Synthesis, antimicrobial evaluation and molecular modeling of some novel phenothiazine derivatives

Abstract

A series of novel phenothiazine derivatives with biologically active moieties were synthesized. Treatment of 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile (2) with carbon disulfide gave 3,3-dimercapto-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (4) which reacted with different reagents to afford novel fused heterocyclic compounds. The newly synthesized compounds were examined for their antimicrobial activity. Among the tested compounds, compounds 2, 4, 5a,b and 13 showed the highest antimicrobial activity. Molecular modeling was evaluated and was in agreement with experimental biological activity results.

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1. Introduction

The chemistry of phenothiazine provides a significant amount of related compounds that have a variety of biological processes. Synthetic phenothiazine and its related compounds are basically effective in the treatment of a number of medical conditions.^1,2 They possess a wide spectrum of pharmacological/biological activities^3–13 and several of their derivatives are in clinical use. Phenothiazine and its related compounds have been reported to possess various biological activities such as antimalarial,¹⁴ antipsychotropic,¹⁵ antimicrobial,¹⁶ antitubercular,^17,18 antitumor^19,20 and analgesic.²¹ Phenothiazine itself is found to be a worming agent for livestock. The pesticidal action of phenothiazine results from the fact that they affect the nervous system of insects by inhibiting the breakdown of acetylcholine.² The carbonyl and the cyano functions of the cyanoacetamides are readily well positioned for reactions with common reagents to form a variety of heterocyclic derivatives. Cyanoacetamides are highly reactive compounds having an active methylene which can take part in a variety of condensation and substitution reactions. In recent years, a number of different quantum mechanical theories arose, for example the Density Functional Theory (DFT). It was considered an effective computational method for studying organic compounds.

The DFT method explains the chemical reactivity and orbitals, energy band gap, electronegativity, and dipole moment.²² Therefore, in the present work we aimed to synthesize new phenothiazine derivatives and perform evaluations for their anticipated antimicrobial activities.

2. Results and discussion

2.1. Chemistry

In the continuation of our research interests, we reported here the behavior of 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile (2) toward some bifunctional reagents as a facile and appropriate route to synthesize some heterocyclic compounds containing a phenothiazine moiety.

The cyanoacetamide 2 was synthesized from the reaction of phenothiazine (1) with a mixture of cyanoacetic acid and acetic anhydride (Scheme 1). Compound 2 was confirmed using both spectral and analytical data. The IR spectrum showed absorption bands at 2259 and 1679 cm⁻¹ corresponding to CN and C=O, respectively. The ¹H-NMR spectrum showed one singlet signal at δ 3.86 ppm attributable to CH₂ protons of the cyanoacetyl group and a multiplet signal at δ 7.46–7.55 ppm due to the aromatic system. Also its mass spectrum showed a molecular ion peak at m/z = 266 (M)⁺ attributable to its correct molecular formula C₁₅H₁₀N₂OS. Reaction of 2 with carbon disulfide afforded the non-isolable intermediate 3 which reacted in situ with dilute hydrochloric acid to afford 3,3-dimercapto-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (4). The ¹H-NMR spectrum of 4 revealed a singlet signal at δ 1.5 ppm attributable to two SH protons. Moreover, the mass spectrum proved the structure through the appearance of a molecular ion peak at m/z = 342 (M)⁺ corresponding to the correct molecular formula C₁₆H₁₀N₂OS₃ (Scheme 1).

Scheme 1 Reaction of compound 2 with carbon disulfide.

The intermediate 3 reacted with different halo-alkanes namely, 1-chlorododecane, 1-chlorohexadecane, 1,3-dibromopropane and 1,5-dibromopentane in a stirring mixture of acetone and N,N-dimethylformamide (1 : 2) containing anhydrous potassium carbonate as a base to afford dithia derivatives 5a,b and 6a,b, respectively (Scheme 2). Compound 5a was confirmed using both elemental analysis and spectral data. The ¹H-NMR spectrum showed two triplet signals at δ 2.10 and 3.50 ppm corresponding to two identical CH₃ groups and two identical S–CH₂ groups, respectively. In addition, a broad signal at δ 3.30–3.89 ppm due to two (CH₂)₁₀ protons and a multiplet signal at δ 7.13–7.43 ppm corresponding to the aromatic system were seen. Moreover, the mass spectrum showed a molecular ion peak at m/z = 679, (M + 1)⁺ and 680 (M + 2)⁺ equivalent to the correct molecular formula C₄₀H₅₈N₂OS₃. The ¹H-NMR spectrum of 5b revealed two triplet signals at δ 0.88 and 3.50 ppm corresponding to two CH₃ and two SCH₂ groups, respectively, in addition to a multiplet signal at δ 1.26–1.42 ppm due to two (CH₂)₁₄ protons and a multiplet signal at δ 7.30–7.67 ppm equivalent to the aromatic system. Moreover, the mass spectrum exhibited a molecular ion peak at m/z = 790 (M)⁺ confirming the correct molecular formula C₄₈H₇₄N₂OS₃. The ¹H-NMR spectrum of 6a displayed a triplet signal at δ 2.89 ppm attributable to SCH₂ protons and a broad signal centered at δ 3.50 ppm corresponding to CH₂ protons. The mass spectrum indicated a molecular ion peak at m/z = 382 (M)⁺ confirming the molecular formula C₁₉H₁₄N₂OS₃ (Scheme 2). Compound 6b was established using both spectral and analytical data. The IR spectrum exhibited absorption frequencies at 2220 and 1698 cm⁻¹ corresponding to CN and C=O groups, respectively. The ¹H-NMR spectrum showed a triplet signal at δ 2.85 ppm corresponding to SCH₂ protons, and a multiplet at δ 1.32–1.95 ppm corresponding to three CH₂ protons. The mass spectrum showed a molecular ion peak at m/z = 410 (M)⁺ attributable to the correct molecular formula C₂₁H₁₈N₂OS₃. Furthermore, the reaction of intermediate 3 with an equimolar amount of dihaloalkanes under the same reaction conditions afforded compounds 7 and 8. The ¹H-NMR spectrum of 7 revealed a singlet signal at δ 4.13 ppm corresponding to two SCH₂ protons. Also, the mass spectrum showed a molecular ion peak at m/z = 443 (M − 1)⁺ corresponding to the molecular formula C₂₄H₁₆N₂OS₃. Structure 8 revealed absorption frequencies at 2239–2218 and 1720–1698 cm⁻¹ attributable to three CN functions and three C=O functions, respectively. Also, the mass spectrum showed a molecular ion peak at m/z = 495 (M)⁺ corresponding to the molecular formula C₂₄H₈N₄O₃S₃ (Scheme 2).

