Vol.04, Issue 01, January 2019 Available Online: www.ajeee.co.in/index.php/AJEEE A STUDY ON COMBRETASTATIN ANALOGUES ALONG WITH RELATED COMPOUNDS
AND EVALUATED THEM FOR ANTICANCER AND ANTIANGIOGENIC ACTIVITY JV’n Richa Bajaj,
Research Scholar (Pharmaceutical Sciences) Dr. Anurekha Jain,
Guide, Jayoti Vidyapeeth Women’s University, Jaipur (Rajasthan) 1 INTRODUCTION
Combretastatins are natural compounds isolated by Pettit and co-workers (1982) from the bark of the South African bush willow tree Combretum caffrum. Out of various stilbene derivatives (termed as combretastatins) isolated from the plant, combretastatin A-4 (CA-4) was found to be most potent (C.M. Lin 1988). CA-4, cis- 1-(3,4,5-trimethoxyphenyl)-2-(3’-hydroxy- 4’-methoxy phenyl) ethene, (1) is active in cis form (G.R. Pettit 1998, 1999). From the structure-activity relationship (SAR) point of view, CA-4 belongs to the class of natural compounds related to biphenyls and contains, as a key structural feature, the cis-stilbene motif. CA-4 exerts a potent cytotoxicity against a variety of human cancer cells including multi drug resistant (MDR) cancer cell lines (E.
Hamel 1983, G.R. Pettit 1989, A.T.
Mcgown 1989, N.H. Nam 1990, 1990, 2003, A.A.E. Al-Zayat 1993, Y. Xia 1998), and also a broad range of preclinical tumor models displays vigorous antitumor (G.R. Pettit 1982, G.R. Pettit 1987, 1995, C.M. Lin 1988, 1989, O.
Boye 1992) as well as substantial antivascular (antiangiogenic) activity in tumor blood flow while causing no significant blood flow retention in normal tissues (D.J. Chaplin 1996, 1999, G.G.
Dark 1997, G.R. Pettit 2000, S. Liekens 2001, G. M. Tozer 2002, P.E. Thorpe 2003). CA-4 does not show in vivo efficacy due to its poor pharmacokinetics resulting from its high lipophilicity and low aquous solubility (K. Oshumi 1998) and also due to isomerization of cis- double bond to the more thermally stable trans-isomer, which is inactive (G.R. Pettit 1998, 1999).
O C H3
O CH3 O
CH3 O
CH3 OH A
B
Fig. 1, (a)(b)
(a) Structural formula and (b) 3D- structure of CA-4.
Till date various Combretastatin analogues have been synthesized and reported to posses cytotoxic activity against several cancer cell lines including murine P-38, murine colon-26, 38,3LL adenocarcinoma, human lung carcinoma A-549, NCI-H522 non-small-cell lung cancer, DMS-273 small-cell lung cancer, COLO-205 colon cancer, human colon adenocarcinoma HT-29, human colon carcinoma HCT-116, human NUGC-3 stomach, MKN-45 stomach, MESSA uterine, MCF-7 breast carcinoma, SKMEL-5 melanoma, MLM melanoma, M- 14 melanoma, leukemia HL-60 cells, CEM leukemia, early pre-B acute lymphoblastic leukemia (Reh), diffuse large cell lymphoma (WSU-DLCL2), chronic lymphocytic leukemia (WSU-CLL), waldenstrom's macroglobulinemia (WSU- WM), apoptosis resistant and BCR-ABL- expressing K-562 cell line, p-glycoprotein expressing cell lines (HL-60R and K- 562ADR), NCI-H460, HCT-15, murine M- 5076 reticulum sarcoma, SF-295 CNS cancer, OVCAR-3 ovarian cancer, CAKI-1 renal cancer and ACHN renal cancer (M.
Cushman 1991, 1993, K. Ohsumi 1998, S.M. Nabha 2000, L. Wang 2002, C. Bailly 2003, J.P. Lieu 2004, A.B.S. Maya 2005, D. Simoni 2005).
2 REVIEW OF LITERATURE
K Aleksandrzak et. al. [1] presented Series of diaryl ethers, amines and amides have been synthesized and tested for antitumor activity. These diaryl
Vol.04, Issue 01, January 2019 Available Online: www.ajeee.co.in/index.php/AJEEE compounds possess some of the
structural features of combretastatin A-4 (a potent antimitotic agent). They were designed to discover whether transferring these structural motifs from stilbenes to hetero substituted diaryl compounds would enhance their biochemical activities. Molecular modeling studies suggested that these diaryl compounds could adopt conformations similar to combretastatin A-4. However, although some agents (5-7) were cytotoxic and others (10 and 12) could interact with tubulin, none were as potent as combretastatin A-4.
Christian Bailly et. al. [2]
proposed the two 4-phenylcoumarin derivatives 3 and 8 have been identified as potent cytotoxic agents. The 3′-OH and a 4′-OCH3 substituents on the 4-phenyl C-ring play an essential role in the cytotoxic action. Compounds 3 and 8 have no effects on human topoisomerases I and II but potently inhibit microtubule assembly. They can therefore be considered as functional analogues of combretastatin A-4, validating thus the design strategy. At the cell level, the two compounds were characterized as proapoptotic agents, but they can also trigger cell death via a nonapoptotic pathway. Compounds 3 and 8 represent interesting candidates for a preclinical development of novel anticancer agents.
D J Chaplin et. al. [3] revealed we have assessed the vascular effects of vinblastine and four other tubulin binding agents (dolastatin 10, dolastatin 15, combretastatin A1 and combretastatin A4), which are awaiting clinical evaluation. All five agents induce a reduction in tumour blood flow as measured by uptake of RbCI 24 h post drug administration. The degree of reduction ranged from 50% with combretastatin A1 to 90% with dolastatin 10. These reductions were similar to that seen with flavone acetic acid (FAA) and indicate that ant vascular effects are a common feature of tubulin binding agents. We subsequently evaluated whether the blood flow reductions, induced by FAA and vinblastine, could be used to enhance the activity of the bioreductive drug tirapazamine. Since the kinetics and extent of blood flow reductions induced by the agents is comparable, similar therapeutic response was expected. Potentiation was only
evident with FAA, indicating that this effect is not directly related to killing of hypoxic tumour cells induced as a consequence of blood flow reduction.
M Cushman et. al. [4] indicated a series of stilbenes has been prepared and tested for cytotoxicity in the five human cancer cell lines A-549 non-small cell lung, MCF-7 breast, HT-29 colon, SKMEL-5 melanoma, and MLM melanoma. The cis stilbenes 6a-f proved to be cytotoxic in all five cell lines, with potencies comparable to that of combretastatin A-4. These cytotoxic compounds were all potent inhibitors of tubulin polymerization. The corresponding trans stilbenes 7b-f were inactive as tubulin polymerization inhibitors and were significantly less cytotoxic in the five cancer cell lines. In the dihydro series, 8b, 8c, and 8f were inactive as tubulin polymerization inhibitors, while 8a, 8d, and 8e were less active than the corresponding cis compounds 6a, 6d, and 6e. The lack of tubulin polymerization inhibitory activity and cytotoxicity displayed by the phenanthrene 23b, which was synthesized as a conformationally rigid analogue of the lead compound 1, indicates that the activity of the stilbenes is not due to a totally planar conformation. Similarly, inactivity of the conformationally restricted analogue 26 suggests that the biologically active conformation of 1a resembles that of the cis alkene 1. Additional inactive compounds prepared include the benzylisoquinoline series 28-32 as well as the protoberberines 38 and 39.
Shortening the two-carbon bridge of 1a to a one-carbon bridge in the diphenylmethane 20 resulted in a decrease in cytotoxicity and tubulin polymerization inhibitory activity.
Although the corresponding benzophenone 18 was as active as 1a as a tubulin polymerization inhibitor, it was less cytotoxic than 1a, and the benzhydrol 19 was essentially inactive. With the exception of the amide 15c, which displayed low antitubulin activity, all of the phenylcinnamic acid derivatives 14a-c and 15a-f were inactive in the tubulin polymerization inhibition assay. The acid 14b and the ester 15a were cytotoxic in several of the cancer cell cultures in spite of their inactivity as tubulin polymerization inhibitors.
