Semi-synthesis and biological activity of γ-lactones analogs of camptothecin

Article abstract of DOI:10.1016/j.bmcl.2008.10.074

A series of E-ring γ-lactone camptothecin derivatives were synthesized by semi-synthesis via a three-step domino reaction. Their biological activity was evaluated on two types of human tumor cell lines A549 and HT-29 with sulforhodamine-B (SRB) method. The antitumor activity of these compounds was lower than SN-38, only compound 12c was found to be close to the activity of Topotecan. The structure-activity relationship (SAR) of these analogs was studied and discussed. Crown Copyright

Full text of DOI:10.1016/j.bmcl.2008.10.074

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6441–6443
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Semi-synthesis and biological activity of c-lactones analogs of camptothecin
a,
Mingzong Li a, Weidong Tang b, Fuxing Zeng a, Liguang Lou b, , Tianpa You
*
*
a Department of Chemistry, University of Science & Technology of China, Hefei, Anhui 230026, PR China
b Chinese Acad. Sci., Shanghai Inst. Mat. Med., 555 Zuchongzhi Rd., Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of E-ring c-lactone camptothecin derivatives were synthesized by semi-synthesis via a three-step
Received 31 August 2008
Revised 13 October 2008
Accepted 16 October 2008
Available online 19 October 2008
domino reaction. Their biological activity was evaluated on two types of human tumor cell lines A549 and
HT-29 with sulforhodamine-B (SRB) method. The antitumor activity of these compounds was lower than
SN-38, only compound 12c was found to be close to the activity of Topotecan. The structure–activity rela-
tionship (SAR) of these analogs was studied and discussed.
Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
Keywords:
Camptothecin
Camptothecin derivatives
Semi-synthesis
Antitumor activity
Topoisomerase I
Camptothecin (CPT, 1, Fig. 1),1 an alkaloid firstly isolated by
Wall and Wani in 1966 from Chinese tree Camptotheca acuminata,
is one of the most famous lead compounds of antitumor agents.
Attributed to the discovery of being a selective inhibitor against
Topoisomerase I (Topo I) in the late 1980s,2 the family of CPT deriv-
atives has been enlarged rapidly during the last two decades.
Among these thousands of CPT analogs, Topotecan3 and Irinotecan4
have been approved as chemotherapeutic drugs in clinical treat-
ment on human cancers, and several other CPT derivatives are in
different phases of clinical trials.5
O
OH
A
N
C
B
N
D
OR
O
E
=
E
O
OH O
OH O
OH O
1
a
b
2
R= H
R= Na
1
Camptothecin CPT
CPTopenedlactoneform
O
O
X
O
HO
O
In early research, a number of E-ring modified CPT analogs,
Y
OH
OH O
OH
O
including ring opened hydroxyl acid 2a or its sodium salt 2b,6
a
b
c
Y= Cl, Br, I
Y= N3
Y= NH2
a
b
X= NH
X= S
CPT lactol 3,7 lactam 4a, thiolactone 4b,8
a
-halo,
a-azide, a-ami-
4
5
6
3
no-lactone 5a–c9,10 and
a
-hydroxy ketoether 611 were reported.
All these compounds were found to be either inactive or less active
than CPT, and once led to a conclusion that the intact six-mem-
OR1
NHR2
a
b
c
d
R1 = H (Hydroxy-amide)
O
R
R
1 = -COR (Ester-amide)
bered a-hydroxy lactone E-ring of CPT was indispensable for anti-
1 = P (conjugat with PEG)
O
tumor activity.12
OH
O
HO
R1 = H, R2 = P
However, further investigations demonstrated that many other
E-ring modified CPT analogs, such as expanded seven-membered
b-hydroxy lactone homo-camptothecin (hCPT) 713, opened lactone
form hydroxy-amide 8a and ester-amide 8b, opened lactone hy-
droxy-amide with conjugation of polyethylene glycol (PEG)
through the C-17 hydroxyl 8c as well as through C-21 carboxylic
acid 8d, were proved to have comparable or even better antitumor
activity than CPT.6b,14,15
8
7
O
O
HO
OH
OH
10
11
9
Figure 1. Structures of camptothecin and some analogs.
Recently, some lactone-free CPT analogs with five-membered
* Corresponding authors. Tel.: +86 551 3606944?
ketone E-ring 9 and 10 were reported. Compound 9 revealed to
E-mail addresses: lglou@mail.shcnc.ac.cn (L. Lou), ytp@ustc.edu.cn (T. You).
0960-894X/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.074

6442
M. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6441–6443
R1
R1
N
be a potent inhibitor of Topoisomerase I and showed interesting
cytotoxic activitie.16 Another lactone-free CPT analog, 22-hydrox-
yacuminatine 11, a CPT structurally-like compound with a substi-
tuted six-membered aromatic system as E-ring, was also found to
have significant cytotoxic activity in vitro on the murine leukemia
P-388 and KB.17
These observations raise the possibility that novel E-ring mod-
ified analogs of CPT might retain or even enhance antitumor activ-
ity. To study this possibility, we synthesized a series of analogs
bearing a five-membered lactone E-ring with branching N-substi-
R2
R2
O
O
N
N
OH
a
N
NHR3
O
13
CPTs
OH O
OH O
b
R1
R1
N
R2
R2
O
O
N
N
OH
O
N
tuted amide at c-position (compound 12, Fig. 2) by semi-synthesis
from CPT. We hoped that these compounds could maintain or en-
hance the cytotoxic activity.
O
OH
NHR3
15
14
NHR3
O
O
To increase the aqueous solubility and enhance the activity,
several N-substituted amides (substituted with terminal hydroxyl
ethyl or propyl and terminal tertiary amino ethyl) analogs 16a–b
and 19a–n were prepared and studied, since in our previous work,
N-propyl morpholine and other terminal tertiary amino groups
were once introduced to a series of hydroxy-amides and proved
to remarkably improve the activity as well as water-solubility.15
The synthesis of lactones 12a–i, 16a–b and 17 is summarized in
Scheme 1. CPTs18 reacted with amine to open the lactone E-ring to
give hydroxy-amide 13, which was unstable in solvents and would
slowly close back to lactone at neutral or acidic condition as re-
ported before,8 but stable as solid after being precipitated out with
ether or petroleum ether. Compound 13 was then converted to the
five-membered lactone 12 in the presence of pyridinium dichro-
mate (PDC) in dichloromethane at room temperature. Similar work
was once carried out by Hertzberg and his co-workers.8 They used
manganese dioxide as oxidizer to give imide in low yield. Accord-
ing to ESI-MS, IR 1H and 13C NMR spectra and compared to that of
12a: R1 = R2 = H, R3
12b
=
n
-Bu
: R1 = Et, R2 = H, R3
= n-Bu
12c: R1 = cyclopentyl, R2 = H, R3
=
n
-Bu
R1
N
R2
O
1
: R = cyclohexyl, R2 = H, R3
= n
-Bu
12d
N
O
12e: R1 = R2 = H, R3 = CH2C6H4-4-OMe
O
NHR3
12
12f: R1, R2 see scheme 2, R3 = CH2CH(OMe)2
O
: R1 = R2 = H, R3 = CH2CH2OTHP
12g
12h: R1 = R2 = H, R3 = CH2CH2CH2OTHP
O
O
b
N
N
OH
NH(CH2)3N
O
x
O
N
N
12i
O
13i
O
NH(CH2)3N
OH O
O
O
imide, product was identified to be c
-amide lactone 12.19 We de-
O
O
N
N
N
duced that this process underwent a three-step tandem reaction
involving an oxidation in aldehyde of 13 following by an intramo-
lecular acetalisation to give semi-acetal 15,20 which in turn, was
immediately oxidized in the presence of PDC to afford lactone 12
(Scheme 1).
The same strategy failed to prepare a more hydrosoluble com-
pound 12i from 13i (Scheme 1) with a terminal tertiary amine
on amide substituent. In this case, the presence of tertiary amine
group, also susceptible to the oxidation, can explain the formation
of a complex mixture. To avoid this problem a different sequence
was applied. Thus, we firstly converted 12f to the corresponding
aldehyde 18 which was then transformed to the desired amine
19 by reductive amination, in the presence of sodium triacetoxy-
borohydride. A series of analogs of compound 19a–n were then
prepared as described in Scheme 2.
c
O
O
O
N
N
16a
: n = 2;
O
12g, h
16b: n = 3.
NH(CH2)nOH
NH(CH2)nOTHP
O
O
O
N
O
O
d
N
N
O
O
12e
17
NH2
NHCH2C6H4-4-OMe
O
O
Scheme 1. Reagents and conditions: (a) R3NH2, neat, 70 °C, 0.5–1.5 h; (b) PDC,
CH2Cl2, rt, 2 h (61–78%, two steps); (c) PPTS 15% equiv, ethanol, 60 °C, over night
(89%); (d) CAN, CH3CN:H2O = 10/1, 20 h (65%).
The antitumor activity of these synthetic compounds was tested
on two human tumor cell lines A549 and HT-29 with SRB method
using SN-38 and Topotecan as references. The result was listed in
Table 1. As can be seen, the resulting five-membered lactone ana-
logs showed lower cytotoxic activity than SN-38, with IC50 values
N-(n-butyl), 12c was the most active compound with IC50 value
close to Topotecan. Amides 19a–n substituted with N-(tertiary
amino ethyl), also brought some benefits compared with the
unsubstituted amide 17. Compounds 19d and 19e which beared
a terminal morpholinyl or 4-methylpiperazinyl showed more ac-
tive than other congeners.
higher than 1
lM.
The SAR study from this series
that cycloalkyls substituted at the 7-position resulted in signifi-
cantly increasing activity when the amide was substituted with
c
-lactone compounds indicated
Several early SAR studies on AB ring modified CPT derivatives
have shown that alkyl substituted at 7-position and hydroxy or
methoxy substituted at 10-position of CPT would significantly en-
hance their activities.21 So we combined this AB ring substituents
with amide N-substituents and hypothesized that the activity of
resulting modified compounds 19g–n could be enhanced. How-
ever, to our disappointment, no combined positive correlation ef-
fect were observed from the results of these in vitro tests (19g,
19i, 19k, 19m, 19n vs 19d; 19h, 19j, 19l vs 19f). The only excep-
tion was 19j which was found to be slightly more active than 19f.
So it seemed that 7- and 10-position substituents in these com-
pounds acted the opposite effect comparing to what we have
learned.
R1
R2
O
N
O
N
O
12
NHR3
O
Figure 2. Structures of compounds 12.

M. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6441–6443
6443
R1
N
12fa: R1 = R2 = H;
12fb
amino ethyl) such as morpholinyl and 4-methylpiperazinyl, the
substituents at 10-position or 7-position or both, became unfavor-
able for their antitumor activity.
R2
O
: R1 = Et, R2 = OMe;
N
O
12fc: R1 = cyclopentyl, R2 = H;
O
12f
Supplementary data
: R1 = cyclohexyl, R2 = H;
12fd
NHCH2CH(OMe)2
O
12fe: R1 = cyclopentyl, R2 = OMe;
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.10.074.
a
12ff: R1 = cyclopropylcarbonyl, R2 = H.
R1
R1
References and notes
R2
R2
O
N
O
N
1. Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. A. J.
Am. Chem. Soc. 1966, 94, 3888.
b
O
O
N
N
2. (a) Hsiang, Y.-H.; Hertzberg, R.; Hecht, S. M.; Liu, L. F. J. Biochem. 1985, 260,
14873; (b) Hsiang, Y.-H.; Lihou, M. G.; Liu, L. F. Cancer Res. 1989, 49, 5077.
3. Sawada, S.; Okajima, S.; Aiyama, R.; Nokata, K. I.; Furuta, T.; Yokokura, T.;
Sugino, E.; Yamaguchi, K.; Miyasaka, T. Chem. Pharm. Bull. 1991, 39, 1446.
4. Kingsbury, W. D.; Boehm, J. C.; Jakas, D. R.; Holden, K. G.; Hecht, S. M.;
Gallagher, G.; Caranfa, M. J.; McCabe, F. L.; Faucette, L. F.; Johnson, R. K.;
Hertzberg, R. P. J. Med. Chem. 1991, 34, 98.
O
O
19
18a-f
NHCH2CH2R4
O
NHCH2CHO
O
: R1 = R2 = H, R4 = NEt2
: R = Et, R2 = OMe, R4
1
19a
19h
N
=
N
N
19b: R1 = R2 = H, R4 = N
: R1 = cyclopentyl, R2 = H, R4
=
19i
O
5. Garcia-Carbonero, R.; Supko, J. G. Clin. Cancer Res. 2002, 8, 641.
6. (a) Adamovics, J. A.; Hutchinson, C. R. J. Med. Chem. 1979, 22, 310; (b) Sawada,
S.; Yaegashi, T.; Furuta, T.; Yokokura, T.; Miyasaka, T. Chem. Pharm. Bull. 1993,
41, 310.
7. Govindachari, T. R.; Ravindranath, K. R.; Viswanathan, N. J. Chem. Soc., Perkin
Trans. 1 1974, 1215.
8. Hertzberg, R. P.; Caranfa, M. J.; Holden, K. G.; Jakas, D. R.; Gallagher, G.; Mattern,
M. R.; Mong, S.-M.; Bartus, J. O.; Johnson, R. K.; Kingsbury, W. D. J. Med. Chem.
1989, 32, 715.
9. Nicholas, A. W.; Wani, M. C.; Manikumar, G.; Wall, M. E.; Kohn, K. W.; Pommier,
Y. J. Med. Chem. 1990, 33, 972.
: R1 = R2 = H, R4
19c
=
19j: R1 = cyclopentyl, R2 = H, R4 =N
19k
N
N
19d: R1 = R2 = H, R4 = N
O
S
: R1 = cyclohexyl, R2 = H, R4
N
=
O
N
N
: R1 = R2 = H, R4 = N
19l: R1 = cyclohexyl, R2 = H, R4 = N
19e
: R1 = R2 = H, R4 = N
19f
N
: R1 = cyclopentyl, R2 = OMe, R4
=
19m
O
19g: R1 = Et, R2 = OMe, R4
: R1 =cyclopropylcarbonyl, R2 = H, R4
=
N
O
N
=
O
19n
10. (a) Ejima, A.; Terasawa, H.; Sugimori, M.; Ohsuki, S.; Matsumoto, K.; Kawato, Y.;
Yasuoka, M.; Tagawa, H. Chem. Pharm. Bull. 1992, 41, 683; (b) Ejima, A.;
Terasawa, H.; Sugimori, M.; Ohsuki, S.; Matsumoto, K.; Kawato, Y.; Tagawa, H.
Chem. Pharm. Bull. 1989, 37, 2253.
Scheme 2. Reagents and conditions: (a) PPTS, acetone, refluxed for over night (76–
91%); (b) R4H, NaBH(OAc)3, CH2Cl2, rt, 0.5–1 h (39–76%).
11. Du, W.; Curran, D. P.; Bevins, R. L.; Zimmer, S. G.; Zhang, J.; Burke, T. G. Bioorg.
Med. Chem. 2002, 10, 103.
12. (a) Wani, M.; Nicholas, A.; Manikumar, G.; Wall, M. J. Med. Chem. 1987, 30,
1774; (b) Wani, M.; Nicholas, A.; Wall, M. J. Org. Chem. 1987, 30, 2317.
13. Lavergne, O.; Lesueur-Ginot, L.; Rodas, F. P.; Bigg, D. C. H. Bioorg. Med. Chem.
Lett. 1997, 7, 2235.
Table 1
Cytotoxicity activity on human tumor cell lines A549 and HT-29.
a
a
Compound
IC50
A549
(l
M)
HT-29
Compound
IC50
A549
(
l
M)
HT-29
14. Yaegashi, T.; Sawada, S.; Nagata, H.; Furuta, T.; Yokokura, T.; Miyasaka, T. Chem.
Pharm. Bull. 1994, 42, 2518.
15. You, T.; Li, M.; Wang, Z.; Geng, Y. Chinese Patent 200510135329X[P], 2007.
16. Hautefaye, P.; Cimetière, B.; Pierré, A.; Léonce, S.; Hickman, J.; Laine, W.; Bailly,
C.; Lavielle, G. Bioorg. Med. Chem. Lett. 2003, 13, 2731.
12a
12b
12c
12d
16a
16b
17
18c
19a
19b
19c
SN-38
70.1
Na
1.28
25.5
Na
Na
Na
Na
65.3
94.3
74.9
0.08
68.3
Na
2.03
28.1
Na
19d
19e
19f
19g
19h
19i
19j
19k
19l
19m
19n
Topotecan
25.6
Na
53.1
Na
Na
Na
11.0
Na
75.7
96.5
Na
39.9
Na
7.25
Na
31.6
Na
16.9
Na
17. (a) Lin, L. Z.; Cordell, G. A. Phytochemistry 1989, 28, 1295; (b) Grillet, F.;
Baumlová, B.; Prévost, G.; Constant, J. F.; Chaumeron, S.; Bigg, D. C. H.; Greene,
A. E.; Kanazawa, A. Bioorg. Med. Chem. Lett. 2008, 18, 2143.
Na
Na
18. CPTs are 7- and 10-substituted CPT analogs with R1 and R2 listed in compounds
12a–i in Scheme 1 and 12fa–f in Scheme 2. 7-Et–10-MeO–CPT was obtained
from SN-38, 7-substituents were induced to CPT or 10-MeO-CPT with the
method of Sawada.3
18.8
31.6
31.6
31.6
0.12
31.6
62.9
Na
19. Structure identification of compound 12a: (a) Data of compound 12a: 1H NMR
(300 MHz, CDCl3, TMS): d 0.91 (3H, t, J = 7.20 Hz), 0.99 (3H, t, J = 7.20 Hz), 1.34
(2H, m), 1.47 (2H, m), 2.15 (1H, m), 2.50 (1H, m), 3.20 (1H, m), 3.34 (1H, m),
5.37 (2H, s), 6.66 (1H, br), 7.72 (1H, t, J = 7.20 Hz), 7.82 (1H, s), 7.87 (1H, t,
J = 7.20 Hz), 7.97 (1H, d, J = 7.80 Hz), 8.27 (1H, d, J = 8.40 Hz), 8.44 (1H, s); 13C
NMR (300 MHz, CDCl3, TMS): d 8.0, 13.7, 20.1, 31.0, 31.5, 39.6, 50.6, 87.9, 96.0,
111.1, 128.3, 128.7, 128.9, 129.8, 130.3, 131.0, 131.4, 149.3, 151.4, 153.6, 156.1,
166.4, 167.3, 167.7; IR (KBr, cmÀ1): 1550, 1672, 1778, 3434; ESI-Ms: m/
z = 418.2 (M+1). (b) The signal of 1H NMR at 6.66 ppm as a broad peak (like
triplet) is the proton of amide NH, which was not observed in Hertzberg’s
work; Infra-Red absorption band at 1778 cmÀ1 displayed that it was a lactone
0.48
0.375
a
Values are measured with SRB method (Na, not active up to 100
SN-38 and Topotecan are the average of two tests.
lM). Values of
In summary, we have synthesized a series of five-membered-c-
amide-lactone from CPT with a key step via a domino reaction.
These compounds were found to be obviously less active than
SN-38 and only compound 12c has the activity close to Topotecan.
The SAR study from these compounds displayed two different sit-
uations: cycloalkyls substituted at the 7-position would signifi-
cantly increase the activity when the amide was substituted with
N-(n-butyl). While the amide was substituted with N-(2-tertiary
carbonyl, not imide carbonyl, so the product was identified as c-amide lactone.
20. Experiments were once designed to capture intermediate 14 or 15. Some
intermediates were detected from TLC in deed, but after being purified by
chromatography on silica gel, only CPT and product 12 were collected.
21. Sawada, S.; Nokata, K. I.; Furuta, T.; Yokokura, T.; Miyasaka, T. Chem. Pharm.
Bull. 1991, 39, 2574.