Scheme 2 Reaction of intermediate 3 with dihalo-reagents.

In addition to that mentioned above, the reaction of intermediate 3 with two moles of 6-chloropyrimidine-2,4-diamine and/or 6-chloro-1,3,5-triazine-2,4-diamine and/or phenacyl bromide in a stirring mixture of acetone and N,N-dimethylformamide (1 : 2) containing anhydrous potassium carbonate as a base afforded the new phenothiazine derivatives 9–11 (Scheme 3). Compounds 9–11 were confirmed on the basis of spectral and analytical data. The IR spectra generally displayed absorption frequencies at 3398–3210, 2220–2192 and 1644–1721 cm⁻¹ corresponding to NH₂, CN and C=O functions, respectively. The ¹H-NMR spectrum of compound 9 revealed three singlet signals at δ 4.48, 6.33 and 6.82 ppm corresponding to two C₅–H pyrimidine protons and four NH₂ protons, respectively. Also, the mass spectrum showed a molecular ion peak at m/z = 556 (M − 2)⁺ corresponding to the molecular formula C₂₄H₁₈N₁₀OS₃. In addition, the ¹H-NMR spectrum of compound 10 revealed two singlet signals at δ 6.94 and 7.12 ppm corresponding to four NH₂ functions. Also, the mass spectrum showed a molecular ion peak at m/z = 560 (M)⁺ equivalent to the molecular formula C₂₂H₁₆N₁₂OS₃. The ¹H-NMR spectrum of compound 11 showed two singlet signals at δ 4.48 and 5.93 ppm consistent with SCH₂ and NH₂ protons, respectively. The mass spectrum revealed a molecular ion peak at m/z = 578 (M)⁺ due to the molecular formula C₃₂H₂₂N₂O₃S₃. On the other hand, the reaction of the intermediate 3 with hydrazine hydrate in a stirring mixture of acetone and N,N-dimethylformamide (1 : 2) containing potassium carbonate as a base at room temperature, afforded compound 12. The IR spectrum revealed absorption frequencies at 3410, 3398, 3111, 3096 and 1698 cm⁻¹ corresponding to two NH₂, two NH and C=O functions, respectively. Moreover, the ¹H-NMR spectrum revealed four singlet signals at δ 5.98, 6.27, 8.46 and 12.33 ppm corresponding to two NH₂ and two NH protons. The mass spectrum displayed a molecular ion peak at m/z = 338 (M)⁺ corresponding to the molecular formula C₁₆H₁₄N₆OS (Scheme 3).

Scheme 3 Reaction of intermediate 3 with haloreagents and hydrazine hydrate.

Furthermore, the reaction of 3 with chloroacetyl chloride in a stirring mixture of acetone and N,N-dimethylformamide (1 : 2) containing anhydrous potassium carbonate as a base afforded compound 13. Treatment of compound 13 with methyl iodide in the presence of sodium ethoxide gave the acyclic compound 14 which underwent cyclization to afford compound 15 (Scheme 4). Compounds 13–15 were established on the bases of spectral and analytical data. Compound 13 displayed absorption frequencies at 2218, 1710 and 1694 cm⁻¹ corresponding to CN and two C=O functions. The ¹H-NMR spectrum of 13 revealed a singlet signal at δ 3.85 ppm corresponding to CH₂ of the dithiolanone ring. Also, the mass spectrum showed the molecular ion peak at m/z = 381 (M)⁺ attributable to the correct molecular formula C₁₈H₁₀N₂O₂S₃. The ¹H-NMR spectrum of compound 14 showed two singlet signals at δ 2.80 and 3.98 ppm attributable to SCH₃ and SCH₂ protons, a triplet signal at δ 1.25 and a quartet at δ 4.13 ppm corresponding to CH₃ and OCH₂ protons, respectively. The mass spectrum showed a molecular ion peak at m/z = 442 (M)⁺ equivalent to the molecular formula C₂₁H₁₈N₂O₃S₃. In addition, the ¹H-NMR spectrum of 15 revealed two singlet signals at δ 2.51 and 5.82 ppm corresponding to SCH₃ and NH₂ protons. Also, the mass spectrum showed a molecular ion peak at m/z = 442 (M)⁺ due to the molecular formula C₂₁H₁₈N₂O₃S₃ (Scheme 4). Also, the intermediate 3 reacted with dimethyl sulfoxide in a stirring mixture of acetone and N,N-dimethylformamide (1 : 2) containing potassium carbonate as a base at room temperature, to afford the S-alkyl derivative 16. Compound 16 was established by both spectral and analytical data. The ¹H-NMR spectrum of compound 16 revealed a singlet signal at δ 2.37 ppm corresponding to two SCH₃ protons. The mass spectrum showed a molecular ion peak at m/z = 371 (M + 1)⁺ confirming the molecular formula C₁₈H₁₄N₂OS₃. When compound 16 was heated with 6-aminothiouracil in refluxing DMF, this afforded the corresponding pyridopyrimidine derivative 17. Structure 17 was suggested for the reaction product on the basis of elemental analysis and spectral evidence. The IR spectrum revealed absorption bands at 3410, 3120, 3098, 1698 and 1680 cm⁻¹ corresponding to NH₂, two NH and two C=O functions. Moreover, the ¹H-NMR spectrum revealed four singlet signals at δ 2.54, 6.35, 11,47 and 12.33 ppm corresponding to SCH₃, NH₂ and two NH protons. In addition to, the mass spectrum showed a molecular ion peak at m/z = 465 (M)⁺ equivalent to the molecular formula C₂₁H₁₅N₅O₂S₃ (Scheme 4).

Scheme 4 Reaction of intermediate 3 with chloroacetyl chloride/methyl iodide and dimethyl sulphate/6-aminothiouracil.

2.2. Biological activity

The newly synthesized target compounds were evaluated for their in vitro antibacterial activity against S. aureus and B. subtilis as examples of Gram-positive bacteria and E. coli and P. aeruginosa as examples of Gram-negative bacteria. They were also evaluated for their in vitro antifungal potential against Candida albicans and F. oxysporum fungal strains.

The disk diffusion technique was used for the determination of the antibacterial and antifungal activity. Ampicillin and clotrimazole were used as reference drugs. The MIC (μg mL⁻¹) values were recorded in Table 1. The results depicted in Table 1 revealed that most of the tested compounds displayed variable inhibitory effects on the growth of the tested Gram-positive and Gram-negative bacterial strains, and also against the antifungal strains. Generally most of the tested compounds revealed better activity against the Gram-positive rather than the Gram-negative bacteria.

Table 1 Minimal inhibitory concentration (MIC, μg mL⁻¹) of some newly synthesized compounds^a

Compound no.	MIC in (μg mL^−1)
	Bacteria				Fungi
	Gram-negative bacteria		Gram-positive bacteria
	E. coli	P. aeruginosa	S. aureus	B. subtilis	C. albicans	F. oxysporum
a MIC: minimal inhibitory concentration values with SEM = 0.02, NA: no activity.
2	125	125	125	125	250	125
4	125	125	125	125	125	62.5
5a	125	62.5	62.5	62.5	NA	NA
5b	125	62.5	57.5	62.5	NA	NA
6a	NA	NA	125	125	NA	NA
6b	NA	NA	NA	NA	NA	NA
7	125	62.5	125	125	250	125
8	125	125	125	62.5	62.5	31.5
9	125	125	125	125	125	125
10	125	125	187.5	125	125	125
11	125	62.5	125	125	NA	NA
12	250	125	250	125	250	125
13	31.5	62.5	NA	NA	NA	NA
15	125	62.5	187.5	125	3.9	3.2
17	250	250	250	250	250	125
Ampicillin	125	125	187.5	125	NA	NA
Clotrimazole	NA	NA	NA	NA	5.8	4.2

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Regarding structure–activity relationships, it was revealed that compounds 2 and 4 which contain the cyanoacetamide moiety were equipotent to ampicillin in inhibiting the growth of E. coli and P. aeruginosa (MIC = 125 μg mL⁻¹), while their activities were 33% more than that of ampicillin against S. aureus (MIC = 125 μg mL⁻¹). Compound 13 recorded an increase in its activity by 75% more than ampicillin against E. coli (MIC = 31.5 μg mL⁻¹) and 50% more than P. aeruginosa (MIC = 62.5 μg mL⁻¹). Compounds 5a and b showed an increase in their activity by more than 66% (MIC = 62.5 μg mL⁻¹) and 69%, (MIC = 57.5 μg mL⁻¹), respectively, over ampicillin against S. aureus while they showed 50% (MIC = 62.5 μg mL⁻¹) more activity than B. subtilis. On the other hand, compound 17 showed very low activity toward most of the tested organisms.