Vol.04, Issue 01, January 2019 Available Online: www.ajeee.co.in/index.php/AJEEE H M He et. al. [5] presented
although certain substituted cis-stilbenes have displayed potent tubulin polymerization inhibitory activity and significant cytotoxicities in cancer cell cultures, these compounds have limited aqueous solubility and are therefore difficult to formulate for in vivo evaluation. A series of water-soluble N- (3,4,5-trimethoxybenzyl)aniline salts has therefore been synthesized in which the olefinic bridge of the stilbenes is replaced by an aminomethylene hydrochloride moiety. A relationship was found between the size of the substituent in the 4- position of the aniline ring and both antitubulin activity and cytotoxicity, such that the smaller the substituent, the greater the potency. The most promising of the newly synthesized compounds was 4- methyl-N- (3, 4, 5-trimethoxybenzyl) aniline hydrochloride, with an IC50 value of 3.5 microM for inhibition of tubulin polymerization and cytotoxicity for a wide variety of cancer cell lines. The cytotoxicities of the benzylaniline hydrochlorides correlated remarkably well with their antitubulin activities.
Cushman Mark et. al. [6]
proposed an array of cis-, trans-, and dihydrostilbenes and some N- arylbenzylamines were synthesized and evaluated for their cytotoxicity in the five cancer cell cultures A-549 lung carcinoma, MCF-7 breast carcinoma, HT- 29 colon adenocarcinoma, SKMEL-5 melanoma, and MLM melanoma. Several cis-stilbenes, structurally similar to combretastatins, were highly cytotoxic in all five cell lines and these were also found to be active as inhibitors of tubulin polymerization. The most active compounds also inhibited the binding of colchicine to tubulin. The most potent of the new compounds, both as a tubulin polymerization inhibitor and as a cytotoxic agent, was (Z)-1-(4-
methoxyphenyl) -2-(3,4,5-
trimethoxyphenyl) ethene (5a). This substance was almost as potent as combretastatin A-4 (1a), the most active of the combretastatins, as a tubulin polymerization inhibitor. Compound 5a was found to be approximately 140 times more cytotoxic against HT-29 colon adenocarcinoma cells and about 10 times more cytotoxic against MCF-7 breast carcinoma cells than combretastatin A-4.
However, 5a was found to be about 20
times less cytotoxic against A-549 lung carcinoma cells, 30 times less cytotoxic against SKMEL-5 melanoma cells, and 7 times less cytotoxic against MLM melanoma cells than combretastatin A-4.
The relative potencies 5a greater than 8a greater than 6a for the cis, dihydro, and trans compounds, respectively, as inhibitors of tubulin polymerization are in agreement with the relative potencies previously observed for combretastatin A- 4 (1a), dihydrocombretastatin A-4 (1c), and trans-combretastatin A-4 (1b). The relative potencies 5a greater than 8a greater than 6a were also reflected in the results of the cytotoxicity assays.
Structure-activity relationships of this group of compounds are also discussed.
Bernard L. Flynn et. al. [7]
revealed multi component coupling of o- iodophenols (or oiodoacetanilides) with terminal alkynes and aryl iodides provides rapid access to potent benzo[b]furan- and indole-based tubulin polymerization inhibitors. These systems represent valuable new leads in the pursuit of anticancer chemotherapies.
Chryso Kanthou et. al. [8]
indicated combretastatin A-4-phosphate (CA-4-P) is a tubulin-binding compound currently in clinical trial as a tumor vascular-targeting agent. In endothelial cells, CA-4-P is known to cause microtubule depolymerization, but little is known about its subsequent effects on cell morphology and function. Here, we demonstrate that within minutes of endothelial cell exposure to CA-4-P, myosin light chain (MLC) was phosphorylated, leading to actinomyosin contractility, assembly of actin stress fibers, and formation of focal adhesions.
These cytoskeletal alterations appeared to be a consequence of Rho activation, as they were abolished by either the Rho inhibitor C3 exoenzyme or Rho-kinase inhibitor Y-27632. In response to CA-4-P, some cells rapidly assumed a blebbing morphology in which F-actin accumulated around surface blebs, stress fibers misassembled into a spherical network surrounding the cytoplasm, and focal adhesions appeared malformed. Blebbing was associated with decreased cell viability and could be inhibited by Rho/Rho-kinase inhibitors or by blocking the CA-4-P-mediated activation of stress- activated protein kinase-2/p38. The extracellular-regulated kinases 1 and 2
Vol.04, Issue 01, January 2019 Available Online: www.ajeee.co.in/index.php/AJEEE (ERK-1/2) were shown to protect against
blebbing since blebbing was attenuated on ERK-1/2 stimulation and was up- regulated by specific inhibition of ERK- 1/2 activation. The use of MLC kinase (MLCK) and myosin adenosine triphosphatase inhibitors led us to propose a role for MLCK and myosin activity independent of MLC phosphorylation in regulating the blebbing process. CA-4-P-mediated contractility and blebbing were associated with a Rho-dependent increase in monolayer permeability to dextrans, suggesting that such functional changes may be important in the rapid response of the tumor endothelium to CA-4-P in vivo.
Sandra Liekens et. al. [9]
presented currently, a large variety of chemotherapeutic drugs are being used to treat cancer. Unfortunately, many compounds hold limited efficacy, due to problems of delivery and penetration and a moderate degree of selectivity for the tumor cells, thereby causing severe damage to healthy tissues. However, the activity of these compounds is mainly restricted by the development of drug resistance. Tumor cells are a rapidly changing target because of their genetic instability, heterogeneity, and high rate of mutation, leading to selection and overgrowth of a drug-resistant tumor cell population [83,181]. Anti-angiogenic therapy, which targets activated endothelial cells, offers several advantages over therapy directed against tumor cells.
First, endothelial cells are a genetically stable, diploid, and homogenous target, and spontaneous mutations rarely occur.
Also, turnover of tumor endothelial cells may be 50 times higher than that of endothelium in normal quiescent tissues, and activated blood vessels express specific markers, like integrin avb3, E- selectin, Tie, and VEGF receptors.
Because anti-angiogenic therapy is directed at activated endothelial cells, its target should be easily accessible by systemic administration. Finally, different tumor cells are sustained by a single capillary, and tumor-associated endothelial cells contribute to both endothelial and tumor cell growth by releasing autocrine and paracrine factors.
Consequently, the activated endothelium presents a more specific target than the tumor cells, and inhibition of a small number of tumor vessels may affect the
growth of many tumor cells [181]. The genomic instability and heterogeneity of tumor cells may also explain the clinical observations that the outcome of patients, with tumors in the same pathological or clinical stage, and their response to anticancer therapy vary considerably.
This points to the importance of establishing an angiogenic profile in patients with cancer and other chronic angiogenic diseases. Indeed, the intratumoral blood vessel density (IVD) was found to be of prognostic value in a variety of solid tumors, including invasive breast [182], lung [183], malignant melanoma, gastrointestinal [184,185], and genitourinary cancers [84]. In these tumors a positive correlation was found between tumor angiogenesis and the risk of metastasis, tumor recurrence, or death.
Furthermore, identification of the angiogenic cytokines or enzymes involved might make it possible in the future to specifically adjust anti-angiogenic therapy to the individual needs of a patient. In this context it should be noted that major progress has been made in the quantitative assessment of measurable parameters directly associated with angiogenesis.
Concepcion Alvarez et. al. [10]
proposed the usually unexplored modification of phenstatins, replacing the 3,4,5-trimethoxyphenyl (A-ring) by the 2,3,4-trimethoxyphenyl arrangement, has been carried out in a series of N-alkyl5- indolyl derivatives. Surprisingly, this modification proved to be very efficient when the keto group is maintained on the bridge and N-methyl-5-indolyl is the B- ring equivalent. The IC50 for the inhibition of tubulin polymerization in the submicromolar range displayed by compound 5 is one of the lower ever described for combretastatin and phenstatin analogues or the reference compounds. When this modification of the A-ring is accompanied by modifications on the bridge, these compounds lack inhibitory activity of tubulin polymerization and cytotoxicity, only maintained for oxime 7. This lack of activity is in agreement with a similar effect observed in previously described analogues maintaining the 3,4,5- trimethoxyphenyl A-ring. As a general trend in this type of compounds, although they display higher inhibition of tubulin polymerization, their cytotoxic effect is
Vol.04, Issue 01, January 2019 Available Online: www.ajeee.co.in/index.php/AJEEE lower than the observed for similar
combretastatins with lower effect on the polymerization of tubulin.
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