Synthesis and antitumor activity of novel substituted uracil-1′(N)-acetic acid ester derivatives of 20(S)-camptothecins

Article abstract of DOI:10.1016/j.ejmech.2016.11.013

A series of novel substituted uracil-1′(N)-acetic acid esters (6–20) of camptothecins (CPTs) were synthesized by the acylation method. These new compounds were evaluated for in vitro antitumor activity against tumor cell lines, A549, Bel7402, BGC-823, HCT-8 and A2780. In vitro results showed that most of the derivatives exhibited comparable or superior cytotoxicity compare to CPT (1) and topotecan (TPT, 2), with 12 and 13 possessing the best efficacy. Four compounds, 9, 12, 13 and 16, were selected to be evaluated for in vivo antitumor activity against H₂₂, BGC-823 and Bel-7402 in mice. In vivo testing results indicated that 12 and 13 had antitumor activity against mouse liver carcinoma H₂₂close to Paclitaxel and cyclophosphamide. 12 had similar antitumor activity against human gastric carcinoma BGC-823 in nude mice compared to irinotecan (3) and possessed better antitumor activity against human hepatocarcinoma Bel-7402 in nude mice than 2. It is also discovered that 12 showed a similar mechanism but better inhibitory activity on topoisomerase I (Topo I) compared to 2. These findings indicate that 20(S)-O-fluorouracil-1′(N)-acetic acid ester derivative of CPTs, 12, could be developed as an antitumor drug candidate for clinical trial.

Full text of DOI:10.1016/j.ejmech.2016.11.013

Accepted Manuscript
Synthesis and antitumor activity of novel substituted uracil-1′(N)-acetic acid ester
derivatives of 20(S)-camptothecins
Di-Zao Li, Qiang-Zhe Zhang, Cun-Ying Wang, Yan-Ling Zhang, Xing-Yu Li, Ji-Tao
Huang, Hong-Yan Liu, Zhao-Di Fu, Hua-Xian Song, Jin-Ping Lin, Teng-Fei Ji, Xian-
Dao Pan
PII:
S0223-5234(16)30951-5
10.1016/j.ejmech.2016.11.013
EJMECH 9046
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 August 2016
Revised Date: 30 October 2016
Accepted Date: 6 November 2016
Please cite this article as: D.-Z. Li, Q.-Z. Zhang, C.-Y. Wang, Y.-L. Zhang, X.-Y. Li, J.-T. Huang, H.-
Y. Liu, Z.-D. Fu, H.-X. Song, J.-P. Lin, T.-F. Ji, X.-D. Pan, Synthesis and antitumor activity of novel
substituted uracil-1′(N)-acetic acid ester derivatives of 20(S)-camptothecins, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.013.
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ACCEPTED MANUSCRIPT
control
Irinotecan (50mg/kg, ip, qwk) (P < 0.001)
L090811-2B (30mg/kg, ip, qwk) (P < 0.001)
L090811-2B (60mg/kg, ip, qwk) (P < 0.001)
3
3
500
000
O
N
N
2500
F
O
2
1
000
500
O
N
O
O
O
N
H
O
1000
5
00
0
12 (L090811-2B)
0
3
6
9
12
15
18
Days
1

ACCEPTED MANUSCRIPT
Synthesis and antitumor activity of novel substituted uracil-1'(N)-acetic acid ester
derivatives of 20(S)-camptothecins
a,b,1
a,1
c, 1
d, 1
a
a
Di-Zao Li , Qiang-Zhe Zhang , Cun-Ying Wang ,Yan-Ling Zhang , Xing-Yu Li , Ji-Tao Huang , Hong-Yan
b
b
e
e
b
b
Liu , Zhao-Di Fu , Hua-Xian Song , Jin-Ping Lin , Teng-Fei Ji* , Xian-Dao Pan*
a: College of Pharmacy and State key Laboratory of Medicinal Chemical Biology, State Key Laboratory of Elemento-Organic Chemistry and
Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, P. R. China;
b: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, P. R. China;
c: Xu Zhou College of Industrial Technology, Xuzhou 221000, China;
d: School of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot, Inner Mongolia, 010021, P. R. China;
e: Beijing Landan Pharm, Beijing 100176, P. R. China.
*
Corresponding Authors
E-mail addresses: xdp@imm.ac.cn (X. P.); jitf@imm.ac.cn (T. J.).
1
These authors contributed equally to this work.
ABSTRACT:
A series of novel substituted uracil-1'(N)-acetic acid esters (6–20) of camptothecins (CPTs) were synthesized by the acylation method. These new
compounds were evaluated for in vitro antitumor activity against tumor cell lines, A549, Bel7402, BGC-823, HCT-8 and A2780. In vitro results
showed that most of the derivatives exhibited comparable or superior cytotoxicity compare to CPT (1) and topotecan (TPT, 2), with 12 and 13
possessing the best efficacy. Four compounds, 9, 12, 13 and 16, were selected to be evaluated for in vivo antitumor activity against H22
,
BGC-823 and Bel-7402 in mice. In vivo testing results indicated that 12 and 13 had antitumor activity against mouse liver carcinoma H22 close to
Paclitaxel and cyclophosphamide. 12 had similar antitumor activity against human gastric carcinoma BGC-823 in nude mice compared to
irinotecan (3) and possessed better antitumor activity against human hepatocarcinoma Bel-7402 in nude mice than 3. It is also discovered that 12
showed a similar mechanism but better inhibitory activity on topoisomerase I (Topo I) compared to 2. These findings indicate that
2
0(S)-O-fluorouracil-1'(N)-acetic acid ester derivative of CPTs, 12, could be developed as an antitumor drug candidate for clinical trial.
Keywords: Camptothecin; Synthesis; Substituted uracil-1'(N)-acetic acid; Antitumor activity; the mechanism of antitumor activity
1. Introduction
1

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Camptothecin (CPT, 1, Figure 1) and its related analogues represent an important class of agents useful in the
treatment of cancer [1–4]. Structure-activity relationship (SAR) studies showed that the ring E lactone and the
natural 20S-configuration of camptothecins (CPTs) are essential for antitumor activity because of their
topoisomerase I (Topo I) inhibitory activity (Figure 2). The modification of CPT on its A- and B-rings, such as
topotecan (TPT, 2, Figure 1), irinotecan (3, Figure 1), rubitecan (9-nitrocamptothecin, 9-NO
2
-CPT, 4, Figure 1),
10-hydroxycamptothecin and 7-ethyl-10-boronic acid camptothecin as prodrug of SN-38, the active metabolite of 3,
the typical Topo I inhibitors, could effectively ablate high-affinity binding of the carboxylate to human serum
albumin (HSA), and improve antitumor activity of CPT [5–14].
Because of the stronger intramolecular hydrogen bonding in polar solvents, the 20(S)-hydroxyl group of CPT is
believed to increase the reaction rate of lactone hydrolysis of CPT at neutral pH by shifting the equilibrium from the
lactone form to the carboxylate form [15]. In this respect, esterification of 20-hydroxyl group is speculated to not
only eliminate the intramolecular hydrogen bonding but also increase the steric hindrance of carbonyl group of
E-ring [16]. Although 20-O-acylated CPTs possess no intrinsic Topo I inhibitory activity, they can be functioned as
prodrugs to release the CPTs in vivo, and improve solubility, pharmacokinetics character and safety of CPTs [17]. A
series of 20-O-acylated CPT derivatives, such as 20-O-glycinate [18], 20-O-alkanoate [19], 20-O-aroylate [20], and
20-O-PG-glycinate [21], were prepared to improve stability and solubility of lactone in CPT. Oxyalkanoic acid
esters and nitrogen-based esters of CPT were synthesized previously, and these esters showed significant antitumor
activity and low toxicity in vivo [ 16, 22]. Another series of nitrogen-based esters of CPT, 20(S)-sulfonylamidine
derivatives of 1, were investigated as potential anticancer agents directly and selectively inhibiting Topo I [23, 24].
These results validated our hypothesis on SAR of CPT [25–27]. Based on this SAR, we designed a series of novel
substituted uracil-1'(N)-acetic acid esters of CPTs to improve their antitumor activity. Herein substituted
uracil-1'(N)-acetic acid esters of CPTs as new compounds were prepared and evaluated for their in vitro and in vivo
antitumor activity.
2. Results and discussion
2.1. Chemistry
To study the antitumor activity of the different substituent groups on 5'-position of uracil-1'(N)-acetic acid esters
of CPTs, we synthesized a series of CPT derivatives (6–20) containing H, F, Cl, Br, and I group on 5'-position of
uracil. Preparation (Scheme 1) and purification of 1'(N)-acetic acid 5a–e were performed according to previously
published procedures [28]. CPTs' ester derivatives 6–20 were prepared in proper yields by the straightforward
acylation of CPTs like 1, 9-NO -CPT or 7-ethylcamptothecin (7-Et-CPT) with the corresponding substituted
2
uracil-1'(N)-acetic acid in the presence of a coupling agent 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide
hydrochloride (EDCI) and a catalyst 4-dimethylaminopyridine (DMAP) in dried pyridine at room temperature (r.t.)
[
29] (Scheme 1).
2.2. In Vitro Cytotoxicity Assay
2

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Cytotoxic activity of these CPTs-20-O-ester derivatives was evaluated against five human tumor cell lines,
A549, Bel7402, BGC-823, HCT-8, and A2780, by using the MTT assay (Table 1) according to the previous method
[
20, 30]. CPT and TPT were used as comparisons. CPT possessed excellent antitumor activities in vitro in
nanomolar concentration, and higher cytotoxic activity than TPT. These ester compounds showed comparable or
higher cytotoxic activity compared to TPT. Most of these compounds displayed less cytotoxicity compared to CPT.
The data exhibiting cytotoxicity of ester were related to that of its parent compound. The ester 6, 8, 9, 11, 12, 13, 14,
16, 17, 18, 19 and 20 had cytotoxic activity (IC50 0.002–0.032 ꢀM) higher than TPT (IC50 0.022–0.100 ꢀM) and
comparable to or even slightly better than CPT (IC50 0.014–0.057 ꢀM) on Bel7402, BGC-823 and HCT-8. The
cytotoxic activity of ester 11 and 16 on A2780, were similar to that of TPT.
The cytotoxicity of the CPT ester could be explained by the structure of heterocyclic acid of uracil and the
substituents on the CPT. The ester containing ethyl or nitro-group on the CPT and F, Cl, and Br on the 5-position of
uracil may strengthen the cytotoxicity, as 12, 13 and 16 possessed the best cytotoxicity. In contrast, the ester
containing iodine atom on the 5-position of uracil might weaken the cytotoxicity because 10 and 15 showed the
weakest cytotoxic activity in all compounds tested with IC50 values almost in micro-molar per liter concentration
level. That is perhaps because the steric hindrance or polarity of iodine is not conducive to form CPTs-DNA-Topo I
complex.
2.3. In Vivo Antitumor Activity Assays
Considering the above analysis of cytotoxicity and structures of the ester derivatives synthesized, four of these
compounds, 9, 12, 13 and 16, were selected to evaluate their in vivo antitumor activity in mice by intra peritoneal
injection, and the tumor model was mouse liver tumor H22 model.
In vivo preliminary evaluation of toxicity and antitumor activity of analogues 9, 12, 13 and 16 were performed
on KM (Kun Ming of China) mice (Table 2). The data revealed that compound 12 and 13 possessed higher tumor
inhibitory rate (TIR, 57.51% and 46.21%) against H22 transplanted mice tumor than that of 16 (11.0%) and 9
(6.39%), lower than that of paclitaxel (73.85%). However, all four esters tested showed lower toxicity in the dose of
40 mg/kg for twice than that of paclitaxel in the dose of 24 mg/kg for three times. In view of these facts, compounds
12 and 13 were further investigated for their doses to be used safely.
During the further investigation of in vivo antitumor activity with different doses (Table 3), both 12 and 13
showed a better dose-efficacy relationship. The in vivo antitumor activity results also showed that TIRs of 13 were
higher than that of 12 in same doses (58.68% vs. 39.12% in 100 mg/kg for once, 76.58% vs. 64.46% in 60 mg/kg
for twice) , but TIRs in 60mg/kg for twice were lower than that of cyclophosphamide (93.66%). Compound 13
possessed higher TIR, but its toxicity was higher than that of 12, with three out of ten mice died when treated by the
dose of 60 mg/kg twice. Therefore, the compound 12 was selected as the candidate compound for further
investigation of antitumor test.
Further evaluations of antitumor activity in vivo showed that 12 had almost same antitumor activity (TIR:
95.97%, T/C: 5.54%, 60mg/kg for twice without death of animals) against human gastric cancer BGC-823 in nude
mice compared to irinotecan (TIR: 94.10%, T/C: 5.48%) (Table 4, Table 5, and Figure 3). Ester 12 possessed
better antitumor activity and lower toxicity (TIR: 81.45%, T/C: 24.53%, 60 mg/kg for twice) against human liver
3

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carcinoma Bel-7402 in nude mice than that of TPT (TIR: 45.48%, T/C: 58.80%, 5 mg/kg for twice) (Table 6, 7, and
Figure 4).
2.4. Mechanism of Action Studies on Compound 12
Two separate assays were carried out to explore the mode of action of compound 12 and TPT. 12 was
preferentially tested for Topo I inhibitory activity using topotecan as a reference drug [31–33]. As shown in Figure
, compounds 12 exhibited strong Topo I inhibitory activity with an almost equal potency compared to TPT with a
5
concentration leading to an inhibition of Topo I from 30ꢀM. Because a typical characteristic of TPT is the ability to
induce cell cycle arrest at G2/M phase [23, 34], we assessed perturbation of the cell cycle on A549 cell using flow
cytometry. It is found that treatment of compound 12 at concentrations from 8 nM to 75 nM caused a marked
increase in the proportion of G2/M phase cells from around 10% to 71.7% (Figure 6.). Moreover, compound 12
showed better Topo I inhibitory effect than TPT.
To study the mechanism of antitumor activity, the apoptosis profile of A-549 human lung adenocarcinoma
epithelial cells treated by 12 was investigated. Firstly, morphological cellular changes of A-549 cells treated with
compound 12 at concentrations from 8 nM to 75 nM were studied. The treated cells showed apoptotic
morphological features, including cell shrinkage and membrane blebbing in concentration-dependent manners
(
Figure 7A). Next, the apoptosis by double staining with FITC-annexin V and propidium iodide was confirmed.
The result of flow cytometry indicated that 12 treatment at concentrations from 8 nM to 75 nM increased the
percentage of apoptotic cells from around 3% to 59% (Figure 7B). The protein of apoptosis signal pathway by
western blot was also studied, showing that anti-apoptotic protein Bcl-2 [35] was reduced, while apoptotic protein
Bax [36] and cleaved Caspase-3 with 12 treatment were enhanced at concentrations from 8 nM to 75 nM from the
proteins without 12 treatment (Figure 7C). These data demonstrated that 12 inhibited A-549 cell growth through
apoptosis induction.
3. Conclusion
Fifteen novel substituted uracil-1'(N)-acetic acid esters of camptothecins were prepared according to a known
method. The in vitro cytotoxicity was evaluated on five human cancer cell lines (A549, Bel7402, BGC-823, HCT-8,
and A2780) by using a MTT assay. In vivo antitumor activity showed that compound 12 possessed higher tumor
inhibitory activity and lower toxicity compared to TPT. The mechanism study revealed that ester 12 was a Topo I
inhibitor (the same as TPT). Compound 12 showed significantly higher in vivo antitumor activity and lower toxicity
compared with TPT, and similar TIR compared to irinotecan. Therefore, it is definitely warranted that compound 12
is a new class of Topo I inhibitor and antitumor clinical trial drug candidate of CPTs.
4. Experimental section
4.1. Chemistry
4

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2
All reactions requiring anhydrous conditions were performed under an Ar or N atmosphere. Chemicals and
solvents were either A.R. grade or purified by standard techniques. Flash column chromatography was carried out
on silica gel 300–400 mesh. Column chromatography was performed by using silica gel with eluent given in
parentheses. Thin layer chromatography (TLC) analyses were carried out on silica gel plates GF254 (Qingdao
Haiyang Chemical Co., Ltd.), compounds were visualized by irradiation with UV light. Melting points were
determined in Yanaco melting point apparatus and are uncorrected. The NMR and MS (ESI) spectra of these
compounds were consistent with their structures. NMR analyses were performed on a Bruker instrument AV 400
(
Bruker Company, USA) by using CDCl or DMSO-d as a solvent with tetramethylsilane (TMS) as reference at
3 6
1
room temperature. HNMR spectra were recorded at 400 MHz and chemical shifts (δ values) are reported in parts
per million (ppm) relative to either a TMS (δ = 0.00 ppm) internal standard or solvent signals and coupling
1
3
constants (J values) are given in Hz, respectively. CNMR spectra were recorded at 100 MHz and chemical shifts
δ values) are reported in ppm measured relative to the solvent peak. MS (ESI) spectra were recorded on Finnigan
(
Lcqadvantage instrument. High-resolution mass spectra (HRMS) were obtained with a VG ZAB-HS spectrometer.
The purity of compound 12 was analyzed compared with 7-Et-CPT by reverse-phase HPLC instrument Agilent
1100 with Kromasil C18 column (250×4.6mm, 5um), detection wavelength 256nm, column temperature 25 °C,
and their relative contents were determined by normalization method of area (Figure 8). Specific optical rotation of
compound 12 was determined by SGW-5 automatic polarimeter, Shanghai, at 20 °C.
4.2. Synthesis of Key Intermediates 5a–e.
Potassium hydroxide (40.0 mmol) was stirred to be dissolved in distilled water (10 mL) at r.t. and the
appropriate uracil or 5'-substituted uracil (10.0 mmol) was added portionwise with vigorous stirring until the
mixture was well-distributed, and then the mixture was heated at 60 °C for 0.5–1 h to be a clear solution To this
.
solution, the solution of bromoacetic acid (12.1–14.5 mmol) in distilled water (8 mL) was added dropwise over 30
minutes. The reaction mixture was heated at 60 °C for another 5 h and was allowed to cool to r.t.. The mixture was
acidified to pH 2 with concentrated HCl aqueous solution and stirred overnight. The resulting precipitate was
collected by filtration to yield a white or pale yellow solid, which was recrystallized from the minimal amount of
distilled water to afford the title compound, an N-acetic acid derivative of the uracils 5a–e in 60-90% yield.
4.3. General Synthetic Procedure for Compounds 6–20.
The corresponding uracil or 5'-substituted uracil 1'(N)-acetic acid, one of the key intermediates 5a–e (1.2 mmol),
was dissolved in 20 mL of anhydrous pyridine which had been pre-treated by 4Å molecular sieves and KOH more
than 48 hours at rt. To this solution, EDCI (1.5 mmol) and DMAP (0.2–0.6 mmol) were added slowly at 0 °C, and
this mixture was stirred for 10 minutes or some longer time until a clear solution was obtained. Under an N
2
atmosphere, CPTs like CPT, 9-NO -CPT or 7-Et-CPT (0.4 mmol) was added in one portion. The reaction mixture
2
was allowed to warm to r.t. and stirred for 12–36 h. After the reaction was completed, as monitored by TLC, the
reaction mixture was diluted by adding CH Cl (40 mL) and aqueous 1 N HCl solution (30 mL). The mixture was
2
2
2 2
stirred for an additional 30 min, and two layers were separated. The aqueous layer was extracted with CH Cl (30
5

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mL × 3). The combined organic layers were dried over Na
residue was purified by flash column chromatography on Silicon gel with CHCl
give 6–20. NMR spectra of all the new compounds 6–20 are provided as Supporting Information.
2
SO
4
, filtered, and concentrated in vacuo. The crude
3
-MeOH (10:1–20:1) as eluent to
4
.3.1. Compound 6
1
Pale yellow solid; yield 84.6%; mp 278–281 °C; H NMR (400 MHz, DMSO-d
6
, TMS) δ: 11.41 (s, 1H, NH),
8
.70 (s, 1H, Ar-H), 8.18 (d, J = 8.4 Hz, 1H, Ar-H), 8.13 (d, J = 8.0 Hz, 1H, Ar-H), 7.88 (t, J = 8.0 Hz, 1H, Ar-H),
.73 (t, J = 7.6 Hz, 1H, Ar-H), 7.67 (d, J = 8.0 Hz, 1H, 6'-H), 7.22 (s, 1H, Ar-H), 5.62 (d, J = 7.6 Hz, 1H, 5'-H), 5.51
7
(
s, 2H, H-17), 5.31 (d, J = 3.2 Hz, 2H, H-5), 4.83 (dd, J = 38.4, 17.6 Hz, 2H, OOCCH
2
-N), 2.16 (q, J = 7.2 Hz, 2H,
, TMS) δ: 167.9, 167.3, 164.1, 157.0,
52.9, 151.3, 148.4, 146.5, 146.0, 132.0, 130.9, 130.3, 129.5, 129.1, 129.0, 128.5, 128.2, 119.4, 101.8, 95.7, 77.4,
1
3
1
1
6
2 3 6
9-CH ), 0.91 (t, J = 7.2 Hz, 3H, 18-CH ); C NMR (100MHz, DMSO-d
20 4 7
6.8, 50.7, 48.8, 30.8, 7.9; HRMS (ESI): m/z calcd for C26H N O +H 501.1405, found 501.1404.
4.3.2. Compound 7
1
Pale yellow solid; yield 86.6%, m p 274–276 °C; H NMR (400MHz, DMSO-d
6
, TMS) δ: 11.98 (d, J = 1.6 Hz,
1
H, NH), 8.70 (s, 1H, Ar-H), 8.17 (m, 3H, 2×Ar-H & 6'-H), 7.89 (m, 1H, Ar-H), 7.73 (m, 1H, Ar-H), 7.22 (s, 1H,
-N),
, TMS) δ:
Ar-H), 5.52 (d, J = 2.0 Hz, 2H, H-17), 5.31 (d, J = 4.0 Hz, 2H, H-5), 4.77 (dd, J = 38.0, 17.6 Hz, 2H, OOCCH
2
1
3
2
1
2 3 6
.17 (dd, J = 14.8, 7.2 Hz, 2H, 19-CH ), 0.91 (t, J = 7.6 Hz, 3H, 18-CH ); C NMR (100MHz, DMSO-d
68.0, 167.1, 166.7, 156.4, 152.4, 149.5, 147.9, 146.0, 144.6, 133.8, 131.5, 130.4, 129.8, 129.0, 128.5, 128.0, 127.7,
−
1
18.9, 115.4, 95.2, 77.0, 66.3, 50.2, 48.4, 30.3, 7.4; MS (ESI): m/z 1035 (2M−H) ; HRMS (ESI): m/z calcd for
26 4 7
C H19FN O +H 519.1311, found 519.1302.
4.3.3. Compound 8
1
Pale yellow solid; yield 85.0%; mp 252–254 °C; H NMR (400 MHz, DMSO-d , TMS) δ: 12.00 (s, 1H, NH),
6
8.70 (s, 1H, Ar-H), 8.25 (s, 1H, 6'-H), 8.18 (m, 2H, Ar-H), 7.89 (t, J = 7.2 Hz, 1H, Ar-H), 7.73 (t, J = 7.2 Hz, 1H,
Ar-H), 7.23 (s, 1H, Ar-H), 5.52 (s, 2H, H-17), 5.30 (d, J = 4.0 Hz, 2H, H-5), 4.77 (dd, J = 33.2, 17.6 Hz, 2H,
13
OOCCH
2 2 3
-N), 2.17 (dd, J = 14.8, 7.6 Hz, 2H, 19-CH ), 0.91 (t, J = 7.6 Hz, 3H, 18-CH ); C NMR (100 MHz,
DMSO-d
6
, TMS) δ: 167.0, 166.7, 159.3, 156.4, 152.4, 149.9, 147.9, 146.0, 144.5, 142.6, 131.5, 130.4, 129.8, 129.0,
−
1
28.5, 128.0, 127.7, 118.9, 106.5, 95.2, 77.0, 66.3, 50.2, 48.5, 30.3, 7.4; MS (ESI): m/z 1067 (2M−H) ; HRMS
(
ESI): m/z calcd for C H ClN O +H 535.1015, found 535.1012.
26 19 4 7
4.3.4. Compound 9
1
Pale yellow solid; yield 82.0%; mp 248–251 °C; H NMR (400 MHz, DMSO-d , TMS) δ: 11.81 (s, 1H, NH),
6
8.69 (s, 1H, Ar-H), 8.29 (s, 1H, 6'-H), 8.18 (m, 2H, Ar-H), 7.88 (t, J = 8.0 Hz, 1H, Ar-H), 7.73 (t, J = 7.6 Hz, 1H,
Ar-H), 7.22 (s, 1H, Ar-H), 5.51 (s, 2H, H-17), 5.30 (d, J = 3.2 Hz, 2H, H-5), 4.87 (dd, J = 33.6, 17.6 Hz, 2H,
13
OOCCH
2 2 3
-N), 2.17 (dd, J = 14.8, 7.2 Hz, 2H, 19-CH ), 0.91 (t, J = 7.2 Hz, 3H, 18-CH ); C NMR (100MHz,
DMSO-d
6
, TMS) δ: 167.2, 166.7, 160.9, 156.4, 152.3, 150.5, 149.6, 147.9, 146.0, 144.5, 131.5, 130.4, 129.8, 129.0,
6

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−
1
28.5, 127.9, 127.7, 118.9, 95.2, 77.0, 68.5, 66.3, 50.2, 48.3, 30.4, 7.4; MS (ESI): m/z 1251 (2M−H) ; HRMS (ESI):
m/z calcd for C26
H19IN
4
O
7
+H 627.0371, found 627.0376.
4.3.5. Compound 10
1
Pale yellow solid; yield 84.0%; mp 238–240 °C; H NMR (400 MHz, DMSO-d
6
, TMS) δ: 11.96 (s, 1H, NH),
8.69 (s, 1H, Ar-H), 8.32 (s, 1H, 6'-H), 8.18 (m, 2H, Ar-H), 7.88 (t, J = 7.6 Hz, 1H, Ar-H), 7.73 (t, J = 7.2 Hz, 1H,
Ar-H), 7.22 (s, 1H, Ar-H), 5.51 (s, 2H, H-17), 5.29 (d, J = 3.2 Hz, 2H, H-5), 4.84 (dd, J = 33.2, 17.6 Hz, 2H,
1
3
OOCCH
DMSO-d
28.5, 127.9, 127.7, 118.9, 95.2, 94.9, 77.0, 66.4, 50.2, 48.5, 30.3, 7.4; HRMS (ESI): m/z calcd for
C H BrN O +H 579.0510, found 579.0513.
2
-N), 2.17 (dd, J = 14.4, 7.2 Hz, 2H, 19-CH
2 3
), 0.92 (t, J = 7.2 Hz, 3H, 18-CH ); C NMR (400 MHz,
6
, TMS) δ: 167.1, 166.7, 159.5, 156.4, 152.3, 150.1, 147.9, 146.0, 145.0, 144.5, 131.5, 130.4, 129.8, 129.0,
1
2
6
19
4
7
4.3.6. Compound 11
1
Pale yellow solid; yield 86.6%; mp 260–262 °C; H NMR (400 MHz, DMSO-d , TMS) δ: 11.41 (s, 1H, NH),
6
8
1
5
7
.25 (d, J = 8.4 Hz, 1H, Ar-H), 8.17 (d, J = 8.4 Hz, 1H, Ar-H), 7.86 (t, J = 7.2 Hz, 1H, Ar-H), 7.73 (t, J = 7.6 Hz,
H, Ar-H), 7.69 (d, J = 8.0 Hz, 1H, 6'-H), 7.20 (s, 1H, Ar-H), 5.63 (d, J = 7.6 Hz, 1H, 5'-H), 5.53 (s, 2H, H-17),
.30 (dd, J = 25.6, 18.8 Hz, 2H, H-5), 4.84 (dd, J = 37.2, 17.6 Hz, 2H, OOCCH
2
-N), 3.20 (d, J = 7.2 Hz, 2H,
);
-CH Me), 2.18 (d, J = 7.2 Hz, 2H, 19-CH ), 1.30 (t, J = 6.8 Hz, 3H, 7-CH CH ), 0.93 (t, J = 6.8 Hz, 3H, 18-CH
2
2
2
3
3
13
C NMR (100MHz, DMSO-d , TMS) δ: 167.4, 166.8, 163.6, 156.4, 151.7, 150.8, 148.4, 146.6, 145.5, 145.4, 144.7,
6
129.9, 129.8, 127.9, 127.6, 126.5, 124.0, 118.8, 101.3, 95.1, 76.9, 66.3, 49.4, 48.3, 30.3, 22.1, 13.9, 7.4; MS (ESI):
+
+
+
24 4 7
m/z 529 (M+H) , 1057 (2M+H) ，1079 (2M+Na) ; HRMS (ESI): m/z calcd for C28H N O +H 529.1718, found
529.1727.
4.3.7. Compound 12
Yellow solid; yield 88.0%; mp > 270 °C (dec.); purity by HPLC 97.3% (flow rate 1.0ml/min, mobile phase
-
3
1
2 3 6
MeOH:H O 59:41); Specific optical rotation [α] -63.2° (in CHCl , c = 9.2×10 M); H NMR (400 MHz, DMSO-d ,
TMS) δ: 12.00 (s, 1H, NH), 8.17 (m, 3H, 2×Ar-H & 6'-H), 7.85 (t, J = 7.6 Hz, 1H, Ar-H), 7.71 (t, J = 7.6 Hz, 1H,
Ar-H), 7.21 (s, 1H, Ar-H), 5.55 (s, 2H, H-17), 5.25 (d, J = 6.4 Hz, 2H, H-5), 4.80 (dd, J = 36.8, 17.6 Hz, 2H,
OOCCH
2
-N), 3.16 (d, J = 6.8 Hz, 2H, 7-CH
2
Me), 2.20 (d, J = 7.2 Hz, 2H, 19-CH
3
2
), 1.29 (t, J = 7.2 Hz, 3H,
, TMS) δ: 167.1, 166.7, 157.5, 157.2,
56.4, 151.6, 149.5, 148.4, 146.5, 145.5, 144.6, 129.9, 129.8, 129.7, 127.8, 127.5, 126.4, 123.9, 118.8, 95.1, 77.0,
1
7
1
6
1
2 3 3 6
-CH CH ), 0.95 (t, J = 7.2 Hz, 3H, 18-CH ); C NMR (100MHz, DMSO-d
+
+
+
−
6.3, 49.4, 48.4, 30.3, 22.1, 13.9, 7.4; MS (ESI): m/z 547 (M+H) , 1093 (2M+H) , 1115 (2M+Na) , 545 (M−H) ,
−
091 (2M−H) ; HRMS (ESI): m/z calcd for C28
H23FN
O
+H 547.1624, found 547.1632.
4
7
4.3.8. Compound 13
1
Pale yellow solid; yield 84.0%; mp 275–277 °C (dec.); H NMR (400MHz, DMSO-d
6
, TMS) δ: 12.00 (s, 1H,
7

ACCEPTED MANUSCRIPT
NH), 8.26 (d, J = 8.4 Hz, 1H, Ar-H), 8.25 (s,1H, 6'-H), 8.17 (d, J = 8.4 Hz, 1H, Ar-H), 7.86 (t, J = 7.6 Hz, 1H,
Ar-H), 7.73 (t, J = 8.0 Hz, 1H, Ar-H), 7.21 (s, 1H, Ar-H), 5.53 (s, 2H, H-17), 5.32 (d, J = 6.8 Hz, 2H, H-5), 4.84 (dd,
J = 32.4, 17.6Hz, 2H, OOCCH -N), 3.22 (dd, J = 14.4, 7.2Hz, 2H, 7-CH Me), 2.18 (dd, J = 14.0, 6.4 Hz, 2H,
2
2
1
3
1
9-CH
2
), 1.30 (t, J = 7.2 Hz, 3H, 7-CH
2
CH
3
3 6
), 0.92 (t, J = 7.2 Hz, 3H, 18-CH ); C NMR (100MHz, DMSO-d ,
TMS) δ: 167.0, 166.7, 159.3, 156.4, 151.7, 149.9, 148.4, 146.6, 145.5, 144.5, 142.6, 130.0, 129.8, 127.9, 127.6,
+
1
26.5, 124.0, 118.8, 106.5, 95.1, 77.0, 66.4, 49.5, 48.5, 30.3, 22.1, 13.9, 7.4; MS (ESI): m/z 563 (M+H) , 1125
+
+
−
−
(
2M+H) , 1147 (2M+Na) , 561 (M−H) , 1123 (2M−H) ; HRMS (ESI): m/z calcd for C28
4 7
H23FN O +H 563.1328,
found 563.1333.
4.3.9. Compound 14
1
Pale yellow solid; yield 80.0%; mp 257–259 °C; H NMR (400 MHz, DMSO-d , TMS) δ: 11.83 (s, 1H, NH),
6
8
7
2
7
1
2
.30 (s, 1H, 6'-H), 8.23 (d, J = 5.6 Hz, 1H, Ar-H), 8.18 (d, J = 8.0 Hz, 1H, Ar-H), 7.85 (t, J = 7.6 Hz, 1H, Ar-H),
.71 (t, J = 8.0 Hz, 1H, Ar-H), 7.20 (s, 1H, Ar-H), 5.54 (s, 2H, H-17), 5.26 (s, 2H, H-5), 4.84 (dd, J = 31.6, 17.6Hz,
H, OOCCH
.2 Hz, 3H, 18-CH
48.4, 146.5, 145.5, 144.6, 129.9, 129.8, 127.8, 127.5, 126.5, 123.9, 118.8, 95.2, 77.0, 68.5, 66.4, 49.4, 48.3, 30.4,
2
-N), 3.18 (m, 2H, 7-CH
2
Me), 2.19 (m, 2H, 19-CH
2 2 3
), 1.29 (t, J = 7.2 Hz, 3H, 7-CH CH ), 0.93 (t, J =
1
3
3
); C NMR (100MHz, DMSO-d , TMS) δ: 167.2, 166.7, 160.9, 156.4, 151.7, 150.5, 149.6,
6
+
+
−
−
2.1, 13.9, 7.4; MS (ESI): m/z 655 (M+H) , 1309 (2M+H) ; 653 (M−H) , 1307 (2M−H) ; HRMS (ESI): m/z calcd
+H 655.0684, found 655.0691.
4 7
for C28H23FN O
4.3.10. Compound 15
1
Pale yellow solid; yield 86.6%; mp > 280 °C (dec.); H NMR (400 MHz, DMSO-d
6
, TMS) δ: 11.96 (s, 1H, NH),
8
7
2
7
1
2
.32 (s, 1H, 6'-H), 8.27 (d, J = 7.6 Hz, 1H, Ar-H), 8.17 (d, J = 8.0 Hz, 1H, Ar-H), 7.86 (t, J = 7.6 Hz, 1H, Ar-H),
.73 (t, J = 6.8 Hz, 1H, Ar-H), 7.21 (s, 1H, Ar-H), 5.53 (s, 2H, H-17), 5.30 (s, 2H, H-5), 4.84 (dd, J = 31.6, 17.6 Hz,
H, OOCCH -N), 3.21 (m, 2H, 7-CH Me), 2.18 (m, 2H, 19-CH ), 1.30 (t, J = 7.2 Hz, 3H, 7-CH CH ), 0.92 (t, J =
2
2
2
2
3
1
3
3 6
.2 Hz, 3H, 18-CH ); C NMR (100MHz, DMSO-d , TMS) δ: 167.1, 166.7, 159.5, 156.4, 151.7, 150.1, 148.4,
46.6, 145.5, 145.0, 144.5, 129.9, 129.8, 127.9, 127.6, 126.5, 124.0, 118.8, 95.2, 94.9, 77.0, 66.4, 49.5, 48.5, 30.3,
2.1, 13.9, 7.4; HRMS (ESI): m/z calcd for C28
H23FN
4
O
7
+H 607.0823, found 607.0824.
4.3.11. Compound 16
1
Dark yellow solid; yield 82.0%; mp > 250 °C (dec.); H NMR (400 MHz, DMSO-d
6
, TMS) δ: 11.43 (s, 1H,
NH), 9.16 (s, 1H, Ar-H), 8.55 (t, J = 8.0 Hz, 2H, Ar-H), 8.05 (t, J = 8.4 Hz, 1H, Ar-H), 7.69 (d, J = 8.0 Hz, 1H,
6
2
=
'-H), 7.28 (s, 1H, Ar-H), 5.63 (dd, J = 7.6, 2.0 Hz, 1H, 5'-H), 5.53 (d, J = 2.0 Hz, 2H, H-17), 5.35 (dd, J = 26.8,
0.0Hz, 2H, H-5), 4.83 (dd, J = 39.2, 17.6 Hz, 2H, OOCCH
2
-N), 2.16 (dd, J = 15.2, 7.2 Hz, 2H, 19-CH
, TMS) δ: 167.4, 166.7, 163.6, 156.4, 153.8, 150.8, 147.8,
45.9, 145.5, 145.1, 144.6, 135.8, 132.8, 129.0, 127.1, 125.5, 120.1, 119.9, 101.3, 96.1, 76.8, 66.3, 50.7, 48.3, 30.3,
2
), 0.92 (t, J
13
3 6
7.2 Hz, 3H, 18-CH ); C NMR (100 MHz, DMSO-d
1
7
−
−
−
−
.4; MS (ESI): m/z 544 (M−H) , 580 (M+Cl) , 1089 (2M−H) , 1125 (2M+Cl) ; HRMS (ESI): m/z calcd for
C H N O +H 546.1256, found 546.1259.
2
6
19
5
9
8

ACCEPTED MANUSCRIPT
4.3.12. Compound 17
1
Bright yellow solid; yield 86.5%; mp 235–237 °C; H NMR (400 MHz, DMSO-d
6
, TMS) δ: 12.00 (s, 1H, NH),
9
7
1
.17 (s, 1H, Ar-H), 8.55 (dd, J = 7.6, 5.6Hz, 2H, Ar-H), 8.17 (d, J = 6.4 Hz, 1H, 6'-H), 8.05 (t, J=8.4 Hz, 1H, Ar-H),
.28 (s, 1H, Ar-H), 5.54 (dd, J = 20.0, 17.2Hz, 2H, H-17), 5.35 (dd, J = 27.2, 20.0 Hz, 2H, H-5), 4.77 (dd, J = 38.4,
13
7.6 Hz, 2H, OOCCH -N), 2.17 (dd, J = 14.8, 7.6 Hz, 2H, 19-CH ), 0.92 (t, J = 7.2 Hz, 3H, 18-CH ); C NMR
2
2
3
(
100 MHz, DMSO-d
6
, TMS) δ: 167.1, 166.6, 157.2, 156.3, 153.8, 149.5, 147.8, 145.9, 145.1, 144.5, 138.3, 135.7,
−
1
5
5
32.8, 129.7, 129.0, 127.1, 125.5, 120.1, 119.9, 96.1, 76.9, 66.3, 50.7, 48.5, 30.3, 7.4; MS (ESI): m/z 562 (M−H) ,
−
−
−
98 (M+Cl) , 1125 (2M−H) , 1223 (2M+HSO
64.1169.
) ; HRMS (ESI): m/z calcd for C26
H18FN
O
+H 564.1161, found
4
5
9
4.3.13. Compound 18
1
Bright yellow solid; yield 83.0%; mp 242–244 °C; H NMR (400MHz, DMSO-d
6
, TMS) δ: 12.01 (s, 1H, NH),
9.17 (s, 1H, Ar-H), 8.55 (t, J = 7.2Hz, 2H, Ar-H), 8.25 (s, 1H, 6'-H), 8.05 (t, J = 8.0 Hz, 1H, Ar-H), 7.29 (s, 1H,
Ar-H), 5.53 (dd, J = 20.0, 17.2 Hz, 2H, H-17), 5.36 (dd, J = 26.8, 19.6 Hz, 2H, H-5), 4.87 (dd, J = 34.0, 17.6 Hz,
1
3
2H, OOCCH
2 2 3
-N), 2.19 (dd, J = 14.8, 6.8 Hz, 2H, 19-CH ), 0.92 (t, J = 7.2 Hz, 3H, 18-CH ); C NMR (100 MHz,
DMSO-d , TMS) δ: 167.0, 166.6, 159.3, 156.3, 153.8, 149.9, 147.8, 145.9, 145.1, 144.5, 142.6, 135.8, 132.9, 129.0,
6
−
−
1
27.1, 125.5, 120.1, 119.9, 106.5, 96.1, 77.0, 66.3, 50.8, 48.5, 30.3, 7.4; MS (ESI): m/z 578 (M−H) , 614 (M+Cl) ,
−
−
1
157 (2M−H) , 1255 (2M+HSO
4
) ; HRMS (ESI): m/z calcd for C26
H18ClN
5
O
9
+H 580.0866, found 580.0868.
4.3.14. Compound 19
1
Bright yellow solid; yield 82.6%; mp 252–254 °C; H NMR (400 MHz, DMSO-d
6
, TMS) δ: 11.82 (s, 1H, NH),
9.16 (s, 1H, Ar-H), 8.54 (t, J = 8.0 Hz, 2H, Ar-H), 8.29 (s, 1H, 6'-H), 8.04 (t, J = 8.0 Hz, 1H, Ar-H), 7.28 (s, 1H,
Ar-H), 5.53 (dd, J = 20.4, 17.2 Hz, 2H, H-17), 5.35 (dd, J = 27.2, 20.0 Hz, 2H, H-5), 4.83 (dd, J = 34.0, 18.0 Hz,
1
3
2H, OOCCH
2 2 3
-N), 2.17 (dd, J = 14.8, 7.2 Hz, 2H, 19-CH ), 0.92 (t, J = 7.2 Hz, 3H, 18-CH ); C NMR (100 MHz,
DMSO-d , TMS) δ: 167.1, 166.6, 160.9, 156.3, 153.7, 150.5, 149.6, 147.8, 145.9, 145.1, 144.5, 135.8, 132.8, 129.0,
6
−
−
1
1
6
27.1, 125.5, 120.1, 119.9, 96.1, 76.9, 68.5, 66.3, 50.7, 48.3, 30.3, 7.4; MS (ESI): m/z 670 (M−H) , 706 (M+Cl) ,
−
−
−
341 (2M−H) , 1377 (2M+Cl) , 1439 (2M+HSO ) ; HRMS (ESI): m/z calcd for C H IN O +H 672.0222, found
4
26 18
5
9
72.0224.
4.3.15. Compound 20
1
Bright yellow solid; yield 84.6%; mp 247–250 °C; H NMR (400 MHz, DMSO-d
6
, TMS) δ: 11.97 (s, 1H, NH),
9.17 (s, 1H, Ar-H), 8.55 (t, J = 7.2 Hz, 2H, Ar-H), 8.32 (s, 1H, 6'-H), 8.05 (t, J = 8.4 Hz, 1H, Ar-H), 7.28 (s, 1H,
Ar-H), 5.53 (dd, J = 20.0, 16.8Hz, 2H, H-17), 5.35 (dd, J = 26.8 Hz, 20.0 Hz, 2H, H-5), 4.83 (dd, J = 33.6, 17.6 Hz,
1
3
2
2 2 3
H, OOCCH -N), 2.17 (dd, J = 14.8, 7.2 Hz, 2H, 19-CH ), 0.92 (t, J = 7.6 Hz, 3H, 18-CH ); C NMR (100 MHz,
DMSO-d , TMS) δ: 167.0, 166.6, 159.5, 156.3, 153.8, 150.2, 147.8, 145.9, 145.1, 145.0, 144.5, 135.8, 132.9, 129.0,
6
9

ACCEPTED MANUSCRIPT
127.1, 125.5, 120.1, 119.9, 96.1, 95.0, 76.9, 66.3, 50.8, 48.5, 30.3, 7.4; HRMS (ESI): m/z calcd for
26 5 9
C H18BrN O +H 624.0361, found 624.0364.
4.4. Cell Lines and In Vitro Cytotoxicity Assays
The human tumor cell lines used in this work were A549 (lung carcinoma), Bel7402 (hepatocarcinoma),
BGC-823 (gastric carcinoma), HCT-8 (colon cancer) and A2780 (ovarian cancer). These cell lines were obtained
from the Peking Union Medical College (PUMC) in China. All cell lines were maintained and assayed in
RPMI-1640 medium (GIBCO product) containing 10% heat-inactivated fetal bovine serum (bought from
Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.) in a humidified atmosphere containing 5% CO in
2
air. Compound stock solutions were prepared at 10 mM in DMSO and diluted with culture medium with the final
DMSO concentration ≤ 0.01% (v/v), a concentration without effect on cell growth. Cells were plated in 96-well
3
microtiter plates at a density of 1–1.5 × 10 /well and incubated in a humidified atmosphere with 5% CO
2
at 37 °C
for 24 h. Test compounds were added onto triplicate wells with different concentrations and 0.1% DMSO for
control. After they had been incubated for 96 h, 100 ꢀL of MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (AMRESCO product) solution (0.4 mg/mL,
[
prepared in RPMI1640) was added to each well and the plate was incubated for an additional 4 h. The supernatant
was discarded before the formazan crystals were dissolved in 150 ꢀL of DMSO for each well. The absorbance (OD)
was quantitated with microplate spectrophotometer (BIORAD type 550 enzyme mark instrument) at 540 nm with
405 nm as the reference wavelength. Wells containing no drugs were used as blanks for the spectrophotometer. The
survival of the cells was expressed as percentage of untreated control wells. The IC50 (ꢀM) values, the
concentrations which causing 50% inhibition of cell growth compared with that of the vehicle control, were
determined according to the literatures' method [37, 38]. Compounds with IC50 ≤ 20 ꢀM were considered effective.
All the experiments were performed three times with duplicated samples.
4.5. Antitumor Activity in Vivo Assay on Compound 12
The compounds were solved in normal saline using Tween-80 as the solubilizer. Four- to six-week-old
BALB-c-nu male mice (Certificate SCXK-Jing-2009-0004, weighing 18–20 g) were obtained from Beijing
Huafukang Bio-tech. Co., Ltd., PRC. Each of them was inoculated subcutaneously at the left flank with a tumor
bulk of human gastric cancer BGC-823 or human liver carcinoma Bel-7402. The mice were divided into
experimental groups after 6 days of inoculation．Groups (n = 8) consisted of control, Irinotecan or TPT and the
analogue 12 with 30 mg/kg and 60 mg/kg two dose-groups. Mice received treatments, ip, at that day for the first
time and then repeated the treatments once per week (QWK) to the end. The change of tumor volumes and body
weights were was observed and recorded every 3 days over the course of treatment. Tumor volumes (TV) were
1
2
monitored by caliper measurement of the length and width and calculated using the formula of TV = /
2
ab , where
the “a” is the tumor length and the “b” is the width [32]. Mice were sacrificed on day 27–30 after implantation of
1
0

ACCEPTED MANUSCRIPT
cells, and tumors were removed and recorded for analysis. Tumor inhibiting rate (%) was calculated when the
control group's median tumor weight reached 1.0–1.4 g and compared with the treatment group's median tumor
weight at that time. Relative tumor volumes (RTV) were calculated using the formula of RTV = V / V , where V is
t
0
0
t
the TV measured at the time of group-dividing and V is the TVs obtained every time from then on. The
evaluation index of antitumor activity is the relative tumor growth rate T/C (%) with the calculating formula as
followed,
T/C (%) ＝ [Treatment Group (T) RTV / Negative Control Group (C) RTV] × 100%
And the standard of curative effect evaluation is: T/C (%) > 40, considered as invalid; T/C (%) ≤ 40, and
after being subjected to the statistical dealing with the result of P < 0.05, considered as effective.
Results were evaluated statistically by t-test [32].
4
4
.6. Mechanism of Action Studies on Compound 12
.6.1. Morphological Observation
Morphological changes of culture cells were observed and examined by using a phase-contrast microscope and
the photographs were taken by using a high-resolution digital camera (Nikon Eclipse Ti, Japan).
4.6.2. Apoptosis Assessment
Annexin V-FITC/propidium iodide double staining kit (BD Biosciences) was used to detect apoptosis. A549
5
cells (5×10 cells/mL) were seeded in six-well plates and treated with 12 at concentrations from 8 nM to 75 nM
for 24 h. The cells were then harvested by trypsinization and washed for twice with ice-cold PBS. After
centrifugation and removal of the supernatants, cells were resuspended in binding buffer which was then added to 5
ꢀ
L of annexin V-FITC and incubated at r.t. for 15 min. After adding 10 ꢀL of propidium iodide (PI) the cells were
incubated at r.t. for another 15 min in the dark. The stained cells were analyzed by a flow cytometer (BD Dickinson)
32, 39].
[
4.6.3. Cell Cycle Arrest Assay
For flow cytometric analysis of cell cycle, A549 cells in exponential growth were treated with 12 and TPT with
concentrations from 8 nM to 75 nM respectively for 48 h. The cells treated with 12 and TPT were collected,
washed twice with PBS, and then fixed with 75% alcohol overnight. The cells were washed with PBS and
eliminated the interference of RNA with 200 mg/mL RNase was added for 30 min. Then, the cells were staining
with 20 mL/L propidium iodide (PI; Sigma) for 30 min. washed and the DNA content was detected by flow
cytometer (BD Dickinson) [32, 39].
11

ACCEPTED MANUSCRIPT
4.6.4. Western Blot Analysis
The treated cells with compound 12 were homogenized and subjected to SDS-PAGE. The proteins were
transferred onto a polyvinylidene difluoride membrane (Roche Molecular Biochemicals, Quebec, Canada), blocked
with 5% milk powder in TBS-T buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, and 0.1% Tween) for 1 h at r.t.,
incubated overnight at 4°C with appropriate primary antibodies against the target proteins, further incubated with
HRP-conjugated secondary antibodies, and developed with the ECL System (Millipore, Billerica, MA).
4.6.5. Topo I-Mediated Plasmid DNA Pcmv-6 Relaxation Assay
DNA cleavage assay was performed as previously described [32, 33]. In brief, pcmv-6 DNA was incubated
with Top I (New England Biolabs, American) in the presence or absence of indicated drugs, 12 and TPT, at 37 °C
for 30 minutes and terminated by the addition of 10% sodium dodecylsulfate (SDS). Proteinase K (200 ꢀg/mL final
concentration) was added before the samples were incubated at 50 °C for 20 min. And 2 ꢀL of 10× gel loading
buffer (0.25% bromophenol blue, 50% glycerol) was added. The mixtures then were analyzed on a 1% agarose gel
(
containing 0.5 ꢀg/mL ethidium bromide) at 8 V/cm for 1 h with TAE (Tris-Acetate-EDTA) as the running buffer.
TPT was used as reference drug. The position of supercoiled DNA (SC) and relaxed DNA (RLX) were visualized
with UV light and indicated with a Gel Doc Ez imager (Bio-Rad Laboratories, Ltd.).
Acknowledgments
This study was financially supported in part by the Fundamental Research Funds for the Central Universities of
China (No. 65011141), the Research Fund for the Doctoral Program of Higher Education of China (Grant No.
20110031120048), the Natural Science Foundation of China (81672747), and Tianjin Municipality Science and
Technology Commission (15JCYBJC50100).
Appendix A. Supplementary data
Supporting Information: NMR spectra of compounds 6–20. This material is available free of charge via the Internet
at http://ees.elsevier.com.
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55 (2012) 9630–9642.
1
5

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O
O
N
6: X = H
N
7
8
:
:
X = F
X = Cl
O
O
X
9: X = Br
10: X = I
O
O
O
O
N
O
HO
O
X
HN
HN
O
O
N
H
O
O
11: X = H
12: X = F
N
N
(i)
13: X = Cl
O
14: X = Br
15: X = I
O
X
O
N
(
ii)
O
X
HO
O
O
N
O
HN
O
HN
NO
2
O
NO
2
OH
O
N
O
O
N
16: X = H
1
7: X = F
5a: X = H
5b: X = F
5c: X = Cl
5d: X = Br
5e: X = I
O
18: X = Cl
19: X = Br
20: X = I
O
X
O
HO
O
O
N
O
O
HN
O
2 2 2
Scheme 1. Synthesis of 5a–e & 6–20. Reagents and conditions: (i) 1. BrCH COOH or ClCH COOH / KOH / H O, 60˚C;
2
2
. HCl / r.t. (ii) RCH COOH (5a–e) / EDCI / DMAP / Pyridine (dried), r.t.
1

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Table 1
Cytotoxicity of substituted uracil-1'(N)-acetic acid esters of CPTs against five human tumor cell lines
IC50 (µM)
A549
0.006
0.007
0.046
0.126
0.054
0.048
6.400
0.065
0. 027
0.038
0.088
0.710
0.051
0.081
0.075
0.090
0.117
Bel 7402
0.014
0.100
0.018
0.086
0.017
0.012
3.283
0.014
0.014
0.012
0.012
4.510
0.018
0.016
0.016
0.013
0.013
BGC-823
0.057
0.078
0.017
0.064
0.016
0.016
7.891
0.017
0.032
0.017
0.031
2.573
0.017
0.017
0.017
0.015
0.016
HCT-8
0.024
0.022
0.014
0.109
0.013
0.006
0.957
0.002
0.007
0.002
<0.002
0.586
0.014
0.013
0.013
0.013
0.014
A2780
0.029
0.078
0.160
0.068
0.110
0.160
0.750
0.086
0.147
0.670
0.182
0.669
0.073
0.115
0.221
0.183
0.200
CPT
TPT
6
7
8
9
1
0
11
1
1
1
1
1
1
1
1
2
2
3
4
5
6
7
8
9
0
A549: human lung cancer; Bel7402: human liver cancer; BGC-823: human stomach cancer; HCT-8: human colon cancer ; A2780: human
ovarian cancer.
2

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Table 2
Preliminary in vivo antitumor activity of substituted uracil-1'(N)-acetic acid ester analogues of CPTs against mouse liver tumor model (H22) (ip)
a
b
c
f
Group
Dose
mg/kg
NS
Number
d0/dn
11/11
10/9
BW /g
TW /g
TIR ％
P value
d0
dn
32.5
18.9
32.1
27.3
27.0
32.0
Control
Paclitaxel
9
21.7
21.8
22.2
21.8
21.6
2.69
0.70
2.52
1.14
1.45
2.39
-
-
<0.01
<0.01
<0.01
<0.01
<0.01
d
24×3
73.85
6.39
e
40×2
10/10
10/10
10/10
10/10
e
1
1
1
2
3
6
40×2
57.51
46.21
11.00
e
40×2
e
40×2
22.2
a
b
c
d
e
BW: Body weight; TW: Tumor weight; TIR: Tumor inhibitory rate; : 24mg/kg in dose for every 3 days and totally for 3 times; : 40mg/kg in
f
dose for the first day and the 5 days later and totally for twice; P: Significant difference compared to control group.
3

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Table 3
Further in vivo antitumor activity of substituted uracil-1'(N)-acetic acid ester analogues of CPTs against mouse liver tumor model (H22) for doses
ip)
(
a
b
c
d
Group
Dose
mg/kg
NS
Number
d0/dn
9/9
BW /g
TW /g
TIR ％
P value
d0
dn
31.1
22.9
29.8
27.2
25.2
26.2
Control
20.6
20.4
20.6
20.6
20.8
20.7
3.63
0.23
2.21
1.29
1.50
0.85
-
-
<0.01
<0.05
<0.01
<0.01
<0.01
Cyclophosphamide
100×2
100×1
60×2
100×1
60×2
9/8
93.66
39.12
64.46
58.68
76.58
1
1
1
1
2
2
3
3
10/8
10/10
10/6
10/7
a
b
c
d
BW: Body weight; TW: Tumor weight; TIR: Tumor inhibitory rate; P: Significant difference compared to control group.
4

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Table 4
Further in vivo antitumor activity of 12 against nude mice human gastric cancer model (BGC-823) for doses by weights (ip)
a
b
c
d
Group
Dose
mg/kg
NS
Number
d0/dn
8/8
BW /g
TW /g
TIR ％
P value
d0
dn
23.6
20.5
21.9
19.1
Control
20.0
19.8
19.9
20.5
3.35
0.18
0.14
0.35
-
-
<0.001
<0.001
<0.001
Irinotecan
50×2
60×2
30×2
8/8
94.10
95.97
89.70
1
1
2
2
8/8
8/8
a
b
c
d
BW: Body weight; TW: Tumor weight; TIR: Tumor inhibitory rate; P: Significant difference compared to control group.
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Table 5
Further in vivo antitumor activity of 12 against nude mice human gastric cancer model (BGC-823) for doses by tumor volumes (ip)
a
b
c
d
Group
Dose
mg/kg
NS
Number
d0/dn
8/8
BV /g
RTV /g
T/C ％
P value
d0
dn
2533
147
Control
119
115
114
125
22.73
1.25
1.26
2.87
-
-
<0.001
<0.001
<0.001
Irinotecan
50×2
60×2
30×2
8/8
5.48
5.54
12.65
1
1
2
2
8/8
143
8/6
334
a
b
c
d
BV: Body volume; RTV: Tumor volume; T/C: Testing group compared to control group; P: Significant difference compared to control group.
6

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Table 6
Further in vivo antitumor activity of 12 against nude mice liver tumor model (Bel-7402) for doses by weights (ip)
a
b
c
d
Group
Dose
mg/kg
NS
Number
d0/dn
8/8
BW /g
TW /g
TIR ％
P value
d0
dn
14.5
14.0
14.0
14.4
Control
TPT
18.6
19.8
18.9
18.6
0.57
0.31
0.11
0.21
-
-
5×2
8/7
45.48
81.45
62.60
<0.05
<0.001
<0.05
1
1
2
2
60×2
30×2
8/8
8/6
a
b
c
d
BW: Body weight; TW: Tumor weight; TIR: Tumor inhibitory rate; P: Significant difference compared to control group.
7

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Table 7
Further in vivo antitumor activity of 12 against nude mice liver tumor model (Bel-7402) for doses by tumor volumes (ip)
a
b
c
d
Group
Dose
mg/kg
NS
Number
d0/dn
8/8
BV /g
RTV /g
T/C ％
P value
d0
dn
511
Control
TPT
113
115
115
119
4.44
2.61
1.09
1.87
-
-
5×2
8/7
302
127
231
58.80
24.53
41.17
<0.01
<0.001
<0.01
1
1
2
2
60×2
30×2
8/8
8/6
a
b
c
d
BV: Body volume; RTV: Tumor volume; T/C: Testing group compared to control group; P: Significant difference compared to control group.
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