Moreover, the experimental results for the newly synthesized compounds were in agreement with theoretical data as shown by determining the energy gap (E_HOMO–E_LUMO) which is an indication of the biological activity. Since molecules with a small energy gap are more polarized and reactive than hard ones, they easily offer electrons to an acceptor. Also, the low values for the energy gap may be due to groups entering into conjugation with those soft molecules.^23–25 Therefore, the energy gap for the compounds with the three highest antimicrobial activities (4, 5b and 13; Table 1) were determined (Table 2). The energy gap of compounds 5b and 13 (Fig. 2 and 3, respectively) were smaller than that of compound 4 (Fig. 1) indicating the ease of charge transfer in compounds 5b and 13 which enhanced their biological activity compared to compound 4 and are in agreement with the experimental data in Table 1. Table 2 shows the HOMO–LUMO energy gap, dipole moment and binding energy of compounds 5b and 13 computed using DFT, BLYP energy functional and the basis set DNP. Compound 13 has the most potent activity against the Gram-negative bacteria, E. coli and P. aeruginosa . This could be related to the lowest value for the dipole moment for compound 13 compared to compounds 4 and 5b. The inverse correlation between the dipole moment and the activity of compound 13 could be clarified in the concept that decreasing the dipole moment will decrease the polarity and increase the lipophilic nature of the compound which favours its permeation more efficiently through the lipid layer of a microorganism.²⁶ The negative results can be attributed either to the inability of these compounds to diffuse through the cell wall of the bacterium and hence are unable to interfere with its biological activity or they can diffuse and are inactivated by an unknown cellular mechanism i.e. bacterial enzymes.²⁷

Table 2 Calculated total energy and binding energy (BLYP/DNP) for compounds (4, 5b and 13) using DFT

Compound	HOMO–LUMO energy gap (eV)	Dipole moment Debye	Binding energy, Hartree
4	−1.877	2.1151	−6.3781553
5b	−2.167	5.6224	−6.7501331
13	−2.488	1.9024	−20.8448839

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Fig. 1 (a) Molecular geometry of compound 4 and (b) calculated HOMO superimposed on the isosurface of the molecular geometry of compound 4.

Fig. 2 (a) Molecular geometry of conformer 5b and (b) calculated HOMO superimposed on the isosurface of the molecular geometry of conformer 5b.

Fig. 3 (a) Molecular geometry of conformer 13 and (b) calculated HOMO superimposed on the isosurface of the molecular geometry of conformer 13.

Also, the isolated compound 13 showed large binding energy values compared to compounds 4 and 5b, which improved the stability of this compound.²⁸

Regarding the activity of the compounds against antifungal strains, the results revealed that compound 15 was 32.7% (MIC = 3.9 μg mL⁻¹) and 24% (MIC = 3.2 μg mL⁻¹) higher than clotrimazole in inhibiting the growth of C. albicans and F. oxysporum, respectively. Compounds 2, 4, 7, 8, 9, 10, 12 and 17 exhibited moderate growth inhibitory activity against C. albicans and F. oxysporum as revealed from their MIC values (MIC = 31.5–250 μg mL⁻¹).

Since the tested compounds were more active against Gram-positive than Gram-negative bacteria, it may be concluded that the antimicrobial activity of the compounds is related to the cell wall structure of the bacteria. It is possible because the cell wall is essential to the survival of bacteria and some antibiotics are able to kill bacteria by inhibiting a step in the synthesis of peptidoglycan. Gram-positive bacteria possess a thick cell wall containing many layers, consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing peptidoglycan and teichoic acids, but in contrast, Gram-negative bacteria have a relatively thin cell wall of lipopolysaccharides and lipoproteins. These differences in cell wall structure can produce differences in antibacterial susceptibility and some antibiotics can kill only Gram-positive bacteria and are inactive against Gram-negative pathogens.²⁹

In conclusion, the present study describes the synthesis and investigates the antimicrobial activities of some newly functionalized phenothiazine derivatives. Also, the HOMO, LUMO, dipole moment and charges on the atoms were used to confirm the geometry of the isolated compounds. Moreover, the compounds were screened for antimicrobial activity. Regarding the structure–activity relationship, compounds 5a and b exhibited the highest biological activity toward Gram positive bacteria, while compound 13 was the highest toward Gram negative bacteria. On the other hand, compound 15 showed the highest activity against pathogenic fungi by C. albicans and F. oxysporum using the MIC method.

2.3. Molecular modeling

All molecular geometries were fully optimized using the DMOL³ program³⁰ implemented in Materials Studio 7.0 (Accelrys, CA).³¹ DMOL³, and BLYP energy functional with the basis set DNP were performed. DFT semi-core pseudopods calculations (dspp) were performed with the double numerical basis sets plus polarization functional (DNP). This basis set was comparable in quality to Gaussian 6-31G sets.^32,33 Delley et al. showed that the DNP basis sets were more accurate than Gaussian basis sets of the same size.³⁴ The RPBE functional was so far the best exchange–correlation functional,^35,36 based on the generalized gradient approximation (GGA), and was employed to take account of the exchange and correlation effects of electrons.

3. Experimental

3.1. Instruments

All melting points are recorded on a Gallenkamp electric melting point apparatus and are uncorrected. The IR spectra ν/cm⁻¹ (KBr) were recorded on a Perkin Elmer Infrared Spectrophotometer Model 157, Grating. The ¹H-NMR spectra were run on a Varian Spectrophotometer at 300 and 75 MHz, respectively, using tetramethylsilane (TMS) as an internal reference and DMSO-d6 as a solvent. The mass spectra (EI) were recorded at 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 University, Giza, Egypt, and the results were found to be in good agreement (±0.3%) with the calculated values.

