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Paper Title:

Synthesis , Characterization and Antimicrobial, Antibacterial Activity

Evaluation of Some New Schiff Bases Derivative Thiocarbohydrazide

Ahmood KH. Jebur, Hiba Hamza Rasheed, Dardaa Aziz Ibrahim

Tikrit University - College of Sciences

A R T I C L E I N F O

Article history:

Received in 2019

Received in revised form 28/12/2019

Accepted 28/12/2019

Keywords:

Thiocarbohydrazide,

Anti-fungal ,

Anti-bacterial,

Schiff base ,

Triazole,

A B S T R A C T

Thiocarbohydrazide (H1) was synthesized by reaction of hydrazine with carbon disulfide CS2 , in the

presence of H2O with subsequent heating to remove H2S, N, N-dithiocarbohydrazone(H2) have been

synthesized from thiocarbohydrazide by reaction with Acetophenone and the later one was underwent the compound (H2) cyclization reaction by ferric chloride as a selective catalyst .to give 5-hydrazineyl-2-

methyl-2-phenyl-2, 3-dihydro-1, 3, 4-thiadiazole[H3]. The compound (H4) 2-mercapto-5- (4-amino

phenyl)-4, 3, 1-triazole was prepared by reaction between the p-amino carboxylic acid and the

thiocarbohydrazide. A series of 4-[4-(benzylidene amino-5-mercapto-4H-[1, 2, 4]triazol-3-yl]-aniline were

synthesized from 4-(4-amino-5-mercapto-4H-[1, 2, 4]triazol-3-yl)-aniline by reaction with different

aromatic aldehydes. The title compounds were evaluated for antibacterial activity against Gram-positive

bacteria (Staphylococcus aureus ) and Gram-negative bacteria (Escherichia coli) and anti-fungal activity against (Candida albicans ) , (Candida tropicalis). The synthesized thiocarbohydrazide derivatives and

Schiff base derivatives were characterized by both physical and spectral data like 1H-NMR, FT-IR. All

the synthesized were characterizing new derivatives by using analytical techniques (FT-IR and 1H NMR ,

as well as by using (C.H.N.S) measurements for these compounds.

© 2020. All rights reserved for Government University of UK.

Introduction

Thiocarbohydrazide(1-5)

is the closest structural analogue of thiosemicarbazide,

derivatives of which are recommended as effective anti-tubercular and antiviral preparations.

Thiocarbohydrazides of the aromatic series exhibit high antiviral and antimicrobial activity (6-

7).Thiocarbohydrazide are hydrazine derivatives of carbonic and thiocarbonic acids.

Thiocarbohydrazides are an important class of compounds which possess applications in both synthetic

organic chemistry and biological fields and has considerable value in many useful applications such as

synthesis of transition metal complexes(8, 9)

and pharmacological studies. Moreover, carbohydrazide

derivatives were widely used as an oxygen scavenger (metal passivation) for water treatment systems,

particularly for boiler-feed systems. To synthesis these active types of heterocyclic compounds , it

must be starting from active materials with wide biological applications, here in this presentation the

active starting material is thiocarbohydrazide [H1] which have best role in heterocyclic synthesis(10)

.

The chemistry of carbohydrazides has grown fast, and has not been reviewed in more than three

decades. Accordingly, it is important to shed more light on the recent literature dealing with that

chemistry, especially in the field of heterocycles. On the other hand, Thiocarbohydrazides are used in

performing a highly selective heavy metal ion adsorbent and as complexing agents for the solvent

extraction separation methods. Thiocarbohydrazide showed structural reinforcement effect in the

preparation of rabbit knee articular cartilage for the scanning electron microscope by enhancing the

binding of osmium tetroxide to it, possibly along with that of other soluble tissue constituents(11)

. Also

effective anti-tubercular.(12)

and antiviral preparations.(13)

exhibit high antiviral(14)

and antimicrobial

activity.(15)

effective fungi static agents, (16)

cytotoxicity of the well-known product melphalan.(17)

Scheme(1): synthesis of Thiocarbohydrazide, hydrazones , triazoles

Experimental Section

MATERIALS AND METHODS

All the chemicals used were supplied by Merck, BDH and Fluka chemicals. FTIR spectra were

recorded on SHIMADZU – FTIR 8400 Fourier transform infrared spectrophotometer using KBr discs.

