Article pubs.acs.org/IECR
Trouble-Free Multicomponent Method for Combinatorial Synthesis of 2‑Amino-4-phenyl-5‑H‑indeno[1,2‑d]pyrimidine-5-one and Their Screening against Cancer Cell Lines Ajinkya A. Patravale, Anil H. Gore, Dipti R. Patil, Govind B. Kolekar, Madhukar B. Deshmukh, and Prashant V. Anbhule* Medicinal Chemistry Research Laboratory, Department of Chemistry, Shivaji University, Kolhapur-416004, Maharashtra, India S Supporting Information *
ABSTRACT: The present study describes an alkaline water−ethanol mediated series of combinatorial synthesis of 2-amino-4phenyl-5-H-indeno[1,2d]pyrimidine-5-one derivatives through sequential multicomponent reaction of 1,3indandione, aromatic aldehydes, and guanidine hydrochloride along with their anticancer evaluation. The effect of sequential addition of the components in the configuration of the desired product has been studied by UV−visible absorption spectroscopy. The synthetic method obeys most of the green chemistry principles in regard to high atom economy and greener, nontoxic, and noncarcinogenic solvent system (water−ethanol). The selected synthesized compounds have been screened against the human breast cancer cell line MCF7, human colon cancer cell line HT29, and normal viro monkey cell line, out of which 2-amino-4-(4methoxyphenyl)-5H-indeno[1,2-d]pyrimidin-5-one showed significant potency toward human breast cancer cell line (MCF7).
■
INTRODUCTION In view of current research, it has been extensively demonstrated that multicomponent reactions (MCRs) prove to be an ideal tool in synthetic organic chemistry because of their high bond-forming efficiency, brevity, and structural diversity.1,2 The 1,3 dicarbonyl compound containing multicomponent reactions have been appreciated as excellent and fruitful strategies for assembling the libraries of heterocyclic scaffolds of druglike molecules.3,4 Multicomponent synthesis also plays a fundamental role in acquiring a green chemistry approach because these reactions are ecofriendly and economically and synthetically effective in the aspects of reaction steps, time, yield, byproducts, and atom economy.5 Pyrimidine is an important class of nitrogen-containing heterocyclic compounds as it constitutes vital structural framework of organic molecules like DNA and RNA;6 it also plays a lead role in the biosynthesis of specific proteins. Along with the peculiar ability of calcium channel modulation,7 pyrimidines are also known to possess noteworthy pharmaceutical activities (anticancer,8,9 anti-HIV,10 antifungal,11 antimalarial,12 antihypertensive,13 antipyretic,14 anti-inflammatory,15,16 antiplatelet aggregation,17 and antitubercular18). Furthermore, the indenonucleolus is a core unit of various natural alkaloids like onychnine (Figure 1) and polyfothine (Figure 2);19 from this class, arylindenopyrimidines have been used as adenosine A2A receptor antagonists,20 which are useful
Figure 2. Polyfothine (left panel) and structure investigated in this work (right panel).
for the treatment of Parkinson’s disease. Moreover, because of the biodynamic properties, recently Shook et al.21 successfully investigated the in vitro and in vivo screening of arylindenopyrimidine molecules22 and several research groups have reported the synthesis of various indenopyrimidine compounds through multicomponent reactions.23,24 However, they suffer from a few demerits; thus, considering the necessity of obeying green chemistry principles and overcoming the limiting factors of previous reports, we report green methodology using a water− ethanol solvent system for achieving a convenient methodology. As is well-known, water is universal green solvent and ethanol also complies with most of green principles25 because of its properties and availability. According to recent reports, it has been found that indeno[1,2-e]pyrimido[4,5-b][1,4]diazepine-5,11-dione26 and indenopyrazole27 are particularly active as anticancer and CDK inhibitors. This literature reveals that indandione-coupled heterocyclic compounds possess remarkable anticancer activity. Thus, development of new druglike molecules against cancer is Received: April 1, 2014 Revised: September 15, 2014 Accepted: September 23, 2014
Figure 1. Onychnine. © XXXX American Chemical Society
A
dx.doi.org/10.1021/ie5013618 | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Industrial & Engineering Chemistry Research
Article
Scheme 1. Synthesis of 2-Amino-4-phenyl-5-H-indeno[1,2]pyrimidine-5-one Derivatives
of major importance in the scientific community. On this basis, we address for the first time 2-amino-4-(4-chlorophenyl)-5-Hindeno[1,2d]pyrimidine-5-one derivatives (Scheme 1) as anticancer agents via a one-pot, green synthesis of aromatic aldehydes, 1,3 indandione, and guanidine hydrochloride in water−ethanol as a continuation of previous work28 using elementary starting materials.
Table 1. Optimization of the Reaction Conditions
■
a
RESULTS AND DISCUSSION In a pilot reaction, p-chloro benzaldehyde (1) (3 mmol) and 1,3-indandione (2) (3 mmol) in 10 mL of water−ethanol, with catalytic amount of aqueous sodium hydroxide, was combined in a 50 mL round-bottom flask. The resultant reaction mixture was stirred at room temperature to complete the Knoevenagel reaction as monitored by thin-layer chromatography (TLC). After the formation of Knoevenagel product (4), guanidine hydrochloride (3) was introduced and the reaction was refluxed in an oil bath until completion (monitored by TLC). The reaction mixture was then poured slowly on crushed ice with constant stirring. The desired product was isolated, filtered, and washed with cold water to remove the excess sodium hydroxide. The resultant pure compound was identified on the basis of spectroscopic data. The infrared (IR) spectra of 2amino-4-(4-chlorophenyl)-5-H-indeno[1, 2-d]pyrimidine-5-one (5) depict two characteristic bands at 3468 and 3310 cm−1, confirming the presence of −NH2. Furthermore, 1706 cm−1 stretching vibration and 1258 cm−1 bending vibration frequency indicate the presence of carbonyl group (see Supporting Information). In the 1H NMR spectrum of the above compound, two doublets appearing between 7.42−7.45 and 8.04−8.07 with uniform coupling constant J = 9.0 are considered to be of the four p-substituted aromatic protons in the aromatic aldehydic moiety. The remaining four aromatic protons of the indano nucleus are exhibited as a multiplet between 7.53 and 7.77, while two protons of −NH2 group appear as a broad singlet at 7.70. The 13C NMR spectrum of the same compound exhibits signals at 165.48 of C−NH2 and 134.78 of C−Cl, and the remaining aromatic carbon signals were observed at 111.23, 121.44, 123.57 (2C), 128.30 (2C), 131.81 (2C), 133.50, 136.10, 136.76, 139.92, 165.85, and 176.23. The only signal appearing at 187.91 of carbonyl carbon in the product verifies the formation of the desired structure (see Supporting Information). Finally, the electrospray ionization mass spectrometry (ESI-MS) analysis which shows molecular ion peak at 308 is found to be in good agreement with the proposed structure. The aggregated spectral data drastically reduces the possibility of any side product formation in significant amounts. With the first successful reaction in hand, to optimize the alkaline medium and the catalytic amount of NaOH, four parallel reactions (Table 1) were carried out with 4-chloro
sr. no.
% of NaOH solution (5 mL)
yield of producta (%)
1 2 3 4 5
1% 2% 3% 4% 5%
40 65 84 92 92
Isolated yield.
benzaldehyde (1) (3 mmol), 1,3-indanedione (2) (3 mmol), and guanidine hydrochloride (3) (4.5 mmol) under the same reaction conditions. It was found that an excellent yield of the desired product was observed in 4% aqueous NaOH (5 mL) solution. To check the scope and limitations, these optimized conditions were further screened with a variety of aldehydes. The aldehydes showed no perceptible electronic bias. The said protocol was found to be robust regardless of the structure and functional alterations on the aldehyde with the exception that aliphatic aldehydes failed. This success of optimized novel reaction encouraged us to produce an atom economy (Table 2). Simultaneously, with the detailed study, we also examined the sequential addition of the components. Surprisingly, we obtained the same products for alterations made in the addition sequence with two different mechanisms (Schemes 2 and 3) which was supported by UV−visible absorption study. As shown in Figure 3 for 2-amino-4-(4-methoxyphenyl)-5Hindeno[1,2-d]pyrimidine-5-one derivative (peak a and b) maximum absorption peak observed at 564 nm synthesized via two different routes. Similar absorption peak pattern was also observed for 2- amino-4-(2-nitrophenyl)-5H-indeno[1,2d]pyrimidine-5-one derivative (peak c and d) absorption peak at 406 nm. Thus, we conclude that both products synthesized through two different routes are identical in nature. All these compounds were isolated in pure form without the necessity of further purifications for spectroscopic investigation. All the physical and analytical data are given in Table 2. In the case of ferrocene 2-aldehyde, the product gives expected protons in the proton NMR (product was partially soluble where only 1H NMR is done in DMSO-d6), but we could not succeed in the respective 13C spectrum because of the requirement of a sample in excess quantity, which creates a solubility problem of the said compound in DMSO-d6 and CDCl3 (Table 2, entry R). A mechanistic pathway of this reaction is proposed in Scheme 2. It is a well-known fact that alkaline medium helps for the formation of Knoevenagel product. The reaction proceeds through the initial formation of the Knoevenagel product of aldehyde (1) and 1,3-indandione (2). The formed Knoevenagel product 4 undergoes Michael addition with guanidine (3) followed by cyclization, isomerization, and aromatization to obtain 2-amino-4-(4-chlorophenyl)-5-H-indeno[1, 2-d] pyrimiB
dx.doi.org/10.1021/ie5013618 | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Industrial & Engineering Chemistry Research
Article
Table 2. Synthesis of 2-Amino-4-phenyl-5H-indeno[1,2-d]pyrimidine-5-one Derivativesa
C
dx.doi.org/10.1021/ie5013618 | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Industrial & Engineering Chemistry Research
Article
Table 2. continued
D
dx.doi.org/10.1021/ie5013618 | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Industrial & Engineering Chemistry Research
Article
Table 2. continued
a
Atom economy is calculated according to (molecular mass of desired product/molecular mass of all reactants) × 100.
Scheme 2. Mechanistic Pathway of the Reaction (Knoevenagel Sequence)
(SRB) assay,29 and the source of the cell lines was the National Cancer Institute (United States). Sulforhodamine B assay was developed by Skehan and colleagues29 to measure drug-induced cytotoxicity and cell proliferation for large-scale drug-screening applications; also, it is a very sensitive and cost-effective method for in vitro anticancer drug screening.30 Initially, four different concentrations of the test compounds varying from 10−7 M to 10−4 M were engaged for the SRB assay. The obtained graph of
dine-5-one (5). In an alternative mechanistic pathway, the aromatic aldehyde initially reacts with the guanidine hydrochloride to form Schiff base 6, which undergoes Michael addition with enolic 1,3-indandione, and it aromatizes to give the desired compound. Anticancer screening was done in Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Mumbai, India. The method of testing was Sulforhodamine B E
dx.doi.org/10.1021/ie5013618 | Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX
Industrial & Engineering Chemistry Research
Article
Scheme 3. Mechanistic Pathway of the Reaction (Schiff Base Sequence)
suggest decrease in the cell count at its expected doubling time proving the anticancer capability of entries F and L at 10−4 M concentration. In addition, the observed 50% inhibition of cell growth (GI50) values for entries F and L were found to be maintained at the lower end (i.e., 22.8 and 27.4 μM, respectively) as compared to the other compounds with GI50 values ranging between 28.9 and 34.4 μM and standard doxorubicin (ADR) having a GI50 value of 0.1 μM. Table 3 Table 3. Anticancer Activity of Synthesized Compounds on Human Breast Cancer Cell Line MCF7
Figure 3. UV−visible absorption spectra of 2-amino-4-(4-methoxyphenyl)-5H-indeno[1,2-d]pyrimidine-5-one (peak a and b) and 2amino-4-(2-nitrophenyl)-5H-indeno[1,2-d]pyrimidine-5-one (peak c and d).
dose−response curve (Figure 4) between the test compounds and different concentrations clearly demonstrated a dose-
compound
LC50a
TGIb
GI50c
entry C entry F entry I entry L entry M ADR (doxorubicin)
>100 >100 >100 >100 >100 >100
>100 92.4 87.7 79.8 >100 19.5
34.4 22.8 28.9 27.4 28.6 100 >100 >100
b
GI50c
>100 >100 34.3
>100 >100 80 >80 >80 >74.7 >80 42.5
>80 >80 77.5 41.4 63.6
