DESIGN AND SYNTHESIS OF NOVEL 1-((DIMETHYLAMINO)METHYL)-3-(4-(3-(SUBSTITUTE DPHENYL)-4-OXOTHIAZOLIDIN-2-YL)PHENYLIMINO)-5-NITROINDOLIN-2-ONES AS POTENT ANTITUBERCULAR AGENTS

Objective: Isatins have emerged as antimicrobial agents due to their broad spectrum of in vitro and in vivo antimicrobial activities. In addition, thiazolidinone also reported to possess various biological activities particularly antimicrobial activity. Due to the importance, we planned to synthesize compounds with isatin functionality coupled with thiazolidinone as possible antitubercular and antimicrobial agents which could furnish better therapeutic results. Methods: In vitro Mycobacterium tuberculosis method and agar streak dilution test are used to estimate antitubercular and antimicrobial potency of title analogs, respectively. Minimum inhibitory concentration of entire title compounds was determined against all tested microorganism such as M. tuberculosis, four Gram-positive, three Gram-negative bacteria, and two fungi. Results: A series of new thiazolidinone substituted Schiff and Mannich bases of 5-nitroisatins were designed and synthesized by a multistep synthesis from isatin. Structures of synthesized compounds are characterized using Fourier-transform infrared, proton nuclear magnetic resonance, mass spectroscopy, and bases of elemental analysis. Mild to good antitubercular and antimicrobial activity was showed by synthesized 5-nitroisatin analogs. The relationship between the biological activity and the functional group variation of the tested compounds was discussed. Conclusion: 3-(4-(3-(4-Aminophenyl)-4-oxothiazolidin-2-yl)phenylimino)-1-((dimethyl amino)methyl)-5-nitroindolin-2-one 6 and 3-(4-(3(2-aminophenyl)-4-oxothiazolidin-2-yl)phenylimino)-1-((dimethylamino)methyl)-5-nitroindolin-2-one 13 were found to be the most potent compounds of this series which might be extended as a novel class of antimicrobial agents.


INTRODUCTION
Bacteria Mycobacterium tuberculosis is the causative organism for tuberculosis (TB). In general, a lung is a major part affected by TB, but other parts can also be affected by TB. Globally within diseases caused by single infectious agent TB is the second largest killer. According to the WHO, every year about 9 million people are affected by TB. Among them, 3 million people are missed by current health systems. TB is one of the top three reasons for the death of women aged between 15 and 44. TB is an airborne disease and is two types, i.e., latent TB and active TB [1]. Chronic cough with sputum containing blood, weight loss, fever, and night sweats is the characteristic symptoms of active TB. It is believed that about one-third of the world's population has latent TB. Latent TB becoming active only in 10% cases, but the risk is much higher in people who have compromised immune systems, i.e., people living with HIV or malnutrition, or people who smoke [2].
Serious life-threatening adverse effect of available antitubercular drugs (anorexia, asthenia, depression, neuritis, hepatotoxicity, etc.) and development of resistance are the two common problems associated with TB treatment [3,4]. In addition, around the world, the emergence and increase of multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis have been alarming authorities. High mortality rates and low cure rates are the two major difficulties faced in the treatment due to these strains [5,6]. Furthermore, in the clinic, drug-resistant TB cases are also reported [7,8]. Hence, a priority task is the development of new drugs with activity against MDR-TB, XDR-TB, and XDR-TB. Moreover, new agents that will shorten the duration of current chemotherapy are also needed. To tackle the menace, there is a need to search for newer therapeutic agents [9].

Materials
The chemicals and reagents used were obtained from various chemical units Qualigens, E. Merck India Ltd., CDH, and SD Fine Chem. These solvents used were of LR grade and purified before their use. The silica

Antitubercular activity
Antitubercular potency of title compounds was estimated by agar dilution method (In vitro M. tuberculosis method) [34,35]. Using OADC growth supplement in Middlebrook 7H11 Agar, slants each test analogs were incorporated in 10-fold serial dilutions. M. tuberculosis H 37 RV inoculums were prepared using OADC growth supplement in fresh Middlebrook 7H11 Agar slants adjusted to 1 mg/ml in 0.05% tween 80 saline diluted to 10 −2 (10 7 CFU/ml concentration approximately). In 7H11 agar tubes per ml 10-fold serial dilutions of test analogs, 5 µl bacterial suspensions were added. At 37°C incubated the tubes and after 28 days, final readings were measured. Results obtained on test tubes (Test analog, medium and H 37 RV) were compared with control tubes (Medium and H 37 RV). The concentration at which complete inhibition of M. tuberculosis growth occurs is known as minimum inhibitory concentration (MIC). INH (Isoniazid) was used as standard drug for comparing MIC of the title analogs and the obtained results are depicted in Table1.

