Synthesis and Preliminarily Cytotoxicity to A 549 , HCT 116 and MCF-7 Cell Lines of Thieno [ 2 , 3-d ] pyrimidine Derivatives Containing Isoxazole Moiety

Under the guidance of our previous achievements, and in order to extend this small molecular library. In current work, other 21 novel structures of thieno[2,3-d]pyrimidines containing isoxazole-moiety (1a-1u) were firstly synthesized and the cytotoxicity to A549, HCT116 and MCF-7 cell lines was evaluated using the MTT method. The results showed that most target compounds exhibited good to excellent cytotoxicity to A549, HCT116 and MCF-7 cell lines, especially 6-Methyl-4-{[3-(4-chlorophenyl)-isoxazol-5-yl-]-methoxy-}-thieno[2,3-d]pyrimidine (1e) exhibited a broad-spectrum and the most potent cytotoxicity to A549, HCT116 and MCF-7 cell lines (IC50s: 2.79, 6.69 and 4.21×10-3 μM, respectively) as compared with the reference drug gefitinib (IC50s: 17.90, 21.55 and 20.68 μM, respectively). 1e can be seen as the best drug candidates for development of anticancer drugs.


Introduction
Cancer is the leading cause of death after cardiovascular disease, and the relative mortality rate caused by cancer is still very high even in developed countries, statically evident around 6-7 million new cases per year [1].Lung cancer is the number one cause of cancer related death globally, with an estimated 13% of total cases and accounting for 18% of total deaths worldwide in 2008 [2].In 2010, there were 222,520 new lung cancer patients diagnosed and 157,300 died [3].However, the non-small cell lung cancer (NSCLC) is usually diagnosed in advanced stages and accounts for 85% of all lung cancer diagnoses in the United States [4].In China, it is reported that lung cancer has replaced the liver cancer as the number one cause of death among people with malignant tumors in 2008 [5].
Breast cancer is also the most common cancer among American women.It was reported that one in every eight women in the United States was found to have breast cancer.According to American Cancer Society, nearly 40,000 women had died of breast cancer in 2011, and about 232,340 new cases of invasive breast cancer will be diagnosed each year.Thus, cancer has been a great threat to human survival, which prompts us to search for new drugs for treatment of cancer.Isoxazole moiety was usually introduced to the drug molecules to improve their biological activities.Some drug molecules containing isoxazole moiety showed remarkable biological activities [27][28][29][30][31][32][33].In our previous work, we introduced the isxoazole moiety at the 4-position of quinazoline parent ring and synthesized some quinazolines containing isoxazole moiety.The biological evaluation showed that most compounds exhibited significant anticancer activity against MCF-7, A549 and HCT116 cell lines [34,35].

Chemistry
To obtain more potent anticancer agent as well as to study their structure-activity relationship (SAR), different substitution patterns at the 6-position of the thieno [2,3-d]pyrimidine core and on the benzene ring of isxoazole moiety were selected based on the substutition's volume and electronic environments, which would affect lipophilicity and steric hindrance, and hence the anticancer activity of the target molecules.Based on these factors, 21 novel structures of thieno [2,3-d]pyrimidine derivatives containing isoxazole-moiety were synthesized and characterized by 1 H NMR, 13 C NMR, IR and mass spectrometry.Their in vitro anticancer activity against lung cancer A549, colorectal cancer HCT116 and breast cancer MCF-7 cell lines was preliminarily evaluated.

Analytical spectral data of compounds 3a-u
The IR spectral data of compounds 3a-u showed a characteristic absorption band at the 3103-3107 cm -1 , which corresponds to the thiophene core, =C-H and Ph-H.The absorption band of the aromatic cycle skeleton, N=C group and the stretching frequency of C=C showed an absorption band at 1548-1567 cm -1 .In the

