cinnoline and Pyrido[3â²,2 - American Chemical Society

Subscriber access provided by KING MONGKUT'S UNIVERSITY OF TECHNOLOGY THONBURI (UniNet)

Article

11H-Pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline and pyrido[3',2':4,5]pyrrolo[1,2c][1,2,3] benzotriazine: two new ring systems with antitumor activity PATRIZIA DIANA, BARBARA PARRINO, ANNA CARBONE, MARINA MUSCARELLA, VIRGINIA SPANO', ALESSANDRA MONTALBANO, PAOLA BARRAJA, ALESSIA SALVADOR, DANIELA VEDALDI, and GIROLAMO CIRRINCIONE J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501244f • Publication Date (Web): 15 Oct 2014 Downloaded from http://pubs.acs.org on October 21, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

11H-Pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline and pyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3] benzotriazine: two new ring systems with antitumor activity Barbara Parrino,&,‡ Anna Carbone,&,‡ Marina Muscarella,& Virginia Spanò,& Alessandra Montalbano,& Paola Barraja,& Alessia Salvador,† Daniela Vedaldi,† Girolamo Cirrincione,& and Patrizia Diana*,& &

Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF),

Università di Palermo, Via Archirafi 32, 90123, Palermo, Italy †

Dipartimento di Scienze del Farmaco, Università di Padova, Via F. Marzolo 5, 35121, Padova,

Italy

KEYWORDS: Cinnolines; Triazines; Pyrido[3',2':4,5]pyrrolo[3,2-c]cinnolines; Pyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazines; Antitumor activity ABSTRACT: Derivatives of new ring systems 11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline and pyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine were prepared from the key intermediates 2(1H-pyrrolo[2,3-b]pyridin-2-yl)anilines in excellent yields (94-99%) and screened by the National Cancer Institute (NCI, Bethesda, USA), on about 60 human tumor cell lines derived

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 58

from nine cancer cell types. Tested compounds exhibited antiproliferative activity against all the human cell lines showing comparable MG_MID range 0.74-1.15 µM. A particular efficacy was observed against the leukemia sub-panel (GI50 range 0.73–0.0090 µM). Flow cytometric analysis of cell cycle demonstrated an increase of percentage of cells in G2/M phase. They cause apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species, and activation of caspase-3, caspase-8 and caspase-9. Moreover, they acted as a topoisomerase I inhibitors.

INTRODUCTION Cinnoline and benzotriazine cores occur in different classes of compounds that exhibited a wide range of pharmacological activities including anticancer, anti-inflammatory, antimalarial, antibacterial, antimicrobial and antifungal properties.1,2 In particular, the antitumor activity of these heterocycles is well discussed in many papers. Dibenzo[c,h]cinnolines having significant cytotoxic activity, topoisomerase I as a target and capable of overcoming MDR1-resistance were reported.3 2,7-Dihydro-3H-dibenzo[de,h]cinnoline-3,7-diones showed in vitro cytotoxic activity against murine (L1210) and human (K562) leukemia cell lines. Moreover, they were active against human leukemia multidrug resistant (K562/DX) cell lines and some of these were also tested in vivo against murine P388 leukemia and proved activity comparable with that of mitoxantrone.4 Anticancer agents as potent topoisomerase I inhibitors and with cytotoxic activity were 11H-isoquino[4,3-c]cinnolin-12-one derivatives; they were also active in the human tumor xenograft model using athymic nude mice.5 Cinnoline derivatives as human tyrosine kinase inhibitors6 and inhibitors of vascular endothelial growth factor (VEGF) receptor tyrosine kinase activity were also reported.7


2

Page 3 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The 3-amino-1,2,4-benzotriazine 1,4-dioxide (TPZ) underwent phase III of clinical studies for the treatment of human cancers for its ability to selectively damage DNA in the hypoxic cells found inside solid tumors; many analogues of TPZ with an interesting antineoplastic activity were reported.8,9 Substituted 1,2,3-benzotriazines with similar structure of vatalanib succinate (PTK787) and vandetanib (ZD6474), used in clinical trials in several type of cancer, exhibited a potency 4-10 fold higher than PTK787 in inhibiting breast, prostate, murine Lewis lung and melanoma cell lines growth.10 Pyrazolo[5,1-c][1,2,4]benzotriazine and its analogues as cytotoxic agents in normoxic and hypoxic conditions11 and a benzotriazine derivative as a potent orally active Src inhibitor with antitumor activity in preclinical assay were also reported.12 In the course of our researches on polycyclic nitrogen systems, we obtained several ring systems bearing pyrrole,13-21 indole,22-29 isoindole30,31 and recently also indazole32 moieties with antitumor activity either in the dark and under light irradiation.


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 58

Chart 1. Cinnolines and Triazines

NO

N N

R

R R1 N H

R2 N N N Indolo[1,2-c]benzo[1,2,3]triazines

R2

Indolo[3,2-c]cinnolines

CN R4

COOEt NC

N N

R R1

N

R2

N R3

R

Pyrrolo[2,1-c][1,2,4]triazines

R1

[1,2,4]Triazino[4,3-a]indoles

R1

Isondolo[2,1-c]benzo[1,2,4]triazines

R2

N R1

11H-pyrido[3',2':4,5]pyrrolo[3,2-c]innolines

R2

N N

R N H

R3

N

N N

N N

N

R1

N N N

R1 R

Pyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazines

1

2,3

We previously reported indolo[3,2-c]cinnoline derivatives which inhibited the proliferation of leukemia, lymphoma and solid tumor-derived cell lines from micromolar to nanomolar concentrations33 (Chart 1). Indolo[1,2-c]benzo[1,2,3]triazines showed antineoplastic activity against a panel of leukemia-, lymphoma-, carcinoma- and neuroblastoma-derived cell lines and inhibitory effect in the proliferation of T and B cell lines at submicromolar/micromolar concentrations.34 Moreover we reported the synthesis of pyrrolo[2,1-c][1,2,4]triazines,35 [1,2,4]triazino[4,3-a]indoles36 and isoindole[2,1-c]benzo[1,2,4]triazines37 with exhibited antitumor activity in the micromolar range. In this paper we describe the synthesis of derivatives of new ring systems 11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline of type 1 and


4

Page 5 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3] benzotriazine of type 2, 3 and report the evaluation of their antitumor activity as well as some investigations directed to the elucidation of their mode of action.

RESULTS AND DISCUSSION CHEMISTRY The key intermediates for the synthesis of the new cinnolines 1, benzotriazines 2 and 3 were substituted 2-(1H-pyrrolo[2,3-b]pyridin-2-yl)anilines of type 4. (Scheme 1) These latter compounds were conveniently prepared by an intramolecular Chichibabin-type reaction between 3-methylpyridine 5 and commercially available 2-aminobenzonitriles 6a-f (45-62% yields) in the presence of lithium diisopropylamide (LDA). Due to the dimerization of the benzonitriles 6, from the reaction mixture dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diimines 7a-f were also isolated (5-10% yields).

Scheme 1. Synthesis of substituted 2-(1H-pyrrolo[2,3-b]pyridin-2-yl)anilines 4a-fa H2N R N Me (a) N 5

CH2 N

R1

CN

R

NH2

N H

R1

4a-f

(b,c)

6a-f

a R = R1 = H; b R = Cl, R1 = H; c R = H, R1 = Cl; d R = Me, R1 = H; e R = OMe; R1 = H; f R = R1 = OMe


HN

H N

R1 R

R1

N H

R

NH 7a-f

5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

Page 6 of 58

Reagents and conditions: (a) LDA, THF, ethylbenzene and heptane, 0 °C, 30 min, (b) THF, 0

°C, 90 min; (c) LDA, THF, ethylbenzene and heptane, 0 °C then 50-60 °C, 4 h.

The diazotization reaction of the obtained derivatives 4a-f was carried out at 0 °C with a stoichiometric amount of sodium nitrite in acetic acid. The diazonium intermediates spontaneously and rapidly undergo intramolecular cyclization on the 3 position of the 7azaindole moiety affording the corresponding 11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnolines 1a-f in excellent yields (94-99%) (Scheme 2).

Scheme 2. Synthesis of substituted 11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnolines 1a-fa

N2

H2N R N

N H

(a)

4a-f

R

R N

R1

N N

N H

R1

N

N H

1a-f

R1

a R = R1 = H; b R = Cl, R1 = H; c R = H, R1 = Cl; d R = Me, R1= H; e R = OMe; R1 = H; f R = R1 = OMe a

Reagents and conditions: (a) NaNO2/H2O, AcOH, 0 °C then rt, 1 h.

To synthesize the desired benzotriazines it was necessary to protect the 3 position of key intermediates 4 to avoid the formation of the azaindolocinnoline ring during the diazotization reaction. Since the nitroso group, in the indole series, resulted a suitable substituent to avoid the undesired cyclization, we decided to use the same group also for the azaindole series.


6

Page 7 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 3. Synthesis of substituted 12-nitrosopyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3] benzotriazines 2a-f and 12-bromopyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazines 3ac,ea H2N R N

N H

NO

AcHN

(a)

R N

R1

4a-f

N H

(b)

R N

R1

8a-f

N H AcHN 9a-f

(d)

Br

R1

(c)

NO

R1

R1 R

R N

N HH N 2

N

11a-c,e

N H

H2N

10a-f

(b)

(b) Br

N

N N N

NO

R1 R

N

3a-c,e

R1

N N N

R

2a-f

a R = R1 = H; b R = Cl, R1 = H; c R = H, R1 = Cl; d R = Me, R1 = H; e R = OMe; R1 = H; f R = R1 = OMe a

Reagents and conditions: (a) Ac2O, rt, 2 h; (b) NaNO2/H2O, AcOH, 0 °C then rt, 1 h; (c) KOH

15%, EtOH, reflux, 24 h; (d) NBS, DMF, rt, 16 h.

The acetylation of compounds 4a-f gave in quantitative yields the corresponding acetamide derivatives 8a-f which were nitrosated with sodium nitrite in acetic acid to give the desired nitroso-acetamides 9a-f in excellent yields (97-99%).


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 58

The subsequent removal of acetyl group using potassium hydroxide led to the amino derivatives 10a-f (94-99% yields). Diazotization of intermediates 10 gave the expected 12nitrosopyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazines 2a-f in excellent yields (91-98%) (Scheme 3). As in indole and pyrrole series a bromine atom was used in the attempt to lock the 3 position of the pyrrole moiety, we brominated compounds 4a-c,e using N-bromosuccinimide in dimethylformamide38 to get the corresponding brominated intermediates 11a-c,e in very good yields (75-91%). In this case, differently from the indole34 and also pyrrole39 series, in which an unusual Japp-Klingemann reaction occurred affording the corresponding cinnoline derivatives, the diazotization of the bromo derivatives 11 gave 12-bromopyrido[3',2':4,5]pyrrolo[1,2c][1,2,3]benzotriazines 3a-c,e in excellent yields (95-99%).

BIOLOGICAL RESULTS AND DISCUSSION Antitumor activity. All the synthesized compounds 1a-f, 2a-f and 3a-c,e were submitted to the National Cancer Institute (NCI; Bethesda MD) and derivatives 1c,e,f, 2c,e,f and 3a,c,f were prescreened according to the NCI protocol at the 10-5 M dose on the full panel of approximately 60 human cancer cell lines derived from 9 human cancer cell types, that have been grouped in disease sub-panels including leukemia, non-small-cell lung, colon, central nervous system, melanoma, ovarian, renal, prostate and breast tumor cell lines. Compounds 1e,f and 2c,e,f satisfied the criteria set by the NCI for activity in this assay and were selected for further screenings at 5 concentrations at 10-fold dilution (10-4-10-8M) on the full panel. The antitumor activity of compounds was given by three parameters for each cell line: GI50 (GI50 is the molar concentration of the compound that inhibits 50% net cell growth), TGI (TGI is the


8

Page 9 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


molar concentration of the compound leading to total inhibition of net cell growth), and LC50 (LC50 is the molar concentration of the compound that induces 50% net cell death). The average values of mean graph midpoint (MG_MID) were calculated for each of these parameters. Table 1. Overview of the in vitro antitumor screening[a] results for derivatives 1e, 1f, 2c, 2e, 2f

GI50 (µM)[b] Compd.

N[c]

N[d]

Range

MG_MID[e]

1e

58

58

0.029-11.80

1.07

1f

59

59

0.018-20.60

1.15

2c

60

60

0.017-8.60

0.74

2e

58

58

0.009-10.80

0.79

2f

60

60

< 0.010-22.70

1.02

[a] Data obtained from the NCI in vitro disease-oriented human tumor cell line screen. [b] GI50: concentration (µM) that inhibit 50% net cell growth. [c] Number of cell lines investigated. [d] Number of cell lines giving positive GI50 values. [e] MG_MID: mean graph midpoint; this is the arithmetic mean value for all tested cancer cell lines. If the indicated effect was not attainable under the concentration range used, the highest tested concentration was used for the calculation.

An evaluation of the data reported in the Table 1 pointed out that compounds 1e,f and 2c,e,f exhibited antiproliferative activity against all the human cell lines showing comparable MG_MID range 0.74-1.15 µM. All selected compounds showed particular efficacy against the leukemia sub-panel having GI50 in the range 0.73–0.0090 µM (Table 2). The different condensation of pyridopyrrolo with the


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 58

other two ring moiety (benzotriazine or cinnoline) did not provoke a clear difference in the antiproliferative power. The most sensitive leukemia cell lines are MOLT-4 (GI50 range 0.029-0.0094 µM), CCRF-CEM (GI50 range 0.035- < 0.010 µM), SR (GI50 range 0.039-0.0090 µM), and HL-60(TB) (GI50 range 0.30-0.040 µM). Moreover derivatives 1e,1f,2c, and 2e exhibited good selectivity against HOP-92 (GI50 range 0.21-0.039 µM) of non-small cell lung sub-panel, HCT-116 (GI50 range 0.21-0.060 µM) of colon sub-panel, CAK-1 (GI50 range 0.18-0.048 µM) of the renal sub-panel, and MDA-MB-468 (GI50 range 0.23-0.032 µM) cell lines of the breast cancer.

