Total synthesis and anticancer activity studies of the stereoisomers of asperphenamate and patriscabratine

Article abstract of DOI:10.1016/j.cclet.2009.10.004

All stereoisomers of asperphenamate 1a and patriscabratine 2a were achieved with a high yield, and total synthesis of 2a is firstly described here. The absolute configuration of patriscabratine was determined as (S,S). The compounds 1a-d and 2a-d have been tested by MTT assay in T47D, MDA-MB231, HL60, Hela and SGC-7901 cell lines in vitro. Among them, the (R,S) stereoisomer shows the strongest anticancer effects, while the (S,R) shows the weakest one.

Full text of DOI:10.1016/j.cclet.2009.10.004

Available online at www.sciencedirect.com
Chinese Chemical Letters 21 (2010) 155–158
www.elsevier.com/locate/cclet
Total synthesis and anticancer activity studies of the
stereoisomers of asperphenamate and patriscabratine
Lei Yuan a, Jin Hui Wang b, Tie Min Sun a,
*
a School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
Received 15 June 2009
Abstract
All stereoisomers of asperphenamate 1a and patriscabratine 2a were achieved with a high yield, and total synthesis of 2a is firstly
described here. The absolute configuration of patriscabratine was determined as (S,S). The compounds 1a–d and 2a–d have been
tested by MTT assay in T47D, MDA-MB231, HL60, Hela and SGC-7901 cell lines in vitro. Among them, the (R,S) stereoisomer
shows the strongest anticancer effects, while the (S,R) shows the weakest one.
# 2009 Tie Min Sun. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: N,N0-substituted phenylalanine-phenylalaninol ester; Asperphenamate; Patriscabratine; Total synthesis; Anticancer activity
Asperphenamate 1a, which has a N,N0-substituted phenylalanine-phenylalaninol ester framework (Fig. 1), was first
isolated by Clark from Aspergillus flavus. The stereochemistry of 1a was established as (S,S) configuration at its chiral
centers [1]. Recently, 1a was isolated by our group from raw malt [2], a traditional medicine for the treatment of
mammary hyperplasia. A preliminary bioassay proved that compound 1a displayed potent antiproliferative activity in
the MCF-7 cell line [3].
Patriscabratine 2a (Fig. 1), which is the analog of asperphenamate, was isolated from ethanol extracts of Patrinia
scabra bunge [4], a traditional medicine for the treatment of acute leukemia. The ethanol extracts displayed significant
cytotoxic activity against acute leukemia as well as inhibitory activity on ehrlich ascites carcinoma [5]. And because of
its similar natural structure to 1a, the compound 2a is expected to show anticancer activity.
The further biological tests are impeded by the low natural abundance of asperphenamate and patriscabratine. The
absolute configuration of patriscabratine was undetermined and its total synthesis was also not developed. In order to
compare the activities among stereoisomers of 1 and 2 and obtain the potent compound, total synthesis of their
stereoisomers and biological evaluation were studied.
The total synthesis of asperphenamate was documented [6,7]. Because of the unsatisfying yields of these methods,
we developed a more efficient method for all stereoisomers of asperphenamate and patriscabratine.
Additionally, all stereoisomers of patriscabratine and asperphenamate have been tested for their antiproliferative
activities in T47D, MDA-MB231, HL60, Hela, and SGC-7901 cell lines in vitro using the MTT method [8].
* Corresponding author.
E-mail address: suntiemin@126.com (T.M. Sun).
1001-8417/$ – see front matter # 2009 Tie Min Sun. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2009.10.004

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L. Yuan et al. / Chinese Chemical Letters 21 (2010) 155–158
Fig. 1. The structure of aperphenamate 1a and patriscabratine 2a.
As shown in Scheme 1, starting from optically pure (L or D)-phenylalanine, (L or D)-phenylalaninol 3 was prepared
according to the method developed by McKennon [9]. N-benzoyl-(L or D)-phenylalaninol 4 was synthesized by
Lewanowicz [10]. N-acetyl-(L or D)-phenylalaninol 5 was readily obtained from acetic anhydride by the same reaction
as the synthesis of 4. Condensation of commercially available N-tert-butoxycarbonyl-(L or D)-phenylalanine with 4 or
5 was promoted by 1,3-dicyclohexylcarbodiimide (DCC) to afford key intermediates N-benzoyl-O-(N0-tert-
butoxycarbonyl-(L or D)-phenylalanyl)-(L or D)-phenylalanin-ol 6 or N-acetyl-O-(N0-tert-butoxycarbonyl-(L or D)-
phenylalanyl)-(L or D)-phenylalaninol 7. Removal of the N-tert-butoxycarbonyl protecting groups of 6 or 7 was
achieved by treatment with 3 equivalents of 1.5 mol/L dry hydrochloride in ethyl acetate, followed by the reaction of
Scheme 1. The synthetic route of patriscabratine and asperphenamate. Condition and reagent: (a) H2SO4/NaBH4, THF, r.t., 98%; (b) 4: benzoyl
chloride, K2CO3, MeOH, r.t., 95%; 5: (CH3CO)2O, K2CO3, MeOH, r.t., 92%; (c) N-tert-butoxycarbonyl-(L or D)-phenylalanine, DCC, CHCl3, r.t.,
65%; (d) (i) 1.5 mol/L HCl/AcOEt, r.t., 76%; (ii) benzoyl chloride, pyridine, r.t., 90%.

L. Yuan et al. / Chinese Chemical Letters 21 (2010) 155–158
157
Table 1
The cytotoxic activities in vitro (IC50 at mmol/L) in five human cancer cell lines.
Compounds
IC50 (mmol/L)
T47D
MDA-MB231
HL60
97.9
Hela
SGC-7901
1aa
1b
1c
92.3
18.7
8.2
96.5
23.1
ntb
ntb
86.7
67.2
94.1
30.7
25.7
46.6
ntb
32.7
20.8
40.9
ntb
11.9
1d
2a
2b
2c
35.8
>100
88.4
83.6
99.7
47.6
>100
90.9
>100
92.4
87.3
97.2
50.9
32.9
67.8
46.7
34.4
58.9
84.7
2d
98.5
a
1a exibited strong activity against MCF-7 and A549 cell lines. The EC50 (mg/mL) value was both 2.5 [3].
Not tested.
b
the deprotected products with benzoyl chloride in pyridine gave the stereoisomers 1a–d and 2a–d the structures were
1
confirmed by IR, H NMR data, 13C NMR, ESIMS and HR-MS [11,12].
The in vitro anticancer activities of 1a–d and 2a–d were investigated by the standard MTT method in T47D, MDA-
MB231, HL60, Hela, and SGC-7901 cell lines. The results are shown in Table 1.
Asperphenamate 1a showed weak activity against T47D, MDA-MB231 and HL60. Patriscabratine 2a was
ineffective against cell lines tested. 1c exhibited the most potent activities against all of cell lines, especially breast
cancer cell lines T47D and MDA-MB231 (IC50 = 8.2 and 11.9 mmol/L, respectively). All stereoisomers showed more
potent activity than reference drug 1a except 2a.
In the stereoisomers of asperphenamate, 1c expressed the strongest anticancer effects, while the 1d displayed the
weakest. On the other hand, 2c showed the broadest activity against all of cell lines and 2d was the weakest in
patriscabratine class of compounds.
It is noteworthy that the benzoyl-substituted compounds (1a–d) are more potent than the acetyl-substituted
compounds (2a–d).
References
[1] A.M. Clark, C.D. Hufford, L.W. Robertson, Lloydia 40 (1977) 146.
[2] J.H. Ling, J.H. Wang, N. Wang, J. Shenyang Pharm. Univ. 22 (2005) 267.
[3] P.L. Wu, F.W. Lin, T.S. Wu, Chem. Pharm. Bull. 52 (2004) 345.
[4] Z.B. Gu, G.J. Yang, W.Y. Liu, Chin. Chem. Lett. 13 (2002) 957.
[5] Committee of National Chinese Medical Manage Bureau ‘‘Chinese Herb’’, Chinese Herb, Shanghai Science and Technology Publishers,
Shanghai, 1999, p. 567.
[6] N.J. Mccorkindale, R.L. Baxter, T.P. Roy, Tetrahedron 34 (1978) 2791.
[7] A.M. Pomini, D.T. Ferreria, R. Braz-filho, Nat. Prod. Res. 20 (2006) 537.
[8] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
[9] M.J. McKennon, A.I. Meyers, K. Drauz, M. Schwarm, J. Org. Chem. 58 (1993) 3568.
´
[10] A. Lewanowicz, J. Lipinski, R. Siedlecka, Tetrahedron 54 (1998) 6571.
20
[11] Compound 1a: m.p. 200–202 8C; ½aꢀD ꢁ98.6 (c 0.76, pyridine); ESIMS m/z: 507.2 [M+1]+, 529.2 [M+Na]+; HR-MS: 529.2098 [M+Na]+
(Calcd. for C32H30N2O4Na, 529.2103); 1H NMR (300 MHz, CDCl3): d 7.08–7.59(m, 20H, ArH), 6.60(s, 1H, 10-NH), 6.51(d, 1H, 7-NH,
J = 6.3), 4.77–4.79(m, 1H, 2-H), 4.45(m, 1H, 5-H), 4.40–4.45(dd, 1H, J = 11.4, 3.0 Hz, 4-Ha), 3.86–3.90(dd, 1H, J = 11.4, 4.2 Hz, 4-Hb),
3.07–3.16(m, 2H, 1-H), 2.84–2.89(dd, 1H, J = 13.2, 7.2 Hz, 6-Ha), 2.75–2.80(dd, 1H, J = 13.2, 8.1 Hz, 6-Hb); 13C NMR (75 MHz, DMSO-
d6): d 171.6, 166.8, 166.3, 138.4, 137.9, 134.6, 133.7, 131.6, 131.3, 129.2 ꢂ 3, 129.1 ꢂ 3, 128.3 ꢂ 3, 127.5 ꢂ 3, 127.3 ꢂ 3, 126.6 ꢂ 2, 126.3,
65.6, 54.6, 50.0, 36.4, 36.1; IR(KBr) (cmꢁ1): 697(dar., mono-suvst.), 1218(nC–O, ester), 1533(dN–H), 1639(nC O, amide), 1751(nC O, ester),
20
3312(nN–H); 1b: m.p. 209–211 8C; ½aꢀD 98.6 (c 0.76, pyridine); IR, ESIMS, HR-MS, 1H NMR and 13C NMR spectra were identical with 1a; 1c:
20
m.p. 210–213 8C; ½aꢀD ꢁ35.8 (c 0.76, pyridine); ESIMS m/z: 507.2 [M+1]+; HR-MS:529.2098 [M+Na]+ (Calcd. for C32H30N2O4Na,
529.2103); 1H NMR (300 MHz, DMSO-d6): d 8.85(d, 1H, 10-NH, J = 7.5), 8.32(d, 1H, 7-NH, J = 8.4), 7.16–7.81(m, 20H, ArH), 4.62–4.68(m,
1H, 2-H), 4.35–4.43(m, 1H, 5-H), 4.09–4.21(m, 2H, 4-H), 3.11–3.18(m, 2H, 1-H), 2.84(m, 2H, 6-H); 13C NMR (75 MHz, DMSO-d6): d 171.3,
166.6, 166.3, 138.4, 136.9, 134.6, 133.7, 131.6, 131.3, 129.2 ꢂ 3, 129.1 ꢂ 3, 128.3 ꢂ 3, 127.5 ꢂ 3, 127.3 ꢂ 3, 126.6 ꢂ 2, 126.3, 65.6, 54.6,
50.0, 36.4, 36.1; IR(KBr) (cmꢁ1): 697(dar., mono-suvst.), 1218(nC–O, ester), 1533(dN–H), 1639(nC O, amide), 1751(nC O, ester), 3312(nN–H);
20
1d: m.p. 212–214 8C; ½aꢀD 35.8 (c 0.76, pyridine); IR, ESIMS, HR-MS, 1H NMR and 13C NMR spectra were identical with 1c.

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L. Yuan et al. / Chinese Chemical Letters 21 (2010) 155–158
20
[12] 2a: m.p. 186–188 8C; ½aꢀD ꢁ32.6 (c 1.0, methanol); ESIMS m/z: 467.9 [M+Na]+; HR-MS: 467.1944 [M+Na]+ (Calcd. for C27H28N2O4Na,
467.1947); 1H NMR (300 MHz, CDCl3): d 7.14–7.73(m, 15H, ArH), 6.61(s, 1H, 10-NH), 6.10(s, 1H, 7-NH), 4.87–4.89(m, 1H, 2-H), 4.41(m,
1H, 5-H), 4.45–4.48(m, 1H, 4-Ha), 3.84–3.87(dd, 1H, J = 11.1, 3.2 Hz, 4-Hb), 3.22–3.29(dd, 1H, J = 6.6 Hz, 13.8 Hz, 1-Ha), 3.18–3.21(dd,
1H, J = 7.1 Hz, 11.9 Hz, 1-Hb), 2.83–2.87(dd, 1H, J = 13.4, 8.4 Hz, 6-Hb), 2.72–2.79(dd, 1H, J = 13.4, 7.2 Hz, 6-Hb), 1.88(s, 3H, CH3); 13
C
NMR (75 MHz, CDCl3): d 171.4, 169.7, 167.2, 136.9, 135.5, 133.0, 131.8, 128.9 ꢂ 3, 128.5 ꢂ 3, 128.4 ꢂ 2, 128.3 ꢂ 2, 127.1, 126.7 ꢂ 2,
126.4,64.8, 54.2, 49.1, 37.2, 37.0, 22.9; IR (KBr) (cmꢁ1): 699 and 747(dar., mono-suvst.), 1213(nC–O, ester), 1448(dCH ), 1539(dN–H),
2
20
1646(nC O, amide), 1746(nC O, ester), 3300(nN–H); 2b: m.p. 188–189 8C; ½aꢀD 33.0 (c 1.0, methanol); IR, ESIMS, HR-MS, 1H NMR and 13
C
20
NMR spectra were identical with 2a; 2c: m.p. 178–181 8C; ½aꢀD ꢁ18.5 (c 1.0, methanol); ESIMS m/z: 467.9 [M+Na]+; HR-MS:467.1944
[M+Na]+ (Calcd. for C27H28N2O4Na, 467.1947); 1H NMR (300 MHz, CDCl3): d 7.06–7.75(m, 15H, ArH), 6.61–6.64(d, 1H, J = 6.8 Hz, 10-
NH), 5.78–5.81(d, 1H, J = 8.4 Hz, 7-NH), 4.96–4.98(m, 1H, 2-H), 4.35-4.37(m, 1H, 5-H), 4.30–4.34(dd, 1H, J = 10.8, 3.4 Hz, 4H-a), 3.93–
3.98(dd, 1H, J = 10.8, 3.6 Hz, 4H-b), 3.25–3.27(m, 2H, 1-H), 2.71–2.76(dd, 1H, J = 13.5, 6.6 Hz, 6H-a), 2.61–2.66(dd, 1H, J = 13.5, 8.1 Hz,
6H-b), 1.92(s, 3H, CH3). 13C NMR (75 MHz, CDCl3): d 171.1, 169.4, 167.2, 136.9, 135.5, 133.0, 132.7, 128.9 ꢂ 3, 128.5 ꢂ 3, 128.4 ꢂ 2,
128.3 ꢂ 2, 127.1, 126.7 ꢂ 2, 126.4,64.8, 54.2, 49.1, 37.2, 37.0, 22.9; IR (KBr) (cmꢁ1): 699 and 747(dar., mono-suvst.), 1213(nC–O, ester),
20
1452(dCH ), 1545(dN–H), 1646(nC O, amide), 1746(nC O, ester), 3300(nN–H); 2d: m.p. 180–184 8C; ½aꢀD 18.5 (c 1.0, methanol); IR, ESIMS,
2
HR-MS, 1H NMR and 13C NMR spectra were identical with 2c.