In vitro anticancer evaluation
We preliminarily evaluated the synthesized compounds 3a-u for in vitro anticancer activity against A549, HCT116 and MCF-7 cell lines.The anticancer efficacy was comparable to the standard drug (gefitinib) ( against A549, HCT116 and MCF-7 cell lines.For 3a-g, it indicates that introducing the methyl and methoxy group to 4-position of the benzene ring of the isoxazole mioety will reduce their anticancer activity (For example, 3a is the most potent compounds among 3a-c, and the active sequence is 3a>3b>3c); while for 3d-g, it indicates that introducing the halogen atom to 4-position of the benzene ring of the isoxazole moiety will increase their anticancer activity, and 4-Cl substituted derivative is more potent than 4-Br substituted derivative; 3e is the most potent compound among 3a-g.In fact, 3e is 6.4-fold more active than gefitinib against A549,  a The IC50 values could not be calculated

Conclusion
In this work, we synthesized 21 novel structures of thieno [2,3-d] Compounds 3a, 3e, 3g and 3h exhibited a broad-spectrum and much stronger inhibitory activity against these tumor cell lines, for which they are considered promising drug candidates for development of anticancer drugs.

Materials and apparatus
Melting points ( o C, uncorrected) were determined on a XT5 micrio melting point apparatus.NMR spectra were recorded on Bruker AVANCE III at 400 MHz for 1 H and 100 MHz for 13 C, using TMS as internal standard and peak multiplicities was designed as follows: singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m).

General procedure for the preparation of the target compounds 3a-u
General synthesis of 3a: 6-methyl-4-chloro-thieno[2,3-d]pyrimidine (0.184 g, 1 mmol) was added into a 25 mL one-necked round-bottom flask with 5mL of dry iso-PrOH, and the mixture was stirred in a cold bath.Then a solution of Et3N (0.101 g, 1 mmol) and (3-phenyl-isoxazole-5-yl)-methanol (0.175 g, 1 mmol) in 10mL dry iso-PrOH was slowly added to the reaction system using a syringe.The mixture was stirred in a cold bath for an additional 30 min, and then temperature was allowed to reach room temperature.After reaction completion (monitored by thin layer chromatography, TLC), the reaction mixture was evaporated under reduced pressure, and the residue was purified by column chromatography on silica gel using petroleum ether and ethyl acetate (5:1→2:1) as eluant.Fractions with similar Rf values were combined to obtain the target compound 3a.Other thieno [2,3-d]pyrimidine derivatives containing isoxazole moiety 3b-u were synthesized using the same process 3a.

Since 1994 ,Fig. 1
Fig. 1 Chemical structures of quinazolines used in clinical or clinical trial

1 H( 6 -
NMR spectra, a singlet at 7.26-7.28ppm in compounds 3a-n belongs to the proton of the 5-position of the thieno[2,3-d]pyrimidine core, while for the compounds 3o-u Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 24 March 2017 doi:10.20944/preprints201703.0190.v1position of the thieno[2,3-d]pyrimidine core was substituted by tert-butyl group), the proton signal of the 5-position of the thieno[2,3-d]pyrimidine core was showed at 7.44 ppm, which is attributed to the steric effects caused by tert-butyl group; a singlet for a proton varying from 8.6-8.8 ppm belonged to the proton of 2-position of the thieno[2,3-d]pyrimidine core; a singlet recorded at 7.1-7.3ppm corresponds to the proton of the isoxazole ring; two protons of CH2 connected isoxazole ring was recorded as a singlet at 5.8-5.9ppm.In 13 C NMR spectra, the carbon of CH2 connected isoxazole-ring appeared at 60-61 ppm, the 3-C, 4-C and 5-C of isoxazole ring appeared at 166, 101 and 158 ppm respectively.The 2-C, 4-C, 8-C and 9-C of thieno[2,3-d]pyrimidine core appeared at 154, 163, 145 and 127ppm respectively, the 6-C of thieno[2,3-d]pyrimidine core of all compounds appeared at 137-138ppm, which indicates that tert-butyl group did not affect the chemical shifts of 5-C and 6-C of thieno[2,3-d]pyrimidine core.Some carbon atoms in the same chemical environment appeared only one signal.All target compounds were also confirmed by MS according to their molecular formulae, most compounds showed the expected molecular ion signal [M] + , but some showed the quasi-molecular ion signal [M+H] + .

Table 1
).It can be seen from the Table 1, the half of compounds exhibit good to excellent anticancer activity Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: