Hence, the IC50 of
1d (9 µM) was twice lower than those of compound 1h
(IC50 = 18 µM).
LY-294002
Figure 3. Structure of compound LY-294002.
always found the most active compounds with an IC50
of 3–4 µM. e fourth fluorobenzimidazole derivative
1h also showed significant antiproliferative activity
(IC50 = 14 µM). Concerning the fused substitutions on the
quinoxaline ring, we noticed a similar biological profile
as those observed against the U937 cell line.
eantiproliferativepotenciesofthesenewderivatives
1a–h were also examined toward the human myeloid leu-
kaemia cell lines K562 and HL60. On K562 cell line, 1e–g
were also found as the most active compounds (IC50 = 3–4
µM). e replacement of the benzylpiperidinyl fluo-
robenzimidazole substituent by a benzylpiperidinyl ben-
zimidazolone group in position 4 of the fused heterocyclic
skeleton (compounds 1a–c) led to a decrease in the activ-
ity (IC50 ~ 46 µM). However, it could be noticed that the
e antiproliferative activities of compounds 1a–h
against the T-lymphocyte Jurkat cell line were similar
to those observed against the HL60 cell line. e same
observation concerning antiproliferative activity of benz-
imidazolone 1d versus fluorobenzimidazole 1h on Jurkat
© 2011 Informa UK, Ltd.

666 V. Desplat et al.
Table 1. In vitro activity of compounds 1a-h on U937, K562, HL60, Jurkat, U266, and MCF7 cells, and cytotoxicity on human peripheral
blood mononuclear cells PBMNC + PHA.
IC50 values (µM)a
Cytotoxicity on activated human
peripheral blood mononuclear cells
(PBMNC)
Compound
U937
14 0.3
>50
K562
HL60
14 0.3
13.5 0.3
>50
Jurkat
U266
46
>50
MCF7
0.5
11 0.5
0.5
>50
> 50
PBMNC + PHA
LY-294002
38
1
22
44
1
1
2
7
>50
>50
>50
1a
1b
1c
1d
1e
1f
46.5
45.5
45.5
1
>50
1
1
>50
>50
0.2
3.5 0.2
0.2
>50
>50
9
>50
>50
41
1
>50
11 0.5
9
4
3
4
0.4
0.2
0.5
0.3
5
> 50
>50
3.5 0.3
3
4
0.2
0.3
3.5 0.3
4
0.3
0.4
0.4
1
6
0.3
0.5
0.3
4
4
0.3
0.3
1
3
4
3
0.3
0.2
3
4
7
4
1g
3.5 0.2
3.5 0.3
1h
42
13 0.5
18 0.5
12 0.5
14 0.5
33
>50
ae IC50 (µM) values correspond to the mean standard deviation from three independent experiments.
cell line could also be noticed (IC50 = 5 µM for 1d versus
12 µM for 1h).
lines Jurkat, and >5.5 for compound 1b against the MCF7
breast adenocarcinoma.
In MCF7 cell line, the pyrrolo[1,2-a]quinoxaline deriv-
atives 1e–g, bearing a benzylpiperidinyl fluorobenzimi-
dazole moiety in position 4 exhibited potent cytotoxicity
with IC50 ranging from 3 to 4 µM. However, the isoindolo
derivative 1h showed low activity (IC50 =33 µM) against
this breast cancer line. Moreover, two of their benzylpip-
eridinyl benzimidazolone analogues bearing an hydroge-
nated isoindole ring (compounds 1a and 1b) have shown
significant antiproliferative activity with an IC50 of 11 and
9 µM, respectively. Aromatization of the tetrahydroisoin-
dole nucleus of 1a (compound 1d) led to a decrease in
the antiproliferative activity; i.e., IC50 =11 µM for 1a and
>50 µM for 1d.
Against each human cancer cell lines, the antiprolif-
erative activities of compounds 1e–g were always found
superior to those of the reference drug LY-294002.
Cytotoxicity. All compounds 1a–h were tested on
activated human peripheral blood mononuclear cells
(Table 1). As expected, most of the pyrrolo[1,2-a]qui-
noxalines 1a–h showed significant level of cytotoxicity
against lymphocytes with IC50 ranging from 4 to >50 µM.
ese preliminary results were used to determine their
respective range of toxic concentration.
Conclusion
In the present report, we described the synthesis of a new
series of substituted isoindolo- or indoloquinoxaline
derivatives and presented their antiproliferative activi-
ties on the human leukemic cell lines U937, K562, Jurkat,
U266, and HL60, and the breast cancer cell line MCF7.
From the biological activity data, some structure–activity
relationships can be inferred. e presence at C-4 posi-
tion of the benzylpiperidinyl fluorobenzimidazole moi-
ety on the isoindolo- and indoloquinoxaline skeletons
resulted in active compounds. Nevertheless, the nature
of this substitution at position 4 in the isoindolo[2,1-a]
quinoxalines 1d and 1h seems less detrimental for the
antiproliferative activity. In addition, the two antiprolif-
erative compounds 1b and 1d showing interesting index
of selectivity may constitute suitable candidates for fur-
ther pharmacological studies. Moreover, it would be also
interesting to enlarge the biological evaluation of these
two new pyrrolo[1,2-a]quinoxaline derivatives 1b and
1d in order to precise now their mechanism of action.
In a general way, the less cytotoxic molecules were
the less active on the various tumor cell lines except
compounds 1d and 1h. Moreover, the benzylpiperidinyl
benzimidazolone derivatives 1a–d were found less cyto-
toxic than their benzylpiperidinyl fluorobenzimidazole
analogues 1e–g in the exception of compound 1h that
presented an IC50 superior to 50 µM against lymphocytes.
Indexes of selectivity were defined as the ratio of the IC50
value on the human mononuclear cells to the IC50 value
on the U937, K562, HL60, Jurkat, U266, or MCF7 lines.
Compounds that demonstrated high selectivity (high
index of selectivity) should offer a potential of safer ther-
apy. is led to identify compounds with index of selec-
tivity >10 for compound 1d on the human leukemic cell
Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
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