Synthesis and biological activities of a pH-dependently activated water-soluble prodrug of a novel hexacyclic camptothecin analog

Article abstract of DOI:10.1016/j.bmcl.2009.03.123

CH0793076 (1) is a novel hexacyclic camptothecin analog showing potent antitumor activity in various human caner xenograft models. To improve the water solubility of 1, water-soluble prodrugs were designed to generate an active drug 1 nonenzymatically, th

Full text of DOI:10.1016/j.bmcl.2009.03.123

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2772–2776
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activities of a pH-dependently activated
water-soluble prodrug of a novel hexacyclic camptothecin analog
Jun Ohwada, Sawako Ozawa, Masami Kohchi, Hiroshi Fukuda, Chikako Murasaki, Hitomi Suda,
Takeshi Murata, Satoshi Niizuma, Masao Tsukazaki, Kazutomo Ori, Kiyoshi Yoshinari, Yoshiko Itezono,
Mika Endo, Masako Ura, Hiromi Tanimura, Yoko Miyazaki, Akira Kawashima, Shunsuke Nagao,
*
Eitarou Namba, Koutarou Ogawa, Kazuko Kobayashi, Hisafumi Okabe, Isao Umeda, Nobuo Shimma
Research Division, Chugai Pharmaceutical Co., Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
CH0793076 (1) is a novel hexacyclic camptothecin analog showing potent antitumor activity in various
human caner xenograft models. To improve the water solubility of 1, water-soluble prodrugs were
designed to generate an active drug 1 nonenzymatically, thus expected to show less interpatient PK var-
iability than CPT-11. Among the prodrugs synthesized, 4c (TP300, hydrochloride) having a glycylsarcosyl
ester at the C-20 position of 1 is highly water-soluble (>10 mg/ml), stable below pH 4 and rapidly gen-
erates 1 at physiological pH in vitro. The rapid (ca. <1 min) generation of 1 after incubation of TP300 with
plasma (mouse, rat, dog and monkey) was also demonstrated. TP300 showed a broader antitumor spec-
trum and more potent antitumor activity than CPT-11 in various human cancer xenograft models.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 18 December 2008
Revised 12 March 2009
Accepted 25 March 2009
Available online 28 March 2009
Keywords:
Camptothecin analog
Topoisomerase I inhibitor
TP300
Antitumor agent
Water-soluble
Prodrug
Drug resistance
BCRP
Camptothecin analogs are potent topoisomerase I inhibitors
showing strong antitumor activity both in vitro and in vivo.1 How-
ever, most have poor water solubility. Two strategies had been ta-
ken to address this issue: (i) synthesis of camptothecin analogs
with an amino functional group, for example, topotecan and Dx-
8951, and (ii) synthesis of water-soluble prodrugs of lipophilic
camptothecin analogs, for example, irinotecan (CPT-11). The com-
pounds in the first category generally show reduced antitumor
activity due to a lower tissue distribution profile. CPT-11 is a
water-soluble prodrug of SN-38 and mainly used clinically for
the treatment of gastrointestinal tumors (Fig. 1). Its clinical utility,
however, is limited due to the drawbacks depicted in Figure 2: high
interpatient variability in pharmacokinetics (due to poor biocon-
version to the active drug SN-38 and SNPs of the metabolizing en-
zyme UGT1A1), severe toxicities (bone marrow and intestine), and
the function of SN-38 as a substrate of the breast cancer resistant
protein (BCRP) efflux pump.2
water solubility of 1 remained to be improved for intravenous
administration.
In this Letter, we report the design, synthesis and biological
activities of a water-soluble prodrug of 1 (4c: named TP300) that
can be pH-dependently converted to 1 and exhibited higher antitu-
mor activity than CPT-11 in various human cancer xenograft
models.
Design and synthesis. The bioconversion of CPT-11 to the active
drug SN-38 by carboxyl esterase is reported to be very low in hu-
man, only 4–5%, and is one of the reasons for the wide interpatient
PK variability of CPT-11. To simultaneously achieve greater water
solubility, conversion efficiency of the prodrug, and tissue distribu-
tion of the active drug, we designed three types of new water-sol-
uble prodrugs of 1: (i) a prodrug (2) activated by peptidase such as
membrane dipeptidase4 overexpressed in gastro intestinal tumors
(Fig. 3), (ii) prodrugs that can be nonenzymatically activated
(Fig. 4). Namely, we designed prodrugs (4a–f) having a dipeptide
ester group at the C-20 position of 1, which were stable at low
pH but rapidly converted to the active drug at physiological pH
by intramolecular cyclization of the dipeptide moiety. Another de-
sign included sulfone prodrugs (6a, 6b) that can be activated by b-
elimination triggered by an intramolecular deprotonation with a
terminal amino group of the promoiety.
We previously reported a new hexacyclic camptothecin analog,
CH0793076 (1),3 that is not a substrate of BCRP and has higher
in vivo antitumor activities than CPT-11, even though the poor
* Corresponding author. Tel.: +81 467 47 2280; fax: +81 467 45 6824.
E-mail address: shinmanbo@chugai-pharm.co.jp (N. Shimma).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.123

J. Ohwada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2772–2776
2773
9
7
N
O
HO
O
A
B
C
N
N
N
O
D
O
N
N
N
E
O
O
O
N
O
OH
O
OH
O
OH
O
SN-38
NH2
Irinotecan
Camptothecin
N
F
HO
O
O
N
N
F
N
N
O
O
OH
O
OH
O
Topotecan
Dx8951
Figure 1. Camptothecin and representative analogs.
CPT-11 (Prodrug)
SN-38 (Active)
Conversion by
carboxylesterase
(4-5%)
N
HO
O
HCl
N
O
O
PK variability by
SNPs
N
N
O
N
N
PK variability
O
O
Glucuronidation by
UGT1A1
Inhibition of
acetylcholine esterase
OH
O
OH
O
Pump-out by BCRP
Early diarrhea
Limited efficacy
PK variability by SNPs
Glucuronide-SN-38
(inactive)
Reconversion to SN-38
in intestine
Late diarrhea
Figure 2. Major drawbacks of CPT-11.
N
N
N
N
N
N
O
O
N
N
N
N
N
O
Peptidase
O
O
N
O
O
O
O
O
O
O
O
O
OH
OH
H2N
N
H
O
2HCl
NH2
O
H2N
O
CH0793076 (1)
3
2
Figure 3. Design of a water-soluble prodrug activated by peptidase.
The synthesisof the prodrugs(4a–f, and6a, 6b) is shownin Figure
5. Prodrugs 4a–f were prepared by esterification of 1 with N-Boc-
dipeptide using N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride (WSCI) in thepresence of 4-(dimethylamino)pyridine
at room temperature followed by removal of the Boc group by 1 N
HCl in AcOH. 6a was prepared in three steps from 1. First, treatment
of 1 with p-nitrophenyl chloroformate, diisopropylethylamine and
4-dimethylaminopyridine in methylene chloride gave the p-nitro-
phenylcarbonate derivative. Then the carbonate was reacted with
2-(2-tert-butoxycarbonylaminoethanesulfonyl)ethanol5 to afford
the sulfone derivative. Removal of the Boc group gave 6a in high
yield. 6b was synthesized via the 2-bromoethylcarbonate
derivative, derived from 1 and 2-bromoethylchloroformate. The
2-bromoethylcarbonate derivative was then reacted with tert-
butoxycarbonyl-cysteine ethyl ester in the presence of potassium
carbonate in acetonitrile to give the sulfide derivative. Oxidation
of the sulfide with OXONE followed by removal of the Boc group
by 1 N HCl in AcOH gave 6b as a yellow powder. The final products
were obtained as hydrochloride salts after lyophilization. The syn-
thesis of the prodrug 2 from 1 is described in Ref. 4.

2774
J. Ohwada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2772–2776
1) Dipeptide type
N
N
Plasma
pH 7.4
O
O
O
N
N
N
R1
R2
R2
R4
R3
N
O
N
N
+
N
O
N
O
R3
N
O
O
R1
O
OH
N
H
O
O
HCl
4a–f
1
5a–f
2) Sulfone type
Plasma
pH 7.4
N
N
N
O
H
N
N
N
N
N
R5
O
N
+
O
S
O
O
O
O
HCl
O
O
O
H2N
O
OH
S
R5
O
O
6a, b
1
7a, b
H2N
R5
S
O
O
Figure 4. Design of water-soluble prodrugs that are pH-dependently activated (non-enzymatic process).
N
N
N
N
N
N
4a: Y= Gly-Gly-
N
N
1) 1N HCl-AcOH
rt, 3 h
2) Lyophilization
N
Boc-dipeptide-OH
WSCI, DMAP, DCM
rt, 1 h
O
4b: Y= Sar-Gly-
O
O
N
N
N
4c: Y= Gly-Sar-
4d: Y= Sar-Sar-
O
O
O
60-70% (2 steps)
4e: Y= Gly-(L)-(N-Me)Ala-
4f: Y= α-di-Me-Gly-Sar-
OH
O
O
O
O
O
Boc-dipeptide
·HCl
CH0793076 (1)
Y
ClCOOPh-p-NO2
DIEA, DMAP/DCM
rt, 3 h
H
N
O
OH
N
N
N
N
N
N
N
N
S
N
1) 1N HCl-AcOH
rt, 1 h
2) Lyophilization
O
O
O
O
O
O
N
N
N
DMAP/DCM rt, 20 h
42%
O
O
O
O
O
O
94%
O
O
O
O
O
O
O
H
N
H2N
O
O
O
O
N+
O
O
S
S
6a: R= -H
·HCl
R
O
O
O
O
6b: R= -COOEt
2-bromoethyl chloroformate
DIEA, DMAP/DCM rt, 20 h
1) 1N HCl-AcOH
rt, 1 h
2) Lyophilization
94%
O
97%
HS
O
O
N
N
N
O
N
N
N
N
H
O
N
N
N
O
N
OXONE
MeOH rt, 2 h 88%
O
K2CO3/CH3CN
rt, 20 h 46%
N
O
O
O
N
O
O
O
O
O
O
O
O
O
O
O
O
S
O
O
O
S
NH
O
O
O
O
Br
HN
O
O
O
Figure 5. Synthesis of water-soluble prodrugs of CH0793076 (1).

J. Ohwada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2772–2776
2775
Table 1
pH stability of prodrugs 4a–f, 6a and 6b
Compound
R1
R2
R3
R4
R5
pH stability
Remaining % of the prodrug in phosphate buffer solution at rt after 3 h
pH 2
N.D.
100
100
95
pH 5
pH 7.4
4a
4b
H
Me
H
Me
H
H
H
H
H
H
H
Me
H
H
Me
Me
Me
Me
H
H
H
H
Me
H
—
—
—
—
—
—
100
100
83
83
47
0
47
73
0
0
0
4c (TP300)
4d
4e
4f
99
78
0
6a
6b
—
—
—
—
—
—
—
—
H
CO2Et
100
100
96
68
4
43
100
80
60
40
20
0
The prodrug (2) was converted to the intermediate (3) smoothly
after i.v. administration, but further conversion to the active drug
(1) was too slow (T1/2 = ꢀ4 h).
TP300, Rat plasma
TP300, Dog plasma
The stability of the prodrugs (4a–f, 6a, 6b) at various pH condi-
tions is summarized in Table 1. The non-N-methylated (R3 = H)
dipeptides (4a, 4b) were stable at pH 7.4 at room temperature
for 3 h. In other words, their conversion to the active drug (1)
was slow at physiological pH. On the other hand, the N-methylated
(R3 = Me) dipeptides (4c–f) were rapidly converted to 1 at pH 7.4.
This rapid conversion can be explained by the contribution of an s-
cis amide configuration that facilitates intramolecular cyclization.
Among the N-methylated dipeptides, the gly-sar ester (4c:
TP300) and the sar-sar ester (4d) were more stable at pH 5 than
TP300, Human plasma
Compound 1, Rat plasma
Compound 1, Dog plasma
Compound 1, Human plasma
the gly-N-Me-(
L
)-ala ester (4e) and
a,a
-di-Me-gly-sar (4f). We se-
0
1
2
3
Incubation time (min)
lected TP300 (4c) as a clinical candidate since it had the highest
stability at pH 2 as well. A sulfone prodrug (6a) also showed favor-
able conversion at pH 7.4 similar to 4c.
Figure 6. Conversion of 14C-TP300 to 14C-Compound 1 in rat, dog and human
plasma.
The conversion of TP300 to 1 occurred very rapidly, in less than
1 min, in plasma from mouse, rat, dog, monkey and human (Fig. 6).
TP300, a hydrochloride, is stable as an amorphous powder and has
sufficient water solubility (>10 mg/ml) for i.v. formulation.
The mechanisms of releasing an active drug (1) from TP300 (4c)
and 6a were confirmed by 1H NMR and MS analysis of the products
(5c and 7a0, respectively)6 derived from the promoieties as de-
picted in Figure 7.
Biological activities. The in vivo antitumor activities of TP300 were
evaluated in human cancer xenograft models of colorectal cancer
HCT116 (BCRP negative) and non-small cell lung cancer NCI-H460
(BCRP positive) and compared with CPT-11 at the maximum toler-
ated doses. The results are shown in Figure 8. TP300 exhibited higher
efficacy than CPT-11 in human cancer xenografts models regardless
N
N
N
N
N
N
O
O
O
N
N
N
O
N
Na2CO3 in CD3OD
N
O
O
HN
N
+
O
N
O
O
O
O
O
HCl
H2N
O
O
N
O
OH
N
O
NH2
O
1
5c
4c
N
N
N
O
H
N
N
N
N
NH2
Na2CO3 in CD3OD
O
N
O
+
N
D
S
O
O
S
O
O
HCl
D+
O
O
O
O
O
H2N
O
OH
S
O
O
7a’
1
6a
Figure 7. Confirmation of the mechanism of releasing active drug (1) from 4c (TP300) and 6a by 1H NMR and MS.

2776
J. Ohwada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2772–2776
HCT116
(colorectal cancer)
BCRP-
NCI-H460
(non-small cell lung cancer)
BCRP+
10000
10000
1000
100
1000
100
*
*
10
20
30
40
5
15
25
35
Days after tumor inoculation
Figure 8. Antitumor effect of TP300 and CPT-11 in human cancer xenograft models. TP300 and CPT-11 were administered at the maximum tolerated dose (MTD) by bolus
intravenous injection once per week for 3 weeks. The MTD was 47 mg/kg for TP300 and 100 mg/kg for CPT-11. Each group consisted of 7 mice. Values for tumor volume are
given as the mean SD. s: vehicle; d: TP300; N: CPT-11. The BCRP protein was detected by Western blotting. *: statistically significantly different in mice treated with TP300
compared with mice treated with CPT-11 (P < 0.05).
2. (a) Chabot, G. G. Clin. Pharmacokinet. 1997, 33, 245; (b) Hecht, J. R. Oncology
of the level of BCRP expression. Data indicating additional antitumor
1998, 12, 72; (c) Dodds, H. M.; Rivory, L. P. Mol. Pharmacol. 1999, 56, 1346;
efficacy of TP300 will be reported in a separate paper.7
(d) Iyer, L.; King, C. D.; Whitington, P. F.; Green, M. D.; Roy, S. K.; Tephly, T.
In summary, we successfully designed and synthesized a water-
soluble prodrug of 1 which can be pH-dependently converted to 1.
TP300 exhibited higher antitumor activity in various human cancer
xenograft models than CPT-11. Because of the non-enzymatic acti-
vation of TP300, less interpatient PK variability than CPT-11 is
expected in clinic. With the unique biological profile of the parent
drug (1) together with improved solubility, TP300 will exhibit bet-
ter efficacy and safety than CPT-11. A Phase 1 trial of TP300 is cur-
rently in progress.
R.; Coffman, B. L.; Ratain, M. J. J. Clin. Invest. 1998, 101, 847; (e) Ando, Y.;
Saka, H.; Asai, G.; Sugiura, S.; Shimokata, K.; Kamataki, T. Ann. Oncol. 1998,
9, 845; (f) Schellens, J. H.; Maliepaard, M.; Scheper, R. J.; Scheffer, G. L.;
Jonker, J. W.; Smit, J. W.; Beijnen, J. H.; Schinkel, A. H. Ann. N.Y. Acad. Sci.
2000, 922, 188.
3. Niizuma, S.; Tsukazaki, M.; Suda, H.; Murata, T.; Ohwada, J.; Ozawa, S.; Fukuda,
H.; Murasaki, C.; Kohchi, M.; Morikami, K.; Yoshinari, K.; Endo, M.; Ura, M.;
Tanimura, H.; Miyazaki, Y.; Takasuka, T.; Kawashima, A.; Namba, E.; Nakano, K,;
Ogawa, K.; Kobayashi, K.; Okabe, H.; Umeda, I.; Shimma, N.; Bioorg. Med. Chem.
Lett. 2009, 19, 2018.
4. Ishitsuka, H.; Okabe, H.; Umeda, I.; Tsukuda, T.; Shimma, N.; 2003,
WO2003043631.
5. Englebretsen, D. R.; Robillard, G. T. Tetrahedron 1999, 55, 6623.
6. Spectral data of 5c: 1H NMR (270 MHz, CD3OD) d ppm 4.00 (2H, s), 3.93 (2H, s),
2.96 (3H, s); Spectral data of 7a0: 1H NMR (500 MHz, CD3OD) d ppm 3.22 (4H, m),
3.02 (3H, m), MS m/z 136 (M+).
7. Endo, M.; Miwa, M.; Ura, M.; Tanimura, H.; Taniguchi, K.; Miyazaki, Y.; Ohwada,
J.; Tsukazaki, M.; Niizuma, S.; Murata, T.; Ozawa, S.; Ogawa, K.; Nanba, E.; Nagao,
S.; Shimma, N.; Yamada-Okabe, H.; Cancer Chemother. Pharmacol., submitted for
publication.
References and notes
1. (a) Kawato, Y.; Aonuma, M.; Hirota, Y.; Kuga, H.; Sato, K. Cancer Res. 1991, 51, 4187;
(b) 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. J. Med. Chem. 1991, 34,
98; (c) Mitsui, I.; Kumazawa, E.; Hirota, Y.; Aonuma, M.; Sugimori, M.; Ohsuki, S.;
Uoto, K.; Ejima, A.; Terasawa, H.; Sato, K. Jpn. J. Cancer Res. 1995, 86, 776.