Synthesis and biological evaluation of novel substituted pyrrolo[1,2-a] quinoxaline derivatives as inhibitors of the human protein kinase CK2

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Graphical Abstract

Synthesis and Biological Evaluation of Novel Substituted Pyrrolo[1,2-a]quinoxaline Derivatives as Inhibitors of

the Human Protein Kinase CK2

*

Jean Guillon, Marc Le Borgne, Charlotte Rimbault, Stéphane Moreau, Solène Savrimoutou, Noël Pinaud, Sophie Baratin,

Mathieu Marchivie, Séverine Roche, Andre Bollacke, Adali Pecci, Lautaro Alvarez, Vanessa Desplat and Joachim Jose

1

2

9

3

N

8

7

4

N

6

5

Pyrrolo[1,2-a]quinoxaline

N

COOH

N

HOOC

N

NH

R

NH

COOH

HOOC

NH

compounds 1a-d

compounds 1e-j

compounds 1k,l

compounds 1m,n

R

R = -Cl,

C

CH

1

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Synthesis and Biological Evaluation of Novel Substituted Pyrrolo[1,2-

a]quinoxaline Derivatives as Inhibitors of the Human Protein Kinase

CK2

a,b*

c

a,b

f

Jean Guillon,

Marc Le Borgne, Charlotte Rimbault, Stéphane Moreau, Solène Savrimoutou,

d

a,b

e

Noël Pinaud, Sophie Baratin, Mathieu Marchivie, Séverine Roche, Andre Bollacke, Adali Pecci,

g

a,b

e

Lautaro Alvarez, Vanessa Desplat and Joachim Jose

a

Université Bordeaux Segalen, Pharmacochimie, FRE 3396, F-33000 Bordeaux, France

CNRS, Pharmacochimie, FRE 3396, F-33000 Bordeaux, France

Université de Lyon, Université Lyon 1, Faculté de Pharmacie - ISPB, EA 4446 Biomolécules Cancer et

b

c

Chimiorésistances, SFR Santé Lyon-Est CNRS UMS3453 - INSERM US7, 8 avenue Rockefeller, F-69373 Lyon Cedex

8

, France

d

ISM – CNRS UMR 5255, Université de Bordeaux, 351 cours de la Libération, F-33405 Talence cedex, France

Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Hittorfstraße

8-62, 48149 Münster, Germany

Departamento de Química Biológica and IFIBYNE (CONICET-UBA), Facultad de Ciencias Exactas y Naturales,

Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina

Departamento de Química Orgánica and UMYMFOR (CONICET-UBA), Facultad de Ciencias Exactas y Naturales,

e

5

f

g

Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EGA Ciudad de Buenos Aires, Argentina

Corresponding author, Tel.: +33 5 57 57 16 52; fax: +33 5 57 57 13 52

E-mail address: jean.guillon@u-bordeaux2.fr

ABSTRACT

Herein we describe the synthesis and properties of substituted phenylaminopyrrolo[1,2-a]quinoxaline-

carboxylic acid derivatives as a novel class of potent inhibitors of the human protein kinase CK2. A set of 15

compounds was designed and synthesized using convenient and straightforward synthesis protocols. The

compounds were tested for inhibition of human protein kinase CK2, which is a potential drug target for

many diseases including inflammatory disorders and cancer. New inhibitors with IC in the micro- and sub-

5

0

micromolar

chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-3-carboxylic acid 1c inhibited human CK2 with an IC50 of

9 nM. Our findings indicate that pyrrolo[1,2-a]quinoxalines are a promising starting scaffold for further

range

were

identified.

The

most

promising

compound,

the

4-[(3-

4

development and optimization of human protein kinase CK2 inhibitors.

Keywords

Pyrrolo[1,2-a]quinoxaline

Protein kinase CK2

Antiproliferative activity

Synthesis

2

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1

. Introduction

Protein kinase CK2, formerly known as Casein Kinase 2, is a ubiquitous eukaryotic serine/threonine protein kinase. CK2

is a highly pleiotropic enzyme which catalyzes the transfer of terminal phosphate from ATP or GTP to various proteins implicated

in a wide variety of cell functions. The catalytic subunits of CK2 (alpha and/or alpha’) are constitutively active either alone or in

combination with the regulatory beta-subunits to give a heterotetrameric protein. A third isoform of the catalytic subunit,

1

designated CK2α’’, was discovered more recently and only very limited information is available. Since its discovery in 1954 by

2

3-5

Burnett and Kennedy, the activity of CK2 in physiological and pathological processes has been largely studied, particularly in

cell proliferation and apoptosis. Overexpression of CK2 creates a favorable environment for tumor development. For example

CK2 is involved (i) to promote abnormal pro-survival signals, (ii) to support neovascularization, (iii) to potentiate MDR

phenotypes and (iv) to upregulate DNA repair. To resume the impact of CK2 on cancer development, recent works demonstrated

that CK2 regulates multiple oncogenic pathways such as EGFR pathways (JAK/STAT, MAPK…), Cdc37/HSP90 pathway, Akt

6

,7

pathway (PTEN, PI3K…). Because of its pro-survival functions and its structural features, a considerable amount of additional

8

works have been devoted to know its druggability. CK2 has now emerged as a relevant therapeutic target for the treatment of

9

cancer (e.g. prostate, mammary gland, lung, kidney, hematologic malignancies). Indeed, diverse strategies are developed to target

1

0

CK2, and to synthesize highly specific, selective and cell-permeable inhibitors. Most of them are directed towards the ATP

binding site, and belong to (i) coumarins (ellagic acid), (ii) polyhalogenated benzimidazoles (TBB), (iii) anthraquinones (emodin),

1

(

iv) pyrazolotriazines, (v) indoloquinazolines (IQA) and naphthyridines and related (CX-4945, CX-5011) (Figure 1). Pre-

clinically, CX-4945 demonstrated single agent potency in suppressing xenograft tumor growth. The studies in BT-474 breast

cancer and BxPC-3 pancreatic xenografts showed robust antitumor activity including partial and complete regression of CX4945.

1

2

These promising results in safety, pharmacokinetics and efficacy studies led to the clinical evaluation of CX -4945.

The pyrrolo[1,2-a]quinoxaline heterocyclic framework constitutes the basis of an important class of compounds

possessing interesting biological activities. These compounds have been reported to serve as key intermediates for the assembly of

1

3

14

15

16-20

several heterocycles including antipsychotic agent, anti-HIV agent, adenosine A

3

receptor modulator, antiparasitic agents,

2

1,22

and antitumor agents.

In this last field, the discovery and development 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-

2

3-25

a]quinoxalines .

Thus, taking into account our experience in the field of the synthesis of new bioactive heterocyclic

1

6-20,23-25

compounds based on our pyrrolo[1,2-a]quinoxaline heterocyclic core,

we used the pyrrolo[1,2-a]quinoxaline moiety as a

template for the design of new isosteres of the CK2 inhibitors CX-4945 and CX-5011 in which the pyrrolo[1,2-a]quinoxaline

nucleus is substituted in different positions by a carboxylic acid function and a 3-substituted aniline in comparison with the

reference compounds (Figure 2). The 3-chloroaniline and 3-ethynylaniline motifs were also conserved in our subsequent

molecules in analogy with these reference derivatives CX-4945 and CX-5011. Our new synthesized compounds were then tested

for inhibition of human protein kinase CK2. The antiproliferative profile of the most potent CK2 inhibitors 1 was then evaluated

in vitro against a panel of four leukemic cell lines: U937, K562, Jurkat and MV-4-11. Moreover, to determine their respective

cytotoxicity, the new pyrrolo[1,2-a]quinoxaline derivatives were tested on activated human peripheral blood mononuclear cells.

Finally, a preliminary molecular modeling study was carried out to enlighten the mechanism of action implicating binding of

pyrroloquinoxalines to the ATP-binding CK2 pocket by using X-ray crystal structure results obtained for compound CX-4945.

2

. Chemistry

3

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The synthesis of the (3-substituted-phenyl)amino-pyrrolo[1,2-a]quinoxaline derivatives 1a-n was accomplished from

commercially available 3-methyl-2-quinoxalinol, from various substituted 1H-pyrrole-2-carboxylic acid alkyl esters, or from

various 2-nitroanilines via their respective lactames 2a-f (Schemes 1-4).

The lactame 2a was prepared in two steps by treatment of commercially available 3-methyl-2-quinoxalinol with

phosphorus oxychloride leading to the chloro derivative 3 following by condensation with ethyl bromopyruvate in dry ethanol

2

4

(

Scheme 1). The preparation of the N-aryl pyrrole 4 was obtained by nucleophilic substitution of the diethyl pyrrole-2,3-

2

6

dicarboxylate 5, previously prepared according to the method of Röder et al., with 2-fluoro-nitrobenzene using cesium carbonate

2

4,25

as the base in refluxing DMF solution (Scheme 1).

produced the spontaneous ring closure onto the ester to afford the desired tricyclic pyrrolo[1,2-a]quinoxaline 2b through a one-pot

Reduction of the nitro moiety of 4 with iron in hot glacial acetic acid

2

4,25

reduction-cyclization step.

Similar substitution of commercialy the methyl pyrrole-2-carboxylate with various ethyl fluoro-

nitrobenzenecarboxylate led to the methyl 1-(4- or 5-ethoxycarbonyl-2-nitrophenyl)pyrrole-2-carboxylates 6a-b. Refluxing

compounds 6a-b in acetic acid with iron powder also gave lactams 2c-d. The synthesis of lactam 2e was achieved from 2-amino-

3

-nitrobenzoic acid according to two pathways. First, refluxing 2-amino-3-nitrobenzoic acid in thionyl chloride gave the

2

7,28

corresponding acid chloride, which was treated with ethanol to give ester 7 in 85% yield.

The Clauson-Kaas reaction of 7 with

2

,5-dimethoxytetrahydrofuran (DMTHF) in acetic acid gave the pyrrolic derivative 8 in 64% yield. The second pathway involved

at first the preparation of the 3-nitro-2-pyrrol-1-ylbenzoic acid 9 according to the Clauson-Kaas reaction. This acid 9 was then

2

9

esterified by treatment with thionyl chloride in ethanol leading to ester 8. The resulting 1-(2-nitrophenyl)pyrrole ester

intermediate 8 was subsequently reduced into the attempted 1-(2-aminophenyl)pyrrole ester 10 using a sodium borohydride-

3

0

copper(II) sulfate in ethanol at room temperature according to the conditions described by Yoo and Lee. This NaBH -CuSO

4

system was found to be quite powerful in reducing our aromatic nitro groups with excellent yield (90%). The reaction of 10 with

1

6

triphosgene in toluene gave the lactam 2e.

These lactames 2a-e were subsequently chlorodehydroxylated with phosphorous oxychloride, leading to the 4-

chloropyrrolo[1,2-a]quinoxalines 11a-e (Scheme 2). These 4-chloropyrrolo[1,2-a]quinoxaline derivatives 11a-e underwent

nucleophilic attack by the 3-chloro or 3-ethynylanilines in refluxing dimethylformamide to give the 4-anilinopyrrolo[1,2-

31

a]quinoxalines 12a-j. The 4-anilinopyrrolo[1,2-a]quinoxalines 12a/c/e/g/i were also prepared in quite good yields (68-85%) by a

direct Buchwald–Hartwig cross-coupling reaction of 4-chloropyrroloquinoxalines 11a-e with 3-chloroaniline using catalytic

32

amounts of Pd(OAc) and BINAP, and Cs CO as a base. The desired acids 1a-j were obtained by alkaline hydrolysis of the

2

3

parent esters 12a-j in good yields. The 3D spatial determination of 1f was established by X-ray crystallography, and confirmed

1

the structure in the solid state as anticipated on the basis of IR and H NMR data (Figure 3).

The 7-anilinopyrroloquinoxaline-4-carboxylic acids 1k-l were synthesized in six or seven steps from commercially

available 4-bromo-2-nitroaniline (Scheme 3). Preparation of 1-(4-bromo-2-nitrophenyl)pyrrole 13 was performed according to the

Clauson-Kaas reaction runned under micro-waves irradiation starting from 4-bromo-2-nitroaniline and 2,5-

4

dimethoxytetrahedrofuran in acetic acid. The resulting phenylpyrrole 13 intermediate was subsequently reduced using a NaBH -

1

8

CuSO

presence of triethylamine provided the ester 15.

prepared by cyclization of the amido-ester 15 in refluxing phosphorus oxychloride. The 3D spatial structure of ethyl 7-

4

treatment into the attempted 1-(2-amino-4-bromophenyl)pyrrole 14. Addition of 14 to ethyl chlorooxoacetate in the

3

4,35

The ethyl 7-bromopyrrolo[1,2-a]quinoxaline-4-carboxylate 16 was then

3

6

3

bromopyrrolo[1,2-a]quinoxaline-4-carboxylate 16 was established by X-ray crystallography and confirmed the structure (Figure

).

4

Then the ethyl 7-anilinopyrrolo[1,2-a]quinoxaline-4-carboxylates 12k-l were synthesized via the Buchwald palladium-

3

7,38

catalyzed cross-coupling reaction of 3-chloroaniline or 3-(trimethylsilylethynyl)aniline

cesium carbonate (Scheme 3). The deprotection of the acetylene moiety of compound 12l by tetrabutylammonium fluoride

with 16 in the presence of BINAP and

3

9,40

(

TBAF) led to anilinopyrrolo[1,2-a]quinoxaline 17.

The palladium-catalyzed cross-coupling reaction of unprotected 3-

4

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ethynylaniline with 16 directly gave the Sonogashira side product 18 (Scheme 4). The hydrolysis of the ester function of

derivatives 12k, 17 and 18 in basic conditions gave the pyrroloquinoxalines 1k,l and 19, respectively.

The synthesis of the 4-anilinopyrrolo[1,2-a]quinoxaline-7-carboxylic acids 1m,n, substituted in position 2 by a pyridine

moiety, was depicted in Scheme 5. The methyl 4-bromopyrrole carboxylate 20 was prepared by regioselective bromination of

24

methyl pyrrole-2-carboxylate. This bromopyrrole ester 20 was then protected as the corresponding tert-butyl carbamate to afford

1 in nearly quantitative yield. The role of the Boc group is not to protect nitrogen but to reduce the electron density of the pyrrole

2

to avoid extensive dehalogenation. Coupling Boc-protected 21 with 3-pyridylboronic acid under Suzuki-Miyaura cross-coupling

conditions proceeded cleanly to afford the 4-phenyl-1H-pyrrole-2-carboxylic acid methyl ester 22 after deprotection of the NH

using a trifluoroacetic acid solution. A X-ray single crystal analysis was also performed on pyrrole 22 in order to confirm the

3

structure (Figure 5). The preparation of N-aryl pyrrole 23 were obtained by nucleophilic substitution of the methyl 4-(3-

pyridyl)pyrrole-2-carboxylate 22 with ethyl 4-fluoro-3-nitrobenzenecarboxylate using cesium carbonate in refluxing DMF

solution (Scheme 5). Reduction of the nitro moiety with iron in hot acetic acid afforded the desired lactame 2f. The lactame 2f was

subsequently chlorodehydroxylated with phosphorous oxychloride, leading to the 4-chloroquinoxalines 11f. Nucleophilic

displacement of the chlorine by various substituted anilines in 11f and ester hydrolysis provided carboxylates 12m,n and acids

1

m,n.

3

. Biological activity

3

.1. Inhibition of human CK2 holoenzyme

These new synthesized pyrrolo[1,2-a]quinoxaline-carboxylic acid derivatives were tested for their inhibitory activity towards

human CK2 holoenzyme (Table 1). The synthetic peptide RRRDDDSDDD was used as the substrate, which is reported to be most

4

1

efficiently phosphorylated by CK2. The purity of the CK2 holoenzyme was superior to 99 % (Figure 6). For initial testing,

inhibition was determined relative to the controls at inhibitor concentrations of 10 µM in DMSO as the solvent. Final

concentration of DMSO never exceeded 1 % of the entire reaction volume. The reaction with pure solvent without inhibitor was

used as negative control and set to 0% inhibition. Reactions without CK2 were used as positive control and set to 100% inhibition.

Compounds with more than 50% inhibition at a concentration of 10 µM were subjected to an IC50 determination. For this purpose,

inhibition was measured at final concentrations ranging from 0.01 to 30 µM in appropriate intervals. IC50 were calculated from the

4

2

resulting dose–response curves. Each value was determined at least in triplicate in independent experiments.

From the 15 synthesized pyrrolo[1,2-a]quinoxalines, we found thirteen potent compounds with an inhibition of more than 50% at

a concentration of 10 µM (1a-h, 1k-n and 19). Nine of them (1c-h, 1k,l and 1n) revealed IC50 in the sub-micromolar range (from

0

.049 to 0.982 µM).

Among compounds 1e-j, which were bearing the 3-substituted aniline in position 4 of the heterocycle and the carboxylic acid

function into the benzene of the quinoxaline moiety, the pyrrolo[1,2-a]quinoxalines 1e and 1f exhibited the best inhibitory activity

towards CK2 (IC₅₀= 0.118 and 0. 078 µM, respectively). Comparison of derivatives 1g,h and 1i,j with 1e,f indicated that the

presence of a carboxylic acid function at C8 position or C9 position yielded significantly less potent inhibitors of CK2, showing

that the carboxylic acid group at C7 position was optimal in analogy with the reference drug CX-4945. Moreover, replacement of

the 3-chloroaniline by a 3-ethynylaniline did not lead to significant modification in the inhibition of CK2 (IC50 = 0.120 µM for 1e

versus 0. 080 µM for 1f, and IC50 = 0.834 µM for both 1g and 1h).

Moreover, in terms of structure-activity relationships discussion, it could be also noticed that the IC50 values of compound 1a

and 1b bearing the carboxylic acid group on the pyrrole moiety, i.e. at position 2 of the heterocyclic skeleton and the 3-substituted

5

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aniline in position 4 (IC50 = 4.28 and 1.89 µM), were found 35 and 24 times lower than those of their 7-substituted analogues 1e,f.

Surprisingly, molecules 1c and 1d, in which the carboxylic acid was moved to the pyrrolic adjacent carbon, were found to be

much more active in their CK2 inhibitory activity, showing that its position on C3 was necessary for potent inhibition. The most

promising compound, the 4-[(3-chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-3-carboxylic acid 1c inhibited human CK2 with an

IC50 of 49 nM. Substitution of the 3-chloroaniline by a 3-ethynylaniline in compound 1d led to a small decrease in the inhibitory

activity (IC50 = 134 nM).

The transposition of the 3-substituted aniline and the carboxylic acid from their respective position 4 and 7 in compounds

1

e,f to position 7 and 4 (compounds 1k,l) induced a strong decrease in the inhibitory activity of CK2 (IC50 0.982 µM for 1k

compared with 0.118 µM for 1e; and IC50 0.850 µM for 1l compared with 0.078 µM for 1f).

In analogy with the reference compound CX-4945, we next introduced a pyridine into the tricyclic pyrrolo[1,2-a]quinoxaline

core with the objective to add a hydrogen bond acceptor. In the hinge region, it was noticed a hydrogen bonding interaction

between the pyrido moiety of CX-4945 and the NH of Val116. However, incorporation of a 3-pyridyl moiety in position 2 of the

pyrroloquinoxaline scaffold (compounds 1e,f) leading to compounds 1m,n induced a 10-fold lower CK2 inhibitory activity, with

IC₅₀from 0.89 (compound 1n) to 1.57 µM (compound 1m).

From a SAR point of view, these preliminary biological results on the inhibitory activity towards human CK2 holoenzyme

revealed the importance of the substitution at the C-4 position of the pyrroloquinoxaline scaffold by a 3-substituted aniline, and

also revealed the need of a carboxylic acid functionality on position 3 of the pyrrole ring. However, in a general way, it could also

be noticed that the nature of the substitution at position 3 in the aniline moiety was less detrimental than the position of the

carboxylic acid function for the CK2 inhibitory activity of the pyrroloquinoxaline derivatives 1.

3

.2. Antiproliferative effect

The antiproliferative profile of the most promising CK2 inhibitors 1 (compounds 1a-h and 1k) at increasing

concentrations (0, 1, 5, 10, 20 and 50 µM) was then evaluated in MTS assay for their in vitro antiproliferative activity against four

human leukemic cell lines (U937, K562, Jurkat and MV-4-11). Compound CX-4945, which showed strong CK2 inhibition, was

applied as a referential cytotoxic agent with potent antiproliferative activity against our cell lines. The results of the

antiproliferative activity studies are summarized in Table 2.

On K562 cell line, all the tested pyrroloquinoxalines 1 were found to be inactive (CC50 > 50 µM), with the exception of

1

c that presented very moderate antiproliferative activity with a CC50 of 42 µM, while CX-4945 presented a CC50 of 7 µM.

Among compounds 1a-h and 1k, the derivatives 1c and 1k exhibited a low antiproliferative activity on the growth of

U937 monocytic cell line (CC50 values of 47.7 and 39.2 µM, respectively). On this U937 cell line, reference CX-4945 showed a

CC50 of 4.2 µM.

Against the Jurkat T-lymphoblastic cell line, we also observed moderate antiproliferative activities for compounds 1b and

1

k which presented CC50 of 41 and 31.4 µM, respectively. These antiproliferative activities were found 10-fold lower than the one

of CX-4945 (CC50 = 4.5 µM).

In the most sensitive acute myeloblastic leukemia cells MV-4-11, compounds 1e,f were active at a 20 µM concentration,

whereas their structural analogue 1k in which the aniline moiety and the carboxylic acid function were inverted between the 4 and

positions of the pyrrroloquinoxaline skeleton was found twice less active (CC50 = 47 µM). For compound CX-4945, a CC50 of 3

µM was observed on MV-4-11, 7-fold lower than the CC s of pyrroloquinoxalines 1e,f.

7

5

0

6

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3

.3. Cytotoxicity

The CK2 inhibitor compounds 1a-h and 1k were tested on activated (PBMNC + PHA) human peripheral blood

mononuclear cells to evaluate their respective cytotoxicity on normal cells (Table 2). As expected, all the tested pyrrolo[1,2-

a]quinoxalines 1 presented a CC50 superior to 50 µM against lymphocytes. Compound CX-4945 exhibited a CC50 of 50 µM. These

preliminary results were used to determine their respective range of toxic concentration.

Indexes of selectivity (IS) were defined as the ratio of the CC50 value on the human mononuclear cells to the CC50 value

on the K562, U937, Jurkat and MV-4-11 lines. Compounds that demonstrated high selectivity (high index of selectivity) should

offer a potential of safer therapy. This led to identify compounds with moderate index of selectivity >2.5 for compounds 1e,f on

the human leukemic cell line MV-4-11, and 1.6 for compound 1k against the Jurkat T-lymphoblastic cell line. The reference

compound CX-4945 showed high selectivity with index of selectivity values noticed between 7.1 and 16.7.

3

.4. Lipophilicity and cell permeability

4

3

As a previous study on design of indeno[1,2-b]indole derivatives as CK2 inhibitors, predictive ADME parameters were

used to determine their biological profile. Then for an evaluation of the cell permeability of pyrroloquinoxalines 1a-h and k-n,

fragment-based partition coefficients (Clog P) and topological polar surface areas (TPSA) were calculated by the molinspiration

4

web services. Compounds 1a-h and k-n presented Clog P values between 3.986 for 1b and 5.94 for 1m. TPSA values were

noticed between 66.633 and 79.525 Å2.

A plot of CK2 inhibitory activity (IC50) versus Clog P values was presented in Figure 7, allowing us to classify compounds 1a-n in

various subsets. The less lipophilic compounds (1b, 1d, 1l and 1h) with Clog P values from 3.986 to 4.373 exhibited moderate

CK2 inhibitory activities (IC₅₀= 0.834-1.89 µM) excepted 1d showing an IC₅₀of 0.134 µM. The most active pyrroloquinoxalines

1

f and 1c with IC₅₀of 0.078 and 0.049 µM presented Clog P of 4.373 and 4.901, respectively. The three compounds (1a, 1n and

k) with Clog P values between 4.901 and 5.065 were found to be less active (IC50 = 0.89-4.28 µM). The reference compound

CX-4945 (IC50 = 0.0037 µM) showed a Clog P of 5.136. At Clog P of 5.288, derivatives 1e and 1g exhibited IC50 of 0.118 and

.834 µM, respectively. Compound 1m, showing the most lipophilic value (Clog P = 5.94), was less active (IC50 = 1.57 µM) on

0

CK2.

4

5-47

Poor absorption and low permeability is predicted for drugs with Clog P ≥ 5 and TPSA ≥ 140 Å2.

Thus, the two most active

compounds 1c and 1f on CK2 showed acceptable lipophilic properties for cell permeability (Clog P < 5) but their cytotoxic

activity on tested leukemia cell lines was limited (e.g. IC50 = 20 µM on MV-4-11 for 1f). Furthermore compound 1k (IC50 = 0.982

µM on CK2) and CX-4945 had similar Clog P values (5.065 and 5.136, respectively) and compound 1k only presented slight

cytotoxicity on three cell lines (31.4 < IC₅₀µM < 47). This suggests that the Clog P estimates did not seem to fit with our

biological results. Further investigations are ongoing to optimize the correlation of physicochemical properties of

pyrroloquinoxalines and their biological potencies.

4

. Molecular dynamics of CK2 complexes

In order to investigate the molecular basis of the interaction of the CK2 ATP binding pocket with the 1f and 1c inhibitors,

Molecular Dynamics (MD) simulation of CK2/1f and CK2/1c complexes were performed. From the protein coordinates of the

4

8

CK2/CX-4945 complex crystal structure (pdb:3pe1) (Figure 8a; see Computational Methods) and 1f and 1c optimized

geometries, two protein/inhibitor systems were constructed and 40 ns MD runs were obtained employing a explicit box of TIP3P

water molecules. Initially, a CK2/CX-4945 MD simulation was carried out as control trajectory, finding that both global complex

7

ACCEPTED MANUSCRIPT

conformation and polar protein-inhibitor interactions observed in the crystal structure resulted stable during the simulation time

scale. Figure 8b shows that the nitrogen atom of the A-ring forms a hydrogen bond with the backbone nitrogen atom of Val116

(

localized at the hinge/αD region), while on the opposite end of the CX-4945 molecule, the carboxyl group from the C-ring

interacts in a strong polar network involving the Lys68, Glu81, Asp175 and two water molecules (positions W1 and W2). The W1

water molecule remained in its position throughout the 40 ns of the trajectory, while exchanges of water molecules at the W2

position were observed. We have also observed that the side chain (D-ring) of the ligand was more flexible than the rest of the

molecule, although the global orientation was conserved. Finally, rotations of the His160 side chain occurred throughout the 40

ns, continually switching from the up to the down conformations. Similar MD results related to the CX-4945 binding mode were

4

9

recently reported by Liu et al. using another initial structure (pdb:3nga).

The MD simulation of the CK2/1f binding mode showed very similar results to those observed in the CX-4945 system

Figure 8c). The main difference resides in the lack of polar interactions between the carbon atoms of the A-ring and the hinge/αD

(

region, which increases the average distance between this protein region and the inhibitor (Table 3, distance A). Similar to the

CX-4945 system, the carboxyl group of 1f is involved in an extensive polar interaction network, with a comparable average

distance to the Lys68 (Table 3, distance B), but with a decrease in the average distance between the Lys68 and the Glu81 residues

(

Table 3, distance C). Moreover, the involvement of the W1 and W2 water molecules in the inhibitor binding mode and the

dynamic behavior of both inhibitor side chain and His160 were similar to the trajectory control observed with CX-4945. All

together these results suggest a very stable binding mode of the inhibitor 1f, highlighting how five-member A-ring inhibitors

could be able to interact within the ATP binding pocket of the CK2 protein.

We also studied the superposition of optimized geometries of 1f and 1c. Remarkably, when the superposition of both

molecules was made through the central B-ring, but with an opposite orientation of the heterocyclic system (i.e. with the 1c A-ring

sited at the position of the 1f C-ring, and vice versa), the position of both carboxyl groups resulted comparable, with a distance of

2

.0 Å between each other (Figure 9). Even more the D-rings and their m-substituents acquired a similar orientation. Afterwards, in

order to carry out the CK2/1c MD simulation, the inhibitor was introduced within the ATP binding pocket in two different initial

configurations: the first one with the heterocyclic system orientated as in the 1f system and the second one with the heterocyclic

system in the opposite orientation. According to the MD results no polar interaction between 1c and receptor residues was

observed when the inhibitor was introduced in the first configuration, resulting in less stable inhibitor trajectory (data not shown).

In consequence, this configuration does not seem to be a suitable binding mode of 1c. When the inhibitor was introduced in the

opposite configuration, a tightly binding mode was established, with the carboxyl group contacting the Lys68 and forming part of

the polar interaction network as in the CX-4945 and 1f systems (Figures 8b-c). Indeed, the interaction between Lys68 and Glu81

residues resulted slightly larger than in the 1f system (Table 3). On the other hand, the average distance between the C-ring and

the nitrogen backbone atom of Val116 resulted larger in the 1c system than in the 1f system. This is expected considering the

larger steric impedance of a six-member ring (1c) in comparison with a five-member ring (1f) orientated to the hinge/αD. Notably

the intramolecular hydrogen bond formed between the carboxyl group and the nitrogen atom of B-ring remained stable during the

4

0ns time scale of the simulation.