Melting point were determined in open capillaries on Thomas Hoover apparatus and were uncorrected

.Finally, used C.H.N.S measurements .

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217

Synthesis Methods:

General Procedure for Synthesis of Thiocarbohydrazide.H1 (18-21)

In a 250 ml round bottomed flask equipped with a magnetic bar stirrer and dropping funnel, a solution

of carbon disulphide (CS2), (13ml, 0.22mol) under stirring to a mixture reaction of hydrazine

hydrate(24ml)and water (15ml), then stirring was continued for 30min at room temperature. The

reaction mixture temperature was then rapidly raised to 100-110oC. and after completion the addition,

the mixture was heated under reflux for 3hrs.on hot plate. The completion of the reaction was

confirmed by TLC, the reaction mixture was cooled and poured in the ice cold water ; the precipitate

was filtered and washed with water, followed by ether and then air dried. the product thus obtained

was recrystallized from water .Yield 80%, m.p.172 -173°C. 1H NMR (CDCl3, δ ppm):

1H-NMR

spectrum of this compound showed signals at δ= 4.49 (NH2) proton and a clear signal at δ= 8.60 ppm

due to (N-H). FT- IR(cm-1

): NH2 (3305), NH (3276, 3205) and C=S (1286).

Synthesis of 5-Aminophenyl-4H-1, 2, 4-triazol-3-yl)thiol H2 (22, 23)

In a 250 ml round bottomed flask equipped with a magnetic bar stirrer dissolve (0.02 mol, 2.4g)

acetophenone, then add (0.02 mol, 2.21g) of thiocarbohydrazide in 20 ml of hydrochloric acid (1N)

and stirring at room temperature for 1hr.The reaction mixture was then poured in ice cold water and

the resulting yellow precipitant solid was washed with water, dried and recrystallized from aq. ethanol,

m.p = 198-200 oC ; yield 85% .

1H NMR (CDCl3, δ ppm): δ (3.8 - 4.2) (s, 2H, NH2), δ 8.9 (s, 1H, NH),

δ (6.9-8.1) (m, 5H, aromatic C-H), δ 8.45 (s, 1H, N=CH). δ 2.15 (s, 3H, CH3). FT- IR (cm-1

): 3323,

3240 (NH2) 3335 (NH), 3012 (ArCH), 1229 (C=S), 1615 (C=N) .

Synthesis of 5-hydrazineyl-2-methyl-2-phenyl-2, 3-dihydro-1, 3, 4-thiadiazole [H3] ( 24)

:

The hydrazone [H2] (0.017 mole, 3.54 g) and FeCl3 (0.005 mole, 1.135 g) were dissolved in absolute

ethanol (25 ml), then the mixture was heated under reflux for (3hrs) in water bath. After completion of

reaction, the reaction mixture was cooled and poured in the ice cold water. The resulting solid was

dried and recrystallized from ethanol. m.p .(113-115 °C) ; Yield 60%; 1H NMR (CDCl3, δ ppm): δ 4.1

(s, 2H, NH2) , δ8.65(s, 1H, NH), δ7.2-7.65(m, 5H, aromaticC-H); FT- IR(cm-1

): 3320, 3240 (NH2)

3330 (NH), 3050 (Ar-CH), 756 (C-S-C), 1610 (C=N).

Synthesis of 4-(4-Amino-5-mercapto-4H-[1, 2, 4] triazol-3-yl)-benzoic acid (H4)(25)

A mixture of thiocarbohydrazide (H1) (0.015 mol) and p-amino benzoic acid (0.01 mol) were taken in a

round bottomed flask. The completion of the reaction was confirmed by TLC. The reaction mixture was

cooled; the precipitate was filtered and washed with water. The product was recrystallized from aq.

ethanol .Yield 80%, m.p. 150 - 153°C.