Table 2. In vitro inhibition of cancer cell line growth by compounds 1e, 1f, 2c, 2e, 2f (µM) Cell lines

GI50 1e

1f

2c

Leukemia CCRF-CEM HL-60(TB) K-562 MOLT-4 RPMI-8226 SR

0.16 0.22 0.029 0.73 0.031

0.35 0.19 0.33 0.018 0.19 0.039

0.023 0.30 0.13 0.018 0.17 0.017

0.043 0.11 0.0094 0.26 0.0090

< 0.010 0.040 0.21 < 0.010 0.11 0.018

Non-Small Cell Lung Cancer A549/ATCC EKVX HOP-62 HOP-92 NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522

7.90 2.82 5.47 0.082 2.62 5.50 10.01 1.93 1.09

7.90 2.43 2.26 0.21 1.94 2.34 20.60 2.46 2.05

2.20 1.83 1.44 0.10 2.19 1.17 8.60 1.33 0.95

10.80 3.38 7.51 0.042 4.14 5.17 9.67 1.67 1.25

13.30 3.31 1.90 0.039 3.70 2.22 22.70 2.61 4.01

Colon Cancer COLO 205

1.13

2.96

1.00

0.68

5.26


2e

2f

10

Page 11 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620

8.23 0.21 2.26 0.75 1.22 0.38

1.95 0.13 1.43 1.34 1.91 0.44

8.40 0.19 0.96 0.91 0.91 0.19

7.23 0.060 3.13 0.62 1.03 0.27

1.00 0.061 1.62 0.79 1.38 0.21

CNS Cancer SF-268 SF-295 SF-539 SNB-19 SNB-75 U251

0.73 1.59 0.64 7.61 0.64 0.93

0.54 2.43 1.73 2.77 1.48 0.92

1.21 2.07 1.89 3.18 1.42 1.27

0.47 4.69 0.47 6.28 0.85 0.72

0.49 2.62 1.82 3.84 2.25 0.60

Melanoma LOX IMVI MALME-3M M14 MDA-MB-435 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62

0.76 1.06 1.07 1.44 1.96 11.80 5.85 7.49 0.91

1.41 2.28 1.79 2.23 11.30 4.49 7.33 3.32 2.49

1.03 1.22 1.19 1.26 2.28 1.06 3.82 3.07 2.36

0.80 0.77 1.57 1.94 2.18 3.48 5.01 8.21 0.85

0.92 2.89 1.71 2.38 17.30 9.10 12.60 4.54 2.92

Ovarian Cancer IGROV1 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 NCI/ADR-RES SK-OV-3

0.31 0.96 1.41 5.72 0.62 1.79 2.69

1.73 1.15 1.60 2.53 1.25 2.29

1.35 0.25 0.98 1.38 1.80 0.43 2.98

0.11 1.11 1.29 8.02 3.90 1.42 1.68

1.94 0.46 1.91 2.69 0.82 0.38 4.02

Renal Cancer 786-0 A498 ACHN CAKI-1 RXF 393 SN12C TK-10 UO-31

0.51 8.00 0.20 0.11 0.66 1.63 4.52 0.12

0.32 2.74 0.33 0.18 0.71 2.27 2.38 0.49

0.84 1.91 0.33 0.13 0.56 1.46 0.94 0.65

0.33 1.27 0.094 0.089 0.31 1.40 5.08 0.076

0.23 15.80 0.25 0.048 0.40 1.80 5.26 0.40

Prostate Cancer


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PC-3 DU-145

Page 12 of 58

1.88

1.49 2.10

0.74 0.61

1.86

2.36 1.99

Breast Cancer MCF7 MDA-MB-231/ATCC HS 578T BT-549 T-47D MDA-MB-468

0.23 1.42 3.47 3.43 0.49 0.067

0.34 2.69 3.70 3.70 0.40 0.23

0.57 0.68 1.05 1.78 0.26 0.14

0.13 1.26 1.17 5.06 0.16 0.032

0.26 2.60 6.80 5.53 0.21 0.16

MG_MD

1.07

1.15

0.74

0.79

1.02

The antiproliferative activity of the most active compounds 1e, 2e and 2f was also evaluated on solid human tumor cell lines (A-431and LoVo) and leukemic cell lines (Jurkat and CEM), whose GI50 values are reported in Table 3.

Table 3. In Vitro Cell Growth Inhibitory Effects of Compounds 1e, 2e and 2f (µM) Compounds

Jurkata

CEM

A-431

LoVo

1e

0.09±0.01

0.65±0.16

2.35±0.32

3.64±0.59

2e

0.09±0.05

0.22±0.09

0.93±0.15

1.28±0.21

2f

0.20±0.11

0.24±0.04

1.41±0.22

3.89±0.75

a

GI50 = compound concentration (µM) required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SEM from the dose−response curves of at least three independent experiments.

A comparison of the antiproliferative activity of the 7-azaindolo[1,2,3]benzotriazine 2c, 2e, and 2f with the triazine derivatives reported in Chart 1 revealed that [1,2,4]triazines and their benzocondensed derivatives fused to pyrrole, indole or isoindole moieties resulted definitely less active


12

Page 13 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


than the fused [1,2,3]triazines. In fact, among the five pyrrolo[2,1-c][1,2,4]triazine derivatives assayed against the NCI 60 human cancer cell lines panel, one resulted devoid of activity; the remaining four showed inhibitory effects in the growth of a wide range of cell lines generally at 10-5 M level.35 Regarding the [1,2,4]triazino[4,3-a]indole derivatives synthesized, only two of them showed antiproliferative activity against 39 - 56 out of the 58 cell lines tested with GI50 mean values in the range 12.5 - 28.7 µM.36 Also isoindolo[2,1-c]benzo[1,2,4]triazines showed similar antiproliferative activity being active against 76 - 88% of the tested cell lines with GI50 mean values in the range 33.9 - 53.7 µM.37 The [1,2,3]triazine system confers to the indolo-fused derivatives a better profile of antiproliferative activity. The seven synthesized compounds, although tested only against a panel of 12 leukemia-, lymphoma-, carcinoma-, and neuroblastoma-derived cell lines, showed GI50 mean values of in the range 0.95 - 19.56 µM.34 The corresponding 7-azaindolo-triazines 2c, 2e, and 2f, tested against the NCI 60 human cancer cell lines panel, resulted active against the totality of the tested cell lines with GI50 mean values of 0.74 - 1.02 µM. Thus, the aza substitution of the indole moiety, independently from the substitution pattern, produced more active compounds. The two series share only one derivative with the same substitution pattern: the 2-chloro substituted ones. The aza-derivative 2e showed to be one magnitude order more active than the deaza-analogue. Both compounds were tested against CCRF-CEM and MOLT-4 leukemia cell lines and the aza-compound was 217 and 111 folds more active than the indolotriazine derivative. Also in the cases of HT-29 of the colon cancer sub panel and ACHN of the renal cancer sub panel the aza-derivative was respectively 26 and 33 folds more active. The indolo[3,2-c]cinnoline series, tested against 12 leukemia-, lymphoma-, and solid tumorderived cell lines, inhibited cell proliferation with GI50 mean values in the 1.42 - 20.94 µM


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 58

range.33 The 7-azaindolo-cinnolines 1e and 1f, tested against the NCI 60 human cancer cell lines panel, showed better activity having mean GI50 values of 1.07 and 1.15 µM respectively. Overall, the 7-aza substitution of the indole moiety led to compounds certainly more active than the deaza analogue previously reported.

Colony formation assay. Treating LoVo cells with compounds 1e, 2e and 2f for 24 h reduced the growth of colonies (7-14 days) in the clonogenic survival assay in a concentration dependent manner. Exposure of cells to 0.625-10 µM of 2e almost completely eliminated the formation of colonies (Figure 1). The other compounds were less efficient in reducing colony formation, since 1e and 2f totally inhibited it up to 2.5 and 5 µM, respectively. However, these results indicate that compounds inhibited long-term survival of LoVo cells at submicromolar-micromolar concentrations.


14

Page 15 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Colony formation assay was performed as described in the experimental section. Results are represented as the percentage of number of colonies compared to untreated cells (0 µM =100 %). Bars, the mean ± SEM of 3 independent experiments.

In vitro antiproliferative activity on drug resistant cell lines. The over-expression of Pglycoprotein (Pgp) is one of the most common mode of cancer resistance. Pgp is a membrane protein which mediates the efflux of various structurally unrelated drugs.40 Therefore, the compounds 1e, 2e and 2f were also tested on two multidrug-resistant cell lines with high levels of Pgp, one derived from a lymphoblastic leukemia (CEMVbl-100), the other derived from a colon carcinoma (LovoDoxo). The three compounds showed comparable potency toward parental cells and cells resistant to vinblastine or doxorubicin exhibiting a resistance index, which is the ratio


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 58

between GI50 values of resistant cells and sensitive cells, in the range 0.33-2.13. (Table 4) Thus, these compounds were not substrates of Pgp.

Table 4. In Vitro Cell Growth Inhibitory Effects of Compounds on Drug Resistant Cell Lines GI50 (µM)a

GI50 (µM)

Compounds

LoVo

LoVodoxo

RIb

CEM

CEMvbl

RI

1e

3.64±0.59

1.21±0.18

0.33

0.65±0.16

1.02±0.11

1.57

2e

1.28±0.21

0.65±0.28

0.51

0.22±0.09

0.44±0.11

2.00

2f

3.89±0.75

4.33±1.31

1.11

0.24±0.04

0.51±0.08

2.13

Doxoc

0.12±0.03

13.50±0.20

112.50

n.d.

n.d.

n.d

Vind

n.d.

n.d.

n.d.

0.004±0.002

0.21±0.03

525

a

GI50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SEM from the dose−response curves of at least three independent experiments; bThe values express the ratio between GI50 determined in resistant and non-resistant cell lines; cDoxo = doxorubicin; dVin = vinblastina

Cell cycle analysis. The effects of two different concentrations of compounds 1e, 2e and 2f on cell cycle progression were examined after 24 h of incubation in Jurkat cells. Representative cell cycle histograms were shown in Figure 2.


16

Page 17 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Effect of compounds 1e (b: 500 nM; c: 250 nM), 2e (d: 500 nM; e: 250 nM) and 2f (f: 500 nM; g: 250 nM) on cell cycle distribution of Jurkat cells (representative histograms). Cells were treated with two different concentrations of the indicated compounds for 24 h. Then the cells were fixed and stained with PI to analyze DNA content by flow cytometry. (a: not treated cells).


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 58

Untreated Jurkat cells presented a typical pattern of proliferating cells: 62.6 %, 15.5 % and 21.9 % of cells were found in G1, S and G2/M phases, respectively (Table 5). When treated with compounds, the percentage of cells distributed in G1 phase clearly dropped with respect of control, whereas an increase of the percentage of cells in G2/M one can be observed after all treatments. Moreover, incubation of cells with these compounds caused a clear increase of the hypodiploid peak, indicative of apoptosis. This latter consists of cells with DNA content lower than G1 phase.

Table 5. Cell cycle analysis of Jurkat cells treated with compounds 1e, 2e and 2f for 24 h % G1a

%S

% G2/M

% SubG1b

Control

62.6

15.5

21.9

5.3

1e (0.5µM)

33.2

28.6

38.2

39.8

1e (0.25µM)

30.2

24.1

45.7

27.8

2e (0.5µM)

28.1

27.2

44.7

39.1

2e (0.25µM)

35.5

25.1

39.4

22.5

2f (0.5µM)

21.0

24.7

54.3

21.7

2f 0.25µM

37.3

15.4

47.3

18.7

a

Percentages of each phase of the cell cycle excluding the subG1 peak Percentage of the cell population with hypodiploid DNA content peak (apoptotic cells)

b

Loss of plasmatic membrane asymmetry in apoptosis and caspase activation. To further characterize the mode of cellular death, we also performed a biparametric cytofluorimetric analysis using PI and Annexin V-FITC, which stain DNA and phosphatidylserine (PS) residues, respectively. PS normally is located in the inner side of plasma membrane but during apoptosis can be translocated in the outer one.41 After 6 h of incubation with compounds 1e, 2e and 2f, no


18

Page 19 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


loss of plasma membrane asymmetry was observed, while this event was clear after 24 h of incubation as an increase of Annexin-V positive cells was detected (Figure 3A).

Figure 3. Panel A. Flow cytofluorimetric analysis of apoptotic cells after the treatment of Jurkat cells with compounds 1e, 2e and 2f. After 6 h and 24 h of incubation with 500 and 250 nM of compounds, cells were labelled with Annexin V-FITC and PI and analysed by flow cytometry. Data are presented as mean ± SEM of 3 independent experiments. C= control. Panel B and C. Caspase activity induced by compounds 1e, 2e and 2f. Jurkat cells were incubated in their presence at 500 nM. After 6 h (B) and 24 h (C) of treatment, cells were lysed and assayed for caspase-3, caspase-8, and caspase-9 activity by colorimetric tests. Data are represented as fold increase of enzymes activity, in comparison to the control (C).


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 58

The percentages of cells with PS exposure were consistent with those of subG1 phase in the previous experiments. Apoptotic cellular changes are largely mediated by caspases, a family of cysteinyl aspartate-speciﬁc proteases, whose target proteins are important indicators of apoptotic cell death. We investigated the activation of three key caspases: caspase-3, an executioner, and caspase-8 and -9, which are initiators associated with extrinsic and intrinsic pathways of apoptosis, respectively.42 Results showed that caspase-3, -8 and -9 were activated by all derivatives (Figures 3B and 3C). In particular, after 6 h of treatment, the activation of initiating caspases is more evident, while after 24 h of treatment an increase in caspase 3 activity can be detected. All together these results suggest an apoptotic cell death.

Mitochondrial involvement in cell death. Mitochondria play an essential role in the propagation of apoptosis.43 It is well established that apoptotic stimuli alter the mitochondrial transmembrane potential (∆ΨM).44 ∆ΨM was monitored by fluorescence of the dye 5,5′,6,6′tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine (JC-1). JC-1 has the unique property of forming orange fluorescent aggregates locally and spontaneously under high mitochondrial ∆ΨM, whereas the monomeric form fluoresces in green.45 When a collapse of ∆ΨM occurs, JC-1 is only found in monomeric form. The percentages of cells with low mitochondrial potential (green fluorescence) were shown in Figure 4A. Treated Jurkat cells in the presence of compounds 1e, 2e and 2f exhibited a dramatic shift in fluorescence in a dose-dependent manner compared to the control cells, indicating depolarization of mitochondrial membrane potential. Since mitochondrial membrane depolarization is associated with mitochondrial production of reactive oxygen species (ROS),46 some other cytofluorimetric tests were performed to detect ROS generation. The probes hydrohethidine (HE) and dihydrodichlorofluorescein diacetate (DCFH2-


20

Page 21 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DA) were used. HE is oxidized by superoxide anion into an ethidium ion, which emits red fluorescence. DCFH2 is susceptible to oxidation by peroxides, generating the green fluorescent product, 2’,7’-dichlorofluorescein (DCF). The results presented in Figure 4B showed that all compounds induced the production of ROS in comparison to the control cells, thus a clear mitochondrial involvement can be hypothesised.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 58

Figure 4. Assessment of mitochondrial dysfunction after treatment with compounds 1e, 2e and 2f. Panel A: induction of loss of mitochondrial membrane potential after 24 h of incubation of Jurkat cells by compounds at the indicated concentrations (nM). Cells were stained with the fluorescent probe JC-1 and analyzed by flow cytometry. Panel B: mitochondrial production of ROS in Jurkat cells. After 24 h of incubation with 500 nM compounds, cells were stained with HE or DCFH2-DA and analyzed by flow cytometry. Data are expressed as mean ± SEM of 3 independent experiments.

Lysosomal involvement in cellular death. Partial lysosomal permeabilization with subsequent release of proteolytic enzymes into the cytosol, and their active contribution to the signaling pathways, has been recently described in several models of apoptosis.47


22

Page 23 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Assessment of lysosomial dysfunction after treatment with compounds 1e, 2e and 2f. Percentages of cells stained with AO (acridine orange) were reported and analyzed by flow cytometry after 24 h of treatment with the indicated two different concentrations (nM). Values are expressed as means ± SEM of at least 3 independent experiments. Flow cytometric analysis was performed, by using the fluorescent dye acridine orange (AO), in order to investigate the integrity of lysosomes. AO is a lysosomotropic base and presents red fluorescence when highly concentrated, as in the case of intact lysosomes. When lysosomes are damaged, AO relocates to the cytosol where it is predominantly in the deprotonated form and it shows green fluorescence.48 The percentage of cells with intact lysosomes was evaluated by assessing AO red fluorescence after 24 h of treatment with two different concentrations of compounds. The percentages of treated cells with lysosomal damage were not significantly increased in comparison with control (Figure 5). Lysosomes are only marginally involved in apoptosis induction by compounds.

DNA binding. As these compounds 1e, 2e and 2f exhibit a planar structure, their interaction with DNA was evaluated with linear dichroism measures. By measuring the flow linear dichroism of the different drug-nucleic acid complexes, it was possible to elucidate the binding geometry of a compound bound to DNA Examination of the Figure 6 revealed two diverse behaviours between the compounds derived from a different condensation of the two main rings. In fact, a clear increase in the values of LD of the DNA band at 260 nm for 1e-DNA complexes was observed, suggesting that the DNA became better oriented in the hydrodynamic field because of a stiffening of the helix upon binding of the drug. The negative LD signals in the long-wavelength absorption (S0-S1 transition, 300-450 nm) of 1e revealed that it was oriented perpendicularly to the flow field, usually caused by an intercalation into the DNA helix. This assumption was further corroborated by the observation that the intensity of the DNA signal increased on


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 58

addition of the drug. The LD spectra of the other two compounds 2e and 2f did not reveal a better orientation of DNA along the hydrodynamic field nor an induced dichroic signal in the compound absorption region, thus 2e and 2f did not interact efficiently with DNA.


24

Page 25 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Linear dichroism LD spectra of mixtures of st-DNA and 1e (panel A), 2e (panel B) and 2f (panel C) at different [compound]/[DNA] ratios a) 0.00, b) 0.01, c) 0.02, d ) 0.03 and e) 0.04 in phosphate buffer.

Inhibition of topoisomerase I. The effect of the compounds 1e, 2e and 2f on the catalytic activity of human topoisomerase I was investigated using a conventional plasmid DNA relaxation assay. Supercoiled plasmid pBR322 (250 µg) was incubated with topoisomerase I in the presence of compounds (5-100 µM) or camptothecin as a reference. The unwinding of the plasmid was monitored by agarose gel electrophoresis separation (Figures 7A and B-upper gels). After run, gels were incubated with ethidium bromide to visualize DNA bands under UV illumination. The relaxation of supercoiled DNA by the enzyme gave a population of topoisomers (lane 2 of the upper gels). In the presence of different concentrations of compounds and of camptothecin, the intensity of the slower bands (corresponding to cleaved DNA and to completely relaxed DNA) significantly increased in comparison to the sample where no compound had been added. To better differentiate the specific (poisoning) and nonspecific effects the same experiments were performed in parallel using ethidium bromide-containing agarose gels. The presence of ethidium bromide is useful to distinguish between the relaxed intact plasmid and the open circular DNA containing permanent single-strand breaks. In fact, the intercalation of high concentrations of ethidium bromide leads to unwinding and to the consequent formation of the positively supercoiled form, whose migration in the gel is slightly faster than that of the initial negatively supercoiled form.49 In contrast, the run of nicked plasmid is essentially unaffected after association of ethidium. Under these conditions, the amount of nicked DNA increases significantly with increasing concentrations of compounds and of camptothecin. This experiment


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 58

indicates DNA single-strand cleavage by the ternary complex of plasmid, topoisomerase, and compounds. Tight binding to DNA is not required for trapping the covalent DNA-topoisomerase I complexes. A comparison of the topoisomerase I inhibition of compounds 1e, 2e, and 2f with the series reported in chart 1 is impossible since no mechanistic studies were performed in those series. However, a search of topoisomerase I inhibitors bearing cinnoline or [1,2,3]triazine moieties revealed that only two series of fused cinnolines targeting topoisomerase I were reported: isoquino[4,3-c]cinnoline-12-ones and dibenzo[c,h]cinnolines.50,51 Instead, the 7-azaindolo[1,2,3]benzotriazine system here reported represents the first case of triazines targeting topoisomerase I.

CONCLUSION Derivatives of new ring systems 11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline and pyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine, obtained in excellent yields from the key intermediates 2-(1H-pyrrolo[2,3-b]pyridin-2-yl)anilines, exhibit high cytotoxic activity, against about 60 human tumor cell lines, with GI50 values reaching nanomolar concentrations. A particular efficacy was observed against the leukemia sub-panel (GI50 range 0.73–0.0090 µM). More importantly, these compounds are also found to be active in cells overexpressing P-gp, suggesting that these derivatives might be useful in treating drug-refractory patients. Flow cytometric analysis of cell cycle demonstrated an increase of percentage of cells in G2/M phase. Further studies showed that they cause apoptosis in Jurkat cell line. The induction of apoptosis is associated with (i) dissipation of the mitochondrial transmembrane potential, (ii) production of reactive oxygen species and cardiolipin oxidation, and (iii) activation of caspase-3, caspase-8 and


26

Page 27 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


caspase-9. At the molecular level, such compounds act through inhibition of topoisomerase I activity. Interestingly, the inhibition of topoisomerase I by compounds bearing the triazine scaffold is unprecedented.

Figure 7. Topoisomerase I inhibition. Effect of increasing concentrations of 1e and camptothecin (Panel A) and of 2f and 2e (Panel B) on the relaxation of plasmid DNA by human topoisomerase


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 58

I in the absence (upper gel of both panels) or in the presence (lower gels of both panel) of ethidium bromide during the electrophoresis. SC, supercoiled form.

EXPERIMENTAL SECTION CHEMISTRY. General Methods. All melting point were taken on a Büchi-Tottoly capillary apparatus and are uncorrected. IR spectra were determined in bromoform with a Jasco FT/IR 5300 spectrophotometer. 1H and 13C NMR spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d6 solution, using a Bruker Avance II series 200 MHz spectrometer. Compounds 1a-f, 2a-f, 3a-c,e 9a-f and 10a-f were characterized only by 1H NMR spectra, for their poor solubility the 13C spectra were not performed. Column chromatography was performed with Merk silica gel 230-400 mesh ASTM or with Büchi Sepacor chromatography module (prepacked cartridge system). Elemental analyses (C, H, N) were within ± 0.4% of theoretical values and were performed with a VARIO EL III elemental analyzer. Purity of all the tested compounds was >95%, determined by HPLC (Agilent 1100 Series).

General procedure for the synthesis of 2-(1H-pyrrolo[2,3-b]pyridin-2-yl)anilines (4a-f). 3-Methylpyridine (13 mmol, 1.2 mL) was added under argon atmosphere to a solution of LDA in THF, ethyl benzene and heptane (10 mmol, 5.0 mL) cooled at 0 °C and in the presence of activated molecular sieves. The resulting suspension was kept under stirring at 0 °C for 30 min. To the reaction mixture a solution of suitable 2-aminobenzonitrile 6a-f (6 mmol) in THF (2.0 mL) keeping the temperature above 10-15 °C was added dropwise under stirring. The reaction mixture was maintained for further 90 minutes at 0 ° C. Then a solution of LDA in THF, ethyl benzene and heptane (40 mmol, 20 mL) was added dropwise under stirring at 0 °C. The reaction mixture was heated at 50-60 °C for 4 h. The suspension obtained was poured into ice water and


28

Page 29 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


then extracted with ethyl acetate (3x100 mL). The organic phase was dried (Na2SO4), evaporated under reduced pressure and the obtained solid recrystallized with ether and drops of ethyl acetate and dichloromethane. The precipitate obtained was filtered off, air-dried to give compounds 4a-f. The organic layer was evaporated and the residue was purified by column chromatography using dichloromethane/ethyl acetate (90/10) as eluent to give compounds 7a-f. 2-(1H-Pyrrolo[2,3-b]pyridin-2-yl)aniline (4a). Brown solid; yield 58%; mp: 185-187 °C; IR (cm-1) 3428-3343; 1H NMR (200 MHz, DMSO-d6) δ: 5.24 (s, 2H, NH2), 6.64-6.69 (m, 2H, H-3’, H-4), 6.83 (d, 1H, J = 7.6 Hz, H-6), 7.05-7.12 (m, 2H, H-5, H-5’), 7.40 (d, 1H, J = 6.9 Hz, H-3), 7.91 (d, 1H, J = 7.2 Hz, H-4’), 8.20 (d, 1H, J = 4.0 Hz, H-6’), 12.30 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 99.2 (d), 116.0 (d), 116.5 (d), 118.2 (s), 119.0 (d), 121.8 (s), 128.4 (d), 129.4 (d), 129.5 (d), 137.0 (s), 142.3 (d), 144.3 (s), 149.2 (s). Anal. Calcd for C13H11N3: C, 74.62; H, 5.30; N, 20.08. Found: C, 74.38; H, 5.42; N, 20.25. Dibenzo[b,f][1,5]diazocine-6,12(5H,11H)-diimine (7a). Yellow solid; yield 6%; mp: 55 °C; IR (cm-1) 3404, 1616; 1H NMR (200 MHz, DMSO-d6) δ: 6.53-6.62 (m, 1H, ArH), 6.76 (dd, 1H, J = 1.0, 8.1 Hz, ArH), 7.08-7.16 (m, 1H, ArH), 7.38-7.47 (m, 3H, 2xNH, ArH), 7.69-7.80 (m, 4H, 2xNH, 2xArH), 8.22 (d, 1H, J = 8.1 Hz, ArH), 8.42 (dd, 1H, J = 1.6, 8.1 Hz, ArH); 13C NMR (50 MHz, DMSO-d6) δ: 112.4 (s), 114.4 (d), 116.4 (d), 117.9 (s), 123.5 (d), 124.7 (d), 127.1 (d), 130.5 (d), 130.6 (d), 132.9 (d), 149.5 (s), 149.9 (s), 161.0 (s), 162.0 (s). Anal. Calcd for C14H12N4: C, 71.17; H, 5.12; N, 23.71. Found: C, 71.38; H, 4.83; N, 23.60. 5-Chloro-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (4b). Brown solid; yield 56%; mp: 237 °C; IR (cm-1) 3446-3359; 1H NMR (200 MHz, DMSO-d6) δ: 5.56 (s, 2H, NH2), 6.67-6.70 (m, 2H, H4, H-6), 6.89 (s, 1H, H-3’), 7.06 (dd, 1H, J = 4.3, 7.3 Hz, H-5’), 7.37 (d, 1H, J = 7.9 Hz, H-3), 7.92 (d, 1H, J = 7.3 Hz, H-4’), 8.20-8.21 (m, 1H, H-6’), 11.84 (s, 1H, NH); 13C NMR (50 MHz,


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 58

DMSO-d6) δ: 98.9 (d), 114.6 (d), 115.1 (s), 115.8 (d), 115.9 (d), 120.7 (s), 127.5 (d), 130.7 (d), 133.1 (s), 133.7 (s), 142.5 (d), 147.4 (s), 148.9 (s). Anal. Calcd for C13H10ClN3: C, 64.07; H, 4.14; N, 17.24. Found: C, 64.22; H, 4.07; N, 17.02. 3,9-Dichlorodibenzo[b,f][1,5]diazocine-6,12(5H,11H)-diimine (7b). Yellow solid; yield 7%; mp: 264-265 °C; IR (cm-1) 3458, 1647; 1H NMR (200 MHz, DMSO-d6) δ: 6.80 (d, 1H, J = 8.8 Hz, ArH), 7.17 (dd, 1H, J = 2.7, 8.8 Hz, ArH), 7.53 (bs, 2H, 2xNH), 7.78 (s, 1H, ArH), 7.79 (s, 1H, ArH), 8.01 (bs, 2xNH), 8.38 (t, 1H, J = 2.7 Hz, ArH), 8.42 (d, 1H, J = 2.7 Hz, ArH); 13C NMR (50 MHz, DMSO-d6) δ: 113.3 (s), 117.7 (s), 118.2 (d), 118.3 (s), 122.8 (d), 128.9 (s), 129.3 (2xd), 130.4 (d), 133.4 (d), 148.1 (s), 148.8 (s), 160.4 (s), 161.0 (s). Anal. Calcd for C14H10Cl2N4: C, 55.10; H, 3.30; N, 18.36. Found: C, 55.00; H, 3.45; N, 18.11. 4-Chloro-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (4c). Brown solid; yield 55%; mp: 222-223 °C; IR (cm-1) 3430-3340; 1H NMR (200 MHz, DMSO-d6) δ: 5.44 (s, 2H, NH2), 6.76 (s, 1H, H3’). 6.84 (d, 1H, J = 8.5 Hz, H-6), 6.89-7.20 (m, 2H, H-5, H-5’), 7.92 (d, 1H, J = 7.3 Hz, H-4’), 8.21-8.22 (m, 1H, H-6’), 11.89 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 99.3 (d), 115.8 (d), 117.3 (d), 117.6 (s), 119.7 (s), 120.7 (s), 127.7 (d), 128.2 (d), 128.3 (d), 135.3 (s), 142.7 (d), 145.0 (s), 148.9 (s). Anal. Calcd for C13H10ClN3: C, 64.07; H, 4.14; N, 17.24. Found: C, 64.40; H, 3.98; N, 17.02. 2,8-Dichlorodibenzo[b,f][1,5]diazocine-6,12(5H,11H)-diimine (7c). Yellow solid; yield 5%; mp: 198-199 °C; IR (cm-1) 3394, 1608; 1H NMR (200 MHz, DMSO-d6) δ: 6.58 (dd, 1H, J = 2.1, 8.7 Hz, ArH), 6.84 (d, 1H, J = 2.1 Hz, ArH), 7.49 (dd, 1H, J = 2.1, 8.8 Hz, ArH), 7.64 (bs, 2H, 2xNH), 7.84 (d, 1H, J = 2.1 Hz, ArH), 7.99 (bs, 2H, 2xNH), 8.24 (d, 1H, J = 8.8 Hz, ArH), 8.39 (d, 1H, J = 8.8 Hz, ArH); 13C NMR (50 MHz, DMSO-d6) δ: 111.1 (s), 114.1 (d), 115.1 (d), 116.1 (s), 125.1 (d), 125.7 (d), 125.9 (d), 132.3 (d), 135.4 (s), 137.6 (s), 150.5 (s), 151.2 (s), 160.9 (s),


30

Page 31 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


162.1 (s). Anal. Calcd for C14H10Cl2N4: C, 55.10; H, 3.30; N, 18.36. Found: C, 54.84; H, 3.47; N, 18.22. 5-Methyl-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (4d). Brown solid; yield 62%; mp: 194 °C; IR (cm-1) 3430-3343; 1H NMR (200 MHz, DMSO-d6) δ: 2.22 (s, 3H, CH3), 5.18 (s, 2H, NH2), 6.51 (d, 1H, J = 7.7 Hz, H-4), 6.64 (bs, 2H, H-3’, H-6), 7.03 (dd, 1H, J = 4.3, 7.3, Hz, H-5’), 7.30 (d, 1H, J = 7.7 Hz, H-3), 7.89 (d, 1H, J = 7.3 Hz, H-4’), 8.17-8.18 (m, 1H, H-6’), 11.76 (1H, s, NH); 13C NMR (50 MHz, DMSO-d6) δ: 21.0 (q), 98.1 (d), 113.8 (s), 115.6 (d), 116.2 (d), 117.6 (d), 120.9 (s), 127.1 (d), 129.0 (d), 137.1 (s), 138.0 (s), 140.0 (d), 145.7 (s), 148.1 (s). Anal. Calcd for C14H13N3: C, 75.31; H, 5.87; N, 18.82. Found: C, 75.62; H, 5.66; N, 18.67. 3,9-Dimethyldibenzo[b,f][1,5]diazocine-6,12(5H,11H)-diimine (7d). Yellow solid; yield 6%; mp: 181 °C; IR (cm-1) 3452, 1614; 1H NMR (200 MHz, DMSO-d6) δ: 2.21 (s, 3H, CH3), 2.46 (s, 3H, CH3), 6.40 (dd, 1H, J = 1.3, 8.2 Hz, ArH), 6.55 (s, 1H, ArH), 7.24 (dd, 1H, J = 1.4, 8.4 Hz, ArH), 7.33 (bs, 2H, 2xNH), 7.49 (s, 1H, ArH), 7.64 (bs, 2H, 2xNH), 8.08 (d, 1H, J = 8.4 Hz, ArH), 8.31 (d, 1H, J = 8.2 Hz, ArH); 13C NMR (50 MHz, DMSO-d6) δ: 21.0 (q), 21.3 (q), 110.3 (s), 115.7 (d), 116.5 (d), 123.3 (d), 126.2 (2xd), 130.6 (d), 140.0 (s), 142.9 (2xs), 149.8 (s), 149.8 (s), 160.8 (s), 162.1 (s). Anal. Calcd for C16H16N4: C, 72.70; H, 6.10; N, 21.20. Found: C, 72.55; H, 6.28; N, 21.02. 5-Methoxy-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (4e). Brown solid; yield 47%; mp: 191192 °C; IR (cm-1) 3433-3348; 1H NMR (200 MHz, DMSO-d6) δ: 3.72 (s, 3H, CH3), 5.26 (s, 2H, NH2), 6.30 (dd, 1H, J = 2.5, 8.5 Hz, H-4), 6.42 (d, 1H, J = 2.5 Hz, H-6), 6.59 (d, 1H, J = 1.8 Hz, H-3’), 7.03 (dd, 1H, J = 4.7, 7.8, Hz, H-5’), 7.32 (d, 1H, J = 8.5 Hz, H-3), 7.87 (dd, 1H, J = 1.5, 7.8 Hz, H-4’), 8.16 (dd, 1H, J = 1.5, 4.7 Hz, H-6’), 11.68 (bs, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 54.8 (q), 97.6 (d), 100.4 (d), 103.0 (d), 109.7 (s), 115.5 (d), 121.0 (s), 126.9 (d),


31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 58

130.2 (d), 137.0 (s), 141.7 (d), 147.3 (s), 148.8 (s), 160.0 (s). Anal. Calcd for C14H13N3O: C, 70.28; H, 5.48; N, 17.56. Found: C, 70.52; H, 5.16; N, 17.33. 3,9-Dimethoxydibenzo[b,f][1,5]diazocine-6,12(5H,11H)-diimine (7e). Yellow solid; yield: 8%; mp: 77-78 °C; IR (cm-1) 3406, 1614; 1H NMR (200 MHz, DMSO-d6) δ: 3.74 (s, 3H, CH3), 3.90 (s, 3H, CH3), 6.18 (dd, 1H, J = 2.5, 9.0 Hz, ArH), 6.30 (d, 1H, J = 2.5 Hz, ArH), 7.53 (dd, 1H, J = 2.5, 8.9 Hz, ArH), 7.09 (d, 1H, J = 2.5 Hz, ArH), 7.49-7.53 (bs, 4H, 4xNH), 8.08 (d, 1H, J = 9.0 Hz, ArH), 8.36 (d, 1H, J = 8.9 Hz, ArH); 13C NMR (50 MHz, DMSO-d6) δ: 54.6 (q), 55.4 (q), 99.2 (d), 102.3 (d), 106.2 (d), 106.4 (s), 111.5 (s), 115.4 (d), 124.9 (d), 132.1 (d), 151.6 (s), 151.9 (s), 160.4 (s), 161.4 (s), 162.3 (s), 162.7 (s). Anal. Calcd for C16H16N4O2: C, 64.85; H, 5.44; N, 18.91. Found: C, 64.59; H, 5.32; N, 18.66. 4,5-Dimethoxy-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (4f). Brown solid; yield 45%; mp: 77-78 °C; IR (cm-1) 3432, 3370; 1H NMR (200 MHz, DMSO-d6) δ: 3.74 (s, 6H, 2xCH3), 4.95 (s, 2H, NH2), 6.53 (s, 1H, H-6), 6.65 (s, 1H, H-3), 7.03-7.09 (m, 2H, H-3’, H-5’), 7.87 (d, 1H, J = 6.9 Hz, H-4’), 8.15-8.16 (m, 1H, H-6’), 11.75 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 55.2 (q), 56.2 (q), 97.7 (d), 101.0 (d), 107.5 (s), 113.3 (d), 115.6 (d), 121.1 (s), 126.8 (s), 126.9 (d), 137.2 (s), 140.6 (s), 141.8 (d), 148.8 (s), 149.7 (s). Anal. Calcd for C15H15N3O2: C, 66.90; H, 5.61; N, 15.60. Found: C, 67.22; H, 5.31; N, 15.39. 2,3,8,9-Tetramethoxydibenzo[b,f][1,5]diazocine-6,12(5H,11H)-diimine (7f). Yellow solid; yield: 8%; mp: 270-271 °C; IR (cm-1) 3381, 1622; 1H NMR (200 MHz, DMSO-d6) δ: 3.72 (s, 3H, CH3), 3.75 (s, 3H, CH3), 3.87 (s, 3H, CH3), 3.92 (s, 3H, CH3), 6.37 (s, 1H, ArH), 7.10-7.13 (m, 3H, 2xNH, ArH), 7.39 (bs, 2H, 2xNH), 7.57 (s, 1H, ArH), 8.01 (s, 1H, ArH); 13C NMR (50 MHz, DMSO-d6) δ: 55.0 (q), 55.6 (q), 55.9 (q), 56.6 (q), 99.9 (d), 102.9 (d), 105.5 (s), 106.6 (d), 109.8 (s), 114.6 (d), 139.1 (s), 145.6 (s), 146.5 (s), 147.4 (s), 151.8 (s), 154.1 (s), 159.6 (s), 160.3


32

Page 33 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(s). Anal. Calcd for C18H20N4O4: C, 60.66; H, 5.66; N, 15.72. Found: C, 60.80; H, 5.42; N, 15.87.

General procedure for the synthesis of N-[2-(1H-pyrrolo[2,3-b]pyridin-2yl)phenyl]acetamides (8a-f). The suitable amino derivative 4a-f (2.3 mmol) was dissolved in acetic anhydride (10 mL) and stirred at room temperature for 2 h. The reaction mixture was then poured into water and the precipitate obtained was filtered off, air-dried and recrystallized from ethanol to afford derivatives 8a-f. N-[2-(1H-Pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (8a). Yellow solid; yield 99%; mp: 184-185 °C; IR (cm-1) 3255, 3166, 1643; 1H NMR (200 MHz, DMSO-d6) δ: 2.04 (s, 3H, CH3), 6.69-7.08 (m, 2H, 2xArH), 7.33-7.34 (m, 2H, 2xArH), 7.64-7.98 (m, 2H, 2xArH), 8.23 (m, 1H, H4’), 9.48 (m, 1H, H-6’), 9.48 (s, 1H, NH), 12.00 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ:

23.5 (q), 99.9 (d), 115.8 (d), 120.7 (s), 120.8 (s), 125.4 (d), 126.6 (d), 126.7 (s), 127.9 (d), 128.1 (d), 129.4 (d), 135.3 (s), 135.7 (s), 142.7 (d), 168.7 (s). Anal. Calcd for C15H13N3O: C, 71.70; H, 5.21; N, 16.72. Found: C, 71.52; H, 5.44; N, 16.63. N-[5-Chloro-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (8b). Yellow solid; yield 99%; mp: 196-197 °C; IR (cm-1) 3257, 3166, 1658; 1H NMR (200 MHz, DMSO-d6) δ: 2.06 (s, 3H, CH3), 6.72 (s, 1H, H-3’), 7.06-7.11 (m, 1H, H-5’), 7.36 (d, 1H, J = 7.7 Hz, H-4), 7.65 (d, 1H, J = 7.7 Hz, H-3), 7.81 (s, 1H, H-6), 7.97-8.00 (m, 1H, H-4’), 8.24-8.25 (m, 1H, H-6’), 9.60 (s, 1H, NH), 12.02 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 23.6 (q), 100.5 (d), 116.0 (d), 120.7 (s), 125.0 (d), 125.2 (s), 125.3 (d), 128.2 (d), 131.0 (d), 132.3 (s), 134.5 (s), 136.6 (s), 143.1 (d), 149.2 (s), 169.0 (s). Anal. Calcd for C15H12ClN3O: C, 63.05; H, 4.23; N, 14.71. Found: C, 63.34; H, 4.01; N, 14.61.


33


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 58

N-[4-Chloro-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (8c). Yellow solid; yield 99%; mp: 220-223 °C; IR (cm-1) 3263, 3128, 1650; 1H NMR (200 MHz, DMSO-d6) δ: 2.05 (s, 3H, CH3), 6.77 (s, 1H, H-3’), 7.06-7.12 (m, 1H, H-5’), 7.42 (d, 1H, J = 7.5 Hz, H-5), 7.65-7.72 (m, 2H, H-3, H-6), 7.99 (d, 1H, J = 7.2 Hz, H-4’), 8.25-8.26 (m, 1H, H-6’), 9.57 (s, 1H, NH), 12.02 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 23.5 (q), 100.8 (d), 116.0 (d), 120.5 (s), 125.8 (d), 128.2 (2xd), 128.7 (d), 129.3 (s), 129.4 (s), 134.1 (s), 134.2 (s), 143.3 (d), 149.1 (s), 168.8 (s). Anal. Calcd for C15H12ClN3O: C, 63.05; H, 4.23; N, 14.71. Found; C, 63.25; H, 4.04; N, 14.52. N-[5-Methyl-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (8d). Yellow solid; yield 99%; mp: 182-184 °C; IR (cm-1) 3276, 3140, 1664; 1H NMR (200 MHz, DMSO-d6) δ: 2.03 (s, 3H, CH3), 2.33 (s, 3H, CH3), 6.64 (s, 1H, H-3’), 7.09-7.13 (m, 2H, H-4, H-5’), 7.43 (s, 1H, H-6), 7.56 (d, 1H, J = 7.3 Hz, H-3), 7.93-7.96 (m, 1H, H-4’), 8.20-8.21 (m, 1H, H-6’), 9.43 (s, 1H, NH), 11.89 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 20.8 (q), 23.5 (q), 99.5 (d), 115.7 (d), 120.7 (s), 123.9 (s), 126.2 (d), 127.2 (d), 127.7 (d), 129.1 (d), 135.0 (s), 135.8 (s), 137.8 (s), 142.5 (d), 149.0 (s), 168.7 (s). Anal. Calcd for C16H15N3O: C, 72.43; H, 5.70; N, 15.84. Found: C, 72.67; H, 5.60; N, 15.68. N-[5-Methoxy-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (8e). Yellow solid; yield 99%, mp: 176-178 °C; IR (cm-1) 3315, 3150, 1673; 1H NMR (200 MHz, DMSO-d6) δ: 2.03 (s, 3H, CH3), 3.79 (s, 3H, CH3), 6.59 (s, 1H, H-3’), 6.89 (d, 1H, J = 8.8 Hz, H-4), 7.05 (dd, 1H, J = 4.9, 7.3 Hz, H-5’), 7.29 (s, 1H, H-6), 7.57 (d, 1H, J = 8.8 Hz, H-3), 7.93 (d, 1H, J = 7.3 Hz, H4’), 8.18-8.20 (m, 1H, H-6’), 9.41 (s, 1H, NH), 11.84 (s, 1H, NH); 13C NMR (50 MHz, DMSOd6) δ: 23.6 (q), 55.3 (q), 99.0 (d), 99.5 (s), 110.9 (d), 111.4 (d), 115.7 (d), 120.8 (s), 127.5 (d),


34

Page 35 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


130.3 (d), 135.8 (s), 136.5 (s), 142.2 (d), 149.0 (s), 159.0 (s), 168.7 (s). Anal. Calcd for C16H15N3O2: C, 68.31; H, 5.37; N, 14.94. Found: C, 68.59; H, 5.12; N 14.72. N-[4,5-Dimethoxy-2-(1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (8f). Yellow solid; yield 99%; mp: 196-197 °C; IR (cm-1) 3276, 3136, 1652; 1H NMR (200 MHz, DMSO-d6) δ: 2.02 (s, 3H, CH3), 3.77 (s, 3H, CH3), 3.85 (s, 3H, CH3), 6.66 (s, 1H, H-3’), 7.05 (dd, 1H, J = 4.7, 7.8 Hz, H-5’), 7.10 (s, 1H, H-3), 7.26 (s, 1H, H-6), 7.94 (dd, 1H, J = 1.5, 7.8 Hz, H-4’), 8.20 (dd,1H, J = 1.5, 4.7 Hz, H-6’), 9.42 (s, 1H, NH), 11.88 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 23.3 (q), 55.6 (q), 55.7 (q), 99.1 (d), 111.5 (d), 111.8 (d), 115.7 (d), 119.5 (s), 120.8 (s), 127.5 (d), 128.3 (s), 135.8 (s), 142.4 (d), 146.5 (s), 148.1 (s), 148.9 (s), 168.7 (s). Anal. Calcd for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50. Found: C, 65.59; H, 5.27; N, 14.72.

