Description:
FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. TITLE OF THE INVENTION – A NOVEL 2H–INDAZOLE DERIVATIVES OF CURCUMIN AND ITS SYNTHESIS
2. APPLICANT(S)
NAME: RK UNIVERSITY
NATIONALITY: INDIAN
ADDRESS: RK University, Bhavnagar Highway, Kasturbadham, Rajkot-360020, Gujarat, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

A NOVEL 2H-INDAZOLE DERIVATIVES OF CURCUMIN AND ITS SYNTHESIS
FIELD OF THE INVENTION
The present invention related to a novel 2H-indazole derivatives of curcumin and its synthesis. The present invention particularly related to a novel 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives of curcumin analogs as potent anticancer agent.

BACKGROUND OF THE INVENTION
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. One defining feature of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs; the latter process is referred to as metastasis. Widespread metastases are the primary cause of death from cancer. Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020, or nearly one in six deaths. The most common cancers are breast, lung, colon and rectum and prostate cancers.

Chemotherapy is normally used for cancer treatment. Since cancer cells lose many of the regulatory functions present in normal cells, they continue to divide when normal cells do not. This feature makes cancer cells susceptible to chemotherapeutic drugs. Approximately five decades of systemic drug discovery and development have resulted in the establishment of a large collection of useful chemotherapeutic agents. However, various kinds of toxicities may occur as a result of chemotherapeutic treatments. In addition to the chemically synthesized anticancer agents, several anticancer compounds with different modes of action have been extracted from plant sources, such as Taxus brevifolia, Catharanthus roseus, Betula alba, Cephalotaxus species, Erythroxylum previllei, Curcuma longa, and many others.

Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. Curcumin has been used extensively in Ayurvedic medicine for centuries, as it is nontoxic and has a variety of therapeutic properties including anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. Over period of research, curcumin was reported to inhibit carcinogen-induced mutations and the formation of tumors in several experimental systems. It also exhibits anti-proliferation capability as a potent tool in cancer therapy. The molecule of Curcumin, [(1E, 6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione] possesses an active methylene group which is a potential site for further derivatization. Monocarbonyl curcumin analogs exhibit a more power full inhibition in various cancer cell lines.

Therefore the inventors of the present invention have synthesized some curcumin analogs bearing a carbonyl group and their further conversion into 2H-indazole derivatives as potent anticancer agents.

OBJECTIVE OF THE INVENTION
The main of the present invention is to provide a novel 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives of curcumin of having below mentioned general formula (4a-4f):

Where, R = 4a - Bromo (Br), 4b - Methyl (CH3), 4c - Nitro (NO2), 4d - Chloro (Cl), 4e - Hydroxy (OH), 4f - Methoxy (-OCH3).

Another objective of the present invention is a novel 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives of curcumin of having below mentioned general formula (4a-4f):


Where, R = 4a - Bromo (Br), 4b - Methyl (CH3), 4c - Nitro (NO2), 4d - Chloro (Cl), 4e – Hydroxy (OH), 4f - Methoxy (-OCH3); useful as potent anticancer agent.

Another objective of the invention is to provide a novel 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives of curcumin of having below mentioned general formula (4a-4f):


Where, R = 4a - Bromo (Br), 4b - Methyl (CH3), 4c - Nitro (NO2), 4d - Chloro (Cl), 4e – Hydroxy (OH), 4f - Methoxy (-OCH3); and method of preparing the same.

SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a novel 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives of curcumin of having below mentioned general formula (4a-4f):


Where, R = 4a - Bromo (Br), 4b - Methyl (CH3), 4c - Nitro (NO2), 4d - Chloro (Cl), 4e - Hydroxy (OH), 4f - Methoxy (-OCH3).

The one more aspect of the present invention is to provide a process for preparation a novel 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives of curcumin as mentioned formula (4a-4f).

BRIEF DESCRIPTION OF DRAWINGS
Fig. 1: Anticancer one dose mean graph for derivative 4a
Fig. 2: Anticancer one dose mean graph for derivative 4c
Fig. 3: Anticancer one dose mean graph for derivative 4e
Fig. 4: Anticancer one dose mean graph for derivative 4d

DESCRIPTION OF THE INVENTION
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

As stated in the present invention herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term “about” is used herein to means approximately, in the region of, roughly, or around.

As stated herein, that it follows in a transitional phrase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a composition, the term “comprising” means that the composition includes at least the recited features or components, but may also include additional features or components.

As per main embodiment, the present invention provides a novel 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives of curcumin of having below mentioned general formula (4a-4f):


Where, R = 4a - Bromo (Br), 4b - Methyl (CH3), 4c - Nitro (NO2), 4d - Chloro (Cl), 4e - Hydroxy (OH), 4f - Methoxy (-OCH3).

As per one embodiment, the general formula (4a-4f) comprises following derivatives:
Derivative No. Chemical structure IUPAC Name
4a 7-(4-Bromobenzylidene)-3-(4-bromophenyl)- 3,3a,4,5,6,7-hexahydro-2H-indazole
4b 7-(4-Methylbenzylidene)-3-(4-methylphenyl)-3,3a,4,5,6,7-hexahydro-2H-indazole
4c 7-(4-Nitrobenzylidene)-3-(4-nitrophesnyl)-3,3a,4,5,6,7 hexahydro-2H-indazole
4d 7-(4-Chlorobenzylidene)-3-(4-chlorophenyl)-3,3a,4,5,6,7-hexahydro-2H-indazole
4e 4-[7-(4-Hydroxybenzylidene)-3,3a,4,5,6,7-
hexahydro-2H-indazol-3-yl]phenol
4f 7-(4-Methoxybenzylidene)-3-(4-methoxyphenyl)-
3,3a,4,5,6,7-hexahydro-2H-indazole

As per another main embodiment, a process for preparation of novel 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives of curcumin, formula (4a-4f) comprises the step of:
a) Mixing various substituted aromatic aldehydes (1a-1f) and cyclohexanone (2) in methanol;
b) Condensing and Heating the solution of step (a) in the presence of boric acid and concentrated HCl catalyst by microwave irradiation (180 W) for 40 seconds in 10-s intervals to give 2,6-dibenzylidenecyclohexan-1-ones (3a–3f);
c) Reacting 2, 6-dibenzylidenecyclohexan-1-ones (3a–3f) of step (b) with hydrazine hydrate (b) under microwave irradiation (180 W) for 1 min in 10-s intervals to get a reaction mixture;
d) Pouring the reaction mixture of step (c) into cold water, filtering and drying the final product, 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives (4a–4f).

The complete reaction scheme of the synthesis of derivatives of the present invention is as follows:

As per one embodiment, the aromatic aldehyde (1a-1f) is selected from 4-Bromobenzaldehyde (1a), 4-Methylbenzaldehyde (1b), 4-nitrobenzaldehyde (1c), 4-Chlorobenzaldehyde (1d), 4-Hydroxybenzaldehyde (1e) and 4-Methoxybenzaldehyde (1f).

As per one embodiment, the intermediate (3a-3f) are 2, 6-dibromobenzylidenecyclohexan-1-one (3a), 2, 6-dimethylbenzylidenecyclohexan-1-one (3b), 2, 6-dinitrobenzylidenecyclohexan-1-one (3c), 2, 6-dichlorobenzylidenecyclohexan-1-one (3d), 2, 6-dihydroxybenzylidenecyclohexan-1-one (3e) and 2, 6-dimethoxybenzylidenecyclohexan-1-one (3f).

EXAMPLES
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLE 1: SYNTHESIS OF 7-(4-SUBSTITUTED BENZYLIDENE)-3-(4 SUBSTITUTED PHENYL)-3, 3A, 4, 5, 6, 7-HEXAHYDRO-2H-INDAZOLE DERIVATIVES (4a-4f)

STEP 1: PREPARATION OF 2, 6-DIBENZYLIDENECYCLOHEXAN-1-ONES (3a–3f)
A solution of 5 mmol of cyclohexanone (2) and 10 mmol of substituted aromatic aldehyde (1a–1f) in 20 mL of methanol was mixed and condensed in the presence of1 mmol Boric acid and 5 ml concentrated aqueous HCl and the mixture was subjected to microwave irradiation (180 W) for 40 seconds in 10-s intervals to give 2, 6-Dibenzylidenecyclohexan-1-ones (3a–3f)

STEP 2: PREPARATION OF 7-(4-SUBSTITUTED BENZYLIDENE)-3-(4 SUBSTITUTED PHENYL)-3, 3A, 4, 5, 6, 7-HEXAHYDRO-2H-INDAZOLE DERIVATIVES (4a-4f)
The hydrazine hydrate (1 mL, 2 mmol) was added to 2, 6-Dibenzylidenecyclohexan-1-ones (3a–3f) of step 1 and the mixture was subjected to microwave irradiation for 1 min in 10-s intervals to get a reaction mixture. The reaction mixture was poured into cold water, filtered and dried to get desired 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives (4a–4f).

EXAMPLE 2: ANALYSIS OF THE DERIVATIVES (4a-4f)

1) 7-(4-Bromobenzylidene)-3-(4-bromophenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4a):
Yield: 78%,
Melting point: 198–200°C.
IR spectrum, ?, cm–1: 3338 (N–H), 1593 (C=N), 1497 (C=C), 1221(C–N).
1H NMR spectrum, d, ppm: 7.26–7.72 m (8H, Harom), 6.42 s (1H, =CH), 5.32 d.d (1H, NCH), 5.00 s (1H, NH), 1.55–3.42 m (7H, CH2, CH).
13C NMR spectrum, d C, ppm: 152.45, 146.07, 139.51, 134.87, 130.91, 129.75,
127.96, 126.20, 123.20, 120.10, 114.40, 69.04, 57.43, 30.24, 29.11, 24.63.
Mass spectrum: m/z 446/448.
Found, %: C 53.80; H 4.02; N 6.24. C20H18N2Br2.
Calculated, %: C 53.84; H 4.07; N 6.28.

2) 7-(4-Methylbenzylidene)-3-(4-methylphenyl) - 3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4b)
Yield: 71%.
Melting point: 168–170°C.
IR spectrum, ?, cm–1: 3328 (N–H), 1585 (C=N), 1490 (C=C), 1224 (C–N).
1H NMR spectrum, d, ppm: 7.35–7.74 m (8H, Harom), 6.48 s (1H, =CH), 5.23 d.d (1H, NCH), 4.98 s (1H, NH), 2.35 s (3H, CH3), 2.10 s (3H, CH3), 1.60–3.38 m (7H, CH2, CH).
13C NMR spectrum, d C, ppm: 150.85, 145.24, 138.51, 134.62, 130.27, 129.44, 127.86, 126.28, 123.72, 120.37, 114.19, 69.46, 57.34, 30.24, 29.81,
28.34, 27.95, 24.28.
Mass spectrum, m/z: 316 [M] +317.
Found, %: C 83.48; H 7.59; N 8.81. C22H24N2.
Calculated, %: C 83.50; H 7.64; N 8.85.

3) 7-(4-Nitrobenzylidene)-3-(4-nitrophenyl) - 3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4c).
Yield: 82 %.
Melting point: 170–71?.
IR spectrum, ?, cm–1: 3330 (N–H), 1590 (C=N), 1450 (-NO2), 1497 (C=C), 1221(C–N).
1H NMR spectrum, d, ppm: 7.41–7.83 m (8H, Harom), 6.50 s (1H, =CH), 5.26 d.d (1H, NCH), 5.02 s (1H, NH), 1.60–3.38 m (7H, CH2, CH).
13C NMR spectrum, d C, ppm: 152.45, 145.07, 139.10, 134.43, 130.73,
129.27, 127.16, 126.89, 122.20, 120.90, 114.85, 69.02,
58.12, 31.02, 30.01, 24.51.
Mass spectrum, m/z: 378 [M] +, 379.
Found, %: C 63.46; H 4.77; N 14.76. C20H18N4O4.
Calculated, %: C 63.49; H 4.80; N 14.81.

4) 7-(4-Chlorobenzylidene)-3-(4-chlorophenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4d)
Yield: 69%.
Melting point: 186–188°C.
IR spectrum ?, cm–1: 3335 (N–H), 1595 (C=N), 1491 (C=C), 1223 (C–N), 853 (C–Cl), 810 (dC–Harom).
1H NMR spectrum, d, ppm: 7.26–7.72 m (8H, Harom), 6.42 s (1H, =CH), 5.30 d.d (1H, NCH), 5.03 s (1H, NH), 1.52–3.44 m (7H, CH2, CH).
13C NMR spectrum, d C, ppm: 150.25, 145.17, 138.53, 134.43, 130.21, 129.25, 127.87, 126.24, 123.68, 119.18, 114.21, 69.71, 57.67, 30.12, 28.54, 26.61.
Mass spectrum: m/z 357/359.
Found, %: C 67.20; H 5.05; N 7.82. C20H18N2Cl2.
Calculated, %: C 67.24; H 5.08; N 7.84.

5) 4-[7-(4-Hydroxybenzylidene)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazol-3-yl]phenol (4e).
Yield: 65%.
Melting point: 166–168°C.
IR spectrum, ?, cm–1: 3410 (O–H), 3335 (N–H), 1589 (C=N), 1491 (C=C), 1221(C–N), 810 (dC–Harom).
1H NMR spectrum, d, ppm: 9.45 s (2H, OH), 7.41–7.83 m (8H, Harom), 6.50 s (1H, =CH), 5.26 d.d (1H, NCH), 5.02 s (1H, NH), 1.60–3.38 m (7H, CH2, CH).
13C NMR spectrum, d C, ppm: 151.95, 146.07, 139.59, 134.24, 131.45, 129.99, 127.74, 126.86, 123.82, 120.63, 114.28, 69.92, 57.19, 30.74, 29.36, 25.55.
Mass spectrum: m/z 320 [M] +, 321.
Found, %: C 74.95; H 6.26; N 8.71. C20H20N2O2.
Calculated, %: C 74.98; H 6.29; N 8.74.

