New Hydrochlorothiazide Derivatives: Synthesis, Characterization and Anti-Microbial Assessments

 

Ayad Kareem Khan, Mohamed Dhiaa Hamdee

Department of Pharmaceutical Chemistry, College of Pharmacy, Mustansiriyah University, Baghdad, Iraq.

 

ABSTRACT:

A new series of hydrochlorothiazide derivatives linked to heterogeneous rings such as 1,3,4-thiadiazine [3], 3,5-dihydroxypyrazolidin [4], 6-hydroxy-2-thioxo-1,3,4-thiadiazinane [5] and diazenyl-5-methyl-2,4-dihydro-3H-pyrazol-3-one [8] were prepared and characterized using FT-IR, ¹H-NMR, ¹³C-NMR spectroscopic techniques and some physicochemical properties. These synthesized compounds were in vitro tested for their antimicrobial activity in the laboratory against several bacterial and fungus isolates. Each screened compounds exhibiting moderate to potent activity.

 

 

1. INTRODUCTION:

One of the important classes of drugs is the diuretic drugs.¹ Human body undergoes a process which leads to the excretion of toxic substances and excess water.² This process is referred to as diuresis. Hydrochlorothiazide is one of the potent diuretic drugs belong to the thiazide class and used for the treatment of hypertension.² Several reports have proved that this drug have antibacterial and anticancer activities.³

 

Several heterocyclic rings have proved antimicrobial, antiviral and anticancer activities in various reports. There are numerous studies regarding the antimicrobial activity of thiadiazine derivatives.^4-7 A series of 1,3,4-thiadiazine derivatives were synthesized and evaluated for their pharmacological activities. They showed promising antibacterial activity against G-positive and G-negative bacteria as well as antifungal properties. Results showed that they have higher activity than levofloxacin.⁸ In addition, Radini (2018) reported the synthesis of a series pyrazolyl 1,3,4-thiadiazine compounds. These derivatives were evaluated against their antibacterial and fungal activity. Only one derivative demonstrated a moderate activity against G positive and fungi.⁹

 

With regard to pyrazolidine related compounds, Idhayadhulla and his coworkers have synthesized novel pyrazolidine-3,5-dione compounds. These derivatives were divided into three subgroups. Results revealed that three of these derivatives have interesting biological properties. The first compound with LD₅₀ of 19.1µg/mL indicated good anticancer profile against MCF-7 breast cancer cell line. The second derivative with MIC of 0.5 µg/mL was evaluated for its antimicrobial activity against Staphylococcus aureus and it was found that it is higher than ciprofloxacin. The last derivative with MIC of 0.5µg/mL was reported to have higher antifungal activity versus clotrimazole against Candida albicans.¹⁰ In addition, another set of novel pyrazolidine derivatives were synthesized and evaluated for their antimicrobial activity.

 

Results showed that these compounds have low to medium level of antibacterial activity against E.coli, P. aeruginosa, S. aureus and B. subtilis compared to ciprofloxacin. The antifungal activity was measured against griseofulvin and the derivatives demonstrated comparable activity towards Candida albicans and Aspergillus niger.¹¹  The last class of heterocyclic rings studied is the pyrazolones. A series of Schiff base pyrozolone derivatives were synthesized and evaluated for their antimicrobial activity. It was found that butyl pyrozolone derivatives have a good activity against Bacillus subtilis and Pseudomonas infections in addition to low antifungal activity.¹²

 

 

In the light of facts and explore to developed the good antimicrobial activities of hydrochlorothiazide derivatives, a series of heterocyclic rings such as 1,3,4-thiadiazine, pyrazolidine-3,5-diol, 6-hydroxy-1,3,4-thiadiazinane-2-thione and [(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)diazenyl]  compounds are designed and synthesized  in the current research.

 

2. EXPERIMENTAL:

2.1. MATERIALS AND METHODS:

All chemicals and raw materials were obtained from the commercial suppliers. Hydrochlorothiazide (HCTZ) was obtained from the State Company of Drug Industries and Medical Appliances (SDI-Iraq). Melting points were set on capillary tubes by the digital STUART apparatus and have not been corrected. Fourier transforms Infrared red (FT-IR) absorption spectra were indicated on SHIMADZU-8400 spectrophotometer utilizing potassium bromide (KBr) pellet and the wave numbers were given in the spectral range (500-4000) cm^-1. The ¹H-NMR and ¹³C-NMR spectra were determined in hexadeutero dimethyl sulfoxide (DMSO-d₆) on a Bruker NMR spectrometer (300 MHz). The chemical shifts are reported in part permillion (δ ppm) using tetramethyl silane (TMS) as an internal reference. The antimicrobial examination was conducted in the department of Biology, College of Science-Baghdad University.

 

2.2. Synthesis of 2-chloro-N-[[6-chloro-1,1-dioxido-3,4-dihydro-2H benzo[e][1,2,4] thiadiazin-7-yl] sulfinyl] acetamide (1).

To a stirred solution of 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4 benzothiadiazine-7-sulfonamide (Hydrochlorothiazide-HCTZ) (2.97g. 0.01mol.)  in dimethyl formamide (DMF) (20ml.), a chloroacetyl chloride (0.8ml. 0.01mol.) was added drop by drop. The reaction carried out by refluxing the reaction mixture for (6hrs.). The resulting solid product then it has been filtered, dried and recrystallized from ethanol.

 

2.3. Synthesis of    N-[[6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4] thiadiazine-7-yl]sulfinyl}-2-hydrazineylacetamide (2).

A mixture of an 2-chloro-N-{[6-chloro-1,1-dioxido-3,4-dihydro-2H benzo[e][1,2,4] thiadiazin-7-yl] sulfinyl] acetamide (1) (3.74g. 0.01mol.) and hydrazine hydrate (99%) (0.5ml. 0.01mol.) has been refluxed to (3hrs.). Separate precipitates were collected washed and re-crystallized from ethanol.

 

2.4. Synthesis of 6-chloro-N-(2-(phenylamino)-4H-1,3,4-thiadiazin-6-yl)-3,4-dihydro-2H-benzo[e] [1,2,4] thiadiazine-7-sulfonamide 1,1-dioxide (3).

To a solution of N-[[6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4] thiadiazin-7-yl]sulfinyl}-2-hydrazineylacetamide (2) (3.69g. 0.01 mol.) in absolute ethanol (20ml.), p-chloro phenylisocyanate (1.69g. 0.01mol.) has been added and refluxed to (4hrs.). The reaction was cooled and the soluble matter was filtered, dried and re-crystallized from ethanol.

 

2.5. Synthesis of N-((6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-7-yl)sulfonyl)-2-(3,5-dihydroxypyrazolidin-1-yl)acetamide (4).

A mixture of N-[[6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4] thiadiazin-7-yl]sulfinyl}-2-hydrazineylacetamide (2) (3.69g. 0.01 mol.), ethyl acetoacetate (1.27ml. 0.01mol.) and absolute ethanol (15ml) basify with sodium hydroxide was mixed carefully then reflexed for (3hrs.). The reaction mixture is then concentrated and cooled with crushed ice to form the solid product, which is eventually filtered and re-crystallized from ethanol-water 1:1.

 

2.6. Synthesis of 6-chloro-N-(6-hydroxy-2-thioxo-1,3,4-thiadiazinan-6-yl)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine-7-sulfonamide 1,1-dioxide (5).

To a stirred solution of potassium hydroxide (0.56g. 0.01mol.) in ethanol (20ml.), N-[[6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-7-yl]sulfinyl}-2-hydrazineylacetamide (2) (3.69g. 0.01mol.) and carbon disulfide (0.6ml. 0.01mol.) was added slowly and refluxed to (3hrs.). The solid precipitate is filtered, washed with ether, dried and crystallized from ethanol.

 

2.7. Synthesis of diazonium chloride salt (6)

Sodium nitrite (0.69 g, 0.01mol.) is gently added to concentrate hydrochloric acid (5ml.) at less than (5°C.) and then hydrochlorothiazide (2.97 g, 0.01 mol.) it was slowly added to the solution over an hour. The reaction mixture was stirred for more time (2 hrs.). It was slowly added to the solution over an hour. The reaction mixture was stirred for more time (2 hrs.).

 

2.8. Synthesis of ethyl 2-(((6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazin-7-yl)sulfonyl)diazenyl)-3-oxobutanoate  (7).

