Synthesis of Some New Arylazothiophene and Arylazopyrazole Derivatives as Antitumor Agents

The starting 1-phenylbutane-1,3-dione (1) was used as key intermediate for the synthesis of several new thiophene and pyrazole derivatives. The newly synthesized compounds were evaluated for in vitro cytotoxicity against an Ehrlich ascites cells and in vivo cytotoxicity for compound 10d using EAC assay and 5-fluorouracil is used as reference drug. Compounds 7c, e and 10c, d showed significant activity in certain cancer cell and have been targeted for further studies, compound 10d is more effective and showed the highest activity. Structures of the newly prepared compounds were confirmed by both spectral, analytical data and molecular calculations.


Introduction
Aryl isothiocyanates are versatile reagents which have been used as synthetic intermediate to prepare biologically active heterocyclic compounds [1].As a part of our program of developing new, simple and efficient procedures for the synthesis of new aromatic compounds using readily available aryl isothiocyanates, we have recently affected recyclization of thiocarbamoyl into pyrazoles and thiazoles [2][3][4][5].This procedure appears to be a fundamental type of type of thiocarbamoyl transformation into pyrazole ring.
In continuation of our studies on the chemistry of thiocarbamoyl and active methylene compounds [6,7] and as a part of our program directed toward developing new approaches to a variety of heterocycles exhibited antitumor activities [8,9], we report here the scope and applicability of 1-phenylbutane-1,3-dione (1) as a unique precursor for the synthesis of some new pyrazole, thiophene and thiocarbamoyl derivatives and their behavior towards different reagents.

General
All melting points are in degree centigrade and were determined on Gallenkamp electric melting point apparatus.The IR spectra ν/cm -1 (KBr) were recorded on Perkin Elmer Infrared Spectrophotometer Model 157, Grating.
The 1 H-NMR spectra were obtained on a Varian Spectrophotometer at 200 MHz using TMS as an internal reference and DMSO-d 6 as solvent.The mass spectra (EI) were recorded on 70 eV with Kratos MS equipment and/ or a Varian MAT 311 A Spectrometer.Elemental analyses (C, H, and N) were carried out at the Micro Analytical Center of Cairo Univ., Giza, Egypt.

Synthesis of Thiocarbamoyl Derivative 3
To a cold suspension of potassium hydroxide (1.4 g, 25 mmol) in DMF (30 mL) was added the benzoyl acetone (4.05 g, 25 mmol), followed by phenyl isothiocyanate (3.375 g, 25 mmol).The mixture was stirred overnight at room temperature and then poured onto ice-cold water.Acidification using dilute HCl until the medium becomes acidic gave solid product 3 which was filtered off, washed with water, dried and crystallized from aqueous ethanol to give compound 3.

Coupling of 3 with Aromatic Diazonium Salts:
Formation of Monoazothiocarbamoyl Derivatives 4a-e General procedure: A well stirred solution of aromatic amines (20 mmol) in concentrated HCl (6 mL) and water (4 mL) was cooled in an ice bath and diazotized with a solution of sodium nitrite (1.39 g, 20 mmol) in water (5 mL).

Synthesis of the Acyclic Intermediate 8a, 9a, 11a
and 13a Equimolecular quantities of 4a (3.59 g, 10 mmol) in ethanol containing potassium carbonate (1.39 g, 10 mmol) and phenacyl bromide and/or ethyl bromoacetate and/or chloroacetonitrile and/or chloroacetone were stirred for 6 hrs at room temperature, then left to stand at the same temperature for 24 h.The separated solid product was washed with water, dried and crystallized from ethanol to give 8a, 9a, 11a and 13a, respectively.8a, Yield 58%; mp 153˚C; IR (KBr): ν/cm

Synthesis of Thiophene Derivatives Pathway 1)
A mixture of equimolecular amounts of 4a-e and α-halo compounds (10 mmol) was stirred in DMF (20 mL) containing potassium carbonate (1.39 g, 10 mmol) overnight.The reaction mixture was poured onto ice-cold water, acidified by dilute HCl, filtered off and recrystallized from ethanol to give the corresponding thiophene derivatives.

Antitumor Activity
Cells of Ehrlich ascites tumor were obtained from National Cancer Institute, Cairo, Egypt.Animals, adult Swiss male albino mice (20 -25 g) were procured from Pharmacology Faculty, Mansoura University, Egypt and used throughout this study.They were housed in microlon boxes in a controlled environment (temperature 25˚C + 2˚C and 12 h dark/light cycle) with standard laboratory diet and water ad libitum (5-Fluorouracil → 20 mg/kg).

Animals Were Divided into Seven Groups as
Follows All animals were inoculated with 2 × 10 6 cells/mouse on day "0" except normal group and treatment started 24 h after inoculation, at 2 doses of 50 and 250 mg/kg/day, i.p.The control group was treated with the same volume of 0.9% sodium chloride solution.All the treatments were given for nine days.

Tumor Reducing Activity
Ehrlich ascites tumor cells (0.2 mL) were injected into the mice peritoneal cavity and different concentrations (1 mg/mL to 0.1 mL/mice on day) of the drug (5 mice/group) were injected from day-1 to day-9 every day.Animals were observed for the development as ascites tumor and death due to tumor burdon.Life span % I LS = (T-C)/C × 100, where T is the number of days the treated animals survived and C is the number of days control animals survived.% I LS more than 25% was considered as significant [10].

Chemistry
We have been particularly interested in studying if reactions of such thiocarbamoyl might be extended to include more general synthesis of other classes of organic compounds and its utility as synthetic intermediate for the synthesis of new heterocyclic compounds.The present work reports on the synthesis of several new arylazothiophene and arylazopyrazole derivatives by the reaction of thiocarbamoyl of the type 4 with compounds containing an active methylene group in the presence of a base.Reactions of this type have not been reported previously, but they were found to give products in excellent yields under very mild conditions.Moreover, the resulting thiophene and pyrazole derivatives have latent functional substituents which have potential for further chemical transformations and new routes for the preparation of substituted thiophene and pyrazole derivatives with possible biological activity.Now, we have extended our synthetic program to the synthesis of otherwise inaccessible heterocyclic ring system utilizing phenyl isothiocyanate as a key starting material.It is known that a great variety from its spectral data.Its 1 H NMR spectrum showed two multiplet signals integrated for (20H) centered at 7.4 and 8.0 (aromatic protons) and a singlet (1H) at δ 10.2.On shaking the compound with D 2 O, the broad band signal at δ 10.2 disappeared.Based on the foregoing data, structure 7a was assigned to this product.The structure 7a was further confirmed by alternative synthesis.Thus, it was found that, stirring of 4a with phenacyl bromide in the presence of potassium carbonate in ethanol at room temperature produced acyclic intermediate 8a.Structure 8a was suggested for the reaction product on the basis of both elemental and spectral analyses (c.f.Experimental part). of reactants bearing the N=C=S fragment undergoes cyclization on reaction with α-halocarbonyl compounds to afford thiazoles, 2,3-dihydrothiazoles [11], which have been shown to exhibit antiprotozoal [12], and fungicidal properties [13].
Thus, the base-prompted reaction of the acidic methylene compound 1 with phenyl isothiocyanate in dry DMF at room temperature in basic medium led to the formation of the non-isolable intermediate 2 which gave thiocarbamoyl derivative 3 upon treatment with dilute HCl.Treatment of 3 with aromatic diazonium salts in the presence of sodium acetate affected acetyl cleavage with the formation of arylazothiocarbamoyl derivatives 4a-e rather than the expected product 5.This result is in agreement with the previously reported work [14][15][16][17][18], (Scheme 1).
Refluxing 8a in ethanol with few drops of TEA led to the formation of a product identical in all respects (m.p. mixed m.p., IR) to 7a.Similarly, compounds 7b-e were synthesized by pathway (1), (Scheme 2).Compound 3 was obtained according to the proposed following mechanism (Figure 1): The addition of two or more equivalents of ethyl bromoacetate, chloroacetonitrile, chloroacetone leads only to thiophenes 10, 12, and 14 in good yields.Thus, condensation of the intermediate salt with an equimolar amount of chloroacetyl chloride or with chloroacetic acid in ethanol, a product that analyzed for C 25 H 23 N 3 O 3 S was isolated in each case in good yield.The acyclic structure 9a was established on the basis of its IR spectrum that showed bands related to NH and CO functions (c.f.Experimental part).
Assignment of the product 3 was based on elemental analysis, IR and 1 H-NMR spectral data. 1 H NMR spectrum of 3 displayed multiplet signals at δ 7.1 -7.5 ppm for aromatic protons and exchangeable proton at 11.9 ppm for NH proton.On the other hand, the structure of the newly prepared hydrazone derivatives 4a-e was based on their correct elemental analysis and spectral data.The 1 H NMR spectrum of compound 4a displayed multiplet signals at δ 6.8 -7.8 ppm for aromatic protons and two exchangeable protons at δ 13.9 and 16.4 ppm for two NH protons.Compounds 4a-e were obtained according to the proposed following mechanism (Figure 2): Alternatively, treatment of the intermediate 6a with ethyl bromoacetate in ethanol gives a single product, which is identical in all respects to 9a (m.p. mixed m.p. and IR spectrum) (c.f.Experimental part).Refluxing of 9a in ethanol with a catalytic amount of TEA or leaving it in DMF containing potassium carbonate at room temperature overnight afforded the corresponding thiophene derivative 10a.In a similar manner, compounds 10a-e were prepared by pathway 1).
In this paper, we describe a generally applicable extension of this synthetic approach, first reported by Hantzsch and Weber [19].Thus, the base-prompted reaction of compounds 4a with potassium carbonate in dry DMF at room temperature affords the non-isolable intermediate 6a.Stirring of 6 with phenacyl bromide in DMF overnight yielded a product 7a, which analyzed correctly for C 29 H 21 N 3 OS.The structure 7a was inferred Similarly, when the intermediate potassium salt 6a is stirred with chloroacetonitrile in ethanol at room tem- perature, the corresponding acyclic intermediate 11a is exclusively isolated in good yield.The structure 11 has been confirmed on the basis of elemental and spectral data (c.f.Experimental part).Furthermore, heating of the intermediate 11a in ethanol containing a catalytic amount of TEA affords the thiophene derivative 12a.The thiophene structure 12a was established on the basis of its IR spectrum which showed bands related to NH and CN functions.Its 1 H NMR spectrum reveals broad signals at δ 14.2 ppm (1H, NH).On the other hand, it has been found that 12a-e are directly formed by treatment of 6a-e with chloroacetonitrile in dimethylformamide and in the pre-sence of potassium carbonate at room temperature overnight by pathway (1), (Scheme 3).
Compound 6a reacted readily with chloroacetone in the presence of ethanol at room temperature to afford the acyclic intermediate 13a by (KCl) elimination.Refluxing 13 in ethanol with a catalytic amount of TEA gave the thiophene derivative 14a whose structure was confirmed by its alternative synthesis.Thus, stirring 6a-e with chloroacetone in DMF overnight affords the thiophene derivative 14a-e in reasonably good yield, (Scheme 4).
On the other hand, treatment of the key intermediate 4b with hydrazine hydrate in DMF gave a single product which analyzed correctly for C 22 H 19 N 5 (16b).The structure of 16b was inferred from its spectral data.The 1 H NMR spectrum revealed a singlet at δ 2.3 assigned for the methyl protons, a broad band located at δ 10.1 and 12.0 assignable to the NH protons, and a multiplet at δ 7.0 -8.2, assigned for aromatic protons.The formation of 16a-e is assumed to proceed via the replacement of the SH group by the hydrazine moiety to give the intermediate 15 which then cyclized via the carbonyl group to afford the final isolable products 16a-e.In fact, the structure of 16 was further confirmed by alternative synthesis.Thus, it has been found that treatment of 4a with hydrazine hydrate for long time in ethanol produced the intermediate 15a.Refluxing 15a in ethanol containing a catalytic amount of TEA or in DMF lead to the formation of a product identical in all respects (m.p., mixed m.p., IR) with 16a [20].Structure 15a is suggested for the reaction product on the basis of both elemental and spectral analyses.The 1 H NMR spectrum revealed a broad signal at δ 6.4 assigned for NH 2 protons, a multiplet at δ 7.2 -7.8 for aromatic protons, and a singlet at δ 12.7 and 14.0 ppm for NH protons, (Scheme 5).
These results indicate that the reaction of thiocar- bamoyls and bifunctional reagents provides an excellent route for the synthesis of five, otherwise not easily accessible arylazothiophenes and pyrazoles.The compounds synthesized will be subjected to biological testing.

