Synthesis and Antiproliferative Effect of Ethyl 4-[4-(4-Substituted Piperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxalinecarboxylate Derivatives on Human Leukemia Cells

Accepted Article

Title: Synthesis and antiproliferative effect of ethyl 4-(4-

substitutedpiperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxaline-

carboxylate derivatives on human leukemic cells

Authors: Jean Guillon, vanessa desplat, marian vincenzi, romain lucas,

stéphane moreau, solène savrimoutou, noel pinaud, david

bigat, elodie enriquez, mathieu marchivie, sylvain routier,

pascal sonnet, filomena rossi, and luisa ronga

This manuscript has been accepted after peer review and appears as an

Accepted Article online prior to editing, proofing, and formal publication

of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

published online in Early View as soon as possible and may be different

to this Accepted Article as a result of editing. Readers should obtain

the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201700049

Link to VoR: http://dx.doi.org/10.1002/cmdc.201700049

A Journal of

www.chemmedchem.org

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Synthesis and antiproliferative effect of ethyl 4-(4-

substitutedpiperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxaline-

carboxylate derivatives on human leukemic cells

Vanessa Desplat⁺,^[a,b]Marian Vincenzi⁺,^[c,d,e]Romain Lucas,^[c,d]Stéphane Moreau,^[c,d]Solène

Savrimoutou,^[c,d]Sandra Rubio,^[c,d]Noël Pinaud,^[f]David Bigat,^[c,d]Elodie Enriquez,^[c,d]Mathieu

Marchivie,^[g]Sylvain Routier,^[h]Pascal Sonnet,^[i]Filomena Rossi,^[e]Luisa Ronga,^[c,d]and Jean

Guillon*^[c,d]

[a]

[b]

[c]

Dr. V. Desplat

Univ. Bordeaux, UFR des Sciences Pharmaceutiques,

F-33076 Bordeaux cedex (France)

Dr. V. Desplat

INSERM U1035, Cellules souches hématopoïétiques normales et leucémiques,

F-33000 Bordeaux (France)

Dr. M. Vincenzi, Dr. R. Lucas, Dr. S. Moreau, S. Savrimoutou, Dr. S. Rubio, D. Bigat, E. Enriquez, Dr. L. Ronga, Prof. J. Guillon

Univ. Bordeaux, UFR des Sciences Pharmaceutiques,

ARNA Laboratory, F-33076 Bordeaux cedex (France)

E-mail: jean.guillon@u-bordeaux.fr

[d]

[e

Dr. M. Vincenzi, Dr. R. Lucas, Dr. S. Moreau, S. Savrimoutou, Dr. S. Rubio, D. Bigat, E. Enriquez, Dr. L. Ronga, Prof. J. Guillon

INSERM U1212, UMR CNRS 5320, ARNA Laboratory,

F-33000 Bordeaux (France)

Dr. M. Vincenzi, Prof. F. Rossi

Department of Pharmacy and CIRPeB,

University of Naples “Federico II”,

Via Mezzocannone, 16 80134 Naples (Italy)

N. Pinaud

[f]

Univ. Bordeaux, ISM – CNRS UMR 5255,

351 cours de la Libération, F-33405 Talence cedex (France)

Dr. M. Marchivie

Univ. Bordeaux, ICMCB CNRS-UPR 9048,

87, Avenue du Docteur Schweitzer, F-33608 Pessac cedex (France)

Prof. S. Routier

[g]

[h]

Institut de Chimie Organique et analytique

Univ. Orleans, CNRS UMR 7311, ICOA,

BP 6759, rue de Chartres, F-45067 Orléans cedex 2 (France)

Prof. P. Sonnet

[i]

Université de Picardie Jules Verne,

Laboratoire de Glycochimie, des Antimicrobiens et des Agroressouces,

UMR CNRS 7378, UFR de Pharmacie, 1 rue des Louvels, F-80037 Amiens cedex 01 (France)

[⁺]

These authors contributed equally to the work

Abstract: Acute leukemia is a hematological malignancy with high

incidence and recurrence rates, and is characterized by an

accumulation of blasts in bone marrow due to proliferation of

immature lymphoid or myeloid cells associated with a blockade of

differentiation. The heterogeneity of leukemia led to look for new

specific molecules for leukemia subtypes or even for therapy-

resistant cases. Among heterocyclic derivatives that attracted

attention due to their large spread biological activities, we have

focused our interest towards the pyrrolo[1,2-a]quinoxaline

heterocyclic framework that has been previously identified as an

interesting scaffold for antiproliferative activities against various

human cancer cell lines. In this work, new ethyl 4-[4-(4-

substitutedpiperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxaline-carboxylate

derivatives 1a-o have been designed, synthesized and evaluated

against five different leukemia cell lines, including Jurkat and U266

(lymphoid cell lines), and K562, U937, HL60 (myeloid cell lines), and

also on normal human peripheral blood mononuclear cells

(PBMNCs). These new pyrrolo[1,2-a]quinoxaline series showed

interesting cytotoxic potential against all tested leukemia cell lines. In

particular, pyrroloquinoxalines 1a and 1m-n seem to be interesting

due to their high activity against leukemia and their low activity

against normal hematopoietic cells leading to high index of

selectivity.

Introduction

Acute leukemia is one of the most common lethal

leukemia which is characterized by an accumulation of blasts in

bone marrow due to the proliferation of immature lymphoid or

myeloid cells associated with a blockade of differentiation. Adult

acute leukemia present a poor prognosis due to a high incidence

of relapses. As treatment has not been modified for many years,

development of new more selective therapeutic approaches is a

challenge, in order to improve the overall survival in patients.^[1]

1

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Heterocyclic framework constitutes the basis of an

yl)]benzyl-pyrrolo[1,2-a]quinoxaline-carboxylate derivatives. So,

by taking into accounts the best results obtained in the previous

series A-B (Figure 1), we decided to refer to the JG564 and

important class of compounds possessing interesting biological

activities. In the recent years, pyrrolo[1,2-a]quinoxalines have

attracted persistent interest due to practically important

properties of some their derivatives, specifically pronounced

biological activities. Thus, these heterocyclic derivatives showed

many biological activities and have been found in many

pharmaceutical molecules such including antipsychotic agents,^[2]

antiviral agents,^[3]adenosine receptor modulators,^[4]antiparasitic

agents,^[5-11]and anticancer agents.^[12-14]Against cancer, the

discovery of new therapeutic agents is one of the most important

goals in medicinal chemistry. Thus, we have recently published

four series of new pyrrolo[1,2-a]quinoxaline derivatives (Figure

1) endowed with interesting activity towards the human leukemia

cells.^[15-18]These antileukemial pyrroloquinoxaline compounds

have been previously reported as new structural analogues of

JG572 pyrrolo[1,2-a]quinoxaline pharmacophores^[15-18]as

a

template for the design of new compounds 1a-o in which the

pyrrolo[1,2-a]quinoxaline ring is substituted in various positions

by the ethyl ester function (Figure 1). In addition, further

modulations on the piperidine core have been also considered,

such as the introduction of new substituted heterocyclic moieties

(benzimidazolone, fluorobenzimidazole and pyridinyltriazole

substituents) in analogy to previously described antileukemial

compounds.^[15-20]The antiproliferative activity of the obtained

derivatives 1a-o was then evaluated in vitro against various

myeloid (U937, HL60, K562) or lymphoid (Jurkat, U266)

leukemic cell lines. To determine their respective cytotoxicity,

the

new

ethyl

4-[4-(4-substitutedpiperidin-1-yl)]benzyl-

derivative A6730,

a

reference Akt inhibitor that presents

pyrrolo[1,2-a]quinoxaline-carboxylate derivatives 1a-o were also

tested on activated human peripheral blood mononuclear cells.

Structure-activity relationships of these new ethyl 4-(4-

substitutedpiperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxaline-

antiproliferative activity against various human leukemia cell

lines.^[15-20]In this context and by considering the biological

activities of previous pyrrolo[1,2-a]quinoxalines on human

leukemic cells, we have undertaken and investigated the

synthesis of a new series of ethyl 4-[4-(4-substitutedpiperidin-1-

carboxylate

derivatives

1a-o

are

also

developed.

Insert Figure 1.

Results and Discussion

Chemistry

Reduction of the nitro moiety of 7 with iron in hot glacial acetic

acid produced the spontaneous ring closure onto the ester to

afford the desired tricyclic pyrrolo[1,2-a]quinoxaline 2a through a

one-pot reduction-cyclization step.^[14,17]The Clauson-Kaas

reaction of 8a-b with 2,5-dimethoxytetrahydrofuran (DMTHF) in

acetic acid gave the pyrrolic derivatives 9a-b. The resulting 1-(2-

nitrophenyl)pyrrole ester intermediates 9a-b were subsequently

reduced into the attempted 1-(2-aminophenyl)pyrrole esters 10a-

b using a sodium borohydride-copper(II) sulfate in ethanol at

0°C.^[10,14,17]This NaBH₄-CuSO₄system was found to be quite

powerful in reducing the aromatic nitro groups. The reaction of

10a-b with triphosgene in toluene gave the lactams 2b-c.

Substitution of commercially methyl pyrrole-2-carboxylate with

various ethyl fluoro-nitrobenzenecarboxylate 11a-b led to the

methyl 1-(4- or 5-ethoxycarbonyl-2-nitrophenyl)pyrrole-2-

carboxylates 12a-b. Refluxing compounds 12a-b in acetic acid

with iron powder also gave lactams 2d-e.

The reported pyrrolo[1,2-a]quinoxaline derivatives 1a-k

were synthesized via different ethyl 4,5-dihydro-4-oxo-5H-

pyrrolo[1,2-a]quinoxaline-carboxylates 2a-e (Schemes 1 and 2).

Different strategies were considered for the synthesis of the

pyrrolo[1,2-a]quinoxaline esters 2a-e in order to introduce the

ester function in various positions of the heterocyclic skeleton.

The synthesis of the ester 2a has been accomplished in six

steps according to the following pathway (Scheme 1): the ethyl

3-hydroxymethylpyrrole-2-carboxylate 3 was prepared by silver-

catalyzed cycloaddition of commercially available propargyl

alcohol with ethyl isocyanoacetate in 1,4-dioxane at 80°C.^[21,22]

The conversion of 3 to ethyl 3-formyl-1H-pyrrole-2-carboxylate 4

was achieved using manganese dioxide (MnO₂) in chloroform.^[21]

The oxidation of the aldehyde function of 4 into a carboxylic acid

group was realized with KMnO₄leading to the pyrrole 5 that was

converted into the diester derivative 6 by treatment with thionyl

chloride in ethanol. The preparation of the N-aryl pyrrole 7 was

obtained by nucleophilic substitution of the diethyl pyrrole-2,3-

Insert Scheme 1.

These lactames 2a-e were subsequently chlorodehydroxylated

with

phosphorous

oxychloride,

leading

to

the

4-

dicarboxylate

6

with 2-fluoro-nitrobenzene using cesium

chloropyrrolo[1,2-a]quinoxalines 13a-e (Scheme 2). 4-

(Pyrrolo[1,2-a]quinoxalin-4-yl)benzaldehydes 14a-e were easily

prepared by a direct Suzuki-Miyaura cross-coupling reaction of

4-chloropyrroloquinoxalines 13a-e with 4-formylphenylboronic

acid performed in the presence of Pd(PPh₃)₄as a catalyst, and

in the presence of potassium carbonate used as the base.^[15-18]

carbonate as the base in refluxing DMF solution.^[14,17]

Insert Scheme 2.

2

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

significant antiproliferative activity with IC₅₀ranging from 6 to 15

M, better than the one found for the reference compound

A6730 (IC₅₀= 17 M). The pyrrolo[1,2-a]quinoxalines 1e-g,

bearing the ethyl ester function in position 7 of the heterocyclic

The 3D structural determination of 1c was established by X-ray

crystallography, and confirmed the structure in the solid state as

anticipated on the basis of NMR data (Figure 2).

skeleton were found to show low antiproliferative activity (IC₅₀

=

34-37 M) or were found inactive (IC₅₀> 50 M). The same

observation could be done for compounds 1h substituted by the

ester group in position 8 and derivative 1o (IC₅₀> 50 M).

Insert Figure 2.

Surprisingly, compound 1n,

a

structural analogue of

The pyrrolo[1,2-a]quinoxaline derivatives 1l-o were synthesized

in six steps from various halogenated 2-nitroaniline according to

Scheme 3. Preparation of 1-(4-bromo-2-nitrophenyl)pyrroles

15a-b was performed according to the Clauson-Kaas reaction

run under micro-waves irradiation starting from halogeno-2-

nitroaniline and 2,5-dimethoxytetrahedrofuran in acetic acid.^[14]

The resulting phenylpyrrole 15a-b intermediates were

subsequently reduced using a NaBH₄-CuSO₄treatment into the

attempted 1-(2-amino-4-bromophenyl)pyrroles 16a-b.^[10,14,17]

Addition of 16a-b to ethyl chlorooxoacetate in the presence of

triethylamine provided the esters 17a-b.^[14,24]The ethyl

halogenopyrrolo[1,2-a]quinoxaline-4-carboxylates 18a-b were

then prepared by cyclization of the amido-esters 17a-b in

refluxing phosphorus oxychloride.^[24]Coupling halogenated

compounds 18a-b with 4-formylphenylboronic acid under

Suzuki–Miyaura cross-coupling conditions proceeded cleanly to

afford the pyrrolo[1,2-a]quinoxaline-esters 19a-b. Reaction of

substituted piperidines with 19a-b and using sodium

cyanoborohydride as reductive agent in methanol gave the

amines 1l-o (Scheme 3).

pyrroloquinoxalines 1l-m, only showed a moderate activity

against the leukaemia cell line K562 with an IC₅₀of 35 M.

Moreover, in terms of structure-activity relationships discussion,

it

could

be

also

noticed

that

the

4-substituted

benzylpiperidinylpyrroloquinoxalines 1a-c, 1i-k and 1d, bearing

the ethyl ester moiety in their position 3, 9 and 6, respectively,

were found the most active compounds with IC₅₀ranging from

0.5 to 15 M. Such observation could also be noticed for

compounds 1l-m, some structural analogues of compound 1d in

which the substitutions of the benzylpiperidinyl moiety and the

ester are simply reversed.

Against the HL60 human acute myeloblastic leukemia

cell line, most of the tested compounds showed antiproliferative

activity with IC₅₀values from 0.5 to 17 M, excepted 1d, 1h and

1n-o that were found less or inactive (IC₅₀> 50 M for 1d, 1h

and 1o; and IC₅₀= 40 M for 1n). The three 4-substituted

benzylpiperidinylpyrroloquinoxalines 1a-c having the ester

function in position 3 (IC₅₀= 1-4 M), and the pyrroloquinoxaline

1j substituted by the ester in C-9 (IC₅₀= 0.5 M) exhibited better

activities than their other homologues. These four last

derivatives showed a better activity that the one noticed for the

reference compound A6730 with an IC₅₀of 5.5 M. Moreover, it

could be noticed that the substitution of the ester at position 7 in

the pyrroloquinoxaline derivatives 1e-g was more detrimental for

the activity (IC₅₀of 7-17 M). In addition to this sub-series, the

replacement of the benzylpiperidinyl benzimidazolone or

benzylpiperidinyl fluorobenzimidazole substituent (compounds

1e-f) by a benzylpiperidinyl triazolylpyridine group in position 4

of the fused heterocyclic skeleton (compound 1g) led to an

increase in the activity (IC₅₀~ 7 M). However, as the

substitution in position 7 by the ethyl ester function led to the

moderate bioactive compounds 1e-g (IC₅₀= 7-17 M), the

substitution at position 8 by this ester (compound 1h) induced a

decrease in the antiproliferative activity upon HL60 cell line with

IC₅₀superior to 50 M. Among the fifteen compounds tested for

antiproliferative activities on HL60 cell line, the pyrrolo[1,2-

Insert Scheme 3.

Biological activity

Cytotoxicity in leukemia cell lines

The fifteen new compounds 1a-o were tested in MTS

assay for their in vitro antiproliferative activity against five human

leukemic cell lines (U937, K562, Jurkat, U266 and HL60). In

addition, compound A6730 (Figure 1) was used in these tests as

the reference standard drug. The biological results are

presented in Table 1. Moreover, compound LY-294002, which

showed antiproliferative activity against the HL60, U937 and

K562 cell lines,^[25-28]was also applied as a referential cytotoxic

agent.

a]quinoxalines 1b, 1f, 1m and 1j, always bearing

a

benzylpiperidinyl fluorobenzimidazole moiety in their 4-position,

were found the most active compounds in each subseries with

an IC₅₀of 0.5-14 M.

Insert Table 1.

Against the T-acute lymphoblastic leukemia Jurkat cell

line, the biological results of compounds 1a-o were quite similar

to those observed against the HL60 cell line in terms of

Structure-activity relationships. These results exhibited a potent

cytotoxicity for pyrrolo[1,2-a]quinoxalines 1a-c (IC₅₀from 1 to 2.1

M) more potent than the one observed for reference A6730

(IC₅₀= 3.5 M). In addition, compounds 1d and 1h, the structural

analogues of pyrroloquinoxalines 1a, bearing the ester group in

Firstly, the antiproliferative potencies of these new

derivatives 1a-o were examined towards the human myeloid

leukaemia cell lines K562 and HL60. Among the fifteen

compounds tested for antiproliferative activities on K562 cell

line, the four pyrrolo[1,2-a]quinoxalines 1a-b, 1j and 1m were

found the most active compounds with an IC₅₀of 0.5-4 M. The

other pyrroloquinoxalines 1c-d, 1i and 1k-l also showed

3

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

position 6 and 8 of the heterocyclic system, were found inactive

(IC₅₀> 50 M). Nevertheless, their homologues pyrrolo[1,2-

a]quinoxalines 1e-g with this ester in the C-7 position showed

moderate antiproliferative activity against the T-lymphocyte

Jurkat cell line with IC₅₀ranging from 12 to 20 M. Surprisingly,

the substitution of this ester function in the C-9 position of the

pyrrolo[1,2-a]quinoxaline skeleton (compounds 1i-k) led to more

active derivatives (IC₅₀= 3-8 M) in comparison to their other

structural analogues 1d-h. We could also noticed that

pyrroloquinoxalines 1l-n, the structural analogues of 1d in which

the substitutions of the benzylpiperidinyl moiety and the ethyl

ester groups are reversed between positions 4 and 6, exhibited

potent cytotoxicity with IC₅₀ranging from 3 to 4 M comparable

to the one of reference A6730. When, the substitution of the

benzylpiperidinyl moiety was displaced in position 7 (compound

1o), the antiproliferative activity against the leukaemia cell line

Jurkat decreased (IC₅₀= 30 M).

Cytotoxicity activity in activated normal peripheral blood

mononuclear cells

The compounds 1a-o were tested on activated

(PBMNC + PHA) human peripheral blood mononuclear cells to

evaluate their respective cytotoxicity on normal cells (Table 1).

In this biological evaluation, most of the pyrroloquinoxalines 1a-

o showed significant level of cytotoxicity against lymphocytes

with IC₅₀ranging from 6 to over 50 M. These results were used

to determine their respective range of toxic concentration.

Indexes of selectivity (IS) were defined as the ratio of the IC₅₀

value on the human mononuclear cells to the IC₅₀value on the

leukemia K562, U937, HL60, Jurkat and U266 lines.

Compounds that demonstrated high selectivity (high index of

selectivity) should offer a potential of safer therapy. This led to

identify compound 1a with index of selectivity >50 on the human

myeloid leukemic cell lines HL60 and Jurkat; and >16.7 on the

U937 cell line. Moreover, we could notice that compounds 1m-n

showed interesting selectivity towards Jurkat CML cell lines (SI >

16.7). These three compounds could now constitute new

Among compounds 1a-o, the benzylpiperidinyl

derivatives 1a-c, bearing the ester in position 3, also exhibited

the best antiproliferative activity on the growth of human myeloid

U937 cell line (IC₅₀from 3 to 6 M) in comparison with their

other analogues 1d-o which were found less active (IC₅₀ranging

from 8 to 39 M) or inactive (IC₅₀> 50 M). Moreover,

pyrroloquinoxaline 1j also showed significant antiproliferative

activity against the U937 cell line, similar the one found for the

reference compound A6730 (IC₅₀= 8 M).

candidates

for

further

pharmacomodulations

and

pharmacological studies.

Study of the mode of action

The reference imidazoquinoxaline A6730, which

showed interesting selectivity on the Jurkat cell line, is also

described as an Akt inhibitor (Akt1 IC₅₀= 58 nM, Akt2 IC₅₀= 10

nM and IC₅₀Akt3 = 2.2 M).^[29]To determine the possible

biological mechanism of action of these new derivatives, we first

evaluated their potency on isolated enzymes such as Akt 1, 2

and 3 as well as on mTOR. These pharmacological tests have

been performed by DiscoverX at the concentrations of 1 and 10

M.^[30]Nevertheless, only low effects have been detected. Akt3

activity was reduced of 40 % with derivative 1b at 10 M. This

enzymatic interaction could not explain alone the cell effects.

Against the human myeloma cell line U266, the

pyrrolo[1,2-a]quinoxalines 1a-b, 1k and 1m, exhibited the most

potent cytotoxicity with IC₅₀ranging from 5 to 7 M. All the other

tested pyrroloquinoxalines 1 were found to present moderate

antiproliferative activity (IC₅₀between 10 to 20 M for

compounds 1c, 1g, 1i, 1l and 1n) or inactive with IC₅₀> 50 M

(derivatives 1d-f, 1h, 1j and 1o).

From a SAR point of view, these preliminary biological

results on these leukemia cell lines enlightened the importance

of the substitution at C-4 position of the pyrroloquinoxaline

scaffold by a benzylpiperidinyl group, and also the need of an

ethyl ester functionalisation in position 3 of the pyrrolo[1,2-

a]quinoxaline ring (compounds 1a-c). Concerning these

bioactive compounds, it could also be noticed that compound 1a

derived from the incorporation of the benzylpiperidinyl

benzimidazolone moiety, which was present in the reference

compound A6730, into the 4-position of the heterocyclic

pyrroloquinoxaline ring was found the most active candidate

against all the leukemia cell lines in comparison to their

benzylpiperidinyl fluorobenzimidazole or benzylpiperidinyl

triazolylpyridine analogues.

Assessment of apoptosis in leukemic cell lines

Apoptosis induces loss of membrane asymmetry resulting

in phosphatidyl serine (PS) exposure detected by annexin V.

The effect on apoptosis of compound 1a, which showed the

most interesting index of selectivity on human myeloid leukemic

cell lines, was studied.The 1a-treatment of HL60 and K562 cells

induces a significantly increase in Annexin V positive cells after

2 and 3 days of incubation (Figure 3). These results could

explain the inhibition of cell proliferation observed with this

compound.

Insert Figure 3.

Against each human cancer cell lines, the

antiproliferative activities of compounds 1 were generally found

superior to those of the other reference drug LY-294002.

4

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

0°C, ethyl isocyanoacetate (27 mmol) was added dropwise. The reaction

mixture was stirred for 10 min at room temperature, then heated 2 h at

80°C. The resulting slurry was concentrated under reduced pressure and

taken up with dichloromethane. The organic layer was washed with brine,

dried with Na₂SO₄, filtered and evaporated under vacuum. The crude

was purified on silica gel (eluent 5:5 AcOEt/petroleum ether) to give 3 as

pale yellow crystals (86%), mp 91°C.^[21,22]

Conclusions

In summary, by taking into account ours previous works

using the pyrrolo[1,2-a]quinoxaline template, we designed and

synthesized series of fifteen new ethyl 4-[4-(4-

a

substitutedpiperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxaline-

carboxylate derivatives 1a-o and then evaluated their

antileukemic activity on the human leukemic cell lines U937,

K562, Jurkat, U266 and HL60. By using these biological results,

Ethyl 3-formyl-1H-pyrrole-2-carboxylate (4)

a

preliminary SAR study has been discussed. The

To a solution of ethyl 3-hydroxymethylpyrrole-2-carboxylate 3

(3.8 mmol) in chloroform (25 mL), was added manganese dioxide (38

mmol). The reaction mixture was then refluxed for 2.5 h. The black solid

was removed by filtration and washed with chloroform. The filtrate and

washings were combined, dried with Na₂SO₄, filtered and evaporated to

dryness under reduced pressure to give 4 as white crystals (70%), mp

104°C.^[21]

pharmacological evaluations of new compounds 1 showed

cytotoxic activity in the different myeloid and lymphoid leukemia

cell lines. Through these new pharmacomodulations on the

pyrroloquinoxaline moiety, biological data demonstrate that

derivatives 1a and 1m-n are interesting compounds due to their

high cytotoxic activity against some leukemia cells (IC₅₀ranging

from 1 to 3 M) and their lower toxicity against the normal

hematopoietic cells (estimated IC₅₀> 50 M). In comparison with

the previously described pyrrolo[1,2-a]quinoxaline series,^[15-18]

these novel antileukemial heterocyclic compounds with high

index of selectivity could constitute more suitable candidates for

further pharmacological modulations and investigations.

Moreover, further studies could be required to know the

mechanism of action of these new bioactive pyrrolo[1,2-

a]quinoxaline derivatives. These studies are currently under

progress.

2-Ethoxycarbonyl-1H-pyrrole-3-carboxylic acid (5)

To a solution of ethyl 3-formyl-1H-pyrrole-2-carboxylate 4 (2.6

mmol) in acetone (6 mL), was added dropwise a solution of KMnO₄(5.2

mmol) in acetone/water (v/v : 1/1). The resulting mixture was then heated

at 40°C for 1 h, then stirred at room temperature for an additional hour.

To the reaction mixture was added an 85% aqueous solution of Na₂S₂O₄

(0.26 mmol). After 10 min of stirring, the reaction solution was filtered on

Celite. The Celite was washed with a 1 M solution of NaOH (7 mL). The

acetone of the filtrate was then evaporated under reduced pressure; the

resulting filtrate was cooled and acidified with a 1 M aqueous solution of

HCl until a precipitate appears. The precipitate was filtered, washed with

water and dried to give 5 as white crystals (31%), mp 184°C. ¹H-NMR ¹H

NMR (CDCl₃) : 12.86 (s, 1H, CO₂H), 12.46 (sl, 1H, NH), 7.07 (t, 1H,

J=5.90 Hz, H-5), 6.60 (t, 1H, J=5.70 Hz, H-4), 4.33 (q, 2H, J=7.20 Hz,

CH₂), 1.31 (t, 3H, J=7.20 Hz, CH₃). Anal. Calcd. for C₈H₉NO₄: C, 52.46;

H, 4.95; N, 7.65. Found: C, 52.63; H, 5.12; N, 7.92.

Experimental Section

Chemistry

Commercially reagents were used as received without

additional purification. Melting points were determined with an SM-LUX-

POL Leitz hot-stage microscope and are uncorrected. IR spectra were

recorded on an NICOLET 380FT-IR spectrophotometer. NMR spectra

were recorded with tetramethylsilane as an internal standard using a

BRUKER AVANCE 300 spectrometer. Splitting patterns have been

reported as follows: s = singlet; bs = broad singlet; d = doublet; t = triplet;

q = quartet; dd = double doublet; ddd = double double doublet; dt =

double triplet; m = multiplet. Analytical TLC were carried out on 0.25

precoated silica gel plates (POLYGRAM SIL G/UV₂₅₄) and visualization

of compounds after UV light irradiation. Silica gel 60 (70-230 mesh) was

used for column chromatography. Microwave experiments were carried

out using a focused microwave reactor (CEM Discover). High resolution

mass spectra (electrospray in positive mode, ESI+) were recorded on a

Waters Q-TOF Ultima apparatus. Elemental analyses were found within

±0.4% of the theoretical values.

Diethyl 1H-pyrrole-2,3-dicarboxylate (6)

To a suspension of 2-ethoxycarbonyl-1H-pyrrole-3-carboxylic acid

5 (0.7 mmol) in ethanol (5 mL) was added thionyl chloride (0.77 mmol).

The reaction mixture was heated under reflux for 3 h. Ethanol was

evaporated under reduced pressure. The residue was triturated with

water and extracted with diethyl ether. The organic layer was then

washed with a saturated aqueous solution of NaHCO₃, dried with Na₂SO₄,

filtered and evaporated to dryness to give 6 as a yellow oil (87%).^[14]

General procedure for ethyl (4-formylphenyl)pyrrolo[1,2-a]quinoxaline-

carboxylate (14a-e, 19a-b)

5.1.1 Ethyl 3-hydroxymethylpyrrole-2-carboxylate (3)

To suspension of compound 13a-e or 18a-b (4.64 mmol), and

Pd(PPh₃)₄(0.232 mmol) in a mixture of toluene/EtOH (75/4.1 mL) under

nitrogen were added K₂CO₃(5.1 mmol) and 4-formylphenylboronic acid

A mixture of propargyl alcohol (17.8 mmol) and Ag₂CO₃(1.8

mmol) in 1,4-dioxane (60 mL) was heated at 80°C for 5 min, then the

mixture was cooling with an ice bath. To this resulting solution cooled at

5

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

(5.1 mmol). The reaction mixture was refluxed for 24 h, and the cooled

suspension was extracted with CH₂Cl₂(3 x 80 mL). The 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. The crude residue was triturated in ethanol. The

resulting precipitate was filtered, washed with ethanol, and purified by

column chromatography on silica gel using dichloromethane as eluent

gave the pure product 14 or 19.

J=7.20 Hz, OCH₂), 1.49 (t, 3H, J=7.20 Hz, CH₃). HRMS-ESI m/z [M+Na]⁺

Calcd for C₂₁H₁₆N₂O₃Na: 367.1059, Found: 367.1059.

Ethyl 6-(4-formylphenyl)-pyrrolo[1,2-a]quinoxaline-4-carboxylate (19a).

Yellow crystals (80%), mp=214°C. IR (KBr) 1720 (COO), 1700 (CHO). ¹H

NMR (CDCl₃) : 10.13 (s, 1H, CHO), 8.11 (dd, 1H, J=2.70 Hz and 1.20

Hz, H-1), 8.05-8.02 (m, 4H, H-2’, H-3’, H-5’ and H-6’), 7.99 (dd, 1H,

J=8.40 Hz and 1.20 Hz, H-9), 7.73 (t, 1H, J=7.50 Hz, H-8), 7.63 (dd, 1H,

J=7.80 Hz and 1.50 Hz, H-7), 7.56 (dd, 1H, J=3.90 Hz and 1.20 Hz, H-3),

7.04 (dd, 1H, J=3.90 Hz and 2.70 Hz, H-2), 4.48 (q, 2H, J=7.20 Hz,

OCH₂), 1.47 (t, 3H, J=7.20 Hz, CH₃). HRMS-ESI m/z [M+Na]⁺Calcd for

C₂₁H₁₆N₂O₃Na: 367.1059, Found: 367.1056.

Ethyl 4-(4-formylphenyl)-pyrrolo[1,2-a]quinoxaline-3-carboxylate (14a).

Pale-yellow crystals (86%), mp=195°C. IR (KBr) 1720 (COO), 1705

(CHO). ¹H NMR (CDCl₃) : 10.13 (s, 1H, CHO), 8.12 (dd, 1H, J=8.00 Hz

and 1.40 Hz, H-6), 8.05 (d, 1H, J=3.00 Hz, H-1), 8.03 (d, 2H, J=7.80 Hz,

H-3’ and H-5’), 7.98 (dd, 1H, J=8.20 Hz and 1.40 Hz, H-9), 7.93 (d, 2H,

J=7.50 Hz, H-2’ and H-6’), 7.67 (td, 1H, J=8.00 Hz and 1.40 Hz, H-7),

7.58 (td, 1H, J=8.00 Hz and 1.40 Hz, H-8), 7.35 (d, 1H, J=3.00 Hz, H-2),

3.83 (q, 2H, J=7.20 Hz, OCH₂), 0.97 (t, 3H, J=7.20 Hz, CH₃). HRMS-ESI

m/z [M+Na]⁺Calcd for C₂₁H₁₆N₂O₃Na: 367.1059, Found: 367.1063.

5.1.5.7 Ethyl 7-(4-formylphenyl)-pyrrolo[1,2-a]quinoxaline-4-carboxylate

(19b). Yellow crystals (58%), mp=123°C. IR (KBr) 1720 (COO), 1700

1

(CHO). H NMR (CDCl₃) : 10.11 (s, 1H, CHO), 8.49 (d, 1H, J=8.20 Hz,

H-8), 8.10 (dd, 1H, J=2.70 Hz and 0.90 Hz, H-1), 8.03 (d, 2H, J=8.20 Hz,

H-3’ and H-5’), 8.01 (s, 1H, H-6), 7.97 (d, 1H, J=8.20 Hz, H-9), 7.90 (d,

2H, J=8.20 Hz, H-2’ and H-6’), 7.61 (dd, 1H, J=3.90 Hz and 0.90 Hz, H-3),

7.07 (dd, 1H, J=3.90 Hz and 2.70 Hz, H-2), 4.63 (q, 2H, J=7.20 Hz,

OCH₂), 1.58 (t, 3H, J=7.20 Hz, CH₃). HRMS-ESI m/z [M+Na]⁺Calcd for

C₂₁H₁₆N₂O₃Na: 367.1059, Found: 367.1070.

Ethyl 4-(4-formylphenyl)-pyrrolo[1,2-a]quinoxaline-6-carboxylate (14b).

Yellow crystals (65%), mp=172°C. IR (KBr) 1715 (COO), 1700 (CHO). ¹H

NMR (CDCl₃) : 10.15 (s, 1H, CHO), 8.29 (d, 2H, J=8.40 Hz, H-3’ and H-

5’), 8.08 (dd, 1H, J=4.20 Hz and 1.20 Hz, H-1), 8.07 (d, 2H, J=8.40 Hz,

H-2’ and H-6’), 8.05 (dd, 1H, J=7.20 Hz and 1.20 Hz, H-7), 7.75 (dd, 1H,

J=7.20 Hz and 1.20 Hz, H-9), 7.60 (t, 1H, J=7.20 Hz, H-8), 7.11 (dd, 1H,

J=3.50 Hz and 1.20 Hz, H-3), 6.99 (dd, 1H, J=4.20 Hz and 3.50 Hz, H-2),

4.54 (q, 2H, J=7.20 Hz, OCH₂), 1.48 (t, 3H, J=7.20 Hz, CH₃). HRMS-ESI

m/z [M+Na]⁺Calcd for C₂₁H₁₆N₂O₃Na: 367.1059, Found: 367.1054.

General

procedure

for

ethyl

4-{4-[(4-(2-oxo-2,3-dihydro-1H-

benzimidazol-1-yl)piperidin-1-yl)benzyl]}-phenylpyrrolo[1,2-a]quinoxaline-

carboxylate, ethyl 4-{4-[(4-(5-fluoro-1H-benzimidazol-2-yl)piperidin-1-

yl)benzyl]}-phenylpyrrolo[1,2-a]quinoxaline-carboxylate, ethyl 4-{4-[4-(3-

(pyridin-2-yl)-1,2,4-triazol-5-yl)piperidin-1-yl)benzyl]}-phenylpyrrolo[1,2-

a]quinoxaline-carboxylate (1a-l), ethyl {4-[(4-(2-oxo-2,3-dihydro-1H-

benzimidazol-1-yl)piperidin-1-yl)benzyl]}-pyrrolo[1,2-a]quinoxaline-4-

carboxylate (1l, 1o), ethyl 6-{4-[(4-(5-fluoro-1H-benzimidazol-2-

Ethyl 4-(4-formylphenyl)-pyrrolo[1,2-a]quinoxaline-7-carboxylate (14c).

Yellow crystals (81%), mp=216°C. IR (KBr) 1715 (COO), 1700 (CHO). ¹H

NMR (CDCl₃) : 10.17 (s, 1H, CHO), 8.80 (dd, 1H, J=2.70 and 1.20 Hz,

H-1), 8.26 (dd, 1H, J=8.70 Hz and 1.80 Hz, H-8), 8.21 (d, 2H, J=7.40 Hz,

H-3’ and H-5’), 8.12 (d, J=1.80 Hz, 1H, H-6), 8.10 (d, 2H, J=7.40 Hz, H-2’

and H-6’), 7.97 (d, 1H, J=8.70 Hz, H-9), 7.07 (dd, 1H, J=4.20 Hz and

1.20 Hz, H-3), 7.02 (dd, 1H, J=4.20 Hz and 2.70 Hz, H-2), 4.46 (q, 2H,

J=7.20 Hz, OCH₂), 1.47 (t, 3H, J=7.20 Hz, CH₃). HRMS-ESI m/z [M+Na]⁺

Calcd for C₂₁H₁₆N₂O₃Na: 367.1059, Found: 367.1054.

yl)piperidin-1-yl)benzyl]}-pyrrolo[1,2-a]quinoxaline-4-carboxylate

(1m)

and ethyl 6-{4-[4-(3-(pyridin-2-yl)-1,2,4-triazol-5-yl)piperidin-1-yl)benzyl]}-

pyrrolo[1,2-a]quinoxaline-4-carboxylate (1n)

The pH of a solution of the aldehyde 14a-e or 19a-b (0.784

mmol) and 4-(2-ketobenzimidazolin-1-yl)piperidine or 4-(5-chloro-2-

ketobenzimidazolin-1-yl)piperidine or 2-(3-piperidin-4-yl-1H-1,2,4-triazol-

5-yl)pyridine (0.941 mmol) in 15 mL methanol was adjusted to 6 by the

dropwise addition of acetic acid. Powered sodium cyanoborohydride

(2.15 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. The 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. This solid was then triturated with diethyl ether,

filtered, washed with diethyl ether and dried under reduced pressure to

give the compounds 1a-o.

Ethyl 4-(4-formylphenyl)-pyrrolo[1,2-a]quinoxaline-8-carboxylate (14d).

Yellow crystals (68%), mp=215°C. IR (KBr) 1720 (COO), 1700 (CHO). ¹H

NMR (CDCl₃) : 10.17 (s, 1H, CHO), 8.65 (dd, 1H, J=2.70 and 0.90 Hz,

H-1), 8.28-8.19 (m, 3H, H-9, H-3’ and H-5’), 8.16 (dd, 1H, J=8.40 Hz and

1.50 Hz, H-7), 8.12-8.04 (m, 3H, H-6, H-2’ and H-6’), 7.07 (dd, 1H,

J=4.20 Hz and 0.90 Hz, H-3), 7.00 (dd, 1H, J=4.20 Hz and 2.70 Hz, H-2),

4.50 (q, 2H, J=7.20 Hz, OCH₂), 1.50 (t, 3H, J=7.20 Hz, CH₃). HRMS-ESI

m/z [M+Na]⁺Calcd for C₂₁H₁₆N₂O₃Na: 367.1059, Found: 367.1062.

Ethyl 4-(4-formylphenyl)-pyrrolo[1,2-a]quinoxaline-9-carboxylate (14e).

