Synthesis of podophyllotoxin derivatives as potential antitumor agents

Article abstract of DOI:10.1007/s10600-014-0972-2

A series of novel podophyllotoxin derivatives was synthesized by coupling 4β-amino-4′-demethyl-4-desoxypodophyllotoxin (3a) or 4β-amino-4-desoxypodophyllotoxin (3b) with N-substituted 5-formylindole (2a-h). Their structures were identied by spectroscopic techniques. These novel derivatives were evaluated for cytotoxicity in vitro against HepG2 and HeLa cell lines. Compared with etoposide, most of the compounds showed more potent cytotoxicities against two tumor cell lines. Judging from the IC50 values, compound 5n is a promising agent, which is about 15 and 5 times more toxic than etoposide against HepG2 and HeLa cell lines, respectively.

Full text of DOI:10.1007/s10600-014-0972-2

Chemistry of Natural Compounds, Vol. 50, No. 3, July, 2014 [Russian original No. 3, May–June, 2014]
SYNTHESIS OF PODOPHYLLOTOXIN DERIVATIVES
AS POTENTIAL ANTITUMOR AGENTS
1,2,3
4,5
4,5*
2,3*
Yanjun Sun,
Hong Chen,
Huiming Hua,
4,5
Yongfeng Liu, and Shi Zhang
A series of novel podophyllotoxin derivatives was synthesized by coupling 4ꢀ-amino-4ꢁ-demethyl-4-
desoxypodophyllotoxin (3a) or 4ꢀ-amino-4-desoxypodophyllotoxin (3b) with N-substituted 5-formylindole
(2a–h). Their structures were identied by spectroscopic techniques. These novel derivatives were evaluated
for cytotoxicity in vitro against HepG2 and HeLa cell lines. Compared with etoposide, most of the compounds
showed more potent cytotoxicities against two tumor cell lines. Judging from the IC values, compound 5n
50
is a promising agent, which is about 15 and 5 times more toxic than etoposide against HepG2 and HeLa cell
lines, respectively.
Keywords: podophyllotoxin, indibulin, cytotoxicity.
Podophyllotoxin is a lignan with potent antiviral and cytotoxic properties [1]. These remarkable properties make
podophyllotoxin an important lead compound for the development of new therapeutic agents. Extensive structural modifications
of podophyllotoxin have been carried out, which culminated in the clinical introduction of two semisynthetic glucoconjugate
analogues of etoposide and teniposide. The chemical modifications lead to the change in the mechanism of action of these
lignans, wherein podophyllotoxin acts as antimicrotubule agent, whereas its aforementioned derivatives act as topoisomerase-II
inhibitors [2]. However, their high toxicity, low water solubility, acquired drug resistance, and gastrointestinal disturbances
have limited the applications of etoposide and teniposide in cancer chemotherapy [3]. In order to obtain better therapeutic
agents, more diverse analogues like NK611, GL-311, NPF, etc. have been synthesized [4–6]. The numerous synthesized
analogs have allowed improvement in the knowledge of structure–activity relationships. One of the major breakthroughs is the
knowledge that the sugar moiety of etoposide is not essential for topoisomerase II inhibition. Synthetic studies on podophyllotoxin
have showed that the O-linked (ethers, esters) and S-linked (thioethers) compounds are less active in comparison to the
N-linked congeners [7].
Indibulin is a novel, synthetic, small molecule with antitumor activity based upon destabilization of microtubules [8].
This clinical candidate is active against a variety of tumors in vitro and in vivo [9]. Furthermore, indibulin lacks the neurotoxicity
typically associated with other tubulin-binding drugs such as the taxanes and vinca alkaloids, which might be related to its
unknown tubulin-binding mode [9]. Both podophyllotoxin and indibulin are potent microtubulin inhibitors, but the indibulin-
binding sites of the two compounds are different [10]. This prompted us to design hybrids of indibulin and podophyllotoxin
with the aim of discovering novel tubulin inhibitors that can lower podophyllotoxinꢁs toxicity by binding to a novel tubulin-
binding domain.
1) School of Pharmacy, Henan University of Traditional Chinese Medicine, 450046, Zhengzhou, P. R. China, e-mail:
sunyanjunily@126.com; 2) Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 110016, Shenyang, P. R. China, e-mail: huimhua@163.com; 3) School of Traditional Chinese
Materia Medica, Shenyang Pharmaceutical University, 110016, Shenyang, P. R. China; 4) Pharmacognosy Division, Medical
College of Chinese Peopleꢁs Armed Police Force, 300162, Tianjin, P. R. China, fax: 86 22 60578193, e-mail:
Chenhongtian06@yahoo.com.cn; 5) Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard,
300162, Tianjin, P. R. China. Published in Khimiya Prirodnykh Soedinenii, No. 3, May–June, 2014, pp. 354–358. Original
article submitted October 16, 2012.
408
0009-3130/14/5003-0408 02014 Springer Science+Business Media New York

TABLE 1. Cytotoxic Activities of Compounds 5a–q against HepG2 and HeLa Cell Lines (IC , ꢂM)*
50
HepG2
25.61 2.69
5.24 0.49
ꢃ
1.74 0.11
ꢃ
Compound
HepG2
HeLa
Compound
HeLa
> 10
4.61 0.38
ꢃ
0.56 0.38
ꢃ
35.70 3.26
ꢃ
0.60 0.04
ꢃ
0.53 0.04
ꢃ
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
9.35 0.86
0.97 0.09
ꢃ
ꢃ
ꢃ
1.72 0.15
3.50 0.23
ꢃ
ꢃ
–
–
77.61 6.75
ꢃ
> 10
3.04 0.20
ꢃ
5.41 0.52
ꢃ
3.97 0.42
ꢃ
0.34 0.02
ꢃ
10.97 0.83
ꢃ
6.91 0.58
ꢃ
3.52 0.34
ꢃ
21.4 0.19
ꢃ
5p
7.52 0.64
2.52 0.02
ꢃ
2.93 0.19
ꢃ
3.16 0.38
ꢃ
ꢃ
1.78 0.15
ꢃ
3.04 0.03
ꢃ
5q
0.76 0.06
ꢃ
2.85 0.27
ꢃ
21.2 2.05
Etoposidea
ꢃ
0.064 0.005
ꢃ
5.17 0.36
ꢃ
______
*IC is defined as the concentration of drug required to inhibit cell growth by 50% compared with untreated control; it is
50
expressed as mean ꢃSD of at least three determinations.
a
Reference control (> 98%).
OHC
OHC
a
R X
2
N
H
N
R
2
X = Br or Cl
1
2a-h
5''
2''
2
NH
H
N
N
2
N
N
5
8
3
7''
R
O
O
O
O
R
OHC
O
O
2
O
O
b
c
O
1
+
N
O
O
O
R
2
2a-h, q
H CO
3
OCH
3
H CO
3
OCH
3
4'
H CO
3
OCH
3
OR
OR
1
1
3a,b
3a: R = H, 3b: R = CH ; 5a: R = R = H; 5b: R = CH , R = H; 5c: R = H, R = 3-Cl-C H CH
2
4a-q
5a-q
OR
1
1
1
3
1
2
1
3
2
1
2
6
4
5d: R = CH , R = 3-Cl-C H CH ; 5e: R = H, R = 2-F-C H CH ; 5f: R = CH , R = 2-F-C H CH
1
3
2
6
4
2
1
2
6
4
2
1
3
2
6
4
2
5g: R = H, R = C H CH ; 5h: R = CH , R = C H CH ; 5i: R = H, R = p-CH OC H CH
1
2
6
5
2
1
3
2
6
5
2
1
2
3
6
4
2
5j: R = CH , R = p-CH OC H CH ; 5k: R = H, R = 4-tert-C H -C H CH
1
3
2
3
6
4
2
1
2
4
9
6
4
2
5l: R = CH , R = 4-tert-C H -C H CH ; 5m: R = H, R = 4-CH -C H CH
1
3
2
4
9
6
4
2
1
2
3
6
4
2
5n: R = CH , R = 4-CH -C H CH ; 5o: R = H, R = C H ; 5p: R = CH , R = C H
1
3
2
3
6
4
2
1
2
2
5
1
3
2
2 5
5q: R = CH , R = 2-Cl-C H CH
1
3
2
6
4
2
a. NaH, DMF, 0ꢄC – r.t., 8 – 12 h; b. acetic acid, anhydrous ethanol, r.t., 3–6 h; c. NaBH , anhydrous ethanol, 0ꢄC, 3–6 h
4
Scheme 1
Scheme 1 outlines the synthetic routes of podophyllotoxin derivatives. The N-substituted 5-formylindoles 2a–h were
readily synthesized by the reaction of 5-formylindole (1) with an aralkyl chloride or bromoethane in the presence of NaH.
N-substituted 5-formylindoles 2a–h were coupled with 4ꢀ-amino-4ꢁ-demethyl-4-desoxypodophyllotoxin 3a or 4ꢀ-amino-
4-desoxypodophyllotoxin 3b in the presence of acetic acid and anhydrous ethanol and then reduced by NaBH to generate the
4
title compounds 5a–q. Compounds 3a and 3b were prepared from 4ꢁ-demethylpodophyllotoxin and podophyllotoxin according
to known procedures [10].
The cytotoxic activities of the synthesized compounds were tested against HepG2 and HeLa cell lines. IC values
50
were obtained by the standard MTT assay. As shown in Table 1, some synthesized compounds exhibited more potent cytotoxic
activities than etoposide. The cytotoxic activities of compounds 5b, 5h, and 5l–q against HepG2 cell line were higher than
etoposide. Compounds 5a, 5h, 5i, 5l, and 5n–q showed comparative or even superior cytotoxic activities against HeLa cell
line than etoposide. On the basis of IC values, compound 5n showed more potent cytotoxic activity than compound 5b,
50
which implied that the introduction of 4-methylbenzyl at N1 of 5-formylindole enhanced the activity of podophyllotoxin
derivatives.
409

In summary, a series of novel podophyllotoxin derivatives has been synthesized and evaluated for their cytotoxic
activities. Most of the synthesized compounds exhibited more potent cytotoxic activities than etoposide. Compound 5n exhibited
promising cytotoxicity against HepG2 and HeLa cell lines (IC values 0.34 and 0.60 ꢂM, respectively). Additional work is
50
under way to study the antitumor effects of compound 5n in vivo.
EXPERIMENTAL
General Experimental. All materials and reagents were obtained from commercial sources and used without further
purification unless stated. Podophyllotoxin (98% purity) and 4ꢁ-demethylpodophyllotoxin (98% purity) were purchased from
Qingze Corporation of Nanjing in China. CH Cl was redistilled over P O . Melting points were determined on an electric
2
2
2 5
X-4 digital visual melting point apparatus and were uncorrected. PMR spectra were obtained using a BrukerARX-300 instrument
(300 MHz) with tetramethylsilane (TMS) as the internal standard. Mass spectral data were obtained on Agilent 6210 TOP-MS
and reported as m/z. Optical rotations were obtained by using a Jasco DIP-370 digital polarimeter.
N-Substituted 5-formylindoles (2a–h) were synthesized by the literature method [10].
General Procedure for the Synthesis of 5a–q. N-Substituted 5-formylindoles 2a–h, q (2.0 mmol) and 4ꢀ-amino-4ꢁ-
demethyl-4-desoxypodophyllotoxin (3a) or 4ꢀ-amino-4-desoxypodophyllotoxin (3b) (2.2 mmol) were dissolved in 5 mL
anhydrous ethanol. To this mixture, 3–5 drops of acetic acid were then added slowly with stirring. After 1–2 h stirring at room
temperature, NaBH (15.0 mmol) was added to the mixture at one time. After an additional 2–3 h stirring at 0ꢄC, the resulting
4
liquid was neutralized by 1 N HCl until pH 7, concentrated under reduced pressure, and then extracted with CH Cl (40 mL ꢅ 3)
2
2
after addition of 30 mL water. The organic phase was washed with water, dried over Na SO , and concentrated in vacuo. The
2
4
residue was chromatographed on silica gel (CH Cl –acetone, 100:1–100:2) to give 5a–q.
2
2
4ꢀ-(((1H-Indol-5-yl)-methyl)amino)-4ꢁ-demethyl-4-desoxypodophyllotoxin (5a) was obtained by silica gel column
25
chromatography (eluent CH Cl –acetone, 100:2). Yield 71%, white solid, mp 184–186ꢄC, [ ] –67.4ꢄ (c 0.23, CHCl ).
2
2
D
3
HR-positive ESI-MS m/z 529.1970 (calcd for C H N O , 529.1975), C H N O .
30 29
2
7
30 28 2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.77 (1H, m, H-3), 3.35 (1H, dd, J = 5.1, 13.8, H-2), 3.74 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.94 (1H, m, H-10ꢁꢁ), 3.95 (1H, d, J = 4.2, H-4), 3.95 (1H, m, H-11ꢀ), 4.31 (1H, t, J = 7.5, H-10ꢁꢁ), 4.41 (1H, dd,
3
J = 8.1, 10.8, H-11 ), 4.51 (1H, d, J = 5.1, H-1), 5.89 (1H, d, J = 1.2, OCH O), 5.90 (1H, d, J = 1.2, OCH O), 6.25 (2H, s,
2
2
H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.49 (1H, s, H-5), 6.52 (1H, t, J = 3.0, H-3ꢁꢁ), 7.16 (1H, dd, J = 1.5, 8.1, H-6ꢁꢁ), 7.23 (1H, t, J = 3.0,
H-2ꢁꢁ), 7.39 (1H, d, J = 8.1, H-7ꢁꢁ), 7.57 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1H-Indol-5-yl)-methyl)amino)-4-desoxypodophyllotoxin (5b) was obtained by silica gel column
25
chromatography (eluent CH Cl –acetone, 100:1). Yield 73%, white solid, mp 185–187ꢄC, [ ] –70.2ꢄ (c 0.29, CHCl ),
2
2
D
3
HR-positive ESI-MS m/z 543.2124 (calcd for C H N O , 543.2131), C H N O .
31 31
2
7
31 30 2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.79 (1H, m, H-3), 3.38 (1H, dd, J = 5.1, 13.8, H-2), 3.72 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.78 (3H, s, 4ꢁ-OCH ), 3.96 (1H, m, H-10ꢁꢁ), 3.98 (1H, d, J = 3.0, H-4), 3.98 (1H, m, H-11ꢀ), 4.32 (1H, t, J = 7.8,
3
3
H-10ꢁꢁ), 4.43 (1H, dd, J = 9.0, 9.6, H-11 ), 4.52 (1H, d, J = 5.1, H-1), 6.24 (2H, s, H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.49 (1H, s,
H-5), 6.54 (1H, d, J = 2.7, H-3ꢁꢁ), 5.91 (2H, s, OCH O), 7.18 (1H, br.d, J = 8.4, H-6ꢁꢁ), 7.22 (1H, d, J = 2.7, H-2ꢁꢁ), 7.40 (1H,
2
d, J = 8.4, H-7ꢁꢁ), 7.58 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(3-Chlorobenzyl)-indol-5-yl)-methyl)amino)-4ꢁ-demethyl-4-desoxypodophyllotoxin (5c) was obtained by
25
silica gel column chromatography (eluent CH Cl –acetone, 100:3). Yield 72%, white solid, mp 179–181ꢄC, [ ] –50.5ꢄ
2
2
D
(c 0.16, CHCl ). HR-positive ESI-MS m/z 653.2050 (calcd for C H ClN O , 653.2055), C H ClN O .
3
37 34
2
7
37 33
2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.78 (1H, m, H-3), 3.34 (1H, dd, J = 5.1, 13.8, H-2), 3.75 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.93 (1H, m, H-11ꢀ), 3.93 (1H, m, H-10ꢁꢁ), 3.96 (1H, br.s, H-4), 4.30 (1H, t, J = 6.9, H-10ꢁꢁ), 4.40 (1H, dd, J = 8.4,
3
9.9, H-11 ), 4.50 (1H, d, J = 5.1, H-1), 5.29 (2H, s, CH ), 5.90 (1H, br.s, OCH O), 5.92 (1H, br.s, OCH O), 6.25 (2H, s, H-2ꢁ,
2
2
2
6ꢁ), 6.43 (1H, s, H-8), 6.50 (1H, s, H-5), 6.54 (1H, d, J = 3.3, H-3ꢁꢁ), 6.93 (1H, m, H-6ꢁꢁꢁ), 7.09 (1H, br.s, H-2ꢁꢁꢁ), 7.14 (1H, d,
J = 3.3, H-2ꢁꢁ), 7.15 (1H, br.d, J = 8.4, H-6ꢁꢁ), 7.20 (1H, d, J = 8.4, H-7ꢁꢁ), 7.21–7.25 (2H, m, H-4ꢁꢁꢁ, 5ꢁꢁꢁ), 7.59 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(3-Chlorobenzyl)-indol-5-yl)-methyl)amino)-4-desoxypodophyllotoxin (5d) was obtained by silica gel
25
column chromatography (eluent CH Cl –acetone, 100:2). Yield 71%, white solid, mp 178–180ꢄC, [ ] –48.3ꢄ (c 0.23,
2
2
D
CHCl ). HR-positive ESI-MS m/z 667.2209 (calcd for C H ClN O , 667.2211), C H ClN O .
3
38 36
2
7
38 35
2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.79 (1H, m, H-3), 3.36 (1H, dd, J = 5.1, 13.8, H-2), 3.72 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.78 (3H, s, 4ꢁ-OCH ), 3.94 (1H, m, H-11ꢀ), 3.94 (1H, m, H-10ꢁꢁ), 3.96 (1H, d, J = 4.2, H-4), 4.31 (1H, t, J = 7.5,
3
3
410

H-10ꢁꢁ), 4.41 (1H, dd, J = 8.1, 9.6, H-11 ), 4.51 (1H, d, J = 5.1, H-1), 5.28 (2H, s, CH ), 5.91 (1H, br.s, OCH O), 5.92 (1H,
2
2
br.s, OCH O), 6.24 (2H, s, H-2ꢁ, 6ꢁ), 6.44 (1H, s, H-8), 6.50 (1H, s, H-5), 6.54 (1H, d, J = 3.3, H-3ꢁꢁ), 6.93 (1H, m, H-6ꢁꢁꢁ), 7.09
2
(1H, br.s, H-2ꢁꢁꢁ), 7.14 (1H, d, J = 3.3, H-2ꢁꢁ), 7.15 (1H, d, J = 8.7, H-6ꢁꢁ), 7.21 (1H, d, J = 8.7, H-7ꢁꢁ), 7.21–7.24 (2H, m, H-4ꢁꢁꢁ,
5ꢁꢁꢁ), 7.59 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(2-Fluorobenzyl)-indol-5-yl)-methyl)amino)-4ꢁ-demethyl-4-desoxypodophyllotoxin (5e) was obtained by
25
silica gel column chromatography (eluent CH Cl –acetone, 100:2). Yield 71%, white solid, mp 178–180ꢄC, [ ] –44.1ꢄ
2
2
D
(c 0.40, CHCl ). HR-positive ESI-MS m/z 637.3242 (calcd for C H FN O , 637.3250), C H FN O .
3
37 34
2
7
37 33
2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.77 (1H, m, H-3), 3.34 (1H, dd, J = 5.1, 13.8, H-2), 3.75 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.94 (1H, m, H-10ꢁꢁ), 3.95 (1H, d, J = 3.9, H-4), 3.95 (1H, m, H-11ꢀ), 4.28 (1H, t, J = 7.8, H-10ꢁꢁ), 4.40 (1H, dd,
3
J = 8.1, 10.8, H-11 ), 4.50 (1H, d, J = 5.1, H-1), 5.37 (2H, s, CH ), 5.89 (1H, d, J = 1.2, OCH O), 5.91 (1H, d, J = 1.2,
2
2
OCH O), 6.25 (2H, s, H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.50 (1H, s, H-5), 6.54 (1H, d, J = 3.3, H-3ꢁꢁ), 6.82 (1H, td, J = 7.5, 1.8,
2
H-3ꢁꢁꢁ), 6.98 (1H, td, J = 7.5, 1.2, H-5ꢁꢁꢁ), 7.07 (1H, td, J = 8.4, 1.2, H-6ꢁꢁꢁ), 7.15 (1H, br.d, J = 8.4, H-6ꢁꢁ), 7.18 (1H, d, J = 3.3,
H-2ꢁꢁ), 7.23 (1H, m, H-4ꢁꢁꢁ), 7.34 (1H, d, J = 8.4, H-7ꢁꢁ), 7.58 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(2-Fluorobenzyl)-indol-5-yl)-methyl)amino)-4-desoxypodophyllotoxin (5f) was obtained by silica gel
25
column chromatography (eluent CH Cl –acetone, 100:1). Yield 69%, white solid, mp 176–178ꢄC, [ ] –43.4ꢄ (c 0.18,
2
2
D
CHCl ). HR-positive ESI-MS m/z 651.2504 (calcd for C H FN O , 651.2507), C H FN O .
3
38 36
2
7
38 35
2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.78 (1H, m, H-3), 3.39 (1H, dd, J = 5.4, 13.8, H-2), 3.71 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.78 (3H, s, 4ꢁ-OCH ), 3.93 (1H, m, H-11ꢀ), 3.93 (1H, m, H-10ꢁꢁ), 3.97 (1H, d, J = 3.9, H-4), 4.29 (1H, t, J = 7.8,
3
3
H-10ꢁꢁ), 4.41 (1H, dd, J = 8.4, 10.8, H-11 ), 4.51 (1H, d, J = 5.4, H-1), 5.37 (2H, s, CH ), 5.90 (1H, s, OCH O), 5.91 (1H, s,
2
2
OCH O), 6.24 (2H, s, H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.51 (1H, s, H-5), 6.53 (1H, d, J = 3.0, H-3ꢁꢁ), 6.82 (1H, td, J = 7.5, 1.5,
2
H-3ꢁꢁꢁ), 6.97 (1H, td, J = 7.5, 1.2, H-5ꢁꢁꢁ), 7.06 (1H, td, J = 8.4, 1.2, H-6ꢁꢁꢁ), 7.16 (1H, br.d, J = 8.4, H-6ꢁꢁ), 7.18 (1H, d, J = 3.0,
H-2ꢁꢁ), 7.23 (1H, m, H-4ꢁꢁꢁ), 7.34 (1H, d, J = 8.4, H-7ꢁꢁ), 7.58 (1H, br.s, H-4ꢁꢁ), .
4ꢀ-(((1-Benzyl-indol-5-yl)-methyl)amino)-4ꢁ-demethyl-4-desoxypodophyllotoxin (5g) was obtained by silica gel
25
column chromatography (eluent CH Cl –acetone, 100:2). Yield 74%, white solid, mp 173–175ꢄC, [ ] –92.0ꢄ (c 0.22,
2
2
D
CHCl ). HR-positive ESI-MS m/z 619.2438 (calcd for C H N O , 619.2444), C H N O .
3
37 35
2
7
37 34 2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.78 (1H, m, H-3), 3.34 (1H, dd, J = 5.1, 13.8, H-2), 3.74 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.95 (1H, m, H-11ꢀ), 3.95 (1H, m, H-10ꢁꢁ), 3.96 (1H, d, J = 3.9, H-4), 4.28 (1H, t, J = 7.8, H-10ꢁꢁ), 4.40 (1H, dd,
3
J = 8.1, 10.8, H-11 ), 4.50 (1H, d, J = 5.1, H-1), 5.28 (2H, s, CH ), 5.89 (1H, d, J = 1.2, OCH O), 5.91 (1H, d, J = 1.2,
2
2
OCH O), 6.25 (2H, s, H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.49 (1H, s, H-5), 6.52 (1H, d, J = 3.0, H-3ꢁꢁ), 7.07 (2H, dd, J = 1.8, 7.8,
2
H-2ꢁꢁꢁ, 6ꢁꢁꢁ), 7.13 (1H, br.d, J = 8.4, H-6ꢁꢁ), 7.15 (1H, d, J = 3.0, H-2ꢁꢁ), 7.25–7.31 (3H, m, H-3ꢁꢁꢁ, 4ꢁꢁꢁ, 5ꢁꢁꢁ), 7.27 (1H, d, J = 8.4,
H-7ꢁꢁ), 7.59 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-Benzyl-indol-5-yl)-methyl)amino)-4-desoxypodophyllotoxin (5h) was obtained by silica gel column
25
chromatography (eluent CH Cl –acetone, 100:1). Yield 72%, white solid, mp 176–178ꢄC, [ ] –79.2ꢄ (c 0.28, CHCl ).
2
2
D
3
HR-positive ESI-MS m/z 633.2595 (calcd for C H N O , 633.2601), C H N O .
38 37
2
7
38 36 2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.79 (1H, m, H-3), 3.35 (1H, dd, J = 5.1, 13.8, H-2), 3.71 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.78 (3H, s, 4ꢁ-OCH ), 3.94 (1H, m, H-11ꢀ), 3.94 (1H, m, H-10ꢁꢁ), 3.95 (1H, d, J = 3.9, H-4), 4.28 (1H, t, J = 7.8,
3
3
H-10ꢁꢁ), 4.40 (1H, dd, J = 8.1, 10.8, H-11 ), 4.50 (1H, d, J = 5.1, H-1), 5.32 (2H, s, CH ), 5.93 (2H, s, OCH O), 6.23 (2H, s,
2
2
H-2ꢁ, 6ꢁ), 6.44 (1H, s, H-8), 6.50 (1H, s, H-5), 6.54 (1H, d, J = 3.0, H-3ꢁꢁ), 7.08 (2H, dd, J = 1.8, 7.8, H-2ꢁꢁꢁ, 6ꢁꢁꢁ), 7.15 (1H, d,
J = 3.0, H-2ꢁꢁ), 7.25 (1H, m, H-6ꢁꢁ), 7.25–7.31 (3H, m, H-3ꢁꢁꢁ, 4ꢁꢁꢁ, 5ꢁꢁꢁ), 7.27 (1H, d, J = 8.4, H-7ꢁꢁ), 7.60 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(4-Methoxybenzyl)-indol-5-yl)-methyl)amino)-4ꢁ-demethyl-4-desoxypodophyllotoxin (5i) was obtained
25
by silica gel column chromatography (eluent CH Cl –acetone, 100:2). Yield 76%, white solid, mp 172–174ꢄC, [ ] –48.4ꢄ
2
2
D
(c 0.20, CHCl ). HR-positive ESI-MS m/z 649.2544 (calcd for C H N O , 649.2550), C H N O .
3
38 37
2
8
38 36 2 8
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.77 (1H, m, H-3), 3.35 (1H, dd, J = 5.1, 13.2, H-2), 3.75 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.76 (3H, s, 4ꢁꢁꢁ-OCH ), 3.94 (1H, m, H-11ꢀ), 3.94 (1H, m, H-10ꢁꢁ), 3.96 (1H, d, J = 3.9, H-4), 4.29 (1H, t,
3
3
J = 7.8, H-10ꢁꢁ), 4.40 (1H, dd, J = 8.4, 10.5, H-11 ), 4.51 (1H, d, J = 5.1, H-1), 5.26 (2H, s, CH ), 5.90 (1H, d, J = 1.2,
2
OCH O), 5.92 (1H, d, J = 1.2, OCH O), 6.25 (2H, s, H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.49 (1H, s, H-5), 6.50 (1H, d, J = 3.0, H-3ꢁꢁ),
2
2
6.80 (2H, d, J = 8.7, H-3ꢁꢁꢁ, 5ꢁꢁꢁ), 7.05 (2H, d, J = 8.7, H-2ꢁꢁꢁ, 6ꢁꢁꢁ), 7.12 (1H, d, J = 3.0, H-2ꢁꢁ), 7.15 (1H, br.d, J = 8.4, H-6ꢁꢁ),
7.30 (1H, d, J = 8.4, H-7ꢁꢁ), 7.57 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(4-Methoxybenzyl)-indol-5-yl)-methyl)amino)-4-desoxypodophyllotoxin (5j) was obtained by silica gel
25
column chromatography (eluent CH Cl –acetone, 100:1). Yield 74%, white solid, mp 174–176ꢄC, [ ] –39.3ꢄ (c 0.17, CHCl ).
2
2
D
3
HR-positive ESI-MS m/z 663.2700 (calcd for C H N O , 663.2706), C H N O .
39 39
2
8
39 38 2 8
411

PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.79 (1H, m, H-3), 3.37 (1H, dd, J = 5.4, 13.8, H-2), 3.71 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.75 (3H, s, 4ꢁꢁꢁ-OCH ), 3.78 (3H, s, 4ꢁ-OCH ), 3.96 (1H, m, H-11ꢀ), 3.96 (1H, m, H-10ꢁꢁ), 3.98 (1H, d, J = 3.9,
3
3
3
H-4), 4.31 (1H, t, J = 7.8, H-10ꢁꢁ), 4.41 (1H, dd, J = 8.1, 10.8, H-11 ), 4.52 (1H, d, J = 5.4, H-1), 5.28 (2H, s, CH ), 5.89 (1H,
2
d, J = 1.2, OCH O), 5.91 (1H, d, J = 1.2, OCH O), 6.24 (2H, s, H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.47 (1H, s, H-5), 6.50 (1H, d,
2
2
J = 3.0, H-3ꢁꢁ), 6.80 (2H, d, J = 8.7, H-3ꢁꢁꢁ, 5ꢁꢁꢁ), 7.05 (2H, d, J = 8.7, H-2ꢁꢁꢁ, 6ꢁꢁꢁ), 7.13 (1H, d, J = 3.0, H-2ꢁꢁ), 7.15 (1H, dd,
J = 8.4, 1.5, H-6ꢁꢁ), 7.29 (1H, d, J = 8.4, H-7ꢁꢁ), 7.57 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(4-tert-Butylbenzyl)-indol-5-yl)-methyl)amino)-4ꢁ-demethyl-4-desoxypodophyllotoxin (5k) was obtained
25
by silica gel column chromatography (eluent CH Cl –acetone, 100:2). Yield 73%, white solid, mp 178–180ꢄC, [ ] –38.5ꢄ
2
2
D
(c 0.27, CHCl ). HR-positive ESI-MS m/z 675.3066 (calcd for C H N O , 675.3070), C H N O .
3
41 43
2
8
41 42 2 8
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 1.28 (9H, s, 4ꢁꢁꢁ-C-CH ), 2.78 (1H, m, H-3), 3.37 (1H, dd, J = 5.1,
3
3
13.8, H-2), 3.75 (6H, s, 3ꢁ, 5ꢁ-OCH ), 3.92 (1H, m, H-11ꢀ), 3.92 (1H, m, H-10ꢁꢁ), 3.98 (1H, d, J = 4.2, H-4), 4.30 (1H, t, J = 7.5,
3
H-10ꢁꢁ), 4.44 (1H, dd, J = 8.4, 9.9, H-11 ), 4.51 (1H, d, J = 5.1, H-1), 5.29 (2H, s, CH ), 5.92 (2H, s, OCH O), 6.25 (2H, s,
2
2
H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.50 (1H, s, H-5), 6.52 (1H, d, J = 3.0, H-3ꢁꢁ), 7.04 (2H, d, J = 8.7, H-2ꢁꢁꢁ, 6ꢁꢁꢁ), 7.14 (1H, d, J = 3.0,
H-2ꢁꢁ), 7.17 (1H, d, J = 8.4, H-6ꢁꢁ), 7.30 (2H, d, J = 8.7, H-3ꢁꢁꢁ, 5ꢁꢁꢁ), 7.31 (1H, d, J = 8.4, H-7ꢁꢁ), 7.58 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(4-tert-Butylbenzyl)-indol-5-yl)-methyl)amino)-4-desoxypodophyllotoxin (5l) was obtained by silica gel
25
column chromatography (eluent CH Cl –acetone, 100:1). Yield 72%, white solid, mp 181–182ꢄC, [ ] –41.6ꢄ (c 0.18,
2
2
D
CHCl ). HR-positive ESI-MS m/z 689.3225 (calcd for C H N O , 689.3227), C H N O .
3
42 45
2
7
42 44 2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 1.27 (9H, s, 4ꢁꢁꢁ-C-CH ), 2.79 (1H, m, H-3), 3.38 (1H, dd, J = 5.1,
3
3
13.8, H-2), 3.72 (6H, s, 3ꢁ, 5ꢁ-OCH ), 3.78 (3H, s, 4ꢁ-OCH ), 3.92 (1H, m, H-11ꢀ), 3.92 (1H, m, H-10ꢁꢁ), 3.98 (1H, d, J = 3.6,
3
3
H-4), 4.32 (1H, t, J = 7.5, H-10ꢁꢁ), 4.43 (1H, dd, J = 9.0, 10.5, H-11 ), 4.51 (1H, d, J = 5.1, H-1), 5.29 (2H, s, CH ), 5.90 (1H,
2
s, OCH O), 5.91 (1H, s, OCH O), 6.24 (2H, s, H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.50 (1H, s, H-5), 6.52 (1H, d, J = 3.0, H-3ꢁꢁ), 7.14
2
2
(1H, d, J = 3.0, H-2ꢁꢁ), 7.16 (1H, J = 8.4, H-6ꢁꢁ), 7.02 (2H, d, J = 8.1, H-2ꢁꢁꢁ, 6ꢁꢁꢁ), 7.28 (2H, d, J = 8.1, H-3ꢁꢁꢁ, 5ꢁꢁꢁ), 7.34 (1H, d,
J = 8.4, H-7ꢁꢁ), 7.58 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(4-Methylbenzyl)-indol-5-yl)-methyl)amino)-4ꢁ-demethyl-4-desoxypodophyllotoxin (5m) was obtained
25
by silica gel column chromatography (eluent CH Cl –acetone, 100:2). Yield 70%, white solid, mp 183–185ꢄC, [ ] –46.1ꢄ
2
2
D
(c 0.19, CHCl ). HR-positive ESI-MS m/z 633.2588 (calcd for C H N O , 633.2601), C H N O .
3
38 37
2
7
38 36 2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.33 (3H, s, 4ꢁꢁꢁ-CH ), 2.78 (1H, m, H-3), 3.38 (1H, dd, J = 4.8,
3
3
14.0, H-2), 3.78 (6H, s, 3ꢁ, 5ꢁ-OCH ), 3.93 (1H, m, H-11ꢀ), 3.93 (1H, m, H-10ꢁꢁ), 3.98 (1H, d, J = 3.3, H-4), 4.31 (1H, t, J = 7.6,
3
H-10ꢁꢁ), 4.44 (1H, dd, J = 8.8, 10.0, H-11 ), 4.53 (1H, d, J = 4.8, H-1), 5.31 (2H, s, CH ), 5.95 (2H, s, OCH O), 6.29 (2H, s,
2
2
H-2ꢁ, 6ꢁ), 6.47 (1H, s, H-8), 6.55 (1H, s, H-5), 6.56 (1H, d, J = 2.4, H-3ꢁꢁ), 7.03 (2H, d, J = 8.0, H-2ꢁꢁꢁ, 6ꢁꢁꢁ), 7.12 (2H, d, J = 8.0,
H-3ꢁꢁꢁ, 5ꢁꢁꢁ), 7.16 (1H, d, J = 2.4, H-2ꢁꢁ), 7.19 (1H, br.d, J = 8.4, H-6ꢁꢁ), 7.32 (1H, d, J = 8.4, H-7ꢁꢁ), 7.62 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(4-Methylbenzyl)-indol-5-yl)-methyl)amino)-4-desoxypodophyllotoxin (5n) was obtained by silica gel
25
column chromatography (eluent CH Cl –acetone, 100:1). Yield 71%, white solid, mp 181–183°C, [ ] –47.8ꢄ (c 0.10,
2
2
D
CHCl ). HR-positive ESI-MS m/z 647.2751 (calcd for C H N O , 647.2757), C H N O .
3
39 39
2
7
39 38 2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.32 (3H, s, 4ꢁꢁꢁ-CH ), 2.80 (1H, m, H-3), 3.38 (1H, dd, J = 4.8,
3
3
14.0, H-2), 3.74 (6H, s, 3ꢁ, 5ꢁ-OCH ), 3.81 (3H, s, 4ꢁ-OCH ), 3.95 (1H, m, H-11ꢀ), 3.95 (1H, m, H-10ꢁꢁ), 3.98 (1H, d, J = 3.2,
3
3
H-4), 4.33 (1H, t, J = 7.6, H-10ꢁꢁ), 4.44 (1H, dd, J = 8.8, 10.0, H-11 ), 4.53 (1H, d, J = 4.8, H-1), 5.31 (2H, s, CH ), 5.93 (1H,
2
s, OCH O), 5.94 (1H, s, OCH O), 6.28 (2H, s, H-2ꢁ, 6ꢁ), 6.46 (1H, s, H-8), 6.54 (1H, s, H-5), 6.54 (1H, br.s, H-3ꢁꢁ), 7.03 (2H,
2
2
d, J = 7.6, H-2ꢁꢁꢁ, 6ꢁꢁꢁ), 7.12 (2H, d, J = 7.6, H-3ꢁꢁꢁ, 5ꢁꢁꢁ), 7.16 (1H, br.s, H-2ꢁꢁ), 7.17 (1H, br.d, J = 8.4, H-6ꢁꢁ), 7.32 (1H, d, J = 8.4,
H-7ꢁꢁ), 7.61 (1H, br.s, H-4ꢁꢁ), .
4ꢀ-(((1-Ethyl-indol-5-yl)-methyl)amino)-4ꢁ-demethyl-4-desoxypodophyllotoxin (5o) was obtained by silica gel
25
column chromatography (eluent CH Cl –acetone, 100:3). Yield 68%, white solid, mp 169–171ꢄC, [ ] –62.4ꢄ (c 0.19,
2
2
D
CHCl ). HR-positive ESI-MS m/z 557.2283 (calcd for C H N O , 557.2288), C H N O .
3
32 33
2
7
32 32 2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 1.47 (3H, t, J = 7.2, H -2ꢁꢁꢁ), 2.77 (1H, m, H -3), 3.38 (1H, dd, J = 5.1,
3
3
3
13.8, H-2), 3.74 (6H, s, 3ꢁ, 5ꢁ-OCH ), 3.92 (1H, m, H-11ꢀ), 3.92 (1H, m, H-10ꢁꢁ), 3.97 (1H, d, J = 3.3, H-4), 4.18 (2H, q,
3
J = 7.2, H -1ꢁꢁꢁ), 4.31 (1H, t, J = 7.5, H-10ꢁꢁ), 4.43 (1H, dd, J = 8.4, 10.5, H-11 ), 4.51 (1H, d, J = 5.1, H-1), 5.90 (2H, s,
2
OCH O), 6.25 (2H, s, H-2ꢁ, 6ꢁ), 6.43 (1H, s, H-8), 6.47 (1H, d, J = 3.3, H-3ꢁꢁ), 6.51 (1H, s, H-5), 7.14 (1H, d, J = 3.3, H-2ꢁꢁ),
2
7.18 (1H, dd, J = 1.2, 8.4, H-6ꢁꢁ), 7.35 (1H, d, J = 8.4, H-7ꢁꢁ), 7.56 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-Ethyl-indol-5-yl)-methyl)amino)-4-desoxypodophyllotoxin (5p) was obtained by silica gel column
25
chromatography (eluent CH Cl –acetone, 100:2). Yield 71%, white solid, mp 172–173ꢄC, [ ] –61.8ꢄ (c 0.18, CHCl ).
2
2
D
3
HR-positive ESI-MS m/z 571.2439 (calcd for C H N O , 571.2444), C H N O .
33 35
2
7
33 34 2 7
412

PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 1.49 (3H, t, J = 7.2, H -2ꢁꢁꢁ), 2.79 (1H, m, H-3), 3.39 (1H, dd, J = 5.6,
3
3
14.0, H-2), 3.72 (6H, s, 3ꢁ, 5ꢁ-OCH ), 3.80 (3H, s, 4ꢁ-OCH ), 3.95 (1H, m, H-11ꢀ), 3.95 (1H, m, H-10ꢁꢁ), 4.00 (1H, d, J = 4.0,
3
3
H-4), 4.21 (2H, q, J = 7.2, H -1ꢁꢁꢁ), 4.34 (1H, t, J = 7.6, H-10ꢁꢁ), 4.45 (1H, dd, J = 8.4, 10.8, H-11 ), 4.54 (1H, d, J = 5.6, H-1),
2
5.93 (2H, s, OCH O), 6.28 (2H, s, H-2ꢁ, 6ꢁ), 6.46 (1H, s, H-8), 6.50 (1H, d, J = 2.8, H-3ꢁꢁ), 6.55 (1H, s, H-5), 7.16 (1H, d,
2
J = 2.8, H-2ꢁꢁ), 7.21 (1H, br.d, J = 8.4, H-6ꢁꢁ), 7.39 (1H, d, J = 8.4, H-7ꢁꢁ), 7.59 (1H, br.s, H-4ꢁꢁ).
4ꢀ-(((1-(2-Chlorobenzyl)-indol-5-yl)-methyl)amino)-4-desoxypodophyllotoxin (5q) was obtained by silica gel
25
column chromatography (eluent CH Cl –acetone, 100:1). Yield 73%, white solid, mp 180–182ꢄC, [ ] –47.5ꢄ (c 0.21,
2
2
D
CHCl ). HR-positive ESI-MS m/z 667.2196 (calcd for C H ClN O , 667.2211), C H ClN O .
3
38 36
2
7
38 35
2 7
PMR spectrum (300 MHz, CDCl , ꢆ, ppm, J/Hz): 2.79 (1H, m, H-3), 3.37 (1H, dd, J = 5.1, 13.8, H-2), 3.72 (6H, s,
3
3ꢁ, 5ꢁ-OCH ), 3.78 (3H, s, 4ꢁ-OCH ), 3.94 (1H, m, H-11ꢀ), 3.96 (1H, m, H-10ꢁꢁ), 3.97 (1H, d, J = 3.9, H-4), 4.31 (1H, t, J = 7.5,
3
3
H-10ꢁꢁ), 4.41 (1H, dd, J = 8.1, 9.6, H-11 ), 4.52 (1H, d, J = 5.1, H-1), 5.29 (2H, s, CH ), 5.91 (1H, s, OCH O), 5.92 (1H, s,
2
2
OCH O), 6.24 (2H, s, H-2ꢁ, 6ꢁ), 6.44 (1H, s, H-8), 6.50 (1H, s, H-5), 6.56 (1H, d, J = 2.7, H-3ꢁꢁ), 7.06 (1H, td, J = 7.5, 1.2,
2
H-6ꢁꢁꢁ), 7.16 (1H, d, J = 2.7, H-2ꢁꢁ), 7.20 (1H, td, J = 7.5, 1.2, H-3ꢁꢁꢁ), 7.24 (2H, m, H-4ꢁꢁꢁ, 5ꢁꢁꢁ), 7.27 (1H, d, J = 7.8, H-6ꢁꢁ), 7.41
(1H, d, J = 7.8, 1.0, H-7ꢁꢁ), 7.61 (1H, br.s, H-4ꢁꢁ).
ACKNOWLEDGMENT
This work was supported by the Doctoral Science Foundation of Henan University of Traditional Chinese Medicine
(No. BSJJ2011-13), the National Natural Science Foundation of China (No. 30873363), the Great Program of Science Foundation
of Tianjin (09ZCKFNC01200), and the Program of the Science Foundation of Tianjin (08JCYBJC070000).
REFERENCES
1.
2.
A. Casreo, J. M. Miguel del Corral, M. Gordaliza, C. Grande, A. Gomez-Zurita, D. Garcia-Gravalos,
and A. San Feliciano, Eur. J. Med. Chem., 38 (1), 65 (2003).
D. M. Reddy, J. Srinivas, G. Chashoo, A. K. Saxena, and H. M. Sampath Kumar, Eur. J. Med. Chem., 46 (6),
1983 (2011).
3.
4.
Y. Q. Liu, L. Yang, and X. Tian, Curr. Bioact. Compd., 3 (1), 37 (2007).
T. Terada, K. Fujimoto, M. Nomura, J. Yamashita, K. Wierzba, R. Yamazaki, J. Shibata, Y. Sugimoto, and Y. Yamada,
J. Med. Chem., 36 (12), 1689 (1993).
5.
6.
7.
Z. Y. Xiao, Y. D. Xiao, J. Feng, A. Golbraikh, A. Tropsha, and K. H. Lee, J. Med. Chem., 45 (11), 2294 (2002).
R. Tawa, M. Takami, Y. Imakura, K. H. Lee, and H. Sakuraiet, Bioorg. Med. Chem. Lett., 7 (4), 489 (1997).
B. A. Bhat, P. B. Reddy, S. K. Agrawal, A. K. Saxena, H. M. Sampath Kumar, and G. N. Qazi, Eur. J. Med. Chem.,
43 (10), 2067 (2008).
8.
9.
R. L. Oostendorp, P. O. Witteveen, B. Schwartz, L. D. Vainchtein, M. Schot, A. Nol, H. Rosing, J. H. Beijnen,
E. E. Voest, and J. H. M. Schellens, Invest. New Drugs, 28 (2), 163 (2010).
P. Marchand, M. Antoine, G. L. Baut, M. Czech, S. Baasner, and E. Gunther, Bioorg. Med. Chem., 17 (18),
6715 (2009).
10.
P. F. Yu, H. Chen, J. Wang, C. X. He, B. Cao, M. Li, N. Yang, Z. Y. Lei, and M. S. Cheng, Chem. Pharm. Bull., 56 (6),
831 (2008).
413