The reaction was performed in round bottom flask and subsequently added the both the benzophenones and hydrazine hydrate in ethanol and water refluxed. After the completion of the reaction (monitored by TLC) reaction mixture was cooled at room temperature, then water was added and solid was formed which was filtered and dried on vacuo to give hydrazones as powdered solid. The general reaction scheme shown in Scheme 2, We tested various substituted benzophenones and all the reaction went nicely to give excellent yields. We can see in scheme 2, the electron donating group like methoxy and methyl 3b, 3c tolerated well and gave very excellent yields. Later on, the electron withdrawing groups like Cl, F tested and gave good yields 3d and 3e. Further, symmetrical and unsymmetrical benzophenones also worked and yielded in good conversion. Halogen compound like Br tolerated and resulted the hydrazones products in good yields. All the synthesized derivatives were confirmed by NMR analysis and further for mass analysis. We have shown here library of ten compounds but this protocol can be extended for series of other derivatives and ample opportunities for modifications and biological study. All the compound well characterized and can be seen in experimental section.

Biological Study:

These all the synthesized aryl hydrazone derivatives were screened for biological activity, since its novel compounds and based on literature observations it can be very potential drug candidates for drug discovery programme. The hydrazone were tested for inhibition of Mycobacterium tuberculosis in invitro MABA assay. The results are summarized in table 1.

In-vitroMycobacterium tuberculosis MABA assay

Entry	Compound	MIC (µM)
1	3a	100
2	3b	12.5
3	3c	15.6
4	3d	50.2
5	3e	66.2
6	3f	76.2
7	3g	32.4
8	3h	22.3
9	3i	6.25
10	3j	8.18

A growth control containing no antibiotic and a sterile control were also prepared on each plate. Sterile water was added to all perimeter wells to avoid evaporation during the incubation. The plate was covered, sealed in plastic bags and incubated at 37 °C in normal atmosphere. After 7 days incubation, 30 µl of alamar blue solution was added to each well, and the plate was re-incubated overnight. A change in colour from blue (oxidised state) to pink (reduced) indicated the growth of bacteria, and the MIC was defined as the lowest concentration of compound that prevented this change in colour.

The biological screening shows the compounds 3b, 3i are shown potent activity against TB cell lines, its great starting point for further exploration of the making more diverse analogues. Other derivatives shown moderate activity against TB cell lines.

CONCLUSIONS:

In summary, the ecofrieldly protocol was developed and applied for the synthesis of hydrazones derivatives at mild temperature. Lots of substrate scope has been shown and it can be further extended. Hydrazone derivatives showed significant inhibition of Mycobacterium tuberculosis. These analogues are chemically tractable and hence provides ample opportunities for further modification to obtain potent antituberculosis agents. The isolated yield of the hydrazones derivatives is excellent, so gram scale synthesis possible. This is starting point for further development.

EXPERIMENTAL SECTION:

Unless otherwise stated, all commercial reagents and solvents were used without additional purification. Analytical thin layer chromatography (TLC) was performed on pre-coated silica gel 60 F₂₅₄ plates. Visualization on TLC was achieved by the use of UV light (254 nm). Column chromatography was undertaken on silica gel (100‒200 mesh) using a proper eluent system. NMR spectra were recorded in chloroform-d and DMSO-d₆at 300 or 400 or 500 MHz for ¹H NMR spectra and 75 MHz or 100 or 125 MHz for ¹³C NMR spectra. Chemical shifts were quoted in parts per million (ppm) referenced to the appropriate solvent peak or 0.0 ppm for tetramethylsilane. The following abbreviations were used to describe peak splitting patterns when appropriate: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, sept = septet, dd = doublet of doublet, td = triplet of doublet, m = multiplet. Coupling constants, J, were reported in hertz unit (Hz). For ¹³C NMR chemical shifts were reported in ppm referenced to the center of a triplet at 77.0 ppm of chloroform-d and 40.0 ppm center for DMSO-d_6.

General procedure for preparation of compounds:

The common procedure for preparation of aryl hydrazones, In round bottom flask charged with benzophenones ( 10 mmol), hydrazine hydrate (10 mmol) then ethanol and water ( 1:1, 20 ml) was added, resulting solution stirred refluxed for 5 h. After completion of the reaction (monitored by TLC) cooled to room temperature, then water was added (20 mL), solid was formed. The reaction mixture then filtered through Buchner funnel, solid was collected and dried on vacuum to give finally dried solid powder. ( All the compounds confirmed by NMR, Mass analysis).

Analytical Data of Synthesized Compounds:

1) (9H-fluoren-9-ylidene)hydrazine (3a):

White solid, Mp 112- 114 °C, yield (90%) , Mass : Cal. 194.2, Observe. 194.1

¹H NMR(400 MHz, CDCl₃) δ 7.87 (d, J = 7.6 Hz, 1H), 7.72 (ddd, J = 10.7, 7.4, 6.5 Hz, 2H), 7.64 (dd, J = 11.8, 5.3 Hz, 1H), 7.47 – 7.37 (m, 1H), 7.37 – 7.25 (m, 3H), 6.52 – 6.27 (m, 2H).

¹³C NMR (100 MHz, CDCl₃) δ 145.56, 141.33, 138.57, 137.76, 130.25, 129.72, 128.52, 127.94, 127.73, 125.51, 120.77, 120.53, 119.56, 77.39, 77.07, 76.76.

2) (bis(4-methoxyphenyl)methylene)hydrazine(3b):

White solid, Mp 120- 122 °C, yield (85%) , Mass : Cal. 256.1, Observe. 256.1

¹H NMR(400 MHz, CDCl₃) δ 7.49, 7.47, 7.42, 7.41, 7.41, 7.40, 7.39, 7.38, 7.33, 7.31, 7.28, 7.26, 7.23, 7.23, 7.22, 7.21, 7.20, 7.05, 7.04, 7.04, 7.03, 7.02, 6.92, 6.89, 6.87, 6.86, 6.84, 6.83, 6.82, 6.81, 6.81, 6.80, 5.32, 3.86, 3.79.

¹³C NMR(101 MHz, CDCl₃) δ 160.73, 159.83, 159.73, 159.21, 149.35, 132.25, 131.72, 131.47, 131.13, 130.37, 130.24, 127.94, 125.10, 114.72, 113.52, 113.37, 113.14, 113.05, 77.39, 77.07, 76.75, 55.34, 55.31.

3) (diphenylmethylene)hydrazine(3c):

White solid, Mp 134- 36 ^°C, yield (80 %) Mass: Cal. 196.1 Observe. 196.2

¹H NMR (400 MHz, CDCl₃) δ 7.65 – 7.40 (m, 5H), 7.40 – 7.21 (m, 5H), 5.42 (s, 2H).

¹³C NMR (100 MHz, CDCl₃) δ 154.60, 152.12, 149.26, 142.39, 139.84, 139.57, 138.45, 133.36, 133.02, 131.26, 130.09, 129.90, 129.44, 128.94, 128.84, 128.16, 127.89, 127.22, 126.48, 120.58, 77.38, 77.06, 76.75.

4) (bis(4-chlorophenyl)methylene)hydrazine (3d):

White solid, Mp 162- 164°C, yield (75%) Mass : Cal. 265.2, Observe. 265.1

¹H NMR (400 MHz, CDCl₃) δ 7.52 (d, J = 8.3 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.31 – 7.16 (m, 2H), 5.46 (s, 1H).

¹³C NMR (101 MHz, CDCl₃) δ 146.50, 138.01, 136.66, 135.25, 134.13, 130.79, 130.36, 129.92, 128.41, 127.59, 117.64, 94.73, 77.39, 77.07, 76.75.

5) (E)-((4-chlorophenyl)(phenyl)methylene) hydrazine (3e):

Brown solid, Mp 170- 172°C, yield (95%) Mass : Cal. 230.1 Observe. 230.2

¹H NMR (400 MHz, CDCl₃) δ 7.58 – 7.48 (m, 1H), 7.47 – 7.38 (m, 1H), 7.34 – 7.21 (m, 3H), 5.43 (s, 1H).

¹³C NMR (101 MHz, CDCl₃) δ 147.87, 138.11, 135.02, 131.30, 130.43, 129.78, 129.57, 129.15, 128.78, 128.31, 128.25, 127.67, 126.42, 77.38, 77.06, 76.75.

6) (di-p-tolylmethylene)hydrazine(3f):

White solid, Mp 115- 118°C, yield (70 %) Mass : Cal. 224.2, Observe.224.1

¹H NMR (400 MHz, CDCl₃) δ 7.34 (dd, J = 16.3, 8.0 Hz, 4H), 7.17 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.1 Hz, 2H), 5.36 (s, 2H), 2.42 (s, 3H), 2.32 (s, 3H).

¹³C NMR (101 MHz, CDCl₃) δ 149.73, 138.71, 137.99, 135.96, 130.04, 128.85, 128.72, 126.50, 77.40, 77.08, 76.76, 21.43, 21.23.