General procedure for the synthesis of N-[2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2yl)phenyl]acetamides (9a-f). To a stirred solution of the suitable derivative 8a-f (2.0 mmol) in acetic acid (2 mL) it was added a solution of sodium nitrite (2.0 mmol, 0.140 g) dissolved in the minimum amount of water at 0 °C. The reaction mixture was stirred at room temperature for 1 h, then poured into water and the precipitate obtained was filtered off, air-dried and recrystallized from ethanol to give derivatives 9a-f. N-[2-(3-Nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (9a). Yellow solid; yield 99%; mp: 240-242 °C; IR (cm-1) 3390, 3147, 1643, 1590 ; 1H NMR (200 MHz, DMSO-d6) δ: 2.09 (s, 3H, CH3), 7.24-7.55 (m, 3H, 3xArH), 8.26-8.49 (m, 4H, 4xArH), 11.5 (s, 1H, NH), 14.3 (s, 1H, NH). Anal. Calcd for C15H12N4O2: C, 64.28; H, 4.32; N, 19.99. Found: C, 64.63; H, 4.07; N, 19.78.


35


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 58

N-[5-Chloro-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (9b). Yellow solid; yield 99%; mp: 243-244 °C; IR (cm-1) 3357, 3188, 1710, 1587; 1H NMR (200 MHz, DMSO-d6) δ: 2.13 (s, 3H, CH3), 7.30-7.41 (m, 2H, H-4, H-5’), 8.34 (d, 1H, J = 8.7 Hz, H-3), 8.42 (d, 1H, J = 7-0 Hz, H-4’), 8.52-8.53 (m, 2H, H-6, H-6’), 11.86 (s, 1H, NH), 14.40 (s, 1H, NH). Anal. Calcd for C15H11ClN4O2: C, 57.24; H, 3.52;N, 17.80. Found: C, 57.45; H, 3.23; N, 17.68. N-[4-Chloro-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (9c). Yellow solid; yield 98%; mp: 257-258 °C; IR (cm-1) 3381, 3156, 1693, 1591; 1H NMR (200 MHz, DMSO-d6) δ: 2.11 (s, 3H. CH3), 7.40 (dd, 1H, J = 5.0, 7.0 Hz, H-5’), 7.62 (d, 1H, J = 7.5 Hz, H5), 8.33-8.44 (m, 3H, H-3, H-4’, H-6), 8.53-8.55 (m, 1H, H-6’), 11.67 (s, 1H, NH), 14.47 (s, 1H, NH). Anal. Calcd for C15H11ClN4O2: C, 57.24; H, 3.52; N, 17,80. Found: C, 57.42; H, 3.65; N, 17.71. N-[5-Methyl-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (9d). Yellow solid; yield 97%; mp: 251-252 °C; IR (cm-1) 3374, 3142, 1703, 1589; 1H NMR (200 MHz, DMSO-d6) δ: 2.11 (s, 3H, CH3), 2.38 (s, 3H, CH3), 7.06 (d, 1H, J = 7.8 Hz, H-4), 7.38 (dd, 1H, J = 5.0, 7.4 Hz, H-5’), 8.20-8.34 (m, 2H, H-3, H-6), 8.42 (d, 1H, J = 7.4 Hz, H-4’), 8.49-8.51 (m, 1H, H-6’), 11.92 (s, 1H, NH), 14.31 (s, 1H, NH). Anal. Calcd for C16H14N4O2: C, 65.30; H, 4.79; N, 19.04. Found: C, 65.56; H, 4.67; N, 19.13. N-[5-Methoxy-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (9e). Yellow solid; yield 99%; mp: 248-249 °C; IR (cm-1) 3326, 3167, 1703, 1589; 1H NMR (200 MHz, DMSO-d6) δ: 2.17 (s, 3H, CH3), 3.85 (s, 3H, CH3), 6.83 (dd, 1H, J = 2.4, 8.9 Hz, H-4), 7.32 (dd, 1H, J = 5.0, 7.2 Hz, H-5’), 8.24 (s, 1H , H-6), 8.41 (d, 1H, J = 7.2 Hz, H-4’), 8.47-8.53 (m, 2H,


36

Page 37 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H-6’ and H-3), 12.50 (s, 1H, NH), 14.30 (s, 1H, NH). Anal. Calcd for C16H14N4O3: C, 61.93; H, 4.55; N, 18.06. Found: C, 61.82; H, 4.41; N, 18.29. N-[4,5-Dimethoxy-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)phenyl]acetamide (9f). Yellow solid; yield 98%, mp: 322-324 °C; IR (cm-1) 3381, 3175, 1708, 1580; 1H NMR (200 MHz, DMSO-d6) δ: 2.14 (s, 3H, CH3), 3.79 (s, 3H, CH3), 3.85 (s, 3H, CH3), 7.30 (dd, 1H, J = 5.1, 7.4 Hz, H-5’), 8.23 (s, 1H, H-3), 8.26 (s, 1H, H-6), 8.41 (dd, 1H, J = 1.7, 7.4 Hz, H-4’), 8.46 (dd, 1H, J = 1.7, 5.1 Hz, H-6’), 11.97 (s, 1H, NH), 12.47 (s, 1H, NH). Anal. Calcd for C17H16N4O4: C, 59.99; H, 4.74; N, 16.46. Found: C, 60.12; H, 4.58; N, 16.30

General procedure for the synthesis of 2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)anilines (10a-f). To a solution of acetylamino derivatives 9a-f (2.0 mmol) in ethanol (10 mL) an aqueous solution of potassium hydroxide (15%, 10 mL) was added. The reaction mixture was refluxed for 24 h. After cooling, the solution was neutralized with hydrochloric acid (1N). The solid was filtered off, air-dried and recrystallized from ethanol to give derivatives 10a-f. 2-(3-Nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (10a). Yellow solid; yield 94%; mp: 220221°C; IR (cm-1) 3433, 3267, 3170, 1592; 1H NMR (200 MHz, DMSO-d6) δ: 6.57-7.23 (m, 4H, 4xArH), 7.81 (s, 2H, NH2), 8.37-8.50 (m, 3H, 3xArH), 14.04 (s, 1H, NH). Anal. Calcd for C13H10N4O: C, 65.54; H, 4.23; N, 23.52. Found: C, 65.32; H, 4.37; N, 23.47. 5-Chloro-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (10b).Yellow solid; yield 98%; mp: 255-256 °C; IR (cm-1) 3438, 3232, 3143, 1598; 1H NMR (200 MHz, DMSO-d6) δ: 6.65 (1H, dd, J = 2.2, 8.8 Hz, H-4), 6.94 (d, 1H, J = 2.2 Hz, H-6), 7.26 (dd,1H, J = 5.2, 7.4 Hz, H-5’), 8.00 (s, 2H, NH2), 8.36-8.51 (m, 3H, H-3, H-4’, H-6’), 14.12 (s, 1H, NH). Anal. Calcd for C13H9ClN4O: C, 57.26; H, 3.33; N, 20.55; Found: C, 57.01; H, 3.50; N, 20.32.


37


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 58

4-Chloro-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (10c). Yellow solid; yield 96%; mp: 356-357 °C; IR (cm-1) 3396, 3246, 3100, 1595; 1H NMR (200 MHz, DMSO-d6) δ: 6.90 (d, 1H, J = 9.0 Hz, H-6), 7.22-7.30 (m, 2H, H-5, H-5’), 7.96 (s, 2H, NH2), 8.39 (dd, 1H, J = 1.7, 7.4 Hz, H-4’), 8.45 (dd, 1H, J = 1.7, 5.1 Hz, H-6’), 8.57 (d, 1H, J = 2.5 Hz, H-3), 14.18 (s, 1H, NH). Anal. Calcd for C13H9ClN4O: C, 57.26; H, 3.33; N, 20.55; Found: C, 57.00; H, 3.52; N, 20.30. 5-Methyl-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (10d). Yellow solid; yield 99%; mp: 344-346 °C; IR (cm-1) 3446, 3300, 3161, 1598; 1H NMR (200 MHz, DMSO-d6) δ: 2.23 (s, 3H, CH3), 6.43 (d, 1H, J = 8.5 Hz, H-4), 6.65 (s, 1H, H-6), 7.21 (dd, 1H, J = 5.2, 7.3 Hz, H-5’), 7.80 (s, 2H, NH2), 8.34-8.42 (m, 3H, H-3, H-4’, H-6’), 13.94 (s, 1H, NH). Anal. Calcd for C14H12N4O: C, 66.65; H, 4.79; N, 22.21. Found: C, 66.35; H, 4.98; N, 22.14. 5-Methoxy-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (10e). Yellow solid; yield 94%, mp: 236-238 °C; IR (cm-1) 3556, 3434, 3162, 1589; 1H NMR (200 MHz, DMSO-d6) δ: 3.77 (s, 3H, CH3), 6.25 (d, 1H, J = 9.0 Hz, H-4), 6.37 (s, 1H, H-6), 7.13-7.19 (m, 2H, H-6' and H-3), 8.03 (s, 2H, NH2), 8.33-8.36 (m, 2H, H-4’, H-5’), 13.88 (s, 1H, NH). Anal. Calcd for C14H12N4O2: C, 62.68; H, 4.51; N, 20.88. Found: C, 62.35; H, 4.79; N, 20.72. 4,5-Dimethoxy-2-(3-nitroso-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (10f). Yellow solid; yield 95%; mp: 234-235 °C; IR (cm-1) 3461, 3216, 3020, 1594; 1H NMR (200 MHz, DMSO-d6) δ: 3.68 (s, 3H, CH3), 3.79 (s, 3H, CH3), 6.43 (s, 1H, H-6), 7.12 (dd, 1H, J = 5.2, 7.2 Hz, H-5’), 7.95 (s, 2H, NH2), 8.22 (s, 1H, H-3), 8.31-8.34(m, 2H, H-4’, H-6’), 13.84 (s, 1H, NH). Anal. Calcd for C15H14N4O3: C, 60.40; H, 4.73; N, 18.78. Found: C, 60.08; H, 4.94; N, 18.63.

General procedure for the synthesis of 2-(3-bromo-1H-pyrrolo[2,3-b]pyridin-2-yl)anilines (11a-c,e). To a solution of appropriate derivatives 4a-c,e (4.3 mmol) in anhydrous DMF (16 mL)


38

Page 39 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a solution of N-bromosuccinimide (4.3 mmol, 0.770 g) in DMF (5 mL) was added dropwise After 16 h at room temperature, the reaction mixture was poured into water and the solid precipitated was filtered off and air-dried to give compounds 11a-c,e that were pure enough to give satisfactory analytical and spectral data. 2-(3-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl)aniline (11a). Yellow solid; yield 75 %; mp: 217218 °C; IR (cm-1) 3417, 3334, 3191; 1H NMR (200 MHz, DMSO-d6) δ: 5.03 (s, 2H, NH2), 6.66 (t, 1H, J = 7.8 Hz, H-4), 6.81 (d, 1H, J = 7.8 Hz, H-6), 7.14-7.21 (m, 3H, H-3, H-5, H-5’), 7.83 (dd, 1H, J = 1.2, 7.8 Hz, H-4’), 8.28 (dd, 1H, J = 1.2, 4.6 Hz, H-6’), 12.17 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 87.3 (s), 114.5 4 (s), 115.1 (d), 115.6 (d), 116.3 (d), 120.0 (s), 126.0 (d), 129.9 (d), 131.2 (d), 135.2 (s), 143.5 (d), 146.7 (s), 147.5 (s). Anal. Calcd for C13H10BrN3: C, 54.19; H, 3.50; N, 14.58. Found: C, 54.33; H, 3.24; N, 14.62. 2-(3-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl)-5-chloroaniline (11b). Yellow solid; yield 91%; mp: 231-234 °C; IR (cm-1) 3401, 3324, 3188; 1H NMR (200 MHz, DMSO-d6) δ: 5.37 (s, 2H, NH2), 6.66 (dd, 1H, J = 2.1, 8.2 Hz, H-4), 6.84 (d, 1H, J = 2.1 Hz, H-6), 7.13 (d, 1H, J = 8.2 Hz, H-3), 7.16-7.22 (m, 1H, H-5’), 7.81-7.88 (m, 2H, H-4’, H-6’), 12.15 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 87.6 (s), 113.2 (s), 113.9 (d), 115.0 (d), 116.4 (d), 119.8 (s), 126.1 (d), 132.8 (d), 133.9 (s), 134.4 (s), 143.6 (d), 147.5 (s), 148.3 (s). Anal. Calcd for C13H9BrClN3: C, 48.40; H, 2.81; N, 13.03. Found: C, 48.08; H, 3.02; N, 13.24. 2-(3-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl)-4-chloroaniline (11c). Yellow solid; yield 78%; mp: 195-197 °C; IR (cm-1) 3446, 3343, 3124; 1H NMR (200 MHz, DMSO-d6) δ: 5.24 (s, 2H, NH2), 6.81 (d, 1H, J = 8.6 Hz, H-6), 7.16-7.22 (m, 3H, H-3, H-5 and H-5’ ), 7.85 (dd, 1H, J = 1.3, 7.8 Hz, H-4’), 8.30 (dd, 1H, J = 1.3, 4.6 Hz, H-6’), 12.21 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 87.7 (s), 115.7 (s), 116.4 (d), 116.5 (d), 118.4 (s), 119.8 (s), 126.2 (2xd), 130.3 (d),


39


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 58

133.7 (s), 143.8 (d), 145.9 (s), 147.5 (s). Anal. Calcd for C13H9BrClN3: C, 48.40; H, 2.81; N, 13.03. Found: C, 48.61; H, 2.63; N, 13.14. 2-(3-Bromo-1H-pyrrolo[2,3-b]pyridin-2-yl)-5-methoxyaniline (11e). Yellow solid; yield 84%; mp: 201-202 ˚C; IR (cm-1) 3411, 3328, 3136; 1H NMR (200 MHz, DMSO-d6) δ: 3.73 (s, 3H, CH3), 5.75 (s, 2H, NH2), 6.27 (dd, 1H, J = 2.4, 8.4, Hz, H-4), 6.38 (d, 1H, J = 2.4 Hz, H-6), 7.06 (d, 1H, J = 8.4 Hz, H-3), 7.16 (dd, 1H, J = 4.7, 7.8 Hz, H-5’), 7.80 (dd, 1H, J = 1.3, 7.8 Hz, H4’), 8.25 (dd, 1H, J = 1.3, 4.7 Hz, H-6’), 12.06 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6) δ: 54.8 (q), 87.1 (s), 99.6 (d), 102.4 (d), 107.6 (s), 116.2 (d), 120.0 (s), 125.78 (d), 132.2 (d), 135.3 (s), 143.2 (d), 147.4 (s), 148.0 (s), 160.9 (s). Anal. Calcd for C14H12BrN3O: C, 52.85; H, 3.80; N, 13.21. Found: C, 52.60; H, 3.98; N, 13.08.

General procedure for the synthesis of 11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnolines (1a-f). To a stirred solution of suitable amines 4a-f (3.0 mmol) in acetic acid (3.0 mL) a solution of sodium nitrite (3.0 mmol, 0.210 g) dissolved in the minimum amount of water at 0 °C was added dropwise. The reaction mixture was stirred at room temperature for 1 h, poured into water and the precipitate obtained was filtered off, air-dried and recrystallized from ethanol to afford derivatives 1a-f. 11H-Pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline (1a). Yellow solid; yield 99%; mp: >400 °C; 1H NMR (200 MHz, DMSO-d6) δ: 7.55 (dd, 1H, J = 5.0, 7.8 Hz, H-8), 7.95-8.03 (m, 2H, 2xArH), 8.58-8.67 (m, 2H, 2xArH), 8.71 (dd, 1H, J = 1.6, 5.0 Hz, H-9), 8.92 (dd, 1H, J = 1.6, 7.8 Hz, H7), 13.53 (s, 1H, NH). Anal. Calcd for C13H8N4: C, 70.90; H, 3.66; N, 25.44. Found: C, 70.69; H, 3.82; N, 25.62. 3-Chloro-11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline (1b) Yellow solid; yield 94%; mp: >400 °C; 1H NMR (200 MHz, DMSO-d6) δ: 7.55 (dd, 1H, J = 4.9, 7.8 Hz, H-8), 8.05 (dd, 1H, J


40

Page 41 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


= 2.0, 8.8 Hz, H-2), 8.63-8.68 (m, 2H, H-1, H-4), 8.72 (dd, 1H, J = 1.5, 4.9 Hz, H-9), 8.93 (dd, 1H, J = 1.5, 7.8 Hz, H-7), 13.70 (s, 1H, NH). Anal. Calcd for C13H7ClN4: C, 61.31; H, 2.77; N, 22.00. Found: C, 61.47; H, 2.59; N, 22.12. 2-Chloro-11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline (1c). Yellow solid; yield 99%; mp: >400 °C; 1H NMR (200 MHz, DMSO-d6) δ: 7.56 (dd, 1H, J= 4.8, 7.8 Hz, H-8), 7.98 (dd, 1H, J = 2.3, 9.1 Hz, H-3), 8.63 (d, 1H, J = 9.1 Hz, H-4), 8.71-8.74 (m, 2H, H-1, H-9), 8.93 (dd, 1H, J = 1.6, 7.8 Hz, H-7), 13.54 (s, 1H, NH). Anal. Calcd for C13H7ClN4: C, 61.31; H, 2.77; N, 22.00. Found: C, 61.47; H, 2.59; N, 22.12. 3-Methyl-11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline (1d). Yellow solid; yield 96%; mp: >400 °C; 1H NMR (200 MHz, DMSO-d6) δ: 2.65 (s, 3H, CH3), 7.48-7.55 (dd, 1H, J = 4.8, 7.8 Hz, H-8), 7.83 (dd, 1H, J = 1.4, 8.5 Hz, H-2), 8.39 (d, 1H, J = 1.4 Hz, H-4), 8.51 (d, 1H, J = 8.5 Hz, H-1), 8.68 (dd, 1H, J = 1.6, 4.8 Hz, H-9), 8.88 (dd, 1H, J = 1.6, 7.8 Hz, H-7), 13.45 (s, 1H, NH). Anal. Calcd for C14H10N4: C, 71.78; H, 4.30; N, 23.92. Found: C, 71.56; H, 4.52; N, 23.87. 3-Methoxy-11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline (1e). Yellow solid; yield 99%; mp: > 400 °C; 1H NMR (200 MHz, DMSO-d6) δ: 4.04 (s, 3H, CH3), 7.54 (dd, 1H, J = 4.7, 7.7 Hz, H8), 7.68 (dd, 1H, J = 2.1, 9.1 Hz, H-2), 7.93 (d, 1H, J = 2.1 Hz, H-4), 8.58 (d, 1H, J = 9.1 Hz, H1), 8.69 (d, 1H, J= 4.7 Hz, H-7), 8.86 (d, 1H, J = 7.7 Hz, H-9), 13.54 (s, 1H, NH). Anal. Calcd for C14H10N4O: C, 67.19; H, 4.03; N, 22.39. Found: C, 67.42; H, 3.87; N, 22.17. 2,3-Dimethoxy-11H-pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline (1f). Yellow solid; yield 99%; mp: >400 °C; 1H NMR (200 MHz, DMSO-d6) δ:4.00 (s, 3H, CH3), 4.05 (s, 3H, CH3), 7.57 (dd, 1H, J = 4.5, 7.5 Hz, H-8), 7.80 (s, 1H, Ar-H), 7.86 (s, 1H, ArH), 8.33-8.37 (m, 1H, H-7), 8.568.64 (m, 1H, H-9), 11.98 (s, 1H, NH). Anal. Calcd for C15H12N4O2: C, 64.28; H, 4.32; N, 19.99. Found: C, 64.43; H, 4.17; N, 19.84.


41


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 58

General procedure for the synthesis of 12-nitrosopyrido[3',2':4,5]pyrrolo[1,2c][1,2,3]benzotriazines(2a-f). To a stirred solution of suitable derivatives 10a-f (3.0 mmol) in acetic acid (3.0 mL) a solution of sodium nitrite (3 mmol, 0.210 g) dissolved in the minimum amount of water at 0 °C was added dropwise. The reaction mixture was stirred at room temperature for 1 h, then poured into water and the precipitate obtained was filtered off, air-dried and recrystallized from ethanol to give derivatives 2a-f. 12-Nitroso-pyrido[3’,2’:4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (2a). Yellow solid; yield 98%; IR (cm-1) 1558; mp: >400 °C; 1H NMR (200 MHz, DMSO-d6) δ: 7.80 (dd, 1H, J = 4.7, 7.9 Hz, H-10), 8.24-8.39 (m, 2H, 2xArH), 8.63-8.71 (m, 2H, 2xArH), 8.75 (dd, 1H, J = 1.6, 4.7 Hz, H9), 9.40 (dd, 1H, J = 1.6, 7.9 Hz, H-11). Anal. Calcd for C13H7N5O: C, 62.65; H, 2.83; N, 28.10. Found: C, 62.47; H, 3.03; N, 28.24. 3-Chloro-12-nitrosopyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (2b). Yellow solid; yield 91%; mp: >400 °C; IR (cm-1) 1562; 1H NMR (200 MHz, DMSO-d6) δ: 7.83 (dd, 1H, J = 4.7, 7.9 Hz, H-10), 8.35 (dd, 1H, J = 2.2, 8.7 Hz, H-2), 8.66 (dd, 1H, J= 1.6, 7.9 Hz, H-11), 8.78 (dd, 1H, J = 1.6, 4.7 Hz, H-9), 8.89 (d, 1H, J = 2.2 Hz, H-4), 9.38 (d, 1H, J =8.7 Hz, H-1). Anal. Calcd for C13H6ClN5O: C, 55.04; H, 2.13; N, 24.69. Found: C, 55.23; H, 2.48; N, 24.82. 2-Chloro-12-nitrosopyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (2c). Yellow solid; yield 92%; mp: >400 °C; IR (cm-1) 1564; 1H NMR (200 MHz, DMSO-d6) δ: 7.85 (dd, 1H, J = 4.6, 7.9 Hz, H-10), 8.40 (dd, 1H, J = 2.3, 8.7 Hz, H-3), 8.66 (dd, 1H, J = 1.6, 7.9 Hz, H-11), 8.74 (d, 1H, J = 8.7 Hz, H-4), 8.80 (dd, 1H, J = 1.6, 4.6 Hz, H-9), 9.31 (d, 1H, J = 2.3 Hz, H-1). Anal. Calcd for C13H6ClN5O: C, 55.04; H, 2.13; N, 24.69. Found: C, 55.31; H, 2.35; N, 24.54. 3-Methyl-12-nitrosopyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (2d). Yellow solid; yield 97%; mp: >400 °C; IR (cm-1) 1571; 1H NMR (200 MHz, DMSO-d6) δ: 2.09 (s, 3H, CH3),


42

Page 43 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7.80 (dd, 1H, J= 4.7, 7.9 Hz, H-10), 8.15 (dd, 1H, J = 2.3, 8.3 Hz, H-2), 8.54 (d, 1H, J = 2.3 Hz, H-4), 8.69 (dd, 1H, J = 1.5 Hz, 7.9 Hz, H-11), 8.75 (dd, 1H, J = 1.5, 4.7 Hz, H-9), 9.30 (d, 1H, J = 8.3 Hz, H-1). Anal. Calcd for C14H9N5O: C, 63.87; H, 3.45; N, 26.60. Found: C, 63.62; H, 3.61; N, 26.54. 3-Methoxy-12-nitrosopyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (2e). Yellow solid; yield 96%, mp: > 400 °C; IR (cm-1) 1568; 1H NMR (200 MHz, DMSO-d6) δ: 4.14 (s, 3H, CH3), 7.77 (dd, 1H, J = 4.8, 7.9 Hz, H-10), 7.91 (dd, 1H, J = 2.3, 8.9 Hz, H-2), 8.19 (d, 1H, J = 2.3 Hz, H-4), 8.67 (dd, 1H, J = 1.6, 7.9 Hz, H-11), 8.72 (dd, 1H, J = 1.6, 4.8 Hz, H-9), 9.30 (d, 1H, J = 8.9 Hz, H-1). Anal. Calcd for C14H9N5O2: C, 60.21; H, 3.25; N, 25.08. Found: C, 60.43; H, 3.02; N, 25.17. 2,3-Dimethoxy-12-nitrosopyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (2f). Yellow solid; yield 98%; mp: > 400 °C; IR (cm-1) 1552; 1H NMR (200 MHz, DMSO-d6) δ: 4.08 (s, 3H, CH3), 4.17 (s, 3H, CH3), 7.78 (dd, 1H, J = 4.8, 7.8 Hz, H-10), 8.19 (s, 1H, Ar-H), 8.68-8.74 (m, 3H, Ar-H, H-9, H-11). Anal. Calcd for C15H11N5O3: C, 58.25; H, 3.58; N, 22.64. Found: C, 58.49; H, 3.22; N, 22.54.

General procedure for the synthesis of 12-bromopyrido[3',2':4,5]pyrrolo[1,2c][1,2,3]benzotriazines (3a-c,e). To a stirred solution of suitable derivatives 11a-c,e (3.0 mmol) in acetic acid (3.0 mL) a solution of sodium nitrite (3.0 mmol, 0.210 g) dissolved in the minimum amount of water at 0 °C was added dropwise. The reaction mixture was stirred at room temperature for 1 h, then poured into water and the precipitate obtained was filtered off, air-dried and recrystallized from ethanol to give derivatives 3a-c,e.


43


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 58

12-Bromopyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (3a). Yellow solid; yield 99%; mp: >400 °C; 1H NMR (200 MHz, DMSO-d6) δ: 7.67 (dd, 1H, J = 4.6, 7.9 Hz, H-10), 7.85-8.02 (m, 2H, H-1, H-4), 8.18 (d, 1H, J = 7.9 Hz, ArH), 8.28 (d, 1H, J = 7.9 Hz, ArH), 8.68 (d, 1H, J = 4.6 Hz, H-9), 8.79 (d, 1H, J = 7.9 Hz, H-11). Anal. Calcd for C13H7BrN4: C, 52.20; H, 2.36; N, 18.73. Found: C, 51.94; H, 2.06; N, 18 .86. 12-Bromo-3-chloropyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (3b). yellow solid; yield 95%; mp: >400 °C; 1H NMR (200 MHz, DMSO-d6) δ: 7.74 (dd, 1H, J = 4.6, 8.1 Hz, H10), 8.10 (dd, 1H, J = 2.2, 8.6 Hz, H-2), 8.29 (dd, 1H, J = 1.5, 8.1 Hz, H-11), 8.45 (d, 1H, J = 2.2 Hz, H-4), 8.74 (dd, 1H, J = 1.5, 4.6 Hz, H-9), 8.85 (d, 1H, J = 8.6 Hz, H-1). Anal. Calcd for C13H6BrClN4: C, 46.81; H, 1.81; N, 16.80. Found: C, 46.61; H, 2.06; N, 16.64. 12-Bromo-2-chloropyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (3c). Yellow solid; yield 96%; mp: >400 °C; 1H NMR (200 MHz, DMSO-d6) δ: 7.74 (dd, 1H, J = 4.6, 8.1 Hz, H10), 7.98 (dd, 1H, J = 2.2, 8.5 Hz, H-3), 8.30 (dd, 1H, J = 1.5, 8.1 Hz, H-11), 8.37 (d, 1H, J = 8.5 Hz, H-4), 8.75 (dd, 1H, J = 1.5, 4.6 Hz, H-9), 8.80 (d, 1H, J = 2.2 Hz, H-1). Anal. Calcd for C13H6BrClN4: C, 46.81; H, 1.81; N, 16.80. Found: C, 47.03; H, 1.68; N, 16.72. 12-Bromo-3-methoxypyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine (3e). Yellow solid; yield 97%; mp: >400°C; 1H NMR (200 MHz, DMSO-d6) δ: 4.01 (s, 3H, CH3), 7.63-7.73 (m, 2H, H-2, H-10), 7.91 (d, 1H, J = 2.7 Hz, H-4), 8.24 (dd, 1H, J = 1.4, 8.0 Hz, H-11), 8.68 (dd, 1H, J = 1.4, 4.4 Hz, H-9), 8.81 (d, 1H, J = 8.9 Hz, H-1). Anal. Calcd for C14H9BrN4O: C, 51.09; H, 2.76; N, 17.02. Found: C, 51.35; H, 2.47; N, 17.14.

