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EurJMC 2015

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EurJMC 2015

  1. 1. Accepted Manuscript A facile synthesis and microtubule-destabilizing properties of 4-(1H- benzo[d]imidazol-2-yl)-furazan-3-amines Andrei I. Stepanov, Alexander A. Astrat’ev, Aleksei B. Sheremetev, Nataliya K. Lagutina, Nadezhda V. Palysaeva, Aleksei Yu. Tyurin, Nataly S. Aleksandrova, Nataliya P. Sadchikova, Kyrill Yu. Suponitsky, Olga P. Atamanenko, Leonid D. Konyushkin, Roman V. Semenov, Sergei I. Firgang, Alex S. Kiselyov, Marina N. Semenova, Victor V. Semenov PII: S0223-5234(15)00147-6 DOI: 10.1016/j.ejmech.2015.02.051 Reference: EJMECH 7733 To appear in: European Journal of Medicinal Chemistry Received Date: 11 October 2014 Revised Date: 18 January 2015 Accepted Date: 27 February 2015 Please cite this article as: A.I. Stepanov, A.A Astrat’ev, A.B Sheremetev, N.K. Lagutina, N.V. Palysaeva, A.Y. Tyurin, N.S. Aleksandrova, N.P. Sadchikova, K.Y. Suponitsky, O.P. Atamanenko, L.D. Konyushkin, R.V. Semenov, S.I. Firgang, A.S. Kiselyov M.N. Semenova, V.V. Semenov, A facile synthesis and microtubule-destabilizing properties of 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.051. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
  2. 2. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 1 A facile synthesis and microtubule-destabilizing properties of 4-(1H- benzo[d]imidazol-2-yl)-furazan-3-amines Andrei I. Stepanov,a Alexander A. Astrat’ev,a Aleksei B. Sheremetev,b Nataliya K. Lagutina,c Nadezhda V. Palysaeva,b Aleksei Yu. Tyurin,b Nataly S. Aleksandrova,b Nataliya P. Sadchikova,c Kyrill Yu. Suponitsky,d Olga P. Atamanenko,b Leonid D. Konyushkin,b Roman V. Semenov,b Sergei I. Firgang,b Alex S. Kiselyov,e Marina N. Semenova,f Victor V. Semenovb,* a Special Design and Construction Bureau SDCB “Technolog”, 33-A Sovetskii Ave., Saint Petersburg, 192076, Russian Federation b N. D. Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 119991 Moscow, Russian Federation c I. M. Sechenov First Moscow State Medical University, Trubetskaya Str. 8-2, 119991 Moscow, Russian Federation d A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov Str., 119991 Moscow, Russian Federation e Department of Biological and Medicinal Chemistry, Moscow Institute of Physics and Technology, Institutsky Per. 9, Dolgoprudny, Moscow Region, 141700, Russian Federation f N. K. Kol’tsov Institute of Developmental Biology, RAS, Vavilov Str., 26, 119334 Moscow, Russian Federation Corresponding author: Victor V. Semenov Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991, Moscow, Russian Federation. Tel.: +7 916 620 9584; fax: +7 499 137 2966. E-mail: vs@zelinsky.ru
  3. 3. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 2 E-mail addresses: Andrei I. Stepanov stepanoff@pisem.net Alexander A. Astrat’ev astrchim@yandex.ru Aleksei B. Sheremetev sab@ioc.ac.ru Nataliya K. Lagutina mpcpr@yandex.ru Nadezhda V. Palysaeva naduasha.85@mail.ru Aleksei Yu. Tyurin tyurin@ioc.ac.ru Nataly S. Aleksandrova natali.aleksandrova.50@mail.ru Nataliya P. Sadchikova cska76@gmail.com Kyrill Yu. Suponitsky kirshik@yahoo.com Olga P. Atamanenko info@chemblock.com Leonid D. Konyushkin LeonidK@chemical-block.com Roman V. Semenov rs@chemical-block.com Sergei I. Firgang sfirgang@yandex.ru Alex S. Kiselyov akiselyov@chemdiv.com Marina N. Semenova ms@chemical-block.com Victor V. Semenov vs@zelinsky.ru
  4. 4. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 3 ABSTRACT A series of 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines (BIFAs) were prepared in good yields (60–90% for each reaction step) via a novel procedure from aminofurazanyl hydroximoyl chlorides and o-diaminobenzenes. The synthetic sequence was run under mild reaction conditions, it was robust and did not require extensive purification of intermediates or final products. Furthermore, there was no need for protection of reactive moieties allowing for the parallel synthesis of diverse BIFA derivatives. Subsequent biological evaluation of the resulting compounds revealed their anti- proliferative effects in the sea urchin embryo model and in cultured human cancer cell lines. The most active compounds showed 0.2–2 µM activities in both assay systems. The unsubstituted benzene ring of the benzoimidazole template as well as the unsubstituted amino group in the furazan ring were essential prerequisites for the antimitotic activity of BIFAs. Compound 57 bearing the 2-chlorophenyl acetamide substituent at the nitrogen atom of the imidazole ring was the most active molecule in the examined set. Keywords: Benzoimidazolfurazanamines Inhibitors of tubulin polymerization Sea urchin embryo Cytotoxicity Abbreviations: BIFA, 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amine; SAR, structure-activity relationship.
  5. 5. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 4 1. Introduction Molecules exhibiting 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amine (BIFA) scaffold have attracted considerable attention of medicinal chemists in the past decade. This core is found in multiple inhibitors of protein kinases, enzymes that represent an important class of cellular drug targets in the treatment of hypertension, neoplastic, autoimmune, neurodegenerative, and inflammatory diseases [1]. BIFA derivatives blocking glycogen synthase kinase GSK-3 signaling were introduced as agents for the treatment of diabetes, Alzheimer’s disease, and as immunomodulators [2]. BIFA-based inhibitors of p60 ribosomal S6 kinase 1 (RSK1) involved in the cell cycle regulation [3] were suggested as promising antitumor agents [4]. BIFAs were reported to work as potent selective modulators of ribosomal p70S6 kinase that control cell growth [5]. They were also shown to suppress the activity of mitogen and stress-activated rho-kinase (MSK-1, ROCK 1 and 2) [6] involved in apoptosis, cell proliferation and migration. Basilea team completed synthesis and screening of a series of BIFAs (Fig. 1, I) [7]. Based on their cytotoxicity and proapoptotic properties, BIFAs were proposed as agents for the treatment of various malignancies and autoimmune disorders [7–9]. BAL27862 (Fig. 1) exhibiting low nM cytotoxicity across multiple cancer cell lines [7,10–12] was selected for further optimization to yield a water-soluble prodrug BAL101553 (Fig. 1) [8]. This compound is currently undergoing phase II clinical trials as both an antimitotic and vascular targeting agent [13]. Insert Fig. 1. N N N O N NH O R1 R2 N N N O N NH O NH2 CN N N N O N NH O NH CN NH2 O NH2 NH N OH N H3CO O H3CO N OH O O O OCH3 H Vinblastine H3CO H3CO H3CO OCH3 O NH O Colchicine I BAL27862 BAL101553 Fig. 1. Structures of reported BIFAs and reference compounds colchicine and vinblastine. Mechanism of BIFAs anti-proliferative activity has been tied to microtubule impairment. For example, BAL27862 caused unique alterations of interphase and mitotic spindle microtubules in cultured cancer cells [10,15]. The compound was shown to inhibit purified tubulin polymerization and to bind tubulin dimers at the colchicine site [16]. Despite of this molecular interaction, the specific effect of BAL27862 on microtubule dynamics differed from those of colchicine and vinblastine suggesting its novel microtubule destabilizing mode of action [16]. It should be noted that of the reported BIFAs the anti-tubulin mechanism was proved only for
  6. 6. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 5 BAL27862. The indirect anti-tubulin effect of BIFAs could be also mediated by GSK-3 known to affect microtubule stability, mitotic spindle formation and orientation [14]. Due to the diverse biological activity of BIFAs, we developed a robust protocol yielding a library of respective derivatives and evaluated their microtubule destabilizing activity using the in vivo sea urchin embryo model. Selected compounds were also studied in vitro using tubulin polymerization assay, cell cycle distribution analysis, and further screened against a panel of human cancer cell lines to assess their cytotoxicity. BIFA scaffold 9 was first reported by Tselinskii et al. in 2001 [17]. To date, there are three main routes towards 9 described in the literature (Scheme 1). Route A [17] involves treatment of (un)substituted o-phenylenediamines 3 with carbimidate 2 [18] easily accessible from the amidoxime 1. Route B [6] is based on recyclization of 5 upon heating with o-phenylenediamines 3 in acetic acid. Route C [2,8] employs condensation of 3 with ethylcyanoacetate at high temperature to yield 6 [19] followed by its sequential conversion to cyano oxime 7, amidoxime 8 and finally, the targeted aminofurazan derivative 9 [20–22]. Insert Scheme 1. CH2(CN)2 NC CN NOH NN O NH2 NH2 NOH NN O NH2 CN NN O NH2 MeO NH N NMe OH NH2 N NMe OH NH2 NO N N N O N Me OH NH2 NHR e g 1 3 8 2 5 3 4 6 7 9 Route A Route B Route C h a b c d N N CN R2 N N CN R2 NOH N N R2 NOH NOH NH2 3 NH2 NHR R1 N N R2 N O N NH2 fa a b e R1 R1R1 R1R1 Scheme 1. Reported syntheses of BIFA scaffold. Reagents and conditions: (a) NaNO2, H+ ; (b) NaOH, NH2OH⋅HCl, H2O, reflux [18]; (c) Pb2O3, AcOH; (d) MeOH, HCl or MeOH, MeONa; (e) o-phenylenediamine 3, EtOH, reflux, 20 h; (f) Pb(OAc)4, AcOH, r.t.; (g) NCCH2CO2Et, 100–190 °C; (h) NaOH, H2O, reflux. All three strategies summarized in Scheme 1 afford poor yields of the targeted compounds 9. They require elevated temperatures, extensive purification of intermediates, convoluted synthetic manipulations and toxic or costly reagents. In this study, we developed a robust and rapid access to
  7. 7. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 6 a library of diverse BIFAs. The described protocol does not employ anhydrous solvents, inert atmosphere, toxic chemicals and chromatographic purification of the targeted molecules. 2. Results and discussion 2.1. Chemistry Benzimidazoles are an important class of biologically active compounds [23–25]. A plethora of methods exist for their syntheses, including condensation of hydroximoyl chloride and unsubstituted o-diaminobenzene. This procedure was originally described by Sasaki et al. for aryl hydroximoyl chloride [26] and further expanded by Paton et al. onto carbohydrate-derived hydroximoyl chloride [27,28]. Considering these results, we turned our attention to optimize the reaction of o-diaminobenzenes 3 and 4-aminofurazan-3-carbohydroximoyl chloride 10 [29,30] (Scheme 2), easily available from amidoxime 1 and sodium nitrite in HCl. We found that the reaction of 10 with o-diaminobenzene (1:2 molar ratio) in ethanol at 60 °C for 0.5 h afforded the benzoimidazole 9a in 81% yield after work-up and recrystallization. Using these conditions, we evaluated the reaction of hydroximoyl chloride 10 with a variety of o-diaminobenzenes 3a–n (Scheme 2). The desired BIFA derivatives 9a–n were obtained in moderate to good yields for both electron-rich and electron-deficient o-diaminobenzenes 3a–n. Condensation of 10 with o-Me- substituted o-diaminobenzenes 3c,d furnished 9c,d as a mixture of two isomers exhibiting a defined proton position(s) at different N-atoms in the imidazole ring. The existence of isomers was further confirmed by a doubling of signals in 1 H and 13 C NMR spectra. N-substituted o-diamines 3o,p were also effective reagents in condensation with hydroximoyl chloride 10 (Scheme 2) to afford 26 and 29. BIFAs 9a,h,i were condensed with 2,5- dimethoxytetrahydrofuran to afford pyrrole derivatives 11a,h,i (Scheme 2). Insert Scheme 2.
  8. 8. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 7 R1 R2 R4 63: H Et pMeO-C6 H4 - 64: Cl H C6H5-CH2- c d e NN O NH2 Cl NOH 10 NN O NH2 NH2 NOH 1 NN O NHR4 Cl HON 62v,w NN O NH2 R4HN NOH 60v-x NN O NHR4 NH2 HON 61v-x 59v-x R4NH2 59-70% 78-82% 60-87% N H N N O N NH2 9a-n NH2 NHR2 3o,p 12'-57' R2Hal NH2 NH2 3a-n 54-92% a 70-90% b a R3COCl or (R3CO)2O 58r-u N N R2 N O N NH2 12-57 g 70-80% N N R2 N O N NH R3 O 14r,16s,19r,20r,24r, 25r,26t,28r,32r,34s, 38r,39r,u,40r,51t R1 N N R2 N O N N 11a,h,i O OMeMeO g R1 R1 R1 R1 R1 3f,q NH2 NHR2 R1 N N R2 N O N R4HN R1 f 59-70% o-Phenylenediamines (3a-q) NH2 NH2 NH2 NH2 F a e NH2 NH2 Cl f NH2 NH2 MeONH2 NH2 Cl hg NH2 NH2 NH2 NH2 NH2 NH2 NH2 NH2 MeO MeOb c d i NH2 NH2 NC NH2 NH F3C OMe NH2 NH2 NH2 NH2 NO N NH2 NH2 MeOOC k l m n NH2 NHMe NH2 NHEt p qo NH2 NH2 HOOC j Acid anhydrates or chloroanhydrates R3COCl, (R3CO)2O (58r-u) u N Cl O F Cl O ts O EtOEt O r Cl O Me O MeOMe O w Amines R4NH2 (59v-x) NH2 MeO MeO NH2 x NH2 v Cl O Et Scheme 2. Facile synthesis of BIFAs. Reagents and conditions: (a) hydroximoyl chloride 10, o- diaminobenzene 3, EtOH, 60 °C, 0.5 h; 5 h for 9c; (b) R2Hal, K2CO3, DMF, 50−90 °C, 4–12 h; (c) R4NH2, NEt3, EtOH, i-PrOH, r.t., 3 h; (d) KOH, (CH2OH)2, reflux, 4 h; (e) NaNO2, HCl, AcOH, ≤10 °C, 3 h, r.t., 1 h; (f) o-phenylenediamine 3f,q, EtOH, reflux, 0.5 h, r.t., 1 h; (g) R3COCl, toluene, reflux, 8–20 h; (R3CO)2O, AlkCOONa, reflux, 3 h. Alkylation of scaffold 9 proceeded regioselectively at a nitrogen atom of the benzimidazole ring, whereas the amino group in the furazan ring was not affected. N-substituted benzimidazoles 12–57 (Table 1) were prepared from the unsubstituted precursors 9a–n and respective alkyl halides 12′′′′–57′′′′ (Scheme 2). BIFAs 12–57 exhibiting free amino group in the furazan ring were coupled with the appropriate acid chlorides or anhydrides 58r–u to form amides 14r, 16s, 19r, 20r, 24r, 25r, 26t, 28r, 32r, 34s, 38r, 39r,u, 40r, and 51t (Scheme 2; Table.1).