Finally, to compare qualitatively the different contributions to the binding energy of 1f and 1c to CK2, we calculated the

electrostatic energy (ele) and Van der Waals contributions (vdw), the sum of which gave the total gas phase binding energy. The

results revealed that vdw contribution is 2.7 Kcal/mol more favorable in the 1f complex while the ele energy is 7.1 Kcal/mol more

favorable in the 1c complex. Thus, without considering the solvation and entropic effects, we found that the binding of 1c to CK2

was significantly stronger than that for 1f. These observations qualitatively correlate with the respective experimental IC50 values.

5

. Conclusion

8

ACCEPTED MANUSCRIPT

In the present report, we showed that substituted (phenylamino)pyrrolo[1,2-a]quinoxaline-carboxylic acid derivatives are

a novel class of potent inhibitors of the human protein kinase CK2. A set of 15 compounds was prepared and tested for their

inhibitory activity. New inhibitors with IC₅₀in the micro- and sub-micromolar range were identified. The most promising

compound, the 4-[(3-chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-3-carboxylic acid 1c, inhibited human CK2 with an IC50 of

4

9 nM. Taken together, these results show that pyrrolo[1,2-a]quinoxalines, such as 1c, are a promising starting point for the

further development and optimization of potent CK2 inhibitors. Moreover, it would be interesting to enlarge the biological

evaluation of the most active pyrrolo[1,2-a]quinoxaline derivatives by measuring the inhibition of endogenous intracellular CK2

activity in leukemic cell lines in comparison with CX-4945.

6

. Experimental

6

.1. 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 designated as follows: s = singlet; bs = broad singlet; d = doublet; t = triplet; q =

quartet; dd = double doublet; 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. Elemental analyses were found within ±0.4% of the theoretical values.

6

.1.1. Diethyl 1-(2-nitrophenyl)pyrrole-2,3-dicarboxylate (4)

To a solution of diethyl pyrrole-2,3-dicarboxylate 5 (4.83 mmol) in 15 mL of DMF was added cesium carbonate (5.80

mmol). The mixture was stirred at room temperature for 10 min, then 1-fluoro-2-nitrobenzene (7.25 mmol) was added. The

reaction mixture was refluxed for 1 h 30, then was diluted in AcOEt (50 mL). The organic layer was washed with water (2 x 30

mL), then brine (40 mL) and dried over sodium sulfate. The organic layer was concentrated under vacuo to give a brown oil. After

triturating in Et

2

O a solid was obtained and filtered off, washed with Et O and dried to give the desired product 4. Orange oil

1

(

92%). IR (KBr) 1700 and 1690 (C=O). H-NMR (CDCl

3

) δ: 8.06 (dd, 1H, J=7.80 and 1.80 Hz, H-3’), 7.74-7.56 (m, 2H, H-4’

and H-5’), 7.40 (dd, 1H, J=7.80 and 1.80 Hz, H-6’), 6.83 (d, 1H, J=3.00 Hz, H-5), 6.69 (d, 1H, J=3.00 Hz, H-4), 4.32 (q, 2H,

J=7.20 Hz, OCH ), 4.10 (q, 2H, J=7.20 Hz, OCH ), 1.36 (t, 3H, J=7.20 Hz, CH ), 1.12 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for

2

3

16 16 2 6

C H N O : C, 57.83; H, 4.85; N, 8.43. Found: C, 57.66; H, 5.03; N, 8.32.

6

.1.2. General procedure for methyl 1-(4-ethoxycarbonyl-2-nitrophenyl)pyrrole-2-carboxylate (6a) and methyl 1-(5-

ethoxycarbonyl-2-nitrophenyl)pyrrole-2-carboxylate (6b)

To a solution of methyl pyrrole-2-carboxylate (24.6 mmol) in 85 mL of DMF was added cesium carbonate (29.5 mmol).

The mixture was stirred at room temperature for 10 min, then ethyl 4-fluoro-3-nitrobenzoate or ethyl 3-fluoro-4-nitrobenzoate

(

7.25 mmol) was added. The reaction mixture was refluxed for 1 h 30 or 4 h (in case of 6b), then was diluted in AcOEt (100 mL).

The organic layer was washed with water (2 x 60 mL), then brine (70 mL) and dried over sodium sulfate. The organic layer was

2 2

concentrated under vacuo to give a brown oil. After triturating in Et O a solid was obtained and filtered off, washed with Et O and

dried to give the desired product 6.

6

.1.2.1. Methyl 1-(4-ethoxycarbonyl-2-nitrophenyl)pyrrole-2-carboxylate (6a)

9

ACCEPTED MANUSCRIPT

1

Yellow crystals (62%); mp 77°C. IR (KBr) 1712 (C=O). H-NMR (CDCl

3

) δ: 8.76 (d, 1H, J=2.00 Hz, H-3’), 8.36 (dd,

1

H, J=8.10 and 2.00 Hz, H-5’), 7.52 (d, 1H, J=8.10 Hz, H-6’), 7.15 (dd, 1H, J=3.95 and 1.65 Hz, H-5), 6.95 (dd, 1H, J=2.85 and

.65 Hz, H-3), 6.43 (dd, 1H, J=3.90 and 2.85 Hz, H-4), 4.47 (q, 2H, J=7.20 Hz, OCH ), 3.70 (s, 3H, OCH ), 1.45 (t, 3H, J=7.20

). Anal. Calcd. for C15 : C, 56.60; H, 4.43; N, 8.80. Found: C, 56.81; H, 4.29; N, 8.96.

2

3

Hz, CH

3

14 2 6

H N O

6

.1.2.2. Methyl 1-(5-ethoxycarbonyl-2-nitrophenyl)pyrrole-2-carboxylate (6b)

1

3

Beige crystals (24%); mp 117°C. IR (KBr) 1710 (C=O). H-NMR (CDCl ) δ: 8.26 (dd, 1H, J=8.40 and 1.80 Hz, H-4’),

8

.15 (d, 1H, J=8.40 Hz, H-3’), 8.09 (d, 1H, J=1.80 Hz, H-6’), 7.15 (dd, 1H, J=3.90 and 1.80 Hz, H-3), 6.97 (dd, 1H, J=2.70 and

.80 Hz, H-5), 6.43 (dd, 1H, J=3.90 and 2.70 Hz, H-4), 4.44 (q, 2H, J=7.20 Hz, OCH ), 3.70 (s, 3H, OCH ), 1.43 (t, 3H, J=7.20

1

2

3

Hz, CH

3

). Anal. Calcd. for C15

H

14

N

2

O

6

: C, 56.60; H, 4.43; N, 8.80. Found: C, 56.52; H, 4.64; N, 9.10.

6

.1.3. Ethyl 2-amino-3-nitrobenzoate (7)

A mixture of 2-amino-3-nitrobenzoic acid (18 mmol) and 50 mL of thionyl chloride was refluxed for 3 h. The mixture

was then evaporated in vacuo to dryness and ethanol (80 mL) was added. The resulting solution was allowed to stir at room

temperature for 15 h. The ethanol was evaporated and water was added. A yellow solid was collected by filtration, washed with

1

water and dried to give 7. Yellow crystals (85%); mp 88°C. IR (KBr) 3380 and 3290 (NH

2

), 1690 (C=O). H-NMR (CDCl

3

) δ:

8

5

.61 (bs, 2H, NH

2

), 8.52 (dd, 1H, J=8.40 and 1.55 Hz, H-6), 8.29 (dd, 1H, J=8.40 and 1.55 Hz, H-4), 6.67 (t, 1H, J=8.40 Hz, H-

), 1.43 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C : C, 51.43; H, 4.80; N, 13.33. Found:

), 4.40 (q, 2H, J=7.20 Hz, OCH

2

3

9 10 2 4

H N O

C, 51.62; H, 4.87; N, 13.58.

6

.1.4. Ethyl 3-nitro-2-pyrrol-1-ylbenzoate (8)

Method A: A mixture of ethyl 2-amino-3-nitrobenzoate 7 (15 mmol) and 2,5-dimethoxytetrahydrofuran (16.6 mmol) in

acetic acid (40 mL) was refluxed for 4 h with vigorous stirring. After cooling, the reaction mixture was poured into water. The

mixture was then extracted twice with diethyl ether. The organic layer was dried over magnesium sulfate and evaporated to

dryness under reduced pressure. The crude residue was then purified by column chromatography using AcOEt/cyclohexane (3/7,

v/v) as eluent to give 8. Method B: To a solution of 3-nitro-2-pyrrol-1-ylbenzoic acid 9 (24.6 mmol) in 80 mL of ethanol was

added thionyl chloride (27 mmol). The reaction mixture was heated under reflux for 2 h. Ethanol was evaporated under reduced

pressure. The residue was triturated with water then extracted with ethyl acetate. The organic layer was washed with a saturated

aqueous sodium bicarbonate solution, dried over sodium sulfate, charcoaled and evaporated to dryness. The crude residue was

then purified by column chromatography using AcOEt/cyclohexane (3/7, v/v) as eluent to give 8. Yellow oil (method A: 64%;

1

method B: 32%). IR (KBr) 1695 (C=O). H-NMR (CDCl

3

) δ: 8.02 (dd, 1H, J=7.80 and 1.50 Hz, H-6), 7.93 (dd, 1H, J=7.80 and

1

4

.50 Hz, H-4), 7.62 (t, 1H, J=7.80 Hz, H-5), 6.72 (dd, 2H, J=2.10 and 2.10 Hz, 2H-α), 6.34 (dd, 2H, J=2.10 and 2.10 Hz, 2H-β),

.15 (q, 2H, J=7.20 Hz, OCH ), 1.12 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C13 : C, 60.00; H, 4.65; N, 10.76. Found:

2

3

12 2 4

H N O

C, 59.84; H, 4.51; N, 10.92.

6

.1.5. 3-Nitro-2-pyrrol-1-ylbenzoic acid (9)

A mixture of 2-amino-3-nitrobenzoic acid (27.5 mmol) and 2,5-dimethoxytetrahydrofuran (30 mmol) in acetic acid (70

mL) was refluxed for 1 h with vigourous stirring. After cooling, the reaction mixture was poured into water. The mixture was then

extracted twice with diethyl ether. The organic layer was dried over sodium sulfate and evaporated to dryness under reduced

pressure. The crude residue was recrystallized from petroleum ether to give 9. Brown crystals (89%); mp 148°C. IR (KBr) 3400-

1

2

4

560 (COOH), 1695 (C=O). H-NMR (CDCl ) δ: 8.15 (dd, 1H, J=7.95 and 1.50 Hz, H-6), 7.97 (dd, 1H, J=7.95 and 1.50 Hz, H-

3

), 7.65 (t, 1H, J=7.95 Hz, H-5), 6.81 (bs, 1H, COOH), 6.73 (dd, 2H, J=2.10 and 2.10 Hz, 2H-α), 6.64 (dd, 2H, J=2.10 and 2.10

8 2 4

Hz, 2H-β). Anal. Calcd. for C11H N O : C, 56.90; H, 3.47; N, 12.06. Found: C, 57.10; H, 3.49; N, 11.84.

10

ACCEPTED MANUSCRIPT

6

.1.6. Ethyl 3-amino-2-pyrrol-1-ylbenzoate (10)

To a solution of ethyl 3-nitro-2-pyrrol1-ylbenzoate 8 (8 mmol) in ethanol (40 mL) was added 6.5 mL of a 2 M aqueous

solution of CuSO . Sodium borohydride (40 mmol) was added portion-wise at 0°C to the reaction mixture which was then stirred

4

at room temperature for 1 h. The reaction mixture was then diluted with ethyl acetate and filtered. The organic layer was

separated, dried over Na SO and evaporated to dryness under reduced pressure to give 10. Orange oil (90%). IR (KBr) 3380 and

2

4

1

3

2

1

290 (NH ), 1695 (C=O). H-NMR (CDCl ) δ: 7.29-7.21 (m, 2H, H-5 and H-6), 6.93 (dd, 1H, J=7.40 and 2.20 Hz, H-4), 6.70 (dd,

2 3

H, J=2.10 and 2.10 Hz, 2H-α), 6.35 (dd, 2H, J=2.10 and 2.10 Hz, 2H-β), 4.10 (q, 2H, J=7.20 Hz, OCH

2 2

), 3.73 (bs, 2H, NH ),

.11 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C H N O : C, 67.81; H, 6.13; N, 12.17. Found: C, 68.03; H, 6.27; N, 12.30.

3

13 14

2

6

.1.7. General procedure for ethyl 4-oxo-5H-pyrrolo[1,2-a]quinoxaline-carboxylates (2b-d) and ethyl 4-oxo-2-(3-pyridyl)-5H-

pyrrolo[1,2-a]quinoxaline-7-carboxylate (2f)

A suspension of 4 or 6a,b or 23 (12.3 mmol) and iron powder (49.1 mmol) in 55 mL of acetic acid was heated under

reflux for 2 h. The reaction mixture was cooled, suspended in 150 mL of a 1 M aqueous solution of HCl, agitated, then filtered

2

off, washed with HCl 1 M (80 mL), water, Et O and dried to give a fluffy white solid.

6

.1.7.1. Ethyl 4-oxo-5H-pyrrolo[1,2-a]quinoxaline-3-carboxylate (2b)

1

Beige crystals (57%); mp 225°C. IR (KBr) 3200-2800 (CONH lactame), 1725 (COO), 1670 (CON). H-NMR (DMSO-

d

6

) δ: 11.46 (s, 1H, NH), 8.25 (d, 1H, J=2.85 Hz, H-1), 8.12 (d, 1H, J=8.10 Hz, H-9), 7.35-7.30 (m, 2H, H-6 and H-8), 7.26-7.21

(

m, 1H, H-7), 6.96 (d, 1H, J=2.85 Hz, H-2), 4.26 (q, 2H, J=7.20 Hz, OCH

2

), 1.30 (t, 3H, J=7.20 Hz, CH

3

). Anal. Calcd. for

C H N O : C, 65.62; H, 4.72; N, 10.93. Found: C, 65.49; H, 4.78; N, 11.04.

1

4

12

2

3

6

.1.7.2. Ethyl 4-oxo-5H-pyrrolo[1,2-a]quinoxaline-7-carboxylate (2c)

1

White crystals (92%); mp 251°C. IR (KBr) 3200-2800 (CONH lactame), 1710 (COO), 1650 (CON). H-NMR (DMSO-

d₆) δ: 11.42 (s, 1H, NH), 8.25 (d, 1H, J=1.95 Hz, H-6), 8.15 (d, 1H, J=8.20 Hz, H-9), 7.94-7.92 (m, 1H, H-1), 7.74 (dd, 1H,

J=8.20 and 1.95 Hz, H-8), 7.09-7.07 (m, 1H, H-3), 6.76-6.73 (m, 1H, H-2), 4.34 (q, 2H, J=7.20 Hz, OCH

2

), 1.33 (t, 3H, J=7.20

Hz, CH ). Anal. Calcd. for C14 : C, 65.62; H, 4.72; N, 10.93. Found: C, 65.74; H, 4.58; N, 11.09.