BIOLOGY.


44

Page 45 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chemicals. All the chemicals were purchased from Sigma-Aldrich (Milan, Italy) if not elsewhere indicated. Cell lines. All employed cell lines presented human origin: Jurkat (T-cell leukemia) and CEM cells (T-cell leukaemia) were grown in RPMI-1640 medium; A-431 (vulvar squamous cell carcinoma) were grown in DMEM medium and LoVo cells (intestinal adenocarcinoma) were grown in HAM'S F12 medium. All media were supplemented with antibiotic (115 units/mL penicillin G and 115 µg/mL streptomycin) and 10% heat-inactivated fetal bovine serum (Invitrogen, Milan, Italy). CEMVbl-100 cells are multidrug-resistant lines selected against vinblastine and they were grown in complete RPMI-1640 medium supplemented with vinblastine (0.1 µg/mL).52 LoVoDoxo cells are doxorubicin resistant subclone of LoVo cells and they were grown in complete HAM’s F12 medium supplemented with doxorubicin (0.1 µg/mL).53 Cellular toxicity. Cellular cytotoxicity test was performed in 96-well tissue culture microtiter plate (Falcon, Becton-Dickinson, Italy), in which 5x103 above cited cells were seeded. Plates were incubated at 37 °C in a humidified 5% incubator for 24 h prior to the experiments. After medium removal, 100 µl of the drug solution, dissolved in DMSO and diluted with the suitable complete medium, were added to each well and incubated at 37 °C for 72 h. DMSO concentration was always lower than 1%. Cell viability was assayed by the MTT [(3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] test as described previously.54 Colony formation assay. LoVo cells were plated at 1000 per well in six-well plates to provide an optimal counting density. Cells were treated with compounds at different concentrations for 24 h. After 24 h, medium was replaced with fresh one and cells were cultured for 1 to 2 weeks until well-defined colonies had formed (replacing culture medium every 2 to 3 days). Cells were


45


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 58

stained with crystal violet and colonies of ≥50 cells were then counted visually.55 Data points represent the average of the three values and error bars represent the SEM. Cell Cycle Analysis. For flow cytometric analysis of DNA content, 106 Jurkat cells in exponential growth were treated at different concentrations of the test compound for 24 h. Cells were centrifuged and fixed with ice-cold ethanol (70%), treated with lysis buffer containing RNAase, and then stained with propidium iodide (PI). Samples were analyzed on a BD FACS Calibur (Becton Dickinson, USA). Externalization of Phosphatidylserine. Surface exposure of phosphatidylserine (PS) by apoptotic cells was measured by flow cytometry with a BD FACS Calibur (Becton Dickinson, USA) by adding Annexin V-FITC to cells according to the manufacturer’s instructions (Annexin-V Fluos, Roche Diagnostic, Indianapolis, IN). Simultaneously, the cells were stained with PI. Excitation was set at 488 nm, and the emission filters were at 525 and 585 nm.56 Caspases assay. The activation of caspases-3, -8 and -9 was quantified with a colorimetric assay following the recommended protocol (Sigma-Aldrich, for caspase 3 and 8 and Calbiochem for caspase-9). Jurkat lisates were incubated with different caspase tetrapeptide substrates conjugated with p-nitroaniline, pNA (Ac- DEVD-pNA for caspase-3, Ac-IETD-pNA for caspase-8 and LEHD-pNA for caspase 9). The formation of pNA was measured at 405 nm using a microtiter plate reader (Biorad). Values were normalized to protein content in cell lysates using Bradford method.57 Mitochondrial dysfunction. The mitochondrial membrane potential was measured with the lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine (JC-1, Molecular Probes, USA), as described elsewhere.45 Briefly, after 24 h of treatment, Jurkat cells were collected by centrifugation and resuspended in Hank’s balanced salt solution (HBSS)


46

Page 47 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


containing 1 µM JC-1. The cytofluorimetric analysis was performed collecting green (FL1) and orange (FL2) fluorescence in at least 10000 events for each sample. The production of ROS was measured by flow cytometry by using hydroethidine (HE, Molecular Probes, USA) and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, USA), respectively.58 After 24 h of treatment, the cells were collected by centrifugation and resuspended in HBSS containing the fluorescence probes 2.5 µM HE or 0.1 µM H2DCFDA. The cells were then incubated for 30 min at 37 °C, centrifuged, and resuspended in HBSS. The fluorescence was directly recorded with the flow cytometer by using as excitation wavelength 488 nm and emission at 585 and 530 nm for HE and H2DCFDA, respectively. Lysosome dysfunction: After 24 h of treatment with different concentrations of test compounds, Jurkat cells were stained with acridine orange AO.59 The fluorescence was directly recorded with a flow cytometer (BD FACS Calibur) using 488 nm wavelength as excitation and emission in the FL3 channel.48 Linear Dichroism. LD measurements were performed with a Jasco J500A circular dichroism spectropolarimeter (Jasco, Cremella, Italy), converted for LD and equipped with an IBM PC and Jasco J interface. For these analyses, the sample orientation was obtained using a flow device designed by Wada and Kozawa,60 which presents a cylindrical rotating cuvette, a 0.14 cm optical path and a constant flow of 800 rpm. LD spectra were performed using different [Compound]/[DNA] ratios, dissolved in phosphate buffer (10 mM, pH 7.2). Topoisomerase I relaxation assay. Topoisomerase I assay was adapted from Bridewell et al..61 Assay buffer was made of 50 mM Tris-HCl (pH=7.5), 50 mM KCl, 0.5 mM dithiothreitol (DTT), 10 mM MgCl2, 0.1 mM EDTA, 30 µg/mL of BSA. Incubation mixtures (20 µL), contained assay buffer, compounds at various concentrations, 0.250 µg pBR322 supercoiled


47


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 58

plasmid and 10 unit of human topoisomerase I, were assembled on ice and topoisomerase reaction was performed at 37 °C for 30 min. Reactions were stopped by the addition of prewarmed SDS (final concentration 1%) followed by proteinase K treatment (final concentration 50 µg/mL) for an additional digestion at 37 °C for 30 min. Relaxation assay samples were analysed by electrophoresis on 0.8% agarose gel in TAE buffer at 20 V overnight and then stained with a solution containing 0.5µg/ml of ethidium bromide. DNA bands were visualized by UV light and photographed through a digital photocamera Kodak DC256. Topoisomerase I Cleavable-Complex Assay. The samples for cleavable complex formation assay were prepared in the same way of the relaxation assay but were loaded into a 0.8% agarose gel containing 0.5 µg/mL of ethidium bromide. The gel was run as previously described and then destained in deionized water. DNA bands were visualized by UV light and photographed through a digital photocamera Kodak DC256.

AUTHOR INFORMATION Corresponding Author *(P.D.) Phone: +39-091-23896815. E-mail: [email protected].

Author Contributions ‡B. Parrino and A. Carbone contributed equally.

ACKNOWLEDGMENT This work was financially supported by Ministero dell’Istruzione dell’Università e della Ricerca (MIUR)


48

Page 49 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES 1) Lewgowd, W.; Stanczak, A. Cinnoline derivatives with biological activity. Arch. Pharm. Chem. Life Sci. 2007, 340, 65-80. 2) Palanki, M. S. S.; Cao, J.; Chow, C. P.; Dneprovskaia, E.; Mak, C. C.; McPherson, A.; Pathak, V. P.; Renick, J.; Soll, R.; Zeng, B.; Noronha, G. Development of novel benzotriazines for drug discovery. Exp. Opin. Drug Discovery 2009, 4, 33-49. 3) Yu, Y.; Singh, S. K.; Liu, A.; Li, T-K.; Liu, L. F.; La Voie, E. J. Substituted dibenzo[c,h]cinnolines: topoisomerase I-targeting anticancer agents. Bioorg. Med. Chem. 2003, 11, 1475-1491. 4) Stefańka, B.; Arciemiuk, M.; Bontemps-Gracz, M. M.; Dzieduszycka, M.; Kupiec, A.; Martelli, S.; Borowski, E. Synthesis and biological evaluation of 2,7-dihydro-3Hdibenzo[de,h]cinnoline-3,7-dione derivatives, a novel group of anticancer agents active on a multidrug resistant cell line. Bioorg. Med. Chem. 2003, 11, 561-572. 5) Ruchelman, A. L.; Singh, S. K.; Ray, A.; Wu, X.; Yang, J-M.; Zhou, N.; Liu, A.; Liu, L. F.; La Voie, E. J. 11H-Isoquino[4,3-c]cinnolin-12-ones: novel anticancer agents with potent topoisomerase I-targeting activity and cytotoxicity. Bioorg. Med. Chem. 2004, 12, 795-806. 6) Rewcastle, G. W.; Denny, W. A.; Bridges, A. J.; Zhou, H.; Cody, D. R.; McMichael, A.; Fry, D. W. Tyrosine kinase inhibitors. 5. Synthesis and structure-activity relationships for 4-[(phenylmethyl)amino]-and 4-(phenylamino)quinazolines as potent adenosine 5’triphosphate binding site inhibitors of the tyrosine kinase domain of the epidermal growth factor receptor. J. Med. Chem. 1995, 38, 3482-3487.


49


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 58

7) Hennequin, L. F.; Thomas, A. P.; Johnstone, C.; Stokes, E. S. E.; Plè, P. A.; Lohmann, J-J. M.; Ogilvie, D. J.; Dukes, M.; Wedge, S. R.; Curwen, J. O.; Kendrew, J.; Lambert-van der Brempt, C. Design and structure-activity relationship of a new class of potent VEGF receptor tyrosine kinase inhibitors. J. Med. Chem. 1999, 42, 5369-5389. 8) Hay, M. P.; Gamage, S. A.; Kovacs, M. S.; Pruijn, F. B.; Anderson, R. F.; Patterson, A. V.; Wilson, W. R.; Brown, J. M.; Denny, W. A. Structure-activity relationships of 1,2,4benzotriazine 1,4-dioxides as hypoxia-selective analogues of tirapazamine. J. Med. Chem. 2003, 46, 169-182. 9) Junnotula, V.; Sarkar, U.; Sinha, S.; Gates, K. S. Initiation of DNA strand cleavage by 1,2,4-benzotriazine 1,4-dioxide antitumor agents: mechanicistic insight from studies of 3methyl-1,2,4-benzotriazine 1,4-dioxide. J. Am. Chem. Soc. 2009, 131, 1015-1024. 10) Lv, J-L.; Wang, R.; Liu, D.; Guo, G.; Jing, Y-K.; Zhao, L-X. Design, synthesis, and antitumor activities of some novel substituted 1,2,3-benzotriazines. Molecules 2008, 13, 1427-1440. 11) Ciciani, G.; Coronnello, M.; Guerrini, G.; Selleri, S.; Cantore, M.; Failli, P.; Mini, E.; Costanzo, A. Synthesis of new pyrazolo[5,1-c][1,2,4]benzotriazines, pyrazolo[5,1c]pyrido[4,3-e][1,2,4]triazines and their open analogues as cytotoxic agents in normoxic and hypoxic conditions. Bioorg. Med. Chem. 2008, 16, 9409-9419. 12) Noronha, G.; Barrett, K.; Boccia, A.; Brodhag, T.; Cao, J.; Chow, C. P.; Dneprovskaia, E.; Doukas, J.; Fine, R.; Gong, X.; Gritzen, C.; Gu, H.; Hanna, E.; Hood, J. D.; Hu, S.; Kang, X.; Key, J.; Klebansky, B.; Kousba, A.; Li, G.; Lohse, D.; Mak, C. C.; McPherson, A.; Palanki, M. S. S.; Pathak, V. P.; Renick, J.; Shi, F.; Soll, R.; Splittgerber, U.; Stoughton, S.; Tang, S.; Yee, S.; Zeng, B.; Zhao, N.; Zhu, H. Discovery of [7-(2,6-


50

Page 51 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dichlorophenyl)-5-methylbenzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-ylethoxy)phenyl]amine a potent, orally active Src kinase inhibitor with antitumor activity in preclinical assays. Bioorg. Med. Chem. Lett. 2007, 17, 602-608. 13) Barraja, P.; Diana, P.; Montalbano, A.; Dattolo, G.; Cirrincione, G.; Viola, G.; Vedaldi, D.; Dall’Acqua, F. Pyrrolo[2,3-h]quinolinones: A new ring system with potent photoantiproliferative activity. Bioorg. Med. Chem. 2006, 14, 8712-8728. 14) Barraja, P.; Caracausi, L.; Diana, P.; Carbone, A.; Montalbano, A.; Cirrincione, G.; Brun, P.; Palù, G.; Castagliuolo, I.; Dall’Acqua, F.; Vedaldi, D.; Salvador A. Synthesis of pyrrolo[3,2-h]quinolinones with good photochemotherapeutic activity and no DNA damage. Bioorg. Med. Chem. 2010, 18, 4830-4843. 15) Barraja, P.; Caracausi, L.; Diana, P.; Montalbano, A.; Carbone, A.; Salvador, A.; Brun, P.; Castagliuolo, I.; Tisi, S.; Dall’Acqua, F.; Vedaldi, D.; Cirrincione, G. Pyrrolo[3,2h]quinazolines as photochemotherapeutic agents. ChemMedChem. 2011, 6, 1238-1248. 16) Barraja, P., Diana, P.; Spanò, V.; Montalbano, A., Carbone, A.; Parrino, B.; Cirrincione G. An efficient synthesis of pyrrolo[3′,2′:4,5]thiopyrano[3,2-b]pyridin-2-one: a new ring system of pharmaceutical interest. Tetrahedron 2012, 68, 5087-5094. 17) Barraja, P.; Spanò, V.; Diana, P.; Carbone, A.; Cirrincione, G. Synthesis of the new ring system 6,8-dihydro-5H-pyrrolo[3,4-h]quinazoline. Tetrahedron Lett. 2009, 50, 5389-5391. 18) Barraja, P.; Diana, P.; Montalbano, A.; Carbone, A.; Viola, G.; Basso, G.; Salvador, A.; Vedaldi, D.; Dall’Acqua, F.; Cirrincione, G. Pyrrolo[3,4-h]quinolinones a new class of photochemotherapeutic agents. Bioorg. Med. Chem. 2011, 19, 2326-2341. 19) Carbone, A.; Pennati, M.; Parrino, B.; Lopergolo, A.; Barraja, P.; Montalbano, A.; Spanò, V.; Sbarra, S.; Doldi, V.; De Cesare, M.; Cirrincione, G.; Diana, P.; Zaffaroni, N. Novel


51


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 58

1H-pyrrolo[2,3-b]pyridine derivatives nortopsentin analogues: synthesis and antitumor activity in peritoneal mesothelioma experimental models. J. Med. Chem. 2013, 56, 70607072. 20) Carbone, A.; Parrino, B.; Barraja, P.; Spanò, V.; Cirrincione, G.; Diana, P.; Maier, A.; Kelter, G.; Fiebig H-H. Synthesis and antiproliferative activity of 2,5-bis(3’indolyl)pyrroles, analogues of the marine alkaloid nortopsentin. Marine Drugs 2013, 11, 643-654. 21) Barraja, P.; Spanò, V.; Diana, P.; Carbone, A.; Cirrincione, G. Synthesis of the new ring system

6,8-dihydro-5H-pyrrolo[3,4-h]quinazoline.

Tetrahedron

Lett.

2009,

50,

5389−5391. 22) Diana, P.; Stagno, A; Barraja, P.; Montalbano, A.; Carbone, A.; Parrino, B.; Cirrincione, G. Synthesis of the new ring system pyrrolizino[2,3-b]indol-4(5H)-one Tetrahedron 2011, 67, 3374-3379. 23) Barraja, P.; Caracausi, L.; Diana, P.; Spanò, V., Montalbano, A.; Carbone, A.; Parrino, B.; Cirrincione, G. Synthesis and antiproliferative activity of the ring system [1,2]ozaxolo[4,5-g]indole. ChemMedChem. 2012, 7, 1901-1904. 24) Diana, P.; Carbone, A.; Barraja, P.; Montalbano, A.; Martorana, A.; Dattolo, G.; Gia, O.; Dalla Via, L.; Cirrincione, G. Synthesis and antitumor properties of 2,5-bis(3’indolyl)thiophenes: analogues of marine alkaloid nortopsentin. Bioorg. Med. Chem. Lett. 2007, 17, 2342-2346. 25) Diana, P.; Carbone, A.; Barraja, P.; Martorana, A.; Gia, O.; Dalla Via, L.; Cirrincione, G. 3,5-Bis(3’-indolyl)pyrazoles, analogues of marine alkaloid nortopsentin: synthesis and antitumor properties. Bioorg. Med. Chem. Lett. 2007, 17, 6134-6137.


52

Page 53 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


26) Diana, P.; Carbone, A.; Barraja, P.; Kelter, G.; Fiebig, H-H.; Cirrincione, G. Synthesis and antitumor activity of 2,5-bis(3’-indolyl)-furans and 3,5-bis(3’-indolyl)-isoxazoles, nortopsentin analogues. Bioorg. Med. Chem. 2010, 18, 4524-4529. 27) Diana, P.; Carbone, A.; Barraja, P.; Montalbano, A.; Parrino, B.; Lopergolo, A.; Pennati, M.; Zaffaroni, N; Cirrincione, G. Synthesis and antitumor activity of 3-(2-phenyl-1,3thiazol-4-yl)-1H-indoles and 3-(2-phenyl-1,3-thiazol-4-yl)-1H-7-azaindoles. ChemMedChem. 2011, 6, 1300-1309. 28) Carbone, A.; Pennati, M.; Barraja, P.; Montalbano, A.; Parrino, B.; Spanò, V.; Lopergolo, A.; Sbarra, S.; Doldi, V.; Zaffaroni, N.; Cirrincione, G.; Diana, P. Synthesis and antiproliferative activity of substituted 3[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1Hpyrrolo[3,2-b]pyridines, marine alkaloid nortopsentin analogues. Curr. Med. Chem. 2014, 21, 1654-1666. 29) Lauria, A.; Patella, C.; Diana, P.; Barraja, P.; Montalbano, A.; Cirrincione, G.; Dattolo G.; Almerico, A. M. A new tetracyclic system of biological interest. Indolo[3,2e][1,2,3]triazolo[1,5-a]pyrimidines through domino reactions of 2-azaindole. Heterocycles 2003, 60, 2669-2675. 30) Barraja, P.; Spanò, V.; Diana, P.; Carbone, A.; Cirrincione, G.; Vedaldi, D.; Salvador, A.; Viola, G.; Dall’Acqua, F. Pyrano[2,3-e]isoindol-2-ones, new angelicin heteroanalogues. Bioorg. Med. Chem. Lett. 2009, 19, 1711-1714. 31) Diana, P.; Martorana, A.; Barraja, P.; Montalbano, A.; Dattolo, G.; Cirrincione, G.; Dall’Acqua, F.; Salvador, A.; Vedaldi, D.; Basso, G.; Viola, G. Isoindolo[2,1a]quinoxaline derivatives, novel potent antitumor agents with dual inhibition of tubulin polymerization and topoisomerase I. J. Med. Chem. 2008, 51, 2387-2399.


53


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 58

32) Barraja, P.; Spanò, V.; Giallombardo, D.; Diana, P.; Montalbano, A.; Carbone, A.; Parrino, B.; Cirrincione G. Synthesis of [1,2]oxazolo[5,4-e]indazoles as antitumour agents. Tetrahedron 2013, 69, 6474-6477. 33) Barraja, P.; Diana, P.; Lauria, A.; Passannanti, A.; Almerico, A. M.; Minnei, C.; Longu, S.; Congiu, D.; Musiu, C.; La Colla, P. Indolo[3,2-c]cinnolines with antiproliferative, antifungal, and antibacterial activity. Bioorg. Med. Chem. 1999, 7, 1591-1596. 34) Cirrincione , G.; Almerico, A. M.; Barraja, P.; Diana, P.; Lauria, A.; Passannanti, A.; Musiu, C.; Pani, A.; Murtas, P.; Minnei, C.; Marongiu, M. E.; La Colla, P. Derivatives of the new ring system indolo[1,2-c]benzo[1,2,3]triazine with potent antitumor and antimicrobial activity. J. Med. Chem. 1999, 42, 2561-2568. 35) Diana, P.; Barraja, P.; Lauria, A.; Montalbano, A.; Almerico, A. M.; Dattolo, G.; Cirrincione, G. Pyrrolo[2,1-c][1,2,4,]triazines from 2-diazopyrroles: synthesis and antiproliferative activity. Eur. J. Med. Chem. 2002, 37, 267-272. 36) Barraja, P.; Diana, P.; Lauria, A.; Montalbano, A; Almerico, A. M.; Dattolo, G.; Cirrincione, G. Synthesis and antiproliferative activity of [1,2,4]triazino[4,3-a]indoles. Anticancer Res. 2004, 24, 3775-3780. 37) Diana, P.; Martorana, A.; Barraja, P.; Lauria, A.; Montalbano, A.; Almerico, A. M.; Dattolo, G.; Cirrincione, G. Isoindolo[2,1-c]benzo[1,2,4]triazines: a new ring system with antiproliferative activity. Bioorg. Med. Chem. 2007, 15, 343-349. 38) Barraja, P.; Diana, P.; Carbone, A.; Cirrincione, G. Nucleophilic reactions in the indole series: displacement of bromine under phase transfer catalysis. Tetrahedron 2008, 64, 11625-11631.


54

Page 55 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


39) Cirrincione, G.; Almerico, A. M.; Aiello, E.; Dattolo, G. Polycondensed nitrogen heterocycles. Part 23. Pyrrole[3,2-c]cinnolines by a Japp-Klingemann type reaction. J. Het. Chem. 1990, 27, 983-985. 40) Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug resistance in cancer: role of ATPdependent transporters. Nat. Rev. Cancer. 2002, 2, 48-58. 41) Fadeel, B.; Xue, D. The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease. Crit. Rev. Biochem. Mol. Biol. 2009, 44, 264-277. 42) Ola, M. S.; Nawaz, M.; Ahsan, H. Role of Bcl-2 family proteins and caspases in the regulation of apoptosis. Mol. Cell. Biochem. 2011, 351, 41-58. 43) Brenner, D.; Mak, T. W. Mitochondrial cell death effectors. Curr. Opin. Cell. Biol. 2009, 21, 871-877. 44) Ly, J. D.; Grubb, D. R.; Lawen, A. The mitochondrial membrane potential (∆ᴪm) in apotosis. Apoptosis 2003, 8, 115-128. 45) Salvioli, S.; Ardizzoni, A.; Franceschi, C.; Cossarizza, A. JC-1 but not DiOC6(3) or rhodamine 123 is a reliable fluorescent probe to asses delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 1997, 411, 77-82. 46) Ott, M.; Gogvadze, V.; Orrenius, S.; Zhivotovsky, B. Mitochondria, oxidative stress and cell death. Apoptosis 2007, 12, 913-922. 47) Johansson, A. C.; Appelqvist, H.; Nilsson, C.; Kågedal, K.; Roberg, K.; Öllinger, K. Regulation of apoptosis-associated lysosomal membrane permeabilization, Apoptosis 2010, 15, 527-540.


55


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 56 of 58

48) Zhao, M.; Eaton, J. W.; Brunk, U.T. Protection against oxidant-mediated lysosomal rupture: a new anti-apoptotic activity of Bcl-2? FEBS Lett. 2000, 485, 104-108. 49) Bailly, C. DNA relaxation and cleavage assays to study topoisomerase inhibitors. Methods Enzymol. 2001, 340, 610-623. 50) Ruchelman, A. L.; Singh, S. K.; Ray, A.; Wu, X.; Yang, J.-M.; Zhou, N.; Liu, A.; Liu, L. F.; LaVoie, E. J. 11H-Isoquino[4,3-c]cinnolin-12-ones; novel anticancer agents with potent topoisomerase I-targeting activity and cytotoxicity. Bioorg. Med. Chem. 2004, 12 795-806. 51) Yu, Y.; Singh, S. K.; Liu, A.; Li, T.-K.; Liu, L. F.; LaVoie, E. Substituted dibenzo[c,h]cinnolines: topoisomerase I-targeting anticancer agents. Bioorg. Med. Chem. 2003, 11, 1475-1491. 52) Dupuis, M. L.; Flego, M.; Molinari, A.; Cianfriglia, M. Saquinavir induces stable and functional expression of the multidrug transporter Pglycoprotein in human CD4 Tlymphoblastoid CEMrev cells. HIV Med. 2003, 4, 338-345. 53) Toffoli, G.; Viel, A.; Tuimoto, I.; Bisconti, G.; Rossi, G.; Boiocchi, M. Pleiotropicresistant phenotype is a multifactorial phenomenon in human colon carcinoma cell lines. Br. J. Cancer 1991, 63, 51-56. 54) Mosmann, T. Rapid colorimetric assays for cellular growth and survival: application to proliferation and citotoxic assay. J. Immunol. Methods 1983 65, 55-63. 55) Diana, P.; Stagno, A.; Barraja, P.; Carbone, A.; Parrino, B.; Dall’Acqua, F.; Vedaldi, D.; Salvador, A.; Brun, P.; Castagliuolo, I.; Issinger, O. G.; Cirrincione, G. Synthesis of triazenoazaindoles: a new class of triazenes with antitumor activity. ChemMedChem 2011, 6, 1291-1299.


56

Page 57 of 58

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


56) Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutellingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 1995, 184, 39-51. 57) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. 58) Rothe, G.; Valet, G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescein. J. Leukocyte Biol. 1990, 47, 440-448. 59) Spanò, V.; Montalbano, A.; Carbone, A.; Parrino, B.; Diana, P.; Cirrincione, G.; Castagliuolo, I.; Brun, P.; Issinger, O. G.; Tisi, S.; Primac, I.; Vedaldi, D.; Salvador, A.; Barraja, P. Synthesis of a new class of pyrrolo[3,4-h]quinazolines with antimitotic activity. Eur. J. Med. Chem. 2014, 74, 340-357. 60) Wada, A.; Kozawa, S. Instrument for the studies of differential flow dichroism of polymer solutions. J. Polym. Sci., Part A 1964, 2, 853-864. 61) Bridewell, D. J. A.; Finlay, G. J.; Baguley, B. C. Mechanism of cytotoxicity of N-[2(dimethylamino)ethyl]acridine-4-carboxamide and of its 7-chloro derivative: the roles of topoisomerases I and II. Cancer Chemother. Pharmacol. 1999, 43, 302-308.


57


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 58 of 58

Table of Contents graphic 11H-Pyrido[3',2':4,5]pyrrolo[3,2-c]cinnoline and pyrido[3',2':4,5]pyrrolo[1,2-c][1,2,3]benzotriazine: two new ring systems with antitumor activity Barbara Parrino, Anna Carbone, Marina Muscarella, Virginia Spanò, Alessandra Montalbano, Paola Barraja, Alessia Salvador, Daniela Vedaldi, Girolamo Cirrincione and Patrizia Diana R2

N

N N N

H2N

R1 R

N N R

R N

N H

R1

N

N H

R1

R = H, Cl, Me, OMe; R1 = H, Cl, OMe; R2 = Br, NO


58

cinnoline and Pyrido[3â²,2 - American Chemical Society

Recommend Documents

cinnoline and Pyrido[3â²,2 - American Chemical Society