English

• Matrine (1) is one of the major active alkaloids isolated from Sophora alopecuroides L, which is a useful Chinese herbal drug. Matrine possesses good biological activities. It not only has been used for the treatment of lipopolysaccharide-induced liver injury, ^[1] but also could regulate immunity, treat cardiac arrhythmia, anti-inflammatory, ^[2] antitumor, ^[3~6] antipyretic^[7] and hepato-protective. The study on biological activity showed that matrine derivatives and sophora acid derivatives have been used in the prevention and treatment of viral diseases of hepatitis B, hepatitis C, human immunodeficiency virus (HIV) and anti-hepatitis B virus (HBV) with greater inhibitory activity than both matrine and sophocarpine.^{[8, 9]} He et al.^[10] has synthesized a series of furoxan-based NO-donating matrine derivatives. These have been evaluated with stronger cytotoxic activities than 5-fluorouracil against human hepatoma cells (HepG2). Some other researchers have synthesized matrinic acid by breaking amide bond.^{[3, 11]}

  Obviously, modification of matrine is becoming one of the most interesting fields for researchers due to the potential pharmacological activities. Matrine derivatives at C-13 position are mainly used as a disinfectant and have effect on Escherichia coli, Staphylococcus aureus, Bacillus subtilis variety with the average sterilization rate of 99.9%. Therefore, introducing a substituent containing N or S atom at C-13 position of matrine is regarded to be important for improving the biological activities. Duan and Zhang et al.^{[12, 13]} had synthesized some matrine derivatives containing S, O or N atom at C-13 position of matrine. However, as we know, the solubility of matrine is not ideal and many matrine derivatives are restricted in experiments on activity measurement and further clinical applications.^[14] Li et al.^[15] successfully synthesized sodium matrine sulfonate which is obviously more hydropholic than matrine. Matrine derivatives with better water solubility are suitable for industrial application and could be promising antitumor drugs in medical fields. And for all we know, there is no research about synthesis of matrine derivatives at C-13 position in aqueous phase by one step. Hence, we describe a simple method for synthesis of matrine derivatives at C-13 position with sophocarpine (2) as the starting material in aqueous phase by one step (Scheme 1). Synthetic method is simple with high yield. Especially, the target compounds a~j have better water solubility.

  Scheme 1

  

  Scheme 1.  Synthesis of compounds a~m

  Reagents and conditions: (ⅰ) sophocarpine (1.25 mmol), amine (2.5 mmol), 2 mL of H₂O, 45~80 ℃, 3~24 h; (ⅱ) sophocarpine (1.25 mmol), amine (5 mmol), CS₂ (10 mmol), MeOH (2 mL)/H₂O (4 mL), 60 ℃, 24 h.

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  1.   Results and discussion

  1.1   Chemistry

  We designed and synthesized thirteen matrine derivatives with matrine as the parent compound. Sophocarpine (2) was used as the starting material and N-containing substituents were introduced via one step classical Michael addition in aqueous phase. Moreover, carbon disulfide participated in the chemical reactions and two S atoms were introduced into compounds k, l and m. The reaction mechanism might be that CS₂ reacted firstly with amine to give thiol compound, which attacked the C-13 position of sophocarpine via Michael addition as shown in Scheme 2. In step two, hydrogen bonds were formed between H₂O and carbonyl group of sophocarpine, and the electrophilicity of β-C in α, β-unsaturated double bond was increased, while hydrogen bonds could also be formed between H atom in thiol group and O atom in H₂O, which increased the nucleophilicity of S atom in thiol. Above all, it could be seen that the reaction yield was increased with H₂O as catalyst.

  Scheme 2

  

  Scheme 2.  Synthetic reaction mechanism of compounds k, l and m

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  The optimization for the synthesis of compound l is listed in Table 1. Firstly, the mixture of pyrrolidine (5.00 mmol), CS₂ (5.00 mmol) and methanol (2 mL) was stirred for 0.5 h at 45 ℃, and then sophocarpine (1.25 mmol) and distilled water (2 mL) were added, and reacted for another 24 h, to generate compound l with a yield of 39%. While the amount of reactants were increased from 5.00 to 10.00 mmol for CS₂ and from 2 to 4 mL for distilled water, the yield was improved to 63%. By changing the temperature, 60 ℃ was found to be the best reaction temperature. The optimized synthetic conditions of compound k were listed in Table 2. Similarly, compound k was synthesized in the same method.

  Table 1

  

  Table 1.  Optimization for the synthesis of compound l^a

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  CS2/mmol	H2O/mL	Temp./℃	Yieldb/%
  5.00	2	45	39
  5.00	4	45	48
  5.00	4	60	67
  10.00	4	60	75
  10.00	4	45	63
  10.00	4	70	60
  10.00	4	80	64
  a Reaction condition: 1.25 mmol of sophocarpine, 5.00 mmol of pyrrolidine, time was 24 h; bIsolated yield.

  Table 2

  

  Table 2.  Optimization for the synthesis of compound k^a

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  Piperidine/mmol	CS2/mmol	Temp./℃	Yieldb/%
  5.00	5.00	45	28
  5.00	5.00	60	55
  5.00	10.00	80	66
  5.00	10.00	60	78
  2.50	10.00	60	64
  aReaction condition: 1.25 mmol of sophocarpine, time was 24 h. b Isolated yield.

  Experimental data of the target compounds were described in detail in Table 3. Their chemical structures were further confirmed by ¹H NMR, ¹³C NMR and HRMS. Moreover, the optical rotations of compounds a, k and m as single crystals were measured, and the values of specific rotation [α]_D²⁰ were +38.1 (c 1.00, CH₃OH), +47.6 (c 1.00, CHCl₃) and +42.6 (c 1.00, CHCl₃), respectively.