3

12 2 3

H N O

6

.1.7.3. Ethyl 4-oxo-5H-pyrrolo[1,2-a]quinoxaline-8-carboxylate (2d)

1

Beige crystals (28%); mp > 275°C. IR (KBr) 3200-2800 (CONH lactame), 1710 (COO), 1650 (CON). H-NMR (DMSO-

d

6

) δ: 11.60 (s, 1H, NH), 8.53 (d, 1H, J=1.80 Hz, H-9), 8.38 (dd, 1H, J=2.90 and 1.90 Hz, H-1), 7.87 (dd, 1H, J=8.40 and 1.80 Hz,

H-7), 7.38 (d, 1H, J=8.40 Hz, H-6), 7.08 (dd, 1H, J=3.95 and 1.90 Hz, H-3), 6.71 (dd, 1H, J=3.95 and 2.90 Hz, H-2) 4.35 (q, 2H,

J=6.90 Hz, OCH ), 1.36 (t, 3H, J=6.90 Hz, CH ). Anal. Calcd. for C14 : C, 65.62; H, 4.72; N, 10.93. Found: C, 65.51; H,

2

3

12 2 3

H N O

4

6

d

.80; N, 11.15.

.1.7.4. Ethyl 4-oxo-2-(3-pyridyl)-5H-pyrrolo[1,2-a]quinoxaline-7-carboxylate (2f)

1

Beige crystals (92%); mp > 270°C. IR (KBr) 3400-3150 (CONH lactame), 1710 (COO), 1685 (CON). H-NMR (DMSO-

6

) δ: 11.58 (s, 1H, NH), 9.34-9.33 (m, 1H, H-1), 9.11 (d, 1H, J=1.80 Hz, H-6), 8.76-8.73 (m, 2H, H-6’ and H-4’), 8.16 (d, 1H,

J=8.40 Hz, H-9), 7.97-7.93 (m, 2H, H-8 and H-2’), 7.85 (dd, 1H, J=7.90 and 5.00 Hz, H-5’), 7.81-7.80 (m, 1H, H-3), 4.36 (q, 2H,

J=6.90 Hz, OCH ), 1.35 (t, 3H, J=6.90 Hz, CH ). Anal. Calcd. for C19 : C, 68.46; H, 4.54; N, 12.61. Found: C, 68.33; H,

2

3

15 3 3

H N O

4

.72; N, 12.78.

6

.1.8. Ethyl 4-oxo-5H-pyrrolo[1,2-a]quinoxaline-9-carboxylate (2e)

11

ACCEPTED MANUSCRIPT

To a solution of compound 10 (7 mmol) in toluene (30 mL) was added triphosgene (2.34 mmol). The reaction mixture

was refluxed for 4 h, and nitrogen was bubbled in to drive off excess of phosgene. The solution was then set aside for 10 min. The

heavy crystalline precipitate was filtered off and washed with diethyl ether to give 2e. White crystals (75%); mp 243°C. IR (KBr)

1

3

1

400-3950 (CONH lactame), 1720 (COO), 1660 (CON). H-NMR (DMSO-d ) δ: 11.52 (s, 1H, NH), 7.47 (dd, 1H, J=3.00 and

6

.20 Hz, H-1), 7.45 (dd, 1H, J=7.80 and 1.80 Hz, H-6), 7.37 (t, 1H, J=7.80 Hz, H-7), 7.30 (dd, 1H, J=7.80 and 1.80 Hz, H-8), 7.11

(

2

dd, 1H, J=3.90 and 1.20 Hz, H-3), 6.70 (dd, 1H, J=3.90 and 3.00 Hz, H-2), 4.46 (q, 2H, J=7.10 Hz, OCH ), 1.33 (t, 3H, J=7.10

3 12 2 3

Hz, CH ). Anal. Calcd. for C14H N O : C, 65.62; H, 4.72; N, 10.93. Found: C, 65.83; H, 4.85; N, 11.10.

6

.1.9. General procedure for ethyl 4-chloropyrrolo[1,2-a]quinoxaline-carboxylates (11b-e) and ethyl 4-chloro-2-(3-

pyridyl)pyrrolo[1,2-a]quinoxaline-7-carboxylate (11f)

3

A solution of 5H-pyrrolo[1,2-a]quinoxalin-4-one 2a-f (4 mmol) in POCl (8 mL) was refluxed for 2 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 sodium carbonate. The precipitate was filtered, dried and recrystallized from ethyl acetate to give 11.

6

.1.9.1. Ethyl 4-chloropyrrolo[1,2-a]quinoxaline-3-carboxylate (11b)

1

White crystals (89%); mp 100°C. IR (KBr) 1710 (COO). H-NMR (CDCl

3

) δ: 7.94 (d, 1H, J=2.55 Hz, H-1), 7.92 (d, 1H,

J=8.10 Hz, H-9), 7.86 (d, 1H, J=8.10 Hz, H-6), 7.62 (t, 1H, J=8.10 Hz, H-8), 7.52 (t, 1H, J=8.10 Hz, H-7), 7.25 (d, 1H, J=2.55

Hz, H-2), 4.44 (q, 2H, J=7.20 Hz, OCH ), 1.45 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C14 : C, 61.21; H, 4.04; N,

0.20. Found: C, 61.13; H, 3.82; N, 10.28.

2

3

2 2

H11ClN O

1

6

1

.1.9.2. Ethyl 4-chloropyrrolo[1,2-a]quinoxaline-7-carboxylate (11c)

1

White crystals (82%); mp 137°C. IR (KBr) 1715 (COO). H-NMR (CDCl

3

) δ: 8.64 (d, 1H, J=2.00 Hz, H-6), 8.24 (dd,

H, J=8.10 and 2.00 Hz, H-8), 8.05-8.04 (m, 1H, H-1), 7.89 (d, 1H, J=8.10 Hz, H-9), 7.14-7.12 (m, 1H, H-3), 6.99-6.96 (m, 1H,

2 3 2 2

H-2), 4.45 (q, 2H, J=7.20 Hz, OCH ), 1.45 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C14H11ClN O : C, 61.21; H, 4.04; N, 10.20.

Found: C, 61.34; H, 3.92; N, 10.36.

6

.1.9.3. Ethyl 4-chloropyrrolo[1,2-a]quinoxaline-8-carboxylate (11d)

1

White crystals (80%); mp 161°C. IR (KBr) 1705 (COO). H-NMR (CDCl

m, 1H, H-7), 7.95 (d, 1H, J=8.70 Hz, H-6), 7.13 (dd, 1H, J=3.90 and 1.90 Hz, H-3), 6.96 (dd, 1H, J=3.90 and 3.00 Hz, H-2), 4.48

q, 2H, J=7.20 Hz, OCH ), 1.48 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C14 : C, 61.21; H, 4.04; N, 10.20. Found: C,

1.09; H, 3.87; N, 10.32.

3

) δ: 8.57 (d, 1H, J=1.80 Hz, H-9), 8.14-8.11

(

2

3

2 2

H11ClN O

6

7

.1.9.4. Ethyl 4-chloropyrrolo[1,2-a]quinoxaline-9-carboxylate (11e)

1

White crystals (80%); mp 84°C. IR (KBr) 1695 (COO). H-NMR (CDCl ) δ: 8.02 (dd, 1H, J=7.80 and 1.50 Hz, H-6),

3

.85 (dd, 1H, J=3.00 and 1.20 Hz, H-1), 7.69 (dd, 1H, J=7.80 and 1.50 Hz, H-8), 7.48 (t, 1H, J=7.80 Hz, H-7), 7.13 (dd, 1H,

J=3.90 and 1.20 Hz, H-3), 6.89 (dd, 1H, J=3.90 and 3.00 Hz, H-2), 4.55 (q, 2H, J=6.90 Hz, OCH

Anal. Calcd. for C14 : C, 61.21; H, 4.04; N, 10.20. Found: C, 61.44; H, 4.19; N, 10.15.

2 3

), 1.46 (t, 3H, J=6.90 Hz, CH ).

2 2

H11ClN O

6

.1.9.5. Ethyl 4-chloro-2-(3-pyridyl)pyrrolo[1,2-a]quinoxaline-7-carboxylate (11f)

1

White crystals (68%); mp 259°C. IR (KBr) 1710 (COO). H-NMR (DMSO-d

H, J=1.80 Hz, H-6), 8.61-8.58 (m, 1H, H-4’), 8.47-8.43 (m, 2H, H-9 and H-6’), 8.36 (d, 1H, J=1.80 Hz, H-2’), 8.23 (dd, 1H,

), 1.38 (t, 3H,

6

) δ: 9.40 (d, 1H, J=1.20 Hz, H-1), 9.23 (d,

1

J=8.40 and 1.80 Hz, H-8), 7.78 (d, 1H, J=1.20 Hz, H-3), 7.65-7.60 (m, 1H, H-5’), 4.39 (q, 2H, J=6.90 Hz, OCH

2

J=6.90 Hz, CH ). Anal. Calcd. for C19 : C, 64.87; H, 4.01; N, 11.94. Found: C, 65.03; H, 4.08; N, 12.14.

3

3 2

H14ClN O

12

ACCEPTED MANUSCRIPT

6

.1.10. General procedure for ethyl 4-[(3-substituted-phenyl)amino]pyrrolo[1,2-a]quinoxaline-carboxylates 12a-j and ethyl 7-

[

(3-substituted-phenyl)amino]pyrrolo[1,2-a]quinoxaline-4-carboxylates 12k,l and ethyl 4-[(3-substituted-phenyl)amino]-2-(3-

pyridyl)pyrrolo[1,2-a]quinoxaline-7-carboxylates 12m,n

Method A: Ethyl 4-chloropyrrolo[1,2-a]quinoxaline-carboxylates 11b-e or ethyl 4-chloro-2-(3-pyridyl)pyrrolo[1,2-

a]quinoxaline-7-carboxylate (11f) (1.5 mmol), 3-substituted aniline (1.95 mmol), and DMF (10 mL) were mixed and heated at

reflux for 2 h 30. The reaction mixture was then diluted with ethyl acetate. The organic layer was washed with water then with

brine, separated, dried over Na

ether, filtered and then purified by column chromatography using AcOEt/cyclohexane (3/7, v/v) as eluent to give 12a-j and

2m,n. Method B: A mixture of 4-chloropyrrolo[1,2-a]quinoxalines 11a/c/e/g/i/m or 7-bromopyrrolo[1,2-a]quinoxaline (16) (1

mmol), 3-substituted aniline (1.2 mmol), Cs CO (1.4 mmol), Pd(OAc) (0.05 mmol) and BINAP (0.075 mmol) in 5 mL of

2 4

SO and evaporated to dryness under reduced pressure. The crude residue was triturated in diethyl

1

2

3

2

toluene was heated at 100°C for 20 h. The reaction mixture was allowed to cool to room temperature, diluted with

dichloromethane and filtered through a pad of Celite and the filter cake was washed with dichloromethane (x3). Concentration and

flash chromatography on silica gel using AcOEt/cyclohexane (3/7, v/v) as eluent afforded 12a/c/e/g/i/m and 12k,l, respectively.

6

.1.10.1. Ethyl 4-[(3-chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-2-carboxylate (12a)

White crystals (method A: 44%; method B: 51%); mp 183°C. IR (KBr) 3390 (NH), 1690 (COO). H-NMR (DMSO-d

δ: 9.45 (s, 1H, NH), 8.93 (d, 1H, J=1.50 Hz, H-1), 8.35 (d, 1H, J=1.80 Hz, H-2’), 8.32 (dd, 1H, J=8.00 and 1.40 Hz, H-9), 8.06

dd, 1H, J=8.20 and 1.65 Hz, H-6’), 7.88 (d, 1H, J=1.50 Hz, H-3), 7.64 (dd, 1H, J=8.00 and 1.40 Hz, H-6), 7.46-7.33 (m, 3H, H-7,

H-8 and H-5’), 7.11-7.07 (m, 1H, H-4’), 4.36 (q, 2H, J=7.20 Hz, OCH ), 1.37 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for

: C, 65.67; H, 4.41; N, 11.49. Found: C, 65.78; H, 4.47; N, 11.62.

1

6

)

(

2

3

20 3 2

C H16ClN O

6

.1.10.2. Ethyl 4-[(3-ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-2-carboxylate (12b)

White crystals (method A: 62%); mp 161°C. IR (KBr) 3395 (NH), 2105 (C≡C), 1685 (COO). H-NMR (CDCl

s, 1H, NH), 8.05 (d, 1H, J=1.40 Hz, H-1), 7.91 (dd, 1H, J=7.90 and 1.50 Hz, H-9), 7.79-7.76 (m, 2H, H-2’ and H-6’), 7.44-7.32

1

3

) δ: 8.37

(

m, 3H, H-6, H-7 and H-8), 7.26-7.22 (m, 2H, H-3 and H-5’), 6.93-6.91 (m, 1H, H-4’), 4.39 (q, 2H, J=7.20 Hz, OCH

2

), 3.11 (s,

1

3 17 3 2

H, C≡CH), 1.42 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C22H N O : C, 74.35; H, 4.82; N, 11.82. Found: C, 74.50; H, 4.97; N,

1.76.

6

.1.10.3. Ethyl 4-[(3-chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-3-carboxylate (12c)

1

Pale-yellow crystals (method A: 45%; method B: 48%); mp 146°C. IR (KBr) 3410 (NH), 1690 (COO). H-NMR

(

DMSO-d

6

) δ: 12.19 (s, 1H, NH), 8.49 (d, 1H, J=3.30 Hz, H-1), 8.35 (d, 1H, J=2.10 Hz, H-2’), 8.27 (dd, 1H, J=8.10 and 1.20 Hz,

H-9), 7.77-7.73 (m, 1H, H-6’), 7.69 (dd, 1H, J=8.10 and 1.20 Hz, H-6), 7.52-7.44 (m, 2H, H-7 and H-8), 7.42 (t, 1H, J=8.10 Hz,

H-5’), 7.33 (d, 1H, J=3.30 Hz, H-2), 7.14-7.10 (m, 1H, H-4’), 4.42 (q, 2H, J=7.20 Hz, OCH

Calcd. for C20 : C, 65.67; H, 4.41; N, 11.49. Found: C, 65.48; H, 4.32; N, 11.53.

2

3

), 1.39 (t, 3H, J=7.20 Hz, CH ). Anal.

3 2

H16ClN O

6

.1.10.4. Ethyl 4-[(3-ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-3-carboxylate (12d)

1

Yellow crystals (method A: 52%); mp 122°C. IR (KBr) 3390 (NH), 2100 (C≡C), 1695 (COO). H-NMR (DMSO-d

6

) δ:

1

6

4

2.07 (s, 1H, NH), 8.44 (d, 1H, J=3.10 Hz, H-1), 8.25-8.17 (m, 2H, H-2’ and H-9), 8.03-8.00 (m, 1H, H-6’), 7.70-7.66 (m, 1H, H-

), 7.51-7.40 (m, 3H, H-5’, H-7 and H-8), 7.31 (d, 1H, J=3.10 Hz, H-2), 7.18-7.16 (m, 1H, H-4’), 4.47 (q, 2H, J=6.90 Hz, OCH₂),

.17 (s, 1H, C≡CH), 1.40 (t, 3H, J=6.90 Hz, CH ). Anal. Calcd. for C H N O : C, 74.35; H, 4.82; N, 11.82. Found: C, 74.17; H,

3

22 17

3

2

.65; N, 11.91.

13

ACCEPTED MANUSCRIPT

6

.1.10.5. Ethyl 4-[(3-chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-7-carboxylate (12e)

1

White crystals (method A: 74%; method B: 69%); mp 236°C. IR (KBr) 3375 (NH), 1685 (COO). H-NMR (DMSO-d

δ: 9.41 (s, 1H, NH), 8.41 (dd, 1H, J=3.00 and 1.20 Hz, H-1), 8.27 (d, 1H, J=1.50 Hz, H-6), 8.23 (d, 1H, J=8.20 Hz, H-9), 8.13 (d,

6

)

1

4

H, J=1.80 Hz, H-2’), 8.05 (dd, 1H, J=8.20 and 1.50 Hz, H-8), 7.87 (dd, 1H, J=8.20 and 1.60 Hz, H-6’), 7.45 (dd, 1H, J=3.60 and

.20 Hz, H-3), 7.41 (t, 1H, J=8.20 Hz, H-5’), 7.10 (dd, 1H, J=8.20 and 1.80 Hz, H-4’), 6.92 (dd, 1H, J=3.60 and 3.00 Hz, H-2),

.37 (q, 2H, J=7.20 Hz, OCH

2

), 1.37 (t, 3H, J=7.20 Hz, CH

3

). Anal. Calcd. for C20

H16ClN

3

O

2

: C, 65.67; H, 4.41; N, 11.49. Found:

C, 65.60; H, 4.54; N, 11.82.

6

.1.10.6. Ethyl 4-[(3-ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-7-carboxylate (12f)

1

White crystals (method A: 78%); mp 183°C. IR (KBr) 3380 (NH), 2105 (C≡C), 1695 (COO). H-NMR (DMSO-d

6

) δ:

9

1

.38 (s, 1H, NH), 8.43 (dd, 1H, J=3.00 and 1.20 Hz, H-1), 8.25 (d, 1H, J=8.70 Hz, H-9), 8.21-8.18 (m, 2H, H-6 and H-8), 8.13 (d,

H, J=1.80 Hz, H-2’), 7.87 (dd, 1H, J=8.40 and 1.80 Hz, H-6’), 7.47 (dd, 1H, J=3.90 and 1.20 Hz, H-3), 7.42 (t, 1H, J=8.400 Hz,

H-5’), 7.17 (dd, 1H, J=8.40 and 1.80 Hz, H-4’), 6.92 (dd, 1H, J=3.90 and 3.00 Hz, H-2), 4.37 (q, 2H, J=7.20 Hz, OCH

2

), 4.02 (s,

1

H, C≡CH), 1.37 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C22H N O : C, 74.35; H, 4.82; N, 11.82. Found: C, 74.21; H, 4.99; N,

3 17 3 2

2.10.

6

.1.10.7. Ethyl 4-[(3-chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-8-carboxylate (12g)

Beige crystals (method A: 69%; method B: 76%); mp 191°C. IR (KBr) 3400 (NH), 1685 (COO). H-NMR (DMSO-d

.53 (s, 1H, NH), 8.63 (d, 1H, J=1.80 Hz, H-9), 8.53 (dd, 1H, J=3.00 and 1.20 Hz, H-1), 8.32 (t, 1H, J=1.90 Hz, H-2’), 8.04-8.01

1

6

) δ:

9

(

m, 1H, H-7), 7.91 (dd, 1H, J=8.20 and 1.60 Hz, H-6’), 7.66 (d, 1H, J=8.40 Hz, H-6), 7.48 (dd, 1H, J=3.90 and 1.20 Hz, H-3),

7

1

.41 (t, 1H, J=8.20 Hz, H-5’), 7.13-7.10 (m, 1H, H-4’), 6.88 (dd, 1H, J=3.90 and 3.00 Hz, H-2), 4.37 (q, 2H, J=7.10 Hz, OCH

2

),

.38 (t, 3H, J=7.10 Hz, CH ). Anal. Calcd. for C20 : C, 65.67; H, 4.41; N, 11.49. Found: C, 65.42; H, 4.29; N, 11.53.

3

3 2

H16ClN O

6

.1.10.8. Ethyl 4-[(3-ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-8-carboxylate (12h)

Beige crystals (method A: 70%); mp 206°C. IR (KBr) 3385 (NH), 2100 (C≡C), 1685 (COO). H-NMR (DMSO-d

1

6

) δ:

9

1

2

1

.48 (s, 1H, NH), 8.63-8.61 (m, 1H, H-9), 8.53-8.52 (m, 1H, H-1), 8.24-8.23 (m, 1H, H-2’), 8.19-8.13 (m, 1H, H-7), 7.92-7.89 (m,

H, H-6’), 7.66-7.62 (m, 1H, H-6), 7.46-7.38 (m, 2H, H-3 and H-5’), 7.20-7.17 (m, 1H, H-4’), 6.88-6.86 (m, 1H, H-2), 4.36 (q,

H, J=7.10 Hz, OCH ), 4.23 (s, 1H, C≡CH), 1.37 (t, 3H, J=7.10 Hz, CH ). Anal. Calcd. for C H N O : C, 74.35; H, 4.82; N,

2

3

22 17

3

2

1.82. Found: C, 74.46; H, 4.87; N, 12.06.

6

.1.10.9. Ethyl 4-[(3-chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-9-carboxylate (12i)

1

Beige crystals (method A: 75%; method B: 65%); mp 172°C. IR (KBr) 3390 (NH), 1690 (COO). H-NMR (DMSO-d ) δ:

6

9

.43 (s, 1H, NH), 8.31 (t, 1H, J=1.60 Hz, H-2’), 8.01 (dd, 1H, J=7.80 and 1.60 Hz, H-6’), 7.79-7.76 (m, 1H, H-6), 7.66 (dd, 1H,

J=3.00 and 1.20, H-1), 7.48 (dd, 1H, J=3.90 and 1.20 Hz, H-3), 7.45-7.43 (m, 2H, H-7 and H-8), 7.40 (t, 1H, J=7.80 Hz, H-5’),

.09 (dd, 1H, J=7.80 and 1.60 Hz, H-4’), 6.88 (dd, 1H, J=3.90 and 3.00 Hz, H-2), 4.48 (q, 2H, J=7.20 Hz, OCH ), 1.35 (t, 3H,

7

2

3 3 2

J=7.20 Hz, CH ). Anal. Calcd. for C20H16ClN O : C, 65.67; H, 4.41; N, 11.49. Found: C, 65.86; H, 4.39; N, 11.64.

6

.1.10.10. Ethyl 4-[(3-ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-9-carboxylate (12j)

1

Beige crystals (method A: 79%); mp 141°C. IR (KBr) 3390 (NH), 2105 (C≡C), 1685 (COO). H-NMR (DMSO-d ) δ:

6

9

1

.34 (s, 1H, NH), 8.21 (t, 1H, J=1.70 Hz, H-2’), 8.15 (d, 1H, J=8.00 Hz, H-6’), 7.77-7.73 (m, 1H, H-6), 7.65 (dd, 1H, J=3.00 and

.20, H-1), 7.48 (dd, 1H, J=3.90 and 1.20 Hz, H-3), 7.44-7.40 (m, 2H, H-7 and H-8), 7.39 (t, 1H, J=8.00 Hz, H-5’), 7.16 (d, 1H,

14

ACCEPTED MANUSCRIPT

2

J=8.00 Hz, H-4’), 6.87 (dd, 1H, J=3.90 and 3.00 Hz, H-2), 4.98 (q, 2H, J=7.20 Hz, OCH ), 4.19 (s, 1H, C≡CH), 1.35 (t, 3H,

J=7.20 Hz, CH ). Anal. Calcd. for C H N O : C, 74.35; H, 4.82; N, 11.82. Found: C, 74.54; H, 4.98; N, 12.14.

3

22 17

3

2

6

.1.10.11. Ethyl 7-[(3-chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-4-carboxylate (12k)

1

Orange crystals (method B: 37%); mp 115°C. IR (KBr) 3425 (NH), 1725 (COO). H-NMR (CDCl

3

) δ: 7.98-7.97 (m, 1H,

H-1), 7.85 (d, 1H, J=1.40 Hz, H-6), 7.83 (d, 1H, J=8.20 Hz, H-9), 7.53-7.52 (m, 1H, H-3), 7.36 (dd, 1H, J=8.20 and 1.40 Hz, H-

8

2

6

), 7.21 (t, 1H, J=8.10 Hz, H-5’), 7.11 (t, 1H, J=1.70 Hz, H-2’), 7.03-6.93 (m, 3H, H-4’, H-6’ and H-2), 5.98 (s, 1H, NH), 4.60 (q,

H, J=7.20 Hz, OCH ), 1.53 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C20 : C, 65.67; H, 4.41; N, 11.49. Found: C,

5.54; H, 4.65; N, 11.57.

2

3

3 2

H16ClN O

6

.1.10.12. Ethyl 7-{[3-(2-trimethylsilylethynyl)phenyl]amino}pyrrolo[1,2-a]quinoxaline-4-carboxylate (12l)

1

Orange crystals (method B: 12%); mp 59°C. IR (KBr) 3410 (NH), 2100 (C≡C), 1720 (COO). H-NMR (CDCl

3

) δ: 8.01

(

dd, 1H, J=2.70 and 1.20 Hz, H-1), 7.89-7.85 (m, 2H, H-6 and H-9), 7.57 (dd, 1H, J=3.90 and 1.20 Hz, H-3), 7.29 (dd, 1H, J=8.30

and 2.40 Hz, H-8), 7.22 (t, 1H, J=1.70 Hz, H-2’), 7.17 (t, 1H, J=7.80 Hz, H-5’), 7.10-7.04 (m, 2H, H-4’ and H-6’), 6.94 (dd, 1H,

J=3.90 and 2.70 Hz, H-2), 6.11 (s, 1H, NH), 4.58 (q, 2H, J=7.10 Hz, OCH ), 1.48 (t, 3H, J=7.10 Hz, CH ). Anal. Calcd. for

Si: C, 70.23; H, 5.89; N, 9.83. Found: C, 70.39; H, 6.02; N, 10.05.

2

3

25 25 3 2

C H N O

6

.1.10.13. Ethyl 4-[(3-chlorophenyl)amino]-2-(3-pyridyl)pyrrolo[1,2-a]quinoxaline-7-carboxylate (12m)

1

Beige crystals (method A: 61%; method B: 67%); mp 234°C. IR (KBr) 3380 (NH), 1710 (COO). H-NMR (DMSO-d

6

) δ:

9

.44 (s, 1H, NH), 9.03-8.99 (m, 2H, H-1 and H-6), 8.52-8.50 (m, 1H, H-4’), 8.27-8.17 (m, 2H, H-6’ and H-9), 8.10-8.03 (m, 3H,

H-2″, H-2’ and H-8), 7.90-7.84 (m, 2H, H-3 and H-6″), 7.52-7.50 (m, 1H, H-5’), 7.45-7.39 (m, 1H, H-5″), 7.10-7.08 (m, 1H, H-

″), 4.35 (q, 2H, J=7.20 Hz, OCH ), 1.37 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C25 : C, 67.80; H, 4.32; N, 12.65.

Found: C, 67.96; H, 4.44; N, 12.80.

4

2

3

4 2

H19ClN O

6

.1.10.14. Ethyl 4-[(3-ethynylphenyl)amino]-2-(3-pyridyl)pyrrolo[1,2-a]quinoxaline-7-carboxylate (12n)

1

6

Beige crystals (method A: 53%); mp 192°C. IR (KBr) 3285 (NH), 2100 (C≡C), 1700 (COO). H-NMR (DMSO-d ) δ:

9

7

.42 (s, 1H, NH), 9.05-9.04 (m, 2H, H-1 and H-6), 8.53-8.51 (m, 1H, H-4’), 8.26-8.11 (m, 5H, H-8, H-9, H-2’, H-6’ and H-2″),

.95-7.88 (m, 2H, H-3 and H-6″), 7.54-7.50 (m, 1H, H-5’), 7.46-7.41 (m, 1H, H-5″), 7.20-7.17 (m, 1H, H-4″), 4.38 (q, 2H, J=6.90

2 3 20 4 2

Hz, OCH ), 4.23 (s, 1H, C≡CH), 1.37 (t, 3H, J=6.90 Hz, CH ). Anal. Calcd. for C27H N O : C, 74.99; H, 4.66; N, 12.95. Found:

C, 75.13; H, 4.78; N, 12.81.

6

.1.11. General procedure for 4-[(3-substituted-phenyl)amino]pyrrolo[1,2-a]quinoxaline-carboxylic acids (1a-j) and ethyl 7-[(3-

substituted-phenyl)amino]pyrrolo[1,2-a]quinoxaline-4-carboxylic acids 1k,l and ethyl 4-[(3-substituted-phenyl)amino]-2-(3-

pyridyl)pyrrolo[1,2-a]quinoxaline-7-carboxylic acids 1m,n

A mixture of ethyl 4-[(3-substituted-phenyl)amino]pyrrolo[1,2-a]quinoxaline-carboxylates 12a-j or ethyl 7-[(3-

substituted-phenyl)amino]pyrrolo[1,2-a]quinoxaline-4-carboxylates 12k,l or ethyl 4-[(3-substituted-phenyl)amino]-2-(3-

pyridyl)pyrrolo[1,2-a]quinoxaline-7-carboxylates 12m,n (0.6 mmol) in methanol (15 mL) and a NaOH aqueous solution (4.8

mmol in 8 mL) was refluxed for 4 h. On cooling, after removal of the solvent under reduced pressure, the residue was diluted with

water and adjusted to pH = 6 with 1 M HCl aqueous solution. The solid precipitate was filtered off and washed with water then

2 2

with diethyl ether, and purified by column chromatography using CH Cl /methanol (9/1, v/v) as eluent to give 1a-n.

6

.1.11.1. 4-[(3-Chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-2-carboxylic acid (1a)

15

ACCEPTED MANUSCRIPT

Pale-yellow crystals (91%); mp > 300°C. IR (KBr) 3290 (NH), 3100-2650 (COOH), 1685 (COO). H-NMR (DMSO-d )

6

1

δ: 12.10 (bs, 1H, COOH), 9.43 (s, 1H, NH), 8.44-8.43 (m, 2H, H-1 and H-2’), 8.16-8.10 (m, 2H, H-9 and H-6’), 7.73 (d, 1H,

J=1.50 Hz, H-3), 7.59 (dd, 1H, J=7.90 and 1.35 Hz, H-6), 7.39-7.30 (m, 3H, H-7, H-8 and H-5’), 7.03 (dd, 1H, J=8.10 and 1.50

13

Hz, H-4’). C-NMR (DMSO-d

6

) δ: 168.1, 146.6, 142.0, 135.0, 132.2, 129.7, 129.5, 126.2, 125.0, 124.7, 123.6, 120.6, 118.6,

17.7, 117.5, 117.0, 113.8, 106.6. Anal. Calcd. for C18 : C, 64.01; H, 3.58; N, 12.44. Found: C, 63.85; H, 3.43; N,

2.77.

1

3 2

H12ClN O

6

.1.11.2. 4-[(3-Ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-2-carboxylic acid (1b)

Beige crystals (90%); mp > 300°C. IR (KBr) 3285 (NH), 3150-2600 (COOH), 2105 (C≡C), 1685 (COO). H-NMR

DMSO-d ) δ: 9.43 (s, 1H, NH), 8.58 (d, 1H, J=1.35 Hz, H-1), 8.35 (d, 1H, J=1.70 Hz, H-2’), 8.23 (d, 1H, J=7.80 Hz, H-9), 8.16

d, 1H, J=7.80 Hz, H-6’), 7.84 (d, 1H, J=1.35 Hz, H-3), 7.58 (d, 1H, J=7.80 Hz, H-6), 7.40-7.28 (m, 3H, H-7, H-8 and H-5’), 7.12

1

(

6

1

3

d, 1H, J=7.80 Hz, H-4’), 5.80 (bs, 1H, COOH), 4.19 (s, 1H, C≡CH). C-NMR (DMSO-d

28.3, 126.3, 125.6, 125.1, 124.7, 123.5, 122.4, 121.2, 120.2, 118.1, 117.9, 113.9, 106.5, 83.4, 79.6. Anal. Calcd. for C20

C, 73.38; H, 4.00; N, 12.84. Found: C, 73.18; H, 3.86; N, 12.70.

6

) δ: 172.9, 167.4, 146.5, 140.5, 135.3,

1

13 3 2

H N O :

6

.1.11.3. 4-[(3-Chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-3-carboxylic acid (1c)

1

Pale-yellow crystals (84%); mp 229°C. IR (KBr) 3300 (NH), 3100-2600 (COOH), 1685 (COO). H-NMR (DMSO-d ) δ:

6

1

5

1

3

3.60 (s, 1H, NH), 8.40 (d, 1H, J=3.00 Hz, H-1), 8.39 (t, 1H, J=2.10 Hz, H-2’), 8.24 (dd, 1H, J=7.90 and 1.50 Hz, H-9), 7.78 (dd,

H, J=8.10 and 1.10 Hz, H-6’), 7.66 (1H, J=7.90 and 1.50 Hz, H-6), 7.47-7.37 (m, 2H, H-7 and H-8), 7.39 (t, 1H, J=8.10 Hz, H-

13

6

’), 7.27 (d, 1H, J=3.00 Hz, H-2), 7.09-7.06 (m, 1H, H-4’). C-NMR (DMSO-d ) δ: 167.9, 145.6, 141.6, 135.2, 132.7, 129.9,

29.8, 126.0, 125.9, 123.9, 123.7, 120.9, 119.3, 118.0, 117.0, 116.4, 115.3, 114.4. Anal. Calcd. for C18

.58; N, 12.44. Found: C, 63.94; H, 3.63; N, 12.67.

H12ClN

3

O

2

: C, 64.01; H,

6

.1.11.4. 4-[(3-Ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-3-carboxylic acid (1d)

1

Yellow crystals (81%); mp 210°C. IR (KBr) 3280 (NH), 3100-2650 (COOH), 2100 (C≡C), 1685 (COO). H-NMR

(

DMSO-d

6

) δ: 13.73 (s, 1H, NH), 8.34 (d, 1H, J=3.00 Hz, H-1), 8.23 (t, 1H, J=1.90 Hz, H-2’), 8.18 (dd, 1H, J=7.80 and 1.50 Hz,

H-9), 8.03 (dd, 1H, J=8.10 and 1.10 Hz, H-6’), 7.64 (dd, 1H, J=7.80 and 1.50 Hz, H-6), 7.43-7.35 (m, 3H, H-7, H-8 and H-5’),

1

3

7

1

.26 (d, 1H, J=3.00 Hz, H-2), 7.14-7.11 (m, 1H, H-4’), 4.14 (s, 1H, C≡CH). C-NMR (DMSO-d

28.8, 128.7, 125.9, 125.8, 124.5, 123.8, 123.5, 121.6, 121.4, 119.2, 119.1, 116.4, 115.0, 114.3, 83.3, 79.8. Anal. Calcd. for

: C, 73.38; H, 4.00; N, 12.84. Found: C, 73.54; H, 3.85; N, 12.98.

6

) δ: 167.9, 145.8, 140.5, 135.4,

20 13 3 2

C H N O

6

.1.11.5. 4-[(3-Chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-7-carboxylic acid (1e)

Beige crystals (96%); mp > 300°C. IR (KBr) 3425 (NH), 3150-2600 (COOH), 1700 (COO). H-NMR (DMSO-d

2.85 (bs, 1H, COOH), 9.47 (s, 1H, NH), 8.41-8.38 (m, 2H, H-1 and H-6), 8.19 (d, 1H, J=8.70 Hz, H-9), 8.16 (d, 1H, J=1.50 Hz,

1

6

) δ:

1

H-2’), 8.06 (dd, 1H, J=8.70 and 1.60 Hz, H-8), 7.88 (dd, 1H, J=8.70 and 1.50 Hz, H-6’), 7.50 (dd, 1H, J=3.65 and 1.20 Hz, H-3),

13

7

.40 (t, 1H, J=8.70 Hz, H-5’), 7.08 (dd, 1H, J=8.70 and 1.50 Hz, H-4’), 6.90 (dd, 1H, J=3.65 and 3.00 Hz, H-2). C-NMR

(

DMSO-d ) δ: 167.2, 146.3, 141.5, 134.4, 132.3, 129.6, 129.1, 127.4, 127.3, 124.3, 121.1, 118.9, 118.4, 117.9, 116.5, 113.7,

6

1

3 2

12.9, 105.4. Anal. Calcd. for C18H12ClN O : C, 64.01; H, 3.58; N, 12.44. Found: C, 64.28; H, 3.70; N, 12.63.

6

.1.11.6. 4-[(3-Ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-7-carboxylic acid (1f)

Beige crystals (65%); mp 280°C. IR (KBr) 3400 (NH), 3250-2500 (COOH), 2100 (C≡C), 1685 (COO). H-NMR

DMSO-d ) δ: 13.07 (s, 1H, COOH), 9.36 (s, 1H, NH), 8.43-8.41 (m, 1H, H-1), 8.28-8.15 (m, 3H, H-9, H-6 and H-8), 8.13 (d, 1H,

1

(

6

16

ACCEPTED MANUSCRIPT

J=1.70 Hz, H-2’), 7.86 (dd, 1H, J=8.30 and 1.70 Hz, H-6’), 7.46 (dd, 1H, J=3.80 and 1.20 Hz, H-3), 7.41 (t, 1H, J=8.30 Hz, H-5’),

1

3

7

1

7

.16 (dd, 1H, J=8.30 and 1.70 Hz, H-4’), 6.91 (dd, 1H, J=3.80 and 2.90 Hz, H-2), 4.22 (s, 1H, C≡CH). C-NMR (DMSO-d

66.6, 146.5, 140.1, 134.7, 128.4, 127.8, 127.4, 127.3, 124.8, 124.1, 122.6, 121.2, 120.4, 118.4, 116.5, 113.9, 113.0, 105.4, 83.2,

9.7. Anal. Calcd. for C20 : C, 73.38; H, 4.00; N, 12.84. Found: C, 73.51; H, 3.93; N, 12.99.

6

) δ:

13 3 2

H N O

6

.1.11.7. 4-[(3-Chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-8-carboxylic acid (1g)

Beige crystals (81%); mp > 300°C. IR (KBr) 3420 (NH), 3100-2600 (COOH), 1690 (COO). H-NMR (DMSO-d

1

6

) δ:

9

7

1

3

1

.50 (s, 1H, NH), 8.61 (d, 1H, J=1.70 Hz, H-9), 8.49-8.47 (m, 1H, H-1), 8.34 (t, 1H, J=1.80 Hz, H-2’), 8.03 (d, 1H, J=8.40 Hz, H-

), 8.03 (d, 1H, J=8.40 Hz, H-7), 7.91 (dd, 1H, J=8.10 and 1.80 Hz, H-6’), 7.62 (d, 1H, J=8.40 Hz, H-6), 7.46 (dd, 1H, J=3.90 and

.10 Hz, H-3), 7.40 (t, 1H, J=8.10 Hz, H-5’), 7.10 (dd, 1H, J=8.10 and 1.80 Hz, H-4’), 6.87 (dd, 1H, J=3.90 and 3.00 Hz, H-2),

1

3

.39 (bs, 1H, COOH). C-NMR (DMSO-d

6

) δ: 166.7, 147.1, 141.3, 138.1, 132.3, 129.6, 129.5, 125.9, 125.7, 124.4, 121.4, 119.3,

: C, 64.01; H, 3.58; N, 12.44. Found: C, 63.86; H, 3.67;

18.3, 118.1, 116.6, 114.8, 112.6, 105.4. Anal. Calcd. for C18

3 2

H12ClN O

N, 12.28.

6

.1.11.8. 4-[(3-Ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-8-carboxylic acid (1h)

White crystals (76%); mp > 300°C. IR (KBr) 3390 (NH), 3150-2600 (COOH), 2100 (C≡C), 1690 (COO). H-NMR

DMSO-d ) δ: 9.43 (s, 1H, NH), 8.62 (d, 1H, J=1.80 Hz, H-9), 8.42-8.41 (m, 1H, H-1), 8.29 (t, 1H, J=1.75 Hz, H-2’), 8.19 (d, 1H,

J=8.20 Hz, H-7), 7.93 (d, 1H, J=8.40 Hz, H-6’), 7.58 (d, 1H, J=8.20 Hz, H-6), 7.54 (dd, 1H, J=3.80 and 1.20 Hz, H-3), 7.38 (t,

H, J=8.40 Hz, H-5’), 7.15 (d, 1H, J=8.40 Hz, H-4’), 6.84 (dd, 1H, J=3.80 and 3.00 Hz, H-2), 4.21 (s, 1H, C≡CH), 3.61 (bs, 1H,

1

(

6

1

3

COOH). C-NMR (DMSO-d

6

) δ: 168.0, 147.0, 140.1, 137.8, 128.5, 128.3, 125.7, 125.6, 124.9, 124.2, 122.8, 121.2, 120.6, 118.2,

16.2, 114.7, 112.5, 105.4, 83.2, 79.7. Anal. Calcd. for C20 : C, 73.38; H, 4.00; N, 12.84. Found: C, 73.44; H, 3.95; N,

2.80.

1

13 3 2

H N O

6

.1.11.9. 4-[(3-Chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-9-carboxylic acid (1i)

Beige crystals (52%); mp > 300°C. IR (KBr) 3410 (NH), 3100-2600 (COOH), 1690 (COO). H-NMR (DMSO-d

.38 (s, 1H, NH), 8.31 (t, 1H, J=1.80 Hz, H-2’), 8.05-8.00 (m, 1H, H-6’), 7.92 (dd, 1H, J=2.90 and 1.20 Hz, H-1), 7.66 (dd, 1H,

1

6

) δ:

9

J=7.50 and 1.65 Hz, H-6), 7.47 (dd, 1H, J=4.05 and 1.20 Hz, H-3), 7.40-7.32 (m, 3H, H-7, H-8 and H-5’), 7.08 (dd, 1H, J=7.50

1

3

and 1.65 Hz, H-4’), 6.86 (dd, 1H, J=4.05 and 2.90 Hz, H-2), 3.43 (bs, 1H, COOH). C-NMR (DMSO-d

6

) δ: 171.1, 147.2, 142.5,

1

37.0, 133.3, 130.6, 129.1, 125.3, 124.5, 124.3, 122.2, 121.9, 120.1, 120.0, 119.7, 119.0, 133.3, 105.8. Anal. Calcd. for

C H ClN O : C, 64.01; H, 3.58; N, 12.44. Found: C, 63.98; H, 3.62; N, 12.37.

1

8

12

3

2

6

.1.11.10. 4-[(3-Ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-9-carboxylic acid (1j)

1

Beige crystals (75%); mp 214°C. IR (KBr) 3385 (NH), 3150-2650 (COOH), 2100 (C≡C), 1690 (COO). H-NMR

(

6

DMSO-d ) δ: 9.20 (s, 1H, NH), 8.38 (dd, 1H, J=2.95 and 1.20 Hz, H-1), 8.27 (t, 1H, J=1.70 Hz, H-2’), 8.18 (dd, 1H, J=7.40 and

1

.50 Hz, H-6’), 7.46-7.40 (m, 2H, H-3 and H-6), 7.36 (t, 1H, J=7.95 Hz, H-7), 7.23 (t, 1H, J=7.65 Hz, H-5’), 7.12-7.10 (m, 1H, H-

8

), 7.07 (dd, 1H, J=7.40 and 1.50 Hz, H-4’), 6.69 (dd, 1H, J=3.90 and 2.95 Hz, H-2), 4.19 (s, 1H, C≡CH), 3.50 (bs, 1H, COOH).

1

3

C-NMR (DMSO-d

6

) δ: 173.0, 146.8, 141.6, 136.6, 133.0, 129.3, 125.4, 125.3, 124.9, 123.3, 122.8, 122.2, 121.1, 120.8, 120.6,

: C, 73.38; H, 4.00; N, 12.84. Found: C, 73.25; H, 3.85; N, 12.93.

1

19.6, 111.9, 105.0, 84.4, 80.6. Anal. Calcd. for C20H N O

13 3 2

6

.1.11.11. 7-[(3-Chlorophenyl)amino]pyrrolo[1,2-a]quinoxaline-4-carboxylic acid (1k)

Red crystals (56%); mp 195°C. IR (KBr) 3405 (NH), 3300-2700 (COOH), 1675 (COO). H-NMR (DMSO-d

H, NH), 8.48-8.47 (m, 1H, H-1), 8.27 (d, 1H, J=6.70 Hz, H-9), 7.66 (d, 1H, J=1.80 Hz, H-6), 7.43 (dd, 1H, J=6.70 and 1.20 Hz,

1

6

) δ: 8.75 (s,

1

17

ACCEPTED MANUSCRIPT

H-8), 7.38-7.36 (m, 1H, H-3), 7.29 (t, 1H, J=6.10 Hz, H-5’), 7.13-7.09 (m, 2H, H-4’ and H-2’), 6.98-6.96 (m, 1H, H-2), 6.89 (d,

1

3

1

H, J=6.10 Hz, H-6’), 3.43 (bs, 1H, COOH). C-NMR (DMSO-d

6

) δ: 165.9, 149.7, 145.0, 139.2, 135.1, 133.1, 130.3, 123.7,

21.6, 119.9, 118.4, 116.2, 115.0, 114.8, 114.6, 113.7, 113.3, 109.2. Anal. Calcd. for C18

H12ClN

3

O

2

: C, 64.01; H, 3.58; N, 12.44.

Found: C, 63.82; H, 3.62; N, 12.40.

6

.1.11.12. 7-[(3-Ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-4-carboxylic acid (1l)

1

Orange crystals (73%); mp > 300°C. IR (KBr) 3355 (NH), 3400-2850 (COOH), 2100 (C≡C), 1630 (COO). H-NMR

(

DMSO-d

6

) δ: 8.49 (s, 1H, NH), 8.26-8.25 (m, 1H, H-1), 8.12 (d, 1H, J=8.70 Hz, H-9), 7.73 (d, 1H, J=2.00 Hz, H-6), 7.29-7.14

m, 5H, H-3, H-2’, H-4’, H-5’ and H-8), 6.91 (d, 1H, J=7.10 Hz, H-6’), 6.81 (dd, 1H, J=3.85 and 2.70 Hz, H-2), 4.10 (s, 1H,

13

C≡CH), 3.41 (bs, 1H, COOH). C-NMR (DMSO-d

19.9, 119.0, 117.4, 116.8, 115.7, 115.0, 113.7, 110.1, 84.3, 80.6. Anal. Calcd. for C20

Found: C, 73.56; H, 3.89; N, 12.93.

6

) δ: 167.5, 154.4, 144.7, 140.1, 136.7, 130.1, 125.1, 123.1, 123.0, 122.4,

1

13 3 2

H N O

: C, 73.38; H, 4.00; N, 12.84.

6

.1.11.13. 4-[(3-Chlorophenyl)amino]-2-(3-pyridyl)pyrrolo[1,2-a]quinoxaline-7-carboxylic acid (1m)

Beige crystals (92%); mp > 300°C. IR (KBr) 3385 (NH), 3600-3000 (COOH), 1690 (COO). H-NMR (DMSO-d

.59 (s, 1H, NH), 9.07-9.06 (m, 1H, H-1), 8.99-8.98 (m, 1H, H-6), 8.49-8.48 (m, 2H, H-4’ and H-9), 8.17-8.16 (m, 2H, H-6’ and

1

6

) δ:

9

H-2’), 8.12-8.10 (m, 2H, H-2″ and H-8), 7.94-7.92 (m, 2H, H-3 and H-6″), 7.51-7.48 (m, 1H, H-5’), 7.42-7.40 (m, 1H, H-5″),

1

3

7

1

.09-7.07 (m, 1H, H-4″), 3.42 (bs, 1H, COOH). C-NMR (DMSO-d

33.1, 130.7, 130.6, 128.5, 126.2, 126.1, 125.0, 124.7, 121.9, 120.6, 119.8, 118.7, 114.4, 113.8, 103.0. Anal. Calcd. for

: C, 66.59; H, 3.64; N, 13.51. Found: C, 66.72; H, 3.62; N, 13.43.

6

) δ: 169.3, 148.4, 147.3, 146.8, 143.0, 139.2, 135.2, 133.5,

23 4 2

C H15ClN O

6

.1.11.14. 4-[(3-Ehtynylphenyl)amino]-2-(3-pyridyl)pyrrolo[1,2-a]quinoxaline-7-carboxylic acid (1n)

Beige crystals (83%); mp > 300°C. IR (KBr) 3355 (NH), 3650-2900 (COOH), 2105 (C≡C), 1695 (COO). H-NMR

DMSO-d ) δ: 13.04 (s, 1H, COOH), 9.54 (s, 1H, NH), 9.07 (s, 1H, H-1), 9.06 (s, 1H, H-6), 8.54-8.53 (m, 1H, H-4’), 8.32 (s, 1H,

1

(

6

H-2’), 8.27-8.22 (m, 2H, H-6’ and H-9), 8.19-8.17 (m, 1H, H-8), 8.14 (s, 1H, H-2″), 8.01 (s, 1H, H-3), 7.92-7.90 (m, 1H, H-6″),

1

3

7

1

.56-7.53 (m, 1H, H-5’), 7.44-7.41 (m, 1H, H-5″), 7.19-7.17 (m, 1H, H-4″), 4.20 (s, 1H, C≡CH). C-NMR (DMSO-d

6

) δ: 167.5,

48.2, 147.4, 146.9, 141.2, 135.9, 133.4, 130.4, 129.6, 128.7, 128.6, 128.4, 126.1, 125.5, 125.4, 124.8, 123.6, 122.4, 121.4, 120.7,

16 4 2

15.2, 115.1, 104.0, 84.4, 80.9. Anal. Calcd. for C25H N O : C, 74.25; H, 3.99; N, 13.85. Found: C, 74.09; H, 3.82; N, 13.75.

6

.1.12. 1-(4-Bromo-2-nitropenyl)pyrrole (13)

A mixture of 4-bromo-2-nitroaniline (23 mmol) and 2,5-dimethoxytetrahydrofuran (25 mmol) in acetic acid (65 mL) was

refluxed for 1 h with vigourous stirring. After cooling, the reaction mixture was poured into water. The mixture was then extracted

twice with diethyl ether. The organic layer was dried over sodium sulfate and evaporated to dryness under reduced pressure to

1

give 13. Orange oil (86%). H-NMR (CDCl ) δ: 8.00 (d, 1H, J=2.20 Hz, H-3), 7.78 (dd, 1H, J=8.55 and 2.20 Hz, H-5), 7.36 (d,

3

1

H, J=8.55 Hz, H-6), 6.78 (dd, 2H, J=1.85 and 1.85 Hz, 2H-α), 6.39 (dd, 2H, J=1.85 and 1.85 Hz, 2H-β). Anal. Calcd. for

C H BrN O : C, 44.97; H, 2.64; N, 10.49. Found: C, 45.12; H, 2.85; N, 10.38.

1

0

7

2

6

.1.13. 1-(2-Amino-4-bromophenyl)pyrrole (14)

To a solution of 1-(4-bromo-2-nitropenyl)pyrrole (13) (19 mmol) in ethanol (95 mL) was added 15 mL of a 2M aqueous

solution of CuSO

at room temperature for 1 h. The reaction mixture was then diluted with ethyl acetate and filtered. The organic layer was

separated, dried over Na SO and evaporated to dryness under reduced pressure to give 14. Orange crystals (90%); mp 67°C. IR

4

. Sodium borohydride (94 mmol) was added portion-wise at 0°C to the reaction mixture which was then stirred

2

4

18

ACCEPTED MANUSCRIPT

1

(

KBr) 3380 and 3300 (NH

J=8.10 and 1.80 Hz, H-5), 6.81 (dd, 2H, J=1.80 and 1.80 Hz, 2H-α), 6.36 (dd, 2H, J=1.80 and 1.80 Hz, 2H-β), 3.81 (bs, 2H,

NH ). Anal. Calcd. for C10 BrN : C, 50.66; H, 3.83; N, 11.82. Found: C, 50.85; H, 4.02; N, 11.97.

2 3

). H-NMR (CDCl ) δ: 7.01 (d, 1H, J=8.10 Hz, H-6), 6.97 (d, 1H, J=1.80 Hz, H-3), 6.92 (dd, 1H,

2

H

9

2

6

.1.14. Ethyl 2-[(5-bromo-2-pyrrolo-1-ylphenyl)amino]-2-oxo-acetate (15)

To a solution containing 1-(2-amino-4-bromophenyl)pyrrole (14) (17 mmol) in 140 mL of THF at 0°C was added Et N

3

(

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 crude residue was cooled, triturated with diethyl ether and filtered to give 15. Beige crystals (43%); mp 70°C.

1

IR (KBr) 3280 (NH), 1690 and 1680 (CO). H-NMR (CDCl

3

) δ: 8.90 (s, 1H, NH), 8.75 (d, 1H, J=1.80 Hz, H-6), 7.40 (dd, 1H,

J=8.40 and 1.80 Hz, H-5), 7.23 (d, 1H, J=8.40 Hz, H-3), 6.80 (dd, 2H, J=1.80 and 1.80 Hz, 2H-α), 6.48 (dd, 2H, J=1.80 and 1.80

Hz, 2H-β), 4.36 (q, 2H, J=7.20 Hz, OCH ), 1.39 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C H BrN O : C, 49.87; H, 3.89; N,

2

3

14 13

2

3

8

.31. Found: C, 50.04; H, 3.73; N, 8.35.

6

.1.15. Ethyl 4-bromopyrrolo[1,2-a]quinoxaline-4-carboxylate (16)

A solution of ethyl 2-[(5-bromo-2-pyrrolo-1-ylphenyl)amino]-2-oxo-acetate (15) (7 mmol) in POCl

3

(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

1

give 16. Yellow crystals (70%); mp 159°C. IR (KBr) 1735 (COO). H-NMR (CDCl ) δ: 8.33 (d, 1H, J=2.10 Hz, H-6), 8.03 (dd,

3

1

H, J=2.80 and 1.20 Hz, H-1), 7.79 (d, 1H, J=8.90 Hz, H-9), 7.72 (dd, 1H, J=8.90 and 2.10 Hz, H-8), 7.57 (dd, 1H, J=3.90 and

.20 Hz, H-3), 7.03 (dd, 1H, J=3.90 and 2.80 Hz, H-2), 4.60 (q, 2H, J=7.20 Hz, OCH ), 1.54 (t, 3H, J=7.20 Hz, CH ). Anal.

2

3

Calcd. for C H BrN O : C, 52.69; H, 3.47; N, 8.78. Found: C, 52.78; H, 3.33; N, 8.89.

1

4

11

2

6

.1.16. Ethyl 7-[(3-ethynylphenyl)amino]pyrrolo[1,2-a]quinoxaline-4-carboxylate (17)

To a solution of ethyl 7-{[3-(2-trimethylsilylethynyl)phenyl]amino}pyrrolo[1,2-a]quinoxaline-4-carboxylate (12l) (0.5

mmol) in THF (5 mL), cooled to -20°C, 0.6 mL of a 1M tetrabutylammonium fluoride solution in THF was added and the

mixture was stirred for 1 h. The mixture was diluted with water and extracted twice with ethyl acetate. The extract was washed

with water, brine, dried with sodium sulfate and evaporated to leave an orange solid which was purified by column

chromatography (silica gel, cyclohexane/ethyl acetate, 7/3, v/v). Orange crystals (69%); mp 68°C. IR (KBr) 3420 (NH), 2105

1

(

C≡C), 1720 (COO). H-NMR (CDCl

3

) δ: 7.95 (dd, 1H, J=2.55 and 1.20 Hz, H-1), 7.80 (d, 1H, J=2.10 Hz, H-6), 7.78 (d, 1H,

J=8.40 Hz, H-9), 7.51 (dd, 1H, J=4.05 and 1.20 Hz, H-3), 7.33 (dd, 1H, J=8.40 and 2.10 Hz, H-8), 7.25 (t, 1H, J=1.70 Hz, H-2’),

.24 (t, 1H, J=8.10 Hz, H-5’), 7.15-7.09 (m, 2H, H-4’ and H-6’), 6.96 (dd, 1H, J=4.05 and 2.55 Hz, H-2), 6.28 (s, 1H, NH), 4.54

q, 2H, J=6.90 Hz, OCH ), 1.46 (t, 3H, J=6.90 Hz, CH ). Anal. Calcd. for C22 : C, 74.35; H, 4.82; N, 11.82. Found: C,

7

(

2

3

17 3 2

H N O

7

4.52; H, 5.06; N, 11.94.

6

.1.17. Ethyl 7-[2-(3-aminophenyl)ethynyl]pyrrolo[1,2-a]quinoxaline-4-carboxylate (18)

A mixture of 7-bromopyrrolo[1,2-a]quinoxaline (16) (3 mmol), 3-ethynylaniline (3.6 mmol), Cs

(0.15 mmol) and BINAP (0.225 mmol) in 15 mL of toluene was heated at 100°C for 20 h. The reaction mixture was

2

3

CO (4.2 mmol),

Pd(OAc)

2

allowed to cool to room temperature, diluted with dichloromethane and filtered through a pad of Celite and the filter cake was

washed with dichloromethane (x3). Concentration and flash chromatography on silica gel using AcOEt/cyclohexane (3/7, v/v) as

1

eluent afforded 18. Orange crystals (22%); mp 145°C. IR (KBr) 3410 and 3330 (NH

2

), 2210 (C≡C), 1725 (COO). H-NMR

19

ACCEPTED MANUSCRIPT

(

CDCl

3

) δ: 8.32 (d, 1H, J=1.80 Hz, H-6), 8.03 (dd, 1H, J=2.70 and 1.20 Hz, H-1), 7.88 (d, 1H, J=8.40 Hz, H-9), 7.76 (dd, 1H,

J=8.10 and 1.80 Hz, H-8), 7.57 (dd, 1H, J=4.05 and 1.20 Hz, H-3), 7.18 (t, 1H, J=8.10 Hz, H-5’), 7.03 (dd, 1H, J=4.05 and 2.70

Hz, H-2), 7.01-6.98 (m, 1H, H-6’), 6.91 (t, 1H, J=1.80 Hz, H-2’), 6.73-6.69 (m, 1H, H-4’), 4.62 (q, 2H, J=7.20 Hz, OCH ), 3.75

s, 2H, NH ), 1.55 (t, 3H, J=7.20 Hz, CH ). Anal. Calcd. for C22 : C, 74.35; H, 4.82; N, 11.82. Found: C, 74.28; H, 5.01;

N, 11.89.