The clear solution of diazonium salt (6) (3.45g, 0.01mol.) was added to solution of ethyl acetoacetate (1.27ml. 0.01mol.) in sodium hydroxide (0.4g. 1mol.). Mixture of reaction was refluxed for (3hrs.). The solid product is filtered, washed with a little hot water, dried and purified from ethanol.

 

2.9. Synthesis of 4-(((6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4] thiadiazin-7-yl)sulfonyl)diazenyl)-5-methyl-2,4-dihydro-3H-pyrazol-3-one (8).

To (4.38g, 0.01mol.) of ethyl 2-(((6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4] thiadiazin-7-yl)sulfonyl)diazenyl)-3-oxobutanoate  (7), hydrazine hydrate (99%) (0.5ml. 0.01mol.) gently added. The reaction mixture was refluxed for (3hrs.) and then cooled to room temperature. The solid precipitate is formed washed, dried and crystalized from ethanol -water1:1.

 

2.10. Antimicrobial activity assessment:

The newly synthesized compounds (3, 4, 5, and 8) were screened for their In vitro antimicrobial activity against different Gram positive bacterial isolates (Staphylococcus aureus, Bacillus subtilis) and Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa). Candida albicans also used in the current evaluation as representatives of yeast-like pathogenic fungus strain. The tested compounds were prepared at (100mg/ml) concentrations using dimethyl sulfoxide (DMSO) as solvent. Disk diffusion method was used to determine the initial activity of the microorganisms. The disks with a diameter of 6.25mm were perforated from Whatman no. 1 filter papers. The batch discs were disbursed to each screw-covered bottle and sterilized by dry heat at (140°C) for one hour. Wells perforated in agar medium seeded with fresh bacteria or fungi separately and filled with 100μl of each concentration. DMSO was used as a control. The incubation was performed at (37°C) for one day. Ampicillin trihydrate and Clotrimazole was used as a standard drug in the current test.  Antimicrobial activities were assessed by measuring the diameter of the observed inhibition region.

3. RESULTS AND DISCUSSION:

3.1. Chemistry:

Synthetic pathways for newly prepared hydrochlorothiazide derivatives (1-3) are presented in Scheme (1).

 

Scheme (1)

 

The target compound (3) having hydrochlorothiazide with 1,3,4-thiadiazine was synthesized by three steps. First step include formation of 2-chloro-N-[[6-chloro-1,1-dioxido-3,4-dihydro-2H benzo[e][1,2,4] thiadiazin-7-yl] sulfinyl] acetamide compound (1) by addition of chloroacetyl chloride to a solution of 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4 benzothiadiazine-7-sulfonamide (Hydrochlorothiazide) in dimethyl formamide as solvent and refluxing mixture.

 

Second step have the reaction between compound (1) with hydrazine hydrate by refluxing process to yield compound (2) N-[[6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4] thiadiazine-7-yl]sulfinyl}-2-hydrazineylacetamide. Final step contains reacting of p-chloro phenylisocyanate with the previous compound (2) absolute ethanol to give the desired product 6-chloro-N-(2-(phenylamino)-4H-1,3,4-thiadiazin-6-yl)-3,4-dihydro-2H-benzo[e] [1,2,4] thiadiazine-7-sulfonamide 1,1-dioxide compound (3).

 

Physicochemical properties of compounds (1-3) and each other synthesized compounds are listed in Table (1).

 

Table-1 Physicochemical behaviors for hydrochlorothiazide derivatives [1-8].

Compound no.	Mol. Formulas	M.W g.mol-1	Yield  (%)	m. p. °C.	Color	Solvent Recryst.
1	C9H9Cl2N3O5S2	374	60	190-192	Off white	ethanol
2	C9H12ClN5O5S2	369	67	137-139	deep brown	ethanol
3	C16H15ClN6O4S3	486	79	125-127	yellow	ethanol
4	C12H16ClN5O7S2	441	71	172-174	pale yellow	ethano-water1:1
5	C10H12ClN5O5S4	445	84	150-152	deep brown	ethanol
6	C7H6Cl2N4O4S2	345	68	140-142	Light orange	ethanol
7	C13H15ClN4O7S2	438	66	161-163	Pale yellow	ethanol
8	C11H11ClN6O5S2	405	58	204-206	brown	ethanol-water1:1