Effect of Drugs on the Viability of Ehrlich
Ascites Cells in Vitro To examine whether these substances have a direct cytotoxic effect on Ehrlich ascites cells (EAC) viability, the percentage of viable cells was estimated by the trypan blue [21], exclusion test.The desired concentration of tumor cells (2 × 10 6 cells per 0.2 mL) was obtained by dilution with saline (0.9% sodium chloride solution).Viability of tumor cells obtained and used in this experiment was always higher than 90%.Below this percentage, the cells were discarded and the entire procedure was ).The other rest of compounds showed weak to no activity.Thus, it would appear that introducing thiophene moiety enhances the cytotoxic properties.By comparing the cytotoxicity results in Table 1, the following structureactivity relationships (SARs) were drawn: 1) Converting the thiocarbamoyl moiety to the corresponding thiophene (7c, e and 10c, d) led to increase cytotoxicity against EAC.Compounds 7e and 10d were the most active, whereas, compounds 7c and 10c exhibited good activity.Thus, the ring substituents affected the activity in the thiophene derivatives; 2) The most of compounds containing electronedonating groups were very weak or completely inactive at ( 3 25 10 ED  ) than the compounds containing electronewithdrawing groups.Thus, the position and nature of substituents on the structure of thiophene derivatives seem to modulate antitumor activity.The reliable criteria for judging the value of any anticancer drug are prolongation of life span and decrease of WBC from blood [8,9].The results of the present study showed an antitumor effect of the compounds against EAC in Swiss albino mice (Table 2).
ED ED ED 3.2.1.2.Effect of Compound 10d on Survival Time [22] The mean survival time (MST) of each group consisting of seven mice was noted.The antitumor efficacy was compared with that of 5-fluorouracil (5-FU), for 9 days.The MST of the treated groups was compared with that of the control group using the following calculation: % Increase in life span over control = (MST of treated group-MST of control group) × 100 -100, where MST= survival time (days of each mouse in a group)/Total no. of mice (Table 2).

Effect of the Sulfur Containing Compounds on
Hematological Parameters To examine the influence of the tested compounds on the hematological status of EAC-bearing mice, a comparison was made among the following groups (n = 7) of mice on the 14 th day after inoculation.The groups comprised of 1) tumor-bearing mice; 2) tumor-bearing mice treated for the first 9 days; and 3) control mice (normal).Blood was drawn from each mouse by the retro orbital plexus method and the white blood cell count (WBC), red blood cell count (RBC) and hemoglobin were determined.From each group take 100 µM sample of Ehrlich ascites cells (from three mice) and make 20 fold dilutions in saline.The cells were stained by Giemsa stain and measure the number of viable cells under microscope.The  viable cells those, which did not take up the stain, where the dead cells (stained cells).
From Table 2; it was noticed that, 1) in the 5 th day after inoculation of Ehrlich cells in mice, increase in body weight and ascites was observed clearly, also the mice became slow and inactive; 2) Mice received compound 10d and 5-FU were more protected against ascites and increase in weight than control mice; 3) Mice received compound 10d showed slight toxic symptoms such as dizziness, dirty look; erection of tail, slow movement.Also the liver of animals was pale and slightly enlarged; 4) Percentage increase of life span over control showed to be high in mice treated with total extracts; 5) Mice that received compound 10d showed comparable results to 5-FU; 6) Hematological parameters of tumor bearing mice on Day 14 showed significant changes when compared with normal mice; 7) Total WBC count was found to increase with a reduction in the hemoglobin content and increase of RBCs; 8) Compound 10d and 5-FU showed minimal ascites and slight increase in body weight unlike control group.

Conclusion
Modification of thiocarbamoyl produced thiophene compounds with potential for further development as anticancer agents.Based on these preliminary screening results, compounds 7c, e and 10c, d showed significant activity in certain cancer cell and have been targeted for further studies, compound 10d is more effective and showed the highest activity.Additional research, including mode of action studies, is planned to accurately establish relative activity for (SAR) and rational design.Recently, the cancer chemopreventive effects of thiophene have been intensively investigated.Thiophene exhibited pronounced antitumor activity by triggering apoptosis inhuman tumor cells [23].Studies are underway to investigate the apoptosis inducing activity of compounds found to be cytotoxic in this study.

Table 1 . Effect of sulfur containing compounds on the vi- ability of Ehrlich ascites cells (EAC) in vitro assay.
Where, ED is the concentration of compounds in μM.