Synthesis and Anticancer activity of

Cyclohexylacetophenone Derivatives

M. Pharm Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore – 560 041

By

Mr. PINAKI BANERJEE,B.Pharm

Under the Guidance of

Mr. GURUBASAVARAJA SWAMY.P.M, M Pharm

Asst. Professor

Department of Pharmaceutical Chemistry,

Acharya & B.M. Reddy College of Pharmacy,

Soldevanahalli, Chikkabanavara Post,

Bangalore -560090.

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1 /

Name of the candidate and address

/

Mr. Pinaki Banerjee

“Gowramma Building”, Ist floor,
Bonemill 4th cross, Nisarga Layout,
Sidedahalli Main Road, , Nagasandra Post,
Bangalore-560 073.
2 / Name of the institution / ACHARYA & B.M. REDDY COLLEGE OF PHARMACY.
89/90, Soldevanahalli, Chikkabanavara post, Hesaraghatta main road,
Bangalore – 560090.
Office: 080-65650815.
3 / Course of study and subject /

MASTER OF PHARMACY

(PHARMACEUTICAL CHEMISTRY)
4 / Date of the admission / 3rd July 2010
5 /

Title of the topic:

“SYNTHESIS AND ANTICANCER ACTIVITY OF CYCLOHEXYL ACETOPHENONE DERIVATIVES.”
6.0 / Brief resume of the intended work
6.1 Need for study:
The biological importance of cyclohexane1-13 has resulted in the synthesis of substituted derivatives which show potent pharmaceutical activities like antibacterial3, antidiabetic1, anticancer29 , antipscychotic4 , anesthetic, expectorant5 activities.
Marketed drugs containing cyclohexane :-
Some of the marketed drugs containing cyclohexane moieties are Cyclacillin10 (antibiotic), Glybenclamide29 (antidiabetic), Lomustine29 , Etoposide29 (anticancer) , ketamine29, phencyclidine10(general anesthetic), Procyclidine HCl29 (anti-parkinsonism), Bromhexidine11(expectorant)





Marketed drugs containing acetophenone :-
Acetophenones14-28 exhibit wide range of biological activities like antipscychotic15, , anti-inflammatory,16,18,21, antidiabetic15 and sedative15 activity. Some of the marketed drugs containing acetophenone moiety are used for the treatment of schizophrenia15 and mania15. Examples:-
Drugs containing acetophenone moiety are also used as psychoactive drugs that are used as anesthetics14 and pain control14 . Example:-

Acetophenone containing drugs are also used as oral hypoglycemic15 agents for the treatment of non insulin dependent diabetes mellitus. Example :-
Benzylidene acetophenone or Chalcone16 (a derivative of acetophenone ) show antibacterial16, antifungal16, antitumor14,15,18 and anti-inflammatory16 activity

Aspalathin29, a C-linked dihydrochalcone glycoside obtained from rooibos plant Aspalathus linearis29 . Aspalathin has shown to possess antimutagenic29 and antioxidant29 properties.

Considering the above facts we planned to Synthesize some new Synthetic heterocyclic compounds of Cyclohexyl acetophenone moiety and to perform anti-cancer activity.
6.2Review of literature:
  1. Kumar S3et al. carried out Antimicrobial and Cytotoxic Activities of Turbinaria conoides. Brown alga , Turbinaria conoides was successively extracted with n-hexane , cyclohexane , methanol and ethanol-water(1:1).Cyclohexane extract possessed a broad array of antibacterial activity and exhibited remarkable antifungal activity.
  2. Synthesis and evaluation of some novel potent type -2 anti diabetic agents was carried out by Velingkar V S1et al.The work involved the synthesis of lead molecule N-[4-{2-(5-chloro-2-methoxy benzimido)ethyl}phenyl sulfonyl]-N-cyclohexyl urea also known as glibenclamide.The synthesized compound was screened for anti –diabetic activity.
  3. Bruins Slot L A4et al Synthesized a potential anti pschyotic with dopamine D2/D3 receptor antagonist and 5HT1Areceptor agonist properties .Brain region specific modulation of immediate – early gene may constitute the marker of anti pschyotic drug – like activity.They investigated the effects of putative antipsychotic drug N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)ethyl]-3-(cyclopent-1-enyl)benzylamine,a compound that targets both dopamine D2 and serotonin 5HT1A receptors, the compound is found to possess potent antipschyotic activity.
  4. Synthesis of cyclo hexane derivatives having expectorant activity was carried out by Moroni A5et al.new esters of N-(2-amino-3,5-dibromobenzyl)trans -4-amino cyclo hexanol,along with certain phenaolic derivatives having expectorant activity.They are therefore useful as active principle in pharmaceutical compositions for oral , topical injection and indicated for treatment of bronchial infections.

