Phosphate ester derivatives of homocamptothecin: Synthesis, solution stabilities and antitumor activities

Article abstract of DOI:10.1016/j.bmc.2010.03.039

Homocamptothecins (hCPTs) represents a new promising class of topoisomerase I inhibitors with enhanced stability and superior antitumor activity. Some phosphodiesters and phosphotriesters homocamptothecin derivatives were designed and synthesized based on our previous synthetic route. The cytotoxicity in vitro on three cancer cell lines and antitumor activity in vivo, and inhibitory properties of topoisomerase I of these derivatives were evaluated. Among them compounds 24e and 24f exhibited higher cytotoxic activity than IRT and the former exhibited the best antitumor activity in vivo and solution stability both at pH 7.4 and pH 3.0.

Full text of DOI:10.1016/j.bmc.2010.03.039

Bioorganic & Medicinal Chemistry 18 (2010) 3140–3146
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Phosphate ester derivatives of homocamptothecin: Synthesis, solution
stabilities and antitumor activities
a
Zhenyuan Miao a, , Jing Zhang a, , Liang You b, , Juan Wang c, Chunquan Sheng a, , Jiangzhong Yao ,
*
a
a
a
a
a
a
Wannian Zhang a, , Hao Feng , Wei Guo , Lei Zhou , Wenfeng Liu , Linjian Zhu , Pengfei Cheng ,
*
Xiaoying Che a, Wenya Wang a, Chuan Luo a, Yulan Xu a, Guoqiang Dong a
a School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
b Department of Pharmacy, Naval general hospital of PLA, 6 Fucheng Road, Beijing 100037, People’s Republic of China
c Department of Chemistry, Fudan University, 220 Handan Road, Shanghai 200433, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Homocamptothecins (hCPTs) represents a new promising class of topoisomerase I inhibitors with
enhanced stability and superior antitumor activity. Some phosphodiesters and phosphotriesters homo-
camptothecin derivatives were designed and synthesized based on our previous synthetic route. The
cytotoxicity in vitro on three cancer cell lines and antitumor activity in vivo, and inhibitory properties
of topoisomerase I of these derivatives were evaluated. Among them compounds 24e and 24f exhibited
higher cytotoxic activity than IRT and the former exhibited the best antitumor activity in vivo and solu-
tion stability both at pH 7.4 and pH 3.0.
Received 10 February 2010
Revised 15 March 2010
Accepted 16 March 2010
Available online 20 March 2010
Keywords:
Homocamptothecins
Antitumor activity
Topoisomerase I
Phosphate
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
9,10-difluoro analog (diflomotecan, 5) is one of the most potent
topoisomerase I inhibitors and was the first homocamptothecin to
20(S)-Camptothecin(CPT, 1, Fig. 1), is a naturally occurring quin-
oline alkaloid from Camptotheca acuminata exhibiting an excellent
antineoplastic activity against a broad spectrum of tumors.1,2 The
moleculartargetof CPTisDNAtopoisomeraseI(TopoI), anuclearen-
zyme that catalyzes the relaxation of supercoiled DNA during DNA
replication. It was found that CPT does not bind to Topo I or to DNA
alone but forms a three-component complex by binding to a cova-
lent DNA–Topo I complex, and thus inhibits DNA relaxation.3–5 The
structure–activity relationship (SAR) studies on CPT derivatives led
to the approval of topotecan (Hycamtin, 2) and irinotecan (Campto-
sar, 3) for ovarian and colon cancers treatment, respectively, and to
the synthesis of several novel CPT derivatives that are currently in
various stages of clinical trials.6–11
Recentstudieshaveshownthatexpansionof thecamptothecin E-
ring to a seven- membered system enhances the solution stability of
the agent and exhibits better biological properties compared to
CPT.12 This new family of very active seven-member hydroxy lac-
tone derivatives represent a new promising class of topoisomerase
I inhibitors. Among these homocamptothecin(hCPT, 4) derivatives,
be selected for clinical trials.13
Our previous work has reported a new synthetic method to form
7-substituted homocamptothecin analogs by couples ring A with
ring C, D, and E. These hCPT compounds exhibited potent antitumor
activity in vitro superior to topotecan.14 But for most compounds,
their in vivo activity does not correlate well with their in vitro data
because of their poor water solubility. In order to obtain the homo-
camptothecin derivatives with the excellent in vivo activity we
designed and synthesized phosphate derivatives of 7-methyl-10-
substituted homocamptothecin and explored whether this modifi-
cation improves the biological properties and solution stability.
