key: cord-345750-dk1exw9l
authors: Kulikov, A. S.; Epishina, M. A.; Batog, L. V.; Rozhkov, V. Yu.; Makhova, N. N.; Konyushkin, L. D.; Semenova, M. N.; Semenov, V. V.
title: Synthesis and antineoplastic properties of (1H-1,2,3-triazol-1-yl)furazans
date: 2014-01-07
journal: Russ Chem Bull
DOI: 10.1007/s11172-013-0113-2
sha: 
doc_id: 345750
cord_uid: dk1exw9l

A method of 3-amino-4-[5-aryl(heteroaryl)-1H-1,2,3-triazol-1-yl)]furazan synthesis was optimized. Condensation of these compounds with 2,5-dimethoxytetrahydrofuran resulted in a series of previously unknown 4-[5-aryl(heteroaryl)-1H-1,2,3-triazol-1-yl)]-3-(pyrrol-1-yl)furazans. All target compounds were evaluated for both antimitotic microtubule destabilizing effect in a phenotypic sea urchin embryo assay and cytotoxicity in a panel of 60 human cancer cell lines. Pyrrolyl derivatives of triazolylfurazans were determined as antiproliferative compounds. The most potent microtubule targeting compounds 7a and 7e are of interest for further trials as antineoplastic agents.

Five membered heterocycles are frequently used in the synthesis of antimitotics that was studied in detail 1 for the analogs of natural compound combretastatin A 4 (CA 4). A water soluble phosphorylated prodrug of CA 4 is cur rently undergoes clinical trials in the USA as antitumor agent. 2, 3 An introduction of 1,2,3 triazole (1,2,3 tri azolocombretastatin) 4, 5 and furazan (combretafurazan) 6 rings into combretastatin framework was regarded as a non isomerizable and metabolically stable bioisosteric replacement of the double bond in cis stilbenes allowing the synthesis of new promising anticancer compounds.

Compared to combretastatin, combretafurazan is a more potent cytotoxic compound in vitro against neuro blastoma cells, yet maintaining similar pharmacokinetic properties. 6 1,2,3 Triazole bridged combretastatin ana log 4,5 exhibits both strong cytotoxicity against ovarian can cer cells and vascular disrupting activity in tumors. 6 More over, this compound is more water soluble than combre tafurazan.

Water soluble biologically active compounds contain ing both cycles, e.g., (1,2,3 triazol 1 yl)furazans 1, ex hibiting other mechanisms of action were synthesized. Thus, compounds with the (1,2,3 triazol 1 yl)furazan moieties inhibit glycogen synthase kinase (GSK 3), a tar get in the treatment of Alzheimer's disease and type 2 diabetes. 7 Other analogs of (1,2,3 triazol 1 yl)furazans inhibit the SARS CoV M pro cysteine protease, an impor tant enzyme responsible for the intracellular replication of severe acute respiratory syndrome coronavirus. 8a Several (1,2,3 triazol 1 yl)furazan derivatives selectively stimu * Dedicated to Academician of the Russian Academy of Sciences I. P. Beletskaya on the occasion of her anniversary. late NO dependent activation of soluble guanylate cy clase (sGC). 8b In the present work we aimed to study a series of (1,2,3 triazol 1 yl)furazans 1 as potential antineoplastic agents, since these compounds can be synthesized by well elabo rated methods. 9-15 Synthesis of these compounds involved 1,3 dipolar cycloaddition of azidofurazans 2 to various dipolarophiles, e.g., acetylenes, morpholinonitroethylene, or compounds with the activated methylene group, e.g., activated nitriles and 1,3 dicarbonyl compounds. The dis advantage of majority of these methods is the formation of 1,2,3 triazole regioisomers. Depending on the type of the substituents, dipolarophiles add differently to the azidofurazans yielding isomeric 4,5 or 5,4 derivatives (Scheme 1).

The regioselective cycloaddition of aroylacetic esters to azidofurazans was described; in the cycloaddition prod ucts, the aryl substituent and the ester group were located, respectively, at the positions 5 and 4 of the triazole ring. 10 However, only two compounds of this type with Ar = Ph and 4 ClC 6 H 4 synthesized from the corresponding aroyl acetates are known. To provide feasible antineoplastic properties, it is necessary to introduce into the triazol ylfurazan structure either alkoxybenzene moieties or heterocyclic pharmacophores and to remove the es ter group.

Thereby, the aim of the present work was the optimi zation of the synthetic procedure for [5 aryl(hetaryl) 1H 1,2,3 triazol 1 yl]furazan derivatives and biological evaluation of the target compounds for antiprolifera tive properties. It was also necessary to clarify whether the regioselectivity of cycloaddition of azidofurazan to aroyl(hetaroyl)acetates 3 with other aromatic or hetero aromatic substituents will be retained. In addition, to extend the scope of triazolylfurazan derivatives with potential antineoplastic activity, we used the Clauson-Kaas pyrrole synthesis involving the reaction of primary amino group of the furazan ring with dimethoxytetra hydrofuran.

For these purposes, aroylacetic esters 3a-g were in volved in the cyclocondensations with aminoazidofur azan 2a under conditions developed earlier. 10 The 1 H and 13 C NMR spectra of synthesized triazolylfurazans 4a-g indicated formation of single regioisomer in high yield; no signals for the second possible regioisomer were detected. The spectral characteristics also indicated high purity of the crude products. Therefore, unpurified esters 4a-g were hydrolyzed to the corresponding acids 5a-g, which also without further purification were subsequently thermally decarboxylated to target 3 amino 4 [5 aryl(hetaryl) 1H 1,2,3 triazol 1 yl]furazans 6a-g in high yields. Thus, we developed a preparative procedure for the synthesis of tri azolylfurazans 6a-g without purification of the interme diates (Scheme 2).

To transform the amino group of triazolylfurazans 6 into the pyrrole ring, compounds 6a-f and 6h 15 were involved in the Clauson-Kaas pyrrole synthesis with 2,5 dimethyltetrahydrofuran. The reaction was carried out in refluxing acetic acid. 14 Pyrrole containing tri azolylfurazans 7a-f,h were obtained in 64-98% yields (Scheme 3). 

The biological activity of seven triazolylfurazan deriv atives bearing amino groups 6a-d,g,i, ester 4c (a precur sor of compound 6c), and seven pyrrole derivatives 7a-f,h was studied. The initial trials were carried out on the sea urchin embryos widely used as a model in screening for compounds with antiproliferative effect. 16,17 Re cently a simple and efficient pheno typic sea urchin embryo assay has been developed. The assay allows identification of compounds with antiproliferative properties and pro vides information about the mech anism of antimitotic activity. 18 Specific changes of sea urchin embryo swimming pattern, namely, settlement to the bottom of the culture vessel and rapid spinning around the animal-vegetal axis, suggest a microtubule destabi lizing activity of a tested compound.* Typical develop mental abnormalities caused by triazolylfurazan 7 are shown in Fig. 1 .

Note that the compounds at effective concentrations leading to the alteration of sea urchin egg cleavage were comparable with the IC 50 for the cultured mammalian and human tumor cells. 18, 19 Target compounds were further selected for cytotoxicity test in 60 human tumor cell lines (Developmental Therapeutics Program at the National Cancer Institute of USA). The results are given in Table 1 .

