Synthesis, characterization and biological evaluation of acetazolamide, cycloserine and isoniazid condensed some novel phthalimide derivatives

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Original Article

Synthesis, characterization and biologicalevaluation of acetazolamide, cycloserine andisoniazid condensed some novel phthalimidederivatives

Karthikeyan Elumalai a,c,*, Mohammed Ashraf Ali a,Manogaran Elumalai b, Kalpana Eluri b, Sivaneswari Srinivasan c,Srinivasan Sivannan d, Sujit Kumar Mohanthi c

aNew Drug Discovery Research, Department of Medicinal Chemistry, Sunrise University, Alwar 301030, Rajasthan,

Indiab Faculty of Pharmaceutical Sciences, UCSI University, Cheras 56000, MalaysiacDepartment of Pharmaceutical Chemistry, Jayamukhi Institute of Pharmaceutical Sciences, Warangal 506 332,

IndiadDepartment of Pharmacy Practice, Dayanantha Sagar College of Pharmacy, Bangalore 560 078, India

a r t i c l e i n f o

Article history:

Received 23 February 2013

Accepted 5 April 2013

Available online 12 June 2013

Keywords:

Phthalimide derivatives

Antimicrobial

Antimycobacterial

Mycobacterium strains

* Corresponding author. New Drug DiscovRajasthan, India. Tel.: þ91 9573396024 (mob

E-mail addresses: karthikeyanelumalai@h0976-1209/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijcas.2013.04.004

a b s t r a c t

Background/aim: In the present study, a series of novel phthalimide derivatives synthesized

because of its potent antimicrobial and antimycobacterial activity.

Method: Structurally modified phthalimide derivatives were prepared through condensa-

tion of substituted phthalic anhydride with corresponding acetazolamide, cycloserine and

isoniazid with variable yields. Synthesized Compounds (6aed), (7aed) and (8aed) analyzed

for their structures, in vitro antimicrobial and antimycobacterial activity.

Result: Among the synthesized compounds, compound 6b, 7b and 8b was found to be the

most potent against Gram-positive bacteria Bacillus subtilis, Gram-negative bacteria

Escherichia coli, Mycobacterium tuberculosis CIP and M. tuberculosis H37Rv.

Conclusion: A series of novel phthalimide derivatives of biological interest were synthesized

and analyzed, suggests that it an interesting compound compared to the current thera-

peutic agents and are considered to investigate further for the same.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction modification is a chemical alteration in a molecule which

Microorganisms are becoming resistance to antibiotics which

resulting in the development of newer compounds. Molecular

ery Research, Departmeile).otmail.com, ekarthikeya2013, JPR Solutions; Publi

could be lead compound or a drug aiming to enhance its

pharmaceutical, pharmacokinetic or pharmacodynamics.

Among the molecular modification used prodrug approach,

nt of Medicinal Chemistry, Sunrise University, Alwar 301030,

[email protected] (K. Elumalai).shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

mailto:[email protected]

mailto:[email protected]

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i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 5 7e6 158

molecular hybridization and bioisosterism are common

methods. Based on literature, molecular hybridization will

leads to potent activity of new molecule. Phthalimide subunit

is important drug candidates with varying biological activities

such as histone deacetylase inhibitor, Tubulin polymerization

inhibitor, human protein kinase CK2 inhibitor, Liver X recep-

tor antagonist and Leukotriene D4 Receptor Antagonist. And

become important as anti inflammatory, analgesics, anti-

convulsant, anti cancer, antimicrobial and antimycobacterial

agents.1e12

Acetazolamide, is a carbonic anhydrase inhibitor that is

used to treat glaucoma, epileptic seizures, Idiopathic intra-

cranial hypertension, cystinuria, periodic paralysis, central

sleep apnea and dural ectasia. Cycloserine is an antibiotic

effective against Mycobacterium tuberculosis, which is consid-

ered as a second line drug for the treatment of tuberculosis.

Isoniazid, also known as isonicotinylhydrazine (INH), is an

organic compound used in first-line medication of prevention

and treatment of tuberculosis. In the present work, attempt

has been taken to synthesize structurally modified phthali-

midederivatives throughcondensationof substitutedphthalic

anhydrides with corresponding acetazolamide, cycloserine

and isoniazid and to evaluate the synthesized compounds for

its antimicrobial and antimycobacterial activities.

2. Materials and methods

The entire chemicalswere supplied by E.Merck (Germany) and

S. D fine Chemicals (India). Melting points were determined by

open tube capillary method and are uncorrected. Purity of the

compounds was checked on thin layer chromatography (TLC)

plates (silica gel G) in the solvent system chloroform and ben-

zeneasamobile phase (70:30), the spotswere locatedunderUV

light. IR spectrums were obtained on a PerkineElmer 1720 FT-

IR spectrometer (KBr Pellets). 1H NMR spectra were recorded

or a Bruker AC 300 MHz spectrometer using TMS as internal

standard in DMSO/CDCl3. Mass spectra were obtained using

Shimadzu LCMS 2010A under ESI ionization technique.

2.1. Synthesis of acetazolamide, isoniazid andcycloserine condensed phthalimide derivatives

Compounds were prepared via a condensation reaction10,11

between commercially available phthalic anhydride, 4,5,6,7-

tetrafluorophthalic anhydride, 4-phenyl ethynyl phthalic an-

hydride and 4,5,6,7-tetrabromophthalic anhydride (10 mmol)

with corresponding acetazolamide, isoniazid and cycloserine

(10 mmol) under reflux for 3.5e5 h in presence of 15 ml of

acetic acid. Then 25mL of ice cold distilledwater was added to

the reaction medium and the compounds filtered and

recrystallized in ethanol. Compound physical properties are

described in analytical data.

2.2. Analytical data

2.2.1. N-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) pyridine-4-carboxamide (6a)Colorless solid, M.P: 224e226 �C, Reaction time e 4 h, Yield e

52%, IR (KBr, cm�1): 3282 (NeH, st), 3076, (CeH, aromatic, st),

1758 (C]NH, imide, st), 1716 (C]O amide), 1722 (C]O, ke-

tone), 1530 (C]C), 1362 (CeN). 1H NMR (DMSO-d6): 6.19e6.51

(m, 4H, ArH), 7.20e7.59 (m, 4H, ArH), 9.93 (s, 1H, NH). MS (m/z):

Mþ calculated 267.06, found, 267.01.

2.2.2. N-(4,5,6,7-tetrafluoro-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) pyridine-4-carboxamide (6b)Colorless solid, M.P: 253e255 �C, Reaction time e 4h, Yield e



tone), 1548 (C]C), 1383 (CeN), 667 (CeF). 1H NMR (DMSO-d6):

6.24e6.68 (m, 4H, ArH), 7.32e7.76 (m, 4H, ArH), 9.86 (s, 1H, NH).

MS (m/z): Mþ calculated 339.02, found, 338.97.

2.2.3. N-[1,3-dioxo-5-(phenylethynyl)-1, 3-dihydro-2H-isoindol-2-yl] pyridine-4-carboxamide (6c)Colorless solid, M.P: 276e279 �C, Reaction time e 4 h, Yield e




(m, 4H, ArH), 7.26e7.40 (m, 4H, ArH), 7.42e7.68 (m, 4H, ArH),

9.83 (s, 1H, NH). MS (m/z): Mþ calculated 367.09, found, 367.03.

2.2.4. N-(4,5,6,7-tetrabromo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) pyridine-4-carboxamide (6d)Colorless solid, M.P: 271e273 �C, Reaction time e 4 h, Yield e



tone), 1542 (C]C), 1370 (CeN), 556(CeBr). 1H NMR (DMSO-d6):

6.14e6.43 (m, 4H, ArH), 7.28e7.57 (m, 4H, ArH), 9.78 (s, 1H, NH).

MS (m/z): Mþ calculated 578.70, found, 578.65.

2.2.5. N-{5-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)sulphonyl]-1,3,4-thiadiazol-2-yl} acetamide (7a)Colorless solid, M.P: 212e214 �C, Reaction time e 5 h, Yield e


2846 (CeH, aliphatic, st) 1738 (C]NH, imide, st), 1717 (C]O

amide), 1728 (C]O, ketone), 1528 (C]C), 1356 (CeN), 1362 (SO2,

st). 1H NMR (DMSO-d6): 2.04 (s, 3H, CH3), 6.38e6.73 (m, 4H,

ArH), 7.43e7.81 (m, 4H, ArH), 9.72 (s, 1H, NH). MS (m/z): Mþ

calculated 351.99, found, 351.94.

2.2.6. N-{5-[(4,5,6,7-tetrafluoro-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)sulfonyl]-1,3,4-thiadiazol-2-yl} acetamide (7b)Colorless solid, M.P: 271e273 �C, Reaction time e 5 h, Yield e




st). 1H NMR (DMSO-d6): 2.04 (s, 3H, CH3), 6.38e6.73 (m, 4H,

ArH), 7.43e7.81 (m, 4H, ArH), 9.72 (s, 1H, NH). MS (m/z): Mþ

calculated 423.95, found, 423.91.

2.2.7. N-(5-{[1,3-dioxo-5-(phenylethynyl)-1,3-dihydro-2H-isoindol-2-yl] sulphonyl}-1,3,4-thiadiazol-2-yl) acetamide (7c)Colorless solid, M.P: 248e251 �C, Reaction time e 5 h, Yield e








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2.2.8. N-{5-[(4,5,6,7-tetrabromo-1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) sulphonyl]-1,3,4-thiadiazol-2-yl} acetamide (7d)Colorless solid, M.P: 256e258 �C, Reaction time e 5 h, Yield e




st), 586 (CeBr). 1H NMR (DMSO-d6): 2.03 (s, 3H, CH3), 6.36e6.71

(m, 4H, ArH), 7.45e7.83 (m, 4H, ArH), 9.88 (s, 1H, NH). MS (m/z):

Mþ calculated 663.63, found, 663.51.

2.2.9. 2-(3-oxo-1,2-oxazolidin-4-yl)-1H-isoindole-1,3(2H)-dione (8a)Colorless solid, M.P: 186e189 �C, Reaction time e 3.5 h, Yield e



amide), 1732 (C]O, ketone), 1546 (C]C), 1368 (CeN). 1H NMR

(DMSO-d6): 6.41e6.75 (m, 4H, ArH), 7.48e7.84 (m, 4H, ArH), 9.91

(s, 1H, NH). MS (m/z): Mþ calculated 232.04, found, 231.96.

2.2.10. 4,5,6,7-tetrafluoro-2-(3-oxo-1,2-oxazolidin-4-yl)-1H-isoindole-1, 3(2H)-dione (8b)Colorless solid, M.P: 194e196 �C, Reaction time e 3.5 h, Yield e



amide), 1734 (C]O, ketone), 1528 (C]C), 1352 (CeN). 1H NMR

(DMSO-d6): 6.32e6.68 (m, 4H, ArH), 7.34e7.72 (m, 4H, ArH), 9.76

(s, 1H, NH). MS (m/z): Mþ calculated 304.01, found, 304.06.

2.2.11. 2-(3-oxo-1,2-oxazolidin-4-yl)-5-(phenylethynyl)-1H-isoindole-1,3(2H)-dione (8c)Colorless solid, M.P: 212e214 �C, Reaction time e 3.5 h, Yield e




Scheme 1 e Reagents and conditions: (a) CH3COOH Reflux 4



2.2.12. 4,5,6,7-tetrabromo-2-(3-oxo-1,2-oxazolidin-4-yl)-1H-isoindole-1,3(2H)-dione (8d)Colorless solid, M.P: 185e188 �C, Reaction time e 3.5 h, Yield e



amide), 1752 (C]O, ketone), 1542 (C]C), 1358 (CeN), 592

(CeBr). 1H NMR (DMSO-d6): 6.34e6.62 (m, 4H, ArH), 7.36e7.76

(m, 4H, ArH), 9.84 (s, 1H, NH). MS (m/z): Mþ calculated 543.68,

found, 543.73.

2.3. In vitro antimicrobial activity

The in vitro antibacterial activities were tested against Gram-

positive bacteria Bacillus subtilis and Gram-negative bacteria

Escherichia coli by standard serial dilutionmethod using a stock

solution of 100 mg/ml concentrations.13,14 Double strength

nutrient broth was used as culture media and dimethyl

sulphoxide (DMSO) was used as solvent control. The stock

solutions of the test compounds were serially diluted in test

tubes containing 1 ml of sterile medium to get the different

concentrations and then inoculated with 100 mL of suspension

of respective microorganism in sterile saline. Norfloxacin was

used as standard drug. The inoculated test tubes were incu-

bated at 37 � 1 �C for 24 h.

2.4. In vitro antimycobacterial activity

Antimycobacterial activity was performed following a proto-

col previously reported.15 Synthesized compounds were pre-

liminarily assayed against to freshly isolate clinical strains,

Mycobacterium fortuitum CA10 and M. tuberculosis B814,

h; (b) CH3COOH Reflux 5h and (c) CH3COOH Reflux 3.5h.



Table 1 e Synthesized phthalimide derivatives: In vitro antimicrobial and antimycobacterial activity.

S. no Compound X1 X2 X3 X4 B. subtilis MIC(mmol/mL)

E. coli MIC(mmol/mL)

M. tuberculosis

CIP MIC(mg/ml)

H37Rv MIC(mg/ml)

1 6a H H H H 0.0288 0.0636 2.65 2.60

2 6b F F F F 0.0109 0.0538 1.17 1.15

3 6c H Phenyl ethynyl H H 0.0198 0.0584 1.56 1.52

4 6d Br Br Br Br 0.0118 0.0664 1.25 1.26

5 7a H H H H 0.0296 0.0648 2.58 2.62

6 7b F F F F 0.0110 0.0526 1.19 1.17


8 7d Br Br Br Br 0.0119 0.0584 1.26 1.28

9 8a H H H H 0.0316 0.0642 3.21 3.17

10 8b F F F F 0.0115 0.0528 1.21 1.19


12 8d Br Br Br Br 0.0119 0.0598 1.26 1.27

13 Norfloxacin 0.0112 0.012 e e

14 Pyrazinamide e e 1.24 1.22

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according to the dilution method in agar. Growth media were

MuellereHilton (Difco) containing 10% of OADC (oleic acid,

albumin and dextrose complex) for M. fortuitum and Middle

brook 7H11 agar (Difco) with 10% of OADC for M. tuberculosis.

