FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
NOVEL CHEMOTHERAPEUTIC AGENTS AGAINST INFLAMMATION AND CANCER"
RELIANCE LIFE SCIENCES PVT.LTD
an Indian Company having its Registered Office at
Chitrakoot, 2nd Floor,
Shree Ram Mills Compound,
Ganpath Rao Kadam Marg,
Worli, Mumbai - 400 013,
Maharashtra, India.
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-

FIELD OF THE INVENTION
The present invention relates to novel compounds having potential application as antitumor and anti-inflammatory agent. The present invention in particular relates to ester derivatives of cinnamic acid, vanillic acid, and 4-hyroxy cinnamic acid; their methods of preparation and compositions and their biological activities for potential use in cancer and inflammation. Further, the present invention also discloses compounds with novel benzofuran lignan structure as a potent antitumor agent and inducer of apoptosis.
BACKGROUND OF THE INVENTION
Phenolic photochemical are diverse group of compounds that exhibit anti-inflammatory, antioxidant, anticarcinogenic, anti-diabetic, anti-atherosclerosis and immunomodulatory activities. These phytochemicals are commonly called chemotherapeutics or chemopreventive agents. Human beings consume such phytochemicals from dietary sources, either as natural components or as synthetic food additives. These phytochemicals may fight disease through suppression of the inflammatory response. Dysregulated inflammation is the cause of a great many diseases including cancer and atherosclerosis [Coussens et al., 2002; Balkwill et al., 2001]. It stands to reason, then, that suppression of inflammation, whether by phytochemicals or by other means, should delay the onset of disease [Craig, 1997; Craig, 1999].
Phenolic compounds widely occur in a variety of plants. Phenolic compounds are ubiquitous in the plant kingdom [Macheix et al., 1990]. These secondary plant metabolites are commonly divided into five major groups, the anthocyanidines, the flavonols/flavones, the flavanones, and the flavan-3-ols and their oligomers and the polymers, the proanthocyanidins. A less common group of flavonoids are chalcones and dihydrochalcones, which are mostly found in individual fruits and vegetables. The fifth group of phenolic compound is hydroxycinnamic acids. The most common hydroxycinnamic acid derivatives are esters of caffeic acid with quinic acid and the caffeic acid phenylethyl ester [Natarajan et al., 1996]. Esters of caffeic acid with quinic acid are the main constituent in coffee, apple juice, artichoke, eggplant, peach , cherry,
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plum , elderberry , apricot etc [Macheix et al.,1990; Clifford et al.,1999]. The caffeic acid phenyl ester (CAPE) is a structural relative of flavanoids that is an active component of propolis from honeybee hives. It has antiviral, anti-inflammatory, and immunomodulatory properties [Grunberger et al., 1988] and has been shown to inhibit the growth of different types of transformed cells [Grunberger et al., 1988; Burke et al., 1995; Su et al., 1994; Su et al., 1991., Hlandon et al., 1980; Guarini et al.,1992]. In the transformed cells, CAPE and phenolic compounds are known to alter the redox'state and induce apoptosis [Chaio et al., 1995]. Although some of the polyphenols are considered to be non-nutritive, interest in these compounds has arisen because of their possible beneficial effects on health.
Several mechanisms have been studied for cancer prevention by polyphenols phytochemical, including modulating cell signaling, inhibiting inflammation, anti-hormone actions, modulating growth factors, antioxidant activities, enhancing apoptosis, and inhibiting cell cycle.
The transcription factors that may be responsible in the inflammation and tumor promotion are NF-KB (protein Nuclear Factor Kappa B), AP-1, CREB, STAT and GATA-3. Most of the inflammatory genes that are over expressed in the inflammation are encoding proinflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes containing K;B sites for NF-KB within their promoters, suggesting that these genes are controlled predominantly by NF-KB [Christmas et al., 2000].
Briefly, NF-KB is a nuclear transcription factor present in all cell types. It is involved in cellular responses to stimuli such as stress, cytokines, free radicals, and bacterial or viral antigens. It is an important mediator of the body's response to infection and the incorrect regulation of NF-KB is associated with the occurrence of cancer and a variety of other diseases. NFKB is present in all cells in a resting state in the cytoplasm, only when activated and translocated to the nucleus is the sequence of events leading to activation initiated [Yamamoto et al., 2001; Aggarwal et al.,2005]. NF-KB is considered a family of Rel domain containing proteins, viz; RelA (also called p65), relB, c-Rel, p50 (also called
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NF-KB 1), and p52 (also called NF-KB2). Similarly, a family of anchorin - domain containing proteins have been identified that keeps the NF-KB in its inactive state within the nucleus. These include IKBCI, IKBP, IKBY, and IKBE, bcl-3, pi05 and pi 00. The activation of NF-KB and its associated kinases like IKK is in most cases dependent on the production of reactive oxygen species by various stress stimuli. The broad spectrum of the function of phenolic antioxidants suggests their multiple targets through which they interfere with various cellular functions and protect against pathological lesions such as cancer and inflammatory diseases.
Currently, there is an increasing interest in therapeutic use of antioxidants to prevent tissue damage induced by overproduction of Reactive Oxygen Inducers (ROI), by reducing free radical formation or by scavenging or promoting the breakdown of these species [Cuzzocrea, S.et al., 2001]. Experiments in different in vitro and in vivo systems have demonstrated the potent anti-oxidant action of plant polyphenols [Damianaki, A.et al., 2000], and it has been suggested that they can prevent oxidative stress related diseases [Aucamp, J. et al., 1997].
A lot has been learned about NF-KB activation in the last decade. Cellular responses to a wide variety of diverse stimuli have been identified, and have shown to lead to the activation of NF-KB [Garg et al., 2002]. These stimuli reveal that NF-KB is a common pathway for cellular adaptation to stress. The stimuli include inflammatory cytokines (TNFa, IL 4 etc), immune related stress such as bacterial infection of S.aureus and their products such as lipopolysaccharide (LPS), viruses such as HIV-1 and their products such as hemagglutinin of the flu virus, physiological stress such as ischemia, physical stress such as UV irradiation, environmental hazards such as cigarette smoke, and many therapeutic drugs such as taxol or haloperidol, apoptotic mediators such as anti Fas, growth factors such as insulin , physiological mediators such as angiotensin II or PAF, oxidative stress such as exposure to hydrogen peroxide etc.
NF-KB regulates expression of a number of genes whose products are involved in Tumorigenisis and Inflammation [Garg et al., 2002]. These include antiapoptotic genes
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(bcl-2 and bcl -xl), cell cycle regulatory genes (eg. Cyclin Dl), proinflammatory genes like Tumor Necrosis Factor, (TNF), Interleukin-1 (IL-1), inducible Nitric Oxide synthase (iNOS), matrix metalloproteinase (MMP-9), urokinase-type plasminogen activator (uPA), and many other chemokines. It also regulates the production of prostaglandins via the proinflammatory gene cyclo-oxygenase-2 (COX-2), which has shown to be overexpressed in a variety of cancers including colorectal cancer and mesothelioma [Kalgutkar et al., 2001; Marrogi et al., 2000].
Cell cycle aberrations and blocking apoptosis provide molecular markers of cancer and cell cycle and apoptosis modulators act as targets for cancer prevention. Indeed a hallmark of cancer is the accumulation of cells with abnormal cell cycle regulation; cell division may be accelerated, cell death slowed, or a combination of these activities. Cell cycle regulatory targets are key cancer therapy targets and numerous cancer therapies induce apoptosis. Cell cycle is an endpoint that is related to cancer development and can be measured in cultured cells. Other endpoints include antioxidant, cellular signaling, etc, that may result in change in cell cycle. The cell cycle in all eukaryotes is composed of five phases, beginning with Gl phase, followed by the DNA synthesis or "S" phase, then the G2 phase, then mitosis or "M" phase, and finally GO, the quiescent state [Hunter,T. and Pines,J.1991]. Cyclin.cyclin-dependent kinase complexes control the two critical checkpoints in the cell cycle at the Gl/S and G2/M transistions by phosphorylating a variety of proteins, such as nuclear lamins and histones for nuclear membrane breakdown and chromosome condensation, as well as proteins leading to the transcription of genes required for proliferation. [Draetta, G. 1990]
While phenolic acids, such as Caffeic acid (3,4,dihydroxycinnamic acid), Cinnamic acid Ferulic acid, Vanillic acid etc are common in many plant foods, only a few examples of their esters with aromatic alcohols (eg. Phenylethyl ferulate, CAPE) are naturally occurring. The esters have antioxidant [Chen et al., 1997], anticancer & antiinflammatory [Li et al., 2003], anti HIV [Burke et al., 1995], antimicrobial activities [Gupta et al., 1979]. Of late the role of mediators in inflammatory and cancer have led to the attention of various scientist in derivation or designing molecules which involve
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novel combinations of these natural compounds for functional therapeutic development. Combinatorial synthesis and subsequent assays for anti-inflammatory and antitumor activities of various novel ester derivatives has been applied in this study, and is proving to be a good approach to bioactive chemotherapeutic development, that could support their rational use as modulators of cell signaling and their use as chemotherapeutic agents against inflammation and carcinogenic diseases.
The inventors of the present invention in addition to recognizing the potential of NF-^B as a therapeutic target, have focused on preparing novel ester derivatives classified into three categories according to the common phenolic acid present in such class. These are derivatives of cinnamic acid as represented in formula 1, derivatives of vannillic acid as represented in formula II and derivatives of hydroxy cinnamic acid as represented by formula III. The aim of such study was to determine whether these novel ester derivatives can suppress NF-KB activation induced by inflammatory agents and carcinogens and block NF-nB regulated gene expression that mediates inflammation and carcinogenesis. It has been surprisingly found by the inventors of the present invention, that these ester derivatives could inhibit NF-^B activation and potentiate apoptosis mediated by chemotherapeutic agents. Furthermore it has also been found that inhihibition of TNF induced ROI generation and inhibition of NF-KB activated gene expression was also observed. The present invention deals with the combinatorial synthesis of wide variety of novel ester derivatives from known phenolic phytochemicals, and their potential use as antitumor and anticancer agents.
Chemotherapy is one of the most common treatments for cancer. It is the main treatment for some types of cancer, such as leukemia, Hodgkin's disease and non-Hodgkin's lymphomas. Cancers of the lung, breast, testes, colon, ovary, and stomach are also treated with chemotherapy. For some patients, chemotherapy may be the only treatment they receive. Majority of the chemotherapeutic agents presently used for cancer treatment were developed by screening in a growth inhibition assay that could inhibit tumor cell proliferation. As described by Stable,P.C, (2004), these chemical substances inhibit the growth of a variety of cancer cells, utilizing a remarkable number of diverse mechanisms
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that include cell cycle arrest, induction of apoptosis, disruption of microtubules, inhibition of angiogenesis, and increasing oxidative damage. Taxols a well-established chemotherapeutic agent used for treating childhood and adult tumors acts by disrupting the microtubule function and causing growth arrest in the G2/M phase of the cell cycle. Thus, chemotherapy becomes effective because the drugs used effect some phase of the cell life cycle. Depending on the drug chosen, chemotherapy can affect malignant cells in one of the three ways: First, damage the DNA of cancer cells so that it can no longer reproduce, thus preventing replication. Second, inhibit the synthesis of new DNA strand so that no cell replication is possible. This is done by blocking the formation of nucleotides that are necessary for new DNA synthesis, hence arresting the cells in S phase. Third, stop the mitotic process by disrupting the microtubule spindle formation.
Apoptosis is the consequence of a series of precisely regulated events that are frequently altered in tumor cells. The mechanism of apoptosis involves a cascade of initiator and effector caspases that are activated sequentially [Kasibhatla,S.et al;2004], followed by chromatin condensation, nuclear fragmentation, plasma membrane blebbing and cell shrinkage. Eventually the cells break into small membrane surrounded bodies (apoptotic bodies), which are eaten up by phagocytes without inciting an inflammatory response in the vicinity. Novel and synthetic molecules capable of modulating cell cycle by targeting G2/M checkpoint followed by induction of apoptosis in multidrug-resistance tumors remain compelling for drug discovery in oncology [Li,Q et.al;1999, Jordan,A et al;1998]. Protein Tyrosine phosphorylation is another central signal pathway involved in mediating various cellular processes such as cell cycle progress, transcriptional regulation, cell transformation, proliferation, differentiation and apoptosis [O'Callaghan et al.,2001; Kalidas et al.,2001]. Several leukemic and breast cancerous cell lines [Sainsbury,J.R.C. et al.,1987] have an elevated phosphotyrosine content suggesting that disruption of balance between phosphorylation and dephosphorylation reactions could have dramatic consequences on normal regulation of cell proliferation.
Recently, a class of Dihydrobenzofuran lignans was shown to posses potential antiproliferative and antitumoral activities [Pieters,L. et. al.,1999]. Synthetic precursors
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and analogues of benzofuran lignans derivatives were synthesized and explored for their potential antiangiogenic and antitubilin/antimitotic activities in the past [Apers.S. et. al. 2001 and Pieters,L. et. al.,1999]. The inventors of the present invention have also been successful in discovering a novel benzofuran lignan structure as a potent antimitotic agent and inducer of apoptosis. The inventors of the present invention have noticed that this novel benzofuranlignan structure, efficiently arrests Jurkat T lymphocytes (p53+/+) in the G2/M phase of the cell cycle and induces apoptosis, thus inhibiting cell growth. The protooncogenes, p53 (tumor suppressor gene), bcl-2 (antiapoptotic gene), bax-a (proapoptotic gene), are known to regulate cell cycle and apoptosis [Hale et. al.,1996]. The influence of p53 gene product, a key element in apoptosis and G2/M arrest, has been characterized in depth [Bunz,F. et. al.,1998]. In this study, the molecular mechanisms of the antiproliferative and apoptotic effects of our novel molecule were investigated to determine whether the transduction signals and/or genes expression are involved and whether it could also affect the tyrosine phosphorylation status. It is for the first time that the synthesis of novel benzofuran lignan structures have shown potential antitumor / antiproliferative activities.
OBJECT OF THE INVENTION
It is the object of the present invention to provide novel compounds for anti-inflammatory and for cancer therapy.
It is the object of the present invention to provide derivatives of cinnamic acid as represented by formula I.
It is the object of the present invention to provide derivatives of vannilic acid as represented by formula II.
It is the object of the present invention to provide derivatives of 4-Hydroxy cinnamic acid as represented by formula III.
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It is the object of the present invention to provide a novel benozfuran lignans derivatives.
It is the object of the present invention to provide methods of preparation of the novel compounds.
It is the object of the present invention to provide compositions and formulations of the novel compounds
It is the object of the present invention to determine the potential use of the novel compounds in cancer and inflammation.
It is the object of the present invention to provide mechanisms of action of these compounds as anti inflammatory and chemotherapeutic agents.
SUMMARY OF THE INVENTION
The present disclosure provides novel compounds, methods, compositions and potential uses for the treatment of cancer and inflammation.
The present invention relates to the combinatorial synthesis of wide variety of novel ester derivatives from known phenolic phytochemicals as represented by Formula I. II and III, and their potential use as antitumor and anticancer agents.
The present invention provides derivatives of Cinnamic acid as represented by the
formula (I):


The present invention provides esters of cinnamic acid of formula I wherein R is selected from aryl, hetero aryl groups.
In the preferred embodiments the present invention includes compounds of formula I
wherein R is selected from vannilic acid, ferulic acid , eugenol, salicylic acid and/ or their
derivatives.
The present invention provides derivatives of Vanillic acid as represented by the formula
(II):

FORMULA II
The present invention provides esters of vanillic acid of formula II wherein R is selected from aryl, hetero aryl groups.
In the preferred embodiments the present invention includes compounds of formula II wherein R is selected from vanillic acid, ferulic acid , eugenol, salicylic acid and/ or their derivatives.
The present invention also provides esters of 4-hyrdroxy cinnamic acid as represented in

formula III.

In the preferred embodiments the present invention includes compounds of formula III wherein R is selected from Vanillic acid, ferulic acid , eugenol, salicylic acid, cinnamic acid and/ or their derivatives.
In one embodiment, the present invention relates to the compounds of formula I, II, III and their derivatives thereof including but not limited to polymorphs, isomers and prodrugs thereof, geometric or optical isomers thereof, and pharmaceutically acceptable esters, ethers, carbamates of such compounds, all solvates and hydrates thereof and all salts thereof.
The present invention further relates to a novel benzofuran lignan structure as a potent antimitotic agent and inducer of apoptosis. The inventors of the present invention have noticed that this novel benzofuranlignan structure, efficiently arrests Jurkat T lymphocytes (p53+/+) in the G2/M phase of the cell cycle and induces apoptosis, thus inhibiting cell growth. It is for the first time that the synthesis of novel benzofuran lignan structures have shown potential antitumor / antiproliferative activities.
In one embodiment, the present invention relates to the compounds of benzofuran lignan structures and their derivatives thereof including but not limited to polymorphs, isomers and prodrugs thereof, geometric or optical isomers thereof, and pharmaceutically acceptable esters, ethers, carbamates of such compounds, all solvates and hydrates thereof and all salts thereof.
In one embodiment the present invention provides methods for preparation of the novel compounds, which includes all conventional methods of esterification of one acid with other phenol. The preferred process involves esterification, protection of all hydroxyl groups followed by hydrolysis to get corresponding acid which reacts with phenolic compound to get corresponding fused ester derivative. The deprotection of hydroxyl groups yields the compound of invention which is then purified and characterized by conventional techniques.
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In one embodiment, the present invention provides the mechanism of action of the compounds of formula I, II, III, benozfuran lignan molecules and derivatives thereof. The present invention in particular has studied the effect of these molecules on NF kappa B modulation.
In on embodiments the present invention provides the pharmaceutical formulations comprising of any of compound of formulas I , II, III, benozfuran lignan molecules and derivatives thereof alone or in combination with a suitable pharmaceutically acceptable excipients. Such formulations are useful in cancer and inllammation. The compounds of the present invention can be administered alone or in combination with other active ingredients.
In one embodiment the present invention provides the method of treatment of cancer by administering to a subject a therapeutically effective amount of the compounds of formulas I, II , III, benozfuran lignan molecules and their derivatives which can be either given alone or in combination with other therapies.
In one embodiment the present invention provides the method of treating inflammation by administering to a subject a therapeutically effective amount of the compounds of formula I, II, III, benozfuran lignan molecules and their derivatives which can be either given alone or in combination with other therapies.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Figure 1: Effect of CAMVE on LPS induced nitrite production. Raw 264.7 cells were pretreated with indicated concentrations of CAMVE for 1 h before being incubated with
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LPS (250ng/ml) for 24 h. The culture supernatant was subsequently isolated and mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride, and 2% phosphoric acid) and incubated at room temperature for 15 min. NaN02 was used to generate a standard curve, and nitrite production was determined by measuring optical density at 540nm. Each column shows the mean ± S.D. of triplicate determinations.
Figure 2: Effect of CAMVE on TNF induced ROI generation (A), and Lipid Peroxidation (B). For A, Jurkat cells were pretreated with indicated concentrations of CAMVE for 1 h. After being stimulated with TNF (1 nM) for 4 h, the relative mean fluorescence intensity (MFI) was measured using a FACS Calibur (BD). The results shown are representive of two independent experiments. For B, Jurkat cells were pretreated with indicated concentrations of CAMVE for 3 h and then incubated with TNF (1 nM) for 1 h and assayed for lipid peroxidation, as described in the "Materials and Methods".
Figure 3: Effect of CAMVE on TNF or LPS dependant NF-KB activation is dose dependent. Jurkat cells were preincubated at 37°C for 3 h with indicated concentrations of CAMVE followed by 30 min incubation with 0.1 nM TNF or lOOng/ml SA-LPS(serum activated LPS). After these treatments, nuclear extracts were prepared and then assayed for NF-KB activation as described in the "Materials and Methods". In the assay performed, the use of specific antibodies against p65 and p50 subunits of the NF-KB heterodimer bound to it's specific oligo coated wells, confirms that the TNF-activated complex consisted of p50 and p65 subunits of the NF-KB transcription factor.
Figure 4: Effect of CAMVE on NF-KB activation in different cell lines. A. HeLa, MCF-7, U937, were incubated at 37°C for 3 h with 15 uM dose of CAMVE and then stimulated with 0.1 nM TNF for 30 min . After these treatments, nuclear extracts were prepared and assayed for NF-KB using BD Transfactor ELISA method. B. RAW 264.7 cells pretreated with indicated doses of CAMVE were stimulated with lOOng/ml LPS for 30 min and assayed for NF-KB activation.
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Figure 5: Effect of CAMVE on TNF or LPS induced nuclear translocation of p65. A.
HeLa cells either untreated or pretreated with 15 uM CAMVE for 3 h at 37 °C were stimulated with 0.1 nM TNF and immunocytochemical analysis were performed as described in "Materials and Methods". B. Jurkat (5 x 105 cells/ml) cells were pretreated with indicated concentrations of CAMVE for 3 h and then stimulated with 0.1 nM of TNF for 30 min or lOOng/ml of SA-LPS (serum activated LPS) for 15 min. After these treatments, cells were washed twice with PBS and nuclei were isolated by incubating the cells with 200ul Pipes-Triton buffer for 30 min at 4°C. Nuclei were stained for p65 and analyzed using FACS. (i). Representiye histogram overlay showing TNF or LPS stimulated (1), basal (2), CAMVE stimulated (3), p65 staining in the nucleus, (ii). Ordinates gives the Isotype - corrected MFI for various treatments.
Figure 6: Effect of CAMVE on TNF or LPS induced COX-2 expression. Cells were pretreated with indicated concentrations of CAMVE for 3 h and then stimulated with either TNF (0.1 nM) or SA-LPS (lOOng/ml) for 12 h. After harvesting the treated cells, the cellular lysates were checked for COX-2 protein expression by an enzyme immunoassay as described in the "Materials and Methods".
Figure 7: Effect of CAMVE on TNF or LPS induced ICAM1 (CD54) expression.
Cells(5xl05cells/ml) were pretreated with different concentrations of CAMVE for 3 h and then treated with TNF (0.1 nM) or SA-LPS (lOOng/ml) for 12 h at 37°C in a C02 incubator. Treated cells were washed and stained with anti human CD54 - FITC conjugated antibody to measure the amount of surface ICAM1 expression. The relative mean fluorescence intensity (MFI) was measured using FACS Calibur (BD).A.Representive histogram overlay showing TNF or LPS stimulated (1), basal (2), CAMVE pretreated (3). B. Values are given as MFI (mean ± S.D.) in percent of basal expression, which was set to 100%.
Figure 8: CAMVE potentiates apoptosis induced by TNF or chemotherapeutic agents. Al. Jurkat cells were incubated at 37°C with TNF, in the presence and absence of
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10 mM of CAMVE for 72 h and the viable cells were assayed using MTT reagent. The results are shown as the mean ± S.D. from triplicate culture. A2. Jurkat cells (lxl06cells/ml) were pretreated with CAMVE for 3 h as indicated and incubated with TNF for 24 hrs, and PARP cleavage was determined by FACS analysis as described in the "Matrials and Methods". M2 gated population represents the percentage apoptotic population B.SA-LPS/Jkt cells pretreated with 10 uM of CAMVE for 3 h, were treated with 1 uM of cis-plastin, doxorubicin, taxol or vincristine for 72 h. The cytotoxicity was then assayed by MTT method. Results are shown as the mean ± S.D. from triplicate samples.
Figure 9: CAMVE induces differential cytotoxicity in different human tumor cell lines. Different human tumor cell lines were cultured either in the presence or absence of CAMVE (30mM) for 72 h. The MTT assay was done and absorbance taken at 570nm. The result indicated are mean O.D. of triplicate culture.
Figure 10: Effect of CAMVE on Cell Cycle distribution. A. 5x105 cells were treated with indicated concentrations of CAMVE as indicated and cell cycle analyses were performed using Flow Cytometry as described in the "Materials and Methods". The percentage of cells in Gl, S, and G2-M phase were calculated using Cell Quest analysis software and are represented within the histograms. The data shown here are from a representative experiment repeated three times with similar results. B. Expression of human Cyclin Dl, was assayed by semi quantitative RT-PCR analysis with GAPDH as internal control. Cells were treated for 24 h with various doses of the compound (0, 5, 10 and 15 mM), then total RNA was extracted and submitted to RT-PCR.
Figure 11: Dose response for compound 27 induced loss of cell viability and cell proliferation in Jurkat cell line. Jurkat cells were treated with 10, 50, 100, 500 nM of the compound, and cell viability was determined by MTT assay 24 h, 48 h after treatment and the GI50 value was estimated. Error bars indicate ± S.D.
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Figure 12: Cell Cycle analysis of Jurkat cells after treatment with various doses of the compound stated as compound 27. 5xl05 cells were treated with different concentration of the compound for 24 h and after staining with PI, cell cycle distribution was analyzed using Flow Cytometer. The data indicate the percentage of cells in each phase of the cell cycle. All experiments were performed in duplicate and gave similar results.
Figure 13: Changes in Cell Cycle distribution with time after treatment of Jurkat cells with the compound stated as compound 27. Jurkat cells were treated with 0.1 uM and 0.5 mM of the compound for 24, 48, 72 h and the percentage of cells in the cell cycle phases (Gl, S, and G2/M) were analyzed by flow cytometry. Results are expressed as means ± S.D.
Figure 14: Induction of Caspase 3 by compound 27. Jurkat cells were treated with indicated concentrations of the compound for 16 and 24 h and harvested in lysis buffer. Cellular lysates were incubated with Ac-DEVD-pNA as described in the "Materials and Methods" for 2 h at 37°C. Absorbance was recorded at 405nm.
Figure 15: Induction of apoptosis and PARP cleavage by compound 27. Jurkat cells were treated with 100 and 500nM of the compound for indicated time period and PARP cleavage was determined using FACS analysis as described in the "Materials and Methods". Percentage apoptotic populations are represented as the M2 gated population.
Figure 16: Compound 27 induced apoptosis in Jurkat cells. A. Morphologicl aspects of propidium iodide stained cells. Jurkat cells were treated for 24 h with different concentrations of the compound and stained with propidium iodide. Arrows identify apoptotic or fragmented nuclei. B. Fragmentations of genomic DNA in cells after treatment for 24 h with indicated concentrations of the compound. Fragmented DNA was extracted and analyzed on 2% agarose gel.
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Figure 17: Differential effect of compound 27 on the cell cycle distribution in U937 cell line. Cells were treated with different concentration of the compound for 24 h and after staining with PI, cell cycle distribution was analyzed using Flow Cytometer. The data indicates the percentage of cells in each phase of the cell cycle.
Figure 18: Effects of compound 27 on p53, Bax, bcl-2 mRNA levels in Jurkat cells.
Expression of human p53, bax-a, bcl-2, was assayed by semi quantitative RT-PCR analysis with GAPDH as internal control. Cells were treated for 24 h with various doses of the compound (0, 50, 100 and 500 nM), then total RNA was extracted and submitted to RT-PCR.
Figure 19: Effect of compound 27 on the apoptosis in cells with different p53 status.
Extent of apoptosis in different cells was measured by staining the cells for PARP cleavage followed by FACS anlalysis. Cells were treated with lOOnM of the compound for 24 h and the level of apoptosis was seen. Percentage apoptotic populations are represented as the M2 gated population.
Figure 20: Suppression of phosphoryrosine levels in Jurkat cells by compound 27.
lxl06 cells were treated with indicated concentrations of the compound for 24 h. Cells were fixed and permeabilized as described in the "Materials and Methods", and the extent of tyrosine phosphorylation in the cells was determined by measuring the increase in fluorescenece produced by the FITC - labeled monoclonal antibody compared to the FITC - labeled isotype control antibody.lOOnM concentration was sufficient to bring significant reduction in tyrosine phosphorylation levels compared to the control values.
DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term " novel compounds" as used herein refers to the compounds derived from the cinnamic acid, tannic acid and gallic acid, more preferably the esters of these acids as represented by some compounds described in the table 1.
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The term "pharmaceutically acceptable" as used herein refers to the substance including carrier, diluent, vehicle excipient, or composition being compatible chemically and/or toxicologically, with the other ingredients comprising a formulation that is not deleterious to the recipient thereof.
The term "aryl" means an aromatic hydrocarbon group having a single (e.g. phenyl) or a fused ring system (e.g. naphthalene, anthracene, phenanthrene, etc.). A typical aryl group is aromatic carbocylic ring having 6, 7, 8, 9 or 10 carbon atoms, such as phenyl, naphthyl, tetrahydronaphthyl or indenyl, which may optionally be substituted with one or more substituents selected from hydroxy, amino, halogen, nitro, cyano, C| to C4 alkyl, C2 to C4 alkenyl, C2 to C4 alkynyl, Ci to C4 alkoxy, C| to C4 dialkylamino, the alkyl moieties having the same meaning as previously defined. The preferred aromatic hydrocarbon group is phenyl.
The term "heteroaryl" means a substituted or unsubstituted aromatic group having at least including one heteroatom selected from N, O and/or S, like imidazolyl, thiadiazolyl, pyridyl, (benzo)thienyl, (benzo)furyl, quinolyl, tetrahydroquinolyl, quinoxalyl or indolyl. The substituents on the heteroaryl group may be selected from the group of substituents listed for the aryl group. The heteroaryl group may be attached via a carbon atom or a heteroatom, if feasible.
The term "heterocyclic group" refers to radicals or groups derived from monocyclic or polycyclic saturated or unsaturated, substituted or unsubstituted heterocyclic nuclei having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms and containing 1, 2 to 3 hetero atoms selected from the group consisting of nitrogen, oxygen or sulfur.
The term substituent is "non-interfering" substituents. By "non-interfering" is meant that the group is suitable chemically and stability wise to occupy the designated position and
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perform the designated or intended role. Thus unsuitable groups are excluded from the definition of "non-interfering".
In addition, compounds of Formula (I), (II), (III), Benozfuran lignan and derivatives thereof may be labeled with an isotope (e.g., 3H, 14C, 35S, 125I, etc.).
A "prodrug" refers to a compounds capable of being converted to compounds of the present invention by reactions of an enzyme, gastric juice, or the like, under physiological conditions in vivo, specifically compounds capable of being converted to compounds of the present invention upon enzymatic oxidation, reduction, hydrolysis, or the like, or a compounds capable of being converted to compounds of the present invention upon hydrolysis or the like by gastric juice or the like.
A "polymorph" refers to a compound that occurs in two or more forms.
The phrase "therapeutically effective amount" means an amount of a compound of the present invention that - treat or prevent the particular disease, condition, or disorder; or attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder; or prevents o delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.
COMPOUNDS OF THE PRESENT INVENTION:
The present invention relates to the compounds of formula (I) and derivatives thereof including but not limited to polymorphs, isomers and prodrugs thereof, geometric or optical isomers thereof, and pharmaceutically acceptable esters, ethers, carbamates of such compounds, all solvates and hydrates thereof and all salts thereof.
However, in accordance with the present invention the R group is selected from Vanillic acid, ferulic acid , eugenol, salicylic acid and/ or their derivatives.
Particularly the present invention provides the compounds of formula I which are represented by structure numbers as follows
1. Methyl 4-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy} -3-methoxy benzoate. (CAMVE)
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2. 2-methoxy-4-[(lE)-3-methoxy-3-oxoprop-l-en-l-yl]phenyl (2E)-3-(3,4 dihydroxy phenyl)acrylate.
3. Methyl 2-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}benzoate.
4. 4-Allyl-2-methoxyphenyl (2E)-3-(3,4-dihydroxyphenyl)acrylate.
5. (±)-2P-[4-0-(3,4,-dihydroxycinnamyl)-3-methoxyphenyl]-3a-methyl-7-methoxy-5 - [(E)-1 -propenyl] -2,3 -dihydrobenzofuran
6. Methyl(E)-3-[2P-{4-0-(3,4-dihydroxycinnamyl)-3-methoxyphenyl}-7-methoxy -3a-methoxycarbonyl-2,3-dihydro-1 -benzofuran-5-yl]propen-2-enate.
7. 2-Methoxy-4-[(l E)-prop-l -en-1 -yljphenyl (2E)-3-(3,4-dihydroxyphenyl)acrylate
8. 4-Formyl-2-methoxyphenyl (2E)-3-(3,4-dihydroxyphenyl)acrylate
The present invention further relates to the compounds of formula (II) and derivatives thereof including but not limited to polymorphs, isomers and prodrugs thereof, geometric or optical isomers thereof, and pharmaceuticaliy acceptable esters, ethers, carbamates of such compounds, all solvates and hydrates thereof and all salts thereof.
However in accordance with the present invention the R group is selected from Vanillic acid, ferulic acid , eugenol, salicylic acid and/ or their derivatives.
Particularly the present invention provides the compounds of formula II which are
9. 4-(Methoxycarbonyl)phenyl 4-hydroxy-3-methoxybenzoate
10. 2-Methoxy -4-(methoxycarbonyl)phenyl 4-hydroxy-3-methoxybenzoate
11. 2-Methoxy-4- [(1 E)-3 -methoxy-3 -oxoprop-1 en-1 yl]phenyl 4—hydroxy-3-methoxy benzoate.
12. Methyl(E)-3-[2p-{4-0-(3-methoxy-4-hydroxyphenyl carbonyl)-3-methoxyphenyl}-7-methoxy-3a-methoxycarbonyl -2,3-dihydro-l-benzofuran-5yl] prop-2-enoate
13. 4-Allyl-2-methoxyphenyl 4-hydroxy -3 -methoxybenzoate.
14. 2-Methoxy-4- [(1 E)-prop-1 en-1 -yljphenyl 4-hydroxy-3 -methoxybenzoate.
20