Triazolylfurazans 6a-d,g,i bearing alkoxybenzene moieties and the unsubstituted amino group, as well as ester 4c, did not affect cell division in both test systems. Their analogs 7a-f,h bearing the pyrrole ring instead of the amino group exhibited moderate activity. The most potent compounds 7a and 7e altered cleavage of the sea urchin eggs at concentration of 50 nmol L -1 . Compound 7e caused the sea urchin embryo spinning suggesting the antitubulin mechanism of action, namely, the ability to destabilize microtubules. Apparently, compound 7a ex hibited similar mechanism of action. Although, compound 7a failed to affect the sea urchin embryo swimming, the arrested eggs acquired tuberculate shape typical of micro tubule destabilizers. 18 The pyrrole ring was shown to be essential for antiproliferative effect, since the related struc tures 6 containing the amino group instead of the pyrrole ring were inactive. It is worth noting that the increase in the number of methoxy groups in the benzene ring (com pounds 7a-d) resulted in reduction of the antimitotic properties. In this respect, triazolylfurazans 7 is distin guished from the known analogs of plant antimitotics com bretastatin and podophyllotoxin interacting with the col chicine site of tubulin. Specifically, trimethoxybenzene According to the data of the National Cancer Institute (NCI) of USA, compounds 7a and 7e inhibited cancer cell growth at relatively low concentrations (GI 50 = 389 and 295 nmol L -1 , respectively). These compounds were referred to the biological expert committee of NCI as promising for further studies. Leukemia SR cells (7a), melanoma MDA MB 435 cells (7a and 7e) , and the co lon cancer cells (7e) were the most sensitive to triazolyl furazans 7a and 7e. The "doze-effect" curves for seven colon cancer cell lines exposed to compound 7e are given in Fig. 2 .

In summary, the preparative synthesis of furazans 6 by 1,3 dipolar cycloaddition of azidoaminofurazan 2a to aroyl(hetaroyl)acetates 3a-g followed by further modifi cation was developed. The procedure does not require pu rification of the intermediates. Subsequent Clauson-Kaas condensation of synthesized triazolylfurazans 6 with dimethoxytetrahydrofuran yielded a series of 4 [5 aryl (hetaryl) 1H 1,2,3 triazol 1 yl] (3 pyrrol 1 yl)furazans 7. The antiproliferative properties of both types of compounds were evaluated using the sea urchin embryo assay. It was found that the amino derivatives of triazolylfurazans 6 failed to affect cell division. However, their analogs 7 bear ing the pyrrole ring exhibited moderate antimitotic activi ty. Two compounds, 7a and 7c, were referred to the NCI biological expert committee as promising compounds for further trials.

NMR spectra were recorded on Bruker WM 250 ( 1 H, 250 MHz) and Bruker AM 300 ( 13 C, 75.5 MHz) spectrometers. Chemical shifts are given in the  scale relative to Me 4 Si (inter nal standard). Mass spectra were obtained on a Varian MAT CH 6 instrument (EI, 70 eV). Thin layer chromatography was per formed on Silufol UV 254 plate (elution with CHCl 3 ), spots were visualized under UV light. Elemental analyses were carried out on a Perkin-Elmer 2400 CHN analyzer.

Ethyl aroylacetates with 4 methoxyphenyl (3a), 3,4 di methoxyphenyl (3b), 3,4,5 trimethoxyphenyl (3c), 4 fluoro phenyl substituents (3f) were commercially available (Aldrich). Synthesis of ethyl 1 (4 aminofurazan 3 yl) 5 R 1H 1,2,3 triazole 4 carboxylates 4a-g (general procedure). A mixture of 4 azidofurazan 3 amine 2a (0.88 g, 7 mol), aroylacetate 3a-g (7 mmol), and MgCO 3 (0.34 g, 4 mmol) in ethanol (20 mL) was refluxed for 8-10 h (until complete consumption of 2a, TLC monitoring). The reaction mixture was filtered hot, the solvent was evaporated in vacuo. The precipitate was filtered off, washed with cold EtOH, and dried in air.

Ethyl 1 (4 aminofurazan 3 yl) 5 (4 methoxyphenyl) 1H 1,2,3 triazole 4 carboxylate (4a). The yield was 96%, m.p. 13 Synthesis of 1 (4 aminofurazan 3 yl) 5 R 1H 1,2,3 tri azole 4 carboxylic acids 5a-g (general procedure). A solution of NaOH (0.5 g, 12.5 mol) in water (50 mL) was added to ethyl 1 (4 aminofurazan 3 yl) 5 R 1H 1,2,3 triazole 4 carboxylate 4a-g (7 mmol). The reaction mixture was refluxed for 1 h, the undissolved residue was filtered off, and the filtrate was acidified with dilute HCl to pH 2. A precipitate was filtered off, washed with water, and dried in air.