Pale yellow crystals (79%), mp=120°C. IR (KBr) 1720 (COO), 1715

1

(CHO). H NMR (CDCl₃) : 10.16 (s, 1H, CHO), 8.21 (d, 2H, J=8.10 Hz,

Ethyl

4-{4-[(4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-

H-3’ and H-5’), 8.19-8.17 (m, 1H, H-8), 8.09 (d, 2H, J=8.10 Hz, H-2’ and

H-6’), 7.88 (dd, 1H, J=2.60 and 1.10 Hz, H-1), 7.74 (dd, 1H, J=7.90 Hz

and 1.50 Hz, H-6), 7.52 (t, 1H, J=7.90 Hz, H-7), 7.03 (dd, 1H, J=3.90 Hz

and 1.10 Hz, H-3), 6.93 (dd, 1H, J=3.90 and 2.60 Hz, H-2), 4.58 (q, 2H,

yl)benzyl]}-pyrrolo[1,2-a]quinoxaline-3-carboxylate (1a). White crystals

(77%), mp 124°C. ¹H NMR (CDCl₃) : 9.87 (s, 1H, NH), 8.12 (d, J=7.80

Hz, 1H, H-6), 8.02 (d, J=2.70 Hz, 1H, H-1), 7.96 (d, J=8.10 Hz, 1H, H-9),

7.77 (d, J=7.80 Hz, 2H, H-2’ and H-6’), 7.64-7.52 (m, 4H, H-7, H-8, H-3’

and H-5’), 7.34-7.28 (m, 2H, H-2 and H-4 benzimid.), 7.12-7.06 (m, 3H,

6

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

H-5 benzimid., H-6 benzimid. and H-7 benzimid.), 4.47-4.39 (m, 1H, CH

pip.), 3.79 (q, J=7.20 Hz, 2H, OCH₂), 3.68 (s, 2H, CH₂N), 3.11 (dl,

J=10.80 Hz, 2H, CH₂pip.), 2.55-2.47 (m, 2H, CH₂pip.), 2.29-2.22 (m,

2H, CH₂pip.), 1.85 (dl, J=11.10 Hz, 2H, CH₂pip.), 0.93 (t, J=7.20 Hz, 3H,

CH₃). ¹³C NMR (CDCl₃) : 166.41 (C=O), 156.77 (C=O benzimid.),

155.71 (C-4), 141.10 (C-3a), 140.58 (C-5a), 137.46 (C-4’), 133.44 (C-7),

131.63 (C-8), 130.51 (C-3’ and C-5’), 130.37 (C-7a benzimid.), 130.01

(C-6), 129.65 (C-1’), 129.35 (C-2’ and C-6’), 127.62 (C-9), 127.36 (C-3a

benzimid.), 124.76 (C-9a), 122.53 (C-5 benzimid.), 122.37 (C-6

benzimid.), 118.15 (C-4 benzimid.), 116.02 (C-3), 115.51 (C-7

benzimid.), 115.31 (C-1), 111.22 (C-2), 64.02 (CH₂), 62.11 (CH₂), 54.52

(NCH₂pip.), 52.06 (CH pip.), 30.56 (CH₂pip.), 15.19 (CH₃). HRMS-ESI

m/z [M+Na]⁺Calcd for C₃₃H₃₁N₅O₃Na: 568.2325, Found: 568.2314.

(dd, J=7.50 and 1.50 Hz, H-7), 7.73 (dd, J=7.50 Hz and 1.50 Hz, 1H, H-

9), 7.57 (d, J=7.80 Hz, 2H, H-3’ and H-5’), 7.51 (t, J=8.70 Hz, 1H, H-8),

7.36-7.31 (m, 1H, H benzimid.), 7.15 (dd, J=3.90 Hz and 1.20 Hz, 1H, H-

3), 7.14-7.04 (m, 3H, H benzimid.), 6.95 (dd, J=3.90 Hz and 2.70 Hz, 1H,

H-2), 4.54 (q, J=7.20 Hz, 2H, OCH₂), 4.47-4.35 (m, 1H, CH pip.), 3.70 (s,

2H, CH₂N), 3.20-3.12 (m, 2H, CH₂pip.), 2.61-2.58 (m, 2H, CH₂pip.),

2.33-2.24 (m, 2H, CH₂pip.), 1.91-1.83 (m, 2H, CH₂pip.), 1.50 (t, J=7.20

Hz, 3H, CH₃). ¹³C NMR (CDCl₃) : 168.10 (C=O), 155.10 (C=O

benzimid.), 154.03 (C-4), 134.18 (C-4’), 133.09 (C-1’ and C-3a

benzimid.), 129.19 (C-3a and C-5a), 129.07 (C-6, C-3’ and C-5’), 128.01

(C-7, C-2’, C-6’ and C-7a benzimid.), 127.31 (C-8), 126.54 (C-9a),

125.22 (C-6 benzimid.), 124.93 (C-5 benzimid.), 116.06 (C-9), 114.30 (C-

1), 109.83 (C-2 and C-3), 109.67 (C-4 benzimid.), 109.00 (C-7

benzimid.), 62.52 (CH₂), 61.50 (CH₂), 53.14 (NCH₂pip.), 50.85 (CH pip.),

29.28 (CH₂pip.), 14.41 (CH₃). HRMS-ESI m/z [M+Na]⁺Calcd for

C₃₃H₃₁N₅O₃Na: 568.2325, Found: 568.2321.

Ethyl

4-{4-[(4-(5-fluoro-1H-benzimidazol-2-yl)piperidin-1-yl)benzyl]}-

pyrrolo[1,2-a]quinoxaline-3-carboxylate (1b). Cream crystals (50%), mp

111°C. IR (KBr) 3140 (NH), 1705 (COO). ¹H NMR (DMSO-d₆) : 11.32 (s,

1H, NH), 8.05 (d, J=7.80 Hz, 1H, H-6), 8.01 (d, J=2.60 Hz, 1H, H-1),

7.95 (d, J=7.80 Hz, 1H, H-9), 7.69 (d, J=7.20 Hz, 2H, H-2’ and H-6’), 7.60

(t, J=7.20 Hz, 1H, H-7), 7.52 (t, J=7.20 Hz, 1H, H-8), 7.44 (sl, 1H, H-4

benzimid.), 7.39 (d, J=7.20 Hz, 2H, H-3’ and H-5’), 7.30 (d, J=2.60 Hz,

1H, H-2), 7.20 (dc, 1H, H-7 benzimid.), 6.94 (td, J=9.00 Hz and 1.20 Hz,

1H, H-6 benzimid.), 3.76 (q, J=7.20 Hz, 2H, OCH₂), 3.56 (s, 2H, CH₂N),

2.95 (d, 2H, J=11.80 Hz, CH₂pip.), 2.92-2.84 (m, 1H, CH pip.), 2.09 (t,

J=10.80 Hz, 2H, CH₂pip.), 2.01 (d, J=12.00 Hz, 2H, CH₂pip.), 1.97-1.88

(m, 2H, CH₂pip.), 0.90 (t, J=7.20 Hz, 3H, CH₃). ¹³C NMR (DMSO-d₆) :

164.95 (C=O), 159.28 (d, J = 236 Hz, C-5 benzimid.), 154.46 (C-4),

139.22 (C-4’), 139.12 (C-1’, C-2 benzimid. and C-3a benzimid.), 136.02

(C-3a, C-5a and C-7a benzimid.), 129.18 (C-3’ and C-5’), 128.55 (C-2’

and C-6’), 127.93 (C-8), 126.38 (C-9a), 123.43 (C-3), 116.91 (C-2),

114.78 (C-9), 114.24 (C-1), 114.04 (C-7 benzimid.), 110.31 (C-4

benzimid.), 110.14 (C-6 benzimid.), 62.77 (CH₂), 60.75 (CH₂), 53.17

(NCH₂pip.), 36.74 (CH pip.), 30.70 (CH₂pip.), 13.78 (CH₃). HRMS-ESI

m/z [M+H]⁺Calcd for C₃₃H₃₀N₅O₂F: 548.2462, Found: 548.2461.

Ethyl

4-{4-[(4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-

yl)benzyl]}-pyrrolo[1,2-a]quinoxaline-7-carboxylate (1e). White crystals

(63%), mp 277°C. IR (KBr) 3180 (NH), 1700 (COO), 1680 (NCON). ¹H

NMR (CDCl₃) : 11.90 (s, 1H,NH), 8.86-8.84 (m, 1H, H-1), 8.78 (d,

J=1.20 Hz, H-6), 8.22 (dd, J=8.70 and 1.20 Hz, H-8), 8.07-8.03 (m, 4H,

H-2’, H-3’, H-5’ and H-6’), 7.95 (d, 1H, H benzimid.), 8.45 (d, J=8.70 Hz,

1H, H-9), 7.64-7.59 (m, 1H, H benzimid.), 7.18-7.05 (m, 4H, H-3 and H

benzimid.), 6.97 (dd, J=3.90 and 2.70 Hz, 1H, H-2), 4.44 (q, J=7.20 Hz,

2H, OCH₂), 4.41-4.39 (m, 1H, CH pip.), 3.72 (s, 2H, CH₂N), 3.16-3.13 (m,

2H, CH₂pip.), 2.56-2.28 (m, 2H, CH₂pip.), 1.90-1.68 (m, 4H, 2 CH₂

pip.), 1.45 (t, J=7.20 Hz, 3H, CH₃). ¹³C NMR (DMSO-d₆) : 165.19 (C=O),

154.15 (C=O benzimid.), 152.98 (C-4), 135.14 (C-4’), 134.047 (C-3a

benzimid.), 135.56 (C-1’), 131.49 (C-3a and C-5a), 130.20 (C-7), 129.93

(C-7a benzimid.), 129.29 (3’ and C-5’), 129.03 (C-6), 128.94 (C-2’ and C-

6’), 127.93 (C-8), 127.75 (C-9a), 124.35 (C-5 benzimid. and C-6

benzimid.), 117.82 (C-9), 116.45 (C-4 benzimid.), 115.11 (C-7

benzimid.), 115.04 (C-3), 110.22 (C-2), 109.71 (C-3), 61.88 (CH₂), 58.90

(CH₂), 51.52 (NCH₂pip.), 47.24 (CH pip.), 25.93 (CH₂pip.), 14.80 (CH₃).

HRMS-ESI m/z [M+Na]⁺Calcd for C₃₃H₃₁N₅O₃Na: 568.2325, Found:

568.2323.

Ethyl

4-{4-[4-(3-(pyridin-2-yl)-1,2,4-triazol-5-yl)piperidin-1-yl)benzyl]}-

pyrrolo[1,2-a]quinoxaline-3-carboxylate (1c). White crystals (58%), mp

125°C. IR (KBr) 3420 (NH), 1705 (COO). ¹H NMR (CDCl₃) : 9.85 (s, 1H,

NH), 8.72 (d, J=4.20 Hz, 1H, H-6 pyr.), 8.19 (d, J=7.80 Hz, 1H, H-5 pyr.),

8.09-8.05 (m, 1H, H-6), 7.96 (d, J=3.00 Hz, 1H, H-1), 7.93-7.86 (m, 1H,

H-9), 7.85-7.76 (m, 1H, H-4 pyr.), 7.70 (d, J=7.80 Hz, 2H, H-2’ and H-6’),

7.61-7.42 (m, 4H, H-7, H-8, H-3’ and H-5’), 7.40-7.29 (m, 1H, H-3 pyr.),

7.26 (d, J=3.00 Hz, 1H, H-2), 3.74 (q, J=7.00 Hz, 2H, OCH₂), 3.62 (s, 2H,

CH₂N), 3.12-2.77 (m, 3H, CH₂pip. and CH pip.), 2.30-1.88 (m, 6H, 3 x

CH₂pip.), 0.88 (t, J=7.00 Hz, 3H, CH₃). ¹³C NMR (CDCl₃) : 165.02

(C=O), 155.60 (C-2 pyr.), 154.48 (C-4 and C-3 triazole), 149.47 (C-6

pyr.), 147.12 (C-5 Triazole), 139.86 (C-4’), 139.15 (C-1’), 137.44 (C-4

pyr.), 136.17 (C-3a and C-5a), 130.37 (C-6), 129.18 (C-3’ and C-5’),

128.32 (C-7), 127.87 (C-2’ and C-6’), 126.18 (C-8), 126.05 (C-9a),

124.59 (C-3 pyr.), 123.42 (C-3), 121.78 (C-5 pyr.), 116.73 (C-2), 114.73

(C-9), 113.98 (C-1), 62.98 (CH₂), 60.72 (CH₂), 53.41 (NCH₂pip.), 35.58

(CH pip.), 30.81 (CH₂pip.), 13.78 (CH₃). HRMS-ESI m/z [M+Na]⁺Calcd

for C₃₃H₃₁N₇O₂Na: 580.2437, Found: 580.2419.

Ethyl

4-{4-[(4-(5-fluoro-1H-benzimidazol-2-yl)piperidin-1-yl)benzyl]}-

pyrrolo[1,2-a]quinoxaline-7-carboxylate (1f). White crystals (61%), mp

132°C. ¹H NMR (DMSO-d₆) : 11.26 (s, 1H,NH), 8.66 (d, J=1.20 Hz, 1H,

H-6), 8.48-8.44 (m, 2H, H-8 and H-1), 8.11 (dd, J=8.20 and 1.20 Hz, H-

8), 8.01 (d, J=7.80 Hz, 2H, H-2’ and H-6’), 7.55 (d, J=7.80 Hz, 2H, H-3’

and H-5’), 7.40-7.22 (m, 2H, 2 H benzimid.), 7.15 (dd, J=3.90 and 1.20

Hz, 1H, H-3), 7.06 (dd, J=3.90 and 2.70 Hz, 1H, H-2), 6.98-6.92 (m, 1H,

H benzimid.), 4.38 (q, J=7.20 Hz, 2H, OCH₂), 3.63 (s, 2H, CH₂N), 3.00-

2.96 (m, 2H, CH₂pip.), 2.87-2.85 (m, 1H, CH pip.), 2.21-2.17 (m, 2H,

CH₂pip.), 2.04-2.00 (m, 2H, CH₂pip.), 1.93-1.86 (m, 2H, CH₂pip.), 1.38

(t, J=7.20 Hz, 3H, CH₃). ¹³C NMR (DMSO-d₆) : 165.96 (C=O), 159.41 (d,

J = 236 Hz, C-5 benzimid.), 154.54 (C-4), 138.27 (C-4’, C-2 benzimid.

and C-3a benzimid.), 135.69 (C-1’ and C-7a benzimid.), 132.12 (C-3a

and C-5a), 130.36 (C-7), 130.18 (C-3’ and C-5’), 129.00 (C-6), 128.55

(C-2’ and C-6’), 127.52 (C-8 and C-9a), 115.50 (C-9), 115.02 (C-1),

113.80 (C-2 and C-7 benzimid.), 110.67 (C-3), 110.50 (C-4 benzimid.),

109.57 (C-6 benzimid.), 62.08 (CH₂), 61.27 (CH₂), 52.74 (NCH₂pip.),

35.76 (CH pip.), 29.43 (CH₂pip.), 14.36 (CH₃). HRMS-ESI m/z [M+H]⁺

Calcd for C₃₃H₃₀N₅O₂F: 548.2462, Found: 548.2455.