Substances were tested at single dose of 100 mg/mL. The active

compoundswere then assayed for inhibitory activity against a

panel of mycobacterial (M. tuberculosis CIP, M. tuberculosis

H37Rv) in Middle brook 7H11 agar by a standard twofold

dilution method. Plates were incubated at 37 �C for 3 or 28

days. Pyrazinamide was used as reference compound. After

cultivation, MICs were read as minimal concentrations of

drugs completely inhibiting visible of mycobacterial growth.

3. Results and discussion

A series of twelve novel phthalimide derivatives were syn-

thesized and characterized by IR, 1H NMR, MS for their

structures. All the compounds were evaluated for antimicro-

bial and antimycobacterial activity.

3.1. Chemistry

Synthesis of phthalimide derivatives by adopting condensa-

tion reaction was performed by following step as outlined in

Scheme 1. In the synthesis phthalic anhydrides (1) reacted

with, isoniazid (2), acetazolamide (3) and cycloserine (4) indi-

vidually in presence of glacial acetic acid under neat condi-

tions resulting in the formation of the product. The reaction

times were found to be between 3.5 and 5 h. Totally, twelve

compounds (6aed; 7aed and 8aed), various substituted

phthalimide derivatives, were synthesized with the yield

ranging from 39 to 61 percent. The present protocol best de-

scribes the synthesis of phthalimide derivative compounds,

were found to be novel and not reported elsewhere.

3.2. Antimicrobial and antimycobacterial activity

Analyzing the activities of the synthesized compounds, the

following structure activity relationships (SARs) were

obtained. The N1 position of phthalimide derivatives

contain pyridine-4-carboxamide (6aed); sulphonyl (1,3,4-

thiadiazol-2-yl) acetamide (7aed) and 3-oxo-1,2-oxazolidin-

4-yl (8aed) groups contributed toward activity against

Gram-positive B. subtilis, Gram-negative E. coli, M. tubercu-

losis CIP and H37RV strain. The third, fourth, fifth and sixth

positions of phthalimide ring contain fluoride atom (6b, 7b

and 8b) showed more potent antimicrobial and anti-

mycobacterial action when compare with unsubstituted,

bromo and 4-phenyl ethynyl substituted ring because it

decreases the electron density in the phthalimide ring due

to inductive effect. Phenyl ethynyl substituted at C5th of

phthalimide derivatives (6c, 7c and 8c) showed moderate

antimicrobial antimycobacterial action when compare with

unsubstituted phthalimide ring due to stearic hindrance

and liphophilicity of the compound. Bromide substituted

phthalimide derivatives (6d, 7d, 8d) showed moderate ac-

tion when compare with unsubstituted phthalimide de-

rivatives due to strong electron withdrawing tendency.

Among the compounds reported here in, compound (6b, 7b

and 8b) is arguably the most potent because it contain fluro

atom at 3, 4, 5 and 6th position of phthalimide derivatives it

enhance the antimycobacterial activity. The results were

shown in Table 1.

4. Conclusion

The phthalimide derivatives were prepared by using glacial

acetic acid as an efficient extended polar solvent. The

importance of substitutions at N1, third, fourth, fifth and sixth

positions of phthalimide derivatives were studied toward the

antimicrobial and antimycobacterial activity. The synthesized

phthalimide derivatives proved to be active against the test

organisms B. subtilis, E. coli, M. tuberculosis CIP and H37RV

strain. Almost all of the titled compounds exhibited weak,

moderate, or high antimicrobial and antimycobacterial ac-

tivity. Our present study makes it an interesting compound

when compared to the current therapeutic agents and are

considered the candidates to investigate further for the same.



i n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 5 7e6 1 61

Conflicts of interest

All authors have none to declare.

Acknowledgments

The authors wish to thank Sunrise University, Alwar for

research support and Tuberculosis Research Center, Chennai,

India. The authors also thank JPR Solutions for partial funding

for the publication.

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Synthesis, characterization and biological evaluation of acetazolamide, cycloserine and isoniazid condensed some novel phthalimide derivatives