2

(

2

3

17 3 2

H N O

6

.1.18. 7-[2-(3-Aminophenyl)ethynyl]pyrrolo[1,2-a]quinoxaline-4-carboxylic acid (19)

A mixture of ethyl 7-[2-(3-aminophenyl)ethynyl]pyrrolo[1,2-a]quinoxaline-4-carboxylate (18) (0.6 mmol) in methanol

12 mL) and a NaOH aqueous solution (4.8 mmol in 8 mL) was refluxed for 4 h. On cooling, after removal of the solvent under

reduced pressure, the residue was diluted with water and adjusted to pH = 6 with 1 M HCl aqueous solution. The solid precipitate

was filtered off and washed with water then with diethyl ether, and purified by column chromatography using CH Cl /methanol

9/1, v/v) as eluent to give 19. Beige crystals (45%); mp > 300°C. IR (KBr) 3500-2900 (COOH), 3400 and 3340 (NH ), 2205

) δ: 10.21 (s, 1H, COOH), 8.42-8.40 (m, 1H, H-1), 8.26 (d, 1H, J=7.80 Hz, H-9), 8.10

s, 1H, H-6), 7.67 (d, 1H, J=7.80 Hz, H-8), 7.30-7.29 (m, 1H, H-3), 7.07 (t, 1H, J=7.60 Hz, H-5’), 6.91-6.90 (m, 1H, H-2), 6.77 (s,

(

2

(

2

1

(

C≡C), 1695 (COO). H-NMR (DMSO-d

6

(

1

3

1

H, H-2’), 6.72 (d, 1H, J=7.60 Hz, H-6’), 6.61 (d, 1H, J=7.60 Hz, H-4’), 5.28 (s, 2H, NH₂). C-NMR (DMSO-d ) δ: 165.8, 148.4,

6

34.7, 131.8, 129.8, 128.8, 126.9, 124.2, 121.9, 118.5, 118.3, 115.7, 115.3, 114.6, 114.2, 113.8, 109.8, 104.1, 89.9, 87.0. Anal.

13 3 2

Calcd. for C20H N O : C, 73.38; H, 4.00; N, 12.84. Found: C, 73.21; H, 4.18; N, 12.94.

6

.1.19. Methyl 4-(3-pyridyl)-1H-pyrrole-2-carboxylate (22)

To suspension of 4-bromopyrrole-1,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (21) (10 mmol), and Pd(PPh

0.5 mmol) in a mixture of toluene/EtOH (120/6 mL) under nitrogen were added K CO (11 mmol) and 3-pyridylboronic acid (11

3 4

)

(

2

3

2 2

mmol). The reaction mixture was refluxed for 24 h, and the cooled suspension was extracted with CH Cl (3 x 100 mL). The

organic layer was washed with a saturated solution of NaCl (150 mL), and the combined organic extracts were dried over sodium

sulfate, filtered, and evaporated under reduced pressure. The crude residue was solubilised in 40 mL of dichloromethane. To this

reaction mixture was added 100 mL of a 10% trifluoroacetic acid solution in dichloromethane. The mixture was stirred at room

temperature for 4 h, then neutralized with 300 mL of a saturated aqueous solution of potassium carbonate and extracted with 120

mL of dichloromethane. The organic layer was washed with water, then brine and dried with anhydrous sodium sulfate. The

solvent was removed under reduced pressure. The residue was triturated in methanol, then the resulting precipitate was filtered,

1

washed with MeOH and dried to give 22. White crystals (34%); mp 177°C. IR (KBr) 3390 (NH), 1700 (COO). H-NMR (CDCl )

3

δ: 10.30 (s, 1H, NH), 8.83 (s, 1H, H-3), 8.48 (d, 1H, J=4.50 Hz, H-4’), 7.80 (d, 1H, J=7.80 Hz, H-6’), 7.33-7.28 (m, 2H, H-2’ and

3 10 2 2

and H-6’), 7.23 (s, 1H, H-5), 3.90 (s, 3H, OCH ). Anal. Calcd. for C11H N O : C, 65.34; H, 4.98; N, 13.85. Found: C, 65.52; H,

5

.06; N, 13.98.

6

.1.20. Methyl 1-(4-ethoxycarbonyl-2-nitrophenyl)-4-(3-pyridyl)-1H-pyrrole-2-carboxylate (23)

To the solution of methyl 4-(3-pyridyl)-1H-pyrrole-2-carboxylate (22) (3.3 mmol) in 12 mL of DMF was added cesium

carbonate (4 mmol). The mixture was stirred at room temperature for 10 min, then ethyl 4-fluoro-3-nitrobenzoate (4 mmol) was

added. The reaction mixture was refluxed for 1 h 30 was then diluted in AcOEt (50 mL), was washed with water (2 x 50 mL), then

brine (50 mL) and dried over sodium sulfate. The organic layer was concentrated under vacuo to give a brown oil. After triturating

in Et

2

O a solid was obtained and filtered off, washed with Et O and dried to give the desired product 23. Orange crystals (69%);

1

mp 163°C. IR (KBr) 1710 (COO). H-NMR (CDCl

3

) δ: 8.85 (s, 1H, H-3), 8.80 (d, 1H, J=1.80 Hz, H-3’), 8.54-8.52 (m, 1H, H-4”),

8

.41 (dd, 1H, J=8.20 and 1.80 Hz, H-5’), 7.84 (d, 1H, J=7.80 Hz, H-6”), 7.59 (d, 1H, J=8.20 Hz, H-6’), 7.45 (d, 1H, J=1.80 Hz,

H-2”), 7.35 (dd, 1H, J=7.80 and 4.90 Hz, H-5”), 7.29 (s, 1H, H-5), 4.49 (q, 2H, J=6.90 Hz, OCH ), 3.75 (s, 3H, OCH ), 1.47 (t,

H, J=6.90 Hz, CH ). Anal. Calcd. for C20 : C, 60.76; H, 4.33; N, 10.63. Found: C, 60.83; H, 4.38; N, 10.52.

2

3

17 3 6

H N O

20

ACCEPTED MANUSCRIPT

6

.2. X-ray Data

The structure of compounds 1f, 16 and 22 has been established by X-ray crystallography (Figures 3-5). Colourless single

3

crystal (0.10 x 0.06 x 0.05 mm ) of 1f was obtained by slow evaporation from methanol/chloroform (30/70) solution: monoclinic,

3

space group P21/n, a=4.8092(4) Å, b=33.851(3) Å, c=9.6672(8) Å, α=90°, β=101.440(6)°, γ=90°, V=1542.5(2) Å , Z=4,

-3

3

δ(calcd)=1.410 Mg.m , FW=327.33 for C20

13 3 2

H N O , F(000)=680. Yellow single crystal (0.12 x 0.07 x 0.07 mm ) of 16 was

obtained by slow evaporation from methanol/dichloromethane (30/70) solution: monoclinic, space group P21/c, a=4.047(3) Å,

3

-3

b=21.371(3) Å, c=14.738(4) Å, α=90°, β=96.03(3)°, γ=90°, V=1267.6(10) Å , Z=4, δ(calcd)=1.672 Mg.m , FW=319.16 for

3

C

14

H11BrN

2

O

2

, F(000)=640. Yellow single crystal (0.15 x 0.10 x 0.10 mm ) of 22 was obtained by slow evaporation from

methanol/chloroform (30/70) solution: monoclinic, space group P21/n, a=5.4906(5) Å, b=14.0619(7) Å, c=12.9743(7) Å, α=90°,

3

-3

β=94.431(3)°, γ=90°, V=998.73(12) Å , Z=4, δ(calcd)=1.345 Mg.m , FW=202.21 for C11

10 2 2

H N O , F(000)=424. Full

crystallographic results have been deposited at the Cambridge Crystallographic Data Centre (CCDC-888291, CCDC-912632 and

3

CCDC-884533), UK, as supplementary Material. The data were corrected for Lorentz and polarization effects and for empirical

5

0

51

52

absorption correction. The structure was solved by direct methods Shelx 86 and refined using Shelx 93 suite of programs.

6

.3. Biological assays

.3.1. Preparation of recombinant human CK2 holoenzyme

The preparation of the human recombinant CK2 holoenzyme was performed according to a protocol described

4

2

previously. For the expression of the α-subunit (CSNK2A1) and β-subunit (CSNK2B) of the human protein kinase CK2 a pT7-7

expression system in Escherichia coli BL21(DE3) was used. Newly transformed starter cultures were grown overnight at 37°C in

LB-medium to the stationary phase. New medium was inoculated with the separate starter cultures for both subunits and cultivated

until an OD500 of 0.6 was reached. Expression was induced by addition of IPTG (1 mM final concentration) and carried out at

3

0°C for 5-6 h for CSNK2A1 and at 37°C for 3 h for CSNK2B. Cells were harvested by centrifugation (6000×g for 10 min at 4°C)

and disrupted by soniﬁcation (three times 30 sec on ice). Preparations were then centrifuged to remove the cells debris and the

bacterial extracts for both subunits were combined and purified by a three-column procedure. Fractions exhibiting CK2 activity

were combined and analyzed by SDS-PAGE and Western Blot.

6

.3.2. Capillary electrophoresis based assay for the testing of inhibitors of the human CK2

The recently established capillary electrophoresis CK2 activity assay was used for testing the inhibitors. Therefore, 2 µL

of the dissolved inhibitors (stock solution in DMSO) were mixed with 78 µL of CK2 supplemented kinase buffer which was

composed of 1 µg CK2 holoenzyme, 50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl and 1 mM DTT. The reaction was

initiated by the addition of 120 µL assay buffer, which was composed of 25 mM Tris/HCl (pH 8.5), 150 mM NaCl, 5 mM MgCl

mM DTT, 100 µM ATP and 0.19 mM of the substrate peptide RRRDDDSDDD. The reaction was carried out for 15 min at 37°C

4

2

,

1

and stopped by the addition of 4 µL EDTA (0.5 M). Subsequently the reaction mixture was analyzed by a PA800 capillary

electrophoresis from Beckman Coulter (Krefeld, Germany). Acetic acid (2 M, adjusted with conc. HCl to a pH of 2.0) was used as

the electrolyte for electrophoretic separation. The separated substrate and product peptide were detected at 214 nm using a DAD-

detector. Pure solvent was used as negative control (0 % inhibition), assays devoid of CK2 were used as positive control (100 %

inhibition). For primary testing an inhibitor concentration of 10 µM was used. Compounds that revealed at least 50 % inhibition at

1

0 µM were used for IC50 determinations. For the determination of IC50 inhibition was determined using nine inhibitor

concentrations ranging from 0.001 µM to 100 µM. IC50 values were calculated from the resulting dose-response curves.

21

ACCEPTED MANUSCRIPT

6

.3.3. Cell culture

The human leukemic cell lines U937, K562, MV-4-11 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% CO in air. The toxicity of various molecules was also evaluated on non-activated,

2

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).

6

.3.4. 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 µL culture media with or without our

various compounds at increasing concentrations (0, 1, 5, 10, 20 and 50 µM) during 1, 2 and 3 days. After 3 hours of incubation at

7°C with 20 µL MTS/well, the plates were read by using an ELISA microplate reader (Thermo, Electrocorporation) at 490 nm

(

3

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 ꢀM ± SD (Microsoft Excell).

6

.4. Computational Methods

The geometries of the 1f and 1c compounds were optimized using the ab initio quantum chemistry program Gaussian 03 and

5

3

the HF/6-31G** basis set. RESP (restraint electrostatic potential) atomic charges of both inhibitors were calculated to generate

the corresponding force field parameters.

5

4

Molecular dynamics (MD) were performed by using the AMBER12 software package. The crystal structure of the

4

8

CK2/CX-4945 complex (pdb:3pe1) was used as starting structure for the MD simulations; having removed the sulfate ions and

water molecules, except those water molecules located within 5 Å of the ligand. The CK2/1f complex was built in silico by

superposing the central ring atoms of the ligand with the corresponding atoms of the CX-4945 compound in the CK2/CX-4945

complex. To build the CK2/1c complex two initial orientations of the ligand were chosen: one with the heterocyclic system

orientated as in the 1f system and the other with the 1c A-ring sited at the position of the 1f C-ring, and vice versa. The complexes

were immersed in an octahedral box of TIP3P water molecules using the Leap module, giving final systems of around 45000

atoms. The systems were initially optimized and then gradually heated to 300 K. Starting from these equilibrated structures 40 ns

MD production runs were performed. All simulations were carried out at 1 atm and 300 K, maintained with the Berendsen

5

barostat and thermostat, using periodic boundary conditions and Ewald sums (grid spacing of 1 Å) for treating long range

electrostatic interactions. The SHAKE algorithm was used to keep bonds involving H atoms at their equilibrium length, allowing

the use of a 2 fs time step for the integration of Newton's equations. The ligand parameters were assigned with the general

AMBER force field (GAFF) and the corresponding RESP charges with the Antechamber module of AMBER. The Amber99 force

5

6

field parameters were used for all residues excepted for the ligands. Computed energetic contributions of 1f and 1c corresponded

to the electrostatic energy and Van der Waals contributions arising from bond, angle and dihedral terms in the force field.

Calculations were performed using the MMPBSA.py method on 500 snapshots of the last 20 ns of the trajectories.

22

ACCEPTED MANUSCRIPT

Acknowledgments

This publication was supported by a grant from Ligue Nationale contre le Cancer (Comité d’Aquitaine-Charentes,

Bordeaux, France). Its content is solely the responsibility of the authors and does not necessarily represent the official views of the

Ligue Nationale contre le Cancer.

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25

ACCEPTED MANUSCRIPT

OH

O

Br

HO

O

OH

O

OH

Br

N

NH

N

NH

O

HO

CH₃

O

OH

Br

O

OH

CN

IQA

emodin

TBB

ellagic acid

O

N

H C

NH

N

3

N

HOOC

N

NH

HOOC

N

NH

N

H

N

H

CX-5011

CX-4945

pyrazolotriazine derivative

C

Cl

CH

Figure 1. Structure of ellagic acid, emodin, TBB, IQA, pyrazolotriazine derivative, CX-4945 and CX-5011.

26

ACCEPTED MANUSCRIPT

1

2

9

6

3

N

8

7

4

N

5

Pyrrolo[1,2-a]quinoxaline

N

COOH

N

HOOC

N

NH

R

NH

COOH

HOOC

NH

compounds 1a-d

compounds 1e-j

compounds 1k,l

compounds 1m,n

R

R = -Cl,

C

CH

Figure 2. General structures of new synthesized pyrrolo[1,2-a]quinoxaline derivatives 1a-n.

27

ACCEPTED MANUSCRIPT

Figure 3. The ORTEP drawing of pyrrolo[1,2-a] quinoxaline 1f with thermal ellipsoids at 30% level.

28

ACCEPTED MANUSCRIPT

Figure 4. The ORTEP drawing of pyrrolo[1,2-a]quinoxaline 16 with thermal ellipsoids at 30% level.

29

Article Doi

Synthesis and biological evaluation of novel substituted pyrrolo[1,2-a] quinoxaline derivatives as inhibitors of the human protein kinase CK2

DOI: 10.1016/j.ejmech.2013.04.051

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Authors:

Article abstract of DOI:10.1016/j.ejmech.2013.04.051

Full text of DOI:10.1016/j.ejmech.2013.04.051

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