6) 7-(4-Methoxybenzylidene)-3-(4-methoxyphenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4f).
Yield: 71%.
Melting point: 170–172°C.
IR spectrum, ?, cm–1: 3335 (N–H), 1589 (C=N), 1491 (C=C), 1221 (C–N), 812 (dC–Harom).
1H NMR spectrum, d, ppm: 7.37–7.80 m (8H, Harom), 6.39 s (1H, =CH), 5.25 d.d (1H, NCH), 5.05 s (1H, NH), 3.58 s (6H, OCH3), 1.59–3.40 m (7H, CH2, CH).
13C NMR spectrum, d C, ppm: 152.45, 146.07, 139.51, 134.87, 130.46, 128.75, 127.09, 125.14, 123.20, 120.10, 114.42, 69.08, 60.75, 57.43, 53.69, 30.24, 29.11, 24.63.
Mass spectrum: m/z 348 [M] +, 349.
Found, %: C 75.80; H 6.91; N 8.02. C22H24N2O2.
Calculated, %: C 75.83; H 6.94; N 8.04.

EXAMPLE 3: ANTICANCER ACTIVITY OF 2-H-INDAZOLE DERIVATIVES
The in vitro anticancer activity of the synthesized compounds was evaluated according to the Developmental Therapeutics Program of the National Cancer Institute (Bethesda, USA). Compounds 4a, 4c, 4d, and 4e were selected and initially screened at a single dose of 10–5 M concentration. The entire 60 human cancer cell lines were organized into nine subpanels derived from nine different human cancer types: leukemia, non-small-cell lung cancer, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast cancer cell lines.

Results:
The results from the single-dose screen was reported as a graph of mean growth percent of the treated cells (Fig.1-4). From the synthesized compounds, 4a shows excellent inhibition against CCRFCEM cell line for leukemia, while 4c exhibits good inhibition against HCT-116 cell line for colon cancer.
, Claims:CLAIMS:
We claim;
1. A novel 2H–indazole derivatives of curcumin with general formula (4a-4f);

Wherein, R = 4a - Bromo (Br), 4b - Methyl (CH3), 4c - Nitro (NO2), 4d -Chloro (Cl), 4e - Hydroxy (OH), 4f - Methoxy (-OCH3).
2. The novel 2H–indazole derivatives of curcumin with general formula (4a-4f) as claimed in claim 1 are:
i. 7-(4-Bromobenzylidene)-3-(4-bromophenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4a),
ii. 7-(4-Methylbenzylidene)-3-(4-methylphenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4b),
iii. 7-(4-Nitrobenzylidene)-3-(4-nitrophenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4c),
iv. 7-(4-Chlorobenzylidene)-3-(4-chlorophenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4d),
v. 4-[7-(4-Hydroxybenzylidene)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazol-3-yl]phenol (4e),
vi. 7-(4-Methoxybenzylidene)-3-(4-methoxyphenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole (4f).
3. The process of preparation of novel 2H–indazole derivatives of curcumin with general formula (4a-4f) as claimed in claim 1 comprises steps of:
a) Mixing substituted aromatic aldehydes and cyclohexanone (2) in methanol;
b) Condensing and Heating the solution of step (a) in the presence of boric acid and concentrated HCl catalyst by microwave irradiation (180 W) for 40 seconds in 10-s intervals to give 2,6-dibenzylidenecyclohexan-1-ones;
c) Reacting 2, 6-dibenzylidenecyclohexan-1-ones of step (b) with hydrazine hydrate (b) under microwave irradiation (180 W) for 1 min in 10-s intervals to get a reaction mixture;
d) Pouring the reaction mixture of step (c) into cold water, filtering and drying the final product, 7-(4-substituted benzylidene)-3-(4-substituted phenyl)-3, 3a, 4, 5, 6, 7-hexahydro-2H-indazole derivatives (4a–4f).
4. The process of preparation of novel 2H–indazole derivatives of curcumin with general formula (4a-4f) as claimed in claim 3, wherein said substituted aromatic aldehydes are selected from 4-Bromobenzaldehyde, 4-Methylbenzaldehyde, 4-nitrobenzaldehyde, 4-Chlorobenzaldehyde, 4-Hydroxybenzaldehyde and 4-Methoxybenzaldehyde.
5. The process of preparation of novel 2H–indazole derivatives of curcumin with general formula (4a-4f) as claimed in claim 3, wherein said 2, 6-dibenzylidenecyclohexan-1-ones are selected from 2, 6-dibromobenzylidenecyclohexan-1-one, 2, 6-dimethylbenzylidenecyclohexan-1-one, 2, 6-dinitrobenzylidenecyclohexan-1-one, 2, 6-dichlorobenzylidenecyclohexan-1-one, 2, 6-dihydroxybenzylidenecyclohexan-1-one and 2, 6-dimethoxybenzylidenecyclohexan-1-one .