Antimicrobial activity
Agar streak dilution method was used to study antimicrobial potency of test analogs. Against four Gram-positive bacteria (Bacillus cereus ATCC 11778, Micrococcus luteus ATCC 4698, Staphylococcus epidermidis ATCC 155, and Staphylococcus aureus ATCC 9144) and three Gram-negative bacteria (Klebsiella pneumoniae ATCC 11298, Pseudomonas aeruginosa ATCC 2853, and Escherichia coli ATCC 25922) synthesized compounds were screened for antibacterial activity. In addition against two fungi (Aspergillus fumigatus ATCC 46645 and Aspergillus niger ATCC 9029) synthesized analogs were tested for antifungal potency. For antibacterial activity, at 37°C various bacterial strains were cultured in Mueller-Hinton broth overnight. Likewise, for antifungal activity, at 30°C, various fungal strains were cultured in YEPDE agar overnight. The final densities of test strains were made to 5 × 10 −5 CFU/ml using nutrient agar medium.

MIC
Agar streak dilution method was used to find out the MIC of test analogs [36]. DMF was used to prepare a stock solution of test analogs. In specified quantity of molten sterile agar (nutrient agar for antibacterial activity and Sabouraud's Dextrose Agar medium for antifungal activity) graded quantities of the synthesized analogs were incorporated. In Petri dish at 40-50°C, a specified quantity of test analogs incorporated medium was poured and allowed to solidify to give a 3-4 mm depth. Latter to the plates containing test analogs (in DMF) and medium, microbial suspension (5×10 −5 CFU/ml) were applied. Finally, Petri plates were incubated at 37°C for 24 h and 48 h for bacteria and fungi, respectively. The lowest concentration at which no visible growth of bacteria or fungi is considered as MIC and the found MIC of test analogs and the standard drug is shown in Table 1.
IR, 1 H-NMR, mass spectra, and elemental analyses data are used to confirm the structures of synthesized novel 5-nitroisatin analogs 5-19. All spectral data are in accordance with the assigned structures. The presence of specific groups in compounds is represented in IR spectra by some characteristic absorption peaks. In IR spectrum appearance of a peak at 1546 and 1328 cm −1 corresponding to aromatic nitro group confirms the formation of 5-nitroisatin 2. Likewise, in IR spectrum appearance of stretching vibration for C=N at 1669 cm −1 and in NMR spectrum a singlet peak for aldehyde proton at δ 10.13 ppm confirms the assigned structure of 4-(5-nitro-2-oxoindolin-3-ylideneamino) benzaldehyde 3. The compound 4 formed through Mannich reaction was confirmed by the absence of absorption bands around 3350 cm −1 corresponds to NH stretching and appearance of a peak at 2944 cm −1 corresponds to CH 3 -CH stretching in its IR spectrum. It is further supported by 1 H-NMR spectrum in which singlet appeared at δ 4.32 ppm for two protons of CH 2 linkage and another singlet appeared for six protons of dimethylamino group at δ 2.17 ppm. Title compounds show absorption bands in IR spectrum at 3001-3029 cm −1 , 2924-2959 cm −1 , 1713-1738 cm −1 , 1650-1678 cm −1 , and 1603-1629 cm −1 , which can be assignable to Ar-H, CH 3 -CH, C=O, C=N, and C=C vibrations, respectively. In addition, nitro group present in title compounds is confirmed by the appearance of two characteristic peaks in IR at 1524-1558 cm −1 , and 1312-1354 cm −1 . The below-mentioned conclusions were made after observing the 1 H-NMR spectra of novel synthesized compounds 5-19. The presence of dimethylamino moiety was evidenced by the appearance of singlet for six protons at δ 2.16-2.43 ppm. A singlet at δ 3.30-3.69 ppm for two protons is due to CH 2 of thiazolidinone. The appearance of singlet for two protons at δ 4.04-4.38 ppm confirms the presence of CH 2 linkage. CH of thiazolidinone was evidenced by appearance of one proton singlet at δ 5.80-6.28 ppm. A group of signals (multiplet) appeared between δ 6.80 and 8.31 ppm corresponds to Ar-H protons. Further mass spectrum confirmed their purity and molecular weight.

Antitubercular activity
In vitro antitubercular activity of all title analogs was screened against M. tuberculosis (H 37 Rv strain) and MIC of entire tested analogs was determined and presented in Table 1 and Fig. 1. Simultaneously, MIC of INH was also measured to control the sensitivity of the test organisms. From the results, it was found that in varying degree synthesized compounds inhibited the growth of M. tuberculosis.

Lahari and Sundararajan
Among various tested compounds, analogs such as 6 and 13 inhibited the growth of M. tuberculosis at a low concentration (MIC: 3.9 µg/ml). Both derivatives possess amino moiety at phenyl ring attached to thiazolidinone ring. MIC of test compounds 7, 8, and 14 was found to be 7.81 µg/ml may be due to the presence of hydroxy and methoxy substituent in phenyl ring. In addition, derivative 15 containing methoxy moiety completely inhibited the growth of M. tuberculosis at 15.62 µg/ml concentration. The MIC of analogs 5, 9, and 16 was found to be 31.25 µg/ml and test compounds 11, 12, and 18 were found to be 62.5 µg/ml. Only at higher concentration remaining synthesized derivatives (compounds 10, 17, and 19) displayed activity (MIC: ≥125 µg/ml).

Antimicrobial activity
In vitro, agar streak dilution method was employed to screen the antimicrobial activity of test derivatives 5-19. Simultaneously, MIC of ciprofloxacin and ketoconazole was also measured to control the sensitivity of the test organisms. The MIC of standard and test analogs was compared effectively in Table 1 and Figs. 2-4. Antimicrobial data indicate that test analog 6 (MIC: 1.95 µg/ml) exhibited superior activity than ciprofloxacin against B. cereus; whereas test analogs 7 and 13 (MIC: 3.9 µg/ml) exhibited similar activity and remaining analogs displayed inferior activity (MIC: ≥7.81 µg/ml). Compared to ciprofloxacin against M. luteus derivative 6 showed higher
Against A. fumigatus and A. niger synthesized compounds were screened for its antifungal activity and found to display a varying degree of antifungal potency. None of the tested analogs displayed superior or equal activity compared to Ketoconazole against both tested fungi (A. fumigatus and A. niger). Out of various tested analogs, compounds 6 and 13 were found to be the most potent compounds of this series. From the study, in general, it was found that compounds 6-8 and 13-15 displayed good antimicrobial activity; compounds 5, 9, and 16 exhibited moderate antimicrobial activity; whereas rest of title compounds (10-12 and 17-19) displayed poor antimicrobial activity.

CONCLUSION
In summing up, from isatin using multistep synthesis various novel Schiff and Mannich bases of 5-nitroisatin was synthesized by substituting different 3-(substitutedphenyl)-2-phenylimino thiazolidinone moiety at C-3 and (dimethylamino)methyl moiety at C-1 of 5-nitroisatin. FT-IR, 1 H-NMR, Mass spectroscopy, and elemental analysis are used to     In vitro antitubercular, antibacterial, and antifungal activities of all novel analogs were estimated by measuring its MIC. This series of analogs displayed a varying degree of antimicrobial activity (mild to good). SAR studies revealed that in determining the antimicrobial activity of novel 5-nitroisatins nature of substituent played a major role than the position of the substituent. Isatin derivatives possessing electron releasing group exhibited superior antimicrobial potency than corresponding isatin derivatives possessing electron withdrawing moieties while unsubstituted analogs displayed intermediate activity. Out of several 5-nitroisatins tested in this series, the potent antibacterial compounds were found to be 3-(4-(3-(4-aminophenyl)-4-oxothiazolidin-2-yl)phenylimino)-1-((dimethylamino)methyl)-5nitroindolin-2-one 6 and 3-(4-(3-(2-aminophenyl)-4-oxothiazolidin-2-yl)phenylimino)-1-((dimethylamino)methyl)-5-nitroindolin-2-one 13. These derivatives displayed potent antibacterial activity which is greater than or almost equal to the tested reference drug. In addition, derivatives 6 and 13 also exhibited some notable antifungal activity against tested fungi. Moreover, these analogs also showed good antitubercular activity. For this reason, these compounds might be extended as a novel class of antimicrobial agents. On the other hand, to enhance the anti-tubercular and antibacterial activity further structural modification is planned.