Synthesis of 4-[4-(arylidene amino-5-mercapto-4H-[1, 2, 4] triazol-3-yl]-sub. benzoic acid H5(a-h)

Suspension of 4-(4-amino-5-mercapto-4H-[1, 2, 4] triazol-3-yl)-benzoic acid (H4) (0.2 mol) with

substituted benzaldehyde were placed in a 500 ml RBF, with 3 to 4 drops of Sulphuric acid was added.

The reaction mixture was refluxed for 2-3h. The reaction mixture was cooled; precipitate was filtered

and washed with water. The product was recrystallized with ethanol. Various substituted aldehyde used

in the reaction are mentioned in table (1)

Synthesis of 4-[4-(arylidene amino-5-mercapto-4H-[1, 2, 4] triazol-3-yl]benzoic acid 5(a)

81% yields as reddish solid; m.p.: 181-183oC;IR (KBr, cm

-1) νmax :3245(O-H str), 1025 (C-S),

1715(C=O str in COOH), 1595 (CH=N), 2921(C-H str); 1H-NMR (400 MHz, DMSO-d6, ppm), δ

(ppm): 13.84(s, 1H, OH), 9.75 (s, 1H, CH=N), 9.65 (s, 1H, C-SH), 7.85-6.85 (m, 9H, Ar-H);

Synthesis of 4-{5-Mercapto-4-[(4-methoxybezylidene)-amino]-4H-[1, 2, 4]-triazol-3-yl-}-benzoic

acid 5(b)

Yield (85%); m. p. 235-237ᵒC; FT-IR (KBr, λmaxcm-1

): 3025(C-H), 2848(OCH3), 1580(CH=N),

960 (C-S), 745 (C-H bend); 1H-NMR (400 MHz, DMSO-d6), δ (ppm), : 9.65 (s, 1H, C-SH), 9.40

(s, 1H, CH=N), 7.80-6.95 (m, 8H, Ar-H), 3.80 (s, 3H, OCH3) .

Synthesis of 4-{5-Mercapto-4-[(3-methyl-bezylidene)-amino]-4H-[1, 2, 4]triazol-3-yl}-benzoic acid

5(c)

Yield(76 %); m. p.245- 248ᵒC; FT-IR (KBr, λmaxcm-1

): 3035 (C-H str), 1583 (CH=N) , 942 (C-S);1H-

NMR (400 MHz, DMSO-d6), δ (ppm), 10.20 (s, 1H, CH=N), 7.85-7.35 (m, 8H, Ar-H), 2.42 (s, 1H,

CH3).

Synthesis of 4-{4-[(4-Bromo-benzylidene)-amino]-5-mercapto-4H-[1, 2, 4] triazol-3-yl}-benzoic

acid 5(d)

Yield (86%); m. p. 254- 256ᵒC; FT-IR (KBr, λmaxcm-1

): 3430(O-H str), 3030(C-H Ar str),

1630(C=C), 1596(CH=N), 965(C-S), 845(C-Br); 1H-NMR (400 MHz, DMSO-d6), δ (ppm),

9.90(s, 1H, C-SH), 7.70-7.30(m, 8H, Ar-H);

Synthesis of 4-{5-Mercapto-4-[(4-N, Ndimethyl-benzylidene)-amino]-4H-[1, 2, 4]triazol-3-yl}-

benzoic acid 5(e)

Yield (75%); m. p. 214-216ᵒC; FT-IR (KBr, , λmaxcm-1

): 3035(C-H Ar str), 2772(C-H str.),

1716(C=O str), 1572(CH=N), 1533(C=C) ; 1H-NMR (400 MHz, DMSO-d6), δ (ppm) 10.15 (s, 1H,

C-SH), 11.2 (1H, -NH), 8.2 CH=N), 8.42-7.50 (m, 8H, Ar-H); 2.38 (s, 6H, CH3).

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Table 1:Physical Properties of the compounds (H5a-e)

Comp.

No. R

Molecular

formula Colour Recrys .sol. M.P(0C)

Percentage

Yield (%)

H5a H C15H13N5S White EtOH 183 - 181 81

H5b OMe C16H15N5OS brown Acetonitrile 235-237 75

H5c CH3 C16H15N5S milky Chloroform 245- 248 86

H5d Br C15H12BrN5S White ACOH 254- 256 76

H5e NMe2 C17H18N6S Dark orange EtOH 214-216 85

RESULTS AND DISCUSSION

The structure of the compounds were confirmed from its melting point in Table (1) and (C.H.N.S)

analysis for some of them in Table (2). Besides FT-IR. Initially(26)

.Thiocarbohydrazide [H1]was

synthesis by reaction of hydrazine with CS2 in aqueous media through direct nucleophilic addition

reaction. Compound [H1] characterized by spectral methods represented by melting point , FT-IR and

1H-NMR, as shown in experimental section. The hydrazone compound [H2]Prepared by condensation

reaction with Acetophenone. This reaction was activated by few drops of (1N) hydrochloric acid, as

shown in (Scheme 2).

The compound (H3) was prepared by aqueous ferric chloride form 2-hydrazino-5-phenyl-5-acetyl.

(H2) . as in the following (scheme).

A novel series of 4-[4-(Arylidene amino-5-mercapto-4H-[1, 2, 4] triazol-3-yl]-benzoic acid complexes

from p-amino benzoic acid and thiocarbohydrazide was synthesized and characterized using analytical

methods like TLC, 1HNMR and FT-IR. The structures of newly synthesized compounds were

characterized on the basis of spectral data and elemental analysis. The physicochemical factors of the

above-synthesized compounds were mentioned in table (1).

The 1H-NMR spectrum of compounds [H5(a-d)] showed signals at δ= (3.50) ppm due to (NH2) group .

A multiple peak was observed on ν scale between 7.85-6.85ppm which showed the presence of the

aromatic ring in compound 5(a-h). Observation of singlet peak on ν scale between 9.75 -9.70 ppm

confirmed the presence of C-SH group in the compounds. The singlet also appeared at d 8.75- 9.75

ppm attributed to one proton of N=CH. Thus, it confirmed the formation of Schiff bases. in addition to

the above analysis of elements as in table 2.

The infrared FT-IR of the synthesized compounds[H5(a-d)] show The spectrum of compounds

appearance of reaction of compound (H4) with carbonyl compound product Schiff bases hydrazones

(H5a-d) showed strong band in the region (1615-1645) cm-1

as due to CH = N stretching vibration, and

disappeared bonds at (3290 and 3210) cm-1

of a symmetric and symmetric N-H stretching. and this is

an excellent proof for the success of hydrazone compounds formation and appearance of the absorption

band of –SH (2600- 2400 cm-1

) .

Analysis of Elements(C.H.N.S)

The physical properties appearance of the synthesized , melting point, Yield% , Molecular formula ,

Recrystallization solution are listed in table (1). The CHNS measurements of compounds that prepared

are refer to correct suggested structure of this research compounds. The elements values are listed in

table (2 ).

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221

Table 2: Elemental analysis ( C.H.N.S) of compounds (H5a-d)

Comp. No.

C % H % N % S %

Cal. Found Cal. Found Cal. Found Cal. Found

H5a 61.71 58.360 4.31 3.810 22.91 23.16 10.57 10.310

H5b 62.68 61.383 4.51 3.364 22.88 23.04 10.32 9.95

H5c 59.27 58.854 4.79 3.975 21.83 22.03 9.90 10.10

H5d 48.84 47.966 3.22 3.922 18.83 19.036 8. 81 7.67

H5e 60.32 60.531 5, 47 5.76 24.54 24.659 9.79 8.580

Anti-microbial activity.

All the newly synthesized titled compounds H5(a-e) were tested for anti-microbial activities by using the

disc diffusion method, (27-29)

.The compound 5(a-h) were assessed against Gram-positive

(Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) and antifungal activity against

Candida albicans, Candida tropicalis, Streptomycin and Griseofulvin were used as a standard .