  9. 9. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 8 Aminofurazans 63 and 64 were synthesized via the recyclization route (Scheme 2). Treatment of hydroximoyl chloride 10 with amines 59v–x in the presence of NEt3 yielded the corresponding amidoximes 60v–x. These were converted to the isomeric amidoximes 61v–x via a recyclization reaction [31–33] in refluxing ethylene glycol with KOH. The corresponding hydroximoyl chlorides 62v,w were synthesized via deazotization of 61v,w with sodium nitrite in HCl. Condensation of the hydroximoyl chloride 62v,w with 1,2-diaminobenzenes in ethanol at 60 °C for 1 h afforded the desired benzoimidazoles 63 and 64 in moderate yields. Cyclization conditions were similar for substituted (3o–q) and unsubstituted (3a–n) o-phenylenediamines (Scheme 2, a and f). Treatment of compound 9a with chloroacetonitrile gave cyanomethyl derivative 40. It was reacted with sodium azide in hot DMF to give tetrazole 65. Alternatively, the nitrile group of compound 40 was coupled with thiosemicarbazide at reflux in trifluoroacetic acid to yield of 2- amino-1,3,4-thiadiazole derivative 66 (Scheme 3). Insert Scheme 3. N N N O N NH2 CN N N N O N NH2 N N NH N N N N O N NH2 N N S NH2 a b 63% 71% 4065 66 Scheme 3. Synthesis of N-(hetarylmethyl) benzoimidazol derivatives 65 and 66. Reagents and conditions: (a) NaN3, NH4Cl, DMF, 100 °C, 8 h; (b) H2NC(S)NHNH2, CF3COOH, reflux, 8 h. The structures of all synthesized products were confirmed by spectroscopy. Both 1 H and 13 C NMR data were consistent with the presence of furazan and benzimidazole moieties. Specifically, for the N-unsubstituted benzimidazole group, there were two distinct signals at ca. 138 ppm and ca. 124–130 ppm for C-2 and C-5/6 respectively, notably signals corresponding to other carbon atoms were broadened and overlapped. This phenomenon has been described for benzimidazoles earlier [34]. It was explained by the rapid proton exchange between N-1 and N-3 atoms. Signals at ca. 140 (C-C-NH2) and ca. 155 ppm (C-NH2) were attributed to carbons of the furazan ring [31,35]. Structures of benzimidazoles 27 and 29 were unequivocally established by X-ray crystallography (Fig. 2). Experimental details are given in Supplementary data. Insert Figure 2.
  10. 10. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 9 Fig. 2. Molecular structure of compounds 27 and 29 showing the atom numbering scheme. Displacement ellipsoids are drawn at the 50% probability level. The N–O bonds in the furazan ring of both structures showed different length (N1–O1 of 1.40–1.41 Å, N2–O1 of 1.37–1.38 Å) affected by substituents [36]. Crystal packing of 27 and 29 and the details of X-ray data collection are presented in Fig. S2 and Table S1, Supplementary data [37]. 2.2. Biological effects 2.2.1. Antiproliferative activity in the sea urchin embryo model The synthesized BIFA analogs were evaluated for their antiproliferative activity using in vivo phenotypic sea urchin embryo assay [38]. This assay has been extensively validated in our lab to afford a reliable insight into specific antimitotic, cytotoxic, and microtubule destabilizing effects of tested compounds. A typical experimental protocol includes (i) fertilized egg test for antimitotic activity displayed by cleavage alteration/arrest, and (ii) swimming pattern observation of blastulae treated by compounds after hatching. The lack of forward movement, settlement to the bottom of the culture vessel, and rapid spinning of embryos around the animal–vegetal axis suggests a microtubule destabilizing activity caused by a molecule (video illustrations are available at http://www.chemblock.com). The attainment of specific tuberculate shape of arrested eggs, which is typical for microtubule destabilizing agents, is considered an indirect evidence of targeting tubulin/microtubules [38–40]. The test results are listed in Table 1. Colchicine and vinblastine sulfate served as reference microtubule destabilizing compounds. Insert Table 1.
  11. 11. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 10 Table 1. Structures of BIFAs and their effects on sea urchin embryos and human cancer cells. Compd R1 R2 R3 Sea urchin embryo effects, EC (µM)a NCI60 screen Cleavage alteration Cleavage arrest Embryo spinning Mean GI50, µMb Mean cell growth, %c Colchicine 50 100 TEd 50 0.132e Vinblastine 0.1 0.2 TEd 2 0.00137f BAL 27862 0.0065–0.017g 9a H H – 2 >4 >4 102.97 11a H H R3C(O)NH=Pyrrole >4 >4 >4 104.11 9b 5-Me H – NDh 9c 4-Me H – NDh 9d 4,5-diMe H – NDh 9e 5-F H – 2 >4 >5 97.83 9f 5-Cl H – 2 >4 >5 97.13 R3COCl or (R3CO)2O 58r-u 9-57 N N R2 N O N NH R3 O 14r,16s,19r,20r,24r, 25r,26t,28r,32r,34s, 38r,39r,u,40r,51t R1N N R N O N H2N 2 1 R 2 4 7 6 5 1 3O OMeOMe N N R2 N O N N 11a,h,i R1
  12. 12. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 11 9g 4-Cl H – 1 >4 >4 99.8 9h 5-OMe H – 0.5 >4 >5 101.01 11h 5-OMe H R3C(O)NH=Pyrrole 2 >4 >4 104.43% 9i 4,7-diOMe H – 2 >4 >4 NDh 11i 4,7-diOMe H R3C(O)NH=Pyrrole NDh 9j 5-COOH H – NDh 9k 5-COOMe H – NDh 9l 5-CN H – >4 >4 >4 107.07% 9m 6 5 H – 0.5 4 >4 NDh 9n NO N 6 7 H – NDh 12 H OH – NDh 102.07 13 H CH – >4 >4 >4 99.56 14 H CH2 – NDh 14r H CH2 Me >4 >4 >4 96.53 15 H F – 1 >4 >4 3.09 16 H F – 0.5 >4 >4 NDh
  13. 13. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 12 16s H F Et 4 >4 >4 7.76 17 H F – 2 >4 >4 8.91 18 H Br – 0.5 >4 2 0.603 19 H F3C – 2 >4 >4 0.676 19r H F3C Me >4 >4 >4 82.18 20 H MeO – 0.2 2 TEd >5 0.457 20r H MeO Me 1 >4 >4 3.98 21 H MeO Me – 0.2 2 TEd >4 0.479 22 H EtO EtO – >4 >4 >4 80.97 23 H Me – NDh 70.38 24 H t-Bu – >4 >4 >4 67.38
  14. 14. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 13 24r H t-Bu Me NDh 73.48 25 H NC – 4 >4 >4 79.81 25r H NC Me >4 >4 >4 94.08 26 H Me – NDh 26t H Me F NDh 99.5 27 H Et – NDh 28 H MeO – NDh 28r H MeO Me >4 >4 >4 84.96 29 5-CF3 MeO – >4 >4 >4 81.06 30 H Cl – NDh 3.39 31 H Cl – >4 >4 >4 84.97 32 H Cl Cl – NDh
  15. 15. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 14 32r H Cl Cl Me NDh 76.68 33 H F Cl – >4 >4 >4 91.9 34 H F Cl – NDh 34s H F Cl Et >4 >4 >4 64.55 35 H Cl O O – >4 >4 >4 88.34 36 H Br O O – >4 >4 >4 86.07 37 H – 0.2 2 TEd 2 NDh 38 H N – 2 >4 >4 89.31 38r H N Me >4 >4 >4 103.6
  16. 16. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 15 39 H N – NDh 96.96 39r H N Me >4 >4 >5 101.38 39u H N N >4 >4 >4 97.07 40 H NC – >4 >4 >4 102.45 40r H NC Me NDh 65 H NN NH N – >4 >4 >4 102.74 66 H NN SNH2 – NDh 41 H N N N NH2 NH2 – >4 >4 >4 101.61 42 H N N N NH2 NMe2 – 2 >4 >4 80.77 43 H N N N NH2 N – 4 >4 >4 87.71
  17. 17. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 16 44 H NH O Me Me – >4 >4 >4 108.54 45 H NH O Me Me – >4 >4 >4 90.2 46 H NH O MeO OMe MeO – 1 4 TEd >5 9.77 47 H NH O F F – 1 >4 >4 3.98 48 H NH O F F – 1 4 >4 3.55 49 H NH O F Cl – 2 >4 >4 70.49 50 H NH O Cl MeO – >4 >4 >4 100.44 51 H NH O Me F – NDh 51t H NH O Me F F NDh 71.77 52 H NH O O F3C – 4 >4 >4 104.79
  18. 18. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 17 53 H NH O O O – 2 4 >4 80.78 54 H NH O Me NHS NH – >4 >4 >4 NDh 55 H NH F O – 4 >4 >4 101.92 56 H NH O Cl – 4 >4 >4 NDh 57 H NH O Cl – 0.05 0.5 TEd >5 0.24 57h 5-OMe NH O Cl – 2 >4 >4 NDh 57h′′′′ 6-OMe NH O Cl – >4 >4 >4 NDh 57i 4,7-diOMe NH O Cl – Not tested due to poor solubility a The sea urchin embryo assay was conducted as described previously [38]. Fertilized eggs and hatched blastulae were exposed to 2-fold decreasing concentrations of compounds. Duplicate measurements showed no differences in effective threshold concentration (EC) values. b GI50: concentration required for 50% cell growth inhibition. c Cell growth percent at 10 µM concentration.
  19. 19. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 18 d TE: tuberculate eggs typical for microtubule destabilizing agents. e NCI60 screen data for colchicine NSC 757. For the structure see Fig. 1. f NCI60 screen data for vinblastine NSC 49842. For the structure see Fig. 1. g Ref. [11]. h ND: Not determined.
  20. 20. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 19 As evidenced from the data, compounds 18 and 37 exhibited antimitotic activity at ca. 0.2– 0.5 µM concentrations. This effect was likely related to their microtubule destabilizing activity, as suggested by embryo spinning. Compounds 20, 21, 46, and 57 were also considered to target tubulin/microtubules. This conclusion was based on the observation that the arrested sea urchin eggs acquired the tuberculate shape typical of the microtubule destabilizers [38–40]. The additional 24 molecules, including 16, 9h, and 9n, that induced cleavage abnormalities at 0.5–4 µM concentration but failed to cause both cleavage arrest and embryo spinning, were classified as tubulin independent antiproliferative agents. It is worth noting that the most active BIFAs were endowed with the unsubstituted phenyl ring in benzimidazole fragment and aminofurazan moiety (R1 = R3 = H). For the R1 substituents, the maximal activity was observed for R1 = 5-MeO. For derivatives with R2 = R3 = H, the activity order was as follows: 5-MeO (9h) = naphtyl (9m)> 4-Cl (9g) > H (9a) = 5-F (9e) = 5-Cl (9f). For the analogs 57, the activity decreased in the order: R1-unsubstituted 57 >6-MeO (57h′)>5-MeO (57h), where 57h was inactive up to 4 µM. For the R2 substituents the order of activity was as follows: R1 = R3 = H; R2 = benzyl, m-MeO (20) = m-MeO-p-Me (21) = o-naphtyl (37) > m-F (16) = m-Br (18) > o-F (15) > p-F (17) > p-CN (25). Compounds substituted with m,p-diEtO (22), p-t-Bu (24), p-Cl (31), o-Cl-methylenedioxy (35), o-Br-methylenedioxy (36) in R2 = benzyl were inactive in the assay. Notably, meta-substitution in R2 with MeO or Hal group was favorable to compound activity. In contrast, presence of bulky groups especially in para-position was deteriorating. Similarly, compounds with R2 = alkyl showed no activity, as evidenced by 13, 14r, 40, and 63. Heterocyclic derivatives with R1 = H; R2 = -CH2-pyridin were inactive up to 4 µM (38r, 39r, and 39u) except for 38 that exhibited antiproliferative effect at 2 µM. For arylacetamide derivatives 44–57 (R2 = -CH2- CO-NH-Ar) compound 57 substituted with o-Cl-phenyl group was the most active microtubule destabilizer. There was a significant reduction in activity for the related p-Cl-phenyl analog 56. In evaluating the effect of R3 substitution (aminofurazane fragment), we found that the best activity was exhibited by the unsubstituted compounds. Specifically, the NH2 derivatives were more active than the respective pyrrole analogs (9a vs 11a; 9h vs 11h). It is worth noting that as opposed to BIFA, their regioisomers 3-amino-4-[5-aryl-1H-1,2,3-triazol-1-yl]furazans I (Fig. 3) were generally inactive in the sea urchin embryo assay. However, the respective 3-pyrrole-substituted furazans II were reported to be potent antimitotic microtubule destabilizing agents [41]. Insert Figure 3. N N N N O N NH2 N N N N O N N (AlkO)n I (AlkO)n II
  21. 21. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 20 Fig. 3. Structures of 3-amino-4-[5-aryl-1H-1,2,3-triazol-1-yl]furazans [41]. Furthermore, unsubstituted NH2 derivatives were generally more active than the respective N-substituted analogs including NHAc (19 vs 19r; 20 vs 20r; 25 vs 25r; 38 vs 38r). Similarly, NH2 derivative was more potent that the COEt compound (16 vs 16s). Thus, the replacement of NH2 in furazan ring with pyrrole, acetyl- or propionamide groups resulted in a marked decrease or loss of the antiproliferative effect. In summary, our assay data suggested that for the synthesized series of BIFAs both the unsubstituted phenyl ring in benzimidazole and the unsubstituted amino group of aminofurazan were essential for the antimitotic activity. In contrast, the nature of R2 was critical to the microtubule destabilizing mode of action. For example, 9h and 9m with R2=H altered cell division in the sea urchin embryos at submicromolar concentrations likely via a tubulin-independent manner (Table 1). 2.2.2. In vitro cytotoxicity The sea urchin embryo test results for the derivatives of 9 correlated well with their cytotoxicity against a panel of human cancer cell lines (NCI60 anticancer drug screen) (Table 1). Compound 57 was the most active in both assays. All potent molecules that exhibited GI50 less than 1 µM (18, 19, 20, 21, and 57) featured the unsubstituted phenyl ring in the benzimidazole fragment and the unsubstituted amino group in the aminofurazan ring. The NCI60 screen mean graphs for these compounds are presented in Supplementary data, Figures S3–S7. These SAR results are in agreement with the cytotoxicity and apoptosis induction data reported previously for the series of related furazanobenzimidazoles [7]. Four human cancer cell lines, namely melanoma MDA-MB- 435, CNS cancer SF 539, renal cancer RXF 393, and ovarian cancer OVCAR-3 cells were the most sensitive to 57 (Table 2). Notably, in melanoma MDA-MB-435 cells, this compound caused total cell growth inhibition and 50% reduction of cell number at concentrations of 0.088 µM and 0.84 µM, respectively. In addition, 57 displayed higher cytotoxicity against NCI/ADR-RES multidrug resistant ovarian cancer cells over expressing P-glycoprotein than against the parent OVCAR-8 cell line (Table 2). Insert Table 2
  22. 22. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 21 Table 2. Effects of compound 57 on human cancer cells Panel Cell line 57 Vinblastinea GI50, µMb TGI, µMc LC50, µMd GI50, µMb TGI, µMc LC50, µMd Melanoma MDA-MB-435 0.028 0.088 0.84 0.00025 0.00025 0.001 Renal cancer RXF 393 0.087 0.366 19.2 0.001 0.05 2.51 CNS cancer SF 539 0.152 0.511 16.9 0.0006 0.0025 0.79 Ovarian cancer OVCAR-3 0.122 0.427 8.05 0.0003 0.0016 0.5 OVCAR-8 0.371 >100 >100 0.0016 0.32 2 NCI/ADR-RESe 0.184 16.0 >100 0.1 0.79 1.58 a NCI60 screen data for vinblastine NSC 49842. b GI50: concentration required for 50% cell growth inhibition. c TGI: concentration required for total (100%) cell growth inhibition. d LC50: concentration required for 50% reduction in cell number. e NCI/ADR-RES: P-glycoprotein-overexpressing multi-drug resistant cell line derived from OVCAR-8.
  23. 23. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 22 2.2.3. Inhibition of tubulin polymerization and cell cycle analysis Compounds 16, 18, 19, 37, and 57 that caused pronounced cleavage alteration/arrest in the sea urchin embryos were evaluated for their anti-tubulin properties using in vitro inhibition of purified tubulin polymerization assay [42] (Table 3). As indicated in Table 1, compounds 18 and 37 induced sea urchin embryo spinning suggestive of their direct microtubule destabilizing activity. These molecules also inhibited tubulin polymerization (Table 3). Compound 57 that caused formation of tuberculate eggs typical for microtubule destabilizers showed IC50 of 7.36 µM in in vitro tubulin polymerization assay. Derivative 16 altered sea urchin embryo cleavage at 0.5 µM (Table 1) whereas the inhibition of tubulin polymerization was observed with the IC50 value of 7.9 µM. Compound 19 was a less potent in vitro tubulin inhibitor. Correspondingly, it exhibited only moderate antiproliferative activity in the sea urchin embryo assay. Insert Table 3. Table 3. Tubulin polymerization inhibition of selected BIFAs. Compd ITP IC50, µMa 16 7.9 18 2.04 19 13.27 37 7.03 57 7.36 Vinblastineb 0.6 a ITP IC50: concentration required for 50% inhibition of in vitro tubulin polymerization. b Data from [43]. Active BIFA derivatives 16, 18, 19, 37, and 57 were further tested for their effects on cell cycle distribution in the mouse fibroblast 3T3 cell line at 1 µM concentration. Molecules 19 and 57 showed G2/M arrest confirming their anti-tubulin mode of action. These compounds were found to induce G2/M block in human epidermoid carcinoma A431 cell line. Namely, A431 cells were treated with 1 µM of 19 and 57 for 24 hrs followed by flow cytometry analysis to display induction of cell cycle arrest of ca. 55% and 60% (percent of cells in G2/M phase, average of 2 experiments with SD < 15%). A subsequent dose-response studies for 19 and 57 yielded EC50 values (the compound concentration that causes 50% cells to arrest, average of 3 experiments with SD < 15%) of ca. 1 µM and 0.5 µM, respectively. Once again, these data correlated well with the sea urchin embryo data and NCI60 GI50 values (0.767 µM and 0.24 µM, respectively, Table 1). In our hands, BIFAs were consistently less cytotoxic than BAL 27862. The sea urchin embryo assay data
  24. 24. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 23 provided insight into the structure-activity relationship for these new series and further confirmed the micritubule-destabilizing mechanism of their activity. 3. Conclusions In summary, 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines (BIFA) were prepared in good yields using a novel robust procedure from aminofurazanyl hydroximoyl chlorides and o- diaminobenzenes (Scheme 2). As opposed to the reported sequences, the developed protocol used mild reaction conditions and accommodated a wide variety of functional groups to afford a diverse array of targeted compounds. Furthermore, our approach to BIFAs requires neither protection of the reactive moieties nor chromatographic purification of the respective intermediates. A subsequent biological evaluation of the resulting library using the sea urchin embryo model and human cancer cell lines revealed the antiproliferative effect of several derivatives. The activity of BIFAs in our assay systems could be attributed to both direct microtubule destabilization and tubulin independent mechanisms. The unsubstituted phenyl ring of benzoimidazole moiety as well as the unsubstituted amino group in the furazan ring were essential prerequisites for the antimitotic activity of BIFAs. The most active compound 57 was substituted with the 2-chlorophenyl acetamide moiety at the N atom of the imidazole fragment. The potent synthetically feasible tubulin-targeting BIFA series will be further evaluated as lead candidates for in vivo experiments. 4. Experimental protocols 4.1. Chemistry and chemical methods Elemental microanalyses were obtained on an Perkin-Elmer 2400 CHN analyzer. Mass spectra were collected on the Varian MAT-CH-6 spectrometer with direct sample injection at an ionization voltage of 70 eV. IR spectra were recorded on IFS-113v Bruker in KBr pellets (1:200); the frequencies were expressed in cm−1 . The 1 H NMR spectra were recorded on Bruker DRX-500 (500 MHz) and Bruker AM-300 (300 MHz) using internal standard with DMSO-D6 as the solvent; the chemical shifts were reported in ppm (δ) and coupling constants (J) values were given in Hertz (Hz). The 13 C, and 15 N NMR spectra were recorded on Bruker AM-300 at 75.47 and 50.7 MHz, respectively. Melting points were measured on a Kofler bench. Completion of the reactions and purity of the obtained products were monitored by thin layer chromatography on the Silufol UV- 254 plates using hexane-acetone mixture (5:3) as an eluent and iodine vapor as a stain. 4.1.1. General procedure for synthesis of benzimidazoles 9a–n from hydroximoyl chloride 10 Hydroximoyl chloride 10 (0.16 g, 1 mmol) was added slowly by small portions to a solution of the appropriate 1,2-diaminobenzene 3 (0.3 g, 1.5 mmol) in ethanol (5–10 mL) at room
  25. 25. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 24 temperature. The mixture was stirred at reflux for 0.5 h followed by an additional hour at room temperature, diluted with water (10 mL) and 0.1 M aq HCl (5 mL). The heterogeneous mixture was stirred for 1 h. The precipitate was filtered, washed with water, and recrystallized from iPrOH/H2O. 4.1.1.1. 3-Amino-4-(1H-benzimidazol-2-yl)-furazan (9a). White solid; yield 0.16 g (80%); mp 268– 269 °C (lit. [17] 264–265 °C); 1 H NMR (DMSO-d6, 500 MHz): 6.84 (s, 2H, NH2), 7.31 (t, J = 7.3 Hz, 1H, H-5), 7.37 (t, J = 7.8 Hz, 1H, H-6), 7.59 (d, J = 7.8 Hz, 1H, H-7), 7.81 (d, J = 7.3 Hz, 1H, H-4), 13.69 (s, 1H, NH); 13 C NMR (DMSO-d6): 112.6 (br), 120.2 (br), 123.8 (br), 135.0 (br), 139.1, 140.8, 143.2 (br), 156.1; EIMS m/z 201 [M]+ (20), 144 (100), 143 (29), 118 (42), 116 (11), 92 (18), 90 (20), 77 (6), 63 (35); Anal. Calcd for C9H7N5O: C 53.73; H 3.51; N 34.81. Found: C 53.61; H 3.47; N 34.93; IR (KBr): ν max 3406, 3303, 1635, 1621, 1604, 1561, 1495, 1459, 1423, 1322, 1278, 1125, 1012, 1000, 955, 900, 864, 748, 732, 697, 611. 4.1.1.2. 3-Amino-4-(4-methyl-1(3)H-benzimidazol-2-yl)-furazan (9c). Ratio of isomers 3:2. White solid; yield 0.16g (73%); mp 213–216 °С; 1 H NMR (DMSO-d6): δ 2.55, 2.59 (s/s = 3/2, 3H, Me-4), 6.88, 6.90 (s/s = 3/2, 2H, NH2), 7.05 (t, J = 7.8 Hz, 2H, H-6,7) and 7.20 (m, 2H, H-6,7), 7.38, 7,58 (2d, J = 7.8 Hz, 1H, H-5), 13.58, 13.64 (s/s = 3/2, 1H, NH); 13 C NMR (DMSO-d6): 16.3, 17.1, 109.5, 117.0, 122.4, 122.6, 122.7, 124.4, 124.9, 129.3, 133.9, 134.0, 138.6, 139.4, 140.2, 142.3, 142.6, 155.6; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.86; H 4.18; N 32.47; IR (KBr): ν max 3430, 3323, 3194, 1635, 1620, 1591, 1516, 1456, 1422, 1327, 1268, 1239, 1157, 1138, 1005, 949, 899, 874, 748, 671, 561. 4.1.1.3. 3-Amino-4-(4,5-dimethyl-1(3)H-benzimidazol-2-yl)-furazan (9d). White solid; yield 0.211g (92%); mp 242–243 °C; 1 H NMR (DMSO-d6): δ 2.32, 2.46 (s/s = 3/1, 6H, Me-4,5), 6.85, 6.88 (2s, 2H, NH2), 7.11 (m, 1H, H-6), 7.27, 7.47 (2d, J = 8.2 Hz, 1H, H-7), 13.43 (br. s, 2H, NH); 13 C NMR (DMSO-d6): 13.6, 14.3, 19.3, 19.6, 109.1, 116.9, 120.5, 125.7, 127.2, 127.5, 129.8, 132.4, 132.6, 135.1, 139.0, 139.8, 140.3, 141.5, 143.2, 156.0; Anal. Calcd for
  26. 26. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 25 C11H11N5O: C 57.63; H 4.84; N 30.55. Found: C 57.69; H 4.81; N 30.48; IR (KBr): ν max 3425, 3284, 3202, 1634, 1619, 1596, 1504, 1458, 1425, 1373, 1323, 1006, 950, 903, 874, 793, 767, 741, 717, 661, 631, 561, 501. 4.1.2. General procedure for the synthesis of pyrroles 11a, 11h, and 11i 2,5-Dimethoxytetrahydrofuran (1.7 mmol) was added to a slurry of respective BIFAs (9a, 9h, 9i) (1.7 mmol) in AcOH (3 mL) at room temperature. The mixture was refluxed for 15 min, cooled to room temperature, and the resulting suspension was stirred for 1 h. The precipitate was filtered, washed with ice water (2×5 mL) followed by 70% iPrOH (2×5 mL) and dried. Crystallization of the crude product from 70% iPrOH afforded pure pyrroles 11a, 11h, and 11i (white solids, 70–90% yield). 4.1.3. General procedure for alkylation of 9a–n 4.1.3.1. Alkylation by benzylhalides 12′′′′–39′′′′ and 41′′′′–43′′′′ A mixture of 3-amino-4-(1H-benzimidazol-2-yl)-furazan 9a–n (0.01 mol), benzylhalide 12′′′′–43′′′′ (0.011 mol), and K2CO3 (1.52 g, 0.011 mol), in dry DMF (30 mL) was stirred at 80–90 °C for 4–5 h (reflux condenser was used for volatile benzylhalides). The mixture was cooled to room temperature and diluted with water (100 mL). The precipitate was filtered and recrystallized from acetic acid. Yields of 12–39 and 41–43 were 70–90%. 4.1.3.2. Synthesis of [2-(4-amino-furazan-3-yl)-benzoimidazol-1-yl]-acetonitrile 40 BIFA 9a (2g, 0.01 mol) was alkylated by ClCH2CN (1.27 g, 0.02 mol) as described in the following procedure 4.1.3.3 at ≤50 °C. The product was isolated and recrystallized from EtOH:AcOH 3:1 v/v to yield 1.56 g (65%) of the targeted nitrile 40 as white solid. 4.1.3.3. Alkylation by N-aryl-acetamides 44′′′′–57′′′′ Alkylation by N-Aryl-acetamides 44′′′′–57′′′′ and separation of products was conducted as alkylation by benzylhalides as described in 4.1.2.1., however at lower temperature (60 °C) and increased reaction time (8–12 h). The precipitate was crystallized from acetic acid or mixture of acetic acid–DMF (10–30% by volume) to give 44–57 (white solid, 70–90% yield). 4.1.3.4. Synthesis of isomeric 2-[2-(4-aminofurazan-3-yl)-5(6)-methoxy-1H-benzimidazol-1-yl]-N- (2-chlorophenyl)acetamides 57h and 57h′′′′
  27. 27. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 26 A mixture of 3-amino-4-(6-methoxy-1H-benzimidazol-2-yl)-furazan 9h (0.23 g, 0.001 mol), 2-chloro-N-(2-chlorophenyl)acetamide 57′′′′ (0.222 g, 0.0011 mol), K2CO3 (0.152 g, 0.0011 mol), KBr (0.03 g), and glym (5 mL) was stirred at reflux for 9 h. The mixture was cooled to room temperature and diluted with water (50 mL). The precipitate was filtered and washed with water (30 mL) to give a mixture of the respective isomers 57h and 57h′′′′. White solids, 91% yield (0.364 g); mp 252–253 °C. Pure isomers were isolated by fractional crystallization from MeCN. Specifically, isomer 57h′′′′ exhibited lower solubility in MeCN and was obtained in 38% yield (0.14 g). More soluble isomer 57h was isolated in 46% yield (0.17 g). 4.1.4. Acylation of 4-(1-R-1H-Benzoimidazol-2-yl)-furazan-3-yl-amines 4.1.4.1. Acylation by anhydrates of aliphatic carbonic acids 58r,s Corresponding BIFA (0.01 mol) was added to the neat acetic or propionic anhydrate (20 mL) and refluxed for 3 h in presence of 5 mmol of dry MeCO2Na or EtCO2Na, respectively. The mixture was cooled to room temperature and diluted with water (100 mL). After a day at room temperature, the precipitate was filtered and recrystallized from DMF–EtOH to yield 80% of the desired products as white solids. 4.1.4.2. Acylation by chloroanhydrates of aromatic carbonic acids (aroylchloroanhydrates) 58t,u i) A mixture of corresponding BIFA (26, 39 or 51) (0.01 mol) and ArCOCl 58t,u (0.015 mol) in toluene (50 mL) was refluxed for 8–20 h until the evolution of HCl stopped. Toluene was evaporated in vacuo, and the residue was recrystallized from DMF–EtOH to yield 80% of the desired products as white solids. ii) A mixture of corresponding BIFA (26, 39 or 51) (0.01 mol) and ArCOCl 58t,u (0.015 mol) in freshly distilled pyridine (30 mL) was refluxed for 3 h, cooled, and diluted with water (100 mL). After 24 h the residue was filtered and recrystallized from DMF–EtOH to afford targeted products as white solids (70% yield). Crude alkylating products 14, 28, 32, 34, and 51 were filtered and without purification acylated to afford 14r, 28r, 32r, 34s, and 51t.