15. 4-Formyl-2-methoxyphenyl 4-hydroxy-3-methoxybenozoate.
16. (±)-2p-[4-0-(3-Hydroxy-4-methoxy)-3-methoxyphenyl]-3a-methyl-7-methoxy-5-
[(E)-l-propenyl]-2,3-dihydrobenzofuran.
The present invention further relates to the compounds of formula (III) and derivatives thereof including but not limited to polymorphs, isomers and prodrugs thereof, geometric or optical isomers thereof, and pharmaceutically acceptable esters, ethers, carbamates of such compounds, all solvates and hydrates thereof and all salts thereof. However in accordance with the present invention the R group is selected from Vanillic acid, ferulic acid, eugenol, salicylic acid and/ or their derivatives.
Particularly the present invention provides the compounds of formula III which are
17. Methyl 4-{[(2E)-3-(4-hydroxyphenyl)prop-2-enoyl]oxy}-3-methoxybenzoate
18. 2-methoxy-4-[(lE)-3-methoxy-3-oxoprop-1 -en-1 -yl]phenyl(2£)-3-(4- hydroxy phenyl) acrylate

19. 4-Formyl-2-methoxyphenyl (2E)-3-(4-hydroxyphenyl)acrylate
20. 2-Methoxyphenyl (2E)-3-(4-hydroxyphenyl)acrylate
21. 4-Allyl-2-methoxyphenyl (2E)-3-(4-hydroxyophenyl)acrylate
22. Methyl [3,4-bis 0-(4-hydroxyphenylacryloyl)]phenylacrylate.
23. 2-Methoxy-4-{(1 E)-prop-1 en-1 yljphenyl (2e)-3-(4-hydroxyphenyl)acrylate
24. Methyl (E)-3-[2p-{4-0-(4-hydroxycinnamoyl)-3-methoxyphenyl}-7-methoxy-3a-methopxycarbonyl-2,3-dihydro-l-benzofuran-5-yl-prop-2-enoate
Particularly the present invention provides the compounds of benzofuran derivatives
25. (±) -2P- {4-0-(3-methoxy-4-hydroxy cinnamoyl)-3-methoxyphenyl]-3a-methyl-7-methoxy-5-[(E)-1 -propenyl]-2,3-dihydrobenzofuran.
26. 2-methoxy-4-(methoxycarbonyl)phenyl 3,4,5-trihydroxybenzoate
21

27. 5-[(£)-2-carboxyvinyl]-2P-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-
dihydro-1 -benzofuran-3cc-carboxylic acid
28. 5-[(E)-2-carboxyvinyl]-7-hydroxy-2P-(4-hydroxy-3- methoxy phenyl)-2,3-
dihydro-1 -benzofuran-3a-carboxylic acid
29. (±) -2P-[4-0-(4-hydroxy cinnamoyl)-3-methoxyphenyl]-3a-methyl-7-methoxy-
5-[(E)-l-propenyl]-2,3-dihydrobenzofuran.
Accordingly, the present invention also encompasses prodrugs of compounds of the present invention. Suitable active metabolites of compounds within the scope of Formulas (I), (II) (III), or benzofuran lignan derivatives in any suitable form, are also included herein.
The compounds of the present invention may contain asymmetric or chiral centers, and therefore may exist in different stereoisomeric forms. All suitable optical isomers and stereoisomeric forms of the compounds of the present invention as well as mixtures thereof, including racemic mixtures, form part of the present invention. In addition, the present invention embraces all geometric and positional isomers. Moreover, some compounds of the present invention may exhibit polymorphism. The present invention includes all polymorphic forms of the compounds according to the invention, which forms the further aspect of the invention. It is to be understood that the present invention encompasses any and all racemic, optically-active, polymorphic and stereoisomeric forms, or mixtures thereof, which form or forms possess properties useful in the treatment of the conditions indicated herein.
Furthermore, the present invention also include isotopically-labeled compounds of the present invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
PREPARATION:
The present invention provides process for preparation of compounds of Formulas I, II, III. Those skilled in the art will understand from this disclosure how to prepare the most preferred compounds of the present invention using any suitable known method.
22

Compounds of Formulas I, II, III and benzofuran lignan derivatives unless otherwise indicated, R, as described above may be conveniently prepared according to general process as given herein later.
In addition, the examples provided herein further illustrate the preparation of the compounds of the present invention. Moreover, those skilled in the art will understand from the present disclosure how to modify Scheme I, and the details of the examples described hereinafter to prepare any specific compound of Formulas I, II, III and benzofuran lignan derivatives of the present invention as desired. It should be understood that Scheme I is provided solely for the purposes of illustration and depicts potential route for synthesizing compounds of Formulas 1, II, III and benzofuran lignan derivatives and does not limit the invention.
Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the compounds of the present invention. Although specific starting materials and reagents are depicted in the examples illustrated below, the suitable substitution can be easily made to provide a variety of derivatives and reaction conditions. In addition, many of the compounds prepared by the method described below can be further modified in light of the disclosure using the conventional chemistry known to those skilled in the art.
FORMULATIONS:
A compound of Formulas (I), (II), (III) or benzofuran lignan derivatives or a derivative thereof can be administered in any conventional form not limited to oral, buccal, nasal, inhalation spray in unit dosage form, parenteral, (for example, intravenous, intramuscular, subcutaneous intrastemal or by infusion techniques), topical (for example, powder, ointment or drop), transdermal, intracisternal, intravaginal, intraperitoneal, intravesical, or rectal,. In another aspect of the invention, the compound of the present invention and at least one other pharmaceutically active agent may be administered either separately or in the pharmaceutical composition comprising both.
The compounds of this invention may also be administered to a mammal other than a human. The method of administration and the dosage to be administered to such a
23

mammal will depend, for example, on the animal species and the disease or disorder being treated. The compounds of this invention may be administered to animals in any suitable manner, e.g., orally, parenterally or transdermally, in any suitable form such as, for example, a capsule, bolus, tablet, pellet, Such formulations are prepared in a conventional manner in accordance with standard veterinary practice
DOSE RANGE
The dose of a compound of Formulas (I), (II), (III) or benzofuran lignan derivatives or derivatives thereof to be administered to a mammal including human or animal for the purposes as mentioned above is not specifically limited. Rather it is widely variable and subject to the pathologies, conditions, symptoms, or age of the subject and judgment of the attending physician or veterinarian.. While it may be practical to administer the daily dose of a compound of this invention, in portions, at various hours of the day, in any given case, the amount of compound of this invention will depend on such factors as the solubility of the compound, prodrug, isomer or pharmaceutically acceptable salt of this invention, the formulation used and the route of administration (e.g., orally, transdermally, parenterally or topically).
The pharmaceutical formulation comprising a compound of Formulas (I), (II), (III) or benzofuran lignan derivatives or the derivatives thereof may be formulated in a conventional manner known to those skilled at the art using one or more pharmaceutically acceptable diluent, carrier, or vehicle.
APPLICATIONS
The present invention provides compounds of formula I, II, III and benzofuran lignan
derivatives for the methods of treatment of diseases or conditions associated with NF kappa B modulation.
As indicated herein the compounds of the present inventions are useful more particularly in inflammatory conditions such as rheumatoid arthritis, inflammatory bowel disease, asthma, dermatosis including psoriasis, atopic dermatitis, and other conditions wherein NF kappa B modulation/activation is indicated.
24

The compounds are also useful in autoimmune diseases, tissue and organ rejections in transplantations, Alzeihmer's diseases, stroke, atherosclerosis, restenosis..
The compounds of the present invention are also useful in cancer such as Hodgkin's disease and other conditions where NF kappa B transcription factor is involved.
The compounds of the present invention are also useful in certain viral infections such as AIDS, osteoarthritis, osteoporosis.
Not limiting to the above said conditions and disorders wherein NF kappa B is modulated, the present invention provides compounds which are useful in other inflammatory and cancer conditions.
The compounds of the present invention are also useful in combination therapies either given along with other medications or therapies.
EXAMPLES
The invention will now be illustrated with the aid of following non-limiting examples. It should be understood, however, that the invention is not limited to the solely to the particular examples given below. It will be apparent that those skill in the art that any modifications, both to the materials and methods, may be practiced without departing from the purpose and interest of this invention
a) all operations which were carried out at room temperature or ambient temperature were in the range of 18 to 25 degree C.
b) Evaporation of the solvent was carried out under reduced pressure (600-4000 pascals;4.5-30mm Hg)with a bath temperature of upto 40 degree C.
c) The course of the reaction was monitored by thin layer chromatography (TLC) and reaction times are given for illustration only.
d) Melting points are uncorrected, the melting points are given for the materials prepared as described, polymorphism may result in isolation of materials with different melting points in some preparations.
25

e) The structure and purity of all final products were assured by at least one of the following techniques: TLC, NMR(nuclear magnetic resonance)spectroscopy, IR(lnfrared spectroscopy), or microanalytical data, and HPLC
f) Yields are given for illustration only.
g) When given, NMR data is in the form of delta (.delta.)values for major diagnostic protons given in parts per million ( ppm) relative to tetramethylsilane (TMS) as internal standard determined at 300 MHz or 400 MHz using the indicated solvent.
h) chemical symbols have their usual meanings; the following abbreviations have also been used: v( volume), w(weight), B.P.( boiling point), M. R. (Melting range), M.pt.(melting point), L(liters), ml(milliliters),gms(grams), mg(milligrams), mol (moles), mmol(millimoles) eq (equivalents) deg C (degree centigrade), cone. HC1( concentrated hydrochloric acid) any other
General Process for the Preparation of Compounds of Formula 1, II and III
The starting material was the appropriate acid used more particularly is the cinnamic acid for formula I, vanillic acid for the formula 11 and 3,4,-dihydroxy cinnamic acid for formula III.
The process involves esterification, protection of all hydroxyl groups as MOM ether followed by hydrolysis to get corresponding acid which reacts with phenolic compound to get corresponding fused ester derivative. The deprotection of hydroxyl groups using methanolic HC1 yields the compound of invention, which is then purified by silica gel column chromatography and characterized by conventional techniques (XH NMR* MASS). The resulting pure compound was then analysed for its melting point, NMR, CMR, Mass Spectroscopy to determine its final structure and purity.
General Process for the Preparation of Compounds of Benzofuran lignan derivatives (Examples 27 and 28) :
The above compounds were prepared by the action of boron tribromide on Methyl (E) -3-[2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-methoxycarbonyl-2,3-dihydro-l-benzofuran-5-yl]prop-2-enoate in dichloromethane at 0°C for 2 hrs. The reaction mixture was decomposed by adding water. The organic layer washed with saturated solution
26

sodium bicarbonate, water, brine and kept over anhydrous sodium sulphate. The organic layer concentrated to yield crude mass which was purified by radial chromatography with increasing concentration of ethyl acetate in petroleum ether.
EXAMPLE 1 : Methyl 4-{[(2E)-3-(3,4-dihydroxypheny!)prop-2-enoyl)oxy}-3-methoxybenzoate.

The above compound was prepared as per the general procedure by condensation of 3,4-
dihydroxy cinnamic acid with methyl vannilate.
LH NMR (CDCI3, 400 MHz) 5ppm:- 3.8 (s, 3H, 1 x Ar-OCH3), 3.85 (s, 3H, 1 x Ar- OCH3),
6.42 (d, 1H, J = 16 Hz), 6.8 - 7.7 (m, Ar-H), 7.8 (d, 1H, J = 16 Hz),
8.05 (brs, 1H, -OH), 8.35 (brs, 1H, -OH).
TOF MS ES :-367 (M++ Na).
Molecular formula : - C1 sH 16O7.
M.R. :-186-189°C.
EXAMPLE 2 : 2-methoxy-4-[(lE)-3-methoxy-3-oxoprop-l-en-l-yl]phenyl (2E)-3-(3,4 dihydroxy phenyl)acrylate.