3.1.1. Synthesis of 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile (2). Phenothiazine (1) (10 mmol) was fused with an equimolar amount of cyanoacetic acid (10 mmol) in acetic anhydride at 80–85 °C in a boiling water-bath for 40 min. The reaction mixture was left for 1 h and then filtered off. The solid product was washed with ethanol (20 mL) to produce 2 as white crystals. Yield: 90%, mp: 215–217 °C. IR (KBr): ν/cm⁻¹ = 2259 (C=N), 1679 (C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 3.86 (s, 2H, CH₂), 7.49–7.55 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 266 (M⁺; 100), 198 (98), 167 (78), 96 (88); chemical formula: C₁₅H₁₀N₂OS: (266.05): calcd: C, 67.65; H, 3.78; N, 10.52%; found: C, 67.82; H, 3.54; N, 10.91%.

3.1.2. Synthesis of 3,3-dimercapto-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (4). To a stirred suspension of potassium carbonate in a mixture of acetone and DMF (2 : 1), N-cyanoacetyl phenothiazine (2) (10 mmol) was added. To the resulting solution, carbon disulfide (10 mmol) was added and the reaction mixture was stirred for 24 h at room temperature to give the nonisolable intermediate 3. The reaction mixture was poured onto crushed ice and neutralized by dilute HCl. The obtained solid product was collected by filtration, washed, dried and crystallized from ethanol to give compound 4.

White crystals; yield 82%, mp 218 °C; IR (KBr): ν/cm⁻¹ = 2218 (CN), 1692 (C=O), 1310 (C=S); ¹H-NMR (DMSO-d6) δ (ppm) = 1.5 (s, 1H, SH), 7.40–7.70 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 342 (M⁺; 65), 241 (34), 187 (45), 172 (26), 93 (100); chemical formula: C₁₆H₁₀N₂OS₃: (342.00); calcd: C, 56.12; H, 2.94; N, 8.18%; found: C, 56.17; H, 2.89; N, 8.24%.

3.2. General procedure for the synthesis of compounds 5a and b

To a stirred mixture of 3 (10 mmol), 1-chlorododecane and/or 1-chlorohexadecane (10 mmol) was added and stirred for 4 h. The reaction mixture was poured onto crushed ice and neutralized by dil. HCl. The obtained solid product was collected by filtration, washed, dried and crystallized from ethanol to give compounds 5a and b.

3.2.1. Synthesis of 3,3-bis(dodecylthio)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (5a). Beige crystals; yield 76%, mp 227 °C; IR (KBr): ν/cm⁻¹ = 2216 (CN), 1720 (C=O), 1317 (C=S); ¹H-NMR (DMSO-d6) δ (ppm) = 2.10 (t, 6H, 2CH₃), 3.49 (t, 4H, 2SCH₂), 3.50 (br, 40H, 2(CH₂)₁₀), 7.13–7.43 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 680 (M⁺ + 2) (18), 391 (34), 327 (21), 276 (47), 77 (100); chemical formula: C₄₀H₅₈N₂OS₃: (678.37): calcd: C, 70.75; H, 8.61; N, 4.13%; found: C, 70.82; H, 8.58; N, 4.19%.

3.2.2. Synthesis of 3,3-bis(hexadecylthio)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (5b). White crystals; yield 72%, mp 218 °C; IR (KBr): ν/cm⁻¹ = 2220 (CN), 1680 (C=O), 1310 (C=S).; ¹H-NMR (DMSO-d6) δ (ppm) = 0.88 (t, 6H, 2CH₃), 1.26–1.42 (m, 56H, 2(CH₂)₁₄), 3.50 (t, 4H, 2SCH₂), 7.30–7.67 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 790 (M⁺) (65), 636 (22), 520 (28), 401 (18), 327 (48), 276 (34), 180 (17), 77 (100); chemical formula: C₄₈H₇₄N₂OS₃: (790.50): calcd: C, 72.86; H, 9.43; N, 3.54%; found: C, 72.92; H, 9.39; N, 3.58%.

3.3. General procedure for the synthesis of compounds 6a and b

To a stirred mixture of 3 (10 mmol), 1,3-dibromopropane and/or 1,5-dibromopentane (10 mmol) was added and stirred for 4 h. The reaction mixture was poured onto crushed ice and neutralized by dil. HCl. The obtained solid product was collected by filtration, washed, dried and crystallized from ethanol to give compounds 6a and b.

3.3.1. Synthesis of 2-(1,3-dithian-2-ylidene)-3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile (6a). Orange crystals; yield 68%, mp 289 °C; IR (KBr): ν/cm⁻¹ = 2221 (CN), 1712 (C=O), 1314 (C=S); ¹H-NMR (DMSO-d6) δ (ppm) = 2.89 (t, 4H, 2SCH₂), 3.50 (br, 2H, CH₂), 6.74–7.67 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 382 (M⁺) (18), 319 (100), 289 (34), 160 (28), 121 (18), 77 (34); chemical formula: C₁₉H₁₄N₂OS₃: (382.03): calcd: C, 59.66; H, 3.69; N, 7.32%; found: C, 59.72; H, 3.72; N, 7.38%.