  28. 28. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 27 4.1.5. 4-Aminofurazan-3-carbox-N-(p-methoxyphenyl)amidoxime (60v). A solution of 4- aminofurazan-3-carbohydroxymoyl chloride 10 (8.1 g, 50 mmol) in EtOH (100 mL) was added drop wise to a solution of p-anizidine 59v (11.9 g, 97 mmol) and NEt3 (6 g, 60 mmol) in iPrOH (50 mL) at 0 °C over 10 min. The mixture was stirred at room temperature for 3 h, the solvent was removed in vacuo and the residue was treated with 80 mL of water. The precipitate was filtered and washed with H2O (2×50 mL) followed by benzene (30 mL). The solid residue (14 g, 86 mmol) was further recrystallized to give 60v. Gray solid, yield 13.3 g, (62%); mp 197–198 °C (from benzene/iPrOH); 1 H NMR (DMSO-d6) δ 3.69 (s, 3H, MeO), 6.18 (s, 2H, NH2), 6.80 (s, 4H, Ar), 8.4 (s, 1H, NH), 11.02 (s, 1H, NOH); 13 C NMR (DMSO-d6) δ 55.1, 113.6, 123.7, 133.2, 140.3, 140.7, 155.3, 155.4; Anal. Calcd for C10H11N5O3 (%): C, 48.19; H, 4.45; N, 28.10. Found (%): C, 48.23; H, 4.51; N, 28.05. IR (KBr, cm-1 ) 3472, 3372, 3272, 2964, 2840, 1652, 1612, 1568, 1536, 1516, 1440, 1400, 1304, 1252, 1156, 1108, 1044, 952. 4.1.6. 4-Aminofurazan-3-carbox-(N-benzylamid)oxime (60w). Gray solid; 7.93 g, 68% yield; mp 124–125 °C (from benzene/iPrOH); 1 H NMR (DMSO-d6) δ 4.63 (d, J = 7.3 Hz, 2H, CH2), 6.27 (s, 2H, NH2), 6.89 (t, J = 7.3 Hz, 1H, NH), 7.21 (m, 5H, Ph), 10.80 (s, 1H, NOH); 13 C NMR (DMSO- d6) δ 46.3 (C4), 126.6 and 128.3 (C6, C7, C8), 139.7 (C5), 140.9 (C2), 144.8 (C3), 155.2 (C1); Anal. Calcd for C10H11N5O2 (%): C, 51.50; H, 4.75; N, 30.03. Found (%): C, 51.55; H, 4.68; N, 28.75. 4.1.7. 4-Aminofurazan-3-carbox-N-(p-methoxybenzylamid)oxime (60x). Light-brown solid; 7.77 g, 59% yield; mp 141–142 °C (from benzene) (lit. [44] 140–142 °C); 1 H NMR (DMSO-d6) δ 3.69 (s, 3H, MeO), 4.54 (d, J = 7.1 Hz, 2H, CH2), 6.26 (s, 2H, NH2), 6.83 (m, 3H, NH, H-3',5'), 7.12 (d, J = 8.4 Hz, 2H, H-2',6'), 10.79 (s, 1H, NOH); 13 C NMR (DMSO-d6) δ 45.8, 55.0, 113.7, 128.1, 132.8, 139.7, 144.8, 155.3, 158.2; EIMS m/z 263 (M+), 233 [M+ –NO]; Anal. Calcd for C11H13N5O3 (%): C, 50.19; H, 4.98; N, 26.60. Found (%): C, 50.27; H, 5.01; N, 26.54. 4.1.8. 4-(p-Methoxyphenylamino)furazan-3-carboxamidoxime (61v). A solution of 60v (13.3 g, 53 mmol) and KOH (2.92 g, 53 mmol) in ethylene glycol (50 mL) was refluxed for 4 h. The reaction mixture was cooled, diluted with water (30 mL), and neutralized with 36% aqueous HCl. The residue was filtered, washed with H2O (100 mL), benzene (15 mL), and recrystallized from benzene–iPrOH to afford 61v. Light-brown solid; yield 10.84 g (82%); mp 191 °C (from benzene/iPrOH) (lit. [44] 190–191 °C); 1 H NMR (DMSO-d6) δ 3.74 (s, 3H, MeO), 6.39 (s, 2H, NH2), 6.97 (d, 2H, J = 8.6 Hz, 2H, ArH-3',5'), 7.38 (d, 2H, J = 8.6 Hz, 2H, ArH-2',6')), 8.79 (s, 1H, NH), 10.71 (s, 1H, NOH); 13 C NMR (DMSO-d6) δ 55.2, 114.5, 118.4, 132.5, 139.7, 143.9, 151.0,
  29. 29. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 28 154.5; EIMS m/z 249 (M+), 219 [M+ –NO]; Anal. Calcd for C10H11N5O3 (%): C, 48.19; H, 4.45; N, 28.10. Found (%): C, 48.22; H, 4.49; N, 28.03. 4.1.9. 4-(Benzylamino)furazan-3-carboxamidoxime (61w). White solid; yield 9.76 g (79%); mp 104 °C; 1 H NMR (DMSO-d6) δ 4.44 (d, J = 7.0 Hz, 2H, CH2), 6.25 (s, 2H, NH2)), 6.51 (t, J = 7.0 Hz, 1H, NH), 7.31 (m, 5H, Ph), 10.50 (s, 1H, NOH)); 13 C NMR (DMSO-d6) δ 47.6 (C4), 127.3, 127.6, 128.5 (C6, C7, C8), 138.4 (C2), 139.7 (C5), 144.0 (C1), 154.9 (C3). 4.1.10. 4-(p-Methoxybenzylamino)furazan-3-carboxamidoxime (61x). A solution of 60x (5.2 g, 20 mmol) and KOH (1.1 g, 20 mmol) in ethylene glycol (15 mL) was refluxed for 4 h. The reaction mixture was cooled, diluted with water (30 mL) and neutralized with 36% aqueous HCl. The residue was filtered, washed with H2O (100 mL), benzene (15 mL), and recrystallized from benzene–iPrOH to furnish 61x. Gray solid; yield 4 g (78%); mp 113–117 °C; 1 H NMR (DMSO-d6) δ 3.72 (s, 3H, MeO), 4.36 (d, J = 6.0 Hz, 2H, CH2), 6.22 (s, 2H, NH2), 6.40 (br. s, 1H, NH), 6.89 (d, J = 8.2 Hz, 2H ArH-3',5'), 7.33 (d, J = 8.2 Hz, 2H, ArH-2',6'), 10.45 (s, 1H, NOH); 13 C NMR (DMSO-d6) δ 47.1, 55.1, 113.9, 129.2, 130.3, 139.7, 144.1, 154.9, 158.7; EIMS m/z 263 (M+); Anal. calcd for C11H13N5O3 (%): C, 50.19; H, 4.98; N, 26.60. Found (%): C, 50.25; H, 5.00; N, 26.52 4.1.11. General procedure for synthesis of 4-(R-amino)furazan-3-carbohydroxymoyl chlorides 62v,w A solution of NaNO2 (0.14 g, 2 mmol) in H2O (2 mL) was added drop wise to a stirred solution of amidoxime 61v or 61w (2 mmol) in a mixture of conc. HCl (3.6 mL), AcOH (6 mL), and H2O (3.2 mL) at < 10 °C. The reaction was allowed to stir for 3 h at 10 °C and for 1 h at room temperature. The solid residue was filtered and washed with H2O (4×5 mL) to give 62v,w (60%– 87% yield) as white crystals. 4.1.11.1. N-hydroxy-4-((4-methoxyphenyl)amino)-1,2,5-oxadiazole-3-carbimidoyl chloride (62v). White solid; yield 0.4 g (60%); mp 202–203 °C; 1 H NMR (DMSO-d6) δ 3.72 (s, 3H, OMe), 6.94 (d, 2H, J = 8.6, ArH-3'',5'' ), 7.38 (d, 2H, J = 8.6, Ar2H, H-2'',6'' ), 8.21 (s, 1H, NOH), 13.62 (s, 1H, NH); 13 C NMR (DMSO-d6) δ 55.1, 114.3, 119.0, 126.1, 132.2, 141.8, 150.6, 154.7; Anal. Calcd for C10H9N4O3: C 44.71; H 3.38; N 20.85. Found (%): C 44.75; H 3.34; N 20.24; IR (KBr): ν max 3363, 3253, 1617, 1602, 1566, 1512, 1466, 1444, 1270, 1231, 1183, 1025, 945, 876, 824, 792, 754, 701, 564, 516.
  30. 30. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 29 4.1.11.2. 4-(Benzylamino)-N-hydroxy-1,2,5-oxadiazole-3-carbimidoyl chloride (62w). White solid; yield 0.44 g (87%); mp 164–165 °C; 1 H NMR (DMSO-d6) δ 4.45 (d, J=5.8 Hz, 2H, CH2), 6.35 (s, 1H, NOH), 7.34 (m, 5H, Ph), 13.41 (s, 1H, NH); 13 C NMR (DMSO-d6) δ 47.8, 126.8, 127.4, 127.7, 128.6, 138.2, 141.6, 154.4; Anal. Calcd for C10H9ClN4O2: C 47.54; H 3.59; N 22.18. Found (%): C 47.58; H 3.52; N 22.01; IR (KBr): ν max 3392, 3263, 1619, 1601, 1562, 1440, 1354, 1233, 1031, 967, 946, 909, 856, 754, 696, 610, 557, 508. 4.1.12. 3-(1-Ethyl-1H-benzimidazol-2-yl)-4-(4-methoxyphenyl)amino-furazan (63). Hydroximoyl chloride 62v (0.27 g, 1 mmol) was added in several portions to a vigorously stirred solution of 1,2- diaminobenzene 3q (0.2 g, 1.5 mmol) in ethanol (5–10 mL) at room temperature. The mixture was stirred at reflux for 0.5 h followed by stirring for 1 h at room temperature, dilution with water (10 mL) and 0.1 M aqueous HCl (5 mL). The heterogeneous mixture was stirred for 1 h, the resulting precipitate was filtered, washed with water, and recrystallized from iPrOH–H2O to afford 63. White solid; yield 0.22 g (65%); mp 135–136 °C; 1 H NMR (DMSO-d6): δ 1.43 (t, J = 7.1 Hz, 3H, CH3), 3.77 (s, 3H, OMe-4''), 4.76 (q, J = 7.1 Hz, 2H, CH2), 7.03 (d, J = 9.0 Hz, 2H, ArH-3'',5''), 7.43 (t, J = 8.0 Hz, 1H, H-5), 7.49 (t, J = 8.2 Hz, 1H, H-6), 7.60 (d, J = 9.0 Hz, 2H, ArH-2'',6''), 7.86 (d, J = 8.2 Hz, 1H, H-7), 7.97 (d, J = 8.0 Hz, 1H, H-4), 9.97 (s, 1H, NH) 13 C NMR (DMSO-d6): δ 15.0, 40.6, 55.6, 110.1, 114.5 (C2), 119.0 (C2), 120.6, 123.5, 124.9, 133.2, 134.9, 137.9, 140.4, 142.1, 152.9, 155.0; 15 N NMR (DMSO-d6): δ 21.3, -20.9, -137.0, -221.7, -297.0; EIMS m/z 335 [M]+ , 295 [M+ - NO]; Anal. Calcd for C18H17N5O2: C 64.47; H 5.11; N 20.88. Found (%): C 64.54; H 5.14; N 20.77; IR (KBr): ν max 3249, 2977, 1623, 1577, 1513, 1439, 1334, 1245, 1180, 1115, 1977, 1035, 1008, 908, 872, 825, 753, 742, 717, 608, 557, 524. 4.1.13. 3-Benzylamino-4-(5-chloro-1H-benzimidazol-2-yl)-furazan (64). White solid; yield 0.19 g (59%); mp 196 °C; 1 H NMR (DMSO-d6): δ 4.56 (d, 2H, J = 5.89 Hz, CH2), 7.34 (m, 6H, Ph, H-6), 7.68 (s, 2H, CH) 13.85 (br, s, 2H, NH); 13 C NMR (DMSO-d6): δ 47.5, 116.0 (br, s), 123.9, 127.1, 127.4, 127.5, 128.3, 128.4, 128.5, 128.4, 137.9, 138.5, 141.3, 155.7; EIMS m/z 325 [M]+ , 295 [M+ - NO], 220, 178, 106, 91; Anal. Calcd for C16H12ClN5O: C 58.99; H 3.71; N 21.50. Found (%): C 59.02; H 3.69; N 21.42; IR (KBr): ν max 3358, 3259, 1630, 1594, 1519, 1496, 1432, 1371, 1332, 1299, 1262, 1236, 1141, 1063, 1029, 986, 955, 927, 855, 815, 741, 705, 640. 4.1.14. Synthesis of 3-amino-4-[1-((1H-tetrazol-5-yl)methyl)-1H-benzimidazol-2-yl]-furazan 65 A solution of nitrile 40 (2.4 g, 0.01 mol), NaN3 (1 g, 0.015 mol), and NH4Cl (0.8g, 0.015 mol) in dry DMF (30 mL) was stirred at 100 °C for 8 h, cooled, and diluted with water (100 mL). The reaction mixture was stirred with activated charcoal (100 mg) for 15 min, filtered, and the pH
  31. 31. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 30 was adjusted to ca. 1 with 36% aqueous HCl. The resulting precipitate of 65 was recrystallized from EtOH to furnish the pure product (white solid, 1.78 g, 63% yield). 4.1.15. Synthesis of 3-Amino-4-[1-((5-amino-1,3,4-thiadiazol-2-yl)methyl)-1H-benzimidazol-2-yl]- furazan 66 A solution of nitrile 40 (2.4 g, 0.01 mol) and thiosemicarbazide (1.4 g, 0.015 mol) in neat CF3COOH (30 mL) was refluxed for 8 h at stirring. The reaction mixture was cooled, diluted with water (100 mL), and stirred for an additional 15 min with activated charcoal (100 mg). The mixture was filtered and neutralized with aqueous NH4OH (25%) to afford the residue of crude 66. It was further recrystallized from EtOH to afford the targeted pure thiadiazol (white solid, 2.23 g, 71% yield). Synthetic and analytical data for the other compounds are presented in Supplementary data. 4.2. Biology. Materials and methods 4.2.1. Phenotypic sea urchin embryo assay [38] Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the Mediterranean Sea on the Cyprus coast and kept in an aerated seawater tank. Gametes were obtained by intracoelomic injection of 0.5 M KCl. Eggs were washed with filtered seawater and fertilized by adding drops of diluted sperm. Embryos were cultured at room temperature under gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered seawater. The embryos were observed with a Biolam light microscope (LOMO, St. Petersburg, Russia). For treatment with the test compounds, 5 mL aliquots of embryo suspension were transferred to six-well plates and incubated as a monolayer at a concentration up to 2000 embryos/mL. Stock solutions of compounds were prepared in DMSO at 10 mM concentration followed by a 10-fold dilution with 96% EtOH. This procedure enhanced the solubility of the test compounds in the salt-containing medium (seawater), as evidenced by microscopic examination of the samples. The maximal tolerated concentrations of DMSO and EtOH in the in vivo assay were determined to be 0.05% and 1%, respectively. Higher concentrations of either DMSO (≥0.1%) or EtOH (>1%) caused nonspecific alteration and retardation of the sea urchin embryo development independent of the treatment stage. The compound solubility in the seawater was estimated by microscopic examination of sample wells. Colchicine and vinblastine sulfate (Sigma-Aldrich) were applied as reference compounds, using 20 mM and 5 mM stock solutions in distilled water, respectively. The antiproliferative activity was assessed by exposing fertilized eggs (8–20 min after fertilization, 45–55 min before the first mitotic cycle completion) to 2-fold decreasing concentrations of the compound. Cleavage alteration and arrest were clearly detected at 2.5–5.5 h
  32. 32. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 31 after fertilization. The effects were estimated quantitatively as an effective threshold concentration, resulting in cleavage alteration and embryo death before hatching or full mitotic arrest. At these concentrations all tested microtubule destabilizers caused 100% cleavage alteration and embryo death before hatching, whereas at 2-fold lower concentrations the compounds failed to produce any effect. For microtubule-destabilizing activity, the compounds were tested on free-swimming blastulae just after hatching (8–10 h after fertilization), which originated from the same embryo culture. Embryo spinning was observed after 15 min to 20 h of treatment, depending on the structure and concentration of the compound. Both spinning and lack of forward movement were interpreted to be the result of the microtubule-destabilizing activity of a molecule. Video illustrations are available at http://www.chemblock.com. Both sea urchin embryo assay and DTP NCI60 cell line activity data are available free of charge via the Internet at http://www.zelinsky.ru. 4.2.2. In vitro tubulin polymerization assay [42] In vitro tubulin polymerization was determined using modified turbidity assay, developed by Cytoskeleton Inc. (Cytodynamix 12), for maximized throughput and maintained sensitivity. Lyophilized bovine tubulin (HTS02, Cytoskeleton Inc.) was re-suspended in G-PEM buffer (80 mM PIPES pH 7, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP, 5% glycerol) to a final concentration of 3 mg/mL and kept at 4 °C. Compounds in 100× stock solutions in DMSO were dotted into pre- warmed 96-well plates (Corning Costar 3696), with the plates immediately transferred to a 37 °C plate reader (SPECTRAmax Plus, Molecular Devices). Cold tubulin was added to the wells, plates were mixed by shaking, and absorbance at 340 nm was read every minute for 30 min. Kinetic curves with 30 points each were collected for tested compound, with a dynamic range between 0 and 0.4 OD units. Percentage inhibition values were calculated using the 30 minute data point, based on control samples (1% DMSO). IC50 values were determined by sigmoidal curve fitting using Excel-based software. 4.2.3. Cell cycle analysis [42] Cell cycle analysis was assessed by flow cytometry. 3T3 mouse fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum, 1 mg/mL L-glutamate, 100 units/mL penicillin G, and 0.2 mg/mL streptomycin sulfate. Cells were plated onto 6-well plates at a final density of 500,000 cells/well at the time of treatment, treated with compounds at a final concentration of 1 µM (0.1% final concentration of DMSO) for 24 h, then trypsinized, collected, rinsed in phosphate buffer saline (PBS), and fixed in 70% cold ethanol overnight at 4 °C. Cells were rinsed in PBS, re- suspended in PBS with 0.2% Tween, incubated with RNAse (final concentration of 1 µg/mL) at 37 °C for 15 min, followed by addition of propidium iodide (final concentration of 50 µg/mL) and 30
  33. 33. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 32 min incubation at room temperature. Cell cycle distribution was determined by flow cytometry using cell sorter Guava PCA-96. A compound was reported as a mitotic arrest inducer when the amount of cells in G2/M phase exposed to 1 µM concentration of an agent was twice or more than in control (DMSO). Acknowledgments The authors acknowledge the compounds screening at the National Cancer Institute (NCI) (Bethesda, MD, USA) by the Developmental Therapeutics Program NCI/NIH (http://dtp.cancer.gov). KYS is thankful to the Russian Scientific Foundation for financial support (project no. 14-13-00884). Supplementary data Supplementary data associated with this article can be found in the online version, at ........................ These data include MOL files, experimental details regarding syntheses, analytical data, and X-ray analysis.
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  36. 36. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 35 [37] Crystallographic information files are also available from the Cambridge Crystallographic Data Center (CCDC) upon request (http://www.ccdc.cam.ac.uk, deposition numbers 1008562–1008563). [38] M.N. Semenova, A.S. Kiselyov, V.V. Semenov, BioTechniques 40 (2006) 765–774. [39] Semenova, M. N.; Kiselyov, A. S.; Titov, I. Y.; Molodtsov, M.; Grishchuck, E.; Spiridonov, I.; Semenov, Chem. Biol. Drug. Des. 70 (2007) 485–490. [40] A.S. Kiselyov, M.N. Semenova, N.B. Chernyshova, A. Leitao, A.V. Samet, K.A. Kislyi, M.M. Raihstat, T. Oprea, H. Lemcke, M. Lantow, D.G. Weiss, N.N. Ikizalp, S.A. Kuznetsov, V.V. Semenov, Eur. J. Med. Chem. 45 (2010) 1683–1697. [41] A.S. Kulikov, M.A. Epishina, L.V. Batog, V.Yu. Rozhkov, N.N. Makhova, L.D. Konyushkin, M.N. Semenova, V.V. Semenov, Russ. Chem. Bull. 62 (2013) 836–843. [42] X. Ouyang, X. Chen, E.L. Piatnitski, A.S. Kiselyov, H.-Y. He, Y. Mao, V. Pattaropong, Y. Yu, K.H. Kim, J. Kincaid, L. Smith II, W.C. Wong, S.P. Lee, D.L. Milligan, A. Malikzay, J. Fleming, J. Gerlak, D. Deevi, J.F. Doody, H.-H. Chiang, S.N. Patel, Y. Wang, R.L. Rolser, P. Kussie, M. Labelle, C. Tuma, Bioorg. Med. Chem. Lett. 15 (2005) 5154–5159. [43] Tubulin polymerization assay using >99% pure tubulin, fluorescence based (BK011P), http://www.cytoskeleton.com/bk011p. [44] A.B. Sheremetev, D.E. Dmitriev, N.K. Lagutina, M.M. Raihstat, A.S. Kiselyov, M.N. Semenova, N.N. Ikizalp, V.V. Semenov, V. V. Mendeleev Commun. 20 (2010) 132–134.
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  44. 44. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S1 Supplementary data A facile synthesis and antiproliferative properties of 4-(1H-benzo[d]imidazol-2-yl)- furazan-3-amines Andrei I. Stepanov,a Alexander A. Astrat’ev,a Aleksei B. Sheremetev,b Nataliya K. Lagutina,c Nadezhda V. Palysaeva,b Aleksei Yu. Tyurin,b Nataly S. Aleksandrova,b Nataliya P. Sadchikova,c Kyrill Yu. Suponitsky,d Olga P. Atamanenko,b Leonid D. Konyushkin,b Roman V. Semenov,b Sergei I. Firgang,b Alex S. Kiselyov,e Marina N. Semenova,f Victor V. Semenovb,* a Special Design and Construction Bureau SDCB “Technolog”, 33-A Sovetskii Ave., Saint Petersburg, 192076, Russian Federation b N. D. Zelinsky Institute of Organic Chemistry, RAS, 47 Leninsky Prospect, 119991 Moscow, Russian Federation c I. M. Sechenov First Moscow State Medical University, Trubetskaya Str. 8-2, 119991 Moscow, Russian Federation d A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov Str., 119991 Moscow, Russian Federation e Department of Biological and Medicinal Chemistry, Moscow Institute of Physics and Technology, Institutsky Per. 9, Dolgoprudny, Moscow Region, 141700, Russian Federation f N. K. Kol’tsov Institute of Developmental Biology, RAS, Vavilov Str., 26, 119334 Moscow, Russian Federation Corresponding author: Victor V. Semenov Address: N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991, Moscow, Russian Federation. Tel.: +7 916 620 9584; fax: +7 499 137 2966. E-mail: vs@zelinsky.ru
  45. 45. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S2 Table of contents 1) Single crystal X-ray crystallography of benzimidazoles 27 and 29 and Fig. S1. Molecular structure of compounds 27 and 29 showing the atom numbering scheme. Page S3 2) Table S1. Crystallographic data for 27 and 29. Page S5 3) Figure S2. Fragment of the crystal packing of 27 and 29. Page S6 4) Figure S3. NCI60 5 dose screen. GI50 Mean Graph for 18. Page S7 5) Figure S4. NCI60 5 dose screen. GI50 Mean Graph for 19. Page S8 6) Figure S5. NCI60 5 dose screen. GI50 Mean Graph for 20. Page S9 7) Figure S6. NCI60 5 dose screen. GI50 Mean Graph for 21. Page S10 8) Figure S7. NCI60 5 dose screen. GI50 Mean Graph for 57. Page S11 9) The Mean Graphs interpretation. Page S12 9) Chemistry. General experimental procedures and synthetic and analytical data for 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines. Pages S13–S45 10) References. Page S46
  46. 46. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S3 Single crystal X-ray crystallography of benzimidazoles 27 and 29. X-ray experiments were carried out using SMART APEX2 CCD (λ(Mo-Kα)=0.71073 Å, graphite monochromator, ω-scans) at 120K. Collected data were analyzed by the SAINT and SADABS software incorporated into APEX2 package (APEX2 and SAINT; Bruker AXS Inc., Madison, Wisconsin, USA, 2009). All structures were solved by the direct methods and refined by the full-matrix least-squares procedure against F2 in anisotropic approximation. The hydrogen atoms of the NH2 groups were found in the difference Fourier synthesis. The H(C) positions were calculated. All the hydrogen atoms were included in the refinement within isotropic approximation by the riding model with the Uiso(H) = 1.5Ueq(Ci) for methyl groups and 1.2Ueq(Ci) for other carbon atoms, where Ueq(C) are equivalent thermal parameters of the parent atoms. The refinement was carried out with the SHELXTL software [i]. The details of data collection and crystal structures refinement are summarized in Table S1. Fig. S1. Molecular structure of compounds 27 and 29 showing the atom numbering scheme. In the molecular structure of 27 (Fig. S1), the aminofurazan moiety is nearly coplanar to the benzimidazole ring (torsion angle C1–C2–C3–N4 is -1.0(7)°) suggesting conjugation between these functionalities and stabilization by the intramolecular hydrogen bond N3–H3A…N4 (H…N 2.35Å, N…N 2.883(5)Å, <NHN 118°). The N–O bonds in the furazan ring showed different length (N1–O1 of 1.402(5)Å, N2–O1 of 1.376(5)Å) affected by substituents [ii]. In the structure of 29 featuring anisole and CF3 substituents, the furazan and benzimidazole rings are also coplanar (torsion angle C1–C2–C3–N4 is -1.0(4)°). For this groups, the conjugation is more pronounced presumably due to the influence of the electron-withdrawing CF3 group as evidenced by the greater difference in the N–O bonds of the furazan ring (N1–O1 of 1.413(3)Å, N2–O1 of 1.368(3)Å). Further stabilization of the planar structure is likely attained by the intramolecular hydrogen bond N3– H3A…N4 (H…N 2.23Å, N…N 2.859(4)Å, <NHN 127°), which is stronger than that in 27. It was
  47. 47. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S4 further determined that the torsion angle C9–N5–C11–C12 of 92.2(3)° is defined by the intramolecular steric effects, while the rotation of the anisole moiety about the C11–C12 bond is most probably induced by crystal packing. In addition to intramolecular H-bond, in the crystal structure of 27 the second hydrogen of the amino group formed H-bond with the nitrogen atom of the furazan ring [N3–H3B…N1(1-x, -0.5+y, 1-z) (H…N 2.14Å, N…N 3.025(5)Å, <NHN 169°)]. However, instead of formation of H-bonded dimers, this resulted in a formation of H-bonded chains along the axis b. In these chains molecules were related by the two-fold axis (Fig. S2). In the crystal structure of 29 the second hydrogen atom of the amino group was bonded to the oxygen atom of the methoxy group [N3-H3B…O2(x, 1+y, z) (H…O 2.23Å, N…O 3.056(3)Å, <NHO 152°)] causing a formation of chains along axis b (Fig. S2). Obviously this H-bond was weaker than the N–H…N bond observed in 27 suggesting better nucleophilic properties of the furazan ring N3 atom as compared to the O-atom of the methoxygroup [ii]. However the N–H…N bond was not observed in 29 that could be a consequence of the cumulative effect of the numerous weak intermolecular interactions contributing to a stabilization of the 3-D crystal structure. Probably, for the same reason the H-bonded chains, other than the H-bonded dimers, were formed in the crystal structure of 27.
  48. 48. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S5 Table S1. Crystallographic data for 27 and 29. Parameter 27 29 Empirical formula Fw Crystal system Space group a, Å b, Å c, Å α, deg β, deg γ, deg V, Å3 Z dcalc, g·cm-3 µ, mm-1 F(000) θ range, deg. Reflections collected Independent reflections Rint Refined parameters Completeness to theta θ, % GOF (F2 ) Reflections with I>2σ(I) R1(F) (I>2σ(I))a wR2(F2 ) (all data)b Largest diff. peak/hole, e⋅Å-3 C11H11N5O 229.25 Monoclinic P21 11.180(2) 4.1948(8) 11.823(2) 90.00 107.621(3) 90.00 528.44(17) 2 1.441 0.100 240 1.81 – 29.03 6268 1591 0.0403 155 99.6 1.021 1304 0.0667 0.1792 0.337 / -0.383 C18H18F3N5O2 393.37 Triclinic P-1 5.0273(6) 11.0378(13) 16.3137(19) 107.435(3) 91.072(3) 97.262(3) 855.20(17) 2 1.528 0.125 408 1.95 – 27.00 9133 3715 0.0531 263 99.2 1.020 2229 0.0609 0.1648 0.735 / -0.443 a R1 = ∑|Fo – |Fc||/∑(Fo). b wR2 = (∑[w(Fo 2 – Fc 2 )2 ]/∑[w(Fo 2 )2 ]1/2 .
  49. 49. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S6 Fig. S2. Fragment of the crystal packing of 27 (top) and 29 (bottom). H-bonded chains are shown.
  50. 50. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S7 Fig. S3. NCI60 5 dose screen. GI50 Mean Graph for 18.
  51. 51. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S8 Fig. S4. NCI60 5 dose screen. GI50 Mean Graph for 19.
  52. 52. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S9 Fig. S5. NCI60 5 dose screen. GI50 Mean Graph for 20.
  53. 53. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S10 Fig. S6. NCI60 5 dose screen. GI50 Mean Graph for 21.
  54. 54. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S11 Fig. S7. NCI60 5 dose screen. GI50 Mean Graph for 57.
  55. 55. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S12 The Mean Graphs interpretation. From: Methodology of the in vitro cancer screen (http://dtp.nci.nih.gov/branches/btb/ivclsp.html Mean graphs facilitate visual scanning of data for potential patterns of selectivity for particular cell lines or for particular subpanels with respect to a selected response parameter. Bars extending to the right represent sensitivity of cell line to the test agent in excess of the average sensitivity of all tested cell lines. Since the bar scale is logarithmic a bar 2 units to the right implies the compound achieved the response parameter (e.g. GI50) for the cell line at a concentration one-hundredth the mean concentration required over all cell lines, and thus the cell line is usually sensitive to that compound. Bars extending to the left correspondingly imply sensitivity less than the mean.
  56. 56. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S13 Alternative representative scale-up procedure for 3-amino-4-(1H-benzimidazol-2-yl)-furazan 9a. Step 1. Synthesis of 1H-benzimidazol-2-yl(hydroxyimino)acetonitrile 7. 7 N H N CN NOH 6 N H N CN A solution of NaNO2 (69 g, 1 mol) in H2O (100 mL) was added dropwise to a vigorously stirred solution of 2-cyanomethylbenzimidazol 6 [iii] (152g, 1 mol) in AcOH (200 mL) at 5–10 °C. The reaction mixture was allowed to stir for additional 0.5 h at 10–15 °C and diluted with water (200 mL). The residue was filtered, washed with cold water, and dried to give crude acetonitrile 7 (150–160 g, 80– 86% yield), which was further used without purification. Step 2. Synthesis of 2-(1H-benzimidazol-2-yl)-N'-hydroxy-2-(hydroxyimino)-ethanimidamide and 2-(1H-benzoimidazol-2-yl)-N-hydroxy-2-hydroxyimino-acetamidine (mixture of isomers of 8). 87 N H N CN NOH N H N NOH NOH NH2 N H N NOH NH NH OH + A solution of NH2OH•HCl (77 g, 1.2 mol) in water (150 mL) was added to a solution of 1H-benzimidazol-2-yl(hydroxyimino)acetonitrile 7 (150 g, 0.8 mol) in EtOH or iPrOH (500 mL) followed by pouring of K2CO3 (60 g, 0.6 mol) portionwise of 5–10 g. The reaction was stirred for 1 h at room temperature, 1 h at 30 °C, 1 h at 40 °C, and 2 h at 50 °C, and cooled to room temperature. Most of EtOH was evaporated in vacuo from the reaction mixture; the residue was diluted with hot water (70–80 °C) and cooled to room temperature. The precipitate was filtered, washed with cold water, and dried in air to give crude glyoxime 8 (120–125 g, 68–71% yield), which was further used without purification. Step 3. Synthesis of N,N'-bis(acetyloxy)-2-[(acetyloxy)imino]-2-(1H-benzimidazol-2-yl)- ethanimidamide and 2-(1H-Benzoimidazol-2-yl)-N-acetoxy-2-acetoxyimino-N'-acetylacetamidine (mixture of isomers of 8-triacetate). 8 isomers N H N NOH NOH NH2 N H N NOAc NOAc AcNH 8-triacetate isomers
  57. 57. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S14 Acetic anhydrate (181 mL, 196 g, 1.92 mol) was added dropwise to a solution of glyoxime 8 (120 g, 0.55 mol) and dry AcONa (10 g 0.12 mol) in acetic acid at 25–30 °C. The reaction mixture was heated to 90–95 °C during 1 h and stirred for 1 h. Acetic acid (ca. 250 mL) was evaporated in vacuo, the residue was diluted with H2O (300 mL) and cooled to room temperature. The precipitate was filtered, washed with cold water, and dried in air to afford crude 8-triacetate (155–165 g, 82–87% yield), which was further used without purification. Step 4. Synthesis of 3-amino-4-(1H-benzimidazol-2-yl)-furazan 9a. N H N NOAc NOAc AcNH 8-triacetate 9{1,1,1} N H N N O N NH2 8-triacetate (150 g, 0.43 mol) was added to a solution of NaOH (86 g, 2.15 mol) in hot H2O (250 mL, 50 °С). The reaction mixture was refluxed for 1 h and cooled to room temperature. The insoluble impurities were removed by filtration, and the pH was adjusted to ca. 6 with acetic acid. The resulting precipitate was filtered, washed with water, and recrystallized from acetic acid to furnish the pure product 9a. White solid; 58–60 g; 67–70% yield; mp 269 °С (lit. [iv] 264–265 °С). 3-Amino-4-(5-methyl-1H-benzimidazol-2-yl)-furazan (9b). N H N N O N NH2 Me White solid; yield 183 mg (85%); mp 268–269 °С; 1 H NMR (DMSO-d6): δ 2.41 (s, 3H, Me-5), 6.84 (s, 2H, NH2), 7.12 (s, 1H, (H-4), 7.50 (m, 2H, ArH-6,7, 13.53 (br. s, 1H, NH); 13 C NMR (DMSO-d6): δ 21.3, 111.6, 119.1, 124.4, 126.0, 134.1, 138.6, 139.8, 140.9, 155.5; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.86; H 4.18; N 32.47; IR (KBr): ν max 3430, 3332, 3171, 1635, 1597, 1457, 1422, 1317, 1274, 1237, 1134, 1005, 952, 897, 877, 798, 761, 732, 668, 597, 562. 3-Amino-4-(4-methyl-1(3)H-benzimidazol-2-yl)-furazan (9c). N N H N O N H2NMe N N H N O N H2N Me Ratio of isomers 3:2 White solid; yield 157 mg (73%); mp 213–216 °С; 1 H NMR (DMSO-d6): δ 2.55, 2.59 (s/s = 3/2, 3H, Me-4), 6.88, 6.90 (s/s = 3/2, 2H, NH2), 7.05 (t, J = 7.8 Hz, 2H, H-6,7) and 7.20 (m, 2H, H-6,7), 7.38,
  58. 58. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S15 7,58 (2d, J = 7.8 Hz, 1H, H-5), 13.58, 13.64 (s/s = 3/2, 1H, NH); 13 C NMR (DMSO-d6): δ 16.3, 17.1, 109.5, 117.0, 122.4, 122.6, 122.7, 124.4, 124.9, 129.3, 133.9, 134.0, 138.6, 139.4, 140.2, 142.3, 142.6, 155.6; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.86; H 4.18; N 32.47; IR (KBr): ν max 3430, 3323, 3194, 1635, 1620, 1591, 1516, 1456, 1422, 1327, 1268, 1239, 1157, 1138, 1005, 949, 899, 874, 748, 671, 561. 3-Amino-4-(4,5-dimethyl-1(3)H-benzimidazol-2-yl)-furazan (9d). N N H N O N H2N Me Me N N H N O N H2N Me Me White solid; yield 211 mg (92%); mp 242–243 °C; 1 H NMR (DMSO-d6): δ 2.32, 2.46 (s/s = 3/1, 6H, Me-4,5), 6.85, 6.88 (2s, 2H, NH2), 7.11 (m, 1H, H-6), 7.27, 7.47 (2d, J = 8.2 Hz, 1H, H-7), 13.43 (br. s, 2H, NH); 13 C NMR (DMSO-d6): δ 13.6, 14.3, 19.3, 19.6, 109.1, 116.9, 120.5, 125.7, 127.2, 127.5, 129.8, 132.4, 132.6, 135.1, 139.0, 139.8, 140.3, 141.5, 143.2, 156.0; Anal. Calcd for C11H11N5O: C 57.63; H 4.84; N 30.55. Found: C 57.69; H 4.81; N 30.48; IR (KBr): ν max 3425, 3284, 3202, 1634, 1619, 1596, 1504, 1458, 1425, 1373, 1323, 1006, 950, 903, 874, 793, 767, 741, 717, 661, 631, 561, 501. 3-Amino-4-(5-fluoro-1H-benzimidazol-2-yl)-furazan (9e). N H N N O N NH2 F White solid; yield 118 mg (54%); mp 271–272 °C; 1 H NMR (DMSO-d6): δ 6.79 (s, 2H, NH2), 7.16 (t, J = 8.2 Hz, 1H, H-6), 7.42 (d, J = 8.2 Hz, 1H, H-7), 7.67 (s, 1H, H-4), 13.76 (s, 1H, NH); 13 C NMR (DMSO-d6): δ 101.3 (br), 111.83, 112.2, 117.5 (br), 138.4, 141.5, 155.5, 157.8, 161.0; 19 F NMR (DMSO-d6): -118.10, -120.20; Anal. Calcd for C9H6FN5O: C 49.32; H 2.76; N 31.95. Found: C 49.38; H 2.80; N 31.86; IR (KBr): ν max 3453, 3418, 3298, 1624, 1601, 1562, 1505, 1491, 1456, 1432, 1415, 1321, 1265, 1224, 1142, 1112, 1005, 954, 904, 861, 808, 771, 732, 699, 633, 614, 513. 3-Amino-4-(5-chloro-1H-benzimidazol-2-yl)-furazan (9f). N NH2 ON H N NCl White solid; yield 148 mg (63%); mp 301–302 °C; 1 H NMR (DMSO-d6): δ 6.80 (s, 2H, NH2), 7.30 (s, 1H, H-6), 7.59 (s, 1H, H-7), 7.74 (s, 1H, H-4), 13.82 (br. s, 1H, NH); 13 C NMR (DMSO-d6): δ 112.7
  59. 59. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S16 (br. d), 120.1 (br. d), 123.9 (br. d), 128.1 (br. d), 134.1 (br. d), 138.4, 141.6, 143.6, 155.7; Anal. Calcd for C9H6ClN5O: C 45.88; H 2.57; N 29.72. Found: C 45.94; H 2.63; N 29.63; IR (KBr): ν max 3439, 3336, 3167, 3113, 632, 1623, 1597, 1454, 1414, 1309, 1271, 1228, 1134, 1057, 1005, 950, 926, 901, 875, 860, 803, 734, 676, 596, 563. 3-Amino-4-(7-chloro-1H-benzimidazol-2-yl)-furazan (9g). N NN H O N NH2 Cl White solid; yield 146 mg (62%); mp 251–252 °C; 1 H NMR (DMSO-d6): δ 6.85 (s, 2H, NH2), 7.32, 7.36 (2t, J = 7.8 Hz, 1H, H-5), 7.41, 7.45 (2d, J = 7.8 Hz, 1H, H-6), 7.57, 7.79 (2d, J = 7.8 Hz, 1H, H- 4), 14.07, 14.24 (2s, 1H, NH); 13 C NMR (DMSO-d6): δ 111.2, 122.3, 123.4, 125.1, 135.4, 138.2, 139.7, 140.9, 155.5; Anal. Calcd for C9H6ClN5O: C 45.88; H 2.57; N 29.72. Found: C 45.93; H 2.62; N 29.61; IR (KBr): ν max 3452, 3339, 3278, 1616, 1589, 1499, 1453, 1453, 1419, 1318, 1257, 1200, 1113, 1004, 975, 948, 897, 778, 739,631, 606, 569. 3-Amino-4-(5-methoxy-1H-benzimidazol-2-yl)-furazan (9h). N NN H O N NH2 MeO White solid; yield 143 mg (62%); mp 210–211°C; 1 H NMR (DMSO-d6): δ 3.79 (s, 3H, OMe-5), 6.81 (s, 2H, NH2), 6.91 (d, J = 8.0, 1H, H-6), 7.04 (s, 1H, H-7), 7.60 (s, 1H, H-4), 13.49 (s, 1H, NH); 13 C NMR (DMSO-d6): δ 56.0 (OMe), 94.7 (br), 113.5 (br), 120.7 (br), 139.0, 155.9; Anal. Calcd for C10H9N5O2: C 51.95; H 3.92; N 30.29. Found: C 52.01; H 3.90; N 30.21; IR (KBr): ν max 3434, 3287, 1624, 1591, 1510, 1462, 1435, 1411, 1270, 1204, 1164, 1119, 1025, 1001, 953, 896, 865, 819, 627. 3-Amino-4-(4,7-dimethoxy-1H-benzimidazol-2-yl)-furazan (9i). N NN H O N NH2 OMe OMe White solid; yield 154 mg (59%); mp 269–270 °C; 1 H NMR (DMSO-d6): δ 3.91 (s, 6H, OMe-4,7, 6.70 (s, 2H, NH2), 6.72 (s, 2H, H-5,6), 13.95 (s, 1H, NH); 13 C NMR (DMSO-d6): δ 55.9 (OMe), 103.2 (br), 105.2 (br), 138.3, 139.0, 155.5; Anal. Calcd for C11H11N5O3: C 50.57; H 4.24; N 26.81. Found: C 52.63; H 4.26; N 26.71; IR (KBr): ν max 3419, 3335, 3208, 1630,1615, 1529,1464, 1449, 1420, 1349, 1275, 1256, 1213, 1176, 1108, 1097, 1004, 983, 951, 895, 855, 778, 748, 721, 681, 567,516, 486.
  60. 60. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S17 2-(4-Aminofurazan-3-yl)-1H-benzimidazole-5-carboxylic acid (9j). White solid; yield 201 mg (82%); mp 319–320 °C; 1 H NMR (DMSO-d6): δ 6.79 (s, 2H, NH2), 7.71 (s, 1H, H-7), 7.91 (d, J = 8.3, 1H, H-6), 8.26 (br. s, 1H, H-4), 12.44 (br. s, 1H, OH), 13.77 (br. s, 1H, NH); 13 C NMR (DMSO-d6): δ 20.9, 113.9 (br. s), 119.0 (br. s), 124.5, 125.8, 138.3, 142.3, 155.5, 167.5, 171.9; Anal. Calcd for C10H7N5O3: C 48.98; H 2.88; N 28.56. Found: C 49.03; H 2.85; N 28.47; IR (KBr): ν max 3473, 3352, 3102, 2629, 2553, 1686, 1635, 1599, 1493, 1455, 1411, 1326, 1287, 1233, 1150,1137, 1088, 1013, 957, 910, 869, 837, 771, 757, 689, 670, 570, 515. 2-(4-Aminofurazan-3-yl)-1H-benzimidazole-5-carboxylic acid mehtyl ester (9k). N N H N O N H2N MeOOC White solid; yield 218 mg (84%); mp 284–285 °C; 1 H NMR (DMSO-d6): δ 3.82 (s, 3H, OMe), 6,77 (s, 2H, NH2), 7.61 (s, 1H, H-7), 7.78 (s, 1H, H-6), 8.12 (s, 1H, H-4), 13.85 (br. s, 1H, NH); Anal. Calcd for C11H9N5O3: C 50.97; H 3.50; N 27.02. Found: C 51.01; H 3.49; N 26.85; IR (KBr): ν max 3542, 3436, 3330, 1692, 1638, 1602, 1435, 1332, 1294, 1248, 1233, 1132, 1092, 1007, 979,952, 900, 863, 826, 772, 751, 567, 501. 2-(4-Aminofurazan-3-yl)-1H-benzimidazole-5-carbonitrile (9l). N NN H O N NH2N White solid; yield 195 mg (86%); mp 288 °C; 1 H NMR (DMSO-d6): δ 6.75 (s, 2H, NH2), 7.62 (d, J = 8.1 Hz, 1H, H-7), 7.74 (d, J = 8.1 Hz, 1H, H-6), 8.13 (s, 1H, H-4), 14.01 (br. .s, 1H, NH); 13 C NMR (DMSO-d6): δ 105.8, 116.1, 120.0, 122.7, 123.5, 127.3, 138.7, 139.7, 143.7, 156.0; Anal. Calcd for C10H6N6O: C 53.10; H 2.67; N 37.15. Found: C 53.14; H 2.64; N 37.07; IR (KBr): ν max 3421, 3311, 2234, 1644, 1624, 1603, 1445, 1407, 1326, 1286, 1236, 1142, 1096, 1012, 956, 907, 872, 826, 754, 628, 571. 3-Amino-4-(1H-naphtho[2,3-d]imidazol-2-yl)-furazan (9m).
  61. 61. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S18 N N H N O N H2N White solid; yield 126 mg (50%); mp 276–278 °C; 1 H NMR (DMSO-d6): δ 6.94 (s, 2H, NH2), 7.42 (s, 2H, H-4,9), 8.18 (m, 4H, H-5,6,7,8), 13.70 (s, 1H, NH); 13 C NMR (DMSO-d6): δ 107.6, 116.6, 123.7, 124.5, 127.8, 127.9, 130.1, 130.9, 134.5, 138.5, 142.8, 144.4, 155.8; Anal. Calcd for C13H9N5O: C 62.15; H 3.61; N 27.87. Found: C 62.27; H 3.66; N 27.75; IR (KBr): ν max 3298, 1626, 1602, 1559, 1469, 1415, 1310, 1267, 1175, 1135, 1006, 954, 906, 874, 861, 735, 614, 477. 3-Amino-4-(6(8)H-imidazo[4',5':3,4]benzo[1,2-c][1,2,5]furazan-7-yl)-furazan (9n). N N H N O N H2N N O N White solid; yield 124 mg (51%); mp 319–320 °C; 1 H NMR (DMSO-d6): δ 6.74 (s, 2H, NH2), 7.84 (d, J = 9.0 Hz, H-4,5), 14.77 (br..s, 1H, NH); 13 C NMR (DMSO-d6): δ 117.6, 127.4 (br), 140.1 (br), 143.2, 144.6, 154.1, 160.3; Anal. Calcd for C9H5N7O2: C 44.45; H 2.07; N 40.32. Found: C 44.49; H 2.01; N 40.25; IR (KBr): ν max 3450, 3354, 3148, 1614, 1627, 1605, 1562, 1525, 1486, 1443, 1414, 1376, 1331, 1261, 1195, 1121, 1081, 1005, 954, 884, 803, 782, 749, 695, 672, 606, 571, 508. 3-(1H-Benzimidazol-2-yl)-4-(1H-pyrrol-1-yl)-furazan (11a). N N ON H N N White solid; yield 333 mg (78%); mp 214 °С; 1 H NMR (DMSO-d6): δ 6.44 (t, J = 2.2 Hz, 2H, H-3'',4''), 7.36 (br. s, 2H, H-5,6), 7.66 (br. s, 1H, H-7), 7.79 (br. s, 1H, H-4), 7.94 (t, J = 2.2 Hz, 2H, H-2'',5''), 13.69 (s, 1H, NH); 13 C NMR (DMSO-d6): δ 111.9, 120.0 (br. s), 122.4, 134.5 (br. s), 138.1, 141.7, 143.0, 151.3; EIMS m/z 251 [M]+ ; Anal. Calcd for C13H9N5O: C 62.15; H 3.61; N 27.87. Found: C 62.19; H 3.63; N 27.81; IR (KBr): ν max 2158-2586, 1565, 1504, 1480, 1435, 1399, 1390, 1374, 1324, 1281, 1232, 1195, 1149, 1116, 1065, 1034, 1003, 967, 919, 905, 886, 737, 604, 583, 484, 456. 3-(5-Methoxy-1H-benzimidazol-2-yl)-4-(1H-pyrrol-1-yl)-furazan (11h).
  62. 62. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S19 N NN H O N MeO N White solid; yield 359 mg (75%); mp 165–166 °C; 1 H NMR (DMSO-d6): δ 3.84 (s, 3H, OMe -5), 6.44 (t, J = 2.2 Hz, 2H, H-3'',4''), 6.94, 7.02 (2dd, J = 2.4 Hz, J = 8.9 Hz, 1H, H-6), 7.04, 7.33 (2d, J = 2.2 Hz, 1H, H-4), 7.50, 7.71 (2d, J = 8.9 Hz, 1H, H-7), 7.94 (t, J = 2.2 Hz, 2H, H-2'',5''), 13.50, 13.56 (2s, 1H, NH); EIMS m/z 281 [M]+ ; Anal. Calcd for C14H11N5O2: C 59.78; H 3.94; N 24.90. Found: C 59.85; H 3.96; N 24.81; IR (KBr): ν max 3144-2672, 1631, 1594, 1566, 1483, 1461, 1430, 1388, 1334, 1276, 1202, 1159, 1113, 1065, 1032, 971, 891, 820, 732, 603. 3-(4,7-Dimethoxy-1H-benzimidazol-2-yl)-4-(1H-pyrrol-1-yl)-furazan (11i). N NN H O N NOMe OMe White solid; yield 402 mg (76%); mp 188 °C; 1 H NMR (DMSO-d6): δ 3.91 (s, 6H, OMe-4,7), 6.40 (br. s, 2H, H-3'',4''), 6.65 (d, J = 7.8 Hz, 1H, CH), 6.76 (d, J = 7.8 Hz, 1H, CH), 7.75 (m, 2H, H-2'',5''), 13.94 (s, 1H, NH). 13 C NMR (DMSO-d6): δ 55.8, 55.9, 103.4, 105.0, 112.0, 121.9, 126.4, 135.0, 136.2, 140.6, 141.4, 145.7, 151.2; Anal. Calcd for C15H13N5O3: C 57.87; H 4.21; N 22.50. Found: C 57.95; H 4.16; N 22.32; IR (KBr): ν max 3244, 3111, 1582, 1561, 1528, 1484, 1461, 1388, 1342, 1270, 1174, 1107, 1095, 1063, 1037, 1005, 986, 970, 911, 892, 858, 787, 741, 722, 666, 598. Alkylation by benzylhalides 12′′′′–43′′′′ (yields 75–90%). 2-[2-(4-Aminofurazan-3-yl)-1H-benzimidazol-1-yl]ethanol (12). N N N O N NH2 OH White solid; yield 1.72 g (70%); mp 173–176 °C; 1 H NMR (DMSO-d6): δ 3.80 (q, J = 4.7 Hz, J = 5.5 Hz, 2H, CH2O), 4.74 (t, J = 5.5Hz, 2H, CH2N), 4.92 (t, J = 4.7 Hz, 1H, OH), 7.00 (s, 2H, NH2), 7.35 (t, J = 8.1 Hz, 1H, H-5), 7.42 (t, J = 8.1 Hz, 1H, H-6), 7.77 (d, J = 8.1 Hz, 1H, H-7), 7.82 (d, J = 8.1 Hz, 1H, H-4); EIMS m/z 245 [M]+ (37), 228 (1), 215 (12), 206 (6), 188 (100), 172 (9), 157 (22), 156 (22),
  63. 63. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S20 144 (96), 131 (5), 118 (12), 102 (6), 77 (10); Anal. Calcd for C11H11N5O2: C 53.87; H 4.52; N 28.56. Found: C 53.82; H 4.50; N 28.61. 3-Amino-4-(1-propargyl-1H-benzimidazol-2-yl)-furazan (13). N N N N O NH2 White solid; yield 1.72 g (72%); mp 210 °C; 1 H NMR (DMSO-d6): 3.33 (br. s, 1H, CH), 5.57, 5.58 (2s, 2H, CH2), 6.88 (br. s, 2H, NH2), 7.40 (t, J = 7.6 Hz, 1H, H-6), 7.49 (t, J = 7.6 Hz, 1H, H-5), 7.82 (d, J = 7.6 Hz, 1H, H-7), 7.86 (d, J = 7.6 Hz, 1H, H-4); EIMS m/z 239 [M]+ (21), 209 (31), 182 (38), 144 (100), 118 (37), 77 (8); Anal. Calcd for C12H9N5O: C 60.25; H 3.79; N 29.27. Found: C 60.29; H 3.82; N 29.18. N-[4-(1-Allyl-1H-benzimidazol-2-yl)-furazan-3-yl]acetamide (14r). N N N N O NH O White solid; yield 2.15 g (76%); mp 160–161 °C; 1 H NMR (DMSO-d6): δ 2.31 (s, 3H, CH3CO), 5.05 (d, J = 17.0 Hz, 1H, CH2=), 5.20 (d, J = 10.0 Hz, 1H, CH2=), 5.30 (s, 2H, CH2), 6.07 (m, 1H, CH=), 7.42 (t, J = 8.0 Hz, 1H, H-6), 7.47 (t, J = 8.0 Hz, 1H, H-5), 7.75 (d, J = 8.0 Hz, 1H, H-7), 7.90 (d, J = 8.0 Hz, 1H, H-4), 10.91 (s, 1H, NH); EIMS m/z 283 [M]+ (3), 268 (5), 253 (1), 241 (1), 227 (1), 211 (10), 200 (7), 194 (4), 184 (45), 182 (5), 169 (5), 156 (5), 144 (6), 129 (2), 116 (3), 102 (5), 90 (4), 77 (9), 43 (99), 41 (100); Anal. Calcd for C14H13N5O2: C 59.36; H 4.63; N 24.72. Found: C 59.43; H 4.66; N 24.60. 3-Amino-4-[1-(2-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan (15). N N N O N NH2 F
  64. 64. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S21 White solid; yield 2.63 g (85%); mp 184–185 °C; 1 H NMR (DMSO-d6): δ 6.03 (s, 2H, CH2), 6.81 (t, J = 7.6 Hz, 1H, H-6''), 6.95 (s, 2H, NH2), 7.06 (t, J = 7.6 Hz, 1H, H-5''), 7.23 (t, J = 8.7 Hz, 1H, H-3''), 7.32 (m, 1H, H-4''), 7.38 (t, J = 7.7 Hz, 1H, H-5), 7.41 (t, J = 7.7 Hz, 1H, H-6), 7.67 (d, J = 7.7 Hz, 1H, H-7), 7.88 (d, J = 7.7 Hz, 1H, H-4; EIMS m/z 309 [M]+ (5), 279 (4), 252 (6), 143 (8), 110 (5), 109 (100), 83 (18); Anal. Calcd for C16H12FN5O: C 62.13; H 3.91; N 22.64. Found: C 62.19; H 3.94; N 22.57. 3-Amino-4-[1-(3-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan (16). N N N O N NH2 F White solid; yield 2.41 g (78%); mp 158–160 °C; 1 H NMR (DMSO-d6): δ 6.00 (s, 2H, CH2), 6.89 (br. s, 2H, NH2), 6.98 (m, 3H, H-4'',5'',6''), 7.30 (t, J = 7.3 Hz, 1H, H-5), 7.36 (m, 2H, H-6,2''), 7.62 (d, J = 7.3 Hz, 1H, H-7), 7.82 (d, J = 7.3 Hz, 1H, H-4); EIMS m/z 309 [M]+ (7), 279 (6), 252 (9), 237 (6), 236 (7), 170 (1), 143 (13), 109 (100), 83 (25); Anal. Calcd for C16H12FN5O: C 62.13; H 3.91; N 22.64. Found: C 62.18; H 3.93; N 22.60. N-(4-[1-(3-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan 3-yl)propionamide (16s). N N N O N NH F O White solid; yield 2.7 g (74%); mp 169–170 °C; 1 H NMR (DMSO-d6): 1.20 (t, J = 7.4 Hz, 3H, CH3CH2), 2.62 (q, J = 7.4 Hz, 2H, CH2CO), 5.96 (s, 2H, CH2), 7.03 (d, J = 7.7 Hz, 1H, H-6''), 7.08 (m, 2H, H-4'',5''), 7.35 (br. k, 1H, J = 6.8 Hz, J =7.7 Hz, H-2''), 7.41 (t, J = 7.7 Hz, 1H, H-5), 7.44 (t, J = 7.7 Hz, 1H, H-6), 7.70 (d, J = 7.7 Hz, 1H, H-7), 7.90 (d, J = 7.7 Hz, 1H, H-4), 11.00 (s, 1H, NH); EIMS m/z 365 [M]+ (6), 335 (7), 309 (11), 308 (13), 237 (8), 170 (2), 143 (15), 109 (100), 83 (23); Anal. Calcd for C19H16FN5O2: C 62.46; H 4.41; N 19.17. Found: C 62.52; H 4.45; N 19.05. 3-Amino-4-[1-(4-fluorobenzyl)-1H-benzimidazol-2-yl]-furazan (17).
  65. 65. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S22 N N N N O NH2 F White solid; yield 2.78 g (90%); mp 176 °C; 1 H NMR (DMSO-d6): δ 5.95 (s, 2H, CH2), 6.94 (s, 2H, NH2), 7.12 (t, J = 8.7 Hz, 2H, H-3'',5''), 7.24 (dd, J = 5.5 Hz, J = 8.7 Hz, 2H, H-2'',6''), 7.37 (t, J = 7.6 Hz, 1H, H-5), 7.41 (t, J =7.6 Hz, 1H, H-6), 7.71 (d, J = 7.8 Hz, 1H, H-7), 7.86 (d, J = 8.1 Hz, 1H, H-4); EIMS m/z 309 [M]+ (6), 379 (7), 263 (1), 143 (6), 110 (6), 109 (100), 83 (16), 63 (6); Anal. Calcd for C16H12FN5O: C 62.13; H 3.91; N 22.64. Found: C 62.05; H 3.88; N 22.69. 3-Amino-4-[1-(3-bromobenzyl)-1H-benzimidazol-2-yl]-furazan (18). N N N O N NH2 Br White solid; yield 2.78 g (75%); mp 176–177 °C; 1 H NMR (DMSO-d6): δ 5.99 (s, 2H, CH2), 6.90 (br. s, 2H, NH2), 7.07 (d, J = 7.7 Hz, 1H, H-6''), 7.22 (t, J = 7.7 Hz, 1H, H-5''), 7.37 (m, 2H, H-5,6)), 7.40 (br. d, J = 7.7 Hz, 1H, H-4''), 7.44 (br. s, 1H, H-2''), 7.62 (br. d, J = 7.3 Hz, 1H, H-7), 7.82 (br. d, J = 7.3 Hz, 1H, H-4); EIMS m/z 371 [M+1]+ (5), 369 [M-1]+ (5), 341 (6), 339 (6), 314 (9), 312 (9), 260 (4), 234 (5), 217 (7), 169 (100), 143 (38), 116 (10), 102 (9), 90 (84), 89 (54), 77 (12), 63 (27); Anal. Calcd for C16H12BrN5O: C 51.91; H 3.27; N 18.92. Found: C 51.87; H 3.24; N 18.89. 3-Amino-4-[1-(3-trifluoromethylbenzyl)-1H-benzimidazol-2-yl]-furazan (19). N N N O N NH2 CF3 White solid; yield 2.91 g (81%); mp 169–170 °C; 1 H NMR (DMSO-d6): δ 6.07 (s, 2H, CH2), 6.92 (s, 2H, NH2), 7.34 (d, J = 7.7 Hz, 1H, H-6''), 7.39 (m, 2H, H-5,6), 7.51 (t, J = 7.7 Hz, 1H, H-5''), 7.60 (d, J = 7.7 Hz, 1H, H-4''), 7.64 (s, 1H, H-2''), 7.70 (d, J = 7.6 Hz, 1H, H-7), 7.86 (d, J = 7.7 Hz, 1H, H-4); EIMS m/z 359 [M]+ (2), 329 (2), 302 (6), 287 (4), 286 (4), 159 (100), 143 (15), 119 (8), 109 (29), 90
  66. 66. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S23 (7), 89 (6), 63 (7); Anal. Calcd for C17H12F3N5O: C 56.83; H 3.37; N 19.49. Found: C 56.77; H 3.35; N 19.55. N-(4-[1-(3-Trifluoromethylbenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (19r). N N N O N NH CF3 O White solid; yield 2.93 g (73%); mp 213–214 °C; 1 H NMR (DMSO-d6): δ 2.32 (s, 3H, CH3CO), 6.03 (s, 2H, CH2), 7.41 (m, 2H, H-5,6,6''), 7.51 (t, J = 7.7 Hz, 1H, H-5''), 7.61 (t, J = 7.7 Hz, 1H, H-4''), 7.68 (br. s, 1H, H-2''), 7.70 (d, J = 7.7 Hz, 1H, H-7), 7.91 (d, J = 7.7 Hz, 1H, H-4), 10.95 (s, 1H, NH); EIMS m/z 401 [M]+ (0.1), 386 (1), 329 (3), 302 (5), 159 (84), 109 (21), 43 (100); Anal. Calcd for C19H14F3N5O2: C 56.36; H 3.52; N 17.45. Found: C 56.22; H 3.48; N 17.57. 3-Amino-4-[1-(3-methoxybenzyl)-1H-benzimidazol-2-yl]-furazan (20). N N N O N NH2 OMe White solid; yield 2.67 g (83%); mp 181–183 °C; 1 H NMR (DMSO-d6): δ 3.68 (s, 3H, OMe -3''), 5.94 (s, 2H, CH2), 6.66 (br. d, J = 7.3 Hz, 1H, H-4''), 6.76 ( br. s, 1H, H-2''), 6.82 (br. d, J = 8.2 Hz, 1H, H- 6''), 6.94 (s, 2H, NH2), 7.20 (t, J = 7.8 Hz, 1H, H-5''), 7.37 (t, J = 7.7 Hz, 1H, H-5), 7.41 (t, J = 7.3 Hz, 1H, H-6), 7.69 (d, J = 7.7 Hz, 1H, H-7), 7.86 (d, J = 7.7 Hz, 1H, H-4); EIMS m/z 321 [M]+ (6), 291 (17), 276 (3), 143 (5), 121 (100), 91 (37), 78 (19), 77 (25); Anal. Calcd for C17H15N5O2: C 63.54; H 4.71; N 21.79. Found: C 63.61; H 4.73; N 21.75. N-(4-[1-(3-Methoxybenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (20r).
  67. 67. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S24 N N N O N NH CF3 O White solid; yield 2.73 g (75%); mp 151 °C; 1 H NMR (DMSO-d6): δ 2.30 (s, 3H, CH3CO), 3.69 (s, 3H, OMe-3''), 5.89 (s, 2H, CH2), 6.72 (br. d, J = 7.5 Hz, 1H, H-4''), 6.81 (br. s, 1H, H-2''), 6.83 (dd, J = 2.1 Hz, J = 8.2 Hz, 1H, H-6''), 7.21 (t, J = 7.9 Hz, 1H, H-5''), 7.40 (t, J = 7.2 Hz, 1H, H-5), 7.43 (t, J = 7.2 Hz, 1H, H-6), 7.69 (d, J = 7.7 Hz, 1H, H-7), 7.91 (d, J = 7.7 Hz, 1H, H-4), 10.94 (s, 1H, NH); EIMS m/z 363 [M]+ (32), 348 (16), 333 (2), 321 (21), 291 (59), 264 (25), 143 (10), 121 (100), 91 (25), 78 (13), 77 (14), 43 (90); Anal. Calcd for C19H17N5O3: C 62.80; H 4.72; N 19.27. Found: C 62.72; H 4.70; N 19.34. 3-Amino-4-[1-(3-methoxy-4-methylbenzyl)-1H-benzimidazol-2-yl)-furazan (21). N N N O N NH2 MeO Me White solid; yield 2.72 g (81%); mp 198–200 °C; 1 H NMR (DMSO-d6): δ 2.11 (s, 3H, Me-4''), 3.84 (s, 3H, OMe-3''), 5.90 (s, 2H, CH2), 6.44 (br. s, 1H, H-2''), 6.79 (s, 2H, NH2), 6.84 (d, J = 8.3 Hz, 1H, H- 5''), 6.99 ( br. d, J = 8.3 Hz, 1H, H-6''), 7.29 (m, 2H, H-5,6), 7.40 (m, 1H, H-7), 7.78 (m, 1H, H-4'); EIMS m/z 335 [M]+ (31), 318 (3), 305 (19), 290 (1), 143 (13), 135 (100), 105 (40), 103 (9), 91 (13), 79 (10), 77 (11); Anal. Calcd for C18H17N5O2: C 64.47; H 5.11; N 20.88. Found: C 64.52; H 5.13; N 20.83. 3-Amino-4-[1-(3,4-diethoxybenzyl)-1H-benzimidazol-2-yl]-furazan (22). N N N O N NH2 EtO OEt White solid; yield 3.26 g (86%); mp 168–170 °C; 1 H NMR (DMSO-d6): δ 1.34 (t, J = 7.0 Hz, 3H, CH3CH2), 1.35 (t, J = 7.0 Hz, 3H, CH3CH2), 3.95 (q, J = 7.0 Hz, 4H, 2OCH2), 5.87 (s, 2H, CH2), 6.63
  68. 68. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S25 (dd, J = 1.6 Hz, J = 8.2 Hz, 1H, H-6''), 6.73 (d, J = 8.2 Hz, 1H, H-5''), 6.81 (s, 2H, NH2), 6.86 (d, J = 1.6 Hz, 1H, H-2''), 7.33 (m, 2H, H-5',6'), 7.59 (d, J = 7.8 Hz, 1H, H-7), 7.78 (d, J = 7.6 Hz, 1H, H-4); EIMS m/z 379 [M]+ (28), 349 (10), 179 (100), 151 (40), 144 (11), 143 (8), 133 (6), 123 (65), 105 (5), 94 (7), 77 (12); Anal. Calcd for C20H21N5O3: C 63.31; H 5.58; N 18.46. Found: C 63.40; H 5.61; N 18.56. 3-Amino-4-[1-(4-methylbenzyl)-1H-benzimidazol-2-yl]-furazan (23). N N N O N NH2 Me White solid; yield 2.32 g (76%); mp 184–185 °C; 1 H NMR (DMSO-d6): 2.23 (s, 3H, Me-4''), 5.93 (s, 2H, CH2), 6.95 (s, 2H, NH2), 7.05 (d, J = 8.0 Hz, 2H, H-3'',5''), 7.10 (d, J = 8.0 Hz, 2H, H-2'',6''), 7.36 (t, J = 7.8 Hz, 1H, H-6'), 7.40 (t, J = 7.8 Hz, 1H, H-5), 7.68 (d, J = 7.8 Hz, 1H, H-7), 7.86 (d, J = 7.8 Hz, 1H, H-4); EIMS m/z 305 [M]+ (18), 275 (16), 259 (1), 248 (5), 232 (7), 106 (7), 105 (100), 103 (8), 77 (10); Anal. Calcd for C17H15N5O: C 66.87; H 4.95; N 22.94. Found: C 66.94; H 4.98; N 22.80. 3-Amino-4-[1-(4-t butylbenzyl)-1H-benzimidazol-2-yl]-furazan (24). N N N O N NH2 t-Bu White solid; yield 2.54 g (73%); mp 175 °C; 1 H NMR (DMSO-d6): δ 1.24 (s, 9H, C(CH3)3-4''), 5.94 (s, 2H, CH2), 6.94 (s, 2H, NH2), 7.10 (d, J = 8.1 Hz, 2H, H-3'',5''), 7.30 (d, J = 8.1 Hz, 2H, H-2'',6''), 7.35 (t, J = 7.8 Hz, 1H, H-6), 7.39 (t, J = 7.8 Hz, 1H, H-5), 7.67 (d, J = 7.8 Hz, 1H, H-7), 7.83 (d, J =7.8 Hz, 1H, H-4); EIMS m/z 347 [M]+ (15), 317 (11), 261 (11), 148 (11), 147 (100), 138 (6), 132 (18), 124 (22), 117 (23), 91 (12); Anal. Calcd for C20H21N5O: C 69.14; H 6.09; N 20.16. Found: C 69.25; H 6.14; N 20.02. N-(4-[1-(4-t Butylbenzyl)-1H-benzimidazol-2-yl]-furazan-3-ylacetamide (24r).
  69. 69. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S26 N N N O N NH t-Bu O White solid; yield 2.8 g (72%); mp 166–167 °C; 1 H NMR (DMSO-d6): 1.24 (s, 9H, C(CH3)3-4''), 2.33 (s, 3H, CH3CO), 5.91 (s, 2H, CH2), 7.14 (d, J = 8.2 Hz, 2H, H-3'',5''), 7.30 (d, J = 8.2 Hz, 2H, H-2'',6''), 7.39 (t, J = 7.6 Hz, 1H, H-6), 7.42 (t, J = 7.6 Hz, 1H, H-5), 7.70 (d, J = 7.6 Hz, 1H, H-7), 7.89 (d, J = 7.6 Hz, 1H, H-4), 10.96 (s, 1H, NH); EIMS m/z 389 [M]+ (3), 374 (1), 347 (2), 317 (14), 261 (6), 147 (100), 132 (19), 124 (6), 119 (7), 117 (21), 105 (9), 91 (11); Anal. Calcd for C22H23N5O2: C 67.85; H 5.95; N 17.98. Found: C 67.95; H 6.00; N 17.87. 3-Amino-4-[1-(4-Cyanobenzyl)-1H-benzimidazol-2-yl]-furazan (25). N N N O N NH2 N White solid; yield 2.66 g (84%); mp 224–225 °C; 1 H NMR (DMSO-d6): δ 6.07 (s, 2H, CH2), 6.91 (s, 2H, NH2), 7.32 (d, J = 8.2 Hz, 2H, H-2'',6''), 7.39 (m, 2H, H-5,6), 7.66 (m, 1H, H-7), 7.73 (d, J = 8.2 Hz, 2H, H-3'',5''), 7.86 (m, 1H, H-4); EIMS m/z 316 [M]+ (5), 286 (4), 259 (8), 143 (8), 117 (9), 116 (100), 89 (24); Anal. Calcd for C17H12N6O: C 64.55; H 3.82; N 26.57. Found: C 64.61; H 3.84; N 26.50. N-(4-[1-(4-Cyanobenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (25r). N N N O N NH O N
  70. 70. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S27 White solid; yield 2.72 g (76%); mp 224–226 °C; 1 H NMR (DMSO-d6): δ 2.32 (s, 3H, CH3CO), 6.03 (s, 2H, CH2), 7.37 (d, J = 8.2 Hz, 2H, H-2'',6''), 7.42 (m, 2H, H-5,6), 7.66 (m, 1H, H-7), 7.73 (d, J = 8.2 Hz, 2H, H-3'',5''), 7.91 (m, 1H, H-4), 10.90 (s, 1H, NH); EIMS m/z 358 [M]+ (18), 344 (6), 343 (28), 341 (1), 316 (11), 286 (27), 259 (41), 244 (5), 117 (8), 116 (100), 89 (12), 43 (15); Anal. Calcd for C19H14N6O2: C 63.68; H 3.94; N 23.45. Found: C 63.79; H 3.97; N 23.32. 3-Amino-4-(1-methyl-1H-benzimidazol-2-yl)-furazan (26). N N Me N O N H2N White solid; yield 1.44 g (67%); mp 206–207 °C; 1 H NMR (DMSO-d6): δ 4.12 (s, 3H, Me), 6.88 (s, 2H, NH2), 7.32 (t, J = 7.8 Hz, 1H, H-5), 7.40 (t, J = 7.8 Hz, 1H, H-6), 7.68 (d, J = 8.2 Hz, 1H, H-7), 7.79 (d, J = 8.2 Hz, 1H, H-4); 13 C NMR (DMSO-d6): δ 31.9, 110.6, 119.5, 122.7, 124.1, 135.6, 138.2, 140.7, 141.4, 155.9; Anal. Calcd for C10H9N5O: C 55.81; H 4.22; N 32.54. Found: C 55.83; H 4.18; N 32.46; IR (KBr): ν max 3410, 3311, 1633, 1598, 1577, 1549, 1472, 1457, 1422, 1327, 1290, 1262, 1231, 1157, 1132, 1071, 998, 905, 866, 813, 754, 740, 717, 604, 568, 546. 4-Fluoro-N-[4-(1-methyl-1H-benzimidazol-2-yl)- furazan-3-yl]benzamide (26t). N N N O N NH Me O F White solid; yield 2.53 g (75%); mp 251–252 °C; 1 H NMR (DMSO-d6): 4.23 (s, 3H, Me), 7.40 (t, J = 7.8 Hz, 1H, H-6), 7.48 (t, J = 7.8 Hz, 1H, H-5), 7.53 (t, J = 8.8 Hz, 2H, H-3'',5''), 7.78 (d, J = 7.8 Hz, 1H, H-7), 7.92 (d, J = 7.8 Hz, 1H, H-4), 8.15 (dd, J = 5.8 Hz, J = 8.8 Hz, 2H, H-2'',6''), 10.14 (s, 1H, NH); EIMS m/z 337 [M]+ (11), 170 (8), 158 (100), 143 (5), 123 (32), 95 (21), 75 (6); Anal. Calcd for C17H12FN5O2: C 60.53; H 3.59; N 20.76. Found: C 60.43; H 3.56; N 20.90. 3-Amino-4-(1-ethyl-1H-benzimidazol-2-yl)-furazan (27). N N N O N NH2 Et White solid; yield 1.95 g (85%); mp 168–169 °C; 1 H NMR (DMSO-d6): δ 1.35 (t, J = 7.0 Hz, 3H, CH3CH2), 4.63 (q, J = 7.0 Hz, 2H, CH2N), 7.01 (s, 2H, NH2), 7.30 (t, J = 7.7 Hz, 1H, H-5), 7.38 (t, J =
  71. 71. M ANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT S28 7.7 Hz, 1H, H-6), 7.70 (d, J = 8.0, 1H, H-7), 7.78 (d, J = 8.0, 1H, H-4); 13 C NMR (DMSO-d6): δ 14.8, 40.1, 110.8, 119.9, 123.0, 124.5, 134.7, 138.1, 140.1, 141.7, 156.2; Anal. Calcd for C11H11N5O: C 57.63; H 4.84; N 30.55. Found: C 57.67; H 4.80; N 30.48; IR (KBr): ν max 3405, 3302, 2988, 2976, 2939, 1635, 1626, 1593, 1575, 1545, 1485, 1472, 1461, 1446, 1410, 1376, 1351, 1330, 1293, 1260, 1202, 1155, 1134, 1088, 1074, 998, 955, 907, 864, 784, 757, 742, 705, 565, 466. N-(4-[1-(4-Methoxybenzyl)-1H-benzimidazol-2-yl]-furazan-3-yl)acetamide (28r). N N N O N NH O MeO White solid; yield 3.02 g (83%); mp 170–173 °C; 1 H NMR (DMSO-d6): δ 2.36 (s, 3H, CH3CO), 3.73 (s, 2H, OMe-4''), 5.89 (s, 2H, CH2), 6.80 (d, J = 8.5 Hz, 2H, H-3'',5''), 7.17 (d, J = 8.5 Hz, 2H, H-2'',6''), 7.36 (d, J = 7.6 Hz, 1H, H-6), 7.39 (t, J = 8.0 Hz, 1H, H-5), 7.64 (d, J = 7.6 Hz, 1H, H-7), 7.85 (d, J = 8.0 Hz, 1H, H-4), 10.95 (s, 1H, NH); EIMS m/z 363 [M]+ (5), 333 (1), 321 (3), 291 (15), 121 (100), 77 (10), 43 (22); Anal. Calcd for C19H17N5O3: C 62.80; H 4.72; N 19.27. Found: C 62.90; H 4.75; N 19.20. 3-Amino-4-(1-(4-methoxybenzyl)-5-(trifluoromethyl)-1H-benzimidazol-2-yl)-furazan (29). N N N O N NH2 MeO F3C White solid; yield 2.3 g (59%); mp 187–188 °C; 1 H NMR (DMSO-d6): δ 3.69 (s, 3H, OMe-4''), 5.95 (s, 2H, CH2), 6.86 (d, J = 8.6 Hz, 2H, H-3'',5''), 7.02 (s, 2H, NH2), 7.16 (d, J = 8.6 Hz, 2H, H-2'',6''), 7.74 (dd, J = 1.6 Hz, J = 8.6 Hz, 1H, H-6), 7.98 (d, J = 8.6 Hz, 1H, H-7), 8.26 (d, J = 1.6 Hz, H-4); 13 C NMR (DMSO-d6): δ 159.3, 156.7,143.3, 141.6, 138.4, 137.8, 128.7, 128.3, 126.9, 124.9, 124.4, 123.3, 121.6, 118.1, 114.6, 113.2, 55.3, 48.4; 19 F NMR (DMSO-d6): -60.24. 15 N NMR (DMSO-d6): 26.70, -18.27, - 131.45, -207.32, -332.63; EIMS m/z 389 [M]+ ; Anal. Calcd for C18H14F3N5O2: C 55.53; H 3.62; N 17.99. Found: C 55.48; H 3.60; N 17.92; IR (KBr): ν max 3442, 3335, 1640, 1614, 1581, 1517, 1464, 1441, 1343, 1331, 1304, 1268, 1255, 1231, 1183, 1158, 1105, 1052, 1033, 1009, 972, 890, 867, 836, 816, 804, 780, 756, 699, 652, 631, 569.

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