 

FTIR spectrum for 2-chloro-N-[[6-chloro-1,1-dioxido-3,4-dihydro-2H benzo[e][1,2,4] thiadiazin-7-yl] sulfinyl] acetamide compound (1) the bands showed good stretch at (3263cm-1) were assigned to the v(N-H) stretching frequency. Besides the appearances of v(C=O) stretching band attributable to amide group at (1689cm-1) and stretching peak at (2835cm-1) back to ν(CH2) and at (1373, 1153cm-1) for asymmetric and symmetric ν(SO2) stretching bands are best proof for the structure give to intended compound as listed in Table (2).

FTIR spectrum of hydrazine carboxamide showed remarkable stretching bands in (3375cm^-1) and (3263cm^-1) which were assigned to the ν(-NHNH₂) group frequency stretch proved the formation of compound (2).

On the other hands disappearance of ν(-NHNH₂) (CH₂) and (C=O) group stretching and appearance of stretching bands ν(C=N) at (1600 cm^-1) for thiadiazine ring consider good proof to formation of compound (3).

 

Table-2 FTIR v(cm^-1) spectral data for hydrochlorothiazide derivatives [1-8].

Comp. No.	ν(N-H)	ν(C-H) Ar.	ν(C-H) Aliph.	ν(C=C) Ar.	ν(SO2) Asym.	ν(SO2) sym.	Others
1	3263	3093	2943	1554	1373	1153	ν(CH2) 2835, ν(C=O) 1689, ν(C-Cl) 771.
2	3267	3067	2970	1519	1334	1161	ν(NH2) 3375, ν(CH2) 2877, ν(C=O) 1670, ν(C-Cl) 775.
3	3267	3086	2924	1543	1342	1165	ν(C=N) 1600.
4	3217	3086	2935	1543	1334	1168	ν(O-H) 3332, ν(CH2) 2889, ν(C-Cl) 779.
5	3267	3093	2947	1519	1319	1180	ν(O-H) 3363, ν(C=S) 1160, ν(C-Cl) 775.
6	3248	3071	2918	1534	1356	1176	ν(C-Cl) 762.
7	3217	3086	2935	1543	1381	1168	ν(CH2) 2889, ν(C=O) 1745, ν(N=N) 1512, ν(C-Cl) 779.
8	3363	3093	2947	1554	1334	1149	ν(C=O) 1674, ν(C=N) 1604, ν(C-Cl) 748.

 

¹H-NMR spectrum of hydrochlorothiazide compounds (1-3), shows the important characteristic chemical shifts (DMSO-d₆, ppm). It displayed signals attributed for hydrochlorothiazide attached to thiadiazine moiety compound (3), two protons of methylene group, one proton of thiadiazine ring, seven aromatic ring protons, one proton of (CH₂-NH-SO₂) sulfonamide, one proton of secondary amine (NH), Two proton proton of different types (NH) thiadiazine respectively as listed in Table (3).

 

Additional spectral method ¹³C-NMR also used for characterization of newly synthesized compounds (1-3). Spectrum of compound (3) showed signals belong to carbon of (NH-CH₂-NH), two carbons of 1,3,4-thiadiazine ring, aromatic carbons and imine carbon (C=N) of 1,3,4-thiadiazine ring respectively as listed in Table-4.

 

Other hydrochlorothiazide derivatives attached with pyrazolidine-3,5-diol rings, 6-hydroxy-1,3,4-thiadiazinane-2-thione moieties compounds (4) and (5) respectively. It was prepared by condensation of compound (2) N-[[6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4] thiadiazin-7-yl]sulfinyl}-2-hydrazineylacetamide (2) with ethylacetoacetate in absolute ethanol to offered compounds (4). On the other hand, intensification of the compound (2) with carbon dioxide in the base medium of potassium hydroxide gives compound (5) as shown in the scheme (2).

 

Scheme (2)

 

FTIR spectrum for pyrazolidine-3,5-diol compound (4) give starching bands for v(O-H) at (3332cm^-1) and v(CH₂) at (2889cm^-1). While pyrazolone compound (5) show starching bands for v(C=S) 1160 beside starching bands for v(O-H) at (3363cm^-1) as listed in Table-2.

