The solvolysis of topotecan in alcohols and acetic anhydride

Article abstract of DOI:10.1016/j.bmcl.2011.02.080

Six derivatives of 10-hydroxycamptothecin were prepared via solvolysis of topotecan in corresponding alcohols and acetic anhydride. We attributed the specific reactivity of topotecan to the internal hydrogen-bonding between 10-hydroxy and the nitrogen atom in position 9. As a result the reaction underwent through an intermediate ortho-quionomethlide species to reach equilibrium. Bioactivity screening data showed all products could potentially inhibit the proliferation of several cancer cell lines in vitro and a bigger size group in 9-position would be favorable for the anti-tumor activities observably.

Full text of DOI:10.1016/j.bmcl.2011.02.080

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2324–2326
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The solvolysis of topotecan in alcohols and acetic anhydride
Jiajun Li a, Guolin Wang b, Mengjie Dong b, , Qian Zhang a,
⇑
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
b PET Center, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Six derivatives of 10-hydroxycamptothecin were prepared via solvolysis of topotecan in corresponding
alcohols and acetic anhydride. We attributed the specific reactivity of topotecan to the internal hydro-
gen-bonding between 10-hydroxy and the nitrogen atom in position 9. As a result the reaction under-
went through an intermediate ortho-quionomethlide species to reach equilibrium. Bioactivity
screening data showed all products could potentially inhibit the proliferation of several cancer cell lines
in vitro and a bigger size group in 9-position would be favorable for the anti-tumor activities observably.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 7 October 2010
Revised 3 February 2011
Accepted 23 February 2011
Available online 26 February 2011
Keywords:
Topotecan
Derivatives of 10-hydroxycamptothecin
Anti-tumor activity
Solvolysis
Camptothecin (CPT, 1, Fig. 1), a pentacyclic alkaloid, was iso-
lated by Wall et al. in 1966 from the Chinese tree Camptotheca
acuminata1 and was reported to possess potent anti-tumor activity.
However, its clinical application has been hindered due to severe
toxicity and extremely poor solubility.2 Until the action mecha-
nism of camptothecin, it can inhibit DNA topoisomerase I, was dis-
covered in 1985, this compound was then revived, which brought a
worldwide attention and led to the successful clinical application
of several CPT derivatives including topotecan (2), irinotecan and
10-hydroxycamptothecin.3,4 Now hundreds of CPT derivatives are
undergoing investigation,5a–c in particular structure modifications
in positions 7, 9 and 10,6a–d some of which even go into various
stages of clinical trials.
N
9
7
5
HO
O
10
11
O
N
N
17
N
N
O
O
O
14
20
O
OH
OH
Camptothecin 1
Topotecan 2
Figure 1. Structures of camptothecin and topotecan.
In our research, a 11C-labeled quaternary ammonium salt of
methanol attacked the carbon-atom in benzyl since the strong
leaving-activity of trimethylamino group.
topotecan,
namely
11C-9-(N,N,N-trimethylaminomethyl)-10-
hydroxycamptothcin, was attempted to prepare as PET (Positron-
Emission Tomography) molecule for tumor diagnosis. In the process
of synthesizing the related reference substance, the methylation of
topotecan was performed via iodomethane and methanol was
selected as solvent due to it could dissolve the substrate very well.
Following the reaction was monitored by TLC until it reached equi-
librium, the main product was separated, purified and determined
via 1H NMR and MS as 10-hydroxy-9-(methoxymethyl)camptothcin
(3)7 unexpectedly, thatis, insteadofmethylationthedimethylamino
group of topotecan was substituted by a methoxyl group. The fact
that the solvent participated in the reaction made us wonder if the
quaternary ammonium salt of topotecan had been formed before
To testify the hypothesis, topotecan was refluxed in methanol
without iodomethane. Out of expectation, the reaction still reached
equilibrium after refluxing 20 h and afforded the same product in
31.7% as before (recovered substrate in 42.0%). From these results,
we attributed the specific chemical behavior of topotecan to the
internal hydrogen-bonding between 10-hydroxy group and the
nitrogen atom in position 9, of which the unshared pair of elec-
trons is locked by hydrogen that makes the methylation of nitro-
gen atom with iodomethane more difficult and enhances the
leaving ability of dimethylamino group as dimethylamine entity.
If that happens, topotecan will transfer into an intermediate
ortho-quinonemethide species as shown in Figure 2. Herein, both
topotecan (2) and 10-hydroxy-9-(methoxymethyl)camptothcin
(3) could be considered as ortho-quinonemethide precursors
(QMPs).8 Dimethylamine or methanol can come off easily to afford
ortho-quinonemethide and inversely they both can react with the
⇑
Corresponding authors. Tel.: +86 21 51980119.
E-mail addresses: mengjiedong@163.com (M. Dong), zhangqian511@shmu.
edu.cn (Q. Zhang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.080

J. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2324–2326
2325
N
O
H
O
O
O
HO
CH3OH
O
O
N
-HN(CH3)2
HN(CH3)2
N
N
N
N
N
-CH3OH
O
O
O
O
O
OH
O
OH
OH
2
3
Figure 2. Mechanism of the solvolysis of topotecan in methanol.
Table 1
N
O
Anti-tumor activity of solvolysis products 3–8 against KB, HepG2 and C26
R
N
HO
Compounds
Cytotoxicity in vitro (IC50
,
l
M)
C26
0.496
100.776
29.106
3.877
HO
O
O
R-OH
N
N
KB
0.829
25.367
19.727
7.246
1.376
0.794
1.151
HepG2
N
reflux
10-Hydroxy CPT
0.981
92.071
3.909
13.480
0.802
O
O
O
O
3
4
5
6
7
8
3 R = CH3
4 R = CH2CH3
5 R = CH(CH3)2
6 R = C(CH3)3
OH
OH
2
0.273
0.157
0.424
1.004
1.306
Scheme 1. Solvolysis of topotecan in different alcohols.
KB, human oral squamous cell carcinoma; HepG2, human liver cancer; C26, mice
colon carcinoma.
N
O
R1
R1O
HO
7 with a yield of 38.2%. While 1 N of DMAP was added into the
reaction, another 10, 20-diacetylation product 8 was separated
besides 7 (Scheme 2). However, the reactions did not afford the rel-
evant products when acetic acid, ethanethiol and water were em-
ployed as solvents, possibly due to their stronger acidity and
weaker nucleophilicity than alcohols.
O
O
N
N
Method A or B
N
N
O
O
O
OH
2 O
OR
2
7 R1 = COCH3, R2 = H
8 R1 = R2 = COCH3
All of these 9-alkoxymethyl and 9-acetoxymethyl derivatives
were screened against three cancer cell lines (KB, HepG2 and
C26) by WST-1 assay and using 10-hydroxycamptothecin (HCTP)
as reference compound. The bioactivity data (shown in Table 1)
demonstrated most of the compounds possessed potential antipro-
liferative activities and concentration dependence (Fig. 3). Espe-
cially, 7 and 6 showed more potent cytotoxic activity on KB and
HepG2, respectively, than that of the reference compound. On
C26 cell line test, 3 derivatives (6, 7 and 8) exhibited stronger activ-
ity than 10-hydroxycamptothecin, in which 7 was even three-fold
more active than the reference compound. Moreover, there was an
evident trend among 9-alkoxymethyl derivatives from the test re-
sults that the bulkier the group in position 9 became, the higher
cytotoxic activity the compound possessed except for compound
4 on HepG2. A possible structure–activity relationship between
9-position group and its bioactivity was suggested that a bigger
size group in the 9-position would be observably favorable for
Scheme 2. Solvolysis of topotecan in acetic anhydride. Reagents and conditions:
Method A: (CH3CO)2O, 50 °C. Method B: (CH3CO)2O/DMAP, CHCl3, reflux.
intermediate as nucleophiles to establish reaction equilibrium as
observed.
Under similar protocol, the solvolysis of topotecan in ethyl alco-
hol, isopropyl alcohol and tert-butyl alcohol afforded 4, 5 and 6,7
respectively, with the yields from 16.0% to 43.2% (Scheme 1). While
cutting off the quantity of starting material recovered from the
reaction, the translation rates would range from 54.6% to 69.8%.
Other than in alcohols, the solvolysis of topotecan in acetic
anhydride, acetic acid, ethanethiol and water was also explored,
respectively. After the solution of topotecan in acetic anhydride
was stirred under 50 °C for 49 h, the reaction afforded compound
KB
HepG2
C26
1.2
1
1.2
1
1.2
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
3
30
300
3000
30000 300000
3
30
6
300
3000
30000 300000
3
30
300
3000
30000 300000
concentration (nM)
concentration (nM)
concentration (nM)
3
4
5
7
8
10-Hydroxycamptothecin
Figure 3. Concentration-versus-cell survival rate curves.

2326
J. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2324–2326
6. (a) Gao, H. Y.; Zhang, X. W.; Chen, Y.; Shen, H. W.; Sun, J.; Huang, M.; Ding, J.; Li,
C.; Lu, W. Bioorg. Med. Chem. Lett. 2005, 15, 2003; (b) Dallavalle, S.; Rocchetta, D.
G.; Musso, L.; Merlini, L.; Morini, G.; Penco, S.; Tinelli, S.; Beretta, G. L.; Zunino, F.
Bioorg. Med. Chem. Lett. 2008, 18, 2781; (c) Li, Q. Y.; Zu, Y. G.; Shi, R. Z.; Yao, L. P.;
Fu, Y. J.; Yang, Z. W.; Li, L. Bioorg. Med. Chem. 2006, 14, 7175; (d) Wang, S.; Li, Y.
Y.; Liu, Y. H.; Lu, A. J.; You, Q. D. Bioorg. Med. Chem. Lett. 2008, 18, 4095.
7. Compounds data: 10-Hydroxy-9-methoxymethyl-(20S)-camptothecin (3). Yield
31.7%, mp >300 °C, 1H NMR (400 MHz, DMSO-d6) d 0.88 (3H, t, J = 7.24 Hz, 18-
CH3), 1.87 (2H, m, 19-CH2), 3.33 (3H, s, O-CH3), 4.87 (2H, s, 9-CH2), 5.26 (2H, s, 5-
CH2), 5.41 (2H, s, 17-CH2), 6.49 (1H, s, 20-OH), 7.26 (1H, s, 14-CH), 7.53 (1H, d,
J = 9.00 Hz, 11-CH), 8.02 (1H, d, J = 9.00 Hz, 12-CH), 8.65 (1H, s, 7-CH), 10.49 (1H,
s, 10-OH); MS-ESI m/z 409.1 (MH+); HRMS: calcd for C22H20N2O6 409.1394,
found: 409.1398.
anti-tumor activities, which was consistent with the bioactivity
screening results of some 9-substituted such as CHO, CN and
CH@NOC(CH3)3 10-hydroxycamptothecins reported by Dallavalle
et al.6b As to 9-acetoxymethyl derivatives, the data of 7 was slightly
smaller than that of 8, indicting that 20-acetyl group was not com-
patible for its bioactivities.
In conclusion, 6 derivatives of 10-hydroxycamptothecin were
obtained via solvolysis of topotecan in corresponding alcohols
and acetic anhydride, and screened on several cancer cell lines as
anti-tumor agents. The mechanism of this kind of reaction is attrib-
uted to the existence of internal hydrogen-bonding between
10-hydroxy and 9-nitrogen atom in topotecan. Consequently the
reaction undergoes through an intermediate ortho-quionomethlide
species to reach an equilibrium. It is worth mentioning that the
lipo-solubility of all the 10-hydroxy 9-alkoxymethyl and 9-
acetoxymethylcamptothecins is improved substantially compared
with 10-hydroxycamptothecin or topotecan base.
9-Ethoxymethyl-10-hydroxy-(20S)-camptothecin (4). Yield 35.0%, mp >300 °C,
1H NMR (400 MHz, DMSO-d6) d 0.89 (3H, t, J = 7.24 Hz, 18-CH3), 1.15 (3H, t,
J = 7.04 Hz, O–CH2–CH3), 1.87 (2H, m, 19-CH2), 3.57 (2H, q, J = 7.04 Hz, O–CH2–
CH3), 4.89 (2H, s, 9-CH2), 5.25 (2H, s, 5-CH2), 5.41 (2H, s, 17-CH2), 6.49 (1H, brs,
20-OH), 7.26 (1H, s, 14-CH), 7.51 (1H, d, J = 9.00 Hz, 11-CH), 8.00 (1H, d,
J = 8.99 Hz, 12-CH), 8.65 (1H, s, 7-CH), 10.42 (1H, s, 10-OH); MS(ESI) m/z 423.1
(MH+); HRMS: calcd for C23H22N2O6 423.1551, found: 423.1556.
10-Hydroxy-9-isopropoxymethyl-(20S)-camptothecin (5). Yield 43.2%, mp >300 °C,
1H NMR (400 MHz, DMSO-d6) d 0.89 (3H, t, J = 7.24 Hz, 18-CH3), 1.16 (3H, s, O–
CH–CH3), 1.17 (3H, s, O–CH–CH30), 1.87 (2H, m, 19-CH2), 3.74 (1H, m, O–CH),
4.89 (2H, s, 9-CH2), 5.26 (2H, s, 5-CH2), 5.42 (2H, s, 17-CH2), 6.51 (1H, s, 20-OH),
7.26 (1H, s, 14-CH), 7.51 (1H, d, J = 9.39 Hz, 11-CH), 7.97 (1H, d, J = 9.00 Hz, 12-
CH), 8.63 (1H, s, 7-CH), 10.41 (1H, s, 10-OH); MS(ESI) m/z 437.2 (MH+); HRMS:
calcd for C24H24N2O6 437.1707, found: 437.1699.
Acknowledgements
10-Hydroxy-9-(tert-butoxymethyl)-(20S)-camptothecin
(6).
Yield
16.0%,
mp >300 °C, 1H NMR (400 MHz, DMSO-d6) d 0.89 (3H, t, J = 7.24 Hz, 18-CH3),
1.28 (9H, s, O–C–(CH3)3), 1.84 (2H, m, 19-CH2), 4.80 (2H, s, 9-CH2), 5.25 (2H, s, 5-
CH2), 5.39 (2H, s, 17-CH2), 6.49 (1H, s, 20-OH), 7.23 (1H, s, 14-CH), 7.47 (1H, d,
J = 9.00 Hz, 11-CH), 7.96 (1H, d, J = 9.39 Hz, 12-CH), 8.58 (1H, s, 7-CH), 10.33 (1H,
s, 10-OH); MS(ESI) m/z 451.1 (MH+); HRMS: calcd for C25H26N2O6 451.1864,
found: 451.1856.
This work was supported by National Drug Innovative Program
(No. 2009ZX09301-011), National Natural Science Foundation
of China (No. 30870730, to Dr. Mengjie Dong) and the Science
and Technology Planning Project of Zhejiang province (No.
2009C33109, to Dr. Mengjie Dong).
10-Acetoxy-9-acetoxymethyl-(20S)-camptothecin (7). Yield 38.2%, mp 236–
239 °C, 1H NMR (400 MHz, CDCl3) d 1.05 (3H, t, J = 7.33 Hz, 18-CH3), 1.90 (2H,
m, 19-CH2), 2.07 (3H, s, 9-COCH3), 2.46 (3H, s, 10-COCH3), 3.82 (1H, s, 20-OH),
5.32 (1H, d, J = 16.49 Hz, 9-CH2), 5.35 (2H, s, 5-CH2), 5.59 (2H, s, 17-CH2), 5.75
(1H, d, J = 16.49 Hz, 9-CH20), 7.58 (1H, d, J = 9.17 Hz, 11-CH), 7.67 (1H, s, 14-CH),
8.26 (1H, d, J = 9.47 Hz, 12-CH), 8.69 (1H, s, 7-CH); MS(ESI) m/z 479.2 (MH+);
HRMS: calcd for C25H22N2O8 479.1449, found: 479.1461.
References and notes
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Chem. Soc. 1966, 88, 3888.
10, 20(S)-Diacetoxy-9-acetoxymethyl-(20S)-camptothecin (8). Yield 8.67%, mp
260–263 °C, 1H NMR (400 MHz, CDCl3) d 0.99 (3H, t, J = 7.55 Hz, 18-CH3), 2.07
(3H, s, 20-COCH3), 2.15 (1H, q, J = 7.15 Hz, 19-CH2), 2.23 (3H, s, 9-COCH3), 2.29
(1H, q, J = 7.15 Hz, 19-CH20), 2.47 (3H, s, 10-COCH3), 5.33 (2H, s, 5-CH2), 5.41 (1H,
d, J = 17.55 Hz, 9-CH2), 5.60 (2H, s, 17-CH2), 5.68 (1H, d, J = 17.06 Hz, 9-CH20),
7.21 (1H, s, 14-CH), 7.60 (1H, d, J = 9.27 Hz, 11-CH), 8.25 (1H, d, J = 8.77 Hz, 12-
CH), 8.70 (1H, s, 7-CH); MS(ESI) m/z 521.2 (MH+); HRMS: calcd for C27H24N2O9
521.1555, found: 521.1567.
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