Letters in Drug Design & Discovery, 2006, 3, 205-209
205
Synthesis and Biological Activity of New 4 -N-Heteroaryl Analogues of Podophyllotoxin Ahmed Kamal* ,a, B. Ashwini Kumara, M. Arifuddina and Sunanda. G. Dastidarb aDivision
of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad - 500007, India
bRanbaxy
Research laboratory, Gorgaon-122001, Haryana, India Received January 18, 2006: Revised February 21, 2006: Accepted February 23, 2006
Abstract- Some new 4β-N-heteroaryl analogues of podophyllotoxin have been prepared by employing Red Phosphorous/I 2 reagent system. These 4β-N-heteroaryl analogues have been evaluated for their cytotoxicity against six human cancer cell lines and some representative ones have shown promising anticancer activity.
Keywords: Epipodophyllotoxin, benzothiazoles, pyrimidines, anticancer activity. INTRODUCTION
Attempts were made to develop new podophyllotoxin derivatives to produce less toxic side effects and during these efforts, VP-16-213 (etoposide) and VM-26 (teniposide) have emerged with potential anticancer activity. Etoposide and teniposide have shown activity for a large number of cancers [9-15]. However, there are certain limitations with the usage of these drugs. To overcome the problems of the drug
The long and fascinating history of podophyllotoxin has attracted the attention of many researchers largely because of the potent anticancer activity of etoposide and teniposide. The semisynthetic derivatives of podophyllotoxin are in clinical use for the treatment of a variety of malignant conditions [1-3]. As part of combination chemotherapy, H R OH O
O O HO
O O OH O
O O
O O
O
H3 CO
O
OCH3
H3CO
OCH3
OCH3 Podophyllotoxin
OH R= CH3
Etoposide
R= Teniposide S
etoposide has become almost a standard in therapies for testicular cancer and small cell lung cancer. Different aspects of the anticancer activity of etoposide against various cancers have been reviewed [4-8].
resistance and poor water availability and to improve their clinical efficacy many researchers have synthesized not only their analogues [16,17] but also prepared structurally modified podophyllotoxin based compounds.
Podophyllotoxin and many of its closely related lignans have attracted considerable interest as human cancer chemotherapeutic agents. Podophyllum and its resin has been used as a folk medicine for the treatment of cancer for over 1000 years.
The structure activity relationship suggested that 4'epimerization and the substituent at 4-position plays an important role [18]. These aspects are equally important even for the DNA topoisomerase II inhibition activity as seen from some of the structurally modified 4-substituted epipodophyllotoxin derivatives like NPF and GL-331 [19,20]. It has been well established that these derivatives exhibit better pharmacological profile than those of etoposide [21]. We have also observed that 4β-anilino and
*Address correspondence to this author at the Division of Organic Chemistry, Indian Institute of Chemical Technology, Hyderabad - 500007, India; Tel: +91-40-27193157; Fax: +91-40-27193189; E-mail:
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Kamal et al.
Bulky groups can be accomodated 4β-Configuration fundamental R A-Ring with methylenedioxy ring is essential for optimal antitumor activity.
O Trans Stereochems itry of lactone is crucial
O O Free rotation of E-ring esential
H3CO
OCH3 OH
3'and 5' E-ring methoxy groups stabilize DNA-topoisomerase II complex
Hydroxy group crucial, 4'-ester phosphate allowed, and 4'-substitution not tolerated
Fig. (1).
imido compounds display improved biological activity and enhanced topoisomerase II inhibition activity [22]. Interestingly, different heterocyclic ring systems have been linked to the podophyllotoxin moiety at 4β-position with a view to improve the biological activity and as well as to overcome the limitations of the etoposide. In this context, some research groups have prepared new podophyllotoxin congeners with substitution at 4-position by different heterocyclic systems. However there are only two compounds reported using benzothiazole and benzimidazole systems. [23, 24] (Fig. 2). These modified podophyllotoxin congeners exhibit superior antitumour activity compared to etoposide. Recently, we synthesized 4β-amido podophyllotoxin using different heterocyclic moieties with better in vitro anticancer activity [25]. In continuation of these efforts, we herein report the synthesis of new podophyllotoxin congeners by using substituted aminopyrimidines and substituted 2aminobenzothiazoles at 4β-position.
aminoheteroaryl substituted compounds with podophyllotoxin. The reaction of podophyllotoxin with red phosphorous/I2 reagent system provides 4βiodopodophyllotoxin or 4β-iodo-4'-Odemethylpodophyllotoxin as intermediates. Since the iodo intermediates are highly reactive and susceptible to nucleophilic attack by even trace of moisture, these have been subjected for the next step without purification to yield the final products as illustrated in Scheme 1.
CHEMISTRY
BIOLOGICAL EVALUATION AND DISCUSSION
These heteroaryl substituted 4β-aminopodophyllotoxin congeners have been synthesized by coupling of various
Some representative 4β-N-heteroaryl analogues of podophyllotoxin have been tested for their biological
It is interesting to note that when this reaction has been carried out in acetonitrile 4'-O-demethylation product is not observed even after long duration of the reaction. However, employing CH2Cl 2 as the solvent in this reaction at room temperature for 5 h provides the 4'-O-demethylation. This finding is similar to the results obtained therefore by changing the solvent, the selectivity of 4'-O-demethylation can be manoeuvred by employing this reagent system. In this manner compounds 2-6 have been synthesized and purified by column chromatography.
N
N HN
S
HN
O O O
O O O
O
H3CO
OCH3 OH
Fig. (2).
N
O
H3 CO
OCH3 OH
Synthesis and Biological Activity of New 4 -N-Heteroaryl Analogues
Letters in Drug Design & Discovery, 2006, Vol. 3, No. 3
I
I
OH Red P/ I 2, MeCN
O O
O
rt, 30 min
O
rt, 5 h
O
O
O
H3CO
Red P / I 2, CH2Cl 2
O
O
O
O
O
OCH3
H3CO
H3 CO
OCH3
OCH3
207
OCH3 OH
OCH3 1
RNH2, THF, BaCO3, rt, 8 h
RNH2, THF, BaCO3, rt, 8 h NHR
NHR O
O
O
O O
O
O
O
H3 CO
H3CO
OCH3 OCH3
5-6
2-4
H6 Cl
OCH3 OH
Cl S
N CH3
N
S
N
2
N
N 3
F
N N 5
4
CH3
H3CS
N
Cl
6
Scheme 1.
activities against 6 human cancer cell lines comprising of different tumor types i.e., DU145 (prostate), HT29 (colon), MCF7 (breast), MCF7ADR (adr. res. breast), NCIH460 (lung) and U251 (CNS). The screening strategy is based on the protocol used by the Developmental Therapeutics Program of the National Cancer Institute, (NCI), Bathesda, USA. In routine screening, each agent is tested over a broad concentration range (ten-fold dilutions starting from > 100 µM to ~10nM) against six human cancer cell lines. Standard compound doxorubicin is tested in each assay as a positive control. The cells are maintained in growing condition in RPMI 1640 medium containing 10% fetal calf serum and incubated at 37 oC under 5% CO 2 atmosphere. All cell lines are inoculated onto a series of standard 96-well microtitre plate on day zero, followed by twenty four hour incubation Table 1.
in the absence of test compound. The inoculation densities used in this screen are as per the Monks et. al., [26] procedure. All NCEs are dissolved in DMSO and diluted further in culture medium. An aliquot of each dilution is added to the growing cells in 96 well plates and incubated for 48 hrs. After incubation the assay is terminated by adding 50 µL of trichloroacetic acid (TCA) and incubating at 4 oC for 30 min. The precipitated cells are washed and stained with sulphorhodamine B dye for 30 min and the excess dye is washed off with acetic acid. Adsorbed dye is solubilised in Tris base (alkaline pH) and quantitated by measuring the OD at 490 nm in an ELISA reader. GI50 (concentration that inhibits the cell growth by 50 %) is calculated according to Boyd M. R [27] method and the results are given in the Table 1.
GI 50 Values of Some of the 4 -N Heteroaryl Analogues of Podophyllotoxin
Compd.
GI50 ( M) DU145 (Prostate)
HT29 (Colon)
MCF7 (Breast)
MCF7ADR (Adr. Res. Breast)
NCIH460 (Lung)
U251 (CNS)
Etoposide
0.8
59
4.3
116
1.1
6.4
3
0.31
0.02
0.06
0.03
0.09
0.11
4