  1. Bernardi L6et al Synthesized D-Nor-7-Ergoline derivatives having anti- Parkinson and anti – pschyotic acitivty and pharmaceutical compositions containing them.
  2. The conformation and local anesthetic activity of carbaniliates Benes L2et al was studied by.They studied the influence of spatial configuration on intensity,onset and duration of anaesthetic effect has been observed in some carbanilates local anaesthetics of fixed conformation.

  1. Stevension C.P1et al. synthesized polyoxygenated cyclohexane derivatives and other constituents from Kaempferiarotunda L. Methanol extracts of K.rotunda rhizomes yielded seven compounds including six polyoxygenated cyclohexane derivatives identified as (-) -6-acetyl zeylenol (1) , 4-acylated derivatives of 1-benzoyloxymethyl-1,6-epoxycyclohexan-2,3,4,5-tetrol(3-6),a Diels alder adduct of 3- benzoyl -1- benzoyloxymethylcyclohexa-4,6-dien-2,3-diol and a triaclyated derivative of salicin. The methanolic extract of the rhizomes of k.rotunda and (-)-2-acetyl-4-benzoyl-1-benzoyloxymethyl-1,6-epoxycyclohexan-2,3,4,5-tetrol had antifeedant activity against larvae of sporoptera littoralis (-) zeylanol also showed antifeedant activity whereas(-)6-acylated zeylenol was inactive.

8. Extraction oxygenated cyclohexanes from PiperCubeb .was carried out by Koul J.L6et al. investigation of the extract of Piper Cubeb yielded two new minor oxygenated cyclohexanes, (-)-rel-2,3,4,5-tetraaetoxy-1-benzoyloxy methylcyclohex-1(6)-ene-2,3,4,5-tetrol and (+)-2,4,5-triacetoxy-1-benzoyloxy methylcyclohex-1(6)-ene-2,3,4,5-tetrol-3-benzoate. In addition , two rare neolignans have been isolated from this species and identified as (-)-kadsurin A and (-)-piperenone.

9.Yuji H3et al. discovery of trans-N-[1-(2-fluorophenyl)-3-pyrazolyl]-3-oxospiro-[6-azaisobenzofuran-1(3H),1’-cyclohexane]-4’-carboxamide,a potent and orally active neuropeptide Y Y5 receptor antagonist. The compounds show high Y5 binding affinity ,metabolic stability and brain and cerebrospinal fluid penetration and low susceptibility to p-glycoprotein transporters