7) (E)-(3,4-dihydronaphthalen-1(2H)-ylidene) hydrazine(3g):

Yellow solid, Mp 180- 182°C, yield (67%) Mass : Cal. 160.2, Observe. 160.1.

¹H NMR (400 MHz,) δ 8.29 (dd, J = 7.6, 1.6 Hz, 1H), 7.36 – 7.21 (m, 2H), 7.21 – 7.12 (m, 1H), 2.87 – 2.79 (m, 2H), 2.80 – 2.69 (m, 2H), 1.97 – 1.85 (m, 2H).

¹³C NMR (101 MHz, 3>) δ 157.13, 140.55, 132.92, 129.49, 128.67, 126.34, 125.54, 77.38, 77.06, 76.74, 29.98, 27.39, 22.17.

8) (E)-(1-(p-tolyl)ethylidene)hydrazine(3h):

White solid, Mp 190- 192°C, yield (72%) Mass : Cal. 224.1, Observe.224.2

¹H NMR (400 MHz, 3>) δ 7.81, 7.79, 7.53, 7.51, 7.22, 7.20, 7.15, 7.13, 5.28, 2.38, 2.33, 2.29, 2.29, 2.09, 2.08.

¹³C NMR (101 MHz, 3>) δ 157.78, 147.73, 139.72, 137.96, 136.64, 135.83, 129.09, 129.04, 126.84, 126.60, 125.48, 77.48, 77.17, 76.85, 21.38, 21.20, 15.00, 11.75.

9) (E)-((4-fluorophenyl)(phenyl)methylene)hydrazine (3i):

White solid, Mp 148- 150°C, yield (82%) Mass : Cal. 214.2 Observe. 214.1

¹H NMR (400 MHz, CDCl₃) δ 7.58 – 7.48 (m, 1H), 7.47 – 7.38 (m, 1H), 7.34 – 7.21 (m, 3H), 5.43 (s, 1H).

¹³C NMR (101 MHz, CDCl₃) δ 147.87, 138.11, 135.02, 131.30, 130.43, 129.78, 129.57, 129.15, 128.78, 128.31, 128.25, 127.67, 126.42, 77.38, 77.06, 76.75.

10) (E)-((4-bromophenyl)(phenyl)methylene) hydrazine (3j):

White solid, Mp 121- 123 °C, yield (65%) Mass : Cal. 274.1, Observe.274.2

¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J = 7.6 Hz, 1H), 7.72 (ddd, J = 10.7, 7.4, 6.5 Hz, 2H), 7.64 (dd, J = 11.8, 5.3 Hz, 1H), 7.47 – 7.37 (m, 1H), 7.37 – 7.25 (m, 3H), 6.52 – 6.27 (m, 2H).

¹³C NMR (101 MHz, CDCl₃) δ 160.73, 159.83, 159.73, 159.21, 149.35, 132.25, 131.72, 131.47, 131.13, 130.37, 130.24, 127.94, 125.10, 114.72, 113.52, 113.37, 113.14, 113.05, 77.39, 77.07, 76.75, 55.34, 55.31.

In-vitroMycobacterium tuberculosis MABA assay

The inoculum was prepared from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone, 0.5% glycerol, albumin, dextrose, supplemented oleic acid, and catalase [OADC]), adjusted to a McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Each drug stock solution was thawed and diluted in 7H9-S at four-fold the final highest concentration tested. Serial two-fold dilutions of each drug were prepared directly in a sterile 96-well microtiter plate using 100 µl 7H9-S. A growth control containing no antibiotic and a sterile control were also prepared on each plate. Sterile water was added to all perimeter wells to avoid evaporation during the incubation. The plate was covered, sealed in plastic bags and incubated at 37 °C in normal atmosphere. After 7 days incubation, 30 µl of alamar blue solution was added to each well, and the plate was re-incubated overnight. A change in colour from blue (oxidised state) to pink (reduced) indicated the growth of bacteria, and the MIC was defined as the lowest concentration of compound that prevented this change in colour.

ACKNOWLEDGMENT:

Authors are thankful to management and principals of respective colleges for providing infrastructural facilities and encouragement. We are also thankful to CSIR-IICT, Hyderabad for providing NMR and Mass data.

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Received on 10.03.2018 Modified on 28.03.2018

Asian J. Research Chem. 2018; 11(2):269-274.

DOI:10.5958/0974-4150.2018.00050.0

INTRODUCTION: