English

• 1. INTRODUCTION

  The human dental tissue-derived mesenchymal stem cells (MSCs) have been proved playing an important role in the clinic treatment^[1], such as the dental pulp MSCs, exfoliated deciduous MSCs, periodontal ligament MSCs, dental follicle progenitor MSCs, alveolar bone-derived MSCs, apical papilla MSCs, tooth germ progenitor MSCs, and gingival MSCs^{[2, 3]}. MSCs can produce a large number of biologically active substances which have the activities of hematopoietic and supporting, activate endogenous stem or progenitor cells, tissue damage repair, immune regulation, anti-apoptosis, anti-oxidation, anti-fibrosis and so on^{[4, 5]}. The most important thing is that the DP-MSCs has the ability to differentiate into different tissue cells, such as neural cells, osteoblasts, adipocytes and chondrocytes^[6]. So, it is reasonable for us to give this hypothesis that the DP-MSCs are suitable candidates for tissue regeneration.

  Dasatinib is a protein tyrosine kinase inhibitor, a Src Kinase inhibitor, and is useful in the treatment of immunologic and oncological diseases^[7], while 2-amino-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (1) which is one of the most important intermediates in the synthesis of Dasatinib fascinated us^[8]. Thus, much attention has been devoted to the synthesis, characterization and crystal structure of compound 1 which has never been reported before^[9].

  The present work deals with the synthesis and characterization of the title compound 1. 2-Chloro-6-methylaniline (2) was used as the starting material to obtain 2-chloro-N(2-chloro-6-methylphenyl) acetamide (3), which then reacted with (E)-N, N-dimethyl-N'-(tritylcarbamothioyl)formimidamide to get N-(2-chloro-6-methylphenyl)-2-(tritylamino)thiazole-5-carboxamide (4). Then 4 was cyclized with HCOOH (Scheme 1) to afford 1. In biological research, to evaluate the induction activity on the dental pulp mesenchymal stem cells (DP-MSCs) differentiation, the DP-MSCs colony formation ability assay and osteogenesis related genes expression assay were determined. The former results indicated that the compound could significantly reduce the colony numbers at the 7^th and 14^th days after compound incubation, and showed a dose dependent relationship. In addition, the data of RT-PCR also confirmed that 1 significantly influences the relative level of genes related with cell osteogenesis and proliferation. All the results indicated that the compound could affect the osteogenic differentiation of DP-MSCs.

  Scheme 1

  

  Scheme 1.  Synthesis route of compound 1

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  2. EXPERIMENTAL

  2.1 Apparatus and materials

  IR spectra (400~4000 cm^-1) were obtained using a Brucker Equinox-55 spectrophotometer. ¹H NMR spectra were obtained using a Varian Inova-400 spectrometer (at 400 MHz). Mass spectra were obtained using a micrOTOF-Q II mass spectrometer. The melting points were taken on a XT-4 micro melting apparatus, and the thermometer was uncorrected. The human promyelocytic leukemia cancer HL-60 cell and the normal cell line HEK-293 were obtained from the Cell Bank of Shanghai Institte of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). These two cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) culture medium according to the protocol. The DMEM culture medium was supplemented with 2% L-glutamine, 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Gibco, Life Technologies). Cells were cultured in a standard cell culture environment (humidified incubator with 5% CO₂ at 37 ℃). The culture media were changed according to the cells growth condition.

  2.2 Synthesis and characterization of compounds 3, 4 and 1

  To a mixture of compound 2 (25.0 g, 176.6 mmol) in CH₂Cl₂ (200 mL) was added pyridine (28.6 mL, 353 mmol) at –10 ℃. After 10 min, chloroacetyl chloride (21.1 mL, 265 mmol) was added. The reaction mixture was allowed to warm to room temperature over a period of 2 h. A solution of 1 N HCl (500 mL) was added and the mixture was stirred for 10 min. The organic phase was separated and the aqueous layer was extracted with CH₂Cl₂ (2 × 300 mL). The combined organic extracts were washed with 1 N HCl solution (400 mL), H₂O (400 mL) and brine (400 mL). It was dried over MgSO₄, filtered and concentrated in vacuo to give the product 3 as a white solid (34.70 g, 90.13% yield). m.p: 162~163 ℃. ¹H NMR (500MHz, CDCl₃): δ 2.28 (s, 3H), 4.25 (s, 2H), 7.14~7.18 (m, 2H), 7.26~7.31 (m, 1H), 8.02 (bs, 1H). IR (KBr pellet, cm⁻¹): 3442m, 3176w, 3054w, 2965m, 2875w, 1598w, 1540s, 1456m, 1397s, 792m, 740s.

  A solution of compound 3 (32.7 g, 150 mmol) and (E)-N, N-dimethyl-N'-(tritylcarbamothioyl) formimidamide (56.1 g, 150 mmol) in EtOH (750 mL) was heated to reflux for 6 h. To the reaction solution obtained in the previous step was added 96% formic acid (150 mL). The reaction mixture was refluxed overnight. The reaction mixture was then partitioned between EtOAc and H₂O (1:1, L). The organic phase was separated and concentrated in vacuo. The residue was subjected to column chromatography using EtOAc in hexane (20%~80%) to give the title compound 1 (24.38 g, 60.74% yield). m.p: 196~197 ℃. ¹H NMR (300MHz, DMSO-d₆): δ 2.21(s, 3H), 7.20~7.22 (m, 2H), 7.37(dd, J = 7.2 and 2.4Hz, 1H), 7.57 (s, 2H), 7.86 (s, 1H), 9.60 (s, 1H). HRMS (ESI ⁺): m/z: calcd. for C₁₁H₁₀ClN₃OS: 290.0131 [M + Na ⁺ ]; found: 290.0119. IR (KBr pellet, cm^-1): 3447m, 3118w, 3088s, 2970m, 2882w, 1707m, 1605s, 1552s, 1418s, 1389m, 783m, 698m.

  2.3 Crystal structure determination

  A suitable single crystal of compound 1 (obtained by slow volatilization of its CH₂Cl₂ solution) was carefully selected under optical microscope and glued on thin glass fibers. The intensity data of 1 were collected on an Oxford Xcalibur E diffractometer. The empirical absorption corrections were applied to the data using the SADABS system^[10]. The structure was solved by direct methods and refined by fullmatrix least-squares techiniques on F² using the SHELXS-97 program. All non-hydrogen atoms of 1 were refined anistropically, and all hydrogen atoms attached to carbon atoms were fixed at their ideal positions. Compound 1 (C₁₁H₁₀ClN₃OS, M_r = 267.73 g/mol) belongs to monoclinic space group P2₁/n with a = 16.7208(7), b = 8.6098(5), c = 16.9367(8) Å, β = 93.496(4)°, V = 2433.7(2) ų, Z = 8, T = 296.15 K, μ(MoKα) = 0.471 mm^-1 and D_c = 1.461 g/cm³. For 1, a total of 11205 reflections were collected (4.80≤2θ≤58.67°), of which 5708 were unique (R_int = 0.0340, R_sigma = 0.0566) and used in all calculations. The final R = 0.0529 (I > 2σ(I)) and wR = 0.1403 (all data).

  2.4 Differentiation of DP-MSCs

  To induce the differentiation of dental pulp mesenchymal stem cells (DP-MSCs), the cells collected were seeded into cell culture plates at the concentration of 1 × 10⁴ cell/mL, then divided into the control and differentiation groups. The cells in the latter group were cultured in culture medium supplemented with transforming growth factor-β (TGF-β) and compound for 20 days, while those in the former group were incubated in basal medium^[11].

  2.5 Cell colon formation

  To evaluate the inhibitory of compound on DP-MSCs differentiation, the cell colony formation assay was performed in this present research. Briefly, the DP-MSCs collected as described were seeded onto 6 well plates at the final destiny of 1 × 10⁵ cell/well, followed by the compound attrition at the concentration of 1, 5 and 10 μg/mL for indicated treatment. At the 7^th and 14^th days after compound treatment, the cells were washed with PBS solution, and the cells remaining on the plates were labeled with crystal violet staining and the numbers were counted. This preformation was conducted at least three times.

  2.6 Genes expression

  Real-time RT-PCR was conducted in this experiment to detect the relative expression of the genes with relationship with the cell osteogenesis and proliferation. In short, the DP-MSCs were isolated from the tissues collected in our hospital, and then the cells were seeded into 24-well plates at the final destiny of 1 × 10⁴ cells/mL. The differentiation of DP-MSCs was induced with the incubation of 5 ng/mL TGF-β, then the compound was added into cells at the concentration of 1, 5 and 10 μg/mL. After 20 days treatment, the cells were harvested, washed with PBS solution and the TRIzol reagent was used for the total RNA isolation. The total RNA was reverse-transcribed into cDNA with cDNA Synthesis Kit after concentration measurement. Subsequently, the Real-Time PCR System was used for the Real-time RT-PCR preformation, and the relative gene expression levels were measured and the data were expressed as mean±SD from three repeats^[12].

  3. RESULTS AND DISCUSSION

  3.1 Molecular structure

  The structure of the title compound was measured by X-ray diffraction crystal structural analysis. The result indicates that the compound is of monoclinic system with space group P2₁/n. It displays a one-dimensional chain structure through two kinds of hydrogen bonds (N–H···N and N–H···O). The molecular structural unit is shown in Fig. 1, in which the angle between the six-membered ring (C(1)~C(6)) and the adjacent five-membered ring (S(1), C(8)~C(10) and N(2)) is 58.06(2)°. The bond lengths of C–C, C–N and C–S fall in the ranges of 1.353(3)~1.453(3), 1.304(3)~1.422(4) and 1.727(3)~1.745(3) Å, respectively. The bond distances of C– F, C–O and C–Cl are 1.651, 1.242 and 1.691 Å, respectively, all falling in their normal scopes^[13-15].

  Figure 1

  

  Figure 1.  Molecular structural unit for the title compound

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  In addition, two kinds of hydrogen bonds (N–H···N and N–H···O) could be found in the packing structure, and their detailed information is given in Table 1. Based on these them, a one-dimensional chain structure is generated, with its crystal packing structure depicted in Fig. 2.

  Table 1

  

  Table 1.  Hydrogen Bonds for the Title Compound

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  D–H···A	d(D–H)	d(H···A)	d(D···A)	∠DHA
  N(1)–H(1)···O(1)a	0.86	2.17	2.928(4)	148
  N(3)–H(3A)···N(2)b	0.87	2.41	3.092(6)	136
  N(3)–H(3B)···O(2)	0.86	2.43	3.265(3)	163
  N(4)–H(4A)···O(2)c	0.86	2.36	3.085(5)	143
  N(6)–H(6A)···N(5)d	0.87	2.35	3.065(5)	140
  N(6)–H(6B)···O(1)e	0.86	2.57	3.160(5)	126
  Symmetry codes: (a) 1/2 – x, 1/2 + y, 1/2 – z; (b) 1 – x, 1 – y, 1 – z; (c) 1/2 – x, 1/2 + y, 3/2 – z; (d) 1 – x, 1 – y, 2 – z; (e) 1/2 + x, 1/2 – y, 1/2 + z

  Figure 2

  

  Figure 2.  Crystal packing structure of the compound along the b axis

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  3.2 Reducd colony formation ability of DP-MSCs after compound treatment

  After the synthesis of the compound, its induction activity DP-MSCs differentiation was evaluated. Firstly, the colony formation assay was conducted to evaluate the influence of compound on DP-MSCs differentiation. After treated with the influence of compound on DP-MSCs differentiation, the cells remaining on the plates were labeled with crystal violet staining. The results in Fig. 3 indicated that the number of cell colonies was much lower than the control group after compound treatment, and the inhibitory effect of the compound on the DP-MSCs colony formation ability was dose dependent. In addition to this, we can also see from the results that the positive control drug GLP-1 showed a slightly weaker activity than the compound. All the results suggested strongly that the colony formation ability of DP-MSCs is significantly reduced by compound.

  Figure 3

  

  Figure 3.  Reduced colony formation ability of DP-MSCs after compound treatment. The DP-MSCs were collected and seeded into plates, followed by compound treatment at different concentrations. The colony formation ability of DP-MSCs was determined by crystal violet staining assay. This experiment was repeated at least three times

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  3.3 Regulated expression of osteogenesis and cell proliferation associated genes after compound treatment

  In the previous research, we have confirmed the reduced colony formation ability of DP-MSCs after compound treatment, while the biological activity needs further exploration. The ability of cell proliferation is the classical character of stem cells. Thus, in this experiment, the RT-PCR was performed to detect the relative expression of cell proliferation associated genes, such as Sox2 and Oct4, which are reported as the key genes involved in the regulation of stem cells proliferation, and the inhibition of them will cause cells apoptosis. Besides, the osteogenesis related transcriptional factors Runx2 and ALP were also measured by RTPCR to detect the induction of osteogenesis of DP-MSCs influenced by compound. As shown in Fig. 4, the relative expression of Runx2 and ALP was significantly up-regulated and the level of cell proliferation associated genes Sox2 and Oct4 was reduced obviously after compound treatment. All the above results indicated the compound could induce the osteogenic differentiation of DP-MSCs.

  Figure 4

  

  Figure 4.  Regulated expression of osteogenesis and cell proliferation associated genes after compound treatment. The DP-MSCs were cultured in basal medium supplemented with 5 ng/mL TGF-β, then different concentrations of compound(1, 5, 10 mg) was added for osteogenic induction for 20 days. The expression of osteogenesis and cell proliferation associated genes were measured by RT-PCR. All the data were expressed as mean±SD

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  4. CONCLUSION

  In conclusion, we synthesized a heterocyclic derivative and characterized it via IR, ¹H NMR, HRMS, and single-crystal X-ray diffraction analysis. In biological research, we aimed to explore the effect of compound in regulating the osteogenesis of DP-MSCs. The results of the colon formation assay indicated that the compound could significantly reduce the colony formation ability of DP-MSCs. In addition, the RT-PCR results indicated the compound significantly up-regulates the expression of osteogenic related transcription factors. All these confirmed that the synthetic compound affects the osteogenic differentiation of DP-MSCs, which laid solid foundation for the regeneration of dentin.

  
  
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• 

  Scheme 1  Synthesis route of compound 1

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  Figure 1  Molecular structural unit for the title compound

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  Figure 2  Crystal packing structure of the compound along the b axis

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  Figure 3  Reduced colony formation ability of DP-MSCs after compound treatment. The DP-MSCs were collected and seeded into plates, followed by compound treatment at different concentrations. The colony formation ability of DP-MSCs was determined by crystal violet staining assay. This experiment was repeated at least three times

  下载: 全尺寸图片 幻灯片
  

  Figure 4  Regulated expression of osteogenesis and cell proliferation associated genes after compound treatment. The DP-MSCs were cultured in basal medium supplemented with 5 ng/mL TGF-β, then different concentrations of compound(1, 5, 10 mg) was added for osteogenic induction for 20 days. The expression of osteogenesis and cell proliferation associated genes were measured by RT-PCR. All the data were expressed as mean±SD

  下载: 全尺寸图片 幻灯片

  Table 1.  Hydrogen Bonds for the Title Compound

  D–H···A	d(D–H)	d(H···A)	d(D···A)	∠DHA
  N(1)–H(1)···O(1)a	0.86	2.17	2.928(4)	148
  N(3)–H(3A)···N(2)b	0.87	2.41	3.092(6)	136
  N(3)–H(3B)···O(2)	0.86	2.43	3.265(3)	163
  N(4)–H(4A)···O(2)c	0.86	2.36	3.085(5)	143
  N(6)–H(6A)···N(5)d	0.87	2.35	3.065(5)	140
  N(6)–H(6B)···O(1)e	0.86	2.57	3.160(5)	126
  Symmetry codes: (a) 1/2 – x, 1/2 + y, 1/2 – z; (b) 1 – x, 1 – y, 1 – z; (c) 1/2 – x, 1/2 + y, 3/2 – z; (d) 1 – x, 1 – y, 2 – z; (e) 1/2 + x, 1/2 – y, 1/2 + z

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