  1. Characterization of cyclohexane and hexane degradation by Rhodococcus sp EC1was carried out byLee E.H9et al. Cyclohexane is a recalcitrant compound that is difficult to degrade than even n-alkanes or mono aromatic hydrocarbons. Rhodococcus sp EC1 was isolated and shown to have maximum cyclohexane degrading ability. In addition to its cyclohexane degradation abilities , it can also degrade hexane . this suggest that EC1 may be important biological resource for the removal of cyclohexane, hexane and other recalcitrant hydrocarbons.
  2. Wang J7et al. carried out oxidation of cyclohexane catalysed by TS-1 in ionic liquid with tert-butyl-hydroperoxide. It is an efficient and environment friendly procedure was described for easy product isolation for the oxidation of cylohexane with tert-butyl hydroperoxide catalysed by titanium silicate 1(TS1) in ambient temperature ionic liquid. Good yield and higher selectivity of the products were found in ionic liquid compared within molecular solvent . the research result showed 13.2% conversion of cyclohexane 97.6% cyclohexanol and cyclohexanone selectivity were obtained in ionic liquid under mild conditions of 90 degrees.
  3. Oxidation of cyclohexane catalysed by manganese oxide octahedral molecular sieves was carried out by Ranjit K4et al. Oxidation of cyclohexane is an important industrial process , its oxidation products , cyclohexanol and cyclohexanone are raw materials for adipic acid and caprolactum synthesis. Acid catalysed cryptomelane type manganese oxide octahedral molecular sieves (H-K-OMS-2) were used for liquid phase oxidation of cyclohexane with tert-butyl hydroperoxide as oxidant. The reactions were carried out in a semi batch reactor at 80 degrees using acetonitrile as solvent . effect of temperature, solvent , effect of tert-butyl hydroperoxide were selectively studied.
  4. Byeong HJ5et al. carried out catalytic dehydrogenation of cyclohexane ,in an FAU type enzyme membrane reactor which was formed on a porous ἀ-Al2O3support tube for use in selective separation of benzene and hydrogen from cyclohexane . The membrane was used for the catalytic dehydrogenation of cyclohexane packed in a membrane reactor with Pt/Al2O3 catalyst .The reaction was carried out at a temperature of 423-523 degree over 1% Pt/Al2O3 catalyst by impregnation method.
  5. The cytotoxic activities of mono and bis Mannich bases derived from acetophenone was studied by Inci Gul H27et al. against Renca and Jurkat cells and found to possess antitumor and cytotoxic activities, 1-phenyl-3-dimethylaminopropan-1-one hydrochloride, and related piperidino and morpholino derivatives, were synthesized as mono mannich bases derived from acetophenone. Conversion of mono to bis Mannich base considerably increase cytotoxicity in most cases. The compounds synthesized were found to be more active than 5-Florouracil.

  1. Rajinder .S17et al. carried out NMR and X-ray characterization of the diastereomeric pinacols derived from photo irradiation of diastereomeric o-alkoxy acetophenones. A simple 1H NMR characterization of diastereomeric pinacols derived from o-alkoxy acetophenones is described . the 1H NMR spectral assignments are based on molecular modeling and substantiated by X-Ray crystallographic structural assignment of the diastereomer.
  2. Synthesis of 3,5-alkylated acetophenone and methyl benzoate derivatives by one pot synthesis was carried out byRoberto .B1et al. via an anionic domino process. 3,5-alkylated acetophenones and methyl benzoate derivatives are key building blocks in the synthesis of retinoic acids , which have potent antiproliferative activity against in cervical cancer cells, HIV-protease activity inhibitors , tyrosine kinase inhibitors , HIV-1 integrase inhibitors , NMDA receptor antagonists.

  1. Katade S14 et al.carried out microwave studies on synthesis of biologically active chalcone derivatives and found that chalcone and their derivatives possess some interesting biological properties such as antibacterial , antifungal , insecticidal, anesthetic, anti-inflammatory, analgesic and ulcerogenic properties. Pyrazole containing chalcone derivative is screened for antimicrobial activity.

  1. Synthesis of a new series of quinolinyl chalcones as anticancer and anti-inflammatory agents was carried out by Kotra .V18et al. . A new series of quinolinyl chalcone derivatives have been synthesized by the reaction by the reaction of quinolinyl and chloroquinolinyl acetophenones with substituted aromatic aldehydes. These compounds have been found to exhibit anticancer and anti-inflammatory activity.

  1. Kuwajima H19et al.isolated an acetophenone glycoside from aerial parts of Exacumaffine and its structure was determined as 2-0-primeverosylpaeonol. The known glycoside , gentiopicroside , 2’-0-p-coumaroyllogenin and glucopaeonol , were also identified.

  1. Isolation of two new cytotoxic acetophenone derivatives from Euphorbia ebracteolata Hayatawas carried out by Ning Zhang20et al., their structures were elucidated as 2,4-dihydroxy-6-methoxyl-methylene-3-methyl-acetophenone and 2-dihydroxy-6-methoxyl-methylene-3-methyl-acetophenone-4-0-β-D-glucopyranoside on the basis of chemical and spectroscopic methods including 1D and 2D-NMR techniques ,and their cytotoxic activities were investigated by using the MTT method.