2. Results and discussion
2.1. Chemistry
The target compounds could be disconnected to ring A and ring
CDE based on our reported synthetic method and preparation of
these compound are illustrated in Schemes 1–3.The key intermedi-
ate CDE ring 19 was synthesized according to the reported route14
and the synthetic process was optimized in our previous studies.
First, six-member hydroxy lactone 15 was obtained from acetone
and ethyl oxalate by 10-step reactions according to our reported
synthetic route.15–17 Then compound 15 was converted to tricyclic
* Corresponding authors. Tel./fax: +86 21 81871243 (C.S.).
E-mail addresses: shengcq@hotmail.com (C. Sheng), zhangwnk@hotmail.com
(W. Zhang).
These three authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.039

Z. Miao et al. / Bioorg. Med. Chem. 18 (2010) 3140–3146
3141
N
N
9
7
HO
O
10
O
N
O
O
A
N
B
C
N
N
D
N
N
O
N
O
E
O
20
O
OH
O
OH
O
OH
O
3
1
2
9
7
F
F
O
O
10
N
A
N
B
N
C
N
D
O
O
E
20
O
O
OH
OH
5
4
Figure 1. Structure of camptothecin and homocamptothecin derivatives.
HO
O
CH3
CH3
O
O
a
e
b-d
+
CH3COCH3
(COOC2H5)2
CH3COCH2COCO2C2H5
N
N
CN
CN
6
7
8
O
O
O
C2H5
O
O
O
O
O
f
O
CH3
CH2COOC2H5
CN
g
CHCOOC2H5
h
i
N
N
N
CN
O
CN
O
11
9
10
C2H5
C2H5
C2H5
O
O
O
N
j
O
O
l
CHCOOC2H5
CHCOOC2H5
k
O
CHCOOC2H5
N
N
CH2NCOCH3
NO
CH2OCOCH3
CH2NHCOCH3
O
13
O
14
O
12
O
O
O
O
OH
O
O
OH
O
OH
O
O
O
n
o
OH
O
m
O
O
N
OCHO
N
OCHO
N
N
O
O
O
O
O
15
16
17
18
OH
O
O
p
O
N
O
19
Scheme 1. Reagents and conditions: (a)Na/CH3OH, rt, 5 h; (b) triethyl orthoformate, p-TSA, 50 °C, 70 min; (c) cyanoacetamide, K2CO3, DMF, 60 °C, 16 h; (d) methyl acrylate,
K2CO3, DMF, 55 °C, 72 h; (e) HCl, CH3COOH, refluxing, N2, 3 h; (f) OHCH2CH2OH, TMSCl, rt, 72 h; (g) diethyl carbonate, t-BuOK, DMF, refluxing, N2, 3 h; (h)CH2CH3I, t-BuOK,
DMF/TMF, À10 °C, 3 h; (i) Raney Ni, H2, Ac2O/HOAc, 50 °C, 12 h; (j) NaNO2, 0 °C, 12 h; (k) CCl4, refluxing, 10 h; (l) Raney Ni, CuCl, rt, 4 h, then H2SO4, rt, 0.5 h; (m) KBH4,
CH3OH, rt, 20 min; (n) CH3COOH, NaIO4, rt, 0.5 h; (o) Zn, BrCH2COOtBu, THF, 75 °C, 3 h; (p) CF3COOH, rt, 10 h.

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Z. Miao et al. / Bioorg. Med. Chem. 18 (2010) 3140–3146
O
O
O
HO
HO
HO
b
a
NH2
NO2
22
21
20
Scheme 2. Reagents and conditions: (a) KNO3, HOAc, 60 °C, 12 h; (b) Pd/C, H2, 35 °C, 8 h.
O
N
O
HO
a
O
O
HO
N
+
O
N
O
NH2
O
OH
O
OH
22
19
23
O
P
O
O
b
R'O
OR
N
N
O
O
OH
24
24a: R=H,R'=Et
24b: R=H,R'=C6H5CH2
24c: R=Et,R'=Et
24d: R=iso-Pr,R'=iso-Pr
24e: R=Bu,R'=Bu
24f: R=pentyl,R'=pentyl
24g: R=cetyl,R'=cetyl
24h: R=CF3CH2,R'=CF3CH2
Scheme 3. Reagents and conditions: (a) toluene, p-TSA, 120 °C, 4 h; (b) POCl3, pyridine, Et3N, a series of alcohols, 0 °C, 1.5 h.
intermediate 19 by reduction, oxidation, Reformatsky and lacton-
ization reaction with a 67.1% yield (four steps).
(Scheme 4). The reaction efficiently yielded the phosphorylated
compounds 24c–h in excellent yield (Fig. 2). Especially, when
phosphorous tripentyl ester was used as phosphorylation agent,
the yield remarkably increased from 12.2% to 96.0%.
Then, in order to avoid using poisonous phosgene, acetic acid
and KNO3 were chosen for nitration reagents. Under this condition,
5-hydroxy-2-nitrohyacetophenone 21 was synthesized with a
33.7% isolated yield (Scheme 2). Hydrogenation of 21 in the pres-
ence of Pd/C afforded the amine 22 (Ring A).18
2.2. In vitro antitumor activities
Finally, Friedlander condensation of the tricyclic 19 with 22 in
the presence of p-toluenesulfonic acid gave 7-methy-10-hydroxy-
homocamptothecin 23, which was reacted with phosphoryl
chloride and different alcohols in pyridine to give the homocam-
ptothecin phosphodiesters 24a, 24b, and phosphotriesters 24c–h
purifying through silica gel column chromatography. These reac-
tions are shown in Scheme 3.
Furthermore, another synthesis method was applied to form
homocamptothecin phosphotriesters by treatment with phospho-
rous trialkyl esters and N,N-dimethylamino-pyridine (DMAP)
All the homocamptothecin analogs were assayed for cytotoxic-
ity against three different human tumor cell lines (A549, MDA-MB-
435, and LOVO) based on MTT assay. The data in Table 1 show that
the antitumor activity of all homocamptothecin phosphates were
much reduced. These phosphates were moderate cytotoxic to all
three cell lines except compound 24g. Among them, compounds
24e and 24f exhibited higher cytotoxic activity against three can-
cer cell lines than IRT. There is a decrease in potency against cancer
cells for compound 24a and 24b indicating that the cytotoxicity of
the phosphodiesters was lower than that of the phosphotriesters.
O
O
HO
O
O
P
a)
R'O
OR
N
N
N
N
O
O
O
O
OH
OH
23
24c-24h
Scheme 4. Reagents and conditions: (a) I2, phosphorous trialkyl esters, DMAP, CH2Cl2, 0–25 °C, 1.5 h.

Z. Miao et al. / Bioorg. Med. Chem. 18 (2010) 3140–3146
3143
Figure 2. Comparison of two synthetic methods of homocamptothecin phos-
photriesters.
Table 1
The in vitro antitumor activity of the target compounds
Compound
IC50 (lM)
A549
MDA-MB-435
LOVO
23
24a
24b
24c
24d
24e
24f
24g
0.03
11.40
29.35
8.05
11.39
2.84
<0.001
5.26
10.77
0.79
3.40
0.51
0.72
>100
0.22
<0.001
1.14
0.01
7.24
9.31
1.26
5.56
0.91
1.19
>100
5.42
0.02
4.99
2.30
>100
15.51
0.04
24h
Topotecan
Irinotecan
Figure 3. Tumor growth and inhibition of compounds 24e (a) and 24f (b) against
A549 xenografts in nude mice administered ip.
4.61
2.5. Solution stability
The SAR analysis of the synthesized compounds revealed that the
alkyl-chain length of phosphates was important to in vitro cytoac-
tivity. For example, the antitumor activity of compound 24e with
the butyl chain was far more than that of compound 24g with
the hexadecyl chain.
Thepreviouslyreportedlactonestabilitystudy inbuffer solutions
showed the homocamptothecin to be considerably more stable than
natural camptothecin product. In this article the phosphotriester
stability of compounds 24e and 24f were determined by modified
HPLC method.19 The results showed that homocamptothecin phos-
photriester derivatives were considerably stable both at pH 3.0
and pH 7.4. For example, after 24 h at pH 3.0, 100% of compounds
24e and 24f remained in phosphate form (some data not shown).
Furthermore, after 24 h at pH 7.4, more than 95% of 24e also re-
mained in the phosphate form while only 91% of compound 24f re-
mained (Fig. 5).
2.3. In vivo antitumor activities
Compound 24e and compound 24f were selected for evaluation
in 5 week tumor growth inhibition in vivo assays using nude mice
implanted with Lewis lung xenografts (Fig. 3). Irinotecan, a clinically
relevant camptothecin, was chosen as a reference drug. Treatment
with irinotecan at 50 mg/kg body weight, however, suppressed tu-
mor growth by 51% as compared with placebo-treated controls.
For compounds 24e and 24f, tumor growth was suppressed by 59%
(25 mg/kg group) and 42% (50 mg/kg group), respectively. In addi-
tion, tumor regression was seen in a dose dependent manner for
compound 24e and compound 24f. For example, the tumor growth
inhibition rate of compound 24e increased from 25% to 59% when
the dose was increased from 6.25 mg/kg to 25 mg/kg.
3. Conclusion
In summary, the data presented here demonstrate that most
phosphotriester derivatives possess potent cytotoxic activity
in vitro and compound 24e has more tumor inhibitory activity than
IRT in the A549 xenograft model. The homocamptothecin phos-
photriester derivative 24e and 24f as potent topoisomerase I inhib-
itors are stable not only at pH 7.4 but also at pH 3.0 as well. These
results make phosphates a new class of homocamptothecin deriv-
atives worthy of further investigation.
2.4. Topoisomerase I-mediated DNA cleavage
The ability of selected homocamptothecin derivatives 23 and
24e to inhibit topoisomerase I was investigated in the cleavable
complex assay. The results indicated that compounds 23 as cyto-
toxic agents were higher potent than CPT (Fig. 4). For example, in-
hibit activity was observed for compound 23 at concentration of
4. Experimental
4.1. General
1
l
M while CPT showed no inhibit activity at the same concentra-
Melting points were measured on an electrically heated XT4A
instrument and are uncorrected. The 1H spectra were recorded at
500 MHz with a Bruker instrument, and reported with TMS as
internal standard and CDCl3 or DMSO-d6 as solvents. Chemical
shifts (d values) and coupling constants (J values) are given in
tion. Furthermore, the increasing concentration of 23 was accom-
panied by a dose-dependent increase in the level of cleavable
complex. Compound 24e also showed potent activity in the DNA
cleavage assay at concentration of 100 lM.

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Z. Miao et al. / Bioorg. Med. Chem. 18 (2010) 3140–3146
CPT
23
24e
DNA Topo I
Figure 4. Effect of compounds 23 and 24e on Topoisomerase I-mediated DNA relaxation. Lane DNA, supercoiled DNA pBR322; Lane Topo I, supercoiled DNA and Topo I. The
samples were reacted with 1, 10, and 50 M drug at 37 °C for 15 min.
l
continued for an additional 4 h. The solution was allowed to cool
to room temperature and the solid was filtered off and washed by
acetone (20 mL) and methanol (20 mL) to give 7-methy-10-
hydroxyhomocamptothecin 23 as yellow solid (0.5 g, 70.9%). mp
>300 °C; 1H NMR (500 MHz, DMSO-d6, dppm): 0.87 (t, 3H,
J = 7.4 Hz), 1.86 (q, 2H, J = 7.4 Hz), 2.76 (s, 3H), 3.06–3.49 (q, 2H,
J = 13.7 Hz), 5.28 (s, 2H), 5.39–5.53 (q, 2H, J = 15.1 Hz), 6.03 (s, 1H),
7.42 (s, 1H), 7.72 (d, 1H, J = 6.6 Hz), 7.87 (s, 1H), 8.15 (d, 1H,
J = 9.2 Hz), 10.32 (s, 1H); 13C NMR (DMSO-d6): 8.60 (1 C), 15.57 (1
C), 36.69 (1 C), 42.92 (1 C), 50.51 (1 C), 61.77 (1 C), 73.49 (1 C),
100.44 (1 C), 111.89 (1 C), 114.39 (1 C), 122.52 (1 C), 128.54 (1 C),
128.79 (1 C), 129.33 (1 C), 139.33 (1 C), 140.20 (1 C), 145.43 (1 C),
149.86 (1 C), 154.32 (1 C), 156.11 (1 C), 159.47 (1 C), 172.27 (1 C);
ESI-MS, m/z: 391.3 [MÀH]À. Anal. Calcd for C22H20N2O5: C, 67.34;
H, 5.14; N, 7.14. Found: C, 67.17; H, 5.15; N, 7.15.
Figure 5. Solution stability of compounds 24e at 37 °C. Each data point represents
the percent of initial concentration of phosphate at pH 3.0 and pH 7.4, respectively.
4.4. General procedure for the preparation of phosphoric acid
diester and triester 7-methylhomocamptothecin-10-yl ester
(24a–h)
ppm and Hz, respectively. ESI mass spectra were performed on an
API-3000 LC–MS spectrometer. TLC analysis was carried out on sil-
ica gel plates GF254 (Qindao Haiyang Chemical, China). Flash col-
umn chromatography was carried out on silica gel 300–400
mesh. Anhydrous solvent and reagents were all analytical pure
and dried through routine protocols.
Toasolutionof7-methy-10-hydroxyhomocamptothecin(39.2 mg,
0.1 mmol) in pyridine (16 mL), POCl3 (0.1 mL, 1.1 mmol) was added
dropwise while held in an ice bath with stirring. The stirring was con-
tinued at ambient temperature for 1.5 h and alcohols or phenol
(10.0 mmol) were added. The mixture was stirred for an additional
1.5 h and water (0.1 mL) was added, the resulted mixture was stirred
at ambient temperature for 1 h and then evaporated to dryness. The
residue was purified by chromatography over silica gel (5% MeOH/
CH2Cl2) to afford diester and triester.
4.2. Preparation of 1-(2-amino-5-hydroxyphenyl)ethanone (22)
4.2.1. Preparation of 1-(5-hydroxy-2-nitrophenyl)ethanone (21)
KNO3 (11.1 g, 0.1 mol) was added to a solution of 3-hydroxyphe-
nylethanone 20 (13.6 g, 0.1 mol) and acetic acid (100 mL) at ambient
temperature. The mixture was stirred at 60 °C for 12 h and poured
into ice-water. Then the solution was extracted with dichlorometh-
ane (3 Â 100 mL). The combined dichloromethane extracts were
dried over sodium sulfate and concentrated under reduced pressure
giving crude material. Recrystallisation from ethyl acetate gave pale
yellow needles 3 (6.1 g, 33.7%), mp 146–147 °C.
4.4.1. Phosphoric acid ethyl ester 7-methylhomocamptothecin-
10-yl ester (24a)
Yellow solid, mp >300 °C. 1H NMR (500 MHz, DMSO-d6, dppm):
0.87 (t, 3H, J = 7.1 Hz), 1.11–1.40 (m, 2H), 1.85 (q, 2H, J = 7.5 Hz),
2.67 (s, 3H), 3.14–3.52 (q, 2H ,J = 14.3 Hz), 3.85 (q, 2H, J = 7.1 Hz),
5.20 (s,2H), 5.41–5.52 (q, 2H, J = 15.1 Hz), 6.08 (s, 1H), 7.32 (s,
1H), 7.72 (d, 1H, J = 6.5 Hz), 7.87 (s, 1H), 8.03 (d, 1H, J = 9.0 Hz);
MS-ESI m/z: 499.6 [MÀ]À.
4.2.2. Preparation of 1-(2-amino-5-hydroxyphenyl)ethanone (22)
A suspension of compound 21 (1 g, 5.5 mmol) and Pd/C (0.1 g) in
ethanol (30 mL) was degassed and refilled with N2 for three times.
Then the mixture was stirred under 20 psi of H2 at 35 °C for 8 h.
The catalyst was filtered and the solvent was removed by evapora-
tion and yellow powder 22 (0.7 g, 83.9%) was obtained used directly
in the next stage, mp 162–165 °C. 1H NMR (500 MHz, DMSO-d6,
dppm): 2.50 (s, 3H), 6.12 (s, 2H), 6.62 (d, 1H, J = 8.8 Hz), 6.82 (d, 1H,
J = 8.8 Hz), 7.07 (s, 1H), 8.66 (s, 1H); ESI-MS, m/z: 152.1 (M+H)+.
4.4.2. Phosphoric acid benzyl ester 7-methylhomocamptothe-
cin-10-yl ester (24b)
Yellow solid, mp >300 °C. 1H NMR (500 MHz, DMSO-d6, dppm):
0.87 (t, 3H, J = 7.0 Hz), 1.30 (s, 2H), 1.84 (q, 2H, J = 7.4 Hz), 2.61
(s, 3H), 3.06–3.49 (q, 2H,J = 14.2 Hz), 5.20 (s, 2H), 5.40–5.51 (q,
2H, J = 15.0 Hz), 6.06 (s, 1H), 7.12–7.46 (m, 7H), 7.73 (s, 1H), 7.84
(s, 1H), 8.02 (s, 1H); MS-ESI m/z: 561.5 [MÀ]À.
4.4.3. Phosphoric acid diethyl ester 7-methylhomocamptothe-
cin-10-yl ester (24c)
4.3. Preparation of 7-methy-10-hydroxyhomocamptothecin (23)
Yellow solid, mp >300 °C. 1H NMR (500 MHz, DMSO-d6, dppm):
0.87 (t, 3H, J = 7.5 Hz), 1.29–1.32 (m, 6H), 1.87 (q, 2H, J = 7.3 Hz),
2.75 (s, 3H), 3.05–3.49 (q, 2H, J = 13.9 Hz), 4.21–4.27 (m, 4H), 5.30
(s, 2H), 5.39–5.55 (q, 2H, J = 15.1 Hz), 6.02 (s, 1H), 7.39 (s, 1H), 7.75
(d, 1H, J = 6.6 Hz), 7.80 (s, 1H), 8.21 (d, 1H, J = 9.2 Hz); MS-ESI m/z:
528.3 [MÀ]À; 31P NMR (500 MHz, DMSO-d6, dppm): À5.27.
A solution of 1-(2-amino-5-hydroxyphenyl)ethanone 22 (0.5 g,
3.3 mmol) and 9-ethyl-9-hydroxy-2,3,8,9-tetrahydro-5H-6-oxa-
3a-aza-cyclohepta[f]indene-1,4,7-trione 1914 (0.5 g, 1.8 mmol) in
toluene (500 mL) was refluxed using a Dean–Stark trap for 30 min.
p-Toluenesulfonic acid (0.1 g) was then added and refluxing was

Z. Miao et al. / Bioorg. Med. Chem. 18 (2010) 3140–3146
3145
4.4.4. Phosphoric acid diisopropyl ester 7-methylhomocampto-
thecin-10-yl ester (24d)
solution was allowed to warm up to room temperature and added
dropwise to a solution of 7-methy-10-hydroxyhomocamptothecin
(39.2 mg, 0.1 mmol) and DMAP (36.6 mg, 0.3 mmol) in dry CH2Cl2
(100 mL) at 0 °C. Then this solution was warmed up to room tem-
perature and was washed with water and brine. The organic layer
was dried over sodium sulfate and concentrated under reduced
pressure giving crude materials which were purified by chroma-
tography over silica gel (5% MeOH/CH2Cl2) to afford trimester with
the yield from 84.4% to 96.0%.
Yellow solid, mp >300 °C. 1H NMR (500 MHz, DMSO-d6, dppm):
0.87 (t, 3H, J = 7.4 Hz), 1.21–1.34 (m, 12H), 1.86 (q, 2H, J = 7.3 Hz),
2.74 (s, 3H), 3.04–3.48 (q, 2H, J = 13.5 Hz), 4.73–4.77 (m, 4H), 5.30
(s, 2H), 5.37–5.53 (q, 2H, J = 15.0 Hz), 6.03 (s, 1H), 7.38 (s, 1H), 7.75
(d, 1H, J = 6.3 Hz), 7.97 (s, 1H), 8.20 (d, 1H, J = 9.0 Hz); MS-ESI m/z:
555.5 [MÀ]À.
4.4.5. Phosphoric acid dibutyl ester 7-methylhomocamptothe-
cin-10-yl ester (24e)
4.6. DNA topoisomerase I activity assays
Yellow solid, mp >300 °C. 1H NMR (500 MHz, DMSO-d6, dppm):
0.86–0.89 (m, 6H), 0.87 (t, 3H, J = 7.3 Hz), 1.34–1.38 (m, 4H), 1.87
(q, 2H, J = 7.4 Hz), 2.75 (s, 3H), 3.04–3.49 (q, 2H, J = 14.1 Hz), 4.16–
4.20 (m, 4H), 5.30 (s, 2H), 5.39–5.55 (q, 2H, J = 15.0 Hz), 6.03 (s,
1H), 7.39 (s, 1H), 7.75 (d, 1H, J = 6.5 Hz), 7.98 (s, 1H), 8.20 (d, 1H,
J = 9.3 Hz); 13C NMR (DMSO-d6): 8.47 (1C), 13.80 (2C), 15.66 (1C),
18.62 (2C), 32.13 (2C), 36.77 (1C), 42.84 (1C), 50.51 (1C), 61.77
(1C), 68.43 (2C), 73.24 (1C), 100.00 (1C), 120.19 (1C), 121.65 (1C),
123.04 (1C), 127.37 (1C), 129.45 (1C), 129.96 (1C), 140.81 (1C),
141.48 (1C), 145.24 (1C), 147.13 (1C), 152.05 (1C), 156.14 (1C),
159.41 (1C), 172.20 (1C); 31P NMR (500 MHz, DMSO-d6, dppm):
À5.38; MS-ESI m/z: 583.4 [MÀ]À. Anal. Calcd for C30H37N2O8 P: C,
61.64; H, 6.38; N, 4.79. Found: C, 61.49; H, 6.39; N, 4.80.
Camptothecin was obtained from the company of Tianzunzez-
hong in China. Topo I (calf thymus), buffer, BSA, loading buffer
and supercoiled DNA pBR322 were all from TaKaRa Biotechnology
Co., Ltd.
All reactions were carried out in 20
distilled water, 2 L DNA TopoI buffer, 2
0.25 g supercoiled DNA, 0.5 U Topo I with or without drug. The
lL volumes (16
lL double
l
lL 0.1% BSA) including
l
reaction was incubated at 37 °C for 15 min. Reactions were stopped
by adding SDS (0.5% final concentration). To the reaction mixtures,
3.5
l
L 6Â loading buffer (0.1 mM EDTA, 7% Glycerol, 0.01% Xylene
Cyanol FF, Bromopenol Blue 0.01%) was added. The mixtures were
electrophoresed in 0.8% agarose gel in TAE buffer for 40 min at
120 V. The gel was stained with ethidium bromide at room tem-
perature and photographed with UV transilluminator.
4.4.6. Phosphoric acid dipentyl ester 7-methylhomocamptothe-
cin-10-yl ester (24f)
Yellow solid, mp >300 °C. 1H NMR (500 MHz, DMSO-d6, dppm):
0.81–0.85 (m, 6H), 0.87 (t, 3H, J = 7.4 Hz), 1.25–1.32 (m, 8H),
1.62–1.66 (m, 4H), 1.86 (q, 2H, J = 7.5 Hz), 2.74 (s, 3H), 3.04–3.49
(q, 2H, J = 13.8 Hz), 4.16–4.20 (m, 4H), 5.30 (s, 2H), 5.39–5.55 (q,
2H, J = 15.1 Hz), 6.03 (s, 1H), 7.39 (s, 1H), 7.75 (d, 1H, J = 6.6 Hz),
7.98 (s, 1H), 8.20 (d, 1H, J = 9.2 Hz); 13C NMR (DMSO-d6): 8.58 (1
C), 14.17 (2C), 15.33 (1C), 22.00 (2C), 27.49 (2C), 29.78 (2C),
36.65 (1C), 42.77 (1C), 50.48 (1C), 61.71 (1C), 68.83 (2C), 73.50
(1C), 99.92 (1C), 113.41 (1C), 122.84 (1C), 124.18 (1C), 128.63
(1C), 129.84 (1C), 132.10 (1C), 140.02 (1C), 145.20 (1C), 146.15
(1C), 149.24 (1C), 151.99 (1C), 156.21 (1C), 159.42 (1C), 172.20
(1C); 31P NMR (500 MHz, DMSO-d6, dppm): À5.58; MS-ESI m/z:
611.8 [MÀ]À. Anal. Calcd for C32H41N2O8 P: C, 62.73; H, 6.75; N,
4.57. Found: C, 61.55; H, 6.76; N, 4.60.
4.7. Cytotoxicity
Cytotoxicity assays are performed on human lung cancer
(A549), breast cancer (MDA-MB-435) and colon cancer (LOVO) cell
lines. Cells (6000–10,000) in 100 lL culture medium per well were
seeded into 96-well microtest plates (Falcon, CA). Cells were trea-
ted in triplicate with gradient concentration of test drugs and incu-
bated at 37 °C for 72 h. For three cell lines, the microculture
tetrazolium [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide, MTT; Sigma, St. Louis, MO] assay was performed to
measure the cytotoxic effects. The drug concentration required
for 50% growth inhibition (IC50) of tumor cells was determined
from the dose–response curve.
4.8. In vivo antitumor activities
4.4.7. Phosphoric acid dicetyl ester 7-methylhomocamptothe-
cin-10-yl ester (24g)
The in vivo antitumor activity of compounds 24e and 24f was
evaluated and IRT was used as reference drug. BALB/C nude male
mice (Certificate SCXK2003-0003, weighing 18–20 g) were ob-
tained from Shanghai Experimental Animal Center, Chinese Acad-
emy of Sciences. A549 lung cancer cell suspensions were
implanted subcutaneously into the right axilla region of mice.
Treatment was begun when implanted tumors had reached a vol-
ume of about 100–300 mm3 (after 17 days). The animals were ran-
domized into appropriate groups (six animals/treatment and eight
animals for the control group) and administered by ip injection
once on day 17 after implantation of cells. Tumor volumes were
monitored by caliper measurement of the length and width and
calculated using the formula of TV = 1/2  a  b2, where a is the tu-
mor length and b is the width. Tumor volumes and body weights
were monitored every 4 days over the course of treatment. Mice
were sacrificed on day 35 after implantation of cells and tumors
were removed and recorded for analysis.
Yellow solid, mp >300 °C. 1H NMR (500 MHz, DMSO-d6, dppm):
0.83–0.88 (m, 6H), 0.87 (t, 3H, J = 7.3 Hz), 1.15–1.27 (m, 52H),
1.62–1.63 (m, 4H), 1.85 (q, 2H,J = 7.2 Hz), 2.75 (s, 3H), 3.05–3.49
(q, 2H, J = 13.9 Hz), 4.16–4.18 (m, 4H), 5.29 (s, 2H), 5.40–5.55 (q,
2H, J = 15.0 Hz), 6.03 (s, 1H), 7.39 (s, 1H), 7.74 (d, 1H, J = 6.2 Hz),
7.97 (s, 1H), 8.20 (d, 1H, J = 9.1 Hz); MS-ESI m/z: 611.8 [MÀ]À.
4.4.8. Phosphoric acid di(2,2,2-trifluoro-ethyl) ester 7-methyl-
homocamptothecin-10-yl ester (24h)
Yellow solid, mp >300 °C. 1H NMR (500 MHz, DMSO-d6, dppm):
0.86 (t, 3H, J = 7.3 Hz), 1.86 (q, 2H, J = 7.4 Hz), 2.75 (s, 3H), 3.05–
3.50 (q, 2H, J = 13.9 Hz), 4.97–5.04 (m, 4H), 5.29 (s, 2H), 5.40–
5.55 (q, 2H, J = 15.0 Hz), 6.03 (s, 1H), 7.39 (s, 1H), 7.74 (d, 1H,
J = 6.5 Hz), 7.97 (s, 1H), 8.20 (d, 1H, J = 9.0 Hz); MS-ESI m/z: 920.8
[MÀ]À; 31P NMR (500 MHz, DMSO-d6, dppm): À6.80.
4.5. General procedure for the preparation of phosphoric acid
triester 7-methylhomocamptothecin-10-yl ester with
phosphorous trialkyl esters (24c–f, 24h)
4.9. Solution stability
A modified HPLC method was adapted as follows for the determi-
Iodine (63.5 mg, 0.3 mmol) was added at 0 °C to a solution of
trialkylphosphite in dry CH2Cl2 (50 mL). After 10 min at 0 °C, the
nation of compounds 24e and 24f.19 To 70
tion in polystyrene tubes and preincubated at 37 °C for 5 min, was
l
L fractions of water solu-

3146
Z. Miao et al. / Bioorg. Med. Chem. 18 (2010) 3140–3146
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2 mM), and the samples were incubated at 37 °C. At defined times
the solution was analyzed and samples were run on a 5 m Nucleosil
C18 column (4.6 Â 250 mm) at 25 °C with a flow rate of 1 mL/min of
an isocratic eluent composed of methanol/water (60:40, v/v), and
the eluted analytes were detected at 254 nm.
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This research was supported in part by the National Natural
Science Foundation of China (No. 30872988), the Key Project of
Science and Technology of Shanghai (No. 09431901700), National
Major Special Project for the Creation of New Drugs (Grant No.
2009ZX09301-011) and Shanghai Leading Academic Discipline
Project (Project No. B906).
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