Synthesis and cytotoxicity evaluation of a novel justicidin G analogue and its phosphate ester

Article abstract of DOI:10.1016/j.cclet.2014.05.042

The novel justicidin G analogue 13 and its phosphate ester 15 were synthesized as potential anticancer agents in several steps starting from commercially available methyl gallate and veratraldehyde. The cytotoxicity of the intermediates was tested against HCT-8, BEL-7402, KETR3, HELA, BGC-823, KB and MCF-7 cell lines by the MTT test, and compound 15 exhibited significant cytotoxicity in HELA and KB cell lines.

Full text of DOI:10.1016/j.cclet.2014.05.042

G Model
CCLET 3023 1–3
Chinese Chemical Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
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Original article
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Synthesis and cytotoxicity evaluation of a novel justicidin G analogue
and its phosphate ester
a
b
b
*
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Q1 Sheng-Peng Wang a, Yuan-Feng Tong a, , Dong-Mei Wang , Nan Wang , Zheng Yan ,
Ping Huang a, Song Wu a,
*
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9
a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese
Academy of Medical Sciences, Beijing 100050, China
b Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica, Peking Union Medical College and Chinese
10
Academy of Medical Sciences, Beijing 100050, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The novel justicidin G analogue 13 and its phosphate ester 15 were synthesized as potential anticancer
agents in several steps starting from commercially available methyl gallate and veratraldehyde. The
cytotoxicity of the intermediates was tested against HCT-8, BEL-7402, KETR3, HELA, BGC-823, KB and
MCF-7 cell lines by the MTT test, and compound 15 exhibited significant cytotoxicity in HELA and KB cell
lines.
Received 31 March 2014
Received in revised form 20 May 2014
Accepted 21 May 2014
Available online xxx
ß 2014 Yuan-Feng Tong and Song Wu. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
Keywords:
Justicidin G
Analogue
Phosphate ester
Antitumor activity
Synthesis
11
12
1. Introduction
Compounds 10–15 were evaluated for their in vitro cytotoxicity 31
against a panel of human tumour cell lines.
32
13
14
15
16
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19
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25
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27
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30
Justicidin G is a natural aryl-naphthalene lignan lactone
isolated from the plant of Justicia procumbens L., which has
shown significant antitumor activity [1–3]. Justicidin G has a
similar chemical structure to that of etoposide and teniposide,
but displays stronger anticancer activity and fewer side effects
and is now being further investigated as a new drug development
candidate [4–7]. Considering the poor solubility of justicidin G
and the successful structural modification experience of etopo-
side phosphate ester [8], we made an attempt to synthesis the
glycosylated and phosphate ester derivatives of justicidin G.
However, the product yields of both derivatives were very low.
Moreover, our previous study of justicidinoside B (the glycosy-
lated product of justicidin G) indicated that derivatives with
substituents at the 60-OH position exhibited hindered rotation
around the aryl-naphthalene bond, which led to difficult
structural modifications and poor chemical stability [9,10].
Hence, we have further investigated the novel justicidin G
analogue 13 with a 50-OH group and its phosphate ester 15.
2. Experimental
33
The synthesis of novel justicidin G analogue 13 and its 34
phosphate ester 15 were realized in several steps starting from 35
commercially available methyl gallate and veratraldehyde 36
(Scheme 1). Compound 3 was synthesized in two steps from 37
veratraldehyde in 90% yield [11]. Compound 8 was obtained in four 38
steps from methyl gallate in 81% yield [12]. Treatment of 39
compound 3 with BuLi and compound 8 under nitrogen atmo- 40
sphere at ꢀ78 8C afforded compound 9 in 70% yield [9]. The 41
synthesis of compound 10 was accomplished via a Diels–Alder 42
reaction from compound 9 and DEADC under acidic conditions [3]. 43
Compound 10 was reduced with NaBH4 in tetrahydrofuran under 44
refluxing to afford compound 11 in 92% yield [10]. Methylation of 45
compound 11 with CH3I/K2CO3 in acetone under refluxing yielded 46
compound 12 [9]. Compound 13 was prepared by treatment of the 47
compound 12 with 10% Pd/C in methanol at ambient temperature. 48
Treatment of compound 13 with EtN(i-Pr)2/CCl4/4-DMAP/ 49
HPO(OBn)2 in acetonitrile at ꢀ30 8C afforded compound 14 in 50
92% yield [8]. Compound 15 was synthesized by treatment of the 51
compound 14 with 10% Pd/C in methanol at ambient temperature. 52
*
Corresponding authors.
E-mail addresses: tongyuanf@imm.ac.cn (Y.-F. Tong), ws@imm.ac.cn (S. Wu).
http://dx.doi.org/10.1016/j.cclet.2014.05.042
1001-8417/ß 2014 Yuan-Feng Tong and Song Wu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: S.-P. Wang, et al., Synthesis and cytotoxicity evaluation of a novel justicidin G analogue and its
phosphate ester, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.05.042

G Model
CCLET 3023 1–3
2
S.-P. Wang et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
Scheme 1. Synthesis of justicidin G analogue and its phosphate ester. Conditions and reagents: (a) Br2, CH3OH, r.t., 12 h, 93%; (b) ethanediol, TsOHꢁH2O, toluene, reflux, 3 h,
96%; (c) NaH, B(OCH3)3, BnBr, DMF, 6 h, 92%; (d) CH2Br2, DMF, 110 8C, 1 h, 96%; (e) LiAlH4, THF, 60 8C, 4 h, 93%; (f) PCC, CH2Cl2, 3 h, 95%; (g) n-BuLi, THF, ꢀ78 8C, 6 h, 70%; (h)
DEADC, AcOH, CH2Cl2, 140 8C, 12 h, 46%; (i) NaBH4, THF, reflux, 3 h, 92%; (j) CH3I, K2CO3, acetone, reflux, 1 h, 96%; (k) H2, 10% Pd/C, CH3OH, r.t., 3 h, 97%; (l) EtN(i-Pr)2, 4-DMAP,
CCl4, HPO(OBn)2, CH3CN, ꢀ30 8C, 5 h, 92%; (m) H2, 10% Pd/C, CH3OH, r.t., 3 h, 93%.
53
54
All these compounds were characterized by NMR and HR-ESI-MS
analyses [13–15].
reaction and screened different solvents, amounts of acidic
reagent, reaction temperatures and time. We found that the
amount of the acidic reagent and reaction temperature were the
main parameters affecting the yield of the production of compound
10. The optimal reaction conditions were found to be 2–3 equiv. of
acetic acid under 140 8C in an appropriate volume of methylene
chloride, whereas excessive solvent will reduce the product yield.
The cytotoxicity of compounds 10–15 was tested against
HCT-8, BEL-7402, KETR3, HELA, BGC-823, KB and MCF-7 cell lines
by the MTT method. The results are summarized in Table 1.
Justicidin G was used as a control compound. From the screening
results, it is evident that all compounds except compound 12
exhibited some level of activity against these cell lines. HCT-8,
HELA and KB were more sensitive than BEL-7402 and KETR3 to
the justicidin G analogues. It is noteworthy that compound 15 as
a phosphate ester not only possessed better water-solubility
(3.5 g/100 mL) [16], but also raised its cytotoxicity against HELA
and KB to nanomolar level.
61
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63
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65
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67
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3. Results and discussion
56
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58
59
60
Herein, we report a novel and facile synthesis of compounds 13
and 15 in good yields. Compound 10 was a key intermediate for the
total synthesis of compound 15. In
a previous study, the
reproducibility of the step converting 9 to 10 was less than
desirable. In this work we optimized the conditions for this
Table 1
Cytotoxicity of compounds 10–15 against human tumour cells (IC50,
m
mol/L).a
Compound Human tumour cells
HCT-8 BEL-7402 KETR3 HELA BGC-823 KB
MCF-7
10
11
12
13
14
15
12.5
26.1
20.7
42.3
38.6
29.9
>100
48.1
41.0
1.7
9.9
7.9
11.0
42.4
>100
45.9
8.0
30.3
24.1
88.4
37.9
>100 >100
>100 >100
>100
>100
19.1
46.0
10.8
1.7
86.1
81.8
18.3
8.8
7.5
4.0
13.1
62.1
4. Conclusion
79
0.019 1.2
0.4 1.2
0.0036 9.7
0.9 8.5
Justicidin G 4.4
5.4
In conclusion, we have synthesized a novel justicidin G
analogue 13 with a 50-OH group and its phosphate ester 15 for
80
81
a
Inhibitory concentration (IC50,
mmol/L) as obtained by the MTT assay.
Please cite this article in press as: S.-P. Wang, et al., Synthesis and cytotoxicity evaluation of a novel justicidin G analogue and its
phosphate ester, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.05.042

G Model
CCLET 3023 1–3
S.-P. Wang et al. / Chinese Chemical Letters xxx (2014) xxx–xxx
3
C–CH2–C), 4.11–3.98 (m, 2H, C–CH2–C), 5.10 (s, 2H, Ar–CH2–O), 5.75 (s, 1H, Ar– 142
CH–O), 5.90 (s, 1H, Ar–CH–Ar), 5.92 (s, 2H, O–CH2–O), 6.47 (s, 1H, Ar–H), 6.70 (s, 143
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83
84
85
86
87
88
the first time, and their structures were characterized by NMR
spectroscopy and HR-ESI-MS. The cytotoxicity of compounds
10–15 was evaluated in seven cell lines by the MTT method and
the results showed that five of them exhibited good antitumor
activity against these cell lines. Moreover, the cytotoxicity of
compound 15 against HELA and KB was improved to nanomolar
level. This result is helpful in discovering new antitumor agents.
1H, Ar–H), 6.92 (s, 1H, Ar–H), 6.97 (s, 1H, Ar–H), 7.48–7.21 (m, 5H, Ar–Hꢂ5). 13
C
144
NMR (125 MHz, DMSO-d6): d 149.7, 148.8, 147.9, 142.1, 140.5, 137.4, 137.1, 145
134.2, 128.8, 128.4, 128.2, 126.7, 111.0, 109.9, 108.3, 101.5, 101.2, 100.5, 71.0, 146
69.3, 65.1, 56.1, 56.0. HRESIMS Calcd. for C26H26NaO8 [M+Na]+: 489.1520, found: 147
489.1512. Compound 10: White solid, mp 162–164 8C. 1H NMR (400 MHz, DMSO- 148
d6): d 0.94 (t, 3H, J = 7.1 Hz, –CH3), 1.25 (t, 3H, J = 7.1 Hz, –CH3), 3.60 (s, 3H, – 149
OCH3), 3.93 (s, 3H, –OCH3), 3.95 (q, 2H, J = 7.1 Hz, C–CH2–C), 4.33 (q, 3H, 150
J = 7.1 Hz, C–CH2–C), 5.14 (s, 2H, Ar–CH2–O), 6.06 (s, 1H, O–CH–O), 6.09 (s, 1H, 151
O–CH–O), 6.45 (s, 1H, Ar–H), 6.56 (s, 1H, Ar–H), 6.71 (s, 1H, Ar–H), 7.48–7.27 (m, 152
5H, Ar–Hꢂ5), 7.63 (s, 1H, Ar–H), 11.92 (s, 1H, Ar–OH). 13C NMR (125 MHz, DMSO- 153
d6): d 169.0, 168.6, 157.3, 152.2, 151.5, 149.1, 148.1, 141.6, 136.7, 134.5, 131.4, 154
130.7, 129.4, 128.4, 127.9, 127.6, 111.7, 105.6, 104.7, 103.2, 102.6, 101.4, 76.9, 155
70.4, 55.5, 55.2, 52.3, 51.6, 13.6, 13.5. HRESIMS Calcd. for C32H31O10 [M+H]+: 156
575.1912, found: 575.1902. Compound 11: White solid, mp 206–207 8C. 1H NMR 157
(300 MHz, DMSO-d6): d 3.58 (s, 3H, –OCH3), 3.92 (s, 3H, –OCH3), 5.13 (s, 2H, Ar– 158
CH2–O), 5.36 (s, 2H, Ar–CH2–O), 6.09 (s, 2H, O–CH2–O), 6.53 (s, 1H, Ar–H), 6.62 (s, 159
1H, Ar–H), 6.90 (s, 1H, Ar–H), 7.46–7.24 (m, 5H, Ar–Hꢂ5), 7.62 (s, 1H, Ar–H), 10.50 160
(s, 1H, Ar–OH). 13C NMR (150 MHz, DMSO-d6): d 169.5, 150.5, 149.7, 148.1, 144.9, 161
141.7, 136.8, 134.7, 129.4, 129.4, 129.3, 128.4, 127.9, 127.7, 123.2, 121.7, 118.7, 162
111.4, 105.5, 105.0, 101.3, 100.7, 70.4, 66.6, 55.5, 55.1. HRESIMS Calcd. for 163
C28H23O8 [M+H]+: 487.1387, found: 487.1391. Compound 12: White solid, mp 164
223–224 8C. 1H NMR (300 MHz, DMSO-d6): d 3.58 (s, 3H, –OCH3), 3.93 (s, 3H, – 165
OCH3), 4.12 (s, 3H, –OCH3), 5.13 (s, 2H, Ar–CH2–O), 5.70 (s, 2H, Ar–CH2–O), 6.10 (s, 166
2H, O–CH2–O), 6.54 (s, 1H, Ar–H), 6.62 (s, 1H, Ar–H), 6.89 (s, 1H, Ar–H), 7.45–7.25 167
(m, 5H, Ar–Hꢂ5), 7.50 (s, 1H, Ar–H). 13C NMR (125 MHz, acetone-d6): d 168.9, 168
151.3, 150.1, 148.3, 147.3, 141.8, 136.9, 134.9, 132.6, 129.5, 129.1, 128.4, 128.0, 169
127.7, 124.9, 123.9, 119.1, 111.4, 105.7, 104.8, 101.4, 100.6, 70.6, 66.8, 59.2, 55.8, 170
55.3. HRESIMS Calcd. for C29H25O8 [M+H]+: 501.1544, found: 501.1535. Com- 171
pound 13: White solid, mp 142–143 8C. 1H NMR (300 MHz, DMSO-d6): d 3.66 (s, 172
3H, –OCH3), 3.93 (s, 3H, –OCH3), 4.12 (s, 3H, –OCH3), 5.69 (s, 2H, Ar–CH2–O), 6.03 173
(s, 2H, O–CH2–O), 6.32 (s, 1H, Ar–H), 6.35 (s, 1H, Ar–H), 6.97 (s, 1H, Ar–H), 7.50 (s, 174
1H, Ar–H), 9.73 (s, 1H, Ar–OH). 13C NMR (100 MHz, DMSO-d6): d 169.6, 151.9, 175
150.6, 148.9, 147.8, 141.2, 134.2, 133.5, 130.1, 129.4, 125.5, 124.4, 119.6, 113.9, 176
106.3, 103.2, 101.5, 101.2, 67.4, 59.7, 56.3, 55.9. HRESIMS Calcd. for C22H19O8 177
[M+H]+: 411.1074, found: 411.1072. Compound 14: Yellowish solid, mp 157– 178
158 8C. 1H NMR (400 MHz, DMSO-d6): d 3.60 (s, 3H–OCH3), 3.95 (s, 3H–OCH3), 179
4.14 (s, 3H–OCH3), 5.14–5.16 (m, 4H, Ar–CH2–Oꢂ2), 5.73 (s, 2H, Ar–CH2–O), 6.12 180
(s, 1H, O–CH–O), 6.13 (s, 1H, O–CH–O), 6.68 (s, 1H, Ar–H), 6.81 (s, 1H, Ar–H), 6.92 181
(s, 1H, Ar–H), 7.39–7.10 (m, 10H, Ar–Hꢂ10), 7.52 (s, 1H, Ar–H). 13C NMR 182
(125 MHz, DMSO-d6) d 169.4, 151.8, 150.3, 150.2, 148.1, 140.1, 139.5, 139.5, 183
133.3, 133.3, 130.4, 129.7, 129.7, 126.8, 126.8, 119.9, 119.9, 113.2, 110.9, 106.3, 184
101.7, 101.4, 98.2, 67.0, 60.2, 56.1, 55.8 31P NMR (200 MHz, DMSO-d6): d ꢀ6.28. 185
89
Acknowledgments
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Q2
We thank National Major Scientific and Technological Special
Project for ‘Significant New Drugs Innovation’ (No.
2012ZX09301002-001), the Union Youth Science & Research Fund
(No. 3332013074) for financial support.
94
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Chem. Lett. 4 (1994) 2567–2572.
HRESIMS Calcd. for C36H31NaO11P [M+Na]+: 693.1496, found: 693.1492.
[15] Compound 15: White solid, mp 212–213 8C. The HRESIMS of 15 established a 187
molecular formula of C22H19O11
[m/z 513.0642 (M+Na)+, Calcd. for 188
186
[9] L. Xiong, M.G. Bi, S. Wu, Y.F. Tong, Total synthesis of 60-hydroxy justicidin A, J.
Asian Nat. Prod. Res. 14 (2012) 322–326.
P
C22H19NaO11P: 513.0640], indicating 14 degrees of unsaturation. 1H NMR spec- 189
trum showed three methoxy groups [dH 3.70 (s, 3H, H-13), 3.94 (s, 3H, H-14), 4.13 190
(s, 3H, H-15)] and four aromatic protons [dH 7.51 (s, 1H, H-5), 7.05 (s, 1H, H-8), 191
6.70 (s, 1H, H-20), 6.83 (s, 1H, H-60)]. 13C NMR and DEPT spectra exhibited 22 192
carbons, including three methyls, two methylenes, sixteen quaternary carbons 193
and one carbonyl. 31P NMR spectrum revealed the existence of one phosphate 194
ester. The HMBC correlations of H-5/C-4, C-7, C-9 and H-8/C-1, C-6, C-10 revealed 195
the present of naphthyl (ring A and ring B). The correlations from H-12 to C-2, C-3, 196
C-4 and C-11 suggested the present of ring C. The correlations of H-13/C-7, H-14/ 197
C-6 and H-15/C-4 gave the substituted positions of three methoxy groups. The 198
linkage of ring B and D was elucidated by the HMBC correlations from H-20 and H- 199
60 to C-1 and C-10. The correlations from H-70 to C-40 and C-50 revealed the 200
presence of ring E. The planar structure of 15 was thus established. 1H NMR 201
(400 MHz, DMSO-d6): d 7.51 (s, 1H, H-5), 7.05 (s, 1H, H-8), 5.71 (s, 2H, H-12), 3.70 202
(s, 3H, H-13), 3.94 (s, 3H, H-14), 4.13 (s, 3H, H-15), 6.70 (s, 1H, H-20), 6.83 (s, 1H, H- 203
60), 6.13 (d, 2H, J = 4.1 Hz, H-70). 13C NMR (125 MHz, DMSO-d6): d 132.3 (C-1), 204
116.9 (C-2), 119.4 (C-3), 147.8 (C-4), 101.3 (C-5), 151.7 (C-6), 150.5 (C-7), 106.0 205
(C-8), 128.6 (C-9), 129.7 (C-10), 169.4 (C-11), 67.3 (C-12), 55.7 (C-13), 56.1 (C-14), 206
59.6 (C-15), 125.3 (C-10), 107.6 (C-20), 148.9 (C-30), 137.2 (C-40), 148.9 (C-50), 116.9 207
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from Bulbophyllum odoratissimum, J. Asian Nat. Prod. Res. 9 (2007) 23–28.
[13] Data of compounds 3 and 8. Compound 3: White solid, mp 95–96 8C. 1H NMR
(300 MHz, DMSO-d6): d 3.74 (s, 3H, –OCH3), 3.77 (s, 3H, –OCH3), 3.98–3.89 (m, 2H,
C–CH2–C), 4.14–4.03 (m, 2H, C–CH2–C), 5.82 (s, 1H, Ar–CH–O), 7.03 (s, 1H, Ar–H),
7.12 (s, 1H, Ar–H). 13C NMR (100 MHz, DMSO-d6): d 190.8, 155.2, 149.3, 126.4,
120.0, 116.6, 111.1, 65.5, 63.5, 57.1, 56.3. HRESIMS Calcd. for C11H14BrO4 [M+H]+:
289.0070, found: 289.0064. Compound 8: White solid, mp 66–68 8C. 1H NMR
(400 MHz, DMSO-d6): d 5.23 (s, 2H, Ar–CH2–O), 6.14 (s, 2H, O–CH2–O), 7.09 (s, 1H,
Ar–H), 7.61–7.19 (m, 6H, Ar–Hꢂ6), 9.75 (s, 1H, –CHO). 13C NMR (100 MHz, DMSO-
d6): d 191.5, 149.9, 143.2, 141.6, 137.0, 132.1, 129.2, 128.8, 128.6, 113.6, 103.4,
103.1, 71.3. HRESIMS Calcd. for C15H13O4 [M+H]+: 257.0808, found: 257.0799.
[14] Data of compounds 9–14. Compound 9: White solid, mp 115–116 8C. 1H NMR
(400 MHz, DMSO-d6): d 3.66 (s, 3H, –OCH3), 3.72 (s, 3H, –OCH3), 3.97–3.84 (m, 2H,
(C-60), 102.1 (C-70). 31P NMR (200 MHz, DMSO-d6): d ꢀ5.83.
208
[16] National Pharmacopoeia Committee, Pharmacopoeia of People’s Republic of 209
China. Part 2, vol. XIV, Chemical Industry Press, Beijing, 2010.
210
Please cite this article in press as: S.-P. Wang, et al., Synthesis and cytotoxicity evaluation of a novel justicidin G analogue and its
phosphate ester, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.05.042