The above compound was prepared as per the general procedure by condensation of 3,4-
dihydroxycinnamic acid with methyl ferulate.
lH NMR (CDCI3,400 MHz) 8ppm :- 3.89 (s, 3H, 1 x Ar-OCH3), 3.92 (s, 3H, 1 x Ar-OCH3), 6.42 (d, 1H, J = 15.7 Hz), 6.86 - 7.69 (m, Ar-H), 7.74 (d, 1H, J = 15.7 Hz), 8.5 (broad hump, 2H, 2 x -OH).
TOF MS ES :- 393 (M+ + Na).
Molecular formula:- C20H18O7.
M.R. :-182-188°C.
EXAMPLE 3 : Methyl 2-{[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}benzoate.

The above compound was prepared as per the general procedure by condensation of 3,4-
dihydroxycinnamic acid with methyl salicylate.
*H NMR (CDCI3,400 MHz) 8ppm:- 3.82 (s, 3H, 1 x Ar-COOCH3), 6.44 (d, 1H, J = 15.8
Hz), 6.8 - 8.2 (m, Ar-H), 7.73 (d, 1H, J = 15.8 Hz), 8.5 (brs, 1H, -OH), 8.8 (brs, 1H, -
OH).
TOF MS ES :- 337 (M+ + Na).
Molecular formula:- C17H14O6.
M. R. :-152-154°C.
28
EXAMPLE 4 : 4-allyl-2-methoxyphenyl (2E)-3-(3,4-dihydroxyphenyl)acrylate.


The above compound was prepared as per the general procedure by condensation of 3,4-dihydroxycinnamic acid with eugenol.
*H NMR (DMSO-ds, 400 MHz) 5ppm :- 3.74 (s, 3H, 1 x Ar-OCH3), 5.0 - 5.2 (m, 2H, Benzylic -CH2), 5.9-6.1 (m, 1H, olefinic proton), 6.47 (d, 1H, J = 15.8 Hz), 6.7 - 7.2 (m, Ar-H), 7.63 (d, 1H, J = 15.8 Hz), TOF MS ES :- 349 (M+ + Na). Molecular formula:- C19H18O5. M.R. :-139-142°C.
EXAMPLE 5: (±)-2P-[4-0-(3,4,-dihydroxycinnamoyl)-3-methoxyphenyI]-3a-methyl-7-methoxy-5-[(E)-l-propenyl]-2,3 dihydrobenzofuran.
The above compound was prepared as per the general procedure by condensation of 3,4-dihydroxycinnamic acid with dehydrodiisoeugenol.

lU NMR (DMSO-d6,400 MHz) 8ppm :-1.38 (d, 3H, J = 6.7 Hz), 1.82 (d, 3H, J = 6.7 H
3.77 (s, 3H, 1 x Ar-OCH3), 3.80 (s, 3H, 1 x Ar-OCH3), 5.21 (d, 1H, J = 10 Hz),
6.0 - 6.2 (m, 1H, olefinic proton), 6.34 (d, 1H, J = 15.8 Hz), 6.49 (d, 1H,
J = 15.8 Hz), 6.6 - 7.4 (m, Ar-H), 7.65 (d, 1H, J = 15.8 Hz).
TOF MS ES :- 489 (M + H).
Molecular formula:- C29H28O7.
M. R. :- 94 - 98 °C.
29

EXAMPLE 6: Methyl (E)-3-[2P-{4-0-(3,4-dihydroxy cinnamoyl)-3-methoxyphenyl}-7-methoxy-3a-methoxycarbonyl-2,3-dihydro-l-benzofuron-5-yl]prop-2-enoate

The above compound was prepared as per the general procedure by condensation of 3,4 dihydroxycinnamic acid with Methyl(E)-3-[2-(4-hydroxy-3-methoxyphenyl)-7-methoxy 3-methoxycarbonyl-2,3-dihydro-l-benzofuran-5-yl]prop-2-enoate.
*H NMR (DMSO-d6, 400 MHz) 5ppm :- 3.71 (s, 6H, 1 x Ar-OCH3), 3.76 (s, 6H, 2 x Ar-COOCH3), 3.86 (s, 3H, 1 x Ar-OCH3), 4.62 (d, 1H, J = 7.3 Hz), 6.06 (d, 1H, J = 7.9 Hz Benzylic proton), 6.4 - 7.6 (m, Ar-H and olefinic protons), 7.63 (d, 1H, J = 15.9 Hz), 7.65 (d, 1H, J = 15.9 Hz). TOF MS ES :- 599 (M+ + Na). Molecular formula : - C31H28011. M.R. :-176-180°C.
30
EXAMPLE 7 : 2-methoxy-4-[(lE)-prop-l-en-l-yl]phenyI(2E)-3-(3,4- dihydroxy phenyl) acrylate


The above compound was prepared as per the general procedure by condensation of 3,4-dihydroxycinnamic acid with isoeugenol.
!H NMR (DMSO-d6, 400 MHz) 8ppm :- 1.85 (d, 1H, J = 6.8 Hz), 3.78 (s, 3H, 1 x Ar-OCH3), 5.57 (d, 1H, J = 7.8 IIz), 6.6 - 7.4 (m, Ar-II,), 7.63 (d, 1H, J = 15.8 Hz), 9.2 (brs, 1H, -OH), 9.7 (brs, 1H, -OH). TOF MS ES :- 349 (M+ + Na). Molecular formula:- C19H18O5. M. R. :-168-172°C.
EXAMPLE 8 : 4-formyl-2-methoxyphenyl (2E)-3-(3,4-dihydroxyphenyl)acrylate

The above compound was prepared as per the general procedure by condensation of 3,4-
dihydroxycinnamic acid with Vanillin.
lH NMR (DMSO-d6,400 MHz) 8ppm :- 3.86 (s, 3H, 1 x Ar-OCH3), 6.52 (d, 1H,
J = 15.8 Hz), 6.7 - 7.5 (m, Ar-H), 7.69 (d, 1H, J = 15.8 Hz), 9.6 (broad hump,
2H, 2 x -OH), 9.99 (s, 1H, -CHO).
TOF MS ES :- 337 (M+ + Na).
Molecular formula:- C17H14O6.
M.R. :-154-159°C.
EXAMPLE 9 : 2-(Methoxycarbonyl)phenyl 4-hydroxy-3-methoxybenzoate
The above compound was prepared as per the general procedure by condensation of vanillic acid with methyl salicylate.
31


'HNMR (CDCI3, 500 MHz) 8ppm :- 3.66 (s, 3H, 1 x Ar-OCH3), 3.87 (s, 3H, 1 x Ar-OCH3), 6.1 (brs, 1H, -OH), 6.9 - 8.0 (m, Ar-H). TOF MS ES :- 325 (M+ + Na). Molecular formula:- C16H14O6. M. R. :- 86 - 88 °C.
EXAMPLE 10 : 2-methoxy-4-(methoxycarbonyl)phenyl 4-hydroxy-3-methoxy benzoate

The above compound was prepared as per the general procedure by condensation of vanillic acid with methyl vanillate.
*H NMR (CDCI3, 300 MHz) 5ppm :- 3.87 (s, 3H, 1 x Ar-OCH3), 3.92 (s, 3H, 1 x Ar-OCH3), 3.97 (s, 3H, 1 x Ar-OCH3), 6.8 - 8.0 (m, Ar-H and -OH). TOF MS ES :- 355 (M+ + Na).
Molecular formula : - C17H16O7.
M. R. :- 131 - 133°C.

EXAMPLE 11 : 2-methoxy-4-[(lE)-3-methoxy-3-oxoprop-l-en-lyl]phenyl 4-hydroxy-3-methoxybenzoate.
The above compound was prepared as per the general procedure by esterification of vanillic acid with methyl ferulate.

lH NMR (CDCh, 400 MHz) 5ppm :- 3.81 (s, 3H, 1 x Ar-OCH3), 3.85 (s, 3H, 1 x Ar-
OCH3), 3.96 (s, 3H, 1 x Ar-OCH3), 6.42 (d, 1H, J = 16.2 Hz), 6.8 - 7.8 (m, Ar-H), 7.68 (d, 1H, J - 16.5 Hz), 8.2 (brs, 1H, -OH).
Molecular formula:- C19H18O7.
TOF MS ES :- 381 (M+ + Na). M.R. :-152-154°C.
EXAMPLE 12 : Methyl(E)-3-[2p-{4-0-(3-methoxy-4-hydroxyphenyl carbonyl)-3-methoxyphenyI}-7-methoxy-3a-methoxycarbonyl-2,3-dihydro-l-benzofuron-5-yl] prop-2-enoate


The above compound was prepared as per the general procedure by condensation of vanillic acid with Methyl (E) -3- [2p-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3cc-methoxycarbonyl-2,3-dihydro-l-benzofuran-5-yl]prop-2-enoate.
*H NMR (CDCI3, 400 MHz) oppm :- 3.81 (s, 611, 1 x Ar-COOCH3), 3.86 (s, 3H, 1 x Ar-OCH3), 3.94 (s, 3H, 1 x Ar-OCU.,), 3.96 (s, 311, 1 x Ar-OCH3), 4.38 (d, 1H, J = 8.4 Hz), 6.21 (d, 1H, J = 7.7 Hz), 6.33 (d, 1H, J = 16Hz), 6.8 - 7.8 (m, Ar-H), 7.65 (d, 1H, J = 16 Hz), 7.9 (brs, 1H, -OH). TOF MS ES :- 587 (M+ + Na). Molecular formula: - C30H28011. M. R. :-217-220°C.
EXAMPLE 13 : 4-allyl-2-methoxyphenyl 4-hydroxy -3-methoxybenzoate.

The above compound was prepared as per the general procedure by esterification of
vanillic acid with eugenol.
lH NMR (DMSO-d6,400 MHz) oppm :- 3.73 (s, 311, 1 x Ar-OCH3), 3.84 (s, 3H,
1 x Ar-OCH3), 5.06 - 5.15 (m, 2H, Benzylic -CH2), 5.94 - 6.03 (m, 1H,
olefinic proton), 6.79 - 7.63 (m, Ar-H and olefinic protons), 10.17 (brs, 111, -OH).
TOF MS ES :- 337 (M+ + Na).
Molecular formula:- CigHi805.
M.R. :- 68-71 °C.


EXAMPLE 14 : 2-Methoxy^-[(lE)-prop-len-l-yl]phenyl4-hydroxy-3-methoxy
The above compound was prepared as per the general procedure by condensation of vanillic acid with isoeugenol.
'H NMR (CDCI3,400 MHz) 8ppm:-1.81 (d, 1H, J = 8.0 Hz), 3.74 (s, 3H, 1 x Ar-OCH3), 3.87 (s, 3H, 1 x Ar-OCH3), 6.0 - 6.2 (m, 1H, olefinic proton), 6.31 (d, 1H, J = 16 Hz), 6.8 - 7.8 (m, Ar-H). TOF MS ES :- 337 (M+ + Na). Molecular formula:- CigHisOs. M.R. :-148-150 °C.


35
EXAMPLE 15 : 4-formyl-2-methoxyphenyl 4-hydroxy-3-methoxybenozoate

EXAMPLE 16 :(±)-2p-[4-0-(3-methoxy-4-hydroxy benzoyl)-3-methoxy phenyl]-3a-methyl-7-methoxy-5-[(E)-l-propenyl]-2,3-dihydrobenzofuran.
OCH3
H3C^^^^>CH3 y^o^YY°cH3
The above compound was prepared as per the general procedure by condensation of
vanillic acid with dehydrodiisoeugenol.
'H NMR (DMSO-d6, 400 MHz) 8ppm :- 1.36 (d, 3H, J = 6.7 Hz), 1.8 (d, 3H, J = 5.5 Hz),
3.76 (s, 3H, 1 x Ar-OCH3), 3.80 (s, 3H, 1 x Ar-OCH3), 3.84 (s, 3H, 1 x Ar-OCH3),
4.0 (m, 1H), 5.23 (d, 1H, J = 8.54 Hz), 6.0 - 6.2 (m, HI, olefinic proton),
6.35 (d, 1H, J = 15.8 Hz), 6.6 - 7.8 (m, Ar-H),), 10.18 (s, 1H, -OH).
TOF MS ES :- 477 (M + H). Molecular formula:- C28H28O7.
M.R. :-136-140°C.
EXAMPLE 17 : methyl 4-{[(2E)-3-(4-hydroxyphenyl)prop-2-enoyl]oxy}-3-methoxy benzoate

The above compound was prepared as per the general procedure by condensation of 4-hydroxycinnamic acid with methyl vanillate.
36

'HNMR (CDCI3,400 MHz) 8ppm:- 3.89 (s, 3H, 1 x Ar-COOCH3), 3.93 (s, 3H, 1 x Ar-OCH3), 6.27 (brs, 1H, -OH).6.59 (d, 1H, J = 15.2 Hz), 6.8 - 7.8 (m, Ar-H), 7.83 (d, 1H, J = 15.8 Hz). TOF MS ES :- 351 (M+ + Na). Molecular formula:- C18H16O6. M.R. :-162-165°C.

The above compound was prepared as per the general procedure by condensation of 4-hydroxy cinnamic acid with methyl ferulate.
'HNMR (CDCI3,400 MHz) 8ppm:- 3.69 (s, 3H, 1 x Ar-OCH3), 3.75 (s, 3H,
1 x Ar-COOCH3), 6.29 (d, 1H, J = 15.8 Hz), 6.34 (d, 1H, J = 15.8 Hz),
6.6 - 7.4 (m, Ar-H), 7.54 (d, 1H, J = 15.8 Hz), 7.67 (d, 1H, J = 15.8 Hz),
9.37 (brs, 1H, -OH).
TOF MS ES :- 377 (M+ + Na).
Molecular formula:- C2oHig06.
M.R. :-174-180°C.


The above compound was prepared as per the general procedure by condensation of 4-hydroxy cinnamic acid with vanillin.
*H NMR (DMSO-d6, 400 MHz) 6ppm :- 3.86 (s, 3H, 1 x Ar-OCH3), 6.65 (d, 1H, J = 16
Hz), 6.7 - 7.7 (m, Ar-H), 7.77 (d, 1H, J = 16 Hz), 10.16 (s, 1H, -CHO), 10.27 (s, 1H, -
OH).
LCMS (Negative Mode, Q1MS) :- 297 (M - H).
Molecular formula :- C17H14O5.
M. R. :-136-138°C.
EXAMPLE 20 : methyl 2-{[(2E)-3-(4-hydroxyphenyI)prop-2-enoylloxy}benzoate

The above compound was prepared as per the general procedure by condensation of 4 hydroxy cinnamic acid with methyl salicylate.
!H NMR (DMSO-d6, 400 MHz) 6ppm:- 3.74 (s, 3H, 1 x Ar-COOCH3), 6.65 (d, 1H, J = 15.8 Hz), 6.8 - 8.0 (m, Ar-H), 7.75 (d, 1H, J = 15.8 Hz), 10.15 (s, 1H, -OH).
38

TOF MS ES :- 321 (M+ + Na). Molecular formula:- C17H14O5. M.R. :-160-163°C.

The above compound was prepared as per the general procedure by condensation of 4-hydroxy cinnamic acid with eugenol.
*H NMR (DMSO-d6, 400 MHz) 5ppm :- 3.74 (s, 3H, 1 x Ar-OCH3), 5.0 - 5.15 (m, 2H, Benzylic -CH2), 5.8 - 6.1 (m, 1H, olefinic proton), 6.61 (d, 1H, J = 15.8 Hz), 6.7 - 7.7 (m, Ar-H), 7.71 (d, 1H, J = 15.8 Hz), 10.12 (s, 1H, -OH).
TOF MS ES :- 333 (M+ + Na). Molecular formula:- C19H18O4.
39
M.R. :-147-151°C.


The above compound was prepared as per the general procedure by condensation of 4-hydroxy cinnamic acid with 3,4-dihydroxy methyl cinnamate.
'H NMR (DMSO-d6, 400 MHz) 8ppm:- 3.74 (s, 3H, 1 x Ar-COOCH3), 6.4 - 8.0 (m, Ar-H and olefinic protons), 10.14 (brs, 2H, 2 x -OH). TOF MS ES :- 513 (M+ + Na). Molecular formula:- C28H22O8. M.R. :-195-200°C.
EXAMPLE 23 : 2-methoxy-4-[(lE)-prop-l-en-l-yl] phenyl (2E)-3-(4-hydroxyphenyl) acrylate

The above compound was prepared as per the general procedure by condensation of 4-hydroxy cinnamic acid with isoeugenol.
lH NMR (DMSO-d6, 400 MHz) 5ppm :- 1.85 (d, 1H, J = 6.1 Hz), 3.78 (s, 3H, 1 x Ar-OCH3), 5.57 (d, 1H, J = 7.8 Hz), 6.2 - 6.5 (m,olefinic proton), 6.61 (d, 1H, J = 16.4 Hz), 6.7 - 7.7 (m, Ar-H), 7.72 (d, 1H, J = 15.8 Hz), 10.12 (brs, 1H, -OH). TOF MS ES :- 333 (M+ + Na). Molecular formula:- C19H18O4. M.R. :-195-200°C.
40

The above compound was prepared as per the general procedure by condensation of ferulic acid and dehydrodiisoeugenol.
*H NMR (CDCb, 400 MHz) 8ppm:- 1.26 (d, 3H, J = 13 Hz), 1.36 (d, 3H, J = 6.7 Hz), 3.4 (m, 1H), 3.76 (s, 3H, 1 x Ar-OCH3), 3.84 (s, 3H, 1 x Ar-OCH3), 3.86 (s, 3H, 1 x Ar-OCH3), 5.11 (d, 1H, J = 9.1 Hz), 6.0 - 6.2 (m, 1H, olefinic proton), 6.27 (d, 1H, J = 2.0 Hz), 6.44 (d, 1H, J = 15.9 Hz), 6.6 - 7.6 (m, Ar-H), 7.73 (d, 1H, J =15.9 Hz). TOF MS ES :- 525 (M+ + Na). Molecular formula:- C30H30O7. M. R. :-115-118°C.

The above compound was prepared as per the general procedure by condensation of gallic acid and methyl vanillate.
'H NMR (DMSO-d6, 400 MHz) Sppm :- 3.82 (s, 3H, 1 x Ar-COOCH3), 3.87 (s, 3H, 1 x Ar-OCH3), 7.0 - 7.8 (m, Ar-H), 9.4 (broad hump, 3H, 1 X 3 -OH). TOF MS ES :- 357 (M+ + Na). Molecular formula:- CieHuOg. M. R. :-193-196°C.

EXAMPLE 27: 5-[(£)-2-carboxyvinyl]-2P-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydro-l-benzofuran-3a-carboxylie acid
The above compound was prepared by the action of boron tribromide on Methyl (E) -3-[2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-methoxycarbonyl-2,3-dihydro-l-benzofuran-5-yl]prop-2-enoate in dichloromethane at 0°C for 2 hrs. The reaction mixture was decomposed by adding water. The organic layer washed with saturated solution sodium bicarbonate, water, brine and kept over anhydrous sodium sulphate. The organic layer concentrated to yield crude mass which was purified by radial chromatography with increasing concentration of ethyl acetate in petroleum ether.

*H NMR (CD3OD, 500 MHz) 8ppm :- 3.66 (s,3H, 1 x Ar-OCH3), 3.71 (s,3H, 1 x Ar-
OCH3), 4.2 (d, 1H, J = 7 Hz), 5.86 (d, 1H, J = 7 Hz), 6.21 (d, 1H, J = 15.5 Hz), 6.5 - 7.2
(m, 5H, ArH), 7.47 (d, 1H, J = 16.0 Hz).
13C NMR (CD3OD, 125 MHz) 8 ppm :- 50.37 (OCH3) ,51.54 (OCH3), 55. 29, 86.85,
112.22 (ArH), 114.13, 114.69 (Olefinic carbon), 115.54 (ArH), 116.39 (ArH), 117.00
(ArH), 125.88, 128.10, 131.35, 141.39, 144.80 (Olefinic carbon), 144.99, 145.21, 149.03,
167.84 (>C=0), 171.15 (>C=0).
DEPT :- 50.37 (CH3), 51.54 (CH3), 55.29 (CH), 86.85 (CH), 112.22 (CH), 114.13 (CH),
114.69 (=CH), 115.54 (CH), 116.39 (CH), 117.00 (CH), 125.88 (>C<), 128.10 (>C<),
131.35 (>C<), 141.39 (>C<), 144.80 (=CH), 144.99 (>C<), 145.21 (>C<), 149.03 (>C<),
167.84 (>C=0), 171.15 (>C=0).
TOF MS ES :- 387 (M + H).
Molecular Formula : C20H18O8. Viscous mass.

EXAMPLE 28: 5-[(E)-2-carboxyvinyl]-7-hydroxy-2P-(4-hydroxy-3-methoxy phenyI)-2,3-dihydro-l-benzofuran-3oc-carboxylic acid

The above compound was prepared by the action of boron tribromide on Methyl (E) -3-[2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-methoxycarbonyl-2,3-C=0), 171.28 (>CO).
DEPT :- 51.70 (CH3), 55.21 (CH), 86.80 (CH), 112.40 (CH), 114.83 (=CH), 115.52 (CH), 115.70 (CH), 116.45 (CH), 117.21 (CH), 125.80 (>C<), 128.36 (>C<), 131.40 (>C<), 141.26 (>C<), 144.51 (=CH), 144.89 (>C<), 145.14 (>C<), 148.80 (>C<), 170.29 (>C=0), 171.28 (>C=0).
TOF MS ES :- 373 (M + H).
Molecular Formula : CigH^Og. Viscous mass.
44

EXAMPLE 29 : (±) -2P-[4-0-(4-hydroxy cinnamoyl)-3-methoxyphenyl]-3a-methyl-7-methoxy-5-[(E)-l-propenyl]-2,3-dihydrobenzofuran.
The above compound was prepared as per the general procedure by condensation of 4-dihydroxycinnamic acid with dehydrodiisoeugenol.

'HNMRCCDCb, 400 MHz) 5ppm :- 1.41 (d, 3H, J = 6.7 Hz), 1.87 (d, 3H, J = 8.25 Hz): 3.48 (m, 1H), 3.82 (s, 3H, 1 x Ar-OCH3), 3.90 (s, 3H, 1 x Ar-OCH3), 4.24
(brs, 1H, -OH), 5.17 (d, 1H, J = 9.1 Hz), 6.0 - 6.2 (m, 1H, olefinic proton), 6.36 (d, 1H, J = 15.9 Hz), 6.50 (d, 1H, J = 15.9 Hz), 6.6 - 7.6 (m, Ar-H),), 7.82 (d, 1H, J =15.9 Hz).
TOF MS ES :- 495 (M+ + Na). Molecular Formula: C29H28O6.
M. R.:- 145 - 148 °C.
BIOLOGICAL EVALUATION OF COMPOUNDS
Cell Lines
The cell lines used in this study were as follows: L929 (mouse fibroblast like cells), RAW 264.7 (mouse macrpphage), U-937 (human histiocytic lymphoma), Jurkat (human T cell leukemia), MCF-7 (human breast cancer cell line), HeLa (human cervical cancer cell line); they were obtained from American Type culture collection (Manassas, VA, USA). L929, U-937, Jurkat, Raw 264.7 were cultured in RPMI 1640, while others iri DMEM supplemented with 10% FBS, penicillin (lOOOU/ml), and streptomycin (lOOug/ml).

Materials
All synthetic chemicals were obtained from commercial sources. Lipopolysaccharide (LPS) , Bovine Serum Albumin (BSA), Phorbol Myristate Acetate (PMA), Propidium Iodide (PI), Actinomycin D (Act D), thiobarbituric acid, sulfanilamide, naphthylenediamine dihydrochloride, tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO etc were obtained from Sigma Aldrich Chemicals (St Louis, MO, USA). Penicillin, streptomycin, neomycin, RPMI 1640 and DMEM medium, fetal bovine serum (FBS) were obtained from Gibco BRL. Purified recombinant human TNF-a (17.5 kDa) was purchased from R&D systems. The ED50 value of TNF-a ranged from 0.02-0.05ng/ml which corresponds to the specific activity of 2.5x107-5xl07 Units/ml. COX-2 ELISA Kit was obtained from Zymed laboratories (Invitrogen immunodetection), Anti human p65 polyclonal antibody (Santa Cruz), Anti CD 54 FITC conjugate (BD) , Anti PARP-F1TC conjugate was from Novus Biologicals, The fluorescent reactive oxygen intermediate probe Dihydrorhodamine 123 (DHR 123) was purchased from Molecular Probes, the NF-KB Transcription Factor assay kit source was from BD Biosciences,Clontech. One step Access RT-PCR kit was purchased from Promega.
Bioassay of cytokine production by RAW 264.7 cells using L929 cell line
Measurement of TNFa in culture medium or supernatant can be done using immunoassay and bioassay. Bioassay is used for the measurement of bioactive TNFa production in the culture medium.
TNFa secretion into the medium by LPS activated macrophage was assayed using L929 tumorigenic murine cells (ATCC) specifically sensitive to TNFa. L929 cell cytotoxicity assay was performed by a modified method [Sano et al.,1999] based on that described elsewhere [Flick,D.A.,and Gifford,G.E.,1984]. Briefly, the L929 cells (log phase cells) were seeded into a flat bottom 96 well plates (6xl04/well) in lOOul volume of RPMI 1640 containing 2% FCS and incubated overnight at 37°C in a 5% CO2 incubator. A working dilution of the culture supernatant collected from LPS (200ng/ml) activated macrophage in a volume of lOOul per well was first tested to obtain 70-75% cytotoxicity equivalent to 75pg/ml of recombinant TNFa standard to the TNFa sensitive L929 cell
46

line. After incubation of the L929 cells appropriate fixed dilution of the culture supernatant collected from the compound treated (lOuM) wells, containing the LPS stimulated macrophages in a volume of lOOul with 2ug/ml Actinomycin D (Act D) was taken and added to the L929 cells and the cells were incubated at room temperature for 15 min followed by 18 h incubation at 37°C overnight with 5% CO2. On the next day the medium was removed and the cells were stained with 0.2 % (w/v) crystal violet for 10 minutes. The wells were gently rinsed with water, and 33% acetic acid (lOOul/well) was added to extract the retained crystal violet. The absorbance at 570 nm was finally measured.
Nitrite Quantification
NO2" accumulation was used as an indicator of Nitric Oxide (NO) production in the medium as described previously [Green et al., 1982]. RAW 264.7 cells were plated at 5xl0scells/ml, and stimulated with LPS (250ng/ml) in the presence or absence of the test compounds for 24h. The isolated supematants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride, and 2% phosphoric acid) and incubated at room temperature for 15 min. NaN02 was used to generate a standard curve, and nitrite production was determined by measuring the optical density at 540nm.
Determination of Thiobarbituric Acid-reactive Substances (TBARS)
Lipid peroxidation was assessed by the TBARS assay, which detects mainly malondialdehyde (MDA), a product of the peroxidation of polyunsaturated fatty acids and related esters. TBARS were measured by a modification of the method described previously, [Ohkawa et al., 1979]. Jurkat cells, 6xl06 cells in 2ml were pretreated with either media or different concentrations of CAMVE (as described in the Figure legends) for 3 h and then stimulated with InM TNF for 1 h. Cells were washed before undergoing three cycles of freeze - thawing in 200ul of water. A 20ul aliquot was subsequently removed for Bradford protein determination, and 800ul of assay mix (0.4% (w/v) thiobarbituric acid, 0.5% (w/v) SDS, 9.4% (v/v) acetic acid, pH 3.5) was added to the remaining sample. Samples were incubated for 60 min at 95°C, cooled at room
47

temperature, and centrifuged at 14,000 x g for 10 min, and the absorbance of the supernatants was read at 532 nm against a standard curve prepared using the MDA standard (lOmM 1,1,3,3-tetramethoxypropane in 20mM Tris-HCL,pH 7.4). Results were calculated as nmol of MDA equivalents/mg of protein and expressed as a percentage of matched control values. Untreated cells showed 0.568 ± 0.08 nmol of TBA-reactive substances/mg protein (subtracting the background absorbance obtained by heating 800 ul of assay mix plus 200ul water).
Measurement of Reactive Oxygen Intermediate ROI
The production of ROI in cells treated with TNFa or LPS was determined by flow cytometer as described by [Manna,S.K.et al.,1999]. Briefly Jurkat cells (5xl05 cells in one ml) were incubated either with RPMI 1640 medium supplemented with 10% FBS or with complete media containing different concentrations of CAMVE for 1 h at 37°C. Cells were then stimulated with InM of TNF for 4 h. After incubation, the cells were washed with D-PBS, and resuspended in 1ml D-PBS. To detect ROI production, cells were exposed to Dihydrorhodamine 123 (5mM stock) at a final concentration of luM for lhr at 37 °C with moderate shaking (lOOrpm) and then washed with D-PBS three times and resuspended in 1ml of D-PBS. Rhodamine 123 fluorescence intensity resulting from Dihydrorhodamine 123 oxidation was measured by FACS Calibur (Becton Dickinson) with excitation at 488nm and was detected between 515 and 550mm. Data was analyzed using Cell Quest software (Becton Dickinson).
Preparation of nuclear extracts
Cell pellet was resuspended in lysis buffer (lOmM HEPES, pH 7.9, 1.5mM MgCl2, 10mM KC1, ImM PMSF, ImMDTT, 0.5% NP 40, O.lmM EGTA and O.lmM EDTA) and allowed to swell on ice for 15 min, followed by centrilugation at 3300xg for 20 min. The cell pellet was resuspended in a volume of lysis buffer equal to the cell pellet volume. The cell suspension was slowly drawn down into a syringe and ejected the content in a single stroke. Disrupted cells were incubated for 15min on ice, and the disrupted cell suspension was centrifuged at 10,000xg for 20 min at 4 °C. Nuclear pellet was resuspended in a volume of extraction buffer (20mM HEPES;pH 7.9, 25% glycerol, 1.5mM MgCl2, 420mM NaCl, O.lmM EDTA, O.lmM EGTA,ImM PMSF and ImM
48

DTT) and incubated on ice for 30 min. The nuclear suspension was centrifuged at 21,000xg for 15 min at 4°C and the supernatant was collected as nuclear extract and stored at -70°C. Protein concentration was estimated using standard Bradford method.
NF-K,B activation assay
To determine NF-KB activation, Transcription profiling was done with the BD Mercury Transfactor kit obtained from BD Biosciences. This method provides rapid, high-throughput detection of specific transcription factors eg NFKB in the nuclear extract. Using an enzyme-linked immunosorbent assay (ELISA)-based format, the Transcription Factor kit detects the DNA binding by specific transcription factors. This method is faster, easier, and significantly more sensitive than eletrophoretic mobility shift assays [EMS A].
The assay was performed by using wells coated with oligonucleotides having the consensus DNA binding sites for the specific transcription factors. 50ug of the nuclear extract proteins were incubated in the wells precoated with their specific oligonucletides, and allowed the activated NFKB to bind to their consensus sequence. Bound transcription factor was detected by a specific Primary Antibody. A horseradish peroxidase -conjugated Secondary Antibody was then used to detect the bound Primary Antibody. After addition of the substrate, the Absorbance was recorded at 655nm.
Nuclear Translocation ofp65 NF-kB by Immunocytochemistry
Hela cells grown on cover slips were washed with 0.1M potassium phosphate buffer (pH 7.4) and fixed with 4% formaldehyde in 0.1 M potassium phosphate buffer (pH 7.4) for lh at room temperature. The cells were permeabilized with 0.1% Triton X - 100 in PBS for lh. It was then incubated with rabbit anti human p65 polyclonal antibody (Santa Cruz) at room temperature for 1 h, and then stained with secondary FITC conjugated goat anti rabbit IgG antibody (Sigma) for 1 h at room temperature. After counterstaing for nuclei with DAPI or Hoechst for 5 mins slides were analyzed under a fluorescence microscope (Labophot-2. Nikon,Tokyo, Japan).
49

Nuclear translocation ofp65 by Flow Cytometry
The assay was performed as described previously [Blaecke et al., 2002]. Briefly after stimulation, cells were washed twice with PBS. Nucleus was prepared by incubating the cells with 200ul Pipes-Triton buffer for 30 min at 4°C. Nuclei staining was performed using mouse anti-human NFKB p65 mAb (Santa Cruz) or with the matching isotype control at 3ug/ml for 30 mins. After washing, the nuclei were incubated with secondary FITC conjugated goat anti-mouse antibody (Sigma) for additional 30 mins at 4°C and analysed using FACS.
COX-2 Protein and gene expression
Quantitative detection of Cox 2 protein expression by activated cells was done by an enzyme-linked immunosorbent sandwich assay using Zymed COX-2 ELISA kit. 1x10 cells were treated with different concentrations of CAMVE and stimulator and incubated at 37°C for 12 hrs. After stimulation, cells were rinsed twice with ice cold PBS, and lOOul of lysis buffer (150mM NaCl, 50mM Tris-HCl pH7.5,500 uM EDTA, lOOuM EGTA, 1.0% Triton X-100 and 1% sodium deoxycholate, ImM PMSF, 10 ug/ml leupeptin, 10ng/ml aprotinin) was added to the pellet. Lysates were sonicated for 20s on ice and centrifuged at 10,000xg for 10 min to sediment the particulate material. The protein concentrations of the supematants were measured by Bradford. 200ng/100ul of the protein was assayed per sample according to the kit protocol. Briefly, lOOul of the sample and the standard were put in the pre antibody coated wells and incubated at lh at 37°C. After three washes lOOul of the HRP conjugated antibody was added and incubated for 30 mis at 4°C. After washing, lOOul of the TMB substrate was added and incubated for 30 min at room temperature in dark. The reaction was stopped and the absorbance was read at 450nm.
ICAM1 (CD 54) gene expression
Cells were pretreated with different concentrations of CAMVE for 3 h and then treated with O.lnM of TNF or lOOng/ml SA-LPS for 12 h at 37°C in a C02 incubator. Extent of ICAM 1 expression was detected by staining the washed cells with FITC-labeled monoclonal antibody which binds to the cells expressing the CD 54 (ICAM 1). Unbound
50

FITC-conjugated antibody is then washed from the cells and the cells were resuspended in 0.5 ml of 1% paraformaldehyde, and analyzed using Flow Cytometer (B D FACS Calibur). Cells CD54 structure is lluorescently stained, with the intensity of staining is directly proportional to the density of CD54.
PARP Cleavage Assay using Flow Cytometry
Extent of PARP cleavage is determined using polyclonal antibody specifically recognizing the 85 kDa fragment of cleaved PARP (NSB 699 Novus Biologicals) and can be used as a marker for detecting apoptotic cells. Treated cells were fixed with 70% chilled ethanol and permeabilized for 30 min at RT ( PBS + 0.5% BSA + 0.02% NaN3 + 0.5% saponin) and stained with anti PARP-FITC (10ul/ 106 cells) for one hour at RT. Cells were washed twice with wash buffer (PBS + 1% heat inactivated FBS) and analysed using FACS.
Cytotoxicity assay
Cytotoxicity was assed by the modified tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described by Plumb et al 1989.
Cell Cycle Analysis
Cells (log phase culture) were treated with vehicles alone (similar volumes of DMSO) or with the compound (various concentrations) to be tested for 24 h. Untreated cells were also included in this experiment for comparison. After treatments, the cells were harvested and washed with cold EDT A/PBS (5mmol/L). Cells were then resuspended in cold EDT A/PBS (300ul) and 100% chilled ethanol (700 ul), vortexed, and incubated at room temperature for 1 h. Samples were centrifuged at 200 x g for 5 minutes and the supernatant was removed. A solution containing propidium iodide (100 ug/ml) and RNase A (lmg/ml) was added to the samples and incubated for 1 h at room temperature. Samples were then transferred to 12x75 mm polysterene tubes and analyzed on flow cytometer. Flow cytometery analyses were done on FACSCalibur (Becton Dickinson, San Jose, CA) and data were analyzed using CellQuest analysis software.
51

Total RNA was isolated using the standard TRIzol method (Gibco BRL). Briefly, 3x10 CellS Were treated With different Concentrations of the compounds as indicated in the
legends to Figures and after harvesting the cell pellet was resuspended in 1 ml of TRIzol with repeated pipetting. The homogenized sample was incubated at RT for 5 min to permit complete dissociation of the nucleoprotein complexes. 200ul of chloroform was
added and the tubeg were shaken vigorously for IS sec by hand and then incubated at RT
for 3 mins. The samples were then eentrifuged at 10,000 RPM for 15 mins at 4°C. The
aqueous phase was transfered to a fresh tube and 500ul of Isopropyl alcohol was added, followed by incubation at RT for 10 mins. The samples were centrifuged at 10,000 RPM for 10 min at 4°C and the RNA pellet was washed with 1ml of 75% ethanol followed by centrifugation at maximum speed at 4°C for 10 mins. Washing was repeated once more and after removing the supernatant, the RNA pellet was dried and dissolved in 20ul of RNase free water (Promega). To ensure total resuspension, tubes were incubated at 55-60 °C for 10 mins. The samples were aliquoted and stored at -70°C.
Semi-quantitative reverse transcriptase (RT)-PCR
Changes in gene expression were verified by semi-quantitative RT-PCR using GAPDH as an internal normalization standard, lug of total RNA (quantified by spectrophotometer) was used to reverse transcribe into cDNA. One step Access RT-PCR kit (Promega) was used for the synthesis of c-DNA followed by the amplification of the gene of interest using gene specific primers. Briefly, 50 ul reaction mixture including lOjul of 5X AMV/7J7 reaction buffer, 0.2mM dNTP mix, 50pmol each of forward and reverse primers, ImM of MgS04 and lug of RNA sample, was subjected to 28 PCR cycles (First strand cDNA synthesis: reverse transcription at 48°C for 45 minutes, AMV RT inactivation and RNA/cDNA/primer denaturation at 94°C for 2 minutes. Second strand synthesis and PCR amplification: denaturation at 94°C for 30 sec, annealing at primer specific temperature for 1 minute and polymerization at 68 C for 2 minutes. Amplification products were separated by agarose gel electrophoresis (2%) and visualized by by ethidium bromide staining. The primer sequences and product sizes were as follows:
)l

1) Cyclin Dl: 402bp 5'-ACCTGGATGCTGGAGGTCTG-3'{forward}; 5'«GAACTTC- ACATCTGTGGCACA-3'{reverse} [Kwon,Y.K. et al; 2005].
2) GAPDH : 239 bp 5'-TGATGACATCAAGAAGGTGGTGAA-3' (forward); 5'-TCCT-TGGAGGCCATGTGGGCCAT-3' {reverse}.
RESULTS AND DISCUSSIONS
All compounds used in these studies were dissolved in DMSO as lOmM stock solution and further dilutions was made in complete medium. The concentration of compounds
used W tke aWion of exposure had minimal effect on the viability Of to CClb as
determined by trypan blue dye exclusion test (data not shown).
Cytotoxity of L929 cells incubated with supernatant from LPS or LPS + Synthetic
compound stimulated macrophage culture
Tumor necrosis factor - a can cause direct cytotoxicity to the lung fibroblast cell line (L929). Bioassy of TNF a is designated on the basis of this cytotoxicity of TNF a, which
cm be used for the identification of murine TNF a activity in tissue «utee supernal
Culture supernatant collected from the LPS activated macrophage produced almost 76% cytotoxicity as shown in Table 1. Culture supernatant collected from the synthetic molecules treated well containing LPS stimulated macrophage modulated the extent of cytotoxicity to the L929 cells [Table 1]. Most of the compounds were able to reduce the cytotoxicity to some extent. However, cytotoxicity of the L929 cells by TNF a was highly reduced by few of the synthetic molecules which suggest that their treatment could inhibit the production of TNF a from macrophages.
Table 1: Cytotoxity of L929 cells incubated with supernatant from LPS or LPS + Synthetic compound stimulated macrophage culture. TNF sensitive L929 cells were treated with supernatant collected from either LPS or LPS + Synthetic compound treated RAW 264.7 cells as described in the "Materials and Methods". Cell viability was assessed by crystal violet staining. Data is represented as percentage cell survival and each value shows the mean ± S.D. of triplicate samples.
53

TREATMENT PERCENTAGE CELL SURVIVAL ± S.D.
L929+ActD control 100
L929 +ActD + LPS +DMSO 99.078 ± 0.490
L929 +ActD + Sup from LPS treated macrophage 24.682 ±1.242
L929 +Act D +TNF (75pg/ml) control 25.700 ±0.812
L929 +ActD + Sup from(LPS+ compound 1) treated macrophage 73.155 ±1.773
L929 +ActD + Sup from(LPS+ compound 2) treated macrophage 68.003 ±1.531
L929 +ActD + Sup from(LPS+ compound 3) treated macrophage 67.048 ±2.301
L929 +ActD + Sup from(LPS+ compound 4) treated macrophage 69.084 ±1.491
L929 +ActD + Sup from(LPS+ compound 5) treated macrophage 67.812 ±0.866
L929 +ActD + Sup from(LPS+ compound 6) treated macrophage 66.571 ±1.485
L929 +ActD + Sup from(LPS+ compound 7) treated macrophage 65.299 ±1.114
L929 +ActD + Sup from(LPS+ compound 8) treated macrophage 67.844 ±1.251
L929 +ActD + Sup from(LPS+ compound 9) treated macrophage 51.081 ±2.768
L929 +ActD + Sup from(LPS+ compound 10) treated macrophage 51.559 ±1.397
L929 +ActD + Sup from(LPS+ compound 11) treated macrophage 52.417± 1.510
L929 +ActD + Sup from(LPS+ compound 12) treated macrophage 51.304 ± 1.208
L929 +ActD + Sup from(LPS+ compound 13) treated macrophage 50.636 ±1.603
L929 +ActD + Sup from(LPS+ compound 14) treated macrophage 50.954 ±1.908
L929 +ActD + Sup from(LPS+ compound 15) treated macrophage 50.859 ±1.843
L929 +ActD + Sup from(LPS+ compound 16) treated macrophage 49.046 ±1.992
L929 +ActD + Sup from(LPS+ compound 17) treated macrophage 51.877 ±1.429
L929 +ActD + Sup from(LPS+ compound 18) treated macrophage 50.350 ± 1.575
L929 +ActD + Sup from(LPS+ compound 19) treated macrophage 51.590 ±1.575
L929 +ActD + Sup from(LPS+ compound 20) treated macrophage 51.908 ± 1.100
L929 +ActD + Sup from(LPS+ compound 21) treated macrophage 52.290 ±1.251
L929 +ActD + Sup from(LPS+ compound 22) treated macrophage 65.553 ±1.326
L929 +ActD + Sup from(LPS+ compound 23) treated macrophage 51.209 ±1.554
L929 +ActD + Sup from(LPS+ compound 24) treated macrophage 50.286 ±1.284
L929 +ActD + Sup from(LPS+ compound 25) treated macrophage 53.690 ± 2.047
54

L929 +ActD + Sup from(LPS+ compound 26) treated macrophage 58.810 ±2.147
L929 +ActD + Sup from(LPS+ compound 27) treated macrophage 46.533 ±1.020
L929 +ActD + Sup from(LPS+ compound 28)treated macrophage 66.444 ±1.106
L929 +ActD + Sup from(LPS+ compound 29 )treated macrophage 50.604 ±1.492
Effect of Synthetic compounds on the nitrite production by LPS stimulated macrophages.
To estimate the anti-inflammatory effects of all the synthetic molecules listed in this study, we measured the accumulation of nitrite, the stable metabolite of NO, in the culture media using Griess reagent. As listed in the Table 2. LPS drastically increased the levels of NO in the culture medium when compared to the basal levels, and this induction was significantly controlled when LPS treatment was given in the presence lOuM of the synthetic molecules. The concentration of LPS induced nitrite accumulation, with or without the presence of any molecule is listed in Table 2. From the data shown, few of the molecules showed very promising results and in order to carry out further detailed studied we selected one of the best amongst them (CAMVE).
Table 2: Effect of Synthetic compounds on the nitrite production by LPS stimulated macrophages. RAW 264.7 cells were plated at 5xl05cells/ml, and stimulated with LPS (200ng/ml) in the presence or absence of test compounds for 24h. The culture supernatants were subsequently isolated and analyzed for nitrite production as described in the "Materials and Methods". Each value shows the mean ± S.D. of triplicate determinations.

TREATMENT CONCENTRATION OF NITRITEuM ± SD
Macrophage +DMSO 0.782 ±0.102
MACROPHAGE + LPS +DMSO 14.560 ±0.133
MACROPHAGE + LPS + compound 1 1.849 ±0.214
MACROPHAGE + LPS + compound 2 2.404 ±0.168
MACROPHAGE + LPS + compound 3 3.022 ± 0.300
MACROPHAGE + LPS + compound 4 3.093 ± 0.437
MACROPHAGE + LPS + compound 5 3.449 ±0.315
55

MACROPHAGE + LPS + compound 6 3.204 ±0.168
MACROPHAGE + LPS + compound 7 3.760 ±0.546
MACROPHAGE + LPS + compound 8 3.893 ±0.133
MACROPHAGE + LPS + compound 9 7.582 ±0.868
MACROPHAGE + LPS + compound 10 6.849 ± 0.806
MACROPHAGE + LPS + compound 11 5.604 ± 0.668
MACROPHAGE + LPS + compound 12 9.582 ±0.482
MACROPHAGE + LPS + compound 13 8.004 ± 0.379
MACROPHAGE + LPS + compound 14 8.382 ±0.278
MACROPHAGE + LPS + compound 15 8.271 ±0.342
MACROPHAGE + LPS + compound 16 8.449 ± 0.234
MACROPHAGE + LPS + compound 17 8.404 ±0.315
MACROPHAGE + LPS + compound 18 7.960 ± 0.437
MACROPHAGE + LPS + compound 19 8.227 ±0.115
MACROPHAGE + LPS + compound 20 8.493 ± 0.267
MACROPHAGE + LPS + compound 21 8.293 ± 0.371
MACROPHAGE + LPS + compound 22 3.982 ±0.301
MACROPHAGE + LPS + compound 23 8.338 ± 0.204
MACROPHAGE + LPS + compound 24 8.582 ±0.214
MACROPHAGE + LPS + compound 25 7.871 ±0.168
MACROPHAGE + LPS + compound 26 6.138 ±0.567
MACROPHAGE + LPS + compound 27 5.027 ± 0.200
MACROPHAGE + LPS + compound 28 7.604 ± 0.204
MACROPHAGE + LPS + compound 29 8.382 ±0.454
Effect of CAMVE (comound 1) on LPS induced nitrite production
To investigate the effect of CAMVE on NO production, we measured the accumulation of nitrite, the stable metabolite of NO, in the culture media using Griess reagent. To investigate the effect of CAMVE on NO production, Raw 264.7 cells, pretreated with indicated concentrations of CAMVE for 1 h were incubated with LPS (250ng/ml) for 24
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h. As shown in Fig.l, LPS alone evoked nitrite productiontion significantly when compared to the naive control, and this induction was inhibited by CAMVE treatment in a dose-dependent manner.
CAMVE blocks TNF induced ROI generation and Lipid Peroxidation
Previous reports have shown that TNF activates NF-KB through generation of ROI [Manna,S.K. et.al.; 1999, Li,N., and Karin,M.;1999]. It's also been reported that ester derivatives of caffeic acid are known as structural relative of flavanoids and displays antioxidant activity [Kimura,Y.,et.al; 1985]. Whether CAMVE could suppress NF-KB activation through suppression of ROI generation was examined by flow cytometry. As shown in Fig.2A, TNF induced ROI generation was suppressed on pretreatment of cells with CAMVE in a dose dependent fashion. Because lipid peroxidation has also been implicated in TNF- induced NF- KB activation [Bowie,A.G. et.al.; 1997], we also examined the effect of CAMVE on TNF induced lipid peroxidation. Results in Fig.2B show that TNF induced lipid peroxidation in Jurkat cells, and this was significantly suppressed by CAMVE in a dose dependent manner. Thus, it is quite likely that CAMVE could prevent oxidative stress induced by various agents and also block TNF signaling through suppression of ROI generation and lipid peroxidation.
CAMVE Inhibits TNF or LPS induced NF-i^B Activation
Jurkat cells were pretreated with the indicated concentrations of CAMVE for 3 h and then stimulated with 0.1 nM TNF or lOOng/ml SA-LPS for 30 min; nuclear extracts were prepared and assayed for NF-K;B by using ELISA based method. As shown in Fig. 3, TNF activated NF-KB almost 4.35 fold (when probed with antibody against p65) and 4.90 fold (when probed with antibody against p50), and CAMVE inhibited this activation in a concentration dependent manner, with maximum inhibition achieved at 15 uM. Similarly, LPS induced NF-KB activation was also blocked by CAMVE as shown in the Fig.3. This result also suggests that CAMVE may act at a step where TNF and LPS converge in the signal transduction pathway.
Various combinations of Rel/NF- KB proteins can constitute an active NF-KB heterodimer that binds to specific sequences in DNA. In this assay, the use of specific antibodies
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against p65 and p50 subunits of the NF-K;B heterodimer bound to it's specific oligo coated wells, suggests that the TNF-activated complex consisted of p50 and p65 subunits of the NF-K;B transcription factor. Furthermore, the use of competitor oligos having the same DNA sequence as the oligo-coated wells, decreases the signal because NF-KB binding decreases as it competes away from the oligo-coated surface of the TransFactor well, indicating the specificity for NF- K,B.
Inhibition ofNF-nfi Activation by CAMVE is not Cell type Specific
As NF- KB activation pathways differ in different cell types, we therefore studied whether CAMVE affects other cell types as well. It has been demonstrated that distinct signal transduction pathways could mediate NF- K;B induction in epithelial and lymphoid cells [Bonizzi,G., et.al;1997]. All the effects of CAMVE was mainly carried out in Jurkat, a human T cell leukemia. In another set of experiments, we found that CAMVE blocks TNF- induced NF- KB activation in HeLa (human cervical cancer), MCF-7 (human breast cancer), U937 (human histiocytic lymphoma) cells as shown in Fig.4A.these results suggest that the effect of CAMVE is not restricted to leukemic T cells but also suppresses NF- K;B activation in other cell types.
As all the cell lines tested are of human origin, we also examined the effect of CAMVE on LPS induced NF- KB activation in murine RAW 264.7 cells. Results shown in Fig.4B, indicates that CAMVE inhibited NF- K;B activation in murine cells too and it's potency was not significantly different from that of human cells. Furthermore, it is well known that NF- KB is an important target for the inducibility of iNOS gene expression by LPS, the inhibition of LPS (lOOng/ml) induced NF- KB activation by CAMVE is very much consistent with the NO (Fig. 1) data. Thus the inhibition of LPS induced NF- KB activation positively correlates to the degree of inhibition of Nitric oxide (NO) production induced by LPS in RAW 264.7 cells.
CAMVE inhibits TNF or LPS induced nuclear translocation ofp6S
Analysis of the p65 translocation was done using flow cytometry and immunofluorescence. Inhibition of TNF induced p65 nuclear translocation by CAMVE in HeLa cell line was also proved by immunofluorescence wherein CAMVE pretreated cells
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did not show p65 signal, otherwise shown by TNF alone treated cells, in the nucleus Fig. 5A.
Flow cytometry analysis of NF- KB translocation in nuclei purified from treated cells is illustrated in Fig. 5 B. Nuclei extracted from Jurkat cells pretreated with CAMVE and stimulated with (O.lnM)TNF or (lOOng/ml) SA-LPS were stained for p65. Staining of p65 in the nuclei of unstimulated cells differed only slightly from the isotype control, indicating a low basal activity of the cells. Basal values were not altered by incubation with CAMVE alone. In our assay, TNF or LPS significantly increased p65 translocation compared to the untreated control (7 fold in case of TNF and 6 fold in case of LPS). The TNF or LPS mediated p65 translocation was blocked with CAMVE pretreatments as shown in Fig.5B. Nuclei population was gated on the basis of PI staining (lug/ml), after doublet elimination by FL-2 Area vs FL-2 Width measurements.
CAMVE inhibits NF- K,B regulated expression of genes associated with Inflammation and Carcinogenesis
Because CAMVE has shown to inhibit TNF and LPS induced NF- KB activation, we examined the expression of NF- KB regulated genes for example adhesion molecule ICAM 1 and COX 2 both known to be major players during inflammation. Cells treated with different concentrations of CAMVE for 3 h and then stimulated with either TNF (0.1 nM) or SA-LPS (lOOng/ml)) for 12 h. After harvesting the treated cells, equal amounts of the cellular lysates were checked for COX-2 protein expression by using COX-2 ELISA kit (Zymed). As shown in Fig.6, COX2 expression induced by NF-K;B activating agents was decreased with increasing concentration of CAMVE treatment. CAMVE alone did not show any induction of COX 2 protein.
Adhesion molecule ICAM 1 expression on TNF or LPS stimulated cells was analyzed by FACS. Cells were pretreated with CAMVE for 3 h and then incubated with TNF (0.1 nM) or SA-LPS (lOOng/ml) for 12h. As shown in Fig.7, TNF or LPS stimulated cells showed a clear-cut increased in ICAM1 expression compared to the untreated control. This induced expression was blocked in CAMVE pretreated cells as shown in the figure. Basal ICAM1 expression was not altered by incubation with CAMVE alone.
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CAMVEpotentiates apoptotic effects of TNF and chemotherapeutic agents
Because NF-K;B regulated gene products, known to have antiapoptotic properties, can also suppress TNF and chemotherapy induced apoptosis, we examined the effects of CAMVE on the apoptotic effects of TNF and other chemotherapeutic drugs. Jurkat cells were treated with variable concentrations of TNF for 72 h either in the absence or presence of lOuM of CAMVE and then examined for cytotoxicity by the MTT method. Results in Fig.8Al,show that the cytotoxic effects of TNF in Jurkat cells were dose dependent and it was further potentiated by treatment of cells with lOuM of CAMVE. To show that the cell death mediated by CAMVE was apoptosis and not necrosis, caspase activation in the form of PARP cleavage was examined using FACS. As shown in Fig.8A2, TNF induced 16.28% of the cells to undergo apoptosis, and CAMVE pretreated cells showed significant potentiation of PARP cleavage in a dose dependent manner. Furthermore, in order to know the effect of chemotherapeutic drugs on NF-KB activated cells, SA-LPS(100ng/mi)activated Jurkat cells were pretreated with lOuM CAMVE for 3 h, were incubated with 1 uM each of cis-platin, doxorubicin, taxol or vincristine for 72 h and cell viability was assessed by the MTT method. As shown in Fig.8B., cytotoxicity induced by various chemotherapeutic agents in NF-KB expressing cells was significantly enhanced by CAMVE pretreatment.
CAMVE induced differential cytotoxicity in different tumor cell lines
As polyphenolic compounds at higher concentrations are also known to alter the redox state and induce apoptosis in transformrd cells [Chaio et al., 1995], we investigated as to whether CAMVE at higher concentration (30 uM ) is also able to mediate cell death in tumor cells derived from different tissue background. The viability of cells after 72 h with or without CAMVE treatment was analyzed using the MTT assay. As shown in Fig.9 it is clear that CAMVE mediated cell death is very much cell type or lineage dependent. U937 (human histiocytic lymphoma) cell line seem to be most sensitive to CAMVE treatment. Thus, CAMVE mediated cytotoxicity is more seen in U937 cells.
CAMVE induces delayed cell cycle progression Polyphenolic compounds are also known to exert their anti-cancer properties by modulating cell cycle progression. To
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verify whether CAMVE modulates cell growth, we examined the effects of different concentrations of CAMVE on the cell cycle distribution of Jurkat cells. According to the cell cycle analysis, Fig.lOA shows that the treatment of Jurkat cells with as low as 5 uM dose of CAMVE for 24 h, resulted in a significant increase of cells in the Gl and compensatory decrease in the S phase of the cell cycle. These results suggest that CAMVE can induce a delayed cell cycle progression during Gl/S transition, resulting in decreased cell proliferation rates.
The transition of Gl/S phase is positively regulated by cell cycle regulatory proteins such as cyclin Dl, cyclin E, cdk2 and cdk4 [Kwon.Y.K. et.al. 2005]. Cyclin Dl is a proto-oncogene that is over expressed in many cancer cell types and known to play a role in cell proliferation through activation of cyclin-dependent kinases [Mukhopadhyay, A. et al.,2002]. Because cyclin Dl plays important roles in progression of Gl phase into the S phase, we investigated the effect of CAMVE on the expression of cyclin Dl mRNA expression using RT-PCR analysis. When cells were treated with various concentrations of CAMVE for 18 h, the expression of cyclin Dl m-RNA was notably decreased as shown in Fig.lOB. Thus, downregulation of cyclin Dl mRNA by CAMVE leads to decreased cell proliferation, supporting the idea that CAMVE can also be used as a promising chemopreventive agent.
BIOLOGICAL EVALUATION OF COMPOUND 27 Cell Lines
The cell lines used in this study were as follows:, Jurkat (human T cell leukemia), MCF-7
(human breast cancer cell line), U-937 (human histiocytic lymphoma) HeLa (human
cervical cancer cell line); they were obtained from American Type culture collection
(Manassas, VA, USA). L929, U-937, Jurkat was cultured in RPMI 1640, while others in
DMEM supplemented with 10% FBS, penicillin (lOOOU/ml), and streptomycin
(lOOug/ml).
Materials
All synthetic chemicals were obtained from commercial sources. Propidium Iodide (PI), tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
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caspase 3 substrate (Ac-DVED-pNA), DMSO etc were obtained from Sigma Aldrich Chemicals (St Louis, MO, USA). Penicillin, streptomycin, neomycin, RPMI 1640 and DMEM medium, fetal bovine serum (FBS) were obtained from Gibco BRL., anti PARP-FITC conjugate was from Novus Biologicals, Monoclonal Anti-Phosphotyrosine FITC conjugate was from Sigma Aldrich (Saint Louis, MO, USA). One step Access RT-PCR kit was purchased from Promega.
Cytotoxicity assay
Cytotoxicity was assed by the modified tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described by Plumb et al 1989.
Cell Cycle Analysis
Cells (log phase culture) were treated with vehicles alone (similar volumes of DMSO) or with the compound (various concentrations) to be tested for 24 h. Untreated cells were also included in this experiment for comparison. After treatments, the cells were harvested and washed with cold PBS. Cells were then resuspended in cold PBS (300ul) and 100% chilled ethanol (700 ul), vortexed, and incubated at room temperature for 1 h. Samples were centrifuged at 200 x g for 5 minutes and the supernatant was removed. A solution containing propidium iodide (100 ng/ml) and RNase A (lmg/ml) was added to the samples and incubated for 1 h at room temperature. Samples were then transferred to 12x75 mm polysterene tubes and analyzed on flow cytometer. Flow cytometery analyses were done on FACSCalibur (Becton Dickinson, San Jose, CA) and data were analyzed using CellQuest analysis software.
Caspase 3 activity assay
To evaluate caspase 3 activity, cell lysates were prepared after their respective treatments with the compounds. 200ug of the cell lysates were incubated with 50 uM caspase 3 substrate (Ac-DVED-pNA) in lOOul reaction buffer (1% NP-40, 20uM tris-HCl, pH7.5, 137mM NaCl, and 10% glycerol) and incubated for 2 h at 37°C. The release of chromophore pNA was monitored spectrophotometrically at 405nm.
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PARP Cleavage Assay using Flow Cytometry
Extent of PARP cleavage is determined using polyclonal antibody specifically recognizing the 85 kDa fragment of cleaved PARP (NSB 699 Novus Biologicals) and can be used as a marker for detecting apoptotic cells. Treated cells were fixed with 70% chilled ethanol and permeabilized for 30 min at RT ( PBS + 0.5% BSA + 0.02% NaN3 + 0.5% saponin) and stained with anti PARP-FITC (10ul/ 106 cells) for one hour at RT. Cells were washed twice with wash buffer (PBS + 1% heat inactivated FBS) and analysed using FACS.
DNA fragmentation Assay
After treatment with the compounds for 24 h, cells were harvested and washed in PBS. The cell pellet was incubated with lysis buffer (lOmM Tris-HCl pH 7.5, ImM EDTA, 1% SDS and 80ug/ml proteinase K) at 37°C overnight. After extraction with phenol/chloroform, the DNA was precipitated with 100% ethanol and then dissolved in Tris-EDTA buffer (pH 8.0) with RNase A at 37°C. The DNA estimation was performed by taking absorbance at 260 / 280 nm, and DNA was resolved in a 1.8% agarose gel, stained with ethidium bromide and visualized under a UV transilluminator.
Nuclear staining assay
After treatment with the compound for 24 h, Jurkat cells were washed once with ice-cold EDTA/PBS (5mM) and fixed with 70% ethanol for 1 h at RT. Fixed cells were placed on slides and stained with PI (5 |4.g/ml) with RNase A (lmg/ml) for 20 mins. After decolourization with water, nuclear morphology of cells was examined by fluorescence microscopy.
RNA extraction
Total RNA was isolated using the standard TRlzol method (Gibco BRL). Briefly, 3 x 10 6 cells were treated with different concentrations of the compounds as indicated in the legends to Figures and after harvesting the cell pellet was resuspended in 1ml of TRlzol with repeated pipetting. The homogenized sample was incubated at RT for 5 min to permit complete dissociation of the nucleoprotein complexes. 200ul of chloroform was
63

added and the tubes were shaken vigorously for 15 sec by hand and then incubated at RT for 3 mins. The samples were then centrifuged at 10,000 RPM for 15 mins at 4°C. The aqueous phase was transfered to a fresh tube and 500ul of Isopropyl alcohol was added, followed by incubation at RT for 10 mins. The samples were centrifuged at 10,000 RPM for 10 min at 4°C and the RNA pellet was washed with 1ml of 75% ethanol followed by centrifugation at maximum speed at 4°C for 10 mins. Washing was repeated once more and after removing the supernatant, the RNA pellet was dried and dissolved in 20ul of RNase free water (Promega). To ensure total resuspension, tubes were incubated at 55-60 °C for 10 mins. The samples were aliquoted and stored at -70°C.
Semi-quantitative reverse transcriptase (RT)-PCR
Changes in gene expression were verified by semi-quantitative RT-PCR using GAPDH as an internal normalization standard, lug of total RNA (quantified by spectrophotometer) was used to reverse transcribe into cDNA. One step Access RT-PCR kit (Promega) was used for the synthesis of c-DNA followed by the amplification of the gene of interest using gene specific primers. Briefly, 50 ul reaction mixture including lOul of 5X AMVITfl reaction buffer, 0.2mM dNTP mix, 50pmol each of forward and reverse primers, ImM of MgSCM and lug of RNA sample, was subjected to 28 PCR cycles (First strand cDNA synthesis: reverse transcription at 48°C for 45 minutes, AMV RT inactivation and RNA/cDNA/primer denaturation at 94 C for 2 minutes. Second strand synthesis and PCR amplification: denaturation at 94 C for 30 sec, annealing at (primer specific temperature) for 1 minute and polymerization at 68 C for 2 minutes. Amplification products were separated by agarose gel electrophoresis (2%) and visualized by by ethidium bromide staining. The primer sequence and product size are as follows: [ Louis,M. et al; 2004] p53: 435bp
5'-ATTCTGGGACAGCCAAGTCT-3' {forward} 5'-GGAGTCTTCCAGTGTGATGA-3' {reverse} bcl-2:127 bp
5'-CTGTGGATGACTGAGTACCT-3' {forward} 5'GAGACAGCCAGGAGAAATCA-3' {reverse}
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Bax-a: 489 bp
5'-GTTTCATCCAGGATCGAGCA-3' {forward} S'-CCATCnCTTCCAGATGGTG-S' {reverse} GAPDH : 239 bp
5'-TGATGACATCAAGAAGGTGGTGAA-3'{forward) 5'-TCCTTGGAGGCCATGTGGGCCAT-3' {reverse}
Tyrosine Phosphorylation assay
Tyrosine Phosphorylation assay was performed by the method described by Far, D.F. et.al., 1994 and ParkJ.B. et.al., 2003. Briefly, cells (106) were washed with ice-cold PBS, pH 7.2 for 30 min at 4°C. After centrifugation and a PBS wash it was treated with 5ml chilled 70 % ethanol. The fixed cells were recovered by centrifugation followed by washing with PBS. The cells were permeabilized with saponin (0.05% in PBS) for 10 min at room temperature. Non specific binding was blocked by incubating the cells for 30 min in PBS, pH 7.6 containing BSA 0.1% and 0.1% (v/v) Tween 20. Thereafter the cells were stained with 20ug/ml of FITC-conjugated anti-phosphotyrosine antibody for 30 min. Extent of tyrosine phosphorylation in the cells was determined by measuring the increase in fluorescence produced by the FITC-labeled monoclonal antibody compared to the FITC-labeled isotype control antibody. Fluorecence events for 10,000 cells were collected and analyzed by flow cytometry (FACSCalibur cytometer with CellQuest software, Becton Dicinson, San Jose, CA).
RESULTS AND DISCUSSIONS
Cell viability and growth inhibition
Because Benzofuran lignans has been reported to contain antiproliferation activity against human tumor cells, the activity of our benzofuran lignan derivative (compound 27) was investigated to determine whether it is capable of inhibiting cell growth of human cancer cells. Jurkat cells were used because this cell line has been used extensively for investigating growth proliferation and cell cycle progression in various cancer studies. The cells were treated for 24 h and 48 h with various concentrations of the compound. As shown in Fig.ll the number of living cells decreased with the increasing concentration of
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the compound. The GI50 representing the concentration of compound causing 50% growth inhibition compared with control cells is approximately around lOOnM.
Effect on Cell Cycle
To understand the mechanism of action, this novel apoptosis-inducing compound was evaluated for its effect on cell cycle by measuring DNA content. We used flow cytometric analysis after treatment of Jurkat cells with varying doses of the compound. As shown in Fig.12 compound specifically arrested cells in the G2-M phase of the cell cycle leading to significant apoptosis as shown in the sub-Gl content. The data shown here confirms that the compound dose as low as lOOnM is effective in significant increase (-50%) of cells in the G2/M phase of the cell cycle. At 50nM (data not shown) no significant increase in G2/M population was achieved. At concentration higher than lOOnM there was further increase in both G2/M and sub Gl population.
Time dependent effects of the G2/Mpromoting doses of compound 27 on the cell, cycle distribution
The time - dependent effects on the cell cycle after treatment with lOOnM and 500nM of the compound over 24h, 48h, 72h time duration was evaluated. As shown in Fig. 13, cells treated with both the concentrations accumulated in G2/M after 24 h with a significant decrease in the Gl and S phase populations. However,after 48 and 72 h of treatment there was a significant decrease in the G2/M population in the cells treated with both doses, followed by an increase in the S and Gl phase population.
Compound 27 induced caspase activation
Caspases are important mediators of apoptosis induced by various apoptotic stimuli. Induction of cell death predominantly occurs after the G2/M cell cycle block in cancer cells. Cell death is related to cellular and molecular events in the cells and occur via two independent cell death processes i.e. necrosis and apoptosis [Park,J.B. et al;2003]. Necrotic cell death is an accidental cell death that does not require any cellular and molecular mechanism and leads to inflammation and tissue injury. Apoptosis, however, does require programmed cellular or molecular events, such as activation of key proteases
66

like caspases. In this study the proteolytic activity of caspase 3 was investigated and quantified by an in vitro assay based on the proteolytic cleavage of DVED-pNA by caspase 3 into the pNA. As shown in Fig.14, Jurkat cells demonstrated a dose dependent increase in DVED-pNA cleavage after 16 and 24 h exposure to the compound. The activity at 24 h was more than the activity at 16 h. This data clearly suggests that G2/M arrest induces caspase activation in cells after treatment with the compound.
Compound 27 induced PARP cleavage and apoptosis is time and dose dependent
An important factor in inducing apoptosis is the enzyme Poly (ADP - ribose) polymerase (PARP) that has been widely studied in vitro. An early transient burst poly (ADP-ribosyl)ation of nuclear proteins was recently shown to be required for apoptosis to proceed in various cell lines followed by cleavage of poly (ADP-ribose) polymerase (PARP), catalyzed by caspases. As shown in Fig.15, significant PARP cleavage was achieved at lOOnM and 500nM and degree of PARP staining increased with time. The PARP cleavage data is very much consistent with the caspase 3 data. Magnitude of caspase 3 activation and sub Gl apoptotic population positively correlates to the degree of PARP cleavage. These data suggests strongly that the compound induces cell death via apoptotic processes.
Compound 27 induced apoptosis in Jurkat cells.
In order to asses the nature of apoptosis induced by the compound, cells treated for 24 h with different concentratons of the compound were examined for their nuclear morphology after propidium iodide staining. As shown in Fig. 16. nucleic acid staining with propidium iodide revealed typical apoptotic nuclei in compound treated cells, but control cells did not show any features of apoptosis. Another hallmark of apoptosis is the degradation of chromosomal DNA at internucleosomal linkages. DNA fragmentation induced by the compound in Jurkat cells was analyzed. Following agarose gel electrophoresis of Jurkat cells treated with various concentrations of the compound for 24 h, a typical ladder pattern of internucleosomal fragmentation was observed (Fig.16). Also the extent of apoptosis was analyzed from the cell cycle data (Fig.12), showing a
markedly increased accumulation ofsub Gl phase population.
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Compound 27 differential cell cycle arrest in cells with differentp53 status.
Since p53 is known to control the G2/M checkpoint, cell differentiation and apoptosis
[Schwartz!)., et al; 1957], we asked whether our compound acted by activatiAB Of a p53 dependent pathway. In order to compare the effects of p53 on compound sensitivity in
tumor cell lines, we next assessed the response to ike comp6und in U937 cells known to
have mutant inactive p53 status. U937 cells were treated for 24 h with 100, 500, 5000, 10000 IlM f/f the compound and cell cycle analysis was performed. As shown in Fig.17,
no significant increase in the proportion of cells in the G2/M phase of the cell cycle was
Seen at any Of the doses. There was also no increase in the sub Gl population of the cells
treated with 100 and 500nM of the compound, clearly stating that these doses were unable to modulate the G2/M checkpoints or cause apoptosis in U937 cell line. However, at higher doses i.e. 5 and 10 uM, there was an increase in the proportion of cells in the S nllflSC Of tll& Wll &Y^ (fr°m ~26% - 35%) accompanied by a slight compensatory
decrease in Gl phase cells. At 10 uM, the sub Gl population increased tO almOSt 30% indicating apoptosis. Thus, these data suggests that the of accumulation of Jurkat cells in the G2/M phase after treatment with lower doses of the compound may indicate that this checkpoint is operational at the G2/M border in Jurkat cells, whereas this checkpoint is not operational in the U937 cell line. This checkpoint may be p53 related, because Jurkat cells have normal p53, whereas U937 cells have mutant inactive p53. However, at higher doses, U937 cells shows a S phase arrest and induction of apoptosis, probably in a p53 independent fashion.
Effect of compound 27 on p53 mRNA expression.
Given the relevance of p53, to the development of the cell cycle arrest and apoptotic response, we next examined it's response to the compound by semi-quantitative RT-PCR in two cell lines with different p53 status. The sensitivity of gene expression in Jurkat (wild type p53) and HeLa (very low level of p53) after treatment with various doses of the compound for 12 h is shown in Fig.18 A. In HeLa cells, significant induction of p53 transcription was seen in a dose dependent fashion. However, there was only slight increase in p53 mRNA level in case of Jurkat cell line. The p53 proapoptotic factor acts
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as a iranscnptional regulator for many pes anil couiu circa to transcription of some of
the other genes involved in the apoptotic pathway [Mansilla, S.et.al; 2003].
Effects on p53 regulated mRNA expression ofbcl-2 and box
p53 is known to regulate the expression of the apoptosis regulating proteins. We investigated the expression of the pro and anti- apoptotic proteins Bax and Bcl-2 in
COmpOld n trCatCu CellS, A3 5hOWn in rig.l8B, semiquantitative RT-PCR analysis
Indicated that the anti-apoptotic bcl-2 mRNA leVeds \»m dOWnregUlated ifl a dOS8 dependent manner by the compound 27. On the other hand, exposure to increasing concentrations of the compound increased the pro-apoptotic bax mRNA levels, in Jurkat cells.
Compound 27 induced differential levels ofapoptois in cells with differentp53 status.
In order to prove that the growth arrest and apoptosis of cancer cells caused by the compound were indeed p53 dependent, we investigated the extent of apoptosis in different cell lines having different p53 status. As shown in Fig.19, the level of apoptosis in MCF-7 was comparable to that of Jurkat cell line. On the other hand HeLa cells known to have very low levels of p53 expression also shows significant induction of apoptosis after treatment with lOOnM of the compound for 24h. This observation is positively correlated with the data shown in Fig 18 A, stating that the compound treatment also leads to an induction in p53 mRNA expression levels in the HeLa cell line. U937 cell line having mutant inactive p53 status does not show induction of apoptosis with the treatment of this compound. Thus, this data clearly proves the significance of p53 in controlling the G2/M checkpoint and subsequent induction of apoptosis. Most of the breast and lung tumors are known to have wild type p53 expression,hence compound 27 would be effective for the killing of such tumor cells.
Suppression of constitutive tyrosine posphorylation by compound 27
The phosphorylation of tyrosine residues of proteins is assumed to be involved in abnormal growth of human tumor cells [Lui,V.W. et.al. 2002]. As reported earlier, chemotherapeutic compounds may act partly through inhibition of protein tyrosine
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phosphorylation to arrest cell cycle progression and induce apoptosis [Chen.H.W., and Huang,H.C; 1998] . Since leukemic and breast cancer cells are reported to have a high level of constitutive phosphotyrosine levels, the effect of this compound on the inhibition of protein tyrosine phosphorylation was investigated. Total tyrosine phophorylation in Jurkat cells was determined by flow cytometry with FITC-labled monoclonal antibody against phosphotyrosine. As shown in Fig.20, the phosphotyrosine level was reduced at all doses. At lOOnM dose itself, significant reduction in the mean fluorescence intensity was achieved when compared with the untreated controls. This data suggests that the ability of the compound to reduce the constitutive phosphotyrosine levels could be one of the mechanisms in regulating cell proliferation and causing cell death. As previously reported [Chen, Z.P.I996], significant induction of p53 message was seen when phosphotyrosine levels were reduced in HeLa cells, giving an additional explanation for the induction p53 expression and regulation of genes involved in cell cycle checkpoints and apoptosis of tumor cells.
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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

Dated this 13th day of October- , 2006
For Reliance Life Sciences Pvt. Ltd.


ABSTRACT
The present invention relates to novel compounds, their methods of preparation and use as chemotherapeutic and anti-inflammatory agents. The present invention relates to esters of compounds particularly cinnamic acid, Vanillic acid and 4-hyrdoxy cinnamic acid derivatives as represented by formulas I, II and III or mixtures thereof optionally their salts when they exist, and preparations thereof. Further the present invention also discloses compounds with novel benzofuran lignan structure as a potent antimitotic agent and inducer of apoptosis. The compounds of the present invention are anti-inflammatory and can also be used for treatment of cancer.