Synthesis and biological evaluation of novel substituted pyrrolo[2,3-d]pyrimidines as anticancer agents

Pyrrolopyrimidines are well known scaffold, which play a critical role as anticancer agents, so it thought of interest to synthesize a series of novel substituted pyrrolo[2,3-d]pyrimidines having diverse groups at position C4 and N7 of the pyrrolo[2,3-d]pyrimidine core and performed in vitro screening against MDA-MB-468 (breast cancer cell line) cell line. The details of the synthetic methods and characterization data of the synthesized compounds have been presented in this study. Compounds 8a, 8h, 8j, 9h, 9i, 9j, 9m, 9n, and 9o showed the excellent anticancer activity compared to standard doxorubicin with an IC50 value of 6.17 μM/ml against MDA-MB-468 (breast cancer cell line), which was non-toxic to normal vero cell line.


INTRODUCTION
Cancer is characterized by the uncontrolled growth of abnormal cells (Grange et al., 2002;Kari and Dave, 2012). The abnormal growth can also be referred to as a neoplasm. Neoplasms can be benign (noncancerous) or malignant (cancerous). Cancerous growths can occur in any organ of the body and are characterized by three distinct properties: the cells replicate rapidly with reduced growth control, the cells lose contact inhibition in vitro, and the resulting neoplasm invades surrounding tissues and may spread to other parts of the body (Foye et al., 1995;Kleinsmith, 2006). The cells of benign tumors have reduced growth control but do not invade surrounding tissues or spread to other parts of the body. Other important aspects of cancer cells include the ability to be self-sufficient and generate local angiogenesis while resisting antigrowth and apoptosis signals. If the spread of a malignant neoplasm is not controlled, it can result in death (Hadfield et al., 2003;Honore et al., 2005;Jordan et al., 1998;Whitman et al., 1995).
Most breast cancers begin either in the breast tissue made up of glands for milk production, called lobules, or in the ducts that connect the lobules to the nipple. The remainder of the breast is made up of fatty, connective, and lymphatic tissues (Edge et al., 2010).
In 2016, an estimated 252,710 new cases of invasive breast cancer will be diagnosed among women and 2,470 cases will be diagnosed in men. Besides, 63,410 cases of in situ breast carcinoma will be diagnosed among women. Approximately 40,610 women and 460 men are expected to die from breast cancer in 2016(Miller et al., 2016. An increasing interest in the biological studies of pyrrolo [2,3-d]pyrimidine in the past decade is a consequence of their wide usage as a pharmaceutically important class of compounds. Pyrrolo[2,3-d]pyrimidine derivatives have a considerable potential in the field of chemotherapy as they were found to exhibit their antitumor activity by inhibiting different types of enzymes such as cyclin-dependent kinase, Src and Abl tyrosine kinase, glycogen synthase kinase-3, adenosine deaminase, and epidermal growth factor receptor protein tyrosine kinase (Ghorab et al., 2010). The derivatives of pyrrolo [2,3-d] pyrimidine have already been discovered as antitumor agents by the NCI (National Cancer Institute, Bethesda, MD) on HCT116 and other cell lines. Some pyrrolo[2,3-d]pyrimidines(1) structurally related with allopurinol (2), 6-mercaptopurine (3), and 1-(1, 1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo [3,4-d] pyrimidin-4-amine (1-NA-PP1) (4) have also been reported as potent inhibitors for the growth of several human tumor cell lines (Gangjee et al., 2010).
In view of these observations, we undertook the synthesis of three series of novel pyrrolo[2,3-d]pyrimidine analogs (Scheme 1) and in vitro screening on breast cancer cell line (8-10).

MATERIALS AND METHODS
Melting points of all the chemical compounds and solvents were determined in open capillaries and are uncorrected ( Table 1). The IR spectra of all compounds were recorded in FT-IR 8400S Shimadzu spectrophotometer using KBr. Mass spectra were obtained using the 2010EV LCMS Shimadzu instrument at 70 eV. 1 H NMR spectra were obtained in DMSO on BRUKER Avance-II 400 MHZ instrument, and the chemical shift was measured as parts per million downfield from tetramethylsilane as an internal standard. The target compounds were synthesized as outlined in Scheme 1.

General method for the synthesis of 2-amino-4,5-diphenyl-1-(substituted)-1H-pyrrole-3-carbonitriles (7a-7o)
A mixture of benzoin (2 g, 0.01 mol), substituted amines (0.01 mol), and concentrated HCl (6-8 drops) in toluene (50 ml) was heated under reflux for 6 hours and cooled. The reaction mixture was filtered, and the resulting residue was dissolved in 30 ml of absolute alcohol. A malononitrile (0.66 mg, 0.01 mol) was added to the previous solution followed by sodium ethoxide (2 g sodium metal in 20 ml of absolute alcohol) as a catalyst. The mixture was refluxed until a solid was formed. The solvent was evaporated under reduced pressure, and the residue was recrystallized from methanol to give the pure yellow crystalline product (Dholakia et al., 2013).

General method for cell line study (MTT assay)
It is a laboratory test and a standard colorimetric assay for measuring cellular growth. It can also be used to determine the cytotoxicity of potential medicinal agents and other toxic materials.
In the present investigation, all the compounds were evaluated against cell lines named MDA-MB 468 (Breast cancer cell line) and vero cell line (normal cell line) for each tested compound as well as standard doxorubicin, and the doseresponse curve (DRC) against MDA-MB 468 (Breast cancer cell line) was plotted with 10 analysis point, i.e., with 10 different drug concentrations. The concentration causing 50% cell growth inhibition (IC 50 ) was determined from DRC using GraphPad prism software (Ver. 5.04) (GraphPad Software, Inc., La Jolla, CA) and Microsoft Excel 2007 (Microsoft Corporation, Redmond, WA) application (Table 2).