Synthesis and Biological Evaluation of 10-Substituted Camptothecin Derivatives with Improved Water Solubility and Activity

Article abstract of DOI:10.1002/cmdc.202000753

Despite remarkable clinical achievements, camptothecin (CPT) still suffers from poor solubility and severe toxicity. Therefore, it is necessary to redevelop CPT derivatives as supplementary antitumor agents with good water solubility and small side effects. In this work, 27 camptothecin derivatives were synthesized and screened for their cytotoxicity against A549 (lung) and HCT-116 (colon) cancer cell lines. Among them, compound B7, 7-ethyl-10-(2-oxo-2-(4-methylpiperidin-1-yl)ethoxy)camptothecin,was demonstrated in vitro to be a more potent antitumor agent than SN-38 by comparison of their inhibitory activities against cell proliferation and colony formation and interference effect on process of cell cycle and cell apoptosis. Additionally, a molecular docking model revealed that B7 can interact with the topoisomerase I–DNA complex, and that the solubility of B7 reached 5.73 μg/mL in water. Moreover, B7 significantly inhibited tumor growth in an A549 xenograft model at dosages of 0.4 and 2.0 mg/kg, and exhibited minimum lethal doses comparable to those of irinotecan. These results indicated that B7, with improved solubility, enhanced activity and acceptable acute toxicity, can be used as a lead compound for the development of novel anticancer agents.

Full text of DOI:10.1002/cmdc.202000753

Accepted Article
Title: Synthesis and biological evaluation of 10-substituted
camptothecin derivatives with improved water solubility and
activity
Authors: Xue-Yan Yang, Hong-Yi Zhao, Hao Lei, Bo Yuan, Shuai Mao,
Minhang Xin, and San-Qi Zhang
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: ChemMedChem 10.1002/cmdc.202000753
Link to VoR: https://doi.org/10.1002/cmdc.202000753
0
1/2020

ChemMedChem
10.1002/cmdc.202000753
FULL PAPER
Synthesis and biological
evaluation
of 10-substituted
camptothecin derivatives with improved water solubility and
activity
[
a]
[a]
[a]
[a]
[a]
[a]
Xue-Yan Yang, Hong-Yi Zhao, Hao Lei, Bo Yuan, Shuai Mao, Minghang Xin, and San-Qi
Zhang*[a]
[
a]
Dr. X.-Y. Yang, Dr. H.-Y. Zhao, B. Yuan, Prof. S. Mao, Prof. M. Xin, Prof. S.-Q. Zhang
Department of Medicinal Chemistry
School of Pharmacy, Xian Jiaotong University
Xian, Shaanxi, 710061, PR China
E-mail: sqzhang@xjtu.edu.cn
Abstract: Despite the remarkable clinical achievement, camptothecin
CPT) still suffers from poor solubility and severe toxicity. Therefore,
successful structural modifications are focused on the A-B rings,
especially at 7-, 9-, 10-, and 11-position of CPT (Figure 1),
intending to improve water solubility and antitumor potency.[10] To
date, structural modification of natural CPT has generated two
antitumor agents approved by FDA, namely topotecan[ and
irinotecan[12], which are now used clinically to treat ovarian, small
cell lung and colon cancers.
(
it is necessary to redevelop CPT derivatives as supplementary
antitumor agents with good water solubility and small side-effects. In
this work, twenty-seven camptothecin derivatives were synthesized
and screened for their cytotoxicity against A549 (lung) and HCT-116
11]
(colon) cancer cell lines. Among them, compound B7, 7-ethyl-10-(2-
oxo-2-(4-methylpiperidin-1-yl)ethoxy)camptothecin, was demonstrated
in vitro to be a more potent antitumor agent than SN-38 by the
comparison of their inhibitory activities against cell proliferation and
colony formation and interference effect on process of cell cycle and
cell apoptosis. Additionally, molecular docking model revealed that B7
can interact with topoisomerase I-DNA complex, and the solubility of
B7 reached 5.73 μg/mL in water. Moreover, B7 significantly inhibited
the tumor growth in A549 xenograft model at the dosage of 0.4 and
SN-38, the active metabolite of irinotecan, displays potent
anti-tumor activity in vitro, but its therapeutic application is
hindered by its poor water solubility.[13] Moreover, intestinal
inflammation caused by SN-38 is of serious concern, because
10-O-glucuronide SN38 (SN-38G), produced by SN-38 and
glucuronic acid in the liver, converts back to the biologically active
compound SN-38 in the presence of bacterial β-glucuronidase in
the intestinal lumen. In this process, SN-38 remaining in the
intestines causes intestinal inflammation, leading to diarrhea. In
fact, the 10-O-substitution strategy was used to avoid the hepatic
metabolism of 10-O-glucuronidation in the liver and its derivatives
have been developed from the medicinal chemistry perspective
to further increase the aqueous solubility and the antitumor
activity.[ Chimmitecan, a case of camptothecin 9-position
modification, was reported in 2007.[15] The allyl substitution at the
2.0 mg/kg. And B7 exhibited minimum lethal doses comparable to
irinotecan. These results indicated that B7, with improved solubility,
enhanced activity and acceptable acute toxicity, can be used as a lead
compound for development of novel anti-cancer agent.
14]
Introduction
9
-position of CPT benefits chimmitecan a salient anti-MDR
activity and oral availability.[ 10-HCPT, another natural alkaloid,
has been approved by CFDA because of its excellent anti-tumor
activity. Nonetheless, side effects such as digestive tract
16]
The contribution of natural products (NPs) to drug discovery
seems impressive.[1] NPs have constantly provided leads
compounds or entered clinical-trials for human disease,
[2]
particularly the cancer. The therapeutic area of oncology has
benefited from numerous NPs, and many of anti-tumor drugs
[3]
currently in clinical use are NPs or designed using NP templates ,
such as vinblastine , paclitaxel and homoharringtonine[6].
Camptothecin (CPT, Figure 1), a quinoline based cytotoxic
alkaloid isolated from the bark of Chinese plant Camptotheca
acuminata, shows significant anticancer activity, but early clinical
trials of CPT were all terminated due to its poor solubility and
stability as well as unpredictable severe toxicity.[7] It was not until
[
4]
[5]
1
985 that the field of camptothecin research was resurrected
because the unique mechanism of CPT was characterized.[8] The
mechanism investigation indicated that CPT can bind to the
transient Topo I/DNA cleavage complex, causing Topo I-
mediated DNA breakage, subsequently blocking intracellular
DNA replication, transcription and repair, and eventually leading
to cell death.[9] Thereafter, thousands of CPT analogues or
derivatives were synthesized and activity evaluated. Most of the
Figure 1. CPT and it’s derivatives with multisubstitution at 7, 9, 10, and 11
positions.
1
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ChemMedChem
10.1002/cmdc.202000753
FULL PAPER
reactions and bone marrow suppression largely circumscribe the
clinical practicability of 10-HCPT.
Previous work from our group concluded that a hydrophobic
moiety, especially a ring fragment, attached to the 10-position of
CPT improved solubility and reduced side-effects.[17] In the
present study, new hydrophobic groups were linked to the 10-
position of 10-HCPT, SN-38 or chimmitecan, and twenty-seven
new CPT derivatives were synthesized and evaluated for their
antitumor activity. It’s demonstrated that the most promising
compound B7 was a potent antitumor agent with improved
solubility, acceptable acute toxicity and potent activity both in vitro
and in vivo.
Results and Discussion
The synthetic routes for the target compounds are illustrated in
Figures 2-3. As shown in Figure 2, 10-HCPT or SN-38 underwent
one or two-step nucleophilic substitution reaction to generate
compounds A1, A8, B1 and B5-B6. Then A1 was reduced to give
A2. 10-HCPT or SN-38 was acylated with alkyl formic acid or
benzoic acid in the presence of condensing agent 2-(7-Aza-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium h e x a f l u o r o -
phoshate (HATU) to afford A3-A7 and B2-B4. In the presence of
anhydrous potassium carbonate, the reaction of SN-38 with ethyl
bromoacetate in DMF gave intermediate 1, which was
subsequently converted to acid 2 via hydrolysis. In the presence
of HATU, acid 2 reacted with 4-methylpiperidine to produce B7
Figure 3. Synthetic pathways for preparation of compounds B8, C1-C11.
Reagents and reaction conditions: (i): 1) allyl bromide, K
acetic acid, Ar, reflux, days; (j): ethyl bromoacetate or 1-bromo-2-
methoxyethane, K CO , DMF, rt, 2-4 h; ethyl 2-bromoisobutyrate, K
acetonitrile, reflux, 5 h; (k): Pd/C, H , 1,4-dioxane, rt, overnight; (l): 1) K
DMF/ H
DIPEA, DCM, rt, 3 h; (m): Pd/C, H
2 3
CO , DMF, rt, 2 h, 2)
5
2
3
2 3
CO ,
2
2 3
CO ,
2
O (1:1, volume ratio), rt, overnight; 2) morpholine or piperidine, HATU,
2
, 1,4-dioxane, rt, overnight; (n): 1) 4-
(
Figure 2).
The intermediate produced by the nucleophilic substitution of
0-HCPT with allyl bromide was rearranged to produce
nitrophenyl chloroformate, DMAP (4-dimethylaminopyridine), dichloromethane,
ice-bath, 3-5 h, 2) SN-38 or chimmitecan, TEA (triethylamine), DMF, rt, 2-3 h.
(o): 1) 4-nitrophenyl chloroformate, DMAP (4-dimethylaminopyridine), DCM,
ice-bath, 3-5 h, 2) chimmitecan, TEA (triethylamine), DMF, rt, 2-3 h. (p): 1) 4-
nitrophenyl chloroformate, DMAP (4-dimethylaminopyridine), DCM, ice-bath, 3-
1
chimmitecan. Then chimmitecan reacted with ethyl bromoacetate,
ethyl 3-bromo-3-methylbutanoate or 1-bromo-2-methoxyethane
to generate C1-C3, respectively. The reaction of hydrolysate of
C1 with piperidine or morpholine produced C6-C7. Subsequently
5
h, 2) chimmitecan, TEA (triethylamine), DMF, rt, 2-3 h.
2
C1-C2 and C6 were reduced by H (catalyst, Pd/C) to give C4-C5
1
16) and human lung adenocarcinoma epithelial cell line (A549)
and C8. SN-38 or chimmitecan reacted with the active carbonic
acid ester which was generated from phenylmethanol and 4-
nitrophenyl carbonochloridate to afford B8 and C9. Similarly,
compounds C10-C11 were synthesized from 4-bromobenzyl
alcohol and benzylamine respectively (Figure 3).
by applying the MTT colorimetric assay (Table 1). 10-HCPT and
SN-38 were selected as the positive controls. As illustrated in
Table 1, all the tested compounds dominantly inhibited growth of
HCT-116 and A549 cell lines. The different antiproliferative effect
suggested that the activity of synthesized compounds was
associated with the structure of substituted groups at the 10-
position of CPT. Among the tested compounds, A1-A7 and B1-
B4 showed activity comparable to that of their parent 10-HCPT
and SN-38 respectively. The introduction of 2-methyl-2-butene
fragment (A1, B1) or 2-methylbutane fragment (B2) into the 10-
position of 10-HCPT or SN-38 had negligible effect on its activity.
Moreover, A3-A7 and B2-B4, the substituent of which at 10-
position was changed to alkyl formyl or benzoyl, also displayed
submicromolar inhibitory activity against the two cell lines.
However, as prodrugs, the activity of A3-A7, B2-B4 and their
corresponding parent drug was nearly indistinguishable.
Subsequently, we elongated the distance between 10-position
oxygen atom of 10-HCPT or SN-38 and the cycloalkane by two
With target compounds in hand, we firstly evaluated their
cytotoxic activity against human colon carcinoma cell line (HCT-
Figure 2. Synthetic pathways for preparation of compounds A1-A8 and B1-B7.
Reagents and reaction conditions: (a): 1-bromo-3-methyl-2-butene, K CO ,
2 3
acetone, reflux 3 h; (b): Pd/C, MeOH, rt, overnight; (c): substituted formic acid,
2
CH to obtain compounds A8 and B5-B6 which displayed reduced
HATU, DIPEA (N,N-diisopropylethylamine), DMF (N,N-dimethylformamide), rt,
activity (IC50 > 1 μM on A549). In our previous work, we found that
the acetamide fragment appended to 10-position of SN-38 would
affect the activity, and the cyclic hydrophobic amine played a
positive role. Therefore, we designed and synthesized compound
B7 which exhibited extremely strong antiproliferative activity
against both A549 and HCT-116 cell lines (IC50 = 0.006, 0.004 μM,
2
-5 h; (d): 1) 1,2-dibromoethane, K
piperidine, K CO , DMF, rt, 2-3 h; (e): ethyl bromoacetate, K
f): K CO , DMF/ H O (1:1, volume ratio), rt, overnight; (g): 4-methylpiperidine,
HATU, DIPEA, dichloromethane, rt, 3 h.
2
CO
3
, DMF, rt, 3 h; 2) morpholine or
2
3
2
CO , DMF, rt, 3 h;
3
(
2
3
2
2
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ChemMedChem
10.1002/cmdc.202000753
FULL PAPER
group at the 9th position of chimmitecan was reduced to a propyl
group or not had almost no impact on the activity (C4-C5, C8).
Substitution of 10-position of chimmitecan with methoxyethyl (C3)
slightly diminished the activity despite the same order of activity
magnitude as chimmitecan. These results indicated that the
introduction of large substituents at the 10-position of
chimmitecan was detrimental to the activity, which might be
caused by the steric hindrance between the 9 and 10 positions.
Subsequently, we synthesized several chimmitecan prodrugs
through the linkage of carbonate or carbamate, all of which
showed comparable activity to chimmitecan, and C11 performed
best (A549, IC50 = 0.011 μM; HCT-116, IC50 = 0.007 μM). Similarly,
B8, as a prodrug of SN-38, also showed a satisfactory anti-
proliferative effect.
As a derivative of SN-38, we anticipated that B7 also has a
broad-spectrum anti-tumor effect, so we determined the cytotoxic
activities of B7 against several different cancer cell lines. The
results were presented in Table 2. As we expected, B7 showed
significantly stronger anti-proliferative activity than SN-38 against
proliferation of Lovo, Colo205, HT-29 (human colon carcinoma
cell lines) and HepG2 (human liver carcinoma cell line).
Table 1. Cytotoxic activities of synthetic compounds against HCT-116 and A549
cell lines (n = 3, ͞x ± SD).
IC50 (μM)
Compds
A549
HCT-116
A1
A2
0.051 ± 0.008
0.064 ± 0.010
0.041 ± 0.023
0.048 ± 0.009
0.053 ± 0.013
0.117 ± 0.086
0.013 ± 0.005
5.152 ± 1.101
0.057 ± 0.010
0.034 ± 0.006
0.043 ± 0.005
0.067 ± 0.011
1.332 ± 0.124
2.469 ± 0.632
0.006 ± 0.001
0.013 ± 0.010
0.018 ± 0.004
0.630 ± 0.101
0.572 ± 0.212
0.050 ± 0.021
0.435 ± 0.119
0.803 ± 0.108
0.214 ± 0.034
0.608 ± 0.008
0.120 ± 0.013
0.028 ± 0.009
0.022 ± 0.005
0.011 ± 0.001
0.065 ± 0.005
0.024 ± 0.007
0.056±0.011
0.058±0.062
0.024±0.002
0.043±0.002
0.064±0.003
0.059±0.009
0.032±0.010
0.524 ± 0.008
0.031 ± 0.007
0.018 ± 0.003
0.020 ± 0.001
0.036 ± 0.001
0.219 ± 0.064
0.299 ± 0.110
0.004 ± 0.001
0.006 ± 0.002
0.007 ± 0.001
1.521 ± 0.675
1.167 ± 0.141
0.073 ± 0.011
0.372 ± 0.021
2.101 ± 0.367
0.278 ± 0.091
0.384 ± 0.085
0.329 ± 0.103
0.005 ± 0.001
0.010 ± 0.001
0.007 ± 0.003
0.041 ± 0.009
0.013 ± 0.002
A3
A4
A5
A6
A7
A8
B1
B2
B3
Table 2. Cytotoxic activities of B7 against different cancer cell lines (n = 3).
B4
IC50 (μM)
B5
Compds
Lovo
Colo205
HT-29
HepG2
B6
B7
0.098 ±
0.103 ±
0.003
0.022 ±
0.003
0.077 ±
0.003
B7
0.010
B8
SN-38
0.328 ±
.091
0.295 ±
0.031
0.072 ±
0.010
0.185 ±
0.036
0
Chimmitecan
C1
Due to their excellent anti-proliferative activities on HCT-116
and A549 cells, B7 and C11 were selected for solubility
determination in different menstruum, and the results were listed
in Table 3. Compared with SN-38, the solubility of B7 in different
solvents was greatly improved and even reached about 11.10
μg/mL in a buffer solution at pH 4.5. We infer that the introduction
of a hydrophobic moiety to the 10-position of CPT may destroy
the rigid planar structure and improve solubility. Tested by HPLC,
C11 showed enhanced solubility in water and pH 4.5 buffer, but
its carbonate structure was hydrolyzed in pH7.4 buffer.
Based on cellular activity and solubility, we selected B7 for
further study. As shown in Figure 4, colony formation assay
confirmed that B7 could remarkably suppress the cell cloning of
A549 cell line at concentrations as low as 0.001 nM, and that the
formation of cell cluster colonies was almost completely inhibited
at a concentration of 10 nM. Compared with the positive control
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
Table 3. The solubility of B7 and C11 in different menstruum (μg/mL).
1
0-HCPT
H
2
O
Buffer solution
SN-38
pH4.5
pH7.4
17.32 ± 0.02
—
respectively).
Next, chimmitecan was synthesized as parent compound and
the introduction of an ester fragment at its 10-position significantly
reduced its activity (C1-C2, IC50 > 1 μM on HCT-116). C6
produced by the reaction of hydrolysate of C1 and piperidine also
showed poor activity. The same result was obtained when
piperidine was replaced with morpholine (C7). Whether the allyl
B7
C11
5.73 ± 0.03
2.11± 0.02
< 0.25
11.10 ± 0.04
5.37± 0.16
< 0.25
SN-38
< 0.25
3
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Figure 4. Colony formation assay. A549 cells were seeded on 6-well plate and
treated with B7 under indicated concentrations for 10 days and then the
generating colonies were stained and counted. The colony formation was
photographed (a) and the numbers of colonies were counted (b). Date are
shown as the mean ± SD of three independent experiments (**P < 0.01
compared with SN-38).
Figure 6. Compound B7 induced apoptosis on A549 cells. Cells were treated
with indicated concentrations for 24 h and the apoptosis rates were assessed
by FITC Annexin V/PI staining and analyzed by flow cytometry (a). The
apoptosis rates were show on the histogram (b). Data shown are the mean ±
SD of three independent experiments (**P < 0.01, compared with SN-38).
SN-38, B7 displayed more potent inhibitory effect on A549 clone
formation at 0.1 nM and 1 nM, which was consistent with the
cytotoxicity results.
To explore whether B7 can affect cell cycle, we applied flow
cytometry assay to determine A549 cell cycle after the treatment
with B7. SN-38 was used as positive control. As shown in Figure
B7 treatment at doses of 0.4 and 2.0 mg/kg led to a significant
tumor growth inhibition. B7-induced inhibition rates of tumor
growth at low and high dosage group reached 57.1% and 63.8%,
respectively. Therefore, we could confirm that B7 exerts a
remarkable antitumor effect in vivo by intraperitoneal
administration treatment.
5
, the results indicated that 10 nM of B7 arrested A549 cells in the
G2 phase.[ In contrast, 100 nM and 1000 nM of B7 blocked
A549 cells in the S phase. And the blocking effect of B7 was much
stronger than SN-38 at same concentration (1 μM).
18]
Figure 7. Antitumor efficacy of B7 administered intraperitoneally in vivo. Balb/C
nude mice bearing A549 cells xenografts were treated with B7 at doses of 0.4
and 2.0 mg/kg once the other day. Tumor size (a) and body weight (b) of the
nude mice were measured every two days. On the last day, mice were sacrificed
and tumors were removed, photographed (c) and weighed (d). (n = 4; *P < 0.05,
Figure 5. Effect of compound B7 on cell cycle progression of A549 cell line.
Cells were exposed to various concentrations of B7 for 24 h and the cell cycle
was assessed by PI/RNase staining and analyzed by flow cytometry (a). The
cell cycle distribution was shown on the histogram (b). Data shown are the mean
*
*P < 0.01, significant difference compared with solvent group).
±
SD of three independent experiments (**P < 0.01, significant difference
compared with control)
In order to investigate the acute toxicity, the minimum lethal
dose of B7 was measured (Table 4). The minimum lethal doses
of B7, SN-38 and Irinotecan in mice determined by intravenous
administration were 35, 40 and 40 mg/kg (or 0.066, 0.102 and
To confirm whether B7 can induce cell apoptosis, the
annexinV/propidium iodide (PI) biparametric cyto-fluorimetric
assay was applied on A549 cells. As depicted in Figure 6, B7
could facilitate A549 cells apoptosis in a concentration-dependent
manner. B7 induced 13.4% and 16.16% of A549 cell line to
undergo apoptosis at concentration of 0.1 and 1 μM, respectively.
Meanwhile, B7-induced apoptosis rates of A549 cells are higher
than the positive drug SN-38 at the concentrations of 0.1 and 1
μM.
The studies in vitro demonstrated that B7 is a more potent
antitumor agent than SN-38. To examine the antitumor efficacy of
B7 in vivo, a test using A549 xenograft model was performed. The
results were shown in Figure 7. Compared with the solvent group,
0
.064 mmol/kg), respectively. The acute toxicity of B7 was
increased compared to SN-38, but was equivalent to Irinotecan.
Moreover, the minimum lethal dose of B7 was much higher than
its inhibitory tumor dose of 0.4-2 mg/kg.
To explore the binding mode of B7 and SN-38 with Topo I-
DNA complex, molecular docking studies were performed. From
the binding model, we noticed that compounds B7 and SN-38
parallelly inserted into the hydrophobic region of DNA, and B7
was more parallel to DNA. As Figure 8A showed, the oxygen atom
in the lactone ring of B7 formed a hydrogen bond interaction with
Thr718 of Topo I, the hydroxyl at 20-position formed hydrogen
bond interaction with Asp533, and nitrogen in the pyridine ring
4
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ChemMedChem
10.1002/cmdc.202000753
FULL PAPER
chromatography- Quadrupole-time of flight Mass Spectrometer (WATERS
I-Class VION IMS Q-TOF).
Table 4. Minimum lethal dose of B7 to mice (n = 6).
B7
SN-38
Irinotecan
Dose
(
1
S)-4-ethyl-4-hydroxy-9-((3-methylbut-2-en-1-yl)oxy)-1,12-dihydro-
4H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A1):
To a round bottom flask were added 10-HCPT (50.0 mg, 0.14 mmol),
CO (22.7 mg, 0.16 mmol), 1-bromo-3-methyl-2-butene (16 μL, 0.14
Dose
Dose
mortality
mortality
mortality
(
mg/kg)
(mg/kg)
25
(mg/kg)
30
K
2
3
25
35
50
0/6
1/6
3/6
0/6
1/6
5/6
0/6
1/6
4/6
mmol) and acetone (10 mL). The solution was refluxed for 3h. Product A1
was isolated using column chromatography (DCM:MeOH = 50:1). Pale
yellow solid, 90% yield. H NMR (400 MHz, DMSO-d
40
40
1
6
) δ 8.51 (s, 1H), 8.05
(
d, J = 9.2 Hz, 1H), 7.54 – 7.46 (m, 2H), 7.28 (s, 1H), 6.52 (s, 1H), 5.54 (t,
J = 6.7 Hz, 1H), 5.42 (s, 2H), 5.25 (s, 2H), 4.71 (d, J = 6.7 Hz, 2H), 1.93 –
.81 (m, 2H), 1.79 (d, J = 2.7 Hz, 6H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR
(100 MHz, DMSO-d ) δ 173.0, 157.7, 157.3, 150.5, 150.5, 146.2, 144.4,
138.3, 130.9, 130.6, 130.3, 129.8, 123.7, 119.9, 118.8, 107.4, 96.5, 72.9,
50
50
formed hydrogen bond interaction with Arg364. Importantly, the
-methylpiperidinyl in B7 extended to the hydrophobic region of
1
4
6
Topo I, which provided additional hydrophobic interaction with the
binding site and enhanced the stability of B7-Topo I-DNA complex.
Therefore, we speculated that B7 was more parallel to DNA and
the additional hydrophobic interaction brought by 4-
methylpiperidine resulted in better activity than SN-38.
6
5.7, 65.4, 50.6, 30.7, 25.9, 18.6, 8.2. HRMS (ESI): m/z calcd for
+
C25H25N2O5: 433.17635 [M+H] ; found: 433.17551.
(
S)-4-ethyl-4-hydroxy-9-(isopentyloxy)-1,12-dihydro-14H-
pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione
Compound A1 (86.2 mg, 0.20 mmol) and Pd/C (8.0 mg, 10%) were mixed
with MeOH (6 mL). The suspension was stirred under H atmosphere at
(A2):
2
room temperature overnight. The mixture was filtered through celite, and
the filtrate was purified by column chromatography (DCM:MeOH = 50:1).
1
Pale yellow solid, 63% yield. H NMR (400 MHz, DMSO-d
6
) δ 8.52 (s, 1H),
8
1
.05 (d, J = 9.2 Hz, 1H), 7.53 (d, J = 2.6 Hz, 1H), 7.49 (dd, J = 9.2, 2.5 Hz,
H), 7.28 (s, 1H), 6.51 (s, 1H), 5.42 (s, 2H), 5.26 (s, 2H), 4.18 (t, J = 6.6
Hz, 2H), 1.89 – 1.84 (m, 2H), 1.78 (d, J = 2.7 Hz, 1H), 1.72 (q, J = 6.6 Hz,
H), 0.98 (d, J = 6.6 Hz, 6H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
DMSO-d ) δ 173.0, 158.0, 157.3, 150.6, 150.5, 146.3, 144.4, 130.9, 130.7,
130.4, 129.9, 123.7, 118.8, 107.3, 96.5, 72.9, 67.0, 65.7, 50.7, 37.8, 30.7,
25.1, 22.9 (2C), 8.2. HRMS (ESI): m/z calcd for C25 : 435.19200
[M+H] ; found: 435.19309.
2
6
Figure 8. Docking of B7 and SN-38 with Topo I-DNA complex (PDB: 1k4t).
Hydrogen bonds are indicated as red dashed lines. The gray residue represents
a piece of DNA.
27 2 5
H N O
+
(
S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl tetrahydro-2H-pyran-4-
carboxylate (A3): To a round bottom flask were added 10-HCPT (50.0
mg, 0.14 mmol), tetrahydropyran-4-yl-carboxylic acid (19.7 mg, 0.15
mmol), HATU (54.0 mg, 0.14 mmol), DIPEA (72 μL, 0.41 mmol) and DMF
Conclusion
In this work, twenty-seven new camptothecin derivatives were
synthesized and screened for their cytotoxicity against A549
(1.5 mL). The resulted mixture was stirred at r.t. for 2 h. Add water (5 mL)
(
lung) and HCT-116 (colon) cancer cell lines. Among them,
to the reaction system, and the precipitated white solid was filtered off and
compound B7 was demonstrated in vitro to be a more potent
antitumor agent than SN-38 by the comparison of their inhibitory
activities on cell proliferation and colony formation and
interference effect on the process of cell apoptosis and cell cycle.
Moreover, the solubility of B7 reached 5.73 μg/mL in water.
Importantly, B7 significantly inhibited the tumor growth in A549
xenograft model at the dosage of 0.4 and 2.0 mg/kg. And B7
exhibited minimum lethal doses comparable to irinotecan.
Additionally, molecular docking model revealed that B7 can
interact with topoisomerase I-DNA complex. These results
indicated that B7, with improved solubility, enhanced activity and
acceptable acute toxicity, can be used as a lead compound for
development of novel anti-cancer agent.
1
recrystallized to obtain A3. Off-white solid, 87% yield. H NMR (400 MHz,
DMSO-d ) δ 8.63 (s, 1H), 8.17 (d, J = 9.1 Hz, 1H), 7.88 (d, J = 2.4 Hz, 1H),
7.63 (dd, J = 9.1, 2.5 Hz, 1H), 7.32 (s, 1H), 6.54 (s, 1H), 5.42 (s, 2H), 5.25
s, 2H), 3.92 (dd, J = 8.0, 3.3 Hz, 2H), 3.46 (td, J = 11.2, 1.8 Hz, 2H), 3.05
6
(
–
2
1
1
2.91 (m, 1H), 2.02 – 1.94 (m, 2H), 1.93 – 1.84 (m, 2H), 1.83 – 1.72 (m,
H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
6
) δ 173.3, 172.9,
57.2, 153.0, 150.4, 149.4, 146.3, 145.7, 131.6, 130.9, 130.8, 128.7, 126.4,
19.6, 119.6, 97.2, 72.8, 66.5 (2C), 65.7, 50.6, 30.8, 28.7 (3C), 8.2. HRMS
+
(ESI): m/z calcd for C26H25N2O7: 477.16618 [M+H] ; found: 477.16600.
(
S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl
cyclohexanecarboxylate (A4): Compound A4 was prepared similarly as
1
described for A3. Off-white solid, 92% yield. H NMR (400 MHz, DMSO-
6
d ) δ 8.68 (s, 1H), 8.21 (d, J = 9.1 Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H), 7.65
(dd, J = 9.1, 2.5 Hz, 1H), 7.35 (s, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.30 (s,
2
1
3
H), 2.77 – 2.64 (m, 1H), 2.11 – 1.98 (m, 2H), 1.96 – 1.81 (m, 2H), 1.82 –
.71 (m, 2H), 1.71 – 1.49 (m, 3H), 1.45 – 1.23 (m, 3H), 0.90 (t, J = 7.3 Hz,
H). 13C NMR (100 MHz, DMSO-d
) δ 174.3, 172.9, 157.2, 152.9, 150.4,
6
Experimental Section
General methods. Unless specified otherwise, all the starting materials,
reagents and solvents are commercially available. All the reactions were
monitored by thin-layer chromatography on silica gel plates (GF254) and
visualized with UV light (254 nm and 365 nm). NMR spectra were recorded
on a 400 Bruker NMR spectrometer with tetramethylsilane (TMS) as an
internal reference. All chemical shifts are reported in parts per million
149.5, 146.3, 145.8, 131.6, 130.9, 130.8, 128.7, 126.4, 119.6, 119.6, 97.1,
72.8, 65.7, 50.6, 42.6, 30.8, 28.9 (2C), 25.8, 25.2 (2C), 8.2. HRMS (ESI):
+
m/z calcd for C27H27N2O6: 475.18691 [M+H]
; found: 475.18670.
(
S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl
(ppm). High resolution mass spectrum (HRMS) was obtained by
cyclopentanecarboxylate (A5): Compound A5 was prepared similarly as
electrospray ionization (positive mode) on an Ultra performance liquid
1
described for A3. Off-white solid, 87% yield. H NMR (400 MHz, DMSO-
5
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000753
FULL PAPER
6
d ) δ 8.61 (s, 1H), 8.15 (d, J = 9.1 Hz, 1H), 7.86 (d, J = 2.3 Hz, 1H), 7.61
(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
(dd, J = 9.1, 2.3 Hz, 1H), 7.32 (s, 1H), 6.53 (s, 1H), 5.42 (s, 2H), 5.24 (s,
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl tetrahydro-2H-pyran-4-
2
4
1
1
H), 3.11 (p, 1H), 2.06 – 1.98 (m, 2H), 1.95 – 1.84 (m, 4H), 1.74 – 1.61 (m,
carboxylate (B2): Compound B2 was prepared similarly as described for
H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
A3. Off-white solid, 81% yield. H NMR (400 MHz, DMSO-d
1
6
) δ 175.1, 172.9,
6
) δ 8.17 (d, J
57.2, 152.9, 150.4, 149.5, 146.3, 145.7, 131.5, 130.8, 130.7, 128.7, 126.4,
= 9.1 Hz, 1H), 7.97 (d, J = 2.3 Hz, 1H), 7.63 (dd, J = 9.1, 2.4 Hz, 1H), 7.31
(s, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.29 (s, 2H), 3.93 (dd, J = 8.1, 3.3 Hz,
2H), 3.46 (qd, J = 11.3, 9.4 Hz, 2H), 3.17 (q, J = 7.4 Hz, 2H), 3.04 – 2.92
(m, 1H), 1.99 (d, J = 11.1 Hz, 2H), 1.94 – 1.73 (m, 4H), 1.29 (t, J = 7.6 Hz,
19.6, 119.5, 97.1, 72.8, 65.7, 50.6, 43.5, 30.8, 29.9 (2C), 25.9 (2C), 8.2.
+
HRMS (ESI): m/z calcd for C26H25N2O6: 461.17126 [M+H] ; found:
461.17077.
3
1
1
1
H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
) δ 173.4, 172.9,
6
57.2, 152.4, 150.5, 149.6, 146.9, 146.3, 145.7, 131.7, 129.0, 127.5, 126.1,
19.5, 115.6, 97.1, 72.8, 66.5 (2C), 65.7, 55.4, 50.0, 30.8, 28.7 (2C), 22.7,
(
S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl
cyclobutanecarboxylate (A6): Compound A6 was prepared similarly as
described for A3. Off-white solid, 85% yield. H NMR (400 MHz, DMSO-
+
4.3, 8.2. HRMS (ESI): m/z calcd for C28H29N2O7: 505.19748 [M+H] ;
1
found: 505.19703.
6
d ) δ 8.63 (s, 1H), 8.17 (d, J = 9.1 Hz, 1H), 7.89 (d, J = 2.4 Hz, 1H), 7.65
(
dd, J = 9.1, 2.5 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.42 (s, 2H), 5.26 (s,
H), 3.54 (p, J = 8.5 Hz, 1H), 2.42 – 2.30 (m, J = 13.8, 12.9, 7.2 Hz, 4H),
.09 – 1.86 (m, 4H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-
(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl
2
2
d
1
cyclohexanecarboxylate (B3): Compound B3 was prepared similarly as
1
6
) δ 173.9, 172.9, 157.2, 152.9, 150.5, 149.5, 146.3, 145.8, 131.6, 130.8,
described for A3. Pale yellow solid, 90% yield. H NMR (400 MHz, DMSO-
30.8, 128.8, 126.5, 119.6, 119.6, 97.1, 72.8, 65.7, 50.7, 37.7, 30.8, 25.2
6
d ) δ 8.17 (d, J = 9.1 Hz, 1H), 7.95 (d, J = 2.3 Hz, 1H), 7.61 (dd, J = 9.1,
(
2C), 18.3, 8.2. HRMS (ESI): m/z calcd for C25H23N2O6: 447.15561
2.4 Hz, 1H), 7.32 (s, 1H), 6.53 (s, 1H), 5.44 (s, 2H), 5.30 (s, 2H), 3.17 (q,
J = 7.2 Hz, 2H), 2.76 – 2.63 (m, 1H), 2.07 (d, J = 10.2 Hz, 2H), 1.94 – 1.83
+
[M+H] ; found: 447.15497.
(
m, 2H), 1.82 – 1.74 (m, 2H), 1.70 – 1.48 (m, 3H), 1.45 – 1.34 (m, 2H),
1
1
1
.29 (t, 4H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
) δ
6
(
S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
74.3, 172.9, 157.3, 152.3, 150.5, 149.7, 146.9, 146.3, 145.7, 131.7, 129.0,
27.5, 126.1, 119.4, 115.6, 97.1, 72.9, 65.7, 55.4, 50.0, 42.6, 30.8, 28.9
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl
cyclopropanecarboxylate (A7): Compound A7 was prepared similarly as
described for A3. Off-white solid, 88% yield. H NMR (400 MHz, DMSO-
1
(2C), 25.2 (2C), 22.7, 14.3, 8.2. HRMS (ESI): m/z calcd for C29H31N2O6:
+
503.21821 [M+H] ; found: 503.21807.
d
(
2
(
6
) δ 8.61 (s, 1H), 8.15 (d, J = 9.1 Hz, 1H), 7.88 (d, J = 2.4 Hz, 1H), 7.63
dd, J = 9.1, 2.5 Hz, 1H), 7.32 (s, 1H), 6.53 (s, 1H), 5.42 (s, 2H), 5.24 (s,
H), 2.02 – 1.95 (m, 1H), 1.93 – 1.83 (m, J = 13.6, 6.8 Hz, 2H), 1.18 – 1.08
m, 4H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
) δ 173.4,
(S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl benzoate (B4):
6
1
72.9, 157.2, 152.9, 150.4, 149.4, 146.3, 145.7, 131.6, 130.8, 130.7, 128.7,
Compound B4 was prepared similarly as described for A3. Pale yellow
1
126.4, 119.6, 119.6, 97.1, 72.8, 65.7, 50.6, 30.8, 13.1, 9.7 (2C), 8.2. HRMS
6
solid, 94% yield. H NMR (400 MHz, DMSO-d ) δ 8.26 – 8.17 (m, 4H), 7.84
+
(ESI): m/z calcd for C24H21N2O6: 433.13996 [M+H] ; found: 433.13959.
– 7.76 (m, 2H), 7.65 (t, J = 7.7 Hz, 2H), 7.34 (s, 1H), 6.54 (s, 1H), 5.44 (s,
H), 5.30 (s, 2H), 3.26 – 3.12 (m, 2H), 1.99 – 1.80 (m, 2H), 1.30 (t, J = 7.5
Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
) δ 172.9,
65.2, 157.3, 152.4, 150.5, 149.8, 147.1, 146.3, 145.9, 134.7, 131.8, 130.4
2C), 129.5 (2C), 129.2, 129.0, 127.6, 126.1, 119.5, 116.0, 97.1, 72.8, 65.7,
2
(
S)-4-ethyl-4-hydroxy-9-(2-morpholinoethoxy)-1,12-dihydro-14H-
6
1
(
pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (A8):
flask was charged with 10-HCPT (50.0 mg, 0.14 mmol), 1,2-
dibromoethane (236 μL, 2.7 mmol), K CO (94.7 mg, 0.69 mmol) and DMF
1mL). The resulted mixture was stirred at 60℃ for 12h. When 10-HCPT
A
5
0.0, 30.8, 22.7, 14.3, 8.2. HRMS (ESI): m/z calcd for C29H25N2O6:
2
3
+
497.17126 [M+H] ; found: 497.17169.
(
was completely consumed, add water (5 mL) to the reaction system, then
the precipitated white solid was filtered off for the next step. To a round
bottom flask were added the precipitated white solid (20.0 mg, 0.04 mmol),
(S)-4,11-diethyl-4-hydroxy-9-(2-morpholinoethoxy)-1,12-dihydro-
14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (B5):
morpholine (19 μL, 0.21 mmol), K
2
CO
3
(29.4 mg, 0.21 mmol) and DMF
Compound B5 was prepared similarly as described for A8. Gray solid, 62%
1
(1.5 mL). The solution was stirred at room temperature for 2h in Argon
6
yield. H NMR (400 MHz, DMSO-d ) δ 8.04 (d, J = 9.9 Hz, 1H), 7.48 (dd,
atmosphere. The solvent was evaporated under vacuum, and the residue
was isolated using column chromatography (DCM:MeOH = 10:1) to give
target compound A8. Yellow solid, 76% yield. H NMR (400 MHz, DMSO-
J = 4.8, 2.1 Hz, 2H), 7.25 (s, 1H), 6.51 (s, 1H), 5.42 (s, 2H), 5.25 (s, 2H),
4.31 (t, J = 5.6 Hz, 2H), 3.61 (t, 4H), 3.17 (dd, J = 14.7, 7.1 Hz, 2H), 2.79
(t, J = 5.6 Hz, 2H), 2.53 (s, 4H), 1.95 – 1.79 (m, 2H), 1.31 (t, J = 7.5 Hz,
1
d
(
4
(
6
) δ 8.52 (s, 1H), 8.06 (d, J = 9.2 Hz, 1H), 7.55 (d, J = 2.6 Hz, 1H), 7.51
d, J = 9.2 Hz, 1H), 7.28 (s, 1H), 6.51 (s, 1H), 5.43 (s, 2H), 5.26 (s, 2H),
.28 (t, J = 5.7 Hz, 2H), 3.65 – 3.56 (m, 4H), 2.80 (t, J = 5.7 Hz, 2H), 2.53
s, 4H), 1.94 – 1.78 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
DMSO-d ) δ 173.0, 157.7, 157.2, 150.5, 150.4, 146.1, 144.3, 130.9, 130.4,
30.3, 129.7, 123.5, 118.8, 107.3, 96.5, 72.9, 66.7 (2C), 66.2, 65.7, 57.4,
3H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
6
) δ 173.0, 157.9,
157.3, 150.5, 150.1, 146.8, 144.9, 144.3, 131.9, 128.8, 128.3, 122.9, 118.7,
103.5, 96.5, 72.9, 66.7 (2C), 66.3, 65.7, 57.5, 54.2 (2C), 50.0, 30.7, 22.7,
+
14.0, 8.2. HRMS (ESI): m/z calcd for C28H32N3O6: 506.22911 [M+H] ;
6
found: 506.22906.
1
5
4
4.1 (2C), 50.6, 30.7, 8.2. HRMS (ESI): m/z calcd for C26H28N3O6:
78.19781 [M+H] ; found: 478.19680.
(
1
S)-4,11-diethyl-4-hydroxy-9-(2-(piperidin-1-yl)ethoxy)-1,12-dihydro-
4H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (B6):
+
Compound B6 was prepared similarly as described for A8. Gray solid, 70%
1
(
S)-4,11-diethyl-4-hydroxy-9-((3-methylbut-2-en-1-yl)oxy)-1,12-
yield. H NMR (400 MHz, DMSO-d
6
) δ 8.01 (d, J = 9.5 Hz, 1H), 7.45 (dd,
dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-
2H), 7.24 (s, 1H), 6.50 (s, 1H), 5.42 (s, 2H), 5.20 (s, 2H), 4.26 (t, J = 5.8
Hz, 2H), 3.14 (dd, J = 14.7, 7.0 Hz, 2H), 2.73 (t, J = 5.8 Hz, 2H), 2.48 (s,
4H), 1.95 – 1.79 (m, 2H), 1.58 – 1.48 (m, 4H), 1.40 (d, J = 4.7 Hz, 2H),
1.30 (t, J = 7.5 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
dione (B1): Compound B1 was prepared similarly as described for A1.
1
Pale yellow solid, 83% yield. H NMR (400 MHz, DMSO-d
6
) δ 8.05 (d, J =
8.9 Hz, 1H), 7.52 – 7.44 (m, 2H), 7.26 (s, 1H), 6.50 (s, 1H), 5.50 (t, J = 6.6
Hz, 1H), 5.42 (s, 2H), 5.27 (s, 2H), 4.77 (d, J = 6.6 Hz, 2H), 3.16 (q, J =
6
DMSO-d ) δ 173.0, 157.9, 157.3, 150.5, 149.9, 146.7, 144.8, 144.2, 131.8,
1
=
1
1
2
4.9, 7.3 Hz, 2H), 1.93 – 1.85 (m, 2H), 1.81 (d, J = 9.7 Hz, 6H), 1.30 (t, J
128.6, 128.3, 122.9, 118.7, 103.4, 96.4, 72.9, 66.6, 65.7, 57.8, 55.0 (2C),
49.9, 30.8, 26.1 (2C), 24.4, 22.6, 13.9, 8.2. HRMS (ESI): m/z calcd for
C29H34N3O5: 504.24985 [M+H] ; found: 504.24928.
7.5 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
6
) δ
+
73.0, 157.8, 157.3, 150.5, 150.0, 146.8, 144.8, 144.2, 138.1, 131.8, 128.8,
28.3, 123.1, 120.1, 118.7, 103.7, 96.4, 72.9, 65.7, 65.4, 50.0, 30.7, 25.9,
2.7, 18.6, 13.9, 8.2. HRMS (ESI): m/z calcd for C27H29N2O5: 461.20765
(
S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
+
[M+H] ; found: 461.20733.
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetate (1): A round
6
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000753
FULL PAPER
bottom flask were charged with 10-HCPT (547.0 mg, 1.5 mmol), K
2
CO
3
(Chimmitecan): A flask was charged with 10-HCPT (50.0 mg, 0.14 mmol),
(415.0 mg, 3.0 mmol), ethyl bromoacetate (200 μL, 1.8 mmol) and DMF
2 3
allyl bromide (24 μL, 0.27 mmol), K CO (37.9 mg, 0.27 mmol) and DMF
(
10 mL). The resulted mixture was stirred at room temperature for 12 h.
(1.5 mL). The resulted mixture was heated in an oil bath at 60°C for 3 h,
then poured into ice water, adjusted to pH = 5 with 1M HCl, and the
precipitated solid was filtered and dried for direct use in the next step. The
solid obtained by suction filtration was added to glacial acetic acid (7 mL)
and refluxed for 4 days under an argon atmosphere. The mixture was
isolated using column chromatography (DCM:MeOH = 20:1) to obtain
The solvent was evaporated by rotary evaporation, and the residue was
added with water (10 mL). A large amount of yellow solid precipitated and
then filtered with suction. The filter cake was washed 3 times with water,
dried and separated by silica gel column chromatography
(DCM:MeOH=100:1~ 40:1) to obtain compound 1. Light yellow powder,
1
chimmitecan. Brown solid, 65% yield. 1H NMR (400 MHz, DMSO-d
7
1% yield. H NMR (400 MHz, DMSO-d6) δ 8.08 (d, J = 9.2 Hz, 1H), 7.53
6
) δ
(
1
dd, J = 9.2, 2.7 Hz, 1H), 7.47 (d, J = 2.7 Hz, 1H), 7.25 (s, 1H), 6.49 (s,
H), 5.41 (s, 2H), 5.27 (s, 2H), 5.05 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.16
10.24 (s, 1H), 8.61 (s, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.53 (d, J = 9.1 Hz,
1H), 7.26 (s, 1H), 6.50 (s, 1H), 6.07 – 5.94 (m, 1H), 5.41 (s, 2H), 5.23 (s,
2H), 4.99 (d, J = 5.0 Hz, 1H), 4.97 (d, J = 13.5 Hz, 1H), 3.78 (d, J = 4.6 Hz,
2H), 1.94 – 1.79 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
(
(
m, 2H), 1.87 (m, 2H), 1.26 (t, J = 7.5 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H), 0.86
t, J = 7.3 Hz, 3H).
6
DMSO-d ) δ 173.0, 157.3, 154.2, 150.5, 149.3, 146.4, 144.0, 136.8, 130.1,
1
3
29.3, 128.9, 126.8, 122.7, 118.5, 117.6, 115.8, 96.3, 72.9, 65.7, 50.8,
(
S)-2-((4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
0.7, 29.0, 8.2. HRMS (ESI): m/z calcd for C23H19N2O5: 405.14505
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)acetic acid (2):
Compound 1 (336.0 mg, 0.75 mmol), K CO (104.0 mg, 0.75 mmol), H
15 mL) and DMF (15 mL) were mixed in a flask. The suspension was
+
[M+H] ; found: 405.14515.
2
3
2
O
(
stirred at room temperature for 14 h. The solvent was evaporated by rotary
evaporation, and the residue was added with water (10 mL). The system
was adjusted to pH 2~3 with 1 M HCl and the precipitated solid was filtered
ethyl
(S)-2-((10-allyl-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-
tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-
yl)oxy)acetate (C1): Compound C1 was prepared similarly as described
for compound 1. Pale yellow solid, 53% yield. H NMR (400 MHz, DMSO-
1
1
and dried by suction to obtain intermediate 2. Dark yellow, 91% yield. H
NMR (400 MHz, DMSO-d6) δ 13.15 (s, 1H), 8.08 (d, J = 9.2 Hz, 1H), 7.53
6
d ) δ 8.70 (s, 1H), 8.05 (d, J = 9.3 Hz, 1H), 7.67 (d, J = 9.4 Hz, 1H), 7.28
(
5
3
m, 1H), 7.47 (d, J = 2.5 Hz, 1H), 7.26 (s, 1H), 6.49 (s, 1H), 5.41 (s, 2H),
.28 (s, 2H,), 4.95 (s, 2H), 3.16 (m, 2H), 1.85 (m, 2H), 1.27 (t, J = 7.5 Hz,
H), 0.86 (t, J = 7.3 Hz, 3H).
(s, 1H), 6.51 (s, 1H), 6.09 – 5.97 (m, 1H), 5.41 (s, 2H), 5.21 (s, 2H), 5.08
– 4.97 (m, 4H), 4.20 (q, J = 7.1 Hz, 2H), 3.88 (d, J = 5.6 Hz, 2H), 1.93 –
1.79 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR
6
(100 MHz, DMSO-d ) δ 173.0, 169.2, 157.3, 154.0, 150.8, 150.4, 146.0,
1
7
44.5, 136.6, 130.4, 129.5, 128.1, 127.6, 121.7, 119.0, 118.8, 116.2, 96.7,
(
S)-4,11-diethyl-4-hydroxy-9-(2-(4-methylpiperidin-1-yl)-2-
2.9, 66.1, 65.7, 61.2, 50.8, 30.7, 29.1, 14.5, 8.2. HRMS (ESI): m/z calcd
oxoethoxy)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-
+
for C27H27N2O7: 491.18183 [M+H] ; found: 491.18194.
b]quinoline-3,14(4H)-dione (B7): Compound 2 (100.0 mg, 0.22 mmol),
HATU (169.0 mg, 0.44 mmol), 4-methylpiperidine (52 μL, 0.44 mmol),
DIPEA (77 μL, 0.44 mmol) and DCM (5 mL) were mixed in a flask. The
mixture was stirred at room temperature for 3.5 h and then isolated using
column chromatography (DCM:MeOH = 75:1~50:1) to produce compound
B7. Yellow solid, 70% yield. H NMR (400 MHz, DMSO-d
ethyl
(S)-2-((10-allyl-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-
tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-
methylpropanoate (C2): To a flask were added chimmitecan (70.0 mg,
0.17 mmol), ethyl 2-bromo-2-methylpropanoate (67.6 mg, 0.35 mmol),
1
6
) δ 8.09 (d, J =
9.2 Hz, 1H), 7.53 (dd, J = 9.2, 2.7 Hz, 1H), 7.43 (d, J = 2.7 Hz, 1H), 7.27
2 3 3
K CO (47.8 mg, 0.35 mmol) and CH CN (10 mL). The reaction mixture
(
s, 1H), 6.52 (s, 1H), 5.43 (s, 2H), 5.29 (s, 2H), 5.09 (q, J = 14.7 Hz, 2H),
was heated at reflux for 5 h and isolated using column chromatography
(DCM:MeOH = 75:1) to obtain compound C2. Yellow solid, 78% yield. H
NMR (400 MHz, DMSO-d ) δ 8.74 (s, 1H), 8.02 (d, J = 9.3 Hz, 1H), 7.36
6
1
4
2
–
0
1
1
1
.32 (d, J = 13.0 Hz, 1H), 3.93 (d, J = 13.7 Hz, 1H), 3.15 (d, J = 7.4 Hz,
H), 3.07 (t, J = 12.0 Hz, 1H), 2.61 (t, J = 11.8 Hz, 1H), 1.87 (q, 2H), 1.77
1.56 (m, 3H), 1.30 (t, J = 7.6 Hz, 3H), 1.16 (d, J = 12.3 Hz, 1H), 1.00 –
(d, J = 9.3 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 6.08 – 5.94 (m, 1H), 5.42 (s,
2H), 5.25 (s, 2H), 5.06 – 4.97 (m, 2H), 4.24 (q, J = 7.1 Hz, 2H), 3.87 (d, J
= 5.3 Hz, 2H), 1.94 – 1.81 (m, J = 13.8, 6.8 Hz, 2H), 1.61 (s, 6H), 1.20 (t,
.86 (m, 7H). 13C NMR (100 MHz, DMSO-d
6
) δ 173.0, 165.6, 157.4, 157.3,
57.0, 150.5, 150.1, 146.7, 144.9, 144.1, 131.8, 128.7, 128.0, 122.6, 118.7,
04.0, 96.5, 72.9, 66.8, 65.7, 49.9, 45.0, 42.1, 34.8, 34.0, 30.8, 22.7, 22.1,
J = 7.1 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
6
) δ
+
3.9, 8.2. HRMS (ESI): m/z calcd for C30H34N3O6: 532.24476 [M+H] ;
173.8, 173.0, 157.3, 151.9, 151.1, 150.5, 146.0, 144.8, 136.6, 130.5, 129.1,
128.3, 128.0, 124.7, 122.5, 119.1, 116.3, 96.8, 80.1, 72.9, 65.7, 61.8, 50.9,
found: 532.24422.
3
0.7, 29.6, 25.9, 25.8, 14.4, 8.2. HRMS (ESI): m/z calcd for C29H31N2O7:
+
519.21313 [M+H] ; found: 519.21320.
(
S)-benzyl
4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-
1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl) carbonate (B8): To
a solution of benzyl alcohol (96 μL, 0.93 mmol) and DMAP (170.0 mg, 1.39
mmol) in DCM (15 mL) were added 4-nitrophenyl carbonochloridate (280.0
mg, 1.39 mmol) in DCM (10 mL) dropwise in an ice bath. After the addition
was complete, the reaction system was stirred at room temperature for 2
h and then the solvent was evaporated. Subsequently, SN-38 (110.0 mg,
(S)-10-allyl-4-ethyl-4-hydroxy-9-(2-methoxyethoxy)-1,12-dihydro-
14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (C3):
Compound C3 was prepared similarly as described for C2. Off-white solid,
1
49% yield. H NMR (400 MHz, DMSO-d
6
) δ 8.69 (s, 1H), 8.07 (d, J = 9.3
Hz, 1H), 7.77 (d, J = 9.4 Hz, 1H), 7.28 (s, 1H), 6.51 (s, 1H), 6.05 – 5.95 (m,
1H), 5.41 (s, 2H), 5.22 (s, 2H), 5.02 (d, J = 6.2 Hz, 1H), 4.99 (s, 1H), 4.33
(t, 2H), 3.84 (d, J = 5.8 Hz, 2H), 3.73 (t, 2H), 3.35 (s, 3H), 1.94 – 1.78 (m,
0.28 mmol), TEA (115 μL, 0.84 mmol) and DMF (3 mL) were added to the
residue. The mixture was stirred at room temperature overnight and
isolated using column chromatography (DCM:MeOH = 50:1) to obtain
2H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
6
) δ 173.0, 157.3,
compound B8. White solid, 83% yield. 1H NMR (400 MHz, DMSO-d
154.9, 150.6, 150.5, 146.1, 144.4, 136.7, 130.3, 129.6, 128.1, 127.5, 121.6,
119.5, 119.0, 116.1, 96.6, 72.9, 71.1, 69.1, 65.7, 58.8, 50.8, 30.7, 29.1,
6
) δ
8
1
2
.20 (d, J = 9.2 Hz, 1H), 8.15 (d, J = 2.4 Hz, 1H), 7.77 (dd, J = 9.2, 2.5 Hz,
+
H), 7.58 – 7.37 (m, 5H), 7.33 (s, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.34 (s,
H), 5.30 (s, 2H), 3.17 (q, J = 7.6 Hz, 2H), 1.89 (q, J = 7.3 Hz, 2H), 1.29 (t,
8.2. HRMS (ESI): m/z calcd for C26H27N2O6: 463.18691 [M+H] ; found:
463.18633.
J = 7.6 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
6
) δ
1
1
6
5
72.9, 157.2, 153.3, 152.6, 150.5, 149.8, 147.0, 146.3, 146.0, 135.4, 131.9,
ethyl
(S)-2-((4-ethyl-4-hydroxy-3,14-dioxo-10-propyl-3,4,12,14-
29.1, 129.1(3C), 128.9(2C), 127.5, 125.4, 119.6, 115.5, 97.2, 72.8, 70.6,
5.7, 50.0, 30.8, 22.7, 14.3, 8.2. HRMS (ESI): m/z calcd for C30H27N2O7:
tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-
yl)oxy)acetate (C4): Compound C4 was prepared similarly as described
for A2. Pale yellow solid, 73% yield. H NMR (400 MHz, DMSO-d
+
27.18183 [M+H] ; found: 527.18308.
1
6
) δ 8.75
(
s, 1H), 8.01 (d, J = 9.3 Hz, 1H), 7.63 (d, J = 9.4 Hz, 1H), 7.28 (s, 1H), 6.50
(
S)-10-allyl-4-ethyl-4,9-dihydroxy-1,12-dihydro-14H-
(s, 1H), 5.41 (s, 2H), 5.22 (s, 2H), 5.03 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H),
3.08 (t, 2H), 1.93 – 1.80 (m, 2H), 1.63 (dq, J = 15.0, 7.4 Hz, 2H), 1.23 (t, J
pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione
7
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000753
FULL PAPER
=
(
7.1 Hz, 3H), 1.01 (t, J = 7.3 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR
100 MHz, DMSO-d ) δ 173.0, 169.3, 157.3, 153.9, 150.7, 150.5, 146.1,
153.3, 152.8, 150.4, 147.6, 147.0, 145.7, 135.6, 135.4, 130.8, 129.7, 129.4,
129.1, 129.1, 128.9, 128.8, 128.7, 127.7, 127.3, 126.4, 119.7, 117.0, 97.2,
72.8, 70.7, 65.7, 50.9, 30.8, 29.6, 8.2. HRMS (ESI): m/z calcd for
6
144.5, 130.3, 128.9, 128.1, 127.6, 124.6, 119.0, 118.5, 96.6, 72.9, 66.0,
+
65.7, 61.2, 50.8, 30.7, 27.0, 23.4, 14.6, 14.5, 8.2. HRMS (ESI): m/z calcd
C31H27N2O7: 539.18183 [M+H] ; found: 539.18168.
+
for C27H29N2O7: 493.19748 [M+H] ; found: 493.19703.
(
S)-10-allyl-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
ethyl
(S)-2-((4-ethyl-4-hydroxy-3,14-dioxo-10-propyl-3,4,12,14-
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl (4-bromobenzyl)
tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)oxy)-2-
methylpropanoate (C5): Compound C5 was prepared similarly as
described for A2. Off-white solid, 89% yield. H NMR (400 MHz, DMSO-
carbonate (C10): Compound C10 was prepared similarly as described for
1
6
B8. White solid, 61% yield. H NMR (400 MHz, DMSO-d ) δ 8.87 (s, 1H),
1
8.13 (d, J = 9.2 Hz, 1H), 7.79 (d, J = 9.2 Hz, 1H), 7.64 (d, J = 8.3 Hz, 2H),
7.45 (d, J = 8.3 Hz, 2H), 7.34 (s, 1H), 6.54 (s, 1H), 5.93 (dq, J = 10.6, 5.9
Hz, 1H), 5.43 (s, 2H), 5.28 (d, J = 14.2 Hz, 4H), 4.96 (q, 2H), 3.80 (d, J =
5.1 Hz, 2H), 1.96 – 1.78 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR (100
d
(
3
(
6
) δ 8.78 (s, 1H), 7.98 (d, J = 9.3 Hz, 1H), 7.32 (d, J = 9.4 Hz, 1H), 7.29
s, 1H), 6.51 (s, 1H), 5.42 (s, 2H), 5.26 (s, 2H), 4.23 (q, J = 7.0 Hz, 2H),
.06 (t, 2H), 1.95 – 1.79 (m, 2H), 1.63 (s, 8H), 1.18 (t, J = 7.1 Hz, 3H), 1.02
t, J = 7.2 Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d
δ 173.8, 173.0, 157.3, 151.7, 150.9, 150.5, 146.1, 144.8, 130.3, 128.5,
6
)
6
MHz, DMSO-d ) δ 172.9, 157.2, 153.2, 152.8, 150.4, 147.5, 147.0, 145.7,
135.6, 134.9, 132.0(2C), 131.0(2C), 130.8, 129.7, 129.4, 127.7, 127.3,
126.3, 122.3, 119.7, 117.0, 97.2, 72.8, 69.8, 65.7, 50.9, 30.8, 29.6, 8.2.
128.3, 127.9, 127.4, 122.1, 119.1, 96.7, 79.8, 72.6, 65.7, 61.8, 50.9, 30.7,
+
2
7.4, 25.9, 25.9, 23.6, 14.6, 14.3, 8.2. HRMS (ESI): m/z calcd for
HRMS (ESI): m/z calcd for C31H26BrN2O7: 617.09234 [M+H] ; found:
+
617.09197 (79Br), 619. 090580 (81Br).
C29H33N2O7: 521.22878 [M+H] ; found: 521.22858.
(
S)-10-allyl-4-ethyl-4-hydroxy-9-(2-oxo-2-(piperidin-1-yl)ethoxy)-1,12-
(S)-10-allyl-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-
dione (C6): Compound C6 was prepared similarly as described for B7.
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl
benzylcarbamate
(C11): Compound C11 was prepared similarly as described for B8. Off-
1
The hydrolysate of C1 was not purified, and condensed with piperidine to
white solid, 52% yield. H NMR (400 MHz, DMSO-d
6
) δ 8.83 (s, 1H), 8.56
1
obtain C6. Pale yellow solid, 69% yield. H NMR (400 MHz, DMSO-d
6
) δ
(t, J = 6.1 Hz, 1H), 8.07 (d, J = 9.1 Hz, 1H), 7.67 (d, J = 9.2 Hz, 1H), 7.42
– 7.35 (m, 4H), 7.34 – 7.26 (m, 2H), 6.53 (s, 1H), 5.98 (dq, J = 10.5, 6.0
Hz, 1H), 5.43 (s, 2H), 5.27 (s, 2H), 5.05 (d, J = 9.9 Hz, 1H), 5.02 (s, 1H),
4.34 (d, J = 6.0 Hz, 2H), 3.83 (d, J = 5.5 Hz, 2H), 1.93 – 1.82 (m, 2H), 0.90
8
6
2
3
.68 (s, 1H), 8.04 (d, J = 9.3 Hz, 1H), 7.64 (d, J = 9.5 Hz, 1H), 7.28 (s, 1H),
.50 (s, 1H), 6.12 – 5.95 (m, J = 11.7, 5.9 Hz, 1H), 5.41 (s, 2H), 5.21 (s,
H), 5.09 (s, 2H), 5.03 (dd, J = 21.8, 5.8 Hz, 2H), 3.88 (d, J = 5.2 Hz, 2H),
.44 (s, 4H), 1.94 – 1.79 (m, 2H), 1.59 (s, 4H), 1.46 (s, 2H), 0.89 (t, J = 7.3
(t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
) δ 172.9, 157.3, 155.1,
6
Hz, 3H). 13C NMR (100 MHz, DMSO-d
152.2, 150.5, 148.0, 146.7, 145.9, 139.6, 136.0, 130.5, 129.1, 128.9(3C),
128.7, 127.8, 127.5(2C), 127.5(2C), 119.5, 117.0, 97.1, 72.9, 65.7, 50.9,
44.6, 30.8, 30.0, 8.3. HRMS (ESI): m/z calcd for C31H28N3O6: 538.19781
6
) δ 173.0, 165.9, 157.3, 154.6,
1
1
2
50.5, 150.5, 150.0, 146.1, 144.3, 136.7, 130.3, 129.3, 128.1, 127.4, 121.0,
18.9, 116.1, 96.6, 72.9, 67.2, 65.7, 50.8, 45.5, 42.7, 30.7, 29.1, 26.4, 25.7,
4.4, 8.3. HRMS (ESI): m/z calcd for C30H32N3O6: 530.22911 [M+H]+;
+
[M+H] ; found: 538.19771.
found: 530.22888.
Solubility determination. B7, C11 or SN-38 was supersaturated in water,
acetate buffer (pH 4.5), or phosphate buffer (pH 7.4) and equilibrated for
24 h at room temperature with shaking. Mixtures were centrifuged for 8
min at 13000 r/min. Supernatants were analyzed by HPLC and quantified
using standard curves generated with pure B7, C11 and SN-38. All
samples underwent isometric elution on a 5 mm Kromasil 100-5-C18
column (4.6 × 150 mm) at 40℃. Eluent composed of 70% methanol and
30% water (0.1% acetic acid) was run at a flow rate of 1 mL/min. Then the
outflows were detected by the ultraviolet detector at 254 nm.
(
S)-10-allyl-4-ethyl-4-hydroxy-9-(2-morpholino-2-oxoethoxy)-1,12-
dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-
dione (C7): Compound C7 was prepared similarly as described for B7.
White solid, 51% yield. H NMR (400 MHz, DMSO-d
1
6
) δ 8.68 (s, 1H), 8.04
(d, J = 9.4 Hz, 1H), 7.66 (d, J = 9.5 Hz, 1H), 7.28 (s, 1H), 6.50 (s, 1H), 6.09
–
5.99 (m, J = 10.3, 6.0 Hz, 1H), 5.41 (s, 2H), 5.21 (s, 2H), 5.12 (s, 2H),
.02 (t, 2H), 3.88 (d, J = 5.4 Hz, 2H), 3.62 (d, J = 19.4 Hz, 4H), 3.55 – 3.44
5
(
m, 4H), 1.93 – 1.80 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz,
DMSO-d ) δ 173.0, 166.5, 157.3, 154.6, 150.5, 150.5, 146.1, 144.4, 136.7,
30.3, 129.3, 128.1, 127.4, 121.1, 119.0, 119.0, 116.1, 96.6, 72.9, 67.0,
6
1
6
Cell culture and MTT assay. The tested six human tumor cell lines
including human colon carcinoma cell lines (HCT-116, Lovo, Colo205 and
HT-29), human lung adenocarcinoma epithelial cell line (A549) and human
liver carcinoma cell line (HepG2) were purchased from Shanghai institute
of life sciences, Chinese Academy of Sciences. All cancer cells were
maintained in DMEM medium supplemented with 10% heat inactivated
fetal bovine serum (FBS) and incubated in a humidified atmosphere
6.5, 66.5, 65.7, 50.8, 45.1, 42.1, 30.7, 29.1, 8.3. HRMS (ESI): m/z calcd
+
for C29H30N3O7: 532.20838 [M+H] ; found: 532.20819.
(
1
3
S)-4-ethyl-4-hydroxy-9-(2-oxo-2-(piperidin-1-yl)ethoxy)-10-propyl-
,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-
,14(4H)-dione (C8): Compound C8 was prepared similarly as described
1
for A2. Off-white solid, 89% yield. H NMR (400 MHz, DMSO-d
6
) δ 8.74 (s,
2
containing 5% CO at 37℃. 10-HCPT (Cool Pharm, Ltd., purity: >98%)
1
H), 8.00 (d, J = 9.3 Hz, 1H), 7.59 (d, J = 9.4 Hz, 1H), 7.28 (s, 1H), 6.50
and SN-38 (Energy Chemical, purity: 98%) were used as positive controls.
Cellular chemosensitivity was determined by using a modified MTT as we
described.[19] Cells were simultaneously treated with final concentrations
of 1.6, 8, 40, 200, 1000, 5000 nM of tested compounds. The IC50 value,
that is, the concentration (μM) of a compound was able to cause 50% cell
death with respect to the control culture, was calculated according to the
inhibition ratios.
(
(
7
1
1
2
s, 1H), 5.42 (s, 2H), 5.23 (s, 2H), 5.07 (s, 2H), 3.45 (s, 4H), 3.12 – 3.02
m, 2H), 1.87 (m, 2H), 1.60 (d, J = 6.5 Hz, 6H), 1.47 (s, 2H), 1.01 (t, J =
.2 Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, DMSO-d
) δ
6
73.0, 166.0, 157.3, 154.5, 150.5, 150.4, 146.2, 144.3, 130.2, 128.7, 128.1,
27.4, 124.0, 119.0, 118.7, 96.6, 72.9, 67.1, 65.7, 50.8, 45.6, 42.7, 30.7,
7.0, 26.5, 25.7, 24.4, 23.4, 14.7, 8.3. HRMS (ESI): m/z calcd for
+
C30H34N3O6: 532.24476 [M+H] ; found: 532.24440.
Colony formation assay. A549 cells were seeded in a six-well plate (300
cells/well), incubated for 24 h and then treated with various concentrations
of B7 or SN-38 for 10 days until colonies were visible. The medium with
drugs were discarded, cells were washed with PBS and then fixed with
methanol for 30 min. Then cells were washed with double distilled water
twice and then dyed with 0.1% crystal violet for 30 min. Afterwards, cells
were washed with water to wipe off the residual stain. The colony
formations were imaged and counted.
(
S)-10-allyl-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-
pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl benzyl carbonate (C9):
Compound C9 was prepared similarly as described for B8. White solid,
6
1
9% yield. H NMR (400 MHz, DMSO-d
6
) δ 8.87 (s, 1H), 8.12 (d, J = 9.2
Hz, 1H), 7.79 (d, J = 9.2 Hz, 1H), 7.52 – 7.37 (m, 5H), 7.34 (s, 1H), 6.54
s, 1H), 5.93 (m, J = 10.3, 5.9 Hz, 1H), 5.43 (s, 2H), 5.33 (s, 2H), 5.26 (s,
(
2
0
H), 5.02 – 4.88 (m, 2H), 3.80 (d, J = 5.5 Hz, 2H), 1.87 (q, J = 7.2 Hz, 2H),
.90 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d
) δ 172.9, 157.2,
6
8
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000753
FULL PAPER
Cell cycle analysis. A549 cells were plated into 6-well plate (1×106
cells/well) and incubated for 24 h and then incubated with various
concentrations of B7 or SN-38 for 24 h. The controls were treated with
vehicle (0.1% DMSO). The cells were then harvested and fixed with 70%
ice-cold ethanol overnight at 4℃. After centrifugation, the cell pellets were
treated with propidium iodide (5 mg/mL) and RNase A in PBS (100 mg/mL)
in PBS for 15 min at room temperature. DNA was observed and analyzed
with a flow cytometer. The independent experiment was repeated three
times.
Conflict of interest
There is no interest conflict to declare.
Keywords: camptothecin derivatives • drug design • cytotoxicity
•
solubility • anti-tumor effect
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8
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Acknowledgements
The financial support from The National Natural Science
Foundation of China (Grant No. 81673354) is gratefully
acknowledged.
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9
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000753
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Entry for the Table of Contents
Twenty-seven camptothecin derivatives were synthesized and cytotoxicity screened. Compound B7, 7-ethyl-10-(2-oxo-2-(4-
methylpiperidin-1-yl)ethoxy)camptothecin, was demonstrated in vitro and in vivo to be a potent antitumor agent. And B7 exhibited
minimum lethal doses comparable to irinotecan. Additionally, the solubility of B7 reached 5.73 μg/mL in water. These results indicated
that B7, with improved solubility, enhanced activity and acceptable acute toxicity, can be used as a lead compound for development of
novel anti-cancer agent.
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