 

¹H-NMR spectrum of pyrazolidine-3,5-diol compound (4) displayed the basic characteristic signals due to four protons of two methylene groups, two protons of -CH₂- pyrazolidine ring, two protons of (2CH) pyrazolidine ring, one proton of NH pyrazolidine ring, two protons of (2OH) hydroxyl of pyrazolidine ring, two protons of aromatic ring, one proton of (CH₂-NH-SO₂) sulfonamide, one proton of secondary amine (NH) and one proton of secondary amide (SO₂-NH-CO) respectively as listed in Table-3.

¹³C-NMR spectrum of pyrazolidine-3,5-diol compound (4) showed the major characteristic signals due to carbon of (CO-CH₂-N), carbon of (CH₂) pyrazolidine ring, carbon of (NH-CH₂-NH), carbons of (CH) pyrazolidine ring, six aromatic carbons, carbon of carbonyl (C=O) group respectively as listed in Table-4.

 

¹H-NMR spectrum of pyrazolone compound (5) detect the signify characteristic chemical shifts were appeared signals suggested the attribution four protons of two methylene groups, one proton of hydroxyl group (–OH), two aromatic ring protons, two protons of (2NH) thiadiazinane, one proton of (CH₂-NH-SO₂) sulfonamide and one proton of secondary amine (NH) respectively as listed in Table-3.

 

¹³C-NMR appeared signals attributed to carbon of (CO-CH₂-N), carbon of (CH₂) thiadiazinane ring, carbon of (C-OH) thiadiazinane ring, six aromatic carbons and carbon of (C=S) group respectively as listed in Table-4.

 

Table-3 ¹H-NMR spectral data (δppm) for hydrochlorothiazide derivatives [1-8].

Comp. No.	1H-NMR parameters (δppm)
1	4.28 (s, 2H, NH-CH2-NH) methylene, 4.80 (s, 2H, CO-CH2-Cl) methylene, 6.97-7.59 (m, 2H, aromatic-H), 7.80 (s, 1H, CH2-NH-SO2) sulfonamide, 8.12 (s, 1H, CH2-NH-C) sec. amine, 12.62 (s, 1H, SO2-NH-CO) sec. amide.
2	3.80 (s, 4H, NHNH2) hydrazine, 4.32 (s, 2H, NH-CH2-NH) methylene, 4.77 (s, 2H, CO-CH2-Cl) methylene, 5.16 (s, 2H, NHNH2) hydrazine, 7.87 (s, 1H, CH2-NH-SO2) sulfonamide, 8.20 (s, 1H, CH2-NH-C) sec. amine, 6.88-7.61 (m, 2H, aromatic-H), 12.91 (s, 1H, SO2-NH-CO) sec. amide.
3	4.14 (s, 2H, NH-CH2-NH) methylene, 4.67 (s, 1H, CH) thiadiazine, 6.90-7.71 (m, 7H, aromatic-H), 7.93 (s, 1H, CH2-NH-SO2) sulfonamide, 8.41 (s, 1H, CH2-NH-C) sec. amine, 8.66 (s, 1H, Ph-NH-C) thiadiazine, 8.88 (s, 2H NH) thiadiazine, 9.63 (s, 1H, NH-Ph).
4	3.35 (s, 4H, CO-CH2-N) methylene, 4.11 (s, 2H, NH-CH2-NH) methylene, 4.67 (s, 2H, CH2) pyrazolidine, 4.90 (t, 2H, 2CH) pyrazolidine, 5.27 (s, 1H, NH) pyrazolidine, 5.81 (s,2H, OH) pyrazolidine, 6.92-7.86 (m, 2H, Aromatic-H), 7.98 (s, 1H, CH2-NH-SO2) sulfonamide, 8.44 (s, 1H, CH2-NH-C) sec. amine, 12.68 (s, 1H, SO2-NH-CO) sec. amide.
5	4.31 (s, 2H, NH-CH2-NH) methylene, 4.66 (s, 2H, CH2) thiadiazinane, 5.50 (s, 1H, OH), 6.46 -7.80 (m, 2H, Aromatic-H), 7.99 (s,2H, 2NH) thiadiazinane, 8.14 (s, 1H, CH2-NH-SO2) sulfonamide, 8.56 (s, 1H, CH2-NH-C) sec. amine.
6	4.55 (s, 2H, NH-CH2-NH) methylene, 6.71 -7.87 (m, 2H, Aromatic-H), 8.22 (s, 1H, CH2-NH-SO2) sulfonamide, 8.73 (s, 1H, CH2-NH-C) sec. amine.
7	1.93 (t, 3H, CH3) methyl, 3.52 (s, 3H, CO-CH3), 4.30 (s, 1H, CH), 4.55 (q, 2H, CH2) methylene, 4.74 (s, 2H, NH-CH2-NH) methylene, 6.96-7.97 (m, 4H, Aromatic-H), 8.32 (s, 1H, CH2-NH-SO2) sulfonamide, 8.77 (s, 1H, CH2-NH-C) sec. amine.
8	1.88 (s, 3H, CH3) pyrazole, 2.20 (s, 1H, CH) pyrazole, 4.51 (s, 2H, NH-CH2-NH) methylene, 6.96-7.75 (m, 2H, Aromatic-H), 8.40 (s, 1H, CH2-NH-SO2) sulfonamide, 8.81 (s, 1H, CH2-NH-C) sec. amine. 12.26 (s, 1H, NH) pyrazole.

 

Diazotization reaction of starting hydrochlorothiazide with sodium nitrite and hydrochloric acid yield the diazonium chloride derivative of hydrochlorothiazide compound (6). Diazonium salt (6) then treated with ethyl acetoacetate in the precene of sodium hydroxide to give ethyl 2-(((6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4] thiadiazin-7-yl)sulfonyl)diazenyl)-3-oxobutanoate derivative (7).

 

Final product of 4-(((6-chloro-1,1-dioxido-3,4-dihydro-2H-benzo[e][1,2,4] thiadiazin-7-yl)sulfonyl)diazenyl)-5-methyl-2,4-dihydro-3H-pyrazol-3-one compound (8) was obtained in good yield from condensation of compound (7) with hydrazine hydrate. The synthetic routes for preparation of mentioned compounds (6-8) are shown in scheme (3).

 

 

Scheme (3)

 

FTIR spectrum for compounds (7) shows the characteristic starching band for v(N-H) at (3217cm^-1) beside v(CH₂) at (2889 cm^-1), ν(C=O) of ester group at (1745 cm^-1) and ν(N=N) at 1512 cm^-1 respectively. While pyrazole compound (8) show starching band for v(N-H) at 3363 cm^-1 beside v(C=O) at (1674cm^-1), ν(C=N) at (1604 cm^-1) as listed in Table-2.

¹H-NMR spectrum of hydrochlorothiazide derivatives (6, 7 and 8) shows the characteristic chemical shifts (DMSO-d₆, ppm) as listed in Table-3.

 

It displayed signals attributed for hydrochlorothiazide linked to pyrazol-3-one moiety compound (8). Three protons of methyl group attached to pyrazolone ring, one proton of (-CH-) pyrazolone ring, two protons of methylene group, two aromatic ring protons, one proton of (CH₂-NH-SO₂) sulfonamide, one proton of secondary amine (NH) and one protone of (NH) pyrazolone ring respectively as listed in Table-3.

 

¹³C-NMR spectrum of hydrochlorothiazide derivatives (6-8) shows the characteristic signals as listed in Table-4. It displayed signals due to hydrochlorothiazide linked to pyrazol-3-one moiety for compound (8) carbon of (CH₃) attached with pyrazolone ring, carbon of (CH₂) thiadiazinane ring, carbon of (-CH-) pyrazolone ring, six aromatic carbons, carbon of imine (C=N) of pyrazolone ring and carbon of (C=O) carboyl group respectively.

 

Table-4 ¹³C-NMR spectral data (δppm) for hydrochlorothiazide derivatives [1-8].

Comp. No.	Compound structure with numbering of carbon atoms	13CNMR Spectral Data (δ ppm)
1		45.59 (C9), 70.81 (C1),121.66-138.32 (C2-C7), 170.41 (C8).
2		42.36 (C9), 71.44 (C1), 120.18-136.91 (C2-C7), 171.06 (C8).
3		68.53 (C1), 115.71 (C8), 177.34 (C10) 118.33-139.29 (C2-C7) (C11-C16), 158.82 (C9).
4		48.21 (C9), 53.88 (C11), 69.35 (C1), 95.55(C10,C12). 122.39-137.06 (C2-C7),174.49 (C8).
5		68.30 (C1), 72.91 (C10), 105.73(C8), 119.69-137.81 (C2-C7), 184.62(C9).
6		70.51 (C1), 122.03-138.79 (C2-C7).
7		18.56 (C13), 29.23 (C10), 68.49 (C1), 73.83 (C12), 86.35 (C8), 121.35-139.61 (C2-C7), 179.71 (C11), 187.44 (C9).
8		20.79 (C11), 69.50 (C1), 72.19 (C8), 122.71-138.45 (C2-C7), 160.53 (C10), 177.45 (C9).

 

3.2. The Antimicrobial Assessments:

The inhibition zone of the newly synthesized hydrochlorothiazide derivatives (3,4,5 and 8) were observed and measured. The biological activates of these prepared compounds were tested against bacterial strains and fungi. Escherichia coli, staphylococcus aureus and candida albicans using ager well diffusion method. The results of this study are summarized in Table (5).

 

 

 

All the synthesized compounds were screened for antibacterial activity against ampicillin trihydrate. Compound (8) showed the highest activity against Staph aureus, whereas it showed the lowest activity against P. aeruginosa together with compound (5).

 

Regarding compound (3), it demonstrated the weak activity against all tested bacteria except P. aeruginosa with no activity against B. subtilis. It was noticed also that the antibacterial activity against Pseudomonas species was similar between compound (3) and (4).

Table-5: Antimicrobial activity for selected hydrochlorothiazide derivatives.

Comp. No.	Staphylococcus aureus Inhibition zone diameter (mm)	Bacillus subtilis Inhibition zone diameter (mm)	Escherichia Coli Inhibition zone diameter (mm)	Pseudomonas aeuroginosa. Inhibition zone diameter (mm)	Candida Albicans Inhibition zone diameter (mm)
3	13	-	15	17	16
4	15	13	17	18	19
5	18	21	21	12	22
8	21	18	19	12	20
Amp. (std.)	30	32	29	27	*
C (std.)	*	*	*	*	25

A (std.) = Ampicillin trihydrate (standard antibiotic drug)

C (std.) = Clotrimazole (standard antifungal drug)

* = not tested

- = no inhibition zone

 

These differences can be rationalized according to the properties of side chain of these derivatives. Compound (3) contains phenyl group in its side chain which may cause steric hindrance causing relatively low overall antibacterial activity. The two hydroxyl groups found in the side chain of compound 4 may contribute to higher water solubility. This could the reason behind the improved activity compared to compound (3).

 

Compound (5) has two interesting features which are the hydroxyl group and the endocyclic secondary amine groups in the side chain. This may impart more affinity due to the formation of two hydrogen bonds.

 

The last compound (8) has methyl group attached to the five-membered ring in the side chain. It is noticed that it has the smallest size in the side chain which may explain its relatively high activity against all bacteria tested due to the less steric hindrance encountered.

 

In addition, these compounds were tested for their antifungal activity using clotrimazole as a reference standard. It was found that compound 8 has the highest activity against Candida albicans whereas the lowest activity was noticed in compound (5).

 

Overall, taking in consideration the activity against all microbes tested, compound 8 is the best among the synthesized compounds.

4. CONCLUSIONS:

Heterocyclic compounds derived from hydrochlorothiazide were successfully prepared and structurally characterized by using different spectroscopic techniques. The synthetic route produced 1,3,4-thiadiazine, 3,5-dihydroxypyrazolidin , 6-hydroxy-2-thioxo-1,3,4-thiadiazinane and diazenyl-5-methyl-2,4-dihydro-3H-pyrazol-3-one moieties attached with hydrochlorothiazide rings. All these finally compounds have been effectively estimated for their anti-microbial activity on four strains of pathogenic bactria and one strain of yeast-like pathogenic fungus.

5. ACKNOWLEDGMENTS:

The authors would like to thank Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad – Iraq for its support in the present work.

 

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Received on 25.05.2019           Modified on 17.06.2019

Accepted on 03.07.2019         © RJPT All right reserved