Synthesis and antitumor activity of a series of lactone-opened camptothecin derivatives

Article abstract of DOI:10.1080/10286020.2017.1392941

A series of E-ring lactone-opened camptothecin (CPT) derivatives bearing with terminal aza-heterocyclic groups were synthesized, and their antitumor activity was evaluated both in vitro and in vivo. Hydroxyl-amide analogues with morpholin-4-yl displayed excellent antitumor activity in vitro and efficient inhibition on tumor xenograph model in nude mice. Ester-amide compounds acted less active in vitro cytotoxicity and lower inhibition activity in vivo. Substitutions at 7- and 10- positions favored the antitumor activity.

Full text of DOI:10.1080/10286020.2017.1392941

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Synthesis and antitumor activity of a series of
lactone-opened camptothecin derivatives
Chao Zheng, Ming-Zong Li, Tian-Pa You, Wei-Ping Tang & Li-Guang Lou
To cite this article: Chao Zheng, Ming-Zong Li, Tian-Pa You, Wei-Ping Tang & Li-Guang Lou
(2017): Synthesis and antitumor activity of a series of lactone-opened camptothecin derivatives,
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2017.1392941
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1392941
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Date: 28 October 2017, At: 17:16

Journal of asian natural Products research, 2017
https://doi.org/10.1080/10286020.2017.1392941
Synthesis and antitumor activity of a series of lactone-opened
camptothecin derivatives
Chao Zhenga#, Ming-Zong Lib#, Tian-Pa Youb, Wei-Ping Tangc and Li-Guang Louc
aKey laboratory of tropical Medicinal Plant chemistry of Ministry of education, collaborative innovation
center of tropical Biological resources, hainan normal university, haikou 571158, china; bdepartment of
c
chemistry, university of science & technology of china, hefei 230026, china; shanghai institute of Materia
Medica, chinese academy of science, shanghai 201203, china
ABSTRACT
ARTICLE HISTORY
received 21 May 2017
accepted 11 october 2017
A series of E-ring lactone-opened camptothecin (CPT) derivatives
bearing with terminal aza-heterocyclic groups were synthesized,
and their antitumor activity was evaluated both in vitro and in vivo.
Hydroxyl-amide analogues with morpholin-4-yl displayed excellent
antitumor activity in vitro and efficient inhibition on tumor xenograph
model in nude mice. Ester-amide compounds acted less active in vitro
cytotoxicity and lower inhibition activity in vivo. Substitutions at 7-
and 10- positions favored the antitumor activity.
KEYWORDS
camptothecin derivatives;
e-ring lactone-opened;
synthesis; antitumor activity
R1
R1
R1
R2
R2
R2
O
O
O
N
OCOR4
NH(CH2
N
N
OH
NH(CH2
N
N
N
3R3
O
)
3R3
)
OH
OH
O
O
OH
O
N
R3
=
N
O
,
N
1. Introduction
Camptothecin (CPT, 1, Figure 1), an natural antitumor alkaloid first isolated from Chinese
tree Camptotheca acuminate by Wall and Wani in 1966 [1], has been drawing long-lasting
interests. As potent inhibitors of DNA Topoisomerase I [2], CPT and some of its analogues
form so-called “cleavable complex” with Topo I and DNA [3], which prevents DNA relega-
tion and induces Topo I-mediated DNA breaks. ree CPT analogues, irinotecan (2) [4],
topotecan (3) [5], and belotecan (4) [6], have been approved as chemotherapeutic drugs for
treatment of human cancers in clinical and several other derivatives are in different phases
of clinical developments [7–9].
e effect of phosphate groups on the hydrolysis of E-ring has been investigated by using
UV-visible RP-HPLC (reverse-phase high-performance liquid chromatography) assay and
kinetic solvent isotope effect (KSIE). It was concluded that P-CPT had a pH-dependent
CONTACT chao Zheng
zhengchao@hainnu.edu.cn; tian-Pa You
ytp@ustc.edu.cn
#these authors contribute equally.
© 2017 informa uK limited, trading as taylor & francis Group

2
C. ZHENG ET AL.
R2 R1
9
7
R3
10
O
A
B
N
C N
17
O
D
E
OH O
3
1 Camptothecin(CPT):R 1 =R 2 =R =H;
3
Irinotecan:R 1 =Et,R 2 =H,R
=
;
2
N
NCOO
3 Topotecan:R 1 =H,R 2 =Me 2NCH2-,R 3 =OH;
Belotecan:R 1 =Me 2CHNHCH2CH2-,R 2 =R 3 =H.
4
Figure 1. cPt and its famous derivatives.
equilibrium between lactone and carboxylate forms, similar to but not identical to CPT.
But the early clinical trails with E-ring sodium salt of CPT (1a, Figure 2) were failed due to
its low activity in vivo and unexpected side effects [10]. So intact lactone E-ring was once
considered to be indispensable to maintain antitumor activity for CPT analogues [11,12].
However, further investigations on lactone-opened form of CPT derivatives demonstrated
that E-ring-opened hydroxyl-amide compounds (5, Figure 2) or ester-amide compounds
(6) were found to have comparable activity with CPT against human tumor cell lines P388,
L1210, and B16 in vivo [13]. More recently, some CPT derivatives with lactone-opened
ester-amide, conjugating with PEG (polyethylene glycol) through the C-21 acid function-
ality or through the 17-hydroxy group of CPT (7, 8), were also exhibited high activity
against a mammary carcinoma (MX-1) xenograph in nude mice [14]. Furthermore, the
O
O
O
N
N
OCOR4
NHR3
N
OH
OH
N
N
N
O-Na+
NHR3
O
OH
O
OH
OH O
1a
5
6
hydroxyl-amide
ester-amide
H
N
H
N
O
O
O
O
O
O
PEG
N
N
O
O
O
O
N
N
O
O
O
O
HN
NH
OH
HO
Me2N
NMe2
7
O
O
O
O
N
N
O
O
N
N
O
O
O
O
HN
NH
OH
HO
O
PEG
O
O
O
N
O
O
N
H
H
8
Figure 2. cPt derivatives with lactone-opened.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
structure–activity relationship studies indicated that substitutions at 7- and 10-positions of
CPT would enhance the antitumor activity [15]. All these survey implied the great potential
in developing novel lactone-opened form of CPT derivatives.
In this paper, we firstly introduced aza-heterocyclic groups such as morpholin-4-yl or
imidazol-1-yl to a series of CPT hydroxyl-amide and ester-amide analogues (11a, 11b,
and 12a–d, Scheme 1). We hoped that these amide groups not only could be converted to
amine salts and hence to enhance their aqueous solubility, but also could offer additional
hydrogen-bonding sites for biological macromolecules, both of which may increase their
biological activity.
2. Chemistry
Synthesis of hydroxyl-amides (11) and ester-amides (12) derivatives is summarized in
Scheme 1. Camptothecin was treated with excess of amines (3-morpholin-4-yl-propylamine
or 3-imidazol-1-yl-propylamine) at 60 °C to give hydroxyl-amides 11. Selective acylation of
17-hydroxy was processed with anhydride-pyridine condition and yielded the 17-esters (12).
Preliminary in vitro antitumor activity evaluation towards compounds 11a-b and 12a-d pro-
vided instructive information for subsequent structure-activity relationships study, which
secured access to the synthesis of compounds 11c-d and 12e-f based on the above mentioned
procedure. e compound 7-cyclohexyl-CPT (9) was prepared with Sawada method [4], and
7-ethyl-10-methoxyl-CPT (10) was obtained from methylation of 7-ethyl-10-hydroxy-CPT
with iodomethane and anhydrous potassium carbonate in refluxing acetone.
Hydroxyl-amides (11a–d) were found to be difficult to purify [13] for being unstable in
acidic or neutral medium. 11a indeed was observed to turn back to CPT in CHCl3 solution.
However, afer procedures optimization, it could be obtained as solid with up to 97.4%
purity (2.1% of CPT as impurity, according to the high-performance liquid chromatography
(HPLC) analysis) in 2 g scale. 11a was stable enough in aqueous solution (5 mg 11a in 1 ml
water, the pH is 10.36), and no more CPT was observed afer 40 h in this solution based
on HPLC analysis. When the compound was dissolved in PBS (phosphate buffer solution,
pH = 7.03), about a half of CPT generated afer 40 h. Compound 12b, which is more stable,
can be purified on a silica gel column to get the product with up to 99.3% purity.
3. Results of biological assay and discussion
e primary test for inhibition rate of compounds 11a–b and 12a–d was performed
on four human cancer cell lines HL-60, A549, BGC-823, and SMMC-7721 by the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay, using mitomycin
and cisplatin as references. e results are summarized in Table 1. Cell lines HL-60 and
A549 are more sensitive to these CPT analogues than cell lines BGC-823 and SMMC-7721.
Hydroxyl-amides 11a and 11b generally showed much higher activity than their esterified
counterparts. Derivatives substituted with morpholin-4-yl have higher inhibition rates than
that with imidazol-1-yl on most of the cell lines. Among the ester-amides, propionate com-
pounds displayed more active than acetate analogues (12b vs. 12a, 12d vs. 12c) in inhibiting
the tumor cell lines. Based on this preceding observation, modification of 7, 10-substituted
CPTs (compounds 9 and 10) was focused on morpholin-4-yl and 17-propionate derivatives.

4
C. ZHENG ET AL.
O
O
N
N
i
N
N
O
O
CPT
9
OH
OH
O
O
HO
MeO
O
O
N
N
ii
N
N
O
O
SN-38
10
OH
OH
O
O
R1
R1
R2
R2
O
O
N
N
OH
NH(CH2)3R3
iii
N
N
O
O
1,9,10
11
OH
OH
O
3
3
:R 1 =R 2 =H,R
1
=
=
11a
N
O
N
11b:R =R 2 =H,R
N
3
:R 1 = c-C5H9,R 2 =H,R
=
N
O
11c
1
2
3
11d:R =Et,R =OMe,R
=
N
O
R1
R2
O
N
OCOR4
NH(CH2)3R3
O
iv
N
12
OH
N
3
:R 1 =R 2 =H,R
1
=
=
=
=
,R 4 =Me
O
12a
3
3
3
12b:R =R 2 =H,R
,R 4 =Et
O
N
N
N
N
:R 1 =R 2 =H,R
1
, R4 =Me
12c
N
12d:R =R 2 =H,R
, R4 =Et
3
4
12e:R 1 = c-C6H11,R 2 =H,R
=
,R =Et
N
O
3
:R 1 =Et,R 2 =OMe,R
=
N
O
,R 4 =Et
12f
Scheme 1. General chemical procedure. reagents and conditions (i): cyclohexanecarbaldehyde, h2o2,
h2so4, hoac-h2o, 0 °c, 3 h; (ii): Mei, K2co3, acetone, reflux over night; (iii) amine, 60 °c; iv: (r4co)2o, Py,
40 °c.
e IC50 values of selected compounds against human cancer cell lines A549/ATCC
and HT29 were listed in Table 2. On these two cell lines, activity of hydroxyl-amide com-
pounds 11 displayed more active than references CPT, SN38, and topotecan, whereas only
compound 12f has comparable activity with the reference drugs for the ester-amide com-
pounds. 7- and 10-substitutions were performed to enhance the activity, particularly on
HT29 cell lines.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Table 1. inhibition rate of lactone-opened cPt derivatives against four human tumor cell lines.
Percentage of tumor cells inhibitiona (%)
compds
Conc. (M)
BGC-823
HL-60
A549
SMMC-7721
11a
1 × 10−7
1 × 10−6
1 × 10−5
1 × 10−7
1 × 10−6
1 × 10−5
1 × 10−7
1 × 10−6
1 × 10−5
1 × 10−7
1 × 10−6
1 × 10−5
1 × 10−7
1 × 10−6
1 × 10−5
1 × 10−7
1 × 10−6
1 × 10−5
1 × 10−7
1 × 10−6
1 × 10−5
1 × 10−7
1 × 10−6
1 × 10−5
31.28
44.70
50.66
33.20
35.72
63.57
19.78
25.53
24.72
12.51
29.16
53.48
26.94
19.27
26.84
19.78
11.81
17.76
70.41
91.82
97.32
55.72
85.50
94.01
1.41
49.88
89.68
28.91
80.54
95.86
5.35
53.88
87.35
99.59
17.14
85.51
96.94
12.24
31.63
87.35
41.84
85.71
95.71
5.71
12.24
63.27
6.73
28.37
90.20
18.42
41.93
89.46
nt
27.63
17.40
33.45
29.62
33.88
53.91
19.03
19.74
17.26
23.30
22.94
55.33
13.14
14.70
17.26
14.91
21.38
4.05
11b
12a
12b
12c
26.28
74.21
0.00
58.35
91.48
12d
Mitomycin
cisplatin
5.03
ntb
nt
49.14
61.68
nt
11.00
40.90
92.94
24.82
46.37
96.79
aevaluation standard of antitumor activity: high activity (inhibition rate % ≥ 50 in conc. 1 × 10−7 M, ≥ 50 in conc. 1 × 10−6 M,
≥ 50 in conc. 1 × 10−5 M); moderate (inhibition rate % < 50 in conc. 1 × 10−7 M, ≥ 50 in conc. 1 × 10−6 M, ≥ 50 in conc.
1 × 10−5 M); low activity (inhibition rate % < 50 in conc. 1 × 10−7 M, < 50 in conc. 1 × 10−6 M, ≥ 50 in conc. 1 × 10−5 M); poor
activity (inhibition rate % < 50 in conc. 1 × 10−7 M, < 50 in conc. 1 × 10−6 M, < 50 in conc. 1 × 10−5 M).
bnt = no tested.
Table 2. cytotoxicity of 7- and 10- modified cPt analogues on human cell lines a549/atcc and ht29.
IC50a (μM)
IC50a (μM)
Compounds
A549/ATCC
HT29
Compds
A549/ATCC
HT29
11a
11c
11d
sn38
cPt
0.03
0.01
0.02
0.06
0.05
0.05
0.01
0.02
0.08
0.12
12a
12e
2.88
1.06
0.2
2.93
1.09
0.27
0.23
12f
topotecan
0.5
aMeasured with sulphorhodamine (srB) method.
Tables 3 and 4 depict the in vivo antitumor efficacy of hydroxyl-amide 11a and ester-
amide 12a, respectively. Compound 11a was tested in a human tumor xenograf model
(A549 cell line, Table 3) in mice using HCPT (10-Hydroxylcamptothecin) as reference drug.
e result indicated that compound 11a had better ability in inhibiting the growth of the
transplanted tumor than HCPT under the same conditions. No mice died in this series of
treatments. Compound 12a was treated on transplanted HT-29 tumor cell line with irino-
tecan as reference. It could markedly inhibit the growth of the tumor, but displayed higher
toxicity (several mice died in the treatment) and lower efficacy in reducing tumor growth
compared with irinotecan.
e activity of lactone-opened hydroxyl-amides was once ascribed to the reform of CPT.
However, according to our investigation, only less than 50% of compound 11a turned back

6
C. ZHENG ET AL.
Table 3. antitumor efficacy of compound 11a against mouse tumor a549 using intravenous injection
(iv).
TVb(X SD, mm3)
Lethalaa
toxicity
Tumor
Compounds Dose(mg/kg)
D0
Dn
RTVc X SD
T/Cd (%)
a549
ns
hcPt
11a
11a
11a
–
8
10
5
0/10
0/5
0/5
0/5
0/5
128 71
131 54
132 134
126 72
120 23
390 342
258 137
211 237
407 365
200 73
2.8 1.0
2.0 0.5
1.5 0.4
2.9 1.3
1.7 0.5
71.4
53.6*
103.6
60.7*
2.5
anumber of dead mice/total number of mice in this series of treatment for 17 days.
btumor volume (tV) of d0 (the day when the chemicals were given) and dn (the day of the optimal treatment, which was the
17th day in the experiments), tV = 1/2 × a× b 2 (in which a is the length of tumors, b is the width of tumors).
crtV is the relative tumor volume, rtV = Vn/V0 (in which V0 is the tV at the day when the chemicals were given and Vn is the
tV of final measurement after 17 days).
dthe evaluation index of antitumor activity was relative tumor growth ratio t/c (%). t/c = trtV/crtV × 100%. (trtV: tested
group’s and positive control group’s rtV; crtV: common negative control group’s rtV).
*p < 0.01 vs. hcPt.
Table 4. antitumor efficacy of compound 12a against mouse tumor ht29 using intravenous injection
(iv).
TVb (X SD, mm3)
Dose(mg/
kg)
Lethalaa
toxicity
Tumor
Compounds
D0
D21
RTVc X SD
T/Cd (%)
ht-29
ns
12a
–
0/10
0/5
3/5
1/5
0/5
0/5
0/5
230 43
218 16
204 27
209 27
219 23
218 22
210 23
1101 287
638 129
486 377
373 153
685 111
541 164
587 222
4.85 1.21
2.98 0.78
2.12 1.70
1.85 0.69
3.19 0.79
2.48 0.69
2.74 0.87
10
20
40
5
10
20
61.4*
43.7*
38.1*
65.8*
51.1*
56.5*
12a
12a
irinotecan
irinotecan
irinotecan
anumber of dead mice/total number of mice in this series of treatment for 21 days.
btumor volume (tV) of d0 (the day when the chemicals were given) and dn (the day of the optimal treatment, which was the
21st day in the experiments), tV = 1/2 × a× b2 (in which a is the length of tumors, b is the width of tumors).
crtV is the relative tumor volume, rtV = Vn/V0 (in which V0 is thetV at the day when the chemicals were given and Vn is the
tV of final measurement after 21 days).
dthe evaluation index of antitumor activity was relative tumor growth ratio t/c (%).t/c = trtV/crtV × 100%. (trtV: tested
group’s and positive control group’s rtV; crtV: common negative control group’s rtV).
*p < 0.01 vs. ns.
to CPT within 40 h in PBS, while the activity of 11a both in vitro and in vivo presented to
be superior to CPT itself (even superior to reference drug HCPT which is considered to be
more efficient than CPT). So, we have reason to believe that hydroxyl-amide 11a does not
only play as a pre-drug of CPT, but also exhibit its own intrinsical antitumor property. It
implies that compound 11a is worthy of further study.
Taken together, a series of E-ring lactone-opened CPT analogues were prepared, and
their biological activity was evaluated. Hydroxyl-amide analogues with terminal morpho-
lin-1-yl group and ester-amide compounds with propionate presented higher inhibition
rate than their congeners. Substitutions at 7- and 10- positions usually enhance the activity.
e superiority of biological evaluation of compound 11a indicated that it had distinct
antitumor property known from CPT itself.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
4. Experimental
IR spectra were obtained on Bruker Equinox 55 spectrophotometer (ermo Scientific,
Massachusett America); NMR spectra were recorded on Bruker 300-MHz or 400-MHz
spectrometer (Bruker Biospin, Billerica America); Chemical shifs (δ values) and coupling
constants are given in ppm and Hz, respectively. MS (ESI) were recorded on a ermo
Finnigan TSQ Quantum Ultra AM mass spectrometer (ermo Finnigan, State of California
America). All reagents and solvents were reagent grade or were purified by standard meth-
ods before use.
4.1. Synthesis of compounds
4.1.1. Synthesis of 7-cyclohexylcamptothecin (9) with Sawada method
To a stirred solution of CPT (35 mg, 0.1 mmol) cooled with ice bath in acetic acid (AcOH)
(1.5 ml), deionized water (1.5 ml) and conc. H2SO4 (0.4 ml) was added cyclohexanecarbal-
dehyde (0.3 mmol) and 30% H2O2 (0.3 mmol). e stirring was continued for additional
3 h. e reaction mixture was then diluted with ice water (6 ml) and extracted with CH2Cl2
(10 ml×3). Combined organic layers were dried on Na2SO4, evaporated under reduced
pressure, and purified on silica gel column chromatography (Eluent: CHCl3~CHCl3/
MeOH = 100/1) to give a yellow powder in 49% yield; 1H NMR (300 MHz, CDCl3, TMS)
δ: 1.20 (t, J = 6.9 Hz, H3-18), 1.43-2.03 (m, H10-cyclohexyl and H2-19), 3.65 (br, H1-OH),
3.73–3.75 (m, H1-cyclohexyl), 5.31 (d, J = 16.5 Hz, H1-17), 5.43 (s, H2-5), 5.76 (d, J = 16.5 Hz,
H1-17), 7.68 (t, J = 7.7 Hz, H1-10), 7.78 (s, H1-14), 7.81 (t, J = 8.1 Hz, H1-11), 8.28–8.30
(m, H1-9 and H1-12); MS (ESI): m/z 431.2[M+H]+.
4.1.2. Synthesis of 7-ethyl-10-methoxylcamptothecin
To a suspension of 7-ethyl-10-hydroxyl-camptothecin (SN38, 100 mg, 0.28 mmol) in 10 ml
acetone, MeI (0.1 ml, 6 equiv.) and K2CO3 (190 mg, 5equiv.) were added. e reaction
mixture was stirred and heated to reflux over night. Afer cooled to room temperature,
the reaction mixture was then diluted with ice water (15 ml) and extracted with CH2Cl2
(10 ml×3). Combined organic layers were dried on Na2SO4, evaporated and purified on
silica gel column chromatography (Eluent: CHCl3~CHCl3/MeOH = 100/1) to give a yel-
1
low powder in 75% yield; H NMR (300 MHz, DMSO-d6 : CDCl3=1:10, TMS) δ: 0.96 (t,
J = 7.3 Hz, H3-18), 1.39 (t, J = 7. 5 Hz, H3-7-Et), 1.88-1.90 (m, H2-19), 3.21 (q, J = 7.5 Hz, H2-
7-Et), 4.00 (s, H3-10-OMe), 5.26 (s, H2-5), 5.29 (d, J = 15.6 Hz, H1-17), 5.54 (d, J = 15.6 Hz,
H1-17), 7.39-7.44 (m, H1-14, H1-11), 7.95 (s, H1-9), 8.06 (d, J = 9.0 Hz, H1-12). MS (ESI):
m/z 407.3 [M+H]+.
4.1.3. General procedure for synthesis of hydroxyl-amides (11a-d)
Suspension of CPTs (1, 9, or 10) in amine (3-morpholin-4-yl-propylamine or 3-imida-
zol-1-yl-propylamine, 5 ml amine per 1 g CPTs) was stirred at 60 °C for 1.5 h (or until total
conversion of CPTs according to TLC analysis) under N2 atmosphere, the clear mixture
was then cooled to room temperature, saturated with diethyl ether (Et2O), and stored in
refrigerator at −4 °C over night then filtrated, and the solid was dried to give hydroxyl-amide.

8
C. ZHENG ET AL.
4.1.3.1. Compound 11a. Yellow powder, yield in 80%; IR (KBr, cm−1) ν: 1651 (CONH); 1H
NMR (300 MHz, CDCl3, TMS) δ: 1.07 (t, J = 7.2 Hz, H3-18), 1.75–1.77 (m, H2-CH2CH2CH2),
2.24–2.26 (m, H1-19), 2.42–2.44 (m, H1-19), 2.43–2.45 (m, H6-NCH2), 3.40–3.42 (m, H2-
CONHCH2), 3.74–3.76 (m, H4-OCH2), 4.88 (d, J = 12.7 Hz, H2-17), 5.04 (s, H2-5), 7.47
(t, J = 8.4 Hz, H1-10), 7.48 (s, H1-14), 7.56 (d, J = 7.2 Hz, H1-9), 7.67 (t, J = 5.2 Hz, H1-11),
7.87 (s, H1-7), 7.97 (d, J = 8.5 Hz, 1H), 8.20 (br, H1-CONH). MS (ESI): m/z 493.4 [M+H]+.
4.1.3.2. Compound 11b. Yellow powder, yield in 93%; IR (KBr, cm−1) ν: 1651 (CONH);
1H NMR (CDCl3, 300 MHz, TMS) δ: 1.06 (t, J = 6.4 Hz, H3-18), 2.04–2.06 (m, H2-19),
2.30–2.39 (m, H2-CH2CH2CH2), 3.32–3.34 (m, H2-CONHCH2), 4.01 (t, J = 7.0 Hz, H2-CH2-
imidazol-1-yl), 4.92 (d, J = 12.6 Hz, H2-17), 5.04 (s, H2-5), 6.94 (s, H1-Imidazo-1-yl), 7.01
(s, H1-Imidazo-1-yl), 7.45 (t, J = 7.2 Hz, H1-10), 7.51 (d, J = 7.1 Hz, H1-9), 7.56 (s, H1-14),
7.65 (d, J = 9.1 Hz, H1-12), 7.70 (t, J = 7.4 Hz, H2-11), 8.03 (br, H1-CONH), 8.05 (s, H1-7).
MS (ESI): m/z 474.2[M+H]+.
4.1.3.3. Compound 11c. Yellow powder, yield in 99%, IR(KBr, cm−1): ν 1655 (CONH); 1H
NMR (CDCl3, 300 MHz, TMS) δ: 1.08 (t, J = 6.6 Hz, H3-18), 1.74–1.76 (m, H2-19), 1.44–2.13
(m, H10-cyclohexyl), 2.29–2.31 (m, H2-CH2CH2CH2), 2.47 (s, H6-NCH2), 3.39-3.43 (m, H1
of cyclohexyl and H2 of CONHCH2), 3.74–3.76 (m, H4-OCH2), 4.80 (d, J = 12.6 Hz, H1-
17), 5.04 (d, J = 12.6 Hz, H1-17), 5.11 (s, H2-5), 7.45 (t, J = 7.5 Hz, H1-10), 7.55 (s, H1-14),
7.60 (t, J = 8.1 Hz, H1-11), 7.92 (d, J = 8.1 Hz, H1-12 and H1-9), 8.21 (br, H1-CONH). MS
(ESI): m/z 575.5[M+H]+.
4.1.3.4. Compound 11d. Yellow powder, yield in 80%, IR (KBr, cm−1): ν 1651 (CONH);
1H NMR (CDCl3, 300 MHz, TMS) δ: 1.08 (t, J = 7.2 Hz, H3-18), 1.25 (3H, t, J = 7.5 Hz,
H3-7-Et), 1.76–1.78 (m, H2-19), 2.27–2.29 (m, H2-CH2CH2CH2), 2.42–2.44 (m, H2-NCH2),
2.49–2.51 (m, H4-NCH2), 2.82 (q, J = 7.5 Hz, H2-7-Et), 3.41-3.43 (m, H2-CONHCH2), 3.76
(t, J = 4.2 Hz, H4-OCH2), 3.93 (s, H3-10-OMe), 4.85 (d, J = 12.9 Hz, H1-17), 4.91 (s, H2-5),
4.94 (d, J = 12.9 Hz, H1-17), 6.89 (d, J = 2.7 Hz, H1-9), 7.34 (d, J = 9.3 Hz, 2.4 Hz, H1-11), 7.46
(s, H1-14), 7.93 (d, J = 9.3 Hz, H1-12), 8.07 (br, H1-CONH). MS (ESI): m/z 551.3 [M+H]+.
4.1.4. General procedure for synthesis of ester-amides (12a-f)
To a stirred solution of hydroxyl-amide (0.30 mmol) in pyridine (3 ml) was added anhydride
(0.2 ml). e mixture was warmed to 40 °C in water bath and stirred over night. e solvents
were removed under reduced pressure, and the residue was purified on silica gel column
chromatography (eluent: ethyl acetate/petroleum ether = 1/1 ~ 3/1) to give ester-amide.
4.1.4.1. Compound 12a. Yellow powder, 82% yield; IR (KBr, cm−1) ν 1734 (MeCOO),
1621 (CONH). 1H NMR (CDCl3, 300 MHz, TMS) δ: 1.10 (t, J = 7.2 Hz, H3-18), 1.76-1.78
(m, H1-19), 2.06 (s, H3-COMe), 2.29–2.31 (m, H2-CH2CH2CH2), 2.29–2.31 (m, H1-19),
2.52–2.54 (m, H1-19, H6-NCH2), 3.31–3.42 (m, H2-CONHCH2), 3.77–3.79 (m, H4-OCH2),
5.15 (d, J = 19.4 Hz, H2-5), 5.47 (d, J = 11.6 Hz, H2-17), 7.43 (t, J = 7.4 Hz, H1-10), 7.58 (s,
H1-14), 7.63 (d, J = 8.1 Hz, H1-9), 7.69 (t, J = 7.2 Hz, H1-11), 8.04 (d, J = 8.3 Hz, H1-12),
8.14 (s, H1-7), 8.17 (br, H1-CONH). MS (ESI): m/z 535.4 [M+H]+.

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9
4.1.4.2. Compound 12b. Yellow powder, 82% yield; IR (KBr, cm−1) ν 1730 (EtCO),
1
1655 (CONH); H NMR (CDCl3, 300 MHz, TMS) δ: 1.11 (t, J = 7.2 Hz, H3-18), 1.21 (t,
J = 7.1 Hz, H3-COEt), 1.76-1.78 (m, H2-19), 2.30–2.32 (m, H1-CH2CH2CH2), 2.40–2.15
(m, H1-CH2CH2CH2), 2.55–2.56 (m, H6-NCH2), 3.31-3.33 (m, H1-CONHCH2), 3.43–3.45
(m, H1-CONHCH2), 3.48 (q, J = 7.0 Hz, H2-COEt), 3.79 (t, J = 4.5 Hz, H4-OCH2), 5.13 (d,
J = 19.2 Hz, H2-5), 5.47 (d, J = 11.6 Hz, H2-17), 7.45 (t, J = 7.4 Hz, H1-10), 7.57 (s, H1-14),
7.60 (d, J = 8.1 Hz, H1-9), 7.68 (t, J = 7.5 Hz, H1-11), 8.03 (d, J = 8.5 Hz, H1-12), 8.12 (s,
H1-7), 8.21 (s, H1-CONH). MS (ESI): m/z 549.3[M+H]+.
4.1.4.3. Compound 12c. Yellow powder, 88% yield. IR (KBr, cm−1) ν 1722 (MeCO), 1651
(CONH); 1H NMR (CDCl3, 300 MHz, TMS) δ: 1.07 (t, J = 7.2 Hz, H3-18), 2.06 (s, H3-COMe),
2.09–2.11 (m, H2-19), 2.32–2.46 (m, H2-CH2CH2CH2), 3.32–3.34 (m, H2-CONHCH2), 4.07
(t, J = 7.0 Hz, H2-CH2-imidazol-1-yl), 5.19–5.21 (m, H2-5), 5.52 (d, J = 11.5 Hz, H2-17),
7.01 (s, H1-imidazol-1-yl), 7.10 (s, H2- imidazol-1-yl), 7.54 (t, J = 7.2 Hz, H1-10), 7.61 (s,
H1-14), 7.76 (d, J = 7.1 Hz, H2-9), 7.79 (t, J = 7.4 Hz, H1-11), 7.82 (br, H1-CONH), 8.12 (d,
J = 9.1 Hz, H1-12), 8.23 (s, H1-7). MS (ESI): m/z 516.2 [M+H]+.
4.1.4.4. Compound 12d. Yellow powder, 74% yield; IR (KBr, cm−1) ν 1726 (EtCO), 1652
(CONH); 1H NMR (CDCl3, 300 MHz, TMS) δ: 1.01 (t, J = 7.1 Hz, H3-18), 1.23 (t, J = 7.1 Hz,
H3-COEt), 2.03–2.05 (m, H2-19), 2.30–2.47 (m, H2-COEt and H2-CH2CH2CH2), 3.30–3.32
(m, H2-CONHCH2), 4.04 (t, J = 7.0 Hz, H2-CH2-imidazol-1-yl), 5.15–5.17 (m, H2-5) 5.52 (d,
J = 11.6 Hz, H2-17), 6.98 (s, H1-imidazol-1-yl), 7.06 (s, H1-imidazol-1-yl), 7.51 (t, J = 7.2 Hz,
H1-10), 7.59 (s, H1-14), 7.60 (br, H1-CONH), 7.67 (d, J = 7.1 Hz, H1-9), 7.73 (t, J = 7.4 Hz,
H1-11), 8.01 (d, J = 9.0 Hz, H1-12), 8.18 (s, H1-7). MS (ESI): m/z 530.3 [M+H+].
4.1.4.5. Compound 12e. Yellow powder, 38% yield; IR (KBr, cm−1): ν 3423, 1728 (EtCO),
1650 (CONH), 1591; 1H NMR (CDCl3, 400 MHz, TMS) δ: 1.07–1.01 (m, H3-18 and H3-
COEt), 1.56–1.94 (m, H10-cyclohexyl), 2.08–2.51 (m, H2-19, H2-COEt and H2-CH2CH2CH2),
2.75 (s, H6-NCH2), 3.31–3.33 (m, H1-CH of cyclohexyl), 3.43–3.45 (m, H2-CONHCH2),
3.89 (s, H4-OCH2), 5.17 (d, J = 18.6 Hz, H1-17), 5.29 (d, J = 18.6 Hz, H1-17), 5.39 (d,
J = 11.6 Hz, H1-5), 5.56 (d, J = 11.6 Hz, H1-5), 7.42 (t, J = 7.3 Hz, H1-10), 7.53 (s, H1-14),
7.63 (t, J = 7.4 Hz, H1-11), 7.90 (d, J = 9.9 Hz, H1-9), 7.96 (d, J = 8.0 Hz, H1-12), 8.09 (s,
H1-CONH). MS (ESI): m/z 631.4 [M+H]+.
4.1.4.6. Compound 12f. Yellow powder, 78% yield; IR (KBr, cm−1) ν: 1726 (EtCO), 1652
(CONH); 1H NMR (CDCl3, 300 MHz, TMS) δ: 1.08–1.10 (m, H3-18 and H3-COEt), 1.27 (t,
J = 6.9 Hz, H3-7-Et), 1.74–1.76 (m, H2-CH2CH2CH2), 2.28–2.30 (m, H2-COEt and H1-19),
2.51–2.53 (m, H1-19 and H6-NCH2), 2.90–2.92 (m, H2-7-Et), 3.19–3.43 (m, H2-CONHCH2),
3.78–3.80 (m, H4-OCH2), 3.89 (s, H3-10-OMe), 4.90–5.03 (d, J = 18.9 Hz, H2-5), 5.23 (d,
J = 11.4 Hz, H1-17), 5.45 (br, H1-OH), 5.61 (d, J = 11.4 Hz, H1-17), 6.73 (s, H1-14), 7.27
(d, J = 9.0 Hz, H1-11), 7.44 (s, H1-9), 7.86 (d, J = 9.0 Hz, H1-12), 8.33 (br, H1-CONH). MS
(ESI): m/z 607.3[M+H]+.

10
C. ZHENG ET AL.
4.2. Assessment of antitumor activity
4.2.1. Cytotoxicity assays
Inhibition rate assays were performed on human lung cancer cell line A549, human gastric
cancer line BGC-823, human liver cancer cell line SMMC-7721, and human leukemia cell
line HL-60. 180 μl (20~40 thousand cell per ml) per well was plated in 96-well plates. Afer
culturing for 24 h, compounds 11a–b and 12a–d were added with different concentrations,
and 0.1% DMSO (dimethyl sulfoxide) was used for control. Afer 48 h of incubation, 20 μl
MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) solution (5 mg/
ml) was added to each well, and afer shaking for 1 min, the plate was incubated for fur-
ther 4 h. Formazan crystals were dissolved with DMSO (150 μl per well). e absorbance
(OD) was quantitated with microplate spectrophotometer at 570 nm. Wells containing no
drugs were used as blanks for the spectrophotometer. e survival of cells was expressed as
percentage of untreated control wells. e prototypical inhibitors mitomycin and cisplatin
were included as reference standards; the results of these assays were used to obtain the
corresponding inhibition rates.
Inhibition of compounds 11c–d, 12 e–f and reference drugs SN38, CPT and topotecan
(different concentrations were: 100, 33, 10, 3.3, 1, 0.33, 0.1, 0.033, 0.01, 0.0033, 0.001, 0.00033
μM) on A549/ATCC and HT29 cell lines were incubated for 72 h with the same method.
IC50 is defined as the inhibitory drug concentration causing a 50% decrease of cell growth
over that of untreated control.
4.2.2. Experimental transplantation tumor in mice
BALA/cA-nude mice, ♀, 6–7 weeks, were purchased from Shanghai SLAC Laboratory
Animal CO. LTD. According to the protocol of transplantation tumor research [16], A549
or HT29 tumor tissues were chopped into 1.5 mm3, which was subcutaneously transplanted
into right axillary fossa of nude mice. e diameter of transplantation tumor was measured
with vernier caliper, and the tumor-transplanted mice were randomly divided into groups
when tumor size grew to 100–300 mm3, i.e. positive control group and treated groups (iv,
3 times per week). Mice in the negative control (normal subject, NS) group were treated
with normal saline.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was supported by the Program for Innovative Research Team in University [grant number
IRT-16R19], Hainan Province Natural Science Foundation of Innovative Research Team Project
[grant number 2016CXTD007] and National Natural Science Foundation of China [grant number
201702039].
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