  Table 3

  

  Table 3.  Experimental data of the target compounds

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  Compd.	Temp./℃	Time/h	Yield/%	m.p./℃
  a	45	4	92	108.9~110.2
  b	45	4	90	—
  c	60	10	93	248.5~249.8
  d	60	6	89	125.2~126.1
  e	60	6	79	51.9~53.1
  f	80	24	76	119.5~120.8
  g	45	4	91	232.8~233.9
  h	45	4	93	79.9~81.7
  i	60	4	69	110.3~111.2
  j	50	3	88	58.7~59.9
  k	60	24	78	208.9~209.9
  l	60	24	75	206.5~208.1
  m	60	24	70	238.2~239.4

  1.2   Crystal data of compounds k, m and a

  We get three single crystals including compounds k, m and a. Their crystal structures were obtained as shown in Figure 1.

  Figure 1

  

  Figure 1.  Chemical and crystal structures of k, m and a

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  In compound k, the structure includes five chiral carbon atoms and has the absolute configuration of 5(S), 6(S), 7(R), 11(R), 13(S) (Figure 1). Single crystal data analysis also reveals that A and B rings present chair conformation and they are perpendicular to the C ring by paralleling with the D ring. There is no stacking interactions exist between the molecules. The three special interactions, C(151)—H(15A)…O(11)[2.716(7) Å , 101°], C(41)—H(41)…O(11) [3.318 (13) Å , 142°] and C(71)—H(71)…S(21) [3.108(6) Å , 113°], were existing in the intermolecule. These bonds are not in agreement with the typical hydrogen bond distances.^[16] But, the bonds have a positive effect on the stability of the crystal. The C＝O and C＝S bond lengths are 1.219(6) and 1.652(5) Å , respectively. The bond angles of N(11)—C(61)—S(11) and S(21)—C(61)—S(11) are 113.0(4)° and 122.3(3)° (Table 4). The crystal system and space group is monoclinic.

  Table 4

  

  Table 4.  Selected bond distances (Å ) and angles (°) for compounds k, m and a^a

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  Compound k C21H33N3OS2
  S(11)—C(61)	1.769(5)	C(61)—S(11)—C(71)	103.7(2)
  S(11)—C(71)	1.804(5)	N(11)—C(61)—S(11)	113.0(4)
  S(21)—C(61)	1.652(5)	S(21)—C(61)—S(11)	122.3(3)
  N(11)—C(61)	1.316(7)	N(11)—C(61)—S(21)	124.7(4)
  Compound m C20H31N3O2S2
  S(1)—C(21)	1.806(4)	C(12)—S(1)—C(16)	104.2(2)
  S(1)—C(16)	1.765(5)	N(3)—C(16)—S(1)	113.2(4)
  S(2)—C(16)	1.659(5)	S(1)—C(16)—S(2)	122.2(3)
  N(3)—C(16)	1.328(7)	N(3)—C(16)—S(2)	124.6(4)
  Compound a C20H33N3•H2O
  C(3)—C(2)	1.528(4)	C(3)—C(4)	1.506(4)
  N(2)—C(3)	1.483(3)	N(1)—C(1)	1.346(4)
  N(2)—C(16)	1.452(4)	N(2)—C(20)	1.461(4)
  O(1)—C(1)	1.229(3)	C(1)—C(2)	1.505(4)
  C(16)—N(2)—C(20)	109.0(3)	C(3)—N(2)—C(20)	111.1(2)
  C(2)—C(3)—N(2)	111.0(2)	N(2)—C(3)—C(4)	111.3(2)
  C(3)—N(2)—C(16)	112.8(2)	C(4)—C(3)—C(2)	107.3(2)
  a In compounds k and m: symmetry code: i＝－x, y+1/2, －z. In compound a: symmetry code: i＝－x+1/2, －y, z+1/2, ii＝x+1/2, －y+1/2, －z, iii＝－x, y+1/2, －z+1/2.

  In compound m, the structure includes five chiral carbon atoms and has the absolute configuration of 5(S), 6(S), 7(R), 11(R), 13(S) (Figure 1). Single crystal data analysis proves that A and B rings present chair conformation and they are perpendicular to the C ring by paralleling with the D ring. Ring D have the chair form and ring C has the half-chair form. Crystal structural investigation reveals the absence of intermolecular hydrogen bonds. A and B rings are at the side of the C ring, but the D ring is at the other side of the C ring. Such structural features can be confirmed by H(1), H(3) and H(4) lying at different sides of C ring. The C＝O and C＝S bond lengths are 1.226(6) and 1.659(5) Å . The bond angles of S(1)—C(16)—S(2) and N(3)—C(16)—S(1) are 122.2(3)° and 113.2(4)° (Table 4). The crystal system and space group are monoclinic.

  In compound a, the structure includes five chiral carbon atoms and has the absolute configuration of 5(S), 6(S), 7(R), 11(R), 13(S) (Figures 1 and 2). A single crystal with approximate dimensions of 0.47 mm×0.27 mm×0.13 mm was chosen for X-ray diffraction (XRD) studies. The molecules are interlinked by two kinds of hydrogen bonds, O(2)—H(21B)…N(2) and O(2)—H(21A)…O(1), with distances of 2.830(3) and 3.038(3) Å which are in the range of classical hydrogen bonds. In addition, the intramolecular hydrogen bond was expressed as C(9)—H(9A)…O(1), the length is 2.749(3) Å , which is in the range of classical hydrogen bonds, too. Their bond angles are 164° [O(2)—H(21A)…O(1)] and 171° [O(2)—H(21B)…N(2)], which is in the range of classic bond angle of 150°~180°. The intramolecular hydrogen bond angle [C(9)—H(9A)…O(1)] is 102°. These strong hydrogen bonds can play a key role in the accumulation of crystals.^{[17, 18]} The four saturated rings have chair form configurations, but the caprolactam ring has a half-chair conformation. Through the data (Tables 4, 5), it is not difficult to find that C(1)—C(2) bond length is longer than O(1)—C(1) bond length. The crystal system space group of compound a is orthorhombic.

  Figure 2

  

  Figure 2.  Hydrogen bonds in compound a

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  Table 5

  

  Table 5.  Hydrogen bonds for the compound a

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  D—H…A	D—H/Å	H…A/Å	D…A/Å	A—H…A/(°)
  O(2)—H(21B)…N(2)a	0.85	2	2.830(3)	171
  O(2)—H(21A)…O(1)a	0.85	2.2	3.038(3)	164
  C(9)—H(9A)…O(1)b	0.97	2.38	2.749(3)	102
  a Hydrogen bonds between compound a and water molecules. bHydrogen bonds between molecules of compound a.

  1.3   Antiproliferation activity

  Antiproliferation activities of synthesized matrine derivatives were evaluated by methyl thiazolyl tetrazolium (MTT) assay. Sophocarpine and matrine were used as positive control. The matrine derivatives (compounds k•HCl, l•HCl, m•HCl) have been prepared as a salt to achieve higher water solubility and improve antiproliferation activity. Among the tested compounds, k•HCl displayed the most effective antiproliferation activity against A431 cell lines with inhibition rates above 94% at the tested concentration of 120 μmol•L^－1. Meanwhile, compound k•HCl displayed inhibition rates above 65% against SW480 cells and above 95% against A549 at 330 μmol•L^－1. Furthermore, the antiproliferation activities of k•HCl, l•HCl, m•HCl against the three cancer cell lines were much stronger than that of matrine.

  Table 6 shows that the resulting compounds a, c, e, f, k and lexhibited stronger antitumor activities against the three cancer cell lines with IC₅₀ values of 1.3 fold to 16 fold higher than that of matrine. Compounds k and l showed stronger inhibitory effect against A549 cells with an IC₅₀ value of 64.2 and 91.2 μmol•L^－1, respectively. It was clear that introduction of substituents containing N or S atom at C-13 position of matrine derivatives could improve the biochemical profile. Moreover, the S atom had better contribution than N atom to the inhibitory effect. Compared to compounds k, l and m, the hydrochloride salts have different inhibition activities, possibly because the different lipophilicity of these derivatives influenced their interaction with tumor cells.^[19] Among all these compounds, k•HCl showed the highest effect against the three cancer cells, with IC₅₀ values of 43.2 μmol•L^－1 against A431 cells.

  Table 6

  

  Table 6.  IC₅₀ of the target compounds against SW480, A549 and A431 cells

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  Compd.	IC50±SD/(μmol•L－1)
  	SW480	A549	A431
  Sophocarpine	> 1000	> 1000	528.2±18.60
  Matrine	> 1000	> 1000	> 1000
  a	175.10±4.81	365.1±29.84	> 100
  c	216.3±13.5	283.2±67.9	405.6±10.4
  e	133.17±21.91	146.3±20.10	281.21±47.71
  f	> 100	> 100	197.78±31.97
  k	> 200	64.23±34.29	> 100
  l	> 200	91.22±28.26	> 100
  k•HCl	221.7±11.67	139.3±1.34	43.2±4.03
  l•HCl	373.6±2.828	332.2±1.20	114.52±58.00
  m•HCl	190.40±21.64	562.80±22.70	191.10±76.43

  2.   Conclusion

  In summary, thirteen matrine derivatives have been successfully synthesized and characterized by ¹H NMR, ¹³C NMR and HRMS. And the preparation method with one step in aqueous phase is simple with mild reaction conditions, low cost and high yields. Organic solvents and expensive catalysts are not needed for the synthesis of these derivatives. According to analysis of single crystal X-ray diffraction, the absolute configurations of the chiral carbon atoms for target compounds a, k and m are 5(S), 6(S), 7(R), 11(R), 13(S). There are no stacking interactions in the three new crystal structures. In compound m, rings A, B and D have chair forms while ring C has the half-chair form. The compound k reveals the existence of interaction between molecules as follows: C(151)—H(15A)…O(11), C(41)—H(41)…O(11), C(71)—H(71)…S(21). These special interaction between molecules can play an important role in the growth of crystals. In compound a, the molecules are interlinked by three kinds of hydrogen bonds: O(2)—H(21B)…N(2), O(2)—H(21A)…O(1) and C(9)—H(9A)…O(1). These interactions link the molecules into a two-dimensional network parallelly. Moreover, the values of specific rotation of compounds a, k and m indicated that the rotatory direction is right handed. Biological studies of the synthetic compounds suggest that introducing substituents containing N or S atom at the C-13 position of matrine has a strong effect on its antiproliferation activity. Matrine derivatives such as compound k•HCl exhibited some activities against three cancer cells including SW480, A549 and A431 cells. The results of our study may be helpful to explore structural modifications of matrine at C-13 position and the synthesis of potential new antitumor agents.

  3.   Experimental

  3.1   General procedure

  Sophocarpine was purchased from Ningxia Doushun Biological Technology Co., Ltd (Yanchi, China). Human colorectal carcinoma cells SW480, human lung cancer cells A549 and human epidermoid carcinoma cells A431 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco. Trypsin, penicillin-streptomycin (P/S) and L-glutamate were purchased from Sigma-Aldrich. All other reagents and solvents were analytical grade, which were supplied by local commercial suppliers and used without further purification unless otherwise noted. ^lH NMR and ¹³C NMR spectra were recorded on 400, 600 MHz (Bruker Avance) instruments. The high-resolution mass spectra were measured using a Bruker Esquire 3000 plus mass spectrometer. Melting points were determined using a digital melting point apparatus (Beijing Tektronix Instrument Co. Ltd., Zhengzhou, China). Optical rotation were measured using America Rudolph (Autopol Ⅳ-T) instruments.

  The crystallographic measurement of the target compounds were collected at 273 K on a Bruker Smart-1000CCD diffractometer with graphite-monochromated Mo Kα radiation (λ＝0.071073 Å ) using ω-2θ scan technique. The structures were solved by direct methods and refined on F² by full matrix least squares with the Bruker's SHELXL-97 program.

  3.2   Synthesis of the target compounds a~j

  Sophocarpine (1.25 mmol) and amines (2.5 mmol) were dissolved in water (2.0 mL) with stirring. The reaction mixture was stirred at different temperatures. After completion of the reaction, water was removed under reduced pressure. The residue was purified with column chromatography on silica gel using ethyl acetate/methanol (V: V＝4:1) as eluent to give a white solid or colorless oil. The target compound was obtained by recrystallization from petroleum ether.

  13-(Piperidin-1-yl)matrine (a): White solid, yield 92%. m.p. 108.9~110.2 ℃; [α]_D²⁰ 38.1 (c 1.00, CH₃OH); ¹H NMR (400 MHz, CDCl₃) δ: 4.28 (dd, J＝12.6, 4.4 Hz, 1H), 4.18~3.95 (m, 1H), 3.07 (t, J＝12.5 Hz, 1H), 2.85~2.74 (m, 2H), 2.62 (dd, J＝11.4, 8.8 Hz, 1H), 2.58~2.35 (m, 7H), 2.14 (s, 1H), 1.96 (ddd, J＝16.0, 15.5, 9.3 Hz, 3H), 1.89~1.74 (m, 2H), 1.74~1.52 (m, 8H), 1.52~1.30 (m, 6H); ¹³C NMR (101 MHz, CDCl₃) δ: 168.9, 65.2, 58.2, 58.1, 55.6, 52.3, 51.3, 51.3, 43.4, 42.0, 36.9, 36.4, 28.6, 27.9, 27.7, 27.0, 27.0, 25.4, 22.1, 21.6; HRMS calcd for C₂₀H₃₄N₃O [M+H]⁺ 332.2703, found 332.2697.

  13-(Pyrrolidin-1-yl)matrine (b): Colorless oil, yield 90%. ¹H NMR (400 MHz, CDCl₃) δ: 4.32 (dd, J＝12.7, 4.3 Hz, 1H), 4.09~3.88 (m, 1H), 3.09 (t, J＝12.6 Hz, 2H), 2.94~2.71 (m, 2H), 2.67~2.37 (m, 7H), 2.13 (s, 1H), 2.06~1.81 (m, 5H), 1.82~1.61 (m, 7H), 1.60~1.49 (m, 2H), 1.50~1.34 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) δ: 167.8, 64.0, 57.2, 57.2, 55.5, 51.8, 50.7, 41.9, 38.5, 35.7, 30.4, 27.7, 26.7, 23.3, 21.1, 20.7; HRMS calcd for C₁₉H₃₂N₃O [M+ H]⁺ 318.2546, found 318.2526.

  13-(Morpholin-4-yl)matrine (c): White solid, yield 93%. m.p. 248.5~249.8 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 4.39~4.27 (m, 1H), 4.01 (dt, J＝10.7, 5.3 Hz, 1H), 3.94~3.61 (m, 4H), 3.26~3.02 (m, 1H), 2.80 (dt, J＝58.7, 29.2 Hz, 2H), 2.67~2.39 (m, 7H), 2.15 (s, 1H), 2.09~1.80 (m, 5H), 1.70 (dd, J＝40.6, 20.2 Hz, 3H), 1.64~1.35 (m, 6H); ¹³C NMR (101 MHz, CDCl₃) δ: 167.5, 67.0, 67.0, 64.1, 57.2, 57.2, 54.9, 50.8, 50.3, 50.3, 42.1, 41.7, 35.8, 33.8, 27.7, 27.6, 26.7, 21.6, 20.2; HRMS calcd for C₁₉H₃₂N₃O₂ [M+H]⁺ 334.2495, found 334.2491.

  13-(4-Methylpiperazin-1-yl)matrine (d): White solid, yield 89%. m.p. 125.2~126.1 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 4.36~4.24 (m, 1H), 4.01 (s, 1H), 3.08 (d, J＝11.2 Hz, 1H), 2.87~2.74 (m, 2H), 2.59 (s, 3H), 2.57~2.50 (m, 3H), 2.43 (dd, J＝16.6, 8.4 Hz, 5H), 2.27 (dd, J＝9.3, 3.3 Hz, 3H), 2.13 (s, 1H), 2.02~1.83 (m, 5H), 1.67 (dd, J＝25.2, 9.1 Hz, 3H), 1.55 (s, 2H), 1.53~1.46 (m, 1H), 1.46~1.32 (m, 3H); ¹³C NMR (151 MHz, CDCl₃) δ: 167.6, 64.2, 57.3, 57.3, 55.3, 55.3, 54.5, 51.0, 49.6, 49.6, 46.0, 42.2, 41.6, 36.1, 35.9, 27.7, 27.7, 26.8, 21.2, 20.8; HRMS calcd for C₂₀H₃₅N₄O [M+H]⁺ 347.2811, found 347.2811.

  13-(Cyclohexylamino)matrine (e): White solid, yield 79%. m.p. 51.9~53.1 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 4.40~4.14 (m, 1H), 3.92 (dt, J＝10.6, 5.4 Hz, 1H), 3.15 (d, J＝2.6 Hz, 1H), 3.01 (td, J＝12.6, 4.1 Hz, 1H), 2.75 (dd, J＝28.4, 9.7 Hz, 2H), 2.53~2.41 (m, 2H), 2.23 (dd, J＝16.9, 5.1 Hz, 1H), 2.06 (s, 1H), 1.96~1.54 (m, 15H), 1.49~1.28 (m, 5H), 1.25~0.91 (m, 5H); ¹³C NMR (151 MHz, CDCl₃) δ: 167.8, 64.0, 57.3, 53.3, 50.5, 44.8, 42.4, 41.8, 39.1, 35.7, 34.1, 33.8, 31.6, 27.7, 26.7, 26.1, 25.1, 21.2, 20.7; HRMS calcd for C₂₁H₃₆N₃O [M+H]⁺ 346.2858, found 346.2862.

  13-(Piperazin-1-yl)matrine (f): White solid, yield 76%. m.p. 119.5~120.8 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 4.27~4.19 (m, 1H), 3.95 (dt, J＝10.6, 5.1 Hz, 1H), 3.01 (dd, J＝22.7, 10.2 Hz, 1H), 2.86 (s, 4H), 2.75 (dd, J＝24.7, 11.2 Hz, 3H), 2.63~2.53 (m, 2H), 2.46 (s, 5H), 2.37 (dd, J＝16.8, 8.6 Hz, 1H), 2.08 (s, 1H), 1.95~1.87 (m, 3H), 1.82 (t, J＝11.9 Hz, 2H), 1.70~1.57 (m, 4H), 1.55~1.49 (m, 2H), 1.48~1.42 (m, 1H), 1.35 (dd, J＝20.9, 9.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ: 167.6, 64.1, 57.2, 54.8, 51.1, 50.2, 45.7, 42.3, 41.5, 35.9, 35.8, 27.7, 27.4, 26.7, 21.2, 20.7; HRMS calcd for C₁₉H₃₃N₄O [M+H]⁺ 333.2654, found 333.2660.

  13-(N, N-Dimethylamino)matrine (g): White solid, yield 91%. m.p. 232.8~233.9 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 4.24 (dd, J＝12.5, 4.2 Hz, 1H), 3.98 (dt, J＝10.6, 5.1 Hz, 1H), 3.03 (t, J＝12.5 Hz, 1H), 2.75 (t, J＝12.9 Hz, 2H), 2.38 (dt, J＝17.2, 10.9 Hz, 3H), 2.20 (s, 6H), 2.08 (s, 1H), 2.00~1.74 (m, 5H), 1.74~1.45 (m, 5H), 1.45~1.23 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ: 166.6, 63.1, 56.3, 56.2, 54.4, 49.9, 41.3, 41.3, 41.2, 40.5, 35.2, 34.9, 27.1, 26.7, 25.8, 20.2, 19.7; HRMS calcd for C₁₇H₃₀N₃O [M+H]⁺ 292.2379, found 292.2371.

  13-(N-Methylamino)matrine (h): White solid, yield 93%. m.p. 79.9~81.7 ℃ (lit.^[9] yield 40%, m.p. 80~81 ℃); ¹H NMR (400 MHz, CDCl₃) δ: 4.26 (dt, J＝15.0, 7.5 Hz, 1H), 3.91 (dt, J＝11.0, 6.9 Hz, 1H), 3.02 (t, J＝12.7 Hz, 1H), 2.92~2.82 (m, 1H), 2.75 (dt, J＝16.3, 11.5 Hz, 2H), 2.56~2.42 (m, 1H), 2.36 (s, 3H), 2.25 (dd, J＝17.0, 5.6 Hz, 1H), 2.05 (s, 2H), 1.96~1.79 (m, 4H), 1.73~1.50 (m, 5H), 1.50~1.41 (m, 2H), 1.42~1.24 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) δ: 167.7, 63.8, 57.2, 57.2, 50.3, 50.1, 42.4, 41.6, 38.4, 35.5, 33.7, 30.5, 27.7, 26.6, 21.1, 20.6; HRMS calcd for C₁₆H₂₈N₃O [M+H]⁺ 278.2232, found 278.2238.

  13-((2-Hydroxyethyl)amino)matrine (i): White solid, yield 69%. m.p. 110.3~111.2 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 4.25 (dd, J＝25.8, 9.6 Hz, 1H), 4.01~3.81 (m, 1H), 3.58 (t, J＝4.9 Hz, 2H), 3.02 (t, J＝12.3 Hz, 2H), 2.82~2.66 (m, 4H), 2.49 (d, J＝16.9 Hz, 1H), 2.28 (dd, J＝16.9, 3.1 Hz, 3H), 2.06 (s, 1H), 1.97~1.86 (m, 3H), 1.82 (d, J＝13.6 Hz, 1H), 1.72~1.50 (m, 5H), 1.44 (d, J＝5.1 Hz, 2H), 1.35 (dd, J＝25.5, 13.4 Hz, 3H); ¹³C NMR (151 MHz, CDCl₃) δ: 167.7, 63.8, 61.3, 57.2, 57.2, 50.2, 48.6, 48.6, 42.5, 41.7, 38.9, 35.5, 31.1, 27.7, 26.6, 21.1, 20.7; HRMS calcd for C₁₇H₃₀N₃O₂ [M+H]⁺ 308.2338, found 308.2342.

  13-((2-Aminoethyl)amino)matrine (j): White solid, yield 88%. m.p. 58.7~59.9 ℃; ¹H NMR (600 MHz CDCl₃) δ: 4.29~4.17 (m, 1H), 3.87 (d, J＝52.2 Hz, 3H), 3.10~2.94 (m, 2H), 2.72 (dd, J＝31.0, 27.2 Hz, 5H), 2.49 (dd, J＝24.6, 10.3 Hz, 1H), 2.33~2.22 (m, 1H), 2.17~2.01 (m, 1H), 1.97~1.77 (m, 6H), 1.73~1.51 (m, 5H), 1.45 (d, J＝3.9 Hz, 2H), 1.35 (t, J＝18.2 Hz, 3H); ¹³C NMR (151 MHz, CDCl₃) δ: 167.7, 63.8, 57.1, 50.2, 48.5, 47.6, 42.4, 41.6, 40.9, 38.7, 35.5, 31.0, 27.7, 26.5, 21.1, 20.6; HRMS calcd for C₁₇H₃₁N₄O [M+H]⁺ 307.2498, found 307.2494.

  3.3   Synthesis of compounds k~m

  Carbon disulfide (10.00 mmol) was added to a mixture of methanol (2.0 mL) and amines (5.0 mmol). The mixture was stirred vigorously at 45 ℃ for 0.5 h and then sophocarpine (1.25 mmol) and water (4.0 mL) were added. The reaction mixture was heated for 24 h at 60 ℃. In the reaction process, a small balloon is sealed at the upper end of the spherical condenser tube in order to avoid the volatile of carbon disulfide. After the end of reaction, MeOH and water were removed under reduced pressure. The residue was purified with column chromatography on silica gel using ethyl acetate/methanol (V:V＝15:1) as eluent to give a white solid. The pure compounds k~m were obtained by recrystallization from ethanol.

  13-(Piperidin-1-yl)carbodithioate matrine (k): White solid, yield 78%. m.p. 208.9~209.9 ℃; [α]_D²⁰ 47.6 (c 1.00, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ: 4.46 (d, J＝2.8 Hz, 1H), 4.40~4.29 (m, 2H), 4.19 (s, 1H), 4.04~3.96 (m, 1H), 3.88 (s, 1H), 3.81 (s, 1H), 3.10 (t, J＝12.6 Hz, 1H), 2.90~2.76 (m, 3H), 2.66 (dd, J＝17.3, 6.7 Hz, 1H), 2.24 (dt, J＝13.5, 6.6 Hz, 1H), 2.20~2.04 (m, 3H), 1.98 (dt, J＝13.0, 6.4 Hz, 2H), 1.80~1.59 (m, 11H), 1.58~1.49 (m, 1H), 1.49~1.35 (m, 3H); ¹³C NMR (101 MHz, CDCl₃)δ: 193.5, 166.6, 64.0, 57.3, 57.3, 52.6, 52.0, 51.4, 42.6, 42.4, 41.8, 37.6, 35.6, 31.8, 27.7, 26.6, 26.0, 25.4, 24.5, 21.2, 20.8; HRMS calcd for C₂₁H₃₄N₃OS₂ [M+H]⁺ 408.2144, found 408.2145.

  13-(Pyrrolidin-1-yl)carbodithioate matrine (l): White solid, yield 75%. m.p. 206.5~208.1 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 4.40 (s, 1H), 4.31 (dd, J＝12.6, 4.0 Hz, 1H), 3.97~3.88 (m, 1H), 3.83 (t, J＝6.8 Hz, 2H), 3.54 (t, J＝6.8 Hz, 2H), 3.03 (t, J＝12.6 Hz, 1H), 2.74 (dd, J＝20.3, 8.0 Hz, 3H), 2.59 (dd, J＝17.3, 6.4 Hz, 1H), 2.19 (dt, J＝13.3, 6.6 Hz, 1H), 2.09 (s, 1H), 2.08~1.96 (m, 4H), 1.97~1.85 (m, 4H), 1.75~1.28 (m, 9H); ¹³C NMR (101 MHz, CDCl₃) δ: 190.7, 166.6, 63.9, 57.3, 57.3, 54.8, 51.9, 50.7, 42.5, 42.0, 41.8, 37.6, 35.6, 31.8, 27.7, 26.6, 26.0, 24.2, 21.2, 20.8; HRMS calcd for C₁₉H₃₂N₃O₂ [M+H]⁺ 394.1988, found 394.1983.

  13-(Morpholin-4-yl)carbodithioate matrine (m): White solid, yield 70%. m.p. 238.2~239.4 ℃; [α]_D²⁰ 1.00, CHCl₃); ¹H NMR (400 MHz, CDCl₃)δ: 4.40 (s, 1H), 4.31 (dd, J＝12.6, 4.0 Hz, 1H), 3.97~3.88 (m, 1H), 3.83 (t, J＝6.8 Hz, 2H), 3.54 (t, J＝6.8 Hz, 2H), 3.03 (t, J＝12.6 Hz, 1H), 2.74 (dd, J＝20.3, 8.0 Hz, 3H), 2.59 (dd, J＝17.3, 6.4 Hz, 1H), 2.19 (dt, J＝13.3, 6.6 Hz, 1H), 2.09 (s, 1H), 2.08~1.96 (m, 4H), 1.97~1.85 (m, 4H), 1.75~1.28 (m, 9H); ¹³C NMR (101 MHz, CDCl₃) δ: 195.4, 166.4, 66.0, 66.0, 63.9, 57.9, 57.3, 51.9, 51.0, 42.7, 42.5, 41.8, 37.6, 31.7, 27.7, 26.6, 21.2, 20.8; HRMS calcd for C₂₀H₃₁N₃O₂S₂ [M+H]⁺ 410.1937, found 410.1914.

  3.4   Antiproliferation activities studies

  The antiproliferation activities of a~m against SW480, A549 and A431 cells were evaluated in vitro by the MTT assay, with slight modifications. SW480, A549 and A431 cells were cultured using DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin and 2 mmol•L^－1 L-glutamate at 37 ℃ and 5% CO₂ with 95% humidity. The cells were collected, and then seeded into 96-well microtiter plates with 100 μL of fresh medium overnight. Solutions of each compound at different concentrations were added to the cells, and cultured for another 72 h. Afterwards, the cells were incubated at 37 ℃ for another 4 h with 20 μL of 5 mg/mL MTT. Finally, the media was washed out, and reduced MTT product (blue formazan product) was solubilized by adding 150 μL of dimethylsulfoxide (DMSO) to each well. Plates were agitated for 10 min at room temperature. The optical density (OD) of the solubilized products was measured using a microplate reader (model Multiskan MK3, Thermo, MA, USA) at 570  nm with background subtraction at 630 nm. The inhibition rate of the compounds was calculated according to the following formula:

  [1－(OD_S－OD_NC)/(OD_PC－OD_NC)]×100%

  where OD_PC and OD_S are the optical densities of the control and test groups at 570 nm, respectively. OD_NC is the optical densities at 630 nm. All assays were done in triplicate. Experimental results are presented as the mean±standard deviation (SD) of three parallel measurements. IC₅₀ values of the compounds against the tumor cell lines were generated using GraphPad Prism 5.

  Supporting Information PDF files of NMR, HRMS spectra and crystallographic data of compounds a~m. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

• 1. [1]

     Zhang, J. P.; Zhang, M.; Zhou, J. P.; Liu, F. T.; Zhou, B.; Xie, W, F.; Guo, C.; Zhang, C.; Qian, D. H. Acta Pharm. Sin. 2001, 22, 183. https://www.sciencedirect.com/science/article/pii/S1572599505800481

  2. [2]

     Hu, H. G.; Wang, S. Z.; Zhang, C. M.; Wang, L.; Ding, L.; Zhang, J. P.; Wu, Q. Y. Bioorg. Med. Chem. Lett. 2010, 20, 7537. doi: 10.1016/j.bmcl.2010.09.075

  3. [3]

     Wang, L. S.; You, Y. J.; Wang, S. Q.; Liu, X.; Liu, B. M.; Wang, J. N.; Lin, X.; Chen, M. S.; Liang, G.; Yang, H. Bioorg. Med. Chem. Lett. 2012, 22, 4100. doi: 10.1016/j.bmcl.2012.04.069

  4. [4]

     Huang, Z. S.; Zhou, X. H. Med. Recapitulate 2009, 15, 1701.

  5. [5]

     Zhang, L. J.; Wang, T. T.; Wen, X. M.; Wei, Y.; Peng, X. C.; Li, H.; Wei, L. Eur. J. Pharmacol. 2007, 563, 69. doi: 10.1016/j.ejphar.2007.01.073

  6. [6]

     Lin, Z.; Huang, C. F.; Liu, X. S.; Jiang, J. H. Basic Clin. Pharmacol. 2010, 108, 304. http://www.ncbi.nlm.nih.gov/pubmed/22193282

  7. [7]

     Chui, C. H.; Lau, F. Y.; Tang, J. C. O.; Kan, K. L.; Cheng, G. Y. M.; Int. J. Mol. Med. 2005, 16, 337. http://www.ncbi.nlm.nih.gov/pubmed/16012772

  8. [8]

     Du, N. N.; Li, X.; Wang, Y. P.; Liu, F. Y.; Liu, X. C.; Li, X.; Peng, Z. G.; Gao, L. M.; Jiang, J. D.; Song, D. Q. Bioorg. Med. Chem. Lett. 2011, 21, 4732. doi: 10.1016/j.bmcl.2011.06.071

  9. [9]

     Gao, L. M.; Han, Y. X.; Wang, Y. P.; Li, Y. H.; Shan, Y. Q.; Li, X.; Peng, Z. G.; Bi, C. W.; Zhang, T.; Du, N. N.; Jiang, J. D.; Song, D. Q. J. Med. Chem. 2011, 54, 869. doi: 10.1021/jm101325h

  10. [10]

      He, L. Q.; Liu, J.; Yin, D. K.; Zhang, Y. H.; Wang, X. S. Chin. Chem. Lett. 2010, 21, 381. doi: 10.1016/j.cclet.2009.11.033

  11. [11]

      Chao, F.; Wang, D. E.; Liu, R.; Tu, Q.; Liu, J. J.; Wang, J. Y. Molecules 2013, 18, 5420. doi: 10.3390/molecules18055420

  12. [12]

      Wang, W.; Duan, Z. H.; Zhang, J. T.; Li, B. L. Z. Kristallogr.-New Cryst. Struct. 2007, 222, 239. http://jbcs.sbq.org.br/imageBank/epub/jbchs_v26n12.epub

  13. [13]

      Wang, W.; Zhang, J. T.; Yan, H. Y.; Li, B. L. Z. Kristallogr.-New Cryst. Struct. 2007, 222, 71. https://www.sciencedirect.com/science/article/pii/S1293255808002999

  14. [14]

      Ci, Y. ; Han, X. L. ; Ma, A. M. ; Wang, L. ; Cheng, C. T. ; Li, J. Y. ; Wen, Y. J. CN 102603744, 2012[Chem. Abstr. 2012, 157, 295336].

  15. [15]

      Li, Z. B. ; Liao, D. D. ; Zhao, C. G. ; Zuo, H. ; He, X. Y. ; Deng, L. ; Chen, M. ; Liu, Q. W. ; Tian, X. ; Yang, J. F. CN 101585838, 2009[Chem. Abstr. 2009, 152, 57453].

  16. [16]

      Kaiser, R. I.; Ochsenfeld, C.; Head-Gordon, M.; Lee, Y. T. J. Chem. Phys. 1999, 110, 2391. http://adsabs.harvard.edu/abs/1999JChPh.110.2391K

  17. [17]

      Hunter, C. A.; Sanders, J. K. M. J. Am. Chem. Soc. 1990, 112, 5525. doi: 10.1021/ja00170a016

  18. [18]

      Kaafarani, B. R.; Pinkerton, A. A.; Neckers, D. C. Tetrahedron Lett. 2001, 42, 8137. doi: 10.1016/S0040-4039(01)01757-9

  19. [19]

      Lo, C. Y.; Hsu, L. C.; Chen, M. S.; Lin, Y. J.; Chen, L. G.; Kuo, C. D.; Wu, J. Y. Bioorg. Med. Chem. Lett. 2013, 23, 305. doi: 10.1016/j.bmcl.2012.10.098

• 

  Scheme 1  Synthesis of compounds a~m

  Reagents and conditions: (ⅰ) sophocarpine (1.25 mmol), amine (2.5 mmol), 2 mL of H₂O, 45~80 ℃, 3~24 h; (ⅱ) sophocarpine (1.25 mmol), amine (5 mmol), CS₂ (10 mmol), MeOH (2 mL)/H₂O (4 mL), 60 ℃, 24 h.

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  Scheme 2  Synthetic reaction mechanism of compounds k, l and m

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  Figure 1  Chemical and crystal structures of k, m and a

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  Figure 2  Hydrogen bonds in compound a

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  Table 1.  Optimization for the synthesis of compound l^a

  CS2/mmol	H2O/mL	Temp./℃	Yieldb/%
  5.00	2	45	39
  5.00	4	45	48
  5.00	4	60	67
  10.00	4	60	75
  10.00	4	45	63
  10.00	4	70	60
  10.00	4	80	64
  a Reaction condition: 1.25 mmol of sophocarpine, 5.00 mmol of pyrrolidine, time was 24 h; bIsolated yield.

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  Table 2.  Optimization for the synthesis of compound k^a

  Piperidine/mmol	CS2/mmol	Temp./℃	Yieldb/%
  5.00	5.00	45	28
  5.00	5.00	60	55
  5.00	10.00	80	66
  5.00	10.00	60	78
  2.50	10.00	60	64
  aReaction condition: 1.25 mmol of sophocarpine, time was 24 h. b Isolated yield.

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  Table 3.  Experimental data of the target compounds

  Compd.	Temp./℃	Time/h	Yield/%	m.p./℃
  a	45	4	92	108.9~110.2
  b	45	4	90	—
  c	60	10	93	248.5~249.8
  d	60	6	89	125.2~126.1
  e	60	6	79	51.9~53.1
  f	80	24	76	119.5~120.8
  g	45	4	91	232.8~233.9
  h	45	4	93	79.9~81.7
  i	60	4	69	110.3~111.2
  j	50	3	88	58.7~59.9
  k	60	24	78	208.9~209.9
  l	60	24	75	206.5~208.1
  m	60	24	70	238.2~239.4

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  Table 4.  Selected bond distances (Å ) and angles (°) for compounds k, m and a^a

  Compound k C21H33N3OS2
  S(11)—C(61)	1.769(5)	C(61)—S(11)—C(71)	103.7(2)
  S(11)—C(71)	1.804(5)	N(11)—C(61)—S(11)	113.0(4)
  S(21)—C(61)	1.652(5)	S(21)—C(61)—S(11)	122.3(3)
  N(11)—C(61)	1.316(7)	N(11)—C(61)—S(21)	124.7(4)
  Compound m C20H31N3O2S2
  S(1)—C(21)	1.806(4)	C(12)—S(1)—C(16)	104.2(2)
  S(1)—C(16)	1.765(5)	N(3)—C(16)—S(1)	113.2(4)
  S(2)—C(16)	1.659(5)	S(1)—C(16)—S(2)	122.2(3)
  N(3)—C(16)	1.328(7)	N(3)—C(16)—S(2)	124.6(4)
  Compound a C20H33N3•H2O
  C(3)—C(2)	1.528(4)	C(3)—C(4)	1.506(4)
  N(2)—C(3)	1.483(3)	N(1)—C(1)	1.346(4)
  N(2)—C(16)	1.452(4)	N(2)—C(20)	1.461(4)
  O(1)—C(1)	1.229(3)	C(1)—C(2)	1.505(4)
  C(16)—N(2)—C(20)	109.0(3)	C(3)—N(2)—C(20)	111.1(2)
  C(2)—C(3)—N(2)	111.0(2)	N(2)—C(3)—C(4)	111.3(2)
  C(3)—N(2)—C(16)	112.8(2)	C(4)—C(3)—C(2)	107.3(2)
  a In compounds k and m: symmetry code: i＝－x, y+1/2, －z. In compound a: symmetry code: i＝－x+1/2, －y, z+1/2, ii＝x+1/2, －y+1/2, －z, iii＝－x, y+1/2, －z+1/2.

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  Table 5.  Hydrogen bonds for the compound a

  D—H…A	D—H/Å	H…A/Å	D…A/Å	A—H…A/(°)
  O(2)—H(21B)…N(2)a	0.85	2	2.830(3)	171
  O(2)—H(21A)…O(1)a	0.85	2.2	3.038(3)	164
  C(9)—H(9A)…O(1)b	0.97	2.38	2.749(3)	102
  a Hydrogen bonds between compound a and water molecules. bHydrogen bonds between molecules of compound a.

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  Table 6.  IC₅₀ of the target compounds against SW480, A549 and A431 cells

  Compd.	IC50±SD/(μmol•L－1)
  	SW480	A549	A431
  Sophocarpine	> 1000	> 1000	528.2±18.60
  Matrine	> 1000	> 1000	> 1000
  a	175.10±4.81	365.1±29.84	> 100
  c	216.3±13.5	283.2±67.9	405.6±10.4
  e	133.17±21.91	146.3±20.10	281.21±47.71
  f	> 100	> 100	197.78±31.97
  k	> 200	64.23±34.29	> 100
  l	> 200	91.22±28.26	> 100
  k•HCl	221.7±11.67	139.3±1.34	43.2±4.03
  l•HCl	373.6±2.828	332.2±1.20	114.52±58.00
  m•HCl	190.40±21.64	562.80±22.70	191.10±76.43

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•