3.3.2. Synthesis of 2-(1,3-dithiolan-2-ylidene)-3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile (6b). Yellow crystals; yield 62%, mp 272 °C; IR (KBr): ν/cm⁻¹ = 2220 (CN), 1698 (C=O), 1321 (C=S); ¹H-NMR (DMSO-d6) δ (ppm) = 1.32–1.95 (m, 6H, 3CH₂), 7.32–7.72 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 410 (M⁺) (21), 346 (27), 279 (42), 175 (58), 77 (100), 65 (78); chemical formula: C₂₁H₁₈N₂OS₃: (410.06): calcd: C, 61.43; H, 4.42; N, 6.82%; found: C, 61.46; H, 4.38; N, 6.86%.

3.4. General procedure for the synthesis of compounds 7 and 8

To a stirred mixture of 3 (10 mmol), 1,2-bis(bromomethyl)benzene (10 mmol) and/or 4,5-dichloro-3,6-dioxocyclohexa-1,4-diene-1,2-dicarbonitrile (10 mmol) was added and stirred for 4 h. The reaction mixture was poured onto crushed ice and neutralized by dil. HCl. The obtained solid product was collected by filtration, washed, dried and crystallized from ethanol to give compounds 7 and 8, respectively.

3.4.1. Synthesis of 2-(1,5-dihydrobenzo[e][1,3]dithiepin-3-ylidene)-3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile (7). Brown crystals; yield 68%, mp 345 °C; IR (KBr): ν/cm⁻¹ = 2222 (CN), 1698 (C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 4.13 (s, 4H, 2SCH₂), 7.02–7.78 (m, 12H, Ar-H); MS (EI, 70 eV): m/z (%) = 443 (M⁺ − 1) (18), 304 (58), 246 (60), 155 (42), 123 (100); chemical formula: C₂₄H₁₆N₂OS₃: (444.04); calcd: C, 64.84; H, 3.63; N, 6.30%; found: C, 64.92; H, 3.68; N, 6.26%.

3.4.2. Synthesis of 2-(1-cyano-2-oxo-2-(10H-phenothiazin-10-yl)ethylidene)-4,7-dioxo-4,7-dihydro benzo[d][1,3]dithiole-5,6-dicarbonitrile (8). Beige crystals; yield 72%, mp 295 °C; IR (KBr): ν/cm⁻¹ = 2239, 2220, 2218 (3CN), 1723, 1720, 1698 (3C=O); ¹H-NMR (DMSO-d₆) δ (ppm) = 6.89–7.02 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 495 (M⁺) (48), 454 (64), 380 (72), 278 (68), 224 (60), 171 (100), 132 (74); chemical formula: C₂₄H₈N₄O₃S₃: (495.98); calcd: C, 58.06; H, 1.62; N, 11.28%; found: C, 57.96; H, 1.89; N, 11.32%.

3.5. General procedure for the synthesis of compounds 9, 10, 12 and 16

To a stirred mixture of 3 (10 mmol), 6-chloropyrimidine-2,4-diamine (20 mmol) and/or 6-chloro-1,3,5-triazine-2,4-diamine (20 mmol) and/or hydrazine hydrate (20 mmol) and/or dimethyl sulfoxide (20 mmol) was added and stirred for 4 h. The reaction mixture was poured onto crushed ice and neutralized by dil. HCl. The obtained solid product was collected by filtration, washed, dried and crystallized from ethanol to give compounds 9, 10, 12 and 16, respectively.

3.5.1. Synthesis of 3,3-bis((2,6-diaminopyrimidin-4-yl)thio)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (9). Beige crystals; yield 60%, mp 289 °C; IR (KBr): ν/cm⁻¹ = 3397, 3315, 3309, 3210 (4NH₂), 2192 (CN), 1644 (C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 4.48 (s, 2H, C₅–H pyrimidine), 6.33 (s, 4H, 2NH₂), 6.82 (s, 4H, 2NH₂), 7.70–7.78 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 556 (M⁺ − 2) (52), 473 (18), 304 (59), 246 (78), 155 (48), 123 (100), 91 (78); chemical formula: C₂₄H₁₈N₁₀OS₃: (558.08): calcd: C, 51.60; H, 3.25; N, 25.07%; found: C, 51.59; H, 3.22; N, 24.97%.

3.5.2. Synthesis of 3,3-bis((4,6-diamino-1,3,5-triazin-2-yl)thio)-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (10). White crystals; yield 58%, mp 335 °C; IR (KBr): ν/cm⁻¹ = 3421, 3402, 3398, 3318 (4NH₂), 2198 (CN), 1689 (C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 6.94 (s, 4H, 2NH₂), 7.12 (s, 4H, 2NH₂), 7.30–7.69 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 560 (M⁺) (38), 453 (28), 332 (29), 266 (36), 198 (100), 156 (32), 80 (67); chemical formula: C₂₂H₁₆N₁₂OS₃: (560.07): calcd: C, 47.13; H, 2.88; N, 29.98%; found: C, 47.29; H, 2.94; N, 30.02%.

3.5.3. Synthesis of (5-hydrazinyl-3-imino-2,3-dihydro-1H-pyrazol-4-yl)(10H-phenothiazin-10-yl)methanone (12). Beige crystals; yield 63% mp 316 °C; IR (KBr): ν/cm⁻¹ = 3410, 3398 (2 NH₂), 3096 (NH), 1698 (C=O).

¹H-NMR (DMSO-d6) δ (ppm) = 5.98 (s, 2H, NH₂), 6.27 (s, 2H, NH₂), 7.34–7.75 (m, 8H, Ar-H), 8.46 (s, 1H, NH), 12.33 (s, 1H, NH); MS (EI, 70 eV): m/z (%) = 338 (M⁺) (72), 289 (87), 250 (100), 182 (62), 75 (78); chemical formula: C₁₆H₁₄N₆OS: (338.03): calcd: C, 58.35; H, 3.81; N, 7.56%; found: C, 58.42; H, 3.79; N, 7.58%.

3.5.4. Synthesis of 3,3-bis(methylthio)-2-(10H-pheno thiazine-10-carbonyl)acrylonitrile (16). Red crystals; yield 63%, mp 316 °C; IR (KBr): ν/cm⁻¹ = 2228 (CN), 1680 (C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 2.37 (s, 6H, 2SCH₃), 7.34–7.75 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 371 (M⁺) (12), 346 (58), 318 (49), 262 (12), 174 (69), 69 (100); chemical formula: C₁₈H₁₄N₂OS₃: (370.03): calcd: C, 58.35; H, 3.81; N, 7.56%; found: C, 58.42; H, 3.79; N, 7.58%.

3.5.5. Synthesis of 2-(3-amino-5-((2-oxo-2-phenyl ethyl)thio)-4-(10H-phenothiazine-10-carbonyl)thiophen-2-yl)-1-phenylethan-1-one (11). To a stirred mixture of 3 (10 mmol), phenacyl bromide (20 mmol) was added and stirred for 4 h. The reaction mixture was refluxed in DMF which contained a catalytic amount of TEA for 6 h. The reaction mixture was allowed to cool to room temperature then poured onto cold ice water and neutralized with conc. HCl. The solid obtained was filtered off and recrystallized from ethanol to yield compound 11.

Brown crystals; yield 68%, mp 345 °C; IR (KBr): ν/cm⁻¹ = 3398 (NH₂), 1721, 1712, 1689 (3C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 4.48 (s, 2H, SCH₂), 5.93 (s, 2H, NH₂), 7.41–7.75 (m, 18H, Ar-H); MS (EI, 70 eV): m/z (%) = 578 (M⁺) (24), 169 (37), 135 (23), 112 (48), 92 (100); chemical formula: C₃₂H₂₂N₂O₃S₃: (578.08); calcd: C, 66.41; H, 3.83; N, 4.84%; found: C, 66.46; H, 3.79; N, 4.88%.

3.5.6. Synthesis of 3-oxo-2-(4-oxo-1,3-dithiolan-2-ylidene)-3-(10H-phenothiazin-10-yl)propanenitrile (13). To a stirred mixture of 3 (10 mmol), phenacyl bromide (20 mmol) was added and stirred for 4 h. The reaction mixture was poured onto crushed ice and neutralized by dil. HCl. The obtained solid product was collected by filtration, washed, dried and crystallized from ethanol to give compound 13.

Orange crystals; yield 70%, mp 226 °C; IR (KBr): ν/cm⁻¹ = 2218 (CN), 1710 (C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 3.85 (s, 2H, CH₂), 7.49–7.67 (m, 18H, Ar-H); MS (EI, 70 eV): m/z (%) = 318 (M⁺) (18), 281 (58), 217 (18), 179 (28), 154 (78), 70 (100); chemical formula: C₁₈H₁₀N₂O₂S₃: (381.99): calcd: C, 56.53; H, 2.64; N, 7.32%; found: C, 56.62; H, 2.81; N, 7.28%.

3.5.7. Synthesis of ethyl-2-((2-cyano-1-(methylthio)-3-oxo-3-(10H-phenothiazin-10-yl)prop-1-en-1-yl)thio)acetate (14). To a solution of sodium ethoxide (prepared from 10 mmol sodium metal in 10 mL ethanol), methyl iodide (10 mmol) was added to compound 13 (10 mmol). The reaction mixture was poured onto crushed ice and neutralized by dil. HCl. The obtained solid product was collected by filtration, washed, dried and crystallized from ethanol to give compound 14.

Orange crystals; yield 62%, mp 265 °C; IR (KBr): ν/cm⁻¹ = 2220 (CN), 1710, 1698 (2C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 1.25 (t, 3H, CH₃), 2.80 (s, 3H, SCH₃), 3.98 (s, 2H, SCH₂), 4.13 (q, 2H, OCH₂), 7.32–7.81 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 442 (M⁺) (18), 364 (37), 286 (18), 213 (29), 154 (78), 77 (100); chemical formula: C₂₁H₁₈N₂O₃S₃: (442.05): calcd: C, 56.99; H, 4.10; N, 6.33%; found: C, 57.01; H, 4.14; N, 6.28%.

3.5.8. Synthesis of ethyl 3-amino-5-(methylthio)-4-(10H-phenothiazine-10-carbonyl)thiophene-2-carboxylate (15). Compound 14 (10 mmol) was refluxed in DMF which contained a catalytic amount of TEA for 6 h. The reaction mixture was allowed to cool to room temperature and was then poured onto cold ice water and neutralized with conc. HCl. The solid obtained was filtered off and recrystallized from ethanol to yield compound 15.

Brown crystals; yield 60%, mp 290 °C; IR (KBr): ν/cm⁻¹ = 3401 (NH₂), 1720, 1698 (2C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 1.34 (t, 3H, CH₃), 2.51 (s, 3H, SCH₃), 4.40 (q, 2H, OCH₂), 5.82 (s, 2H, NH₂), 7.30–7.67 (m, 8H, Ar-H); MS (EI, 70 eV): m/z (%) = 442 (M⁺) (21), 364 (37), 316 (18), 288 (48), 211 (59), 169 (76), 92 (100); chemical formula: C₂₁H₁₈N₂O₃S₃: (442.05): calcd: C, 56.99; H, 4.10; N, 6.33%; found: C, 57.01; H, 4.14; N, 6.28%.

3.5.9. Synthesis of 5-amino-7-(methylthio)-6-(10H-phenothiazine-10-carbonyl)-2-thioxo-2,3-dihydro pyrido[2,3-d]pyrimidin-4(1H)-one (17). A mixture of compounds 16 (10 mmol) and 6-aminothiouracil (10 mmol) were refluxed in DMF which contained a catalytic amount of TEA for 6 h. The reaction mixture was allowed to cool to room temperature and was then poured onto cold ice water and neutralized with conc. HCl. The solid obtained was filtered off and recrystallized from ethanol to yield compound 17.

Brown crystals; yield 78% mp, over 350 °C; IR (KBr): ν/cm⁻¹ = 3410 (NH₂), 3120, 3098 (2NH), 1698, 1680 (2C=O); ¹H-NMR (DMSO-d6) δ (ppm) = 2.54 (s, 3H, SCH₃), 6.35 (s, 2H, NH₂), 7.29–7.68 (m, 8H, Ar-H), 11.47 (s, 1H, NH), 12.33 (s, 1H, NH); MS (EI, 70 eV): m/z (%) = 465 (M⁺) (48), 262 (12), 217 (18), 174 (58), 108 (39), 69 (100); chemical formula: C₂₁H₁₅N₅O₂S₃: (465.04); calcd: C, 54.18; H, 3.25; N, 15.04%; found: C, 54.21; H, 3.29; N, 14.98%.

3.6. Biological activity

3.6.1. Antibacterial assay. Antibacterial studies of the newly synthesized compounds were carried out against the representative panel of Gram-positive S. aureus and B. subtilis and Gram-negative E. coli and P. aeruginosa. The activity of the compounds was determined as per the National Committee for Clinical Laboratory Standards (NCCLS) protocol using Müller-Hinton Broth. Primary screening was done first for antibacterial activity in six sets against E. coli, and S. aureus at different concentrations of 1000, 500, and 250 μg mL⁻¹. The compounds found to be active in the primary screening were similarly diluted to obtain concentrations of 200, 125, 100, 62.5, 50, 25, and 12.5 μg mL⁻¹ for a secondary screening to test in a second set of dilutions against all microorganisms. The inoculum size for the test strain was adjusted to 106 CFU mL⁻¹ (Colony Forming Unit per milliliter) by comparing the turbidity (turbidimetric method). The Müller-Hinton Broth was used as a nutrient medium to grow and dilute the compound suspension for test organisms. 2% DMSO was used as a diluent/vehicle to obtain the desired concentration of synthesized compounds and standard drugs to test on the standard microbial strains. Synthesized compounds were diluted to 1000 μg mL⁻¹ concentration as a stock solution. The control tube containing no antibiotic was immediately sub cultured (before inoculation) by spreading a lapful evenly over quarter of plate of medium suitable for the growth of test organisms. The culture tubes were then incubated for 24 h at 36 °C and the growth was monitored visually and spectrophotometrically. 10 μg mL⁻¹ suspensions were further inoculated on an appropriate media and growth was noted after 24 h and 48 h. The lowest concentration (highest dilution) required to arrest the growth of bacteria was regarded as the minimal inhibitory concentration (MIC) i.e. the amount of growth from the control tube before incubation (which represents the original inoculum) was compared. The solvent had no influence on strain growth. The result of this was greatly affected by the size of the inoculum. The test mixture should contain 106 CFU mL⁻¹ of organisms. DMSO and sterilized distilled water were used as negative controls while Ampicillin antibiotic (IU strength) was used as a positive control. The standard drug used in the present study was “ampicillin” for evaluating antibacterial activity.

3.6.2. Antifungal assay. The newly prepared compounds were screened for their antifungal activity as a primary screening in six sets against Candida albicans and F. oxysporum at various concentrations of 1000, 500, and 250 μg mL⁻¹. The primary active compounds were similarly diluted to obtain 200, 125, 100, 62.5, 50, 25, and 12.5 μg mL⁻¹ concentrations for a secondary screening to test a second set of dilutions against fungi. The fungal activity of each compound was compared with clotrimazole as a standard drug, which showed a MIC of 5.8 μg mL⁻¹ against C. albicans. For fungal growth, in the present protocol, we have used Sabouraud’s Dextrose Broth at 28 °C in aerobic condition for 48 h. DMSO and sterilized distilled water were used as negative controls while clotrimazole (IU strength) was used as a positive control.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra14723a

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