Efficient Synthesis of a New Class of N-Nucleosides of 4 H-Thiochromeno [ 2 , 3-d ] pyrimidine-10-Sulfone as Potential Anticancer and Antibacterial Agents

A highly practical and efficient preparation of 6-methy-4H-thiochromene and 7-methyl-thiochromene[2,3-d]pyrimidine derivatives was developed via a multi-component reaction of 3-methyl-thiophenol (1), aldehydes (2), and malononitrile (3). A series of pyrimidine nucleoside, thiochromene[2,3-d]pyrimidine and thiochromene[2,3-d]pyrimidine-10-sulfone was efficiently obtained. These hybrid compounds were evaluated as potential antibacterial and anticancer agents and showed encouraging biological activities. Some of these derivatives showed broad-spectrum antitumour activity against the nine tumour subpanels tested, and demonstrated significant activity in the in vitro antitumour screening expressed by MG-MID log10GI50 value of −4.55, −4.67 and −4.73 of compounds 9a, 9b and 9c, respectively.


Introduction
Derivation of thiopyranopyrimidine and pyrimidine nucleosides has attracted much attention because of potent antitumor and antiviral activity [1].Pyrimidine nucleosides substituted at the C-5 position constitute a class of biologically significant molecules [2].The well-known cancer chemotherapeutic 5-fluorouracil and antiviral agents, such as 5-iodo-2'-deoxyuridine and 5-(trifluoro methyl)-2'-deoxyuridine, have been in clinical use for several years [3].Meanwhile, in recent years, there have been significant interests in the potential usages of the C-5-substituted pyrimidine nucleosides in synthetic oligonucleotide probes as a tether site for linking reporter groups to nucleic acids [4].When the modified nucleosides are incorporated into the duplex B-DNA, C-5-substituents are located in the major groove [5], and so do not disrupt Watson-Crick base pairing.As a result, the methodologies for constructing suitable linker arms and generating bonds to C-5 are important for the synthesis of potential therapeutic agents and synthetic oligonucleotide probes.Therefore, the 5-position of pyrimidine nucleosides has been the target of extensive studies on modifications [1].
Also, 1-benzothiopyran-4-ones are the thioanaloggues of flavones [6] which are a class of naturally occurring pharmacologically active compounds.The thioflavones also exhibit various pharmacological activities [7] such as antimalarial, antimicrobial, and antifungal activity and are useful as potent inhibitors of steroid sulfatase [8].In general, 4H-1-benzothio pyran-4-ones are synthesized by the condensation of thiophenols with ethyl benzoylacetates in polyphosphoric acid, but the yields are low to moderate [9].The cyclization of ethyl β-(aryl thio)-cinnamates, derived from Michael addition of thiophenols to ethyl phenylpropiolates, with stannic chloride or phosphorus pentoxide, methanesulfonic acid, gives 2-phenyl-4H-1-benzo-thiopyran-4-ones [10].However, this method could not be applied for the synthesis of methoxy substituted thioflavones because competitive cyclization into the cinnamyl aromatic ring, rather than the sulfur-bearing ring, occurs when cinnamyl ring is activated by methoxy substituent.The reaction of S-aroyl derivatives of thio salicylic acid with N-phenyl-(triphenylphosphoranylidene) ethemine [11] or (trimethylsilyl)-methylene-triphenylphosphorane [12] leads to the acylphosphoranes which undergo subsequent intramolecular Wittig cyclization to afford 2-phenyl-4H-1-benzothiopyran-4-ones, but the separation of phenyl isocyanate is tedious.Alternatively, the condensation of methyl thiosalicylate with trilithiated acetoacetanilides [13] or dilithiated N-benzoyl hydrazones [14] with excess lithium diisopropylamide gives the C-acylated intermediates which undergo subsequent cyclo-dehydration and hydrolysis with HCl to afford 2phenyl-4H-1-benzothiopyran-4-ones in moderate to high yields.With the aim to develop and identify novel active compounds, we carried out a number of modifications on the structure of organic reagents.
I was devoid of any activity [15], first obtaining the planer isosteric derivative II (Figure 1).Also, as a part of our research program devoted to the preparation and evaluation of new anticancer agents, we extensively studied several polycyclic chromophores [16][17][18][19] among which we recently disclosed that the pyrido [2,3-d] pyrimidine, pyrimido-quinolines, thieno [2,3-d] pyrimidine and triazolo [4,3-a] pyrimidin-6-sulfonamide showed a detectable cytotoxic activity on human tumor cell lines which was ascribed to its ability to interfere with mitochondrial functions.