It was found that the compounds containing the bromo group, methyl , N, N-dimethyl and methoxy

substitution along with the Schiff bases systems. exhibited significant antimicrobial, antifungal

activity. Whereas, other derivatives containing other groups like methyl , methoxy and N, N-di methyl

group showed moderate to weak anti-microbial activity. It was found that of newly Schiff base

derivatives Compounds H5b, H5c , H 5(d) and H5(e), showed significant activity against Gram-

positive (Staphylococcus aureus whereas compounds H5b, H5d, and H5e showed significant

antifungal activity against Candida albicans and Candida tropicalis. While H5(d), H5(b) showed

moderate activity against bacterial strains. Anti-microbial data of all the synthesized compounds were

shown in table (3). The presence of bromine atom at para position in H5(d) has increased the

antimicrobial activity of the compound H5b. The similarly presence of methoxy group at para position

increased the antibacterial activity of H5(c), whereas, the remarkable antifungal activity of H5(e)

observed due to the presence of substituted amino group at para position. Similarly, the good anti-

bacterial activity of H5(d) was also reported due to the presence of Chloro group at para position

compound H5d. Antimicrobial activity depends on the nature of bacterial strain, the solvent and

chelating ability of the Schiff base. It is believed that Schiff bases act by forming a chelate with the

bacterial strain , Electron withdrawing ability of bromine yielded a compound with significant anti-

bacterial activity, as shown in the pictures in the below.

CONCLUSION

In conclusion, the synthesis of the Schiff base compounds Substituted thiocarbohydrate derivatives

(H5a-d) by using thiocarbohydrazide, p-amino benzoic acid and substituted aromatic aldehydes in

presence of ethanol. All the derivatives prepared by this method are analyzed by 1HNMR and IR. The

title compounds were evaluated for antibacterial activity against Gram-positive bacteria

(Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) While, anti-fungal activity

against (Candida albicans) and. (Candida tropicalis). As in table (3, 4). Results revealed that the

compound H5b showed very good antibacterial activity while, H5c and H5d, H5e, showed good

antibacterial activity. On the other hand antifungal activity against (Candida albicans) and. (Candida

tropicalis). Results revealed that the compounds H5c , H5d, H5e showed very good antifungal activity

.The study would be a fruitful matrix for the development of Substituted thiocarbohydrate derivatives

for further bio-evaluation. From the results of various biological activities it is clear that these

compounds would be of better use in drug development.

Table 3. Antifungal , Antibacterial activity of synthesis N, N-dithiocarbohydrazone derivatives

compounds Conc.

in µg/mL

Zone of inhibition in (mm) Zone of inhibition in (mm)

Staphylococcus

aureus

Escherichia

colis

Candida

tropicalis

Candida

tropicalis

H5a

A 0.010 22 16

A 0.050 22 14 16 71

A 0.0150 19 14 14 71

H5b

A6 0.010 34 13 18 71

A60.050 30 11 20 13

A60.0150 32 11 16 71

H5c

A9 0.010 21 14 21 71

A90.050 20 13 16 02

A9 0.0150 20 14 17 14

H5d

B7 0.010 25 16 19 18

B70.050 19 17 16 15

B70.0150 20 18 17 18

H5e

D30.010 20 16 19 19

H3 0.050 13 13 16 15

K5 0.0150 20 14 18 16

Streptomycin std. - 02 02 15 18

Griseofulvin std. - 01 01

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الفعالية التثبيطية للمركب (H4a) Staphylococcus aurea

الفعالية التثبيطية للمركب(H5b ) Staphylococcus aurea

الفعالية التثبيطية للمركب(H5c ) Staphylococcus aurea

الفعالية التثبيطية للمركب(H5d ) Staphylococcus aurea

لفعالية التثبيطية للمركب (H5a ) Esheriechia coli

لفعالية التثبيطية للمركب (H5b ) Esheriechia coli

لفعالية التثبيطية للمركب (H5c ) Esheriechia coli

لفعالية التثبيطية للمركب(H5d ) Esheriechia coli

لفعالية التثبيطية للمركب

(H5e ) Esheriechia coli

( H5a) الفعالية التثبيطية للمركباتCandida albicans

( H5b) الفعالية التثبيطية للمركباتCandida albicans

( H5c) الفعالية التثبيطية للمركباتCandida albicans

( H5d) الفعالية التثبيطية للمركباتCandida albicans s

( H5e) الفعالية التثبيطية للمركباتCandida albicans

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