When crude carboxylic acids 4a-g (unwashed with EtOH) were used, the yields of products 5a-g were virtually the same. 

82 (br.s, 4 H, 2 H in Ar and 2 H, NH 2 ). 13 C NMR (DMSO d 6 ), : 56.03 (OMe)

Aminofurazan 3 yl) 5 (3,4 methylenedioxyphenyl) 1H

3 triazole 4 carboxylic acid (5d)

Aminofurazan 3 yl) 5 (4 ethoxyphenyl) 1H 1,2,3 tri azole 4 carboxylic acid (5e). The yield was 89%, m

13 C NMR (DMSO d 6 ), : 13.72 (Me)

Aminofurazan 3 yl) 5 (4 fluorophenyl) 1H 1,2,3 tri azole 4 carboxylic acid (5f). The yield was 79%, m

Aminofurazan 3 yl) 5 (2 thienyl) 1H 1,2,3 triazole 4 carboxylic acid (5g). The yield was 66%, m

28; N, 30.12. C 9 H 6 N 6 O 3 S

17; N, 30.20. MS, m/z (I rel (%)

2 Hz); 7.73 (d, 1 H, C(5) thiophene ring, 3 J = 5.2 Hz)

The yield was 71%, m

.81; N, 32.43. C 11 H 10 N 6 O 2

: 3.79 (s, 3 H, OMe); 6.65 (s

13 C NMR (DMSO d 6 )

OMe)

: 3.70 (s, 3 H, OMe); 3.73 (s, 6 H, 2 OMe); 6.74 (s, 2 H in Ar)

DMSO d 6 ), : 56.04 (OMe); 193 C

.96; N, 30.87. MS, m/z (I rel

: 6.08 (s, 2 H, CH 2 )

The yield was 63%, m

The yield was 68%, m.p. 191-192 C

: 6.63 (s, 2 H, NH 2 ); 7.22, 7.49 (both d, 2 H each

The yield was 73%, m.p

) thiophene ring

.79; N, 27.39. C 15 H 12 N 6 O 2

68 (br.s, 2 H, C(2) and C(5) of pyrrole ring)

13 C NMR (CDCl

: 56.09 (OMe)103 C

) and C(5) of pyrrole ring

: 1.31 (t, 3 H, Me, 3 J = 7.0 Hz); 4.05 (q, 2 H, CH 2 , 3 J = 7.0 Hz); 6.35 (s, 2 H, C(3) and C(4) of pyrrole ring); 6.83 (s, 2 H, C(2) and C(5) of pyrrole ring)

37 (s, 1 H, triazole ring)

98; N, 28.19. C 14 H 9 FN 6 O

35 (s, 2 H, C(3) and C(4) of pyrrole ring); 6.83 (s, 2 H, C(2) and C(5) of pyrrole ring)

Institute of Developmental Bi ology of RAS in Cyprus. Adult sea urchins

was carried out at the following devel opmental steps: (1) fertilized eggs, 8-15 min after fertilization

47, 3275; (b) Pat. RF 2158265, Byul. Isobret

Modern Analysis of Antibiotics

Bioorganic Marine Chemistry

BioTech niques

Cytotoxicity in 60 human cancer cell lines was studied at the National Cancer Institute of USA according to the procedure described at http://dtp.nci.nih.gov/branches/btb/ivclsp.html.The authors are grateful to the National Cancer Insti tute (Bethesda, Maryland, USA) for the screening of com pounds in the framework of the Developmental Thera peutics Program for the search of antineoplastic medici nal agents; http://dtp.cancer.gov).