  1. Mahmoud. A21et al. Investigated the aerial parts of Ethuliaconyzoidesfrom Egypt by examining two new monoterpene 5-methylcoumarin .,namely 5’-epi-isoethuliacoumarin B and 5’-epi-isoethuliacoumarin A,and a new monoterpene acetophenone derivative ,ethuliaconyzophenone,in addition to the five known compounds ,ethuliacoumarin, cycloethuliacoumarin, isoethuliacoumarin A, isoethuliacoumarin B and 4-hydroxy-5-methyl-coumarin-4-0-β-D-glycopyranoside.

  1. Isolation of acetophenone C-glycoside and stilbene O-glucosides in Upuna borneensis was carried out by Zulfiqar A22 et al. Three acetophenone C-glycosides ; 2,4,6-tihydroxyacetophenone 3-C-β-(2’-O-p-hydroxybenzoyl)-glucopyranoside ,2,4,6-trihydroxyacetophenone3-C-β-(2’-O-E-coumaroyl)-glucopyranoside,2,4,6-trihydroxyacetophenone 3-C-β(2’-O-E-cinnamoyl)-glucopyranoside.They are found to possess various biological activities like cytotoxicity , anti viral , and anti-inflammatory activity.
  2. Guarna A23et al. synthesized a new enantiopure bicyclic γ/δ amino acid (BTKa) derived from tartaric acid and alpha-amino acetophenone. This amino acid has a rigid skeleton and carries substituents at the 3,5 and7 positions and finds different applications in organic and peptidometric synthesis.

  1. Synthesis, evaluation and computational studies on a series of acetophenone was carried out by Alka . B13et al. based 1-(aryloxypropyl)-4-(chloroaryl)piperazines as potential atypical antipsychotics. It has been found by an online software program that all these compounds have a good brain penetration.

  1. Kuang HX28et al. isolated a new phenolic glycoside,3,5-dimethyl-6-hydroxy-2-methoxy-4-O-D-glucopyranosyl-oxy-acetophenone from the aerial parts of Dryopteris fragrans.. Aerial parts were used for the treatment of skin diseases . such as psoriasis, rash, dermatitis, dermatophytosis and chronic eczema.

  1. ATP-Dependent Carboxylation of Acetophenone by a Novel Type of Carboxylase was carried out by Jobst B24et al. . Anaerobic ethylbenzene metabolism in the betaproteobacterium Aromatoleum aromaticum is initiated by anaerobic oxidation to acetophenone via (S)-1-phenyletthanol . carboxylation is catalysed by an acetophenone induced enzyme. The same enzyme is involved in actophenone metabolism in absence of ethylbenzene. Acetophenone has been assumed to be carboxylated to benzoylacetate during anaerobic ethylbenzene metabolism.

6.3 Objectives of the study
1)To synthesize some newer derivatives of cyclohexyl acetophenone
2)To characterize the synthesized compounds by different analytical techniques such as IR, NMR and Mass spectral data.
3)To screen the synthesized compounds for their in vivoanti-cancer activity.
4)To publish the research works in peer reviewed journals.
ACTIVITIES / DURATION
Literature survey / Till the completion of project
Synthesis and collection of analytical data / 6 Months
Pharmacological activities / 2 Months
Typing of thesis book and sending for publication / 1 Month
Total / 9 Months
7.0
8.0 / Materials and methods:
7.1 Sources of data
Databases like Chemical abstracts, Biological abstracts, Medline, and Journal of Chemistry section B, Indian Journal of Heterocyclic Chemistry, European Journal of Medicinal Chemistry, Bioorganic and Medicinal Chemistry Letters, Acta crystallographica, through Helinet of RUGHS etc.
7.2 Method of collection of Data
A) Synthesis of the compounds:
Chemicals and other reagents required for the synthesis will be procured from standard company sources. Compounds will be synthesized by using standard procedures. The reactions will be monitored by TLC and purification of the compounds will be carried out by recrystallization method using suitable solvent.

B) Characterization of the compounds:
The synthesized compounds will be characterized by preliminary laboratory techniques such as melting point, boiling point etc. Compounds synthesized will be confirmed by FTIR, Mass Spectroscopy and NMR spectral data.
C) Screening of anti-cancer activity:
Anticancer activity studies: Against Ehrlich Ascites Carcinoma (EAC cells)
Method Used: In-vivo anticancer activity.
Animals Used: Swiss Albino mice.
Number of animals used: 72 numbers.
Anticancer activity against Ehrlich Ascites Carcinoma:
The animals will be classified into twelve groups of 6 animals each. The EAC cell containing phosphate buffer saline (106 cells/0.1 mL) will be injected into the peritoneal cavity of test group animals and treatment will be started 24 h after inoculation of tumor cells, (once daily as single dose) for 10 days. Group I will serve as control and will receive 0.3% CMC suspension. Group II will serve as standard and will receive vincristine (ip, 520 µg/kg body weight). Groups III-XII will serve as test groups and receive test compound, administered intraperitoneally. Antitumor activity will be screened by determining different parameters like body weight analysis, mean survival time and percent increase in life span.
7.3 Does the study require any investigation or interventions to be conducted on animals?
YES
7.4 Has ethical clearance been obtained from your institution in case of
7.3?
Enclosed
Reference:
1)Velingkar V S,Dandekar V D,Muruganathan K.Sythesis and pharmacological evaluation of some novel potent type II anti diabetic agents.Int.J.Pharm.sci. 2009 July-Sep;1(1):149-58.
2)Benes L,Stolc S,Bauer V,Tichy M.Conformation and local anaesthetic activity of carbanilates.Experientia 1982 December;38:1348-49.
3)Sadish K, kumar Y,Khan M S Y,Gupta V, Clercq E D.Antimicrobial and cytotoxic activity of Turbinaria conoides.Iran J Pharma.Sci. 2010;9(4):209-14.
4)Slot L A,Lestienne F,Barret C G,Tancredi A N,Cussac D.F15063, apotential anti pschyotic with dopamine D2/D3 receptor antagonist and 5HT1A receptor agonist properties.Eur.J.Pharm 2009 August;620:27-35.
5)Moroni A.Cyclohexane derivatives having expectorant activity , the procedure for their preparation and pharmaceutical compositions which contain them Pneumonologie,1985 July 9;147(1):62-74.
6)Bernardi L,Chiodini L ,Temperilli A.D-Nor-7-ergoline derivatives having anti Parkinson and anti – pschyotic acitivity and pharmaceutical compositions containing them.Chem.Abst,1989 Aug.29;108:38179.
7)boxamide,a potent and orally active neuropeptide Y Y5 receptor antagonist. Bioorg Med Chem 2009 June-August;17:6971-82.
8)Kumar R, Sithambaram S, Suib S.L. Cyclohexane oxidation catalysed by manganese dioxide octahedral molecular sieves. J. Catal 2009 March;262:304-13.
9)Jeong B.H, Sotowa K.I, Kusakabe K. Catalytic dehydrogenation of cyclohexane in a FAU type zeolite membrane reactor. ...Indian J Med Sci 2003 October;224:151-158.
10)Chen JR, Chen SK. Experimental study of ignition and explosion in cyclohexane liquid under oxygen oxidation conditions.J Loss Prevent Proc 2005 March;18:97-106.
11)Wang J, Zhao H, Zhang X, Liu R, Hu Y. Oxidation of cyclohexane catalysed by TS-1 in ionic liquid with tert-butyl hydroperoxide. Chi J Chem Eng 2008 June;16:373-5.
12)Wen Y, Potter O.E, Sridhar T. Uncatalysed reaction of cyclohexane in a continuous reactor. Chem Eng Science. 1997 December;52:4593-4605.
13)Lee E.H, Cho K.S. Characterization of cyclohexane and hexane degradation by Rhodococcus Sp. EC1.Chemosphere .2008 April;71:1738-44.
14)Ballini R, Barboni L, Fiorini D, Giarlo G, Palmieri A . one pot synthesis of 3,5-alkylated acetophenone and methyl benzoate derivatives via an anionic domino process.Chem.Commun 2005 Jan-April: 2633-4
15)Bali A, Sharma K, Bhalla A, Bala S, Reddy D, Singh A et al . Synthesis , evaluation and computational studies on a series of acetophenone based 1-(aryloxypropyl)-4-(chloroaryl) piperazines as potential atypical antipsychotics. Eur J Med Chem 2010January-February;45:2656-62.
16)Kadate S, Phalgune U , Biswas S, Wakharkar R, DespandeN. Microwave studies on synthesis of biologically active chalcone derivatives. Indian J Chem 2008 June;47B:927-31
17)Singh R, Garg Pk , Hundal MS, Ishar MPS.NMR and X-ray characterization of the diastereomeric pinacols derived from the photo–irradiation of o-alkoxyacetophenones.Indian J Chem 2006 Feb;45B:506-9
18)KotraV, Ganapaty S, Adapa R S. Synthesis of a new series of quinolinyl chalcones as anticancer and anti-inflammatory agents. Indian J Chem 2010 August;49B:1109-16
19)Kuwajima H, Shibano N, Baba T, Takaishi K, Inouet K, Shingu T. An acetophenone glycoside from Exacum affine. Phytochemistry;41:289-92.
20)Zhang N, Cai H, Cai B, Yang H, Li J, Yang G. Two new cytotoxic acetophenone derivatives from Euphorbia ebracteolata Hayata. Fitoterapia 2010 ;81:385-88.
21)Mahmoud A, A, Inuma M, Tanaka T. Further monoterpene 5-Methylcoumarins and an acetophenone derivative from Ethulia Conyzoides. Phytochemistry. September 1997;48:543-46.
22)Ali Z, Ito T, Tanaka T, Nakaya K, Mutura J, Darnaedi D et al . Isolation of acetophenone C-glucosides and stilbene O-glucosides in Upuna borneensis. Phytochemistry 2004 January-April;65:2141-46.
23)Guarna A, Bucelli H, Machetti F, Menchi F, Occhiato E.G, Scarpi D et al . Synthesis of a new enantiopure bicyclic γ/δ –amino acid (BTKa) derived from tartaric acid and ἀ-amino acetophenone. Tetrahedron 2002 July-October;58:9865-70.
24)Jobst B, Schuhle K, Linne U, Heider J. ATP-Dependent Carboxylation of acetophenone by a novel type of carboxylase. J Bacteriol. 2010 March;192(5):1387-94.
25)Onda M, Kohama Y, Suga K, Yamaguchi I. Microwave spectrum and molecular planarity of acetophenone. J.mol.sc 1998 February;442:19-22.
26)Masson J., Cividino P, Court J. Selective hydrogenation of acetophenone on chromium promoted Raney Nickel catalysis III. Applied catalysis A: general.1997 November;161:191-97.
27)Gul H. I, Vepsalainen J, Gul M, Erciyas E, Hannimen O.Cytotoxic activities of mono and bis Mannich bases against Renca and Jurkat cells. Pharmaceutica Acta Helvatiae 2000 August-december;74:393-98.
28)Kuang H , Zhang Y, li G.Y, Zeng W.M, Wang H.R, Song Q.Y .Isolation of a new phenolic glycoside from the aerial parts of Dryopterisfragrans . Fitoterapia 2008 January;79:319-20.
29)Wilson and Gisvold. Text book of Organic and Medicinal and Pharmaceutical Chemistry.10th ed. (New York).Lippincott-Raven Publishers;1998;342-93.
9.0 /

Signature of the Candidate

10.0 / Remarks of the Guide
11.0 / Name and Designation of
11.1Guide

11.2 Signature

/ Mr. GURUBASAVARAJA SWAMY.P.M, M.Pharm
Asst. Professor,
Department of Pharmaceutical Chemistry,
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli,
Bangalore-90

11.3 Co-Guide

11.4 Signature

/ NIL

11.5 Head of the Department

11.6 Signature

/ Dr. Amit Kumar Das
Professor,
Department of Pharmaceutical Chemistry,
Acharya & B.M. Reddy College of Pharmacy,
Soldevanahalli,
Bangalore-90
12.0 /

12.1Remarks of Principal

12.2 Name of the Principal
12.3 Signature / Dr. Divakar Goli
Principal,
Acharya and B.M. Reddy College of Pharmacy,
Soldevanahalli,
Bangalore-90

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