Chemistry
In this paper, we describe a new efficient synthesis of 2amino-3-cyano-4H-thiochromene-4-aryl 4 via cyclocondensation of arylidenemalononitriles (aldehyde + malono nitrile) with 3-methyl-thio-phenol in high yields under the mild conditions, in which the thiopyran moiety gave us the potential to insert a pendant amino and cyano groups both at the 2-and in the 3-position of the chromophore.The reactivity of the new derivatives towards the organic reagents such as formic acid has been studied.The preparation of the thiochromene and the 4-aryl substituted derivatives were performed following the synthetic route described in Scheme 1.The starting 2amino-4-(4-aryl)-6-methyl-4H-thiochromeno-3-carbonitrile (4a-f) were prepared by the following described procedure via the condensation of thiophenol derivatives 1 with aldehydes and malononitrile 3 in ethanol/piperidine [19].
The compounds were purified by recrystalization and Characterized by analytical and spectral data.In particular, the most discriminating features of the 1  spectra of compounds 6a-f was a singlet at ≈4.50 ppm attributed to the proton in the 4-position of the thiopyran, also at ≈8.80 ppm the resonated proton of the pyrimidine at 2-position.The synthetic sequence leading to the Nglycosides substituted thiochormen [2,3-d] pyrimidine (Scheme 2) involved the conversion of the pyrimidine moiety of (6a-f) into the intermediate potassium salt, in which the reactive NH functionality was protected as previously reported by us [16].The subsequent addition of potassium salt of 6 with the appropriate bromo furanosyl/pyranosyl, in anhydrous acetone at room temperature, involves the removing of KBr as a first step Scheme 2.
The glycosylation of 6d-f with 2,3,5-tri-O-acetyl-β-Darabinopyranosyl bromide 11 or 2,3,4,6-tetra-O-acetyl -α-D-glycosyl bromide 12a,b in acetone and in the presence of aqueous potassium hydroxide afforded the corresponding acetylated nucleosides 7a-c, 8a-f respectively in good yields ≈65% -70% Scheme 2. Thin layer chromatography (chloroform: methanol, 8:2) indicated the formation of the pure compounds.The structures of 7a-c, 8a-f were confirmed by elemental analysis and spectral data (IR, 1 H NMR, 13 C.NMR) (cf.Exp.).The 1 H NMR spectrum of compound 8a as an example, showed the anomeric proton of the glucose moiety as a doublet at  5.95 ppm with a coupling constant J 1'-2' = 10.67   10a-f respectively.The structures of free nucleosides 9 and 10 have been established on the basis of their spectral data and elemental analyses.Thus, the 1 H NMR spectrum of 10a showed the anomeric proton as a doublet at  6.04 ppm, J

Antibacterial Activity
The compounds 9a-c, 10a-f was evaluated for their efficacy as antibacterial in vitro by disc diffusion method against various bacterial strains.The antibacterial activity has been compared to some standard antibacterial agents like sulfanilamide and sulfadiazine that contain a pamino benzene sulfonamide moiety.From the results in Table 1 Compound (9a-c) exhibited excellent activity toward Gram(+ve) and Gram(−ve) bacteria E. coli, P.aeruginosa, S. epidermidis, B. subtilis and Vibrio species, as compared with the reference drug (sulfa-diazine, This may be due to the presence of 4-methyl-piprazine on p-positions of 4-thiochromene ring and arabinofuranos ring at N-1of the pyrimidine, while the other compounds exhibited mild to moderate activity compared to sulfadiazine against B. subtilis.All of compounds exhibited excellent activity toward Vibrio species.Compounds 10a, 10d exhibited high activity toward Pseudomonos aeruginosa while the other compounds exhibited mild to moderate activity.Also all of compounds exhibited excellent activity toward E. coli as compared with the reference drug (sulfadiazine) (Table 2).

Antitumour Activity
Amongst the substituted 4-piperazino/morpholine-Nnucleoside thiochromene derivatives synthesized, compounds 9a-c and 10a-f were chosen by National Cancer Institute (NCI) as prototypes for preliminary test and were evaluated in three cell lines one dose prescreen [20][21][22] comprising of MCF-7 (breast), NCI-H460 (lung) and SF-268 (CNS) cell lines.These have been in use by DTP (Development Therapeutic Program) for several years to evaluate combinatorial libraries and have proven to be an effective test of agents, which exhibited some capability level to inhibit the growth of human tumour cells in culture.The compounds were added at a single

5,6d
N N H concentration (10 −4 M) and the culture was incubated for 48 h.End point determination were Compounds 9a, 9b and 9c were further evaluated at 10-fold dilutions of five concentrations ranging from 10 −4 to 10 −8 M against 60 different human tumour cell lines organized in subpanels representing melanoma, leukaemia and cancers of breast, prostate, lung, colon, ovary, CNS and kidney.The details of the cell lines used are shown in Table 3 and the experimental procedures have been described in the literature in detail [23][24][25].Three dose response parameters were calculated for each experimental agent: the compound concentration required to carry 50% of net cell growth (GI 50 ) which signifies the growth inhibitory power of the test agents, the compound concentrations resulting in total growth inhibition (TGI) which signifies the cytostatic effect of the test agent, and the concentration of the compound leading to the 50% of net cell death (LC 50 ) which signifies the cytotoxic effect of the test agent.The log 10 GI 50 , log 10 TGI and log 10 LC 50 were then determined defined as the means of the log 10 's of the individual GI 50 , TGI and LC 50 value as shown in  chreduce the growth of any one of the cell lines to 32% or less, were selected for further evaluation in the full panel of 60 human tumour cell lines.Compound 9a has shown 0% of growth inhibition against all the three cell lines.Similarly, 9b and 9c presented the 1% and 31% of growth inhibition for the NCI-H460 cell line, respectively.However, compounds 10a-f have not reduced the growth of any cell lines by 32% or less.Therefore, only three compounds 9a, 9b and 9c have been selected for 60-cell line panel assay (Table 3).

8,10d
From Table 4, we can conclude that, all the active compounds in this test showed broad-spectrum antitumour activity against the nine tumour subpanels tested, and demonstrated significant activity in the in vitro antitumour screening expressed by MG-MID log 10 GI 50 value of −4.55, −4.67 and −4.73 of compounds 9a, 9b and 9c, respectively, whereas compounds 10a-f were inactive (log 10 GI 50 > −4).Substitution of N-glucopyranosyl (10a-c) and N-galactopyranosyl (10e-f) on thiochromen [2,3-d] pyrimidine ring has reduced the activity.Log 10 GI 50 value of compound 9a is −4.50 and −4.54 in the breast cancer cell lines (MCF-7 and MDA-MB-231, respectively), whereas this value in case of E7010 is −6.14 and −5.07 for the same cell lines [25].Substitution of arabinofuranosyl group on the N-1 position of pyrimidine, and piperazine, 4-methylpiperazine and morpholine at 4 positions of thiochromene, respectively, (9a-c) also exhibits similar log 10 GI 50 value for these cell lines, that is, −4.45 and −4.72, however the activity is enhanced particularly in case of leukaemia CCRF-CEM cell line (log 10 GI 50 −5.18,GI 50 is 6.62 µM).In case of compound 9c log 10 GI 50 value is −5.36 and −4.38 in the breast cancer cell lines (MCF-7 and MDA-MB-231 cell lines, respectively) and also demonstrated significant activity in other cell lines.

Conclusion
The synthesis and screening of anticancer and antibacterial activities of a novel series of N-nucleoside-thiochromene-5-piperazino/5-methyl-piperazino/and or 5morpholino have been investigated.Compounds 9a-c were screened against 60 human cancer cell lines and exhibited a broad spectrum of activity against almost all the cancer cell lines and in the case of certain cancer cell lines, the activity was comparable to E7010.Furthermore, most of the compounds showed better activity than the controls in antibacterial screening except against P. aeruginosa.

Experimental
All starting materials, solvents, and reagents were very pure grade.Chromatography solvents were HPLC grade.Reactions were monitored by thin layer chromatography (TLC), (Silica gel 60 F 254 ).Melting points were determined on the Electrothermal 9100 melting point apparatus and are uncorrected.The IR spectra (KBr) were recorded on a FT-IR NEXCES spectrophotometer (Shimadzu, Japan).The NMR spectra were measured with a Jeol ECA 500 MHz.Mass spectra (EI) were run at 70 eV with a Finnigan SSQ 7000 spectrometer.Compounds were properly characterized by elemental analyses.The Pharmacological evaluations of the products were carried out in Pharmacological Unit Pharmacology department, (NCI, Cairo University, Egypt).

Scheme 2 .
Scheme 2. Synthesis of the nucleosides of thiochromen derivatives.

Table 3 . Primary in vitro growth inhibition assay results at 10 −4 M concentration. PTC a Compound MCF-7 b NCI-H460 c SF-268 d
a PTC, percent test cell growth compared with untreated control cells.b Breast cell line.c Lung cell line.d CNS cell line.
Data obtained from the NCI's in vitro disease-oriented human tumour cells screen.b Log 10 GI 50 = log of molar concentration that inhibits 50% net cell growth.c Log 10 TGI = log of molar concentration that produces a total growth inhibition.d Log 10 LC 50 = log of molar concentration that leads to 50% net cell death.