Ethyl

4-{4-[(4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-

(1d). Pale-yellow

yl)benzyl]}-pyrrolo[1,2-a]quinoxaline-6-carboxylate

crystals (47%), mp 125°C. ¹H NMR (CDCl₃) : 10.58 (s, 1H, NH), 8.13 (d,

J=7.80 Hz, 2H, H-2’ and H-6’), 8.04 (dd, J=2.70 and 1.20 Hz, H-1), 8.01

7

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Ethyl

4-{4-[4-(3-(pyridin-2-yl)-1,2,4-triazol-5-yl)piperidin-1-yl)benzyl]}-

Ethyl

4-{4-[(4-(5-fluoro-1H-benzimidazol-2-yl)piperidin-1-yl)benzyl]}-

pyrrolo[1,2-a]quinoxaline-7-carboxylate (1g). Pale yellow crystals (62%),

mp 134°C. ¹H NMR (CDCl₃) : 9.85 (s, 1H, NH), 8.77-8.64 (m, 2H, H-6

and H-6 pyr.), 8.22-8.18 (m, 2H, H-1 and H-5 pyr.), 8.04-7.99 (m, 1H, H-

8), 7.98 (d, J=7.80 Hz, H-2’ and H-6’), 7.91 (d, J=8.40 Hz, H-9), 7.86-7.83

(m, 1H, H-4 pyr.), 7.55 (d, 2H, J=7.80 Hz, H-3’ and H-5’), 7.41-7.35 (m,

1H, H-3 pyr.), 7.08 (dd, J=3.90 and 1.20 Hz, 1H, H-3), 6.96 (dd, J=3.90

and 2.70 Hz, 1H, H-2), 4.46 (q, J=7.20 Hz, 2H, OCH₂), 3.69 (s, 2H,

CH₂N), 3.10-30.6 (m, 2H, 2H, CH₂pip.), 2.94-2.89 (m, 1H, CH pip.), 2.29-

2.01 (m, 6H, 3 CH₂pip.), 1.45 (t, J=7.20 Hz, 3H, CH₃). ¹³C NMR (CDCl₃)

: 165.99 (C=O), 155.98 (C-2 pyr.), 154.91 (C-4 and C-3 Triazole),

149.43 (C-6 pyr.), 147.33 (C-5 triazole), 140.15 (C-4’), 137.39 (C-4 pyr.),

135.65 (C-1’), 131.95 (C-3a and C-5a), 130.05 (C-7), 129.56 (C-3’ and C-

5’), 128.53 (C-6), 128.19 (C-2’ and C-6’), 127.26 (C-8 and C-9a), 124.48

(C-3 pyr.), 121.84 (C-5 pyr.), 115.32 (C-9), 114.84 (C-1), 113.67 (C-2),

109.63 (C-3), 62.91 (CH₂), 62.21 (CH₂), 53.38 (NCH₂pip.), 35.38 (CH

pip.), 30.67 (CH₂pip.), 14.35 (CH₃). HRMS-ESI m/z [M+Na]⁺Calcd for

C₃₃H₃₁N₇O₂Na: 580.2437, Found: 580.2426.

pyrrolo[1,2-a]quinoxaline-9-carboxylate (1j). Pale-yellow crystals (33%),

mp 98°C. ¹H NMR (CDCl₃) : 11.15 (s, 1H, NH), 8.14 (dd, J=7.80 Hz and

1.50 Hz, 1H, H-8), 7.99 (d, J=8.10 Hz, 2H, H-2’ and H-6’), 7.84 (dd,

J=2.70 Hz and 1.20 Hz, 1H, H-1), 7.69 (dd, J=7.80 Hz and 1.50 Hz, 1H,

H-6), 7.55 (d, J=8.10 Hz, 2H, H-3’ and H-5’), 7.52-7.45 (m, 1H, H

benzimid.), 7.48 (t, J=7.80 Hz, H-7), 7.28-7.25 (m, 1H, H benzimid.), 7.05

(dd, J=4.20 Hz and 1.20 Hz, 1H, H-3), 7.00-6.96 (m, 1H, H benzimid.),

6.90 (dd, J=4.20 Hz and 2.70 Hz, 1H, H-2), 4.58 (q, J=7.00 Hz, 2H,

OCH₂), 3.71 (s, 2H, CH₂N), 3.11-2.98 (m, 2H, CH₂pip.), 3.02-2.85 (m,

1H, CH pip.), 2.25-1.94 (m, 6H, 3 CH₂pip.), 1.48 (t, J=7.00 Hz, 3H,

CH₃). ¹³C NMR (CDCl₃) : 168.97 (C=O), 159.31 (d, J = 236 Hz, C-5

benzimid.), 155.00 (C-4), 140.34 (C-4’), 137.34 (C-2 benzimid. and C-3a

benzimid.), 137.97 (C-1’), 136.42 (C-2 benzimid. and C-3a benzimid.),

136.67 (C-1’), 132.59 (C-8, C-3a and C-5a), 130.99 (C-9a, C-3’ and C-

5’), 129.35 (C-6), 128.86 (C-7), 128.45 (C-2’ and C-6’), 126.32 (C-7a

benzimid.), 124.69 (C-9), 121.86 (C-1), 120.03 (C-4 benzimid.), 114.20

(H-2 and C-7 benzimid.), 110.17 (C-6 benzimid.), 109.60 (C-3), 64.69

(CH₂), 62.41 (CH₂), 53.21 (NCH₂pip.), 36.76 (CH pip.), 30.76 (CH₂pip.),

14.23 (CH₃). HRMS-ESI m/z [M+H]⁺Calcd for C₃₃H₃₀N₅O₂F: 548.2462,

Found: 548.2469.

Ethyl

4-{4-[(4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-

yl)benzyl]}-pyrrolo[1,2-a]quinoxaline-8-carboxylate (1h). Yellow crystals

(48%), mp 277°C. ¹H NMR (CDCl₃) : 13.40 (s, 1H, NH), 8.67-8.62 (m,

2H, H-7 and H-1), 8.20-8.00 (m, 5H, H-9, H-6, H-2’, H-6’ and H

benzimid.), 7.61-7.56 (m, 2H, H-3’ and H-5’), 7.35-7.25 (m, 2H, H

benzimid.), 7.18-6.95 (m, 2H, H-3 and H benzimid.), 6.96 (dd, J=3.90 and

2.70 Hz, H-2), 4.49 (q, J=7.20 Hz, 2H, OCH₂), 4.40-4.32 (m, 1H, CH

pip.), 3.70 (s, 2H, CH₂N), 3.15-3.10 (m, 2H, CH₂pip.), 2.51-2.46 (m, 2H,

CH₂pip.), 2.36-2.20 (m, 2H, CH₂pip.), 1.95-1.81 (m, 2H, CH₂pip.), 1.48

(t, J=7.20 Hz, 3H, CH₃). ¹³C NMR (DMSO-d₆+ 2 drops of CF₃COOD) :

166.37 (C=O), 155.35 (C=O benzimid.), 154.18 (C-4), 135.60 (C-3a),

134.78 (C-5a), 133.72 (C-3’ and C-5’), 131.79 (C-2’ and C-6’), 130.59 (C-

4’), 130.16 (C-7a benzimid.), 130.05 (C-1’), 128.77 (C-9), 128.03 (C-8

and C-3a benzimid.), 126.46 (C-6), 125.50 (C-9a), 122.64 (C-7), 122.01

C-5 and C-6 benzimid.), 119.81 (C-4 and C-7 benzimid.), 119.11 (C-1),

110.81 (C-2), 110.17 (C-3), 63.21 (CH₂), 60.44 (CH₂), 53.31 (NCH₂pip.)

and 48.60 (CH pip.), 27.36 (CH₂pip.), 15.60 (CH₃). HRMS-ESI m/z

[M+Na]⁺Calcd for C₃₃H₃₁N₅O₃Na: 568.2325, Found: 568.2327.

Ethyl

4-{4-[4-(3-(pyridin-2-yl)-1,2,4-triazol-5-yl)piperidin-1-yl)benzyl]}-

pyrrolo[1,2-a]quinoxaline-9-carboxylate (1k). Pale-yellow crystals (41%),

mp 87°C. ¹H NMR (CDCl₃) : 9.75 (s, 1H, NH), 8.71 (d, J=4.50 Hz, 1H,

H-6 pyr.), 8.20 (d, J=8.10 Hz, 1H, H-8), 8.16 (dd, J=8.10 Hz and 1.50 Hz,

1H, H-5 pyr.), 7.99 (d, J=8.10 Hz, 2H, H-2’ and H-6’), 7.87 (dd, J=7.80 Hz

and 1.50 Hz, 1H, H-4 pyr.), 7.82 (dd, J=3.00 and 1.20 Hz, 1H, H-1), 7.68

(dd, J=7.50 Hz and 1.50 Hz, 1H, H-6), 7.57 (d, J=8.10 Hz, 2H, H-3’ and

H-5’), 7.48 (t, J=7.80 Hz, 1H, H-7), 7.39 (dd, J=7.50 Hz and 5.10 Hz, 1H,

H-3 pyr.), 7.05 (dd, J=3.90 Hz and 1.20 Hz, 1H, H-3), 6.89 (dd, J=3.90

Hz and 3.00 Hz, 1H, H-2), 4.58 (q, J=7.20 Hz, 2H, OCH₂), 3.71 (s, 2H,

CH₂N), 3.11-3.07 (m, 2H, CH₂pip.), 2.98-2.90 (m, 1H, CH pip.), 2.32-

1.99 (m, 6H, 3 CH₂pip.), 1.49 (t, J=7.20 Hz, 3H, CH₃). ¹³C NMR (CDCl₃)

: 168.98 (C=O), 155.97 (C-2 pyr.), 155.94 (C-4), 154.90 (C-3 Triazole),

149.45 (C-6 pyr.), 147.34 (C-5 Triazole), 140.27 (C-4’), 137.41 (C-4 pyr.),

136.82 (C-1’), 133.03 (C-8, C-3a and C-5a), 129.49 (C-6), 128.85 (C-7),

128.64 (C-2’ and C-6’), 124.67 (C-9), 124.50 (C-3 pyr.), 121.83 (C-5 pyr.),

121.73 (C-1), 114.07 (C-2), 109.26 (C-3), 62.89 (CH₂), 62.30 (CH₂),

53.37 (NCH₂pip.), 35.37 (CH pip.), 30.68 (CH₂pip.), 14.22 (CH₃).

HRMS-ESI m/z [M+Na]⁺Calcd for C₃₃H₃₁N₇O₂Na: 580.2437, Found:

580.2421.

Ethyl

4-{4-[(4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-

yl)benzyl]}-pyrrolo[1,2-a]quinoxaline-9-carboxylate (1i). White crystals

(62%), mp 151°C. ¹H NMR (DMSO-d₆) : 10.89 (s, 1H, NH), 8.10 (dd,

J=7.80 and 1.50 Hz, 1H, H-8), 8.00 (d, J=8.10 Hz, 2H, H-2’ and H-6’),

7.85-7.83 (m, 1H, H-1), 7.74 (dd, J=8.20 and 1.20 Hz, 1H, H-6), 7.61-

7.58 (m, 1H, H-8), 7.57 (d, J=8.10 Hz, 2H, H-3’ and H-5’), 7.26-7.23 (m,

1H, H benzimid.), 7.11-7.09 (m, 1H, H-3), 7.03-6.95 (m, 4H, H-2 and 3 x

H benzimid.), 4.51 (q, J=6.90 Hz, 2H, OCH₂), 4.23-4.14 (m, 1H, CH pip.),

3.64 (s, 2H, CH₂N), 3.03-2.98 (m, 2H, CH₂pip.), 2.42-2.34 (m, 2H, CH₂

pip.), 2.22-2.08 (m, 2H, CH₂pip.), 1.69-1.65 (m, 2H, CH₂pip.), 1.36 (t,

J=6.90 Hz, 3H, CH₃). ¹³C NMR (CDCl₃) : 169.02 (C=O), 155.35 (C=O

benzimid.), 154.81 (C-4), 140.60 (C-4’), 137.50 (C-1’), 136.88 (C-3a

benzimid.), 133.08 (C-3a and C-5a), 129.37 (C-7, C-8, C-9a and C-7a

benzimid.), 129.14 (C-3’ and C-5’), 128.71 (C-6), 128.24 (C-2’ and C-6’),

126.37 (C-5 benzimid.), 124.70 (C-9), 121.74 (C-6 benzimid.), 114.03 (C-

4 benzimid.), 109.81 (C-7 benzimid. and C-1), 109.78 (C-2), 109.27 (C-

3), 62.68 (CH₂), 62.34 (CH₂), 53.22 (CH pip.), 50.81 (NCH₂pip.), 29.28

(CH₂pip.), 14.23 (CH₃). HRMS-ESI m/z [M+Na]⁺Calcd for

C₃₃H₃₁N₅O₃Na: 568.2325, Found: 568.2318.

Ethyl

6-{4-[(4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-

yl)benzyl]}-pyrrolo[1,2-a]quinoxaline-4-carboxylate (1l). Yellow crystals

(70%), mp 180°C. ¹H NMR (CDCl₃) : 8.63 (s, 1H, NH), 8.08 (dd, J=2.70

and 1.20 Hz, 1H, H-1), 7.93 (dd, J=8.10 and 1.30 Hz, 1H, H-9), 7.87 (d,

J=8.10 Hz, 2H, H-2’ and H-6’), 7.71 (t, J=8.10 Hz, 1H, H-8), 7.65 (d,

J=7.20 Hz, 1H, H benzimid.), 7.58-7.51 (m, 3H, H-7, H-3’ and H-5’), 7.39-

7.34 (m, 1H, H benzimid.), 7.19-7.14 (m, 1H, H benzimid.), 7.14-7.07 (m,

2H, H-3 and H benzimid.), 7.04 (dd, J=3.90 and 2.70 Hz, 1H, H-2), 4.49

(q, J=7.20 Hz, 2H, OCH₂), 4.45-4.41 (m, 1H, CH pip.), 3.68 (s, 2H, CH₂N),

3.20-3.16 (m, 2H, CH₂pip.), 2.67-2.50 (m, 2H, CH₂pip.), 2.29 (m, 2H,

CH₂pip.), 1.95-1.84 (m, 2H, CH₂pip.), 1.50 (t, J=7.20 Hz, 3H, CH₃). ¹³C

NMR (CDCl₃) : 165.24 (C=O), 156.14 (C=O benzimid.), 143.43 (C-4),

143.24 (C-3a), 138.87 (C-5a), 138.18 (C-1’ and C-4’), 133.18 (C-6 and C-

3a benzimid.), 132.27 (C-2’ and C-6’), 130.45 (C-8), 130.17 (C-9a),

129.54 (C-7a benzimid.), 129.42 (C-3’ and C-5’), 127.63 (C-7), 122.11

(C-5 benzimid.), 122.00 (C-6 benzimid.), 115.97 (C-9), 113.85 (C-4

8

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

benzimid.), 110.81 (C-7 benzimid. and C-1), 110.69 (C-2), 110.05 (C-3),

63.75 (CH₂), 62.75 (CH₂), 54.22 (NCH₂pip.), 51.90 (CH pip.), 30.34 (CH₂

pip.), 15.11 (CH₃). HRMS-ESI m/z [M+Na]⁺Calcd for C₃₃H₃₁N₅O₃Na:

568.2325, Found: 568.2308.

(C=O benzimid.), 142.79 (C-3a and C-4), 137.00 (C-5a), 136.90 (C-1’

and C-4’), 134.07 (C-7 and C-3a benzimid.), 129.24 (C-2’, C-6’ and C-7a

benzimid.), 128.72 (C-6, C-9a, C-3’ and C-5’), 128.08 (C-8), 123.14 (C-5

benzimid. and C-6 benzimid.), 116.62 (C-1), 114.71 (C-9), 108.92 (C-2

and C-3), 108.46 (C-7 benzimid.), 108.35 (C-4 benzimid.), 61.37 (CH₂),

61.02 (CH₂), 52.15 (NCH₂pip.), 49.62 (CH pip.), 28.11 (CH₂pip.), 13.68

(CH₃). HRMS-ESI m/z [M+Na]⁺Calcd for C₃₃H₃₁N₅O₃Na: 568.2325,

Found: 568.2314.

Ethyl

6-{4-[(4-(5-fluoro-1H-benzimidazol-2-yl)piperidin-1-yl)benzyl]}-

pyrrolo[1,2-a]quinoxaline-4-carboxylate (1m). Yellow crystals (63%), mp

192°C. IR (KBr) 3140 (NH), 1715 (COO). ¹H NMR (CDCl₃) : 9.55 (s, 1H,

NH), 8.08 (dd, J=2.70 Hz and 1.20 Hz, 1H, H-1), 7.93 (dd, J=8.40 Hz and

1.20 Hz, 1H, H-9), 7.82 (d, J=8.10 Hz, 2H, H-2’ and H-6’), 7.69 (t, J=8.40

Hz, 1H, H-8), 7.61 (dd, J=8.40 and 1.20 Hz, 1H, H-7), 7.53 (dd, J=4.20

and 1.20 Hz, H-3), 7.46 (d, 2H, J=8.10 Hz, H-3’ and H-5’), 7.50-7.47 (m,

1H, H benzimid.), 7.28-7.24 (m, 1H, H benzimid.), 7.02 (dd, J=4.20 and

2.70 Hz, H-2), 7.00-6.93 (m, 1H, H benzimid.), 4.49 (q, J=7.20, 2H,

OCH₂), 3.68 (s, 2H, CH₂N), 3.34-3.20 (m, 2H, CH₂pip.), 3.05-2.94 (m,

1H, CH pip.), 2.31-2.22 (m, 2H, CH₂pip.), 2.19-1.99 (m, 4H, 2 CH₂pip.),

1.48 (t, J=7.20 Hz, 3H, CH₃). ¹³C NMR (CDCl₃) : 165.31 (C=O), 160.38

(d, J = 235 Hz, C-5 benzimid.), 143.33 (C-4), 143.31 (C-3a), 138.37 (C-

5a, C-2 benzimid. and C-3a benzimid.), 138.11 (C-4’ ), 138.07 (C-1’),

134.31 (C-7a benzimid.), 133.17 (C-6), 132.27 (C-2’ and C-6’), 130.47

(C-8, C-3’ and C-5’), 129.60 (C-9a), 1127.70 (C-7), 116.01 (C-1), 115.98

(C-4 benzimid.), 113.93 (C-9), 110.14 (C-2, C-3, C-6 benzimid. and C-7

benzimid.), 63.88 (CH₂), 62.83 (CH₂), 54.24 (NCH₂pip.), 37.69 (CH pip.),

31.74 (CH₂pip.), 15.15 (CH₃). HRMS-ESI m/z [M+H]⁺Calcd for

C₃₃H₃₁N₅O₂F: 548.2462, Found: 548.2448.

1-(3-Chloro-2-nitrophenyl)pyrrole (15a)

A mixture of commercial 3-chloro-2-nitroaniline (30 mmol) and

2,5-dimethoxytetrahydrofuran (33 mmol) in acetic acid (65 mL) was

refluxed for 10 min by microwave with vigorous stirring. The irradiation

was programmed to maintain a constant temperature (120°C) with a

maximal output power of 200 W. After cooling, the reaction mixture was

poured into water 0°C. The mixture was then extracted twice with diethyl

ether. The organic layer was dried over Na₂SO₄and filtered. Solvent was

removed under reduced pressure and the crude product was purified by

flash chromatography (eluent 7:3 cyclohexane/AcOEt) to give 15a. Beige

crystals (86%); mp 61°C. ¹H-NMR ¹H NMR (CDCl₃) : 7.53 (d, 1H,

J=7.95 Hz, H-4), 7.51 (d, 1H, J= 7.95 Hz, H-6), 7.40 (t, 1H, J= 7.95 Hz,

H-5), 6.84 (dd, 2H, J=1.95 and 1.95 Hz, H-), 6.37 (dd, 2H, J=1.95 and

1.95 Hz, H-). Anal. Calcd for C₁₀H₇ClN₂O₂: C, 53.95; H, 3.17; N, 12.58.

Found: C, 54.06; H, 3.32; N, 12.68.

Ethyl

6-{4-[4-(3-(pyridin-2-yl)-1,2,4-triazol-5-yl)piperidin-1-yl)benzyl]}-

pyrrolo[1,2-a]quinoxaline-4-carboxylate (1n). Yellow crystals (70%), mp

1-(2-Amino-3-chlorophenyl)pyrrole (16a)

1

187°C. H NMR (DMSO-d₆) : 8.75-8.60 (m, 2H, NH and H-6 pyr.), 8.37

(dd, J=8.40 Hz and 1.20 Hz, 1H, H-5 pyr.), 8.05 (d, J=7.50 Hz, 1H, H-9),

7.95-7.93 (m, 1H, H-1), 7.79 (t, J=7.80 Hz, 1H, H-8), 7.73 (d, J=7.80 Hz,

2H, H-2’ and H-6’), 7.65-7.61 (m, 2H, H-7 and H-4 pyr.), 7.45-7.42 (m, 3H,

H-3’, H-5’ and H-3 pyr.), 7.36 (dd, J=4.00 Hz and 1.20 Hz, 1H, H-3), 7.07

(dd, J=4.00 and 2.70 Hz, 1H, H-2), 4.38 (q, J=6.90, 2H, OCH₂), 3.59 (s,

2H, CH₂N), 3.08-2.94 (m, 2H, CH₂pip.), 2.87-2.75 (m, 1H, CH pip.), 2.25-

2.15 (m, 2H, CH₂pip.), 2.06-1.95 (m, 2H, CH₂pip.), 1.90-1.71 (m, 2H,

CH₂pip.), 1.35 (t, J=6.90 Hz, 3H, CH₃). ¹³C NMR (DMSO-d₆) : 164.07

(C=O), 154.45 (C-3 Triazole and C-2 pyr.), 149.98 (C-6 pyr.), 147.15 (C-5

Triazole), 142.50 (C-4), 141.80 (C-3a), 138.08 (C-5a, C-4’ and C-4 pyr.),

137.53 (C-1’), 131.83 (C-6), 131.34 (C-2’ and C-6’), 130.44 (C-8, C-9a,

C-3’ and C-5’), 128.71 (C-7), 123.86 (C-3 pyr.), 121.77 (C-5 pyr.), 115.55

(C-1), 114.70 (C-9), 109.14 (C-2 and C-3), 62.50 (CH₂), 62.00 (CH₂),

56.93 (NCH₂pip.), 53.28 (CH pip.), 30.93 (CH₂pip.), 14.42 (CH₃).

HRMS-ESI m/z [M+Na]⁺Calcd for C₃₃H₃₁N₇O₂Na: 580.2437, Found:

580.2413.

To a solution of 1-(3-chloro-2-nitrophenyl)pyrrole (15a) (28

mmol) in ethanol (120 mL) was added 19 mL of a 2M aqueous solution of

CuSO₄. Sodium borohydride (138 mmol) was added portion-wise at 0°C

to the reaction mixture which was then stirred at room temperature for 1

h. The reaction mixture was then diluted with ethyl acetate and filtered.

The organic layer was separated, dried over Na₂SO₄and evaporated to

dryness under reduced pressure to give 16a after purification by flash

chromatography (eluent 9:1 CH₂Cl₂/MeOH). Yellow oil (74%); mp 79°C.

IR (KBr) 3455, 3375 (NH₂). ¹H-NMR ¹H NMR (CDCl₃) : 7.31 (d, 1H,

J=7.80 Hz, H-4), 7.11 (d, 1H, J=7.80 Hz, H-6), 6.86 (dd, 2H, J=1.95 and

1.95 Hz, H-), 6.75 (t, 1H, J=7.80 Hz, H-5), 6.40 (dd, 2H, J=1.95 and

1.95 Hz, H-), 4.16 (s, 2H, NH₂). Anal. Calcd for C₁₀H₉ClN₂: C, 62.35; H,

4.71; N, 14.54. Found: C, 62.23; H, 5.02; N, 14.57.

Ethyl 2-[(6-chloro-2-pyrrolo-1-ylphenyl)amino]-2-oxo-acetate (17a)

Ethyl

7-{4-[(4-(2-oxo-2,3-dihydro-1H-benzimidazol-1-yl)piperidin-1-

yl)benzyl]}-pyrrolo[1,2-a]quinoxaline-4-carboxylate (1o). White crystals

(48%), mp 138°C. ¹H NMR (DMSO-d₆) : 10.86 (s, 1H, NH), 8.67 (dd,

J=2.70 and 1.20 Hz, 1H, H-1), 8.48 (d, J=8.70 Hz, 1H, H-9), 8.31 (d,

J=1.80 Hz, 1H, H-6), 8.02 (dd, J=8.40 Hz and 2.10 Hz, 1H, H-8), 7.86 (d,

J=8.10 Hz, 2H, H-2’ and H-6’), 7.49 (d, J=8.10 Hz, 2H, H-3’ and H-5’),

7.41 (dd, J=3.90 and 1.20 Hz, 1H, H-3), 7.26-7.24 (m, 1H, H benzimid.),

7.10-7.08 (dd, J=3.90 and 2.70 Hz, 1H, H-2), 7.04-6.95 (m, 3H, 3 x H

benzimid.), 4.48 (q, J=6.70 Hz, 2H, OCH₂), 4.20-4.15 (m, 1H, CH pip.),

3.61 (s, 2H, CH₂N), 3.08-2.95 (m, 2H, CH₂pip.), 2.42-2.27 (m, 2H, CH₂

pip.), 2.18-2.15 (m, 2H, CH₂pip.), 1.75-1.60 (m, 2H, CH₂pip.), 1.41 (t,

J=6.70 Hz, 3H, CH₃). ¹³C NMR (DMSO-d₆) : 163.16 (C=O), 153.34

To a solution containing 1-(2-amino-4-bromophenyl)pyrrole

(16a) (17 mmol) in 140 mL of THF at 0°C was added Et₃N (17 mmol)

followed by ethyl oxalylchloride (17 mmol) dropwise over 15 minutes. The

reaction mixture was warmed to room temperature and stirred for 14 h.

The reaction mixture was filtered and the filter cake was washed with

THF then with ethyl acetate. The organic phase was washed twice with

25 mL of a 1M HCl aqueous solution, dried over sodium sulfate, filtered,

and concentrated. The residue was cooled, triturated with diethyl ether,

decanted; and the crude product was purified by flash chromatography

(eluent 8:2 cyclohexane/AcOEt) to give 17a. Yellow oil (94%). IR (KBr)

9

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

3280 (NH), 1690 and 1680 (CO). ¹H-NMR ¹H NMR (CDCl₃) : 8.45 (s, 1H,

NH), 7.53-7.27 (m, 2H, H-4 and H-6), 6.84-6.73 (m, 3H, H-5 and H-),

6.31 (dd, 2H, J=1.95 and 1.95 Hz, H-), 4.39-4.27 (m, 2H, CH₂), 1.43-

1.30 (m, 3H, CH₃). Anal. Calcd for C₁₄H₁₃ClN₂O₃: C, 57.45; H, 4.48; N,

9.57. Found: C, 57.25; H, 4.66; N, 9.74.

Cytotoxicity Test

The MTS cell proliferation assay (Promega, France) is a

colorimetric assay system, which measures the reduction of a tetrazolium

component (MTS) into formazan produced by the mitochondria of viable

cells. Cells were washed twice in PBS (Phosphate Buffer Saline) and

plated in quadruplicate into microtiter-plate wells in 100 L culture media

with or without our various compounds at increasing concentrations (0, 1,

5, 10, 20 and 50 M) during 1, 2 and 3 days. After 3 hours of incubation

at 37°C with 20 L MTS/well, the plates were read by using an ELISA

microplate reader (Thermo, Electrocorporation) at 490 nm wavelength.

The amount of colour produced was directly proportional to the number

of viable cells. The results are expressed as the concentrations inhibiting

cell growth by 50% after a 3 days incubation period. The 50% cytotoxic

concentrations (CC₅₀) were determined by linear regression analysis,

expressed in μM ± SD (Microsoft Excel).

Ethyl 6-chloropyrrolo[1,2-a]quinoxaline-4-carboxylate (18a)

A solution of ethyl 2-[(6-chloro-2-pyrrolo-1-ylphenyl)amino]-2-oxo-acetate

(17a) (7 mmol) in POCl₃(10 mL) was refluxed for 20 min. 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 ammonium

hydroxide. The precipitate was filtered, washed with diethyl ether, and

dried to give 18a which was purified by flash chromatography (eluent

CH₂Cl₂). Yellow crystals (57%); mp 110°C. IR (KBr) 1735 (COO). ¹H

NMR (CDCl₃) : 8.03 (dd, 1H, J=2.70 and 1.20 Hz, H-1), 7.81 (d, 1H,

J=7.80 Hz, H-9), 7.60-7.49 (m, 3H, H-3, H-7 and H-8), 7.02 (dd, 1H, J=

3.90 and 2.70 Hz, H-2), 4.58 (q, 2H, J=7.20 Hz, CH₂), 1.54 (t, 3H, J=7.20

Hz, CH₃). Anal. Calcd for C₁₄H₁₁ClN₂O₂: C, 61.21; H, 4.04; N, 10.20.

Found: C, 61.14; H, 3.89; N, 10.04.

Annexin V staining by flow cytometry

Cells (10⁵) were incubated for 3 days with increasing doses of 1a (0, 1, 5,

and 10 µM diluated with Dimethyl sulfoxide (DMSO)). Experiments were

X-ray Data

performed with APC-Annexin

V (Biolegend, CA) according to the

manufacturer's instructions. Briefly, cells (2x10⁴) were incubated with 5

µL of APC-Annexin V resuspended in 295 µL of 1X binding buffer for 10

min at room temperature in the dark. Then, cells were analyzed by flow

cytometry. The APC-Annexin V-positive cells were considered as

apoptotic. Flow cytometry analysis was performed with a BD Accuri

(Becton-Dickinson, France) and experiments were analysed using the

BD Accuri C6 software.

The structure of compound 1c has been established by X-ray

crystallography (Figure 2). Colorless single crystal of 1c was obtained by

slow evaporation from a methanol/chloroform solution (v/v : 20/80) :

triclinic, space group P-1, a=10.5656(7) Å, b=14.2740(10) Å,

c=22.6078(14)

Å,

=83.453(4)°,

=89.521(4)°,

=81.077(5)°,

V=3346.2(4)Å³, Z=4, (calcd)= 1.304 Mg.m^-3, FW=656.74 for

C₃₃H₃₁N₇O₂5.5H₂O, F(000)=1396. Full crystallographic results have

been deposited at the Cambridge Crystallographic Data Centre (CCDC-

888294), UK, as supplementary material.^[35]The data were corrected for

Lorentz and polarization effects and for empirical absorption

correction.^[36]The structure was solved by direct methods Shelx 2013^[37]

and refined using Shelx 2013^[37]suite of programs.

Acknowledgements

This publication was supported by a grant from Ligue Nationale

contre le Cancer (Comité Aquitaine-Charentes, Bordeaux,

France). Marian Vincenzi thanks the Università Italo Francese

(UIF) for financial support (project C2-1, 2014/38723, cap.

6.01.1810 UIF).

Biology

Cell culture

Keywords: pyrrolo[1,2-a]quinoxaline • synthesis • cytotoxic

activity • cancer • leukemia

The human leukemic cell lines U937, K562, HL60, U266 and

Jurkat were grown in RPMI 1640 medium (Life Technology, France)

supplemented with 10% fetal calf serum (FCS), antibiotics (100 U/mL

penicillin, 100 µg/mL streptomycin) and L-glutamin, (Eurobio, France) at

37°C, 5% CO₂in air. The toxicity of various molecules was also

evaluated on non-activated, freshly isolated normal human peripheral

blood mononuclear cells (PBMNC), as well as phytohemagglutinin (T

lymphoproliferative agent) (PHA)-induced cells. PBMNC from blood of

healthy volunteers were obtained following centrifugation on Ficoll

gradient. Cells were then incubated in medium alone or induced to enter

cell cycle by the addition of PHA (5 µg/mL, Murex Biotech Limited,

Dartford, UK).

References:

[1]

[2]

M. A. Lichtman. Battling the Hematological Malignancies: The 200

Years’ War. The Oncologist 2008, 13, 126-138.

G. Campiani, S. Butini, C. Fattorusso, F. Trotta, S. Franceschina,

M. De Angelis, K.S. Nielsen. Novel aryl piperazine derivatives with

medical utility. 2006, WO2006072608.

[3]

G. Campiani, F. Aiello, M. Fabbrini, E. Morelli, A. Ramunno, S.

Armaroli, V. Nacci, A. Garofalo, G. Greco, E. Novellino, G. Maga, S.

Spadari, A. Bergamini, L. Ventura, B. Bongiovanni, M. Capozzi, F.

10

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Bolacchi, S. Marini, M. Coletta, G. Guiso, S. Caccia.

[17]

[18]

V. Desplat, S. Moreau, S. Belisle-Fabre, D. Thiolat, J. Uranga, R.

Lucas, S. Massip, L. de Moor, C. Jarry, D. Mossalayi, P. Sonnet, G.

Déléris, J. Guillon, Synthesis and Evaluation of the Antiproliferative

Activity of Novel Isoindolo[2,1-a]quinoxaline and Indolo[1,2-

a]quinoxaline Derivatives. J. Enzyme Inhib. Med. Chem. 2011, 26,

657-667.

Quinoxalinylethylpyridylthioureas (QXPTs) as potent non-

nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Further

SAR studies and identification of

a novel orally bioavailable

hydrazine-based antiviral agent. J. Med. Chem. 2001, 44, 305-315.

S. Schann, S. Mayer, S. Gardan. Pyrrolo[1,2-a]quinoxaline

derivatives as Adenosine A3 receptor modulators and uses thereof.

2007, EP1798233.

[4]

[5]

V. Desplat, M. Vincenzi, R. Lucas, S. Moreau, S. Savrimoutou, N.

Pinaud, J. Lesbordes, E. Peyrilles, M. Marchivie, S. Routier, P.

Sonnet, F. Rossi, L. Ronga, J. Guillon, Synthesis and evaluation of

the cytotoxic activity of novel ethyl 4-[4-(4-substitutedpiperidin-1-

yl)]benzyl-phenylpyrrolo[1,2-a]quinoxaline-carboxylate derivatives

in myeloid and lymphoid leukemia cell lines. Eur. J. Med. Chem.

2016, 113, 214-227.

J. Guillon, P. Grellier, M. Labaied, P. Sonnet, J.-M. Léger, R.

Déprez-Poulain, I. Forfar-Bares, P. Dallemagne, N. Lemaître, F.

Péhourcq, J. Rochette, C. Sergheraert, C. Jarry, Synthesis,

antimalarial activity and molecular modeling of new pyrrolo[1,2-

a]quinoxalines,

bispyrrolo[1,2-a]quinoxalines,

bispyrido[3,2-

e]pyrrolo[1,2-a]pyrazines

and

bispyrrolo[1,2-a]thieno[3,2-

[19]

C.W. Lindsley, Z. Zhao, W.H. Leister, R.G. Robinson, S.F. Barnett,

D. Defeo-Jones, R.E. Jones, G.D. Hartman, J.R. Huff, H.E. Huber,

M.E. Duggan, Allosteric Akt (PKB) inhibitors: discovery and SAR of

isozyme selective inhibitors. Bioorg. Med. Chem. Lett. 2005, 15,

761-764

e]pyrazines. J. Med. Chem. 2004, 47, 1997-2009.

[6]

J. Guillon, I. Forfar, M. Mamani-Matsuda, V. Desplat, M. Saliège, D.

Thiolat, S. Massip, A. Tabourier , J.-M. Léger, B. Dufaure, G.

Haumont, C. Jarry, D. Mossalayi, Synthesis, Analytical Behaviour

and Biological Evaluation of New 4-Substituted Pyrrolo[1,2-

a]quinoxalines as Antileishmanial Agents. Bioorg. Med. Chem.

2007, 15, 194-210.

[20]

[21]

[22]

[23]

[24]

H. A. Jensen, R. P. Bunaciu, J. D. Varner, A. Yen, GW5074 and

PP2 kinase inhibitors implicate nontraditional c-Raf and Lyn

function as drivers of retinoic acid-induced maturation. Cell. Signal.

2015, 27, 1666-1675.

[7]

[8]

[9]

J. Guillon, S. Moreau, L. Ronga, L. Basmaciyan, A. Cohen, S.

Rubio, G. Bentzinger, N. Azas, C. Mullié, P. Sonnet, Design,

Synthesis and Antimalarial Activity of Some New Aminoalcohol-

pyrrolo[1,2-a]quinoxaline Derivatives. Lett. Drug Des. Discovery

2016, 13, 932-942..

J. Liu, Z. Fang, Q. Zhang, Q. Liu, X. Bi, Silver-Catalyzed

Isocyanide-Alkyne Cycloaddition: A General and Practical Method

to Oligosubstituted Pyrroles. Angew. Chem., Int. Ed. 2013, 52,

6953-6957.

J. Guillon, S. Moreau, E. Mouray, V. Sinou, I. Forfar, S. Belisle-

Fabre, V. Desplat, P. Millet, D. Parzy, C. Jarry, P. Grellier, New

Ferrocenic Pyrrolo[1,2-a]quinoxaline Derivatives : Synthesis, and

In Vitro Antimalarial Activity. Bioorg. Med. Chem. 2008, 16, 9133-

9144.

M. Gao, C. He, H. Chen, R. Bai, B. Cheng, A. Lei, Synthesis of

Pyrroles by Click Reaction: Silver-Catalyzed Cycloaddition of

Terminal Alkynes with Isocyanides. Angew. Chem., Int. Ed. 2013,

52, 6958-6961.

J. Guillon, P. Dallemagne, B. Pfeiffer, P. Renard, D. Manechez, A.

Kervran, S. Rault, Synthesis of New Pyrrolo[1,2-a]quinoxalines :

Potential Non-peptide Glucagon Receptor Antagonists. Eur. J. Med.

Chem. 1998, 33, 293-308.

J. Guillon, E. Mouray, S. Moreau, C. Mullié, I. Forfar, V. Desplat, S.

Belisle-Fabre, F. Ravanello, A. Le-Naour, N. Pinaud, J.-M. Léger,

G. Gosmann, C. Jarry, G. Déléris, P. Sonnet, P. Grellier, New

Ferrocenic Pyrrolo[1,2-a]quinoxaline Derivatives: Synthesis, and in

Vitro Antimalarial Activity – Part II. Eur. J. Med. Chem. 2011, 46,

2310-2326.

J. Guillon, R. C. Reynolds, J.-M. Leger, M.-A. Guie, S. Massip, P.

Dallemagne, C. Jarry, Synthesis and Preliminary In Vitro

Evaluation of Antimycobacterial Activity of New Pyrrolo[1,2-

a]quinoxaline-carboxylic Acid Hydrazide Derivatives. J. Enzyme

Inhib. Med. Chem. 2004, 19, 489-495.

[10]

[11]

L. Ronga, M. Del Favero, A. Cohen, C. Soum, P. Le Pape, S.

Savrimoutou, N. Pinaud, C. Mullié, S. Daulouede, P. Vincendeau,

N. Farvacques, P. Agnamey, F. Pagniez, S. Hutter, N. Azas, P.

Sonnet, J. Guillon, Design, Synthesis and Biological Evaluation of

Novel 4-Alkapolyenylpyrrolo[1,2-a]quinoxalines as Antileishmanial

Agents – Part III. Eur. J. Med. Chem. 2014, 81, 378-393.

N. Primas, P. Suzanne, P. Verhaeghe, S. Hutter, C. Kieffer, M.

Laget, A. Cohen, J. Broggi, J.-C. Lancelot, A. Lesnard, P.

Dallemagne, P. Rathelot, S. Rault, P. Vanelle, N. Azas, Synthesis

[25]

P. Liu, B. Xu, J. Li, H. Lu. LY294002 inhibits leukemia cell invasion

and migration through early growth response gene 1 induction

independent of phosphatidylinositol 3-kinase-Akt pathway.

Biochem. Biophys. Res. Commun. 2008, 377, 187-190.

S. Matsuda, A. Nakanishi, Y. Wada, Y. Kitagishi. Roles of

PI3K/AKT/PTEN Pathway as a Target for Pharmaceutical Therapy.

Open Med. Chem. J. 2013, 7, 23-29.

[26]

[27]

and

in

vitro

evaluation

of

4-trichloromethylpyrrolo[1,2-

H.G. Jørgensen, E.K. Allan, S.M. Graham, J.L. Godden, L.

Richmond, M.A. Elliott, J.C. Mountford, C.J. Eaves, T.L. Holyoake.

Lonafarnib reduces the resistance of primitive quiescent CML cells

to imatinib mesylate in vitro. Leukemia 2005, 19, 1184-1191.

S. Zhao, M. Konopleva, M. Cabreira-Hansen, Z. Xie, W. Hu, M.

Milella, Z. Estrov, G.B. Mills, M. Andreeff. Inhibition of

phosphatidylinositol 3-kinase dephosphorylates BAD and promotes

apoptosis in myeloid leukemias. Leukemia 2004, 18, 267-275.

C.W. Lindsley. The Akt/PKB Family of Protein Kinases: A Review of

Small Molecule Inhibitors and Progress Towards Target Validation:

A 2009 Update. Curr. Top. Med. Chem. 2010, 10, 458-477.

R. Boulahjar, A. Ouach, M. Chiurato, S. Bourg, M. Ravache, R. Le

Guével, S. Marionneau-Lambot, T. Oullier, O. Lozach, L. Meijer, C.

Guguen-Guillouzo, S. Lazar, M. Akssira, Y. Troin, G. Guillaumet, S.

a]quinoxalines as new antiplasmodial agents. Eur. J. Med. Chem.

2014, 83, 26-35.

[12]

[13]

J. Milne, K.D. Normington, M. Milburn. Tetrahydroquinoxalinone

sirtuin modulators. 2006, WO2006094210.

[28]

F. Grande, F. Aiello, O. De Grazia, A. Brizzi, A. Garofalo, N.

Meamati. Synthesis and antitumor activities of a series of novel

quinoxalinhydrazides. Bioorg. Med. Chem. 2007, 15, 288-294.

J. Guillon, M. Le Borgne, C. Rimbault, S. Moreau, S. Savrimoutou,

N. Pinaud, S. Baratin, M. Marchivie, S. Roche, A. Bollacke, A.

Pecci, L. Alvarez, V. Desplat, J. Jose, Synthesis and Biological

Evaluation of Novel Substituted Pyrrolo[1,2-a]quinoxaline

Derivatives as Inhibitors of the Human Protein Kinase CK2. Eur. J.

Med. Chem. 2013, 65, 205-222.

[14]

[29]

[30]

[15]

[16]

V. Desplat, A. Geneste, M.-A. Begorre, S. Belisle Fabre, S. Brajot,

S. Massip, D. Thiolat, D. Mossalayi, C. Jarry, J. Guillon, Synthesis

of New Pyrrolo[1,2-a]quinoxaline Derivatives as Potential Inhibitors

of Akt Kinase. J. Enzyme Inhib. Med. Chem. 2008, 23, 648-658.

V. Desplat, S. Moreau, A. Gay, S. Belisle-Fabre, D. Thiolat, S.

Massip, D. Mossalayi, C. Jarry, J. Guillon, Synthesis and

Evaluation of the Antiproliferative Activity of Novel Pyrrolo[1,2-

a]quinoxaline Derivatives, Potential Inhibitors of Akt Kinase. Part II.

J. Enzyme Inhib. Med. Chem. 2010, 25, 204-215.

Routier.

Novel tetrahydropyrido[1,2-a]isoindolone derivatives

(Valmerins) : potent cyclin dependant kinase / glycogen synthase

kinase 3 inhibitors with antiproliferative activities and antitumor

effects in human tumor xenografts. J. Med. Chem. 2012, 55, 9589-

9606.

[31]

A. Pawlak, A. Rapak, I. Zbyryt, B. Obmińska-Mrukowicz. The Effect

of Common Antineoplastic Agents on Induction of Apoptosis in

Canine Lymphoma and Leukemia Cell Lines. In vivo 2014, 28, 843-

850.

11

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

[32]

[33]

F. Li, J. Liu, J. Shi, Y. Luan. Synthesis and biological evaluation of

a fatty acyl di-cytarabine prodrug. RSC Adv. 2015, 5, 25382-25388.

A. Katsoulasa, Z. Rachida, F. Brahimia, J. McNameeb, B. J. Jean-

Claude. Cytokinetics and mechanism of action of AKO4: a novel

nitrogen mustard targeted to bcr-abl. Leukemia Res. 2005, 29,

565–572.

[34]

[35]

H. N. Nagesh, N. Suresh, G. V. S. B. Prakash, S. Gupta, J. V. Ra,

K. V. G. C. Sekhar. Synthesis and biological evaluation of novel

phenanthridinyl piperazine triazoles via click chemistry as anti-

proliferative agents. Med. Chem. Res. 2015, 24, 523–532.

Supplementary

Crystallographic Data Centre, University Chemical Lab, Lensfield

Road, Cambridge, CB2 1EW, UK; E-mail:

deposit@chemcrys.cam.ac.uk.

X-ray

crystallographic

data:

Cambridge

[36]

[37]

G. M. Sheldrick (1996) SADABS, University of Göttingen, Germany.

G. M. Sheldrick, Acta Crystallogr. Sect. A 2008, 64, 112-122.

12

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Figure 1. Structure of A6730 and bioactive compounds of previously described series A-B, and general structure of new synthesized substituted pyrrolo[1,2-

a]quinoxaline derivatives 1a-o.

13

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Figure 2. The ORTEP drawing of pyrrolo[1,2-a]quinoxaline 1c with thermal ellipsoids at 30% level.

14

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

60

50

40

30

20

10

0

*

Day 0

Day 2

Day 3

0

0,5 µM

1 µM

HL60

5 µM

10 µM

30

25

20

15

10

5

*

Day 0

Day 2

Day 3

0

0,5 µM

1 µM

K652

5 µM

10 µM

Figure 3. Effect of pyrrolo[1,2-a]quinoxaline 1a on apoptosis of HL60 and K562 cells. Cells were cultured with or without increasing doses (0.5 µM, 1 µM, 5 µM,

10 µM) of compound 1a for 2 and 3 days in culture medium then stained with APC-Annexin V, and analyzed by flow cytometry. Results are expressed as Mean ±

Standard Error (SEM) of two independent experiments. * p< 0.05 compared with controls (t-test).

15

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Scheme 1. Synthesis of ethyl 4,5-dihydro-4-oxo-5H-pyrrolo[1,2-a]quinoxaline-carboxylates 2a-e. Reagents and conditions: a) Ag₂CO₃, dioxane, 80°C, 2 h, 86%;

b) MnO₂, CHCl₃, reflux, 2.5 h, 70%; c) KMnO4, Acetone/H₂O, 40°C/1 h then RT/1 h, 31%; d) EtOH, SOCl₂, reflux, 3 h, 87%; e) 2-fluoro-nitrobenzene, Cs₂CO₃,

DMF, reflux, 1.5 h, 92%; f) Fe, CH₃COOH, reflux, 2 h, 28-92%; g) DMTHF, AcOH, reflux, 1 h, 64-93%; h) CuSO₄/NaBH₄, EtOH, 0°C, 1 h, 88-90%; i) (Cl₃CO)₂CO,

toluene, reflux, 4 h, 48-81%; j) methyl pyrrole-2-carboxylate; Cs₂CO₃, DMF, reflux, 2 h, 24-62%.

16

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Scheme 2. Synthesis of pyrrolo[1,2-a]quinoxalines 1a-k. Reagents and conditions: a) POCl₃, reflux, 2 h, 68-89%; b) OHC-C₆H₄-B(OH)₂, Pd[P(C₆H₅)₃]₄, K₂CO₃,

toluene, EtOH, reflux, 24 h, 65-86%; c) 4-(2-ketobenzimidazolin-1-yl)piperidine or 4-(5-fluorobenzimidazolin-2-yl)piperidine or 2-(3-piperidin-4-yl-1H-1,2,4-triazol-5-

yl]pyridine, NaBH₃CN, MeOH, reflux, 5 h, 33-77%.

17

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Scheme 3. Synthesis of pyrrolo[1,2-a]quinoxalines 1l-o. Reagents and conditions: a) DMTHF, AcOH, reflux, MW, 10 min, 82-86%; b) CuSO₄/NaBH₄, EtOH, 0°C

to RT, 1 h, 74-80%; c) H₅C₂-OOC-CO-Cl, TEA, THF, RT, 14 h, 85-94%; d) POCl₃, reflux, 20 min, 57-64%; e) OHC-C₆H₄-B(OH)₂, Pd[P(C₆H₅)₃]₄, K₂CO₃, toluene,

EtOH, reflux, 24 h, 58-80%; f) 4-(2-ketobenzimidazolin-1-yl)piperidine or 4-(5-fluorobenzimidazolin-2-yl)piperidine or 2-(3-piperidin-4-yl-1H-1,2,4-triazol-5-

yl]pyridine, NaBH₃CN, MeOH, reflux, 5 h, 48-70%.

18

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

Table 1. In vitro activity of compounds 1a-o on U937, K562, HL60, Jurkat and U266 cells, and cytotoxicity on human peripheral blood mononuclear cells PBMNC

+ PHA.

IC₅₀values (M)^[a]

Cytotoxicity on

activated human

Compound

A6730

Structure

K562

U937

HL60

Jurkat

U266

peripheral blood

mononuclear cells

(PBMNC)

PBMNC + PHA

>50

17  0.3

8  0.2

5.5 

3.5 

n.d.^[b]

0.2

LY-294002

38 ± 1

14 ±

0.3

14 ±

0.3

22 ± 1

46 ± 2

>50

1a

1b

1c

1d

4 ± 0.1

3 ± 0.2

9 ± 1

3 ± 0.1

4 ± 0.2

6 ± 0.2

>50

1 ±

0.1

1 ± 0.1

5 ±

0.2

>50

9 ± 0.6

17 ± 1.2

>50

1.5 ±

0.1

7 ±

0.2

4 ±

0.2

2.1 ±

0.1

17 ±

1.5

7 ± 0.1

>50

1e

1f

34 ± 2

>50

17 ±

1.2

20 ±

1.3

>50

37 ± 1.2

33 ±

1.7

17 ±

1.6

14 ±

0.6

>50

1g

1h

>50

20 ±

1.2

7 ±

0.3

12 ±

0.5

10 ± 1

>50

1i

15 ± 0.3

39 ± 2

16 ± 2 8 ± 0.4

10 ±

1.2

>50

1j

0.5 ± 0.1

7 ± 0.1

8 ± 0.2

0.5 ±

0.1

3 ± 0.1

7 ± 0.5

>50

6 ± 0.3

1k

13 ±

0.5

12 ±

1.1

6 ±

0.2

18 ± 1.3

19

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

1l

6 ± 0.2

23 ±

1.3

15 ±

2.2

4 ± 0.2

20 ± 2

>50

1m

3 ± 0.2

12 ±

0.5

7 ±

0.3

3 ± 0.1

5 ±

0.2

>50

1n

35 ± 2.1

36 ±

2.1

40 ± 2 3 ± 0.2

16 ±

1.5

>50

1o

>50

15 ±

1.5

>50

30 ±

1.6

>50

Cytarabine^[31,32]

60.05

8.84

1.29 ±

0.45

Chlorambucil^[31,33]

Etoposide^[31,34]

79.04

36.16

35.01

± 3.32

10.26 ±

0.20

14.10

± 0.54

6.01 ±

1.66

^[a]The IC₅₀(µM) values correspond to the mean +/- standard deviation from 3 independent experiments.

^[b]n.d. = not determined.

20

10.1002/cmdc.201700049

ChemMedChem

FULL PAPER

List of captions: