Synthesis and biological evaluation of novel 10-substituted-7- Ethyl-10-hydroxycamptothecin (SN-38) prodrugs

Article abstract of DOI:10.3390/molecules191219718

In an attempt to improve the antitumor activity and reduce the side effects of irinotecan (2), novel prodrugs of SN-38 ( 3) were prepared by conjugating amino acids or dipeptides to the 10-hydroxyl group of SN-38 via a carbamate linkage. The synthesized compounds completely generated SN-38 in pH 7.4 buffer or in human plasma, while remaining stable under acidic conditions. All prodrug compounds demonstrated much greater in vitro antitumor activities against HeLa cells and SGC-7901 cells than irinotecan. The most active compounds, 5h, 7c, 7d, and 7f, exhibited IC50 values that were 1000 times lower against HeLa cells and 30 times lower against SGC-7901 cells than those of irinotecan, and the inhibitory activities of these prodrugs against acetylcholinesterase (AchE) were significantly reduced, with IC50 values more than 6.8 times greater than that of irinotecan. In addition, compound 5e exhibited the same level of tumor growth inhibitory activity as irinotecan (CPT-11) in a human colon xenograft model in vivo.

Full text of DOI:10.3390/molecules191219718

Molecules 2014, 19, 19718-19731; doi:10.3390/molecules191219718
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of Novel 10-Substituted-7-
ethyl-10-hydroxycamptothecin (SN-38) Prodrugs
Mo Zhou 1,†, Meixia Liu 1,†, Xinhua He 1, Hong Yu 2, Di Wu 3, Yishan Yao 1, Shiyong Fan 1,
Ping Zhang 1, Weiguo Shi 1,* and Bohua Zhong 1,*
1
Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China;
E-Mails: michelle_zm2011@163.com (M.Z.); liumeixia@163.com (M.L.);
hexinhua01@163.com (X.H.); spray_yao123456@hotmail.com (Y.Y.);
fsyn1996@163.com (S.F.); gundnir@163.com (P.Z.)
2
Cell Biology Laboratory of Jilin Province Tumor Institute, No 1018 Huguang Road,
Changchun 130012, China; E-Mail: yuhong123@163.com
3
Tumor Centre, No.1 Hospital, Jilin University, 71 Xin-Min Street, Changchun 130012, China;
E-Mail: wd78@163.com
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed;
E-Mails: bohuazhong0501@163.com (B.Z.); shiwg1988@126.com (W.S.);
Tel.: +86-10-6693-1639 (B.Z.); +86-10-6687-4612 (W.S.); Fax: +86-10-6821-1656 (B.Z. & W.S.).
External Editor: Jean Jacques Vanden Eynde
Received: 29 October 2014; in revised form: 19 November 2014 / Accepted: 19 November 2014 /
Published: 27 November 2014
Abstract: In an attempt to improve the antitumor activity and reduce the side effects of
irinotecan (2), novel prodrugs of SN-38 (3) were prepared by conjugating amino acids or
dipeptides to the 10-hydroxyl group of SN-38 via a carbamate linkage. The synthesized
compounds completely generated SN-38 in pH 7.4 buffer or in human plasma, while
remaining stable under acidic conditions. All prodrug compounds demonstrated much
greater in vitro antitumor activities against HeLa cells and SGC-7901 cells than irinotecan.
The most active compounds, 5h, 7c, 7d, and 7f, exhibited IC50 values that were 1000 times
lower against HeLa cells and 30 times lower against SGC-7901 cells than those of irinotecan,
and the inhibitory activities of these prodrugs against acetylcholinesterase (AchE) were
significantly reduced, with IC50 values more than 6.8 times greater than that of irinotecan. In

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addition, compound 5e exhibited the same level of tumor growth inhibitory activity as
irinotecan (CPT-11) in a human colon xenograft model in vivo.
Keywords: antitumor agent; camptothecins; prodrugs; SN-38
1. Introduction
Camptothecin (CPT 1, Figure 1) derivatives are potent topoisomerase I inhibitors with strong
antitumor activities both in vitro and in vivo and are the only antitumor agents with topoisomerase I
inhibitory activity used in the clinic [1,2].
Figure 1. Structure of camptothecin and its analogs.
3
1
2
SN-38
Irinotecan
Camptothecin
Irinotecan (2) is a water-soluble prodrug of CPT derivative SN-38 (3). It is the most widely used CPT
derivative for the treatment of colorectal cancer that has been previously treated with 5-fluorouracil; in
addition, it has activity against a wide range of other cancers either as a single agent or in combination
with other antitumor agents [3–6]. However, high individual variation in efficacy and toxicity as well as
side effects, including acute cholinergic diarrhea, preclude its clinical use. It is believed that the low
bioconversion efficiency (4%–5%) from irinotecan to the active form SN-38 is responsible for high
interpatient variability in terms of the pharmacokinetics, which leads to considerable individual variation
in efficacy and toxicity [7–9]. Moreover, the 4-piperidinopiperidine moiety of irinotecan is responsible for
inhibiting acetylcholinesterase (AChE) activity, causing acute cholinergic diarrhea [10–12].
In this study, novel water soluble prodrugs of SN-38, with reduced AChE inhibitory activity, that
could be completely converted to SN-38 were designed and synthesized. Linear amino acids or
dipeptides with less steric effects were used to replace the 4-piperidinopiperidine moiety of irinotecan.
Firstly, amino acids are considered to have well physiological compatibility and ability to penetrate the
biological membrane by active transport, which make them the ideal prodrug carriers. Secondly, the
carboxyl groups(-COOH) in amino acids or dipeptides can be easily converted to sodium or potassium
salt to improve water solubility of prodrugs.The target compounds were prepared by conjugating an
amino acid or dipeptide to the 10-hydroxyl group of SN-38 via a carbamate linker.
These prodrugs were stable in aqueous solutions at acidic pH levels but were rapidly converted to
active SN-38 in pH 7.4 buffer or in human plasma (Figure 2). These new CPT prodrugs exhibited much
greater antitumor activity and less AChE inhibitory activity than irinotecan in vitro.

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Figure 2. Conversion of target compound to SN-38.
2. Results and Discussion
2.1. Chemistry
The synthesis of the prodrugs is shown in Scheme 1. The benzyl ester of the amino acid was initially
converted in good yield to the corresponding isocyanate in the presence of bis(trichloromethyl)
carbonate. Next, the isocyanate was reacted with SN-38 to give coupled compounds 4 in 83%–89%
yield. Deprotection of 4 by catalytic hydrogenation yielded compounds 5a–h.
Scheme 1. Synthesis of prodrugs 5a–h.
5a R1 = H
5b R1 = CH3
5c R1 = CH(CH3)2
5d R1 = (CH2)2COOH
5e R1 = CH2CH(CH3)2
5f R1 = CH(CH3)CH2CH3
5g R1 = L-CH2COOH (R)
5h R1 = D-CH2COOH (S)
Reagents and conditions: (a) (i) Et3N, THF, rt, 30 min; (ii) isocyanate, THF, 40 °C, overnight;
(b) Pd/C, H2, THF/EtOH, rt, overnight.
Next, the dipeptide derivatives 7a–f were synthesized by further conjugating the amino acid
derivatives 5 with another amino acid in THF under room temperature for 24 h, as depicted in
Scheme 2.

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Scheme 2. Synthesis of prodrugs 7a–f.
7a R1 = H
7b R1 = H
7c R1 = H
R2 = D-CH2COOH
R2 = L- CH2COOH
R2 = (CH2)2COOH
7d R1 = CH3 R2 = D-CH2COOH
7e R1 = CH3 R2 = L-CH2COOH
7f R1 = CH3 R2 = (CH2)2COOH
Reagents and conditions: (a) (i) Et3N, THF, rt, 30 min; (ii) isocyanate, THF, 40 °C, overnight;
(b) Pd/C, H2, THF/EtOH, rt, overnight.
2.2. Cytotoxicity
The in vitro antitumor activities were evaluated on human cancer cell lines SGC-7901 and HeLa by
the MTT assay, using irinotecan as the reference compound. The results are summarized in Table 1. The
prodrugs 5a–h and 7a–f all exhibited much more potent antiproliferative activities against HeLa cells
than irinotecan. Compounds 5e–5h and 7b–7f also showed greater antitumor activities against SGC-7901
cells than irinotecan. The most active compounds 5h, 7c, 7d, and 7f exhibited IC50 values that were less
than 3.2 nM against HeLa cells, providing an approximately 1000-fold increase in potency compared to
irinotecan. Meanwhile, they all showed antiproliferative activity that was 30-fold greater than that of
irinotecan against SGC-7901 cells. Compared with the amino acid derivatives, the dipeptide derivatives
exhibited greater antitumor activities.
Table 1. Antiproliferative activities of the derivatives against human cancer cell lines.
IC50 (μM)
HeLa
Compound
R1
SGC-7901
5a
H
2.69 ± 0.11 (30.4 ± 47.0) × 10−3
1.75 ± 0.60 (9.85 ± 11.1) × 10−3
1.88 ± 0.72 (5.61 ± 40.3) × 10−3
0.50 ± 0.19 (1.82 ± 64.6) × 10−3
5b
CH3
5c
CH(CH3)2
CH2CH(CH3)2
CH(CH3)CH2CH3 0.76 ± 0.34 (3.64 ± 1.03) × 10−3
5e
5f
5g
CH2COOH (R)
D-CH2COOH (S) 0.24 ± 0.95 <(3.20 ± 64.3) × 10−3
7.38 ± 1.24 1.32 ± 0.13
0.98 ± 0.05 (9.07 ± 33.2) × 10−3
5h
Irinotecan

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Table 1. Cont.
IC50 (μM)
HeLa
Compound R1
R2
SGC-7901
7a
7b
7c
7d
7e
7f
H
H
H
D-CH2COOH 1.04 ± 0.90 <(3.20 ± 12.0) × 10−3
L-CH2COOH 0.61 ± 0.68 (14.8 ± 135.0) × 10−3
(CH2)2COOH 0.20 ± 0.07 <(3.20 ± 0.40) × 10−3
CH3 D-CH2COOH 0.26 ± 0.64 <(3.20 ± 2.0) × 10−3
CH3 L-CH2COOH 0.21 ± 0.55 (12.9 ± 133.0) × 10−3
CH3 (CH2)2COOH 0.27 ± 0.18 (1.57 ± 3.10) × 10−3
Irinotecan
7.38 ± 1.24
1.32 ± 0.13
2.3. AChE Inhibition Assay
Irinotecan has been reported to be a potent inhibiter of AChE, and inhibition of this enzyme causes
acute cholinergic diarrhea. Unlike irinotecan, the compounds we assayed were only weak inhibitors of
AChE. As shown in Table 2, the IC50 value of irinotecan is 0.2 μM, while compounds 5a, 5f, 5g, 5h, 7a,
7c, 7d, and 7f exhibited IC50 values greater than 1.36 μM. One possible explanation is that the active site
of AChE is present at the bottom of a gorge that is lined with hydrophobic amino acid residues [13]. The
terminal dipiperidino moiety in CPT-11 that interacts with amino acid residues within AChE plays a
major role in enzyme inhibition, which has been confirmed by X-ray crystallographic studies [14].
Hence, the hydrophobic side chain moiety of isoleucine in 5f was attracted into the gorge where catalysis
occurs, while the others with hydrophilic moiety had a lower binding affinity with it.
Table 2. AChE inhibitory activity.
Compound IC50 (μM) Compound IC50 (μM)
5a
5f
19.79
1.36
7c
7d
>100
4.44
5g
5h
7a
3.08
7f
36.08
0.20
7.43
Irinotecan
115.7
2.4. Stability and Conversion
The chemical stability of the prodrugs at pH 4.6 or 7.4 in PBS at 37 °C is summarized in Table 3. All
the target compounds showed pH-dependent stability. They were stable at pH 4.6 at 37 °C for 12 h.
Except for compound 5h, less than 35% of the prodrugs remained after incubation for 12 h in pH 7.4
buffer. The dipepetide derivatives 7a–f showed much less stability than the amino acid derivatives
5a–h. For example, less than 30% of the prodrugs 7a–f remained, while more than 85% of the prodrugs
5a–h remained after incubation for 1 h in pH 7.4 buffer.

Molecules 2014, 19
Table 3. The chemical stability of the prodrugs in pH 4.6 and pH 7.4 conditions.
19723
Remaining Prodrugs (%)
1 h 12 h
pH 4.6 pH 7.4 pH 4.6 pH 7.4
Compound
5a
5d
5g
5h
7a
7b
7c
7d
7e
7f
100
98.1
100
100
100
100
100
100
100
100
85.3
85.2
93.9
93.4
26.0
17.6
29.4
26.3
23.4
14.4
98.4
92.5
94.5
94.5
96.8
96.9
96.4
95.5
94.5
94.7
14.8
8.6
34.1
52.2
0
0
0
0
0
0
The conversions of the prodrugs to SN-38 in human plasma are summarized in Table 4. All the
compounds could be rapidly converted to their active form in plasma. Except for compound 5h, the
conversion rate of the prodrugs was more than 35% after incubation for 1 h and complete conversion
was observed after incubation for 12 h in plasma. As at pH 7.4, the conversion rate of the dipeptide
derivatives was faster than that of the amino acid derivatives.
Table 4. Conversion of the prodrugs to SN-38 in human plasma.
Conversion (%)
Compound
1 h
3 h
74.7
72.5
99.4
68.9
99.7
99.4
64.2
37.4
-
12 h
5a
5b
5c
5d
5e
5f
38.8
36.5
80.1
38.0
77.5
74.6
36.8
15.3
95.3
99.6
99.2
97.6
99.6
98.7
100.0
100.0
-
100.0
-
-
5g
5h
7a
7b
7c
7d
7e
7f
98.5
85.4
-
-
-
-
-
-
-
-
-
-
-
2.5. Tumor Growth Inhibitory Activity of 5e in a Human Colon Xenograft Model in Vivo
The antitumor activity of 5e, compared with CPT-11 and SN-38, was investigated in human colon
adenocarcinoma SW1116 xenografts in nude mice. The model animals were divided into four
experimental groups, eight animals per group. Tested compouds were each administered by injection in
the tumor section once per day for contiguous 6 days. The injection doses of test compound 5e, CPT-11,

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and SN-38 were 60 mg/kg, respectively. Using the vehicle group as the control, we tested the inhibitory
activities of these compounds for 6 days and measured the changes in body weight and tumor size every
2 days. The results showed that 5e inhibited tumor growth by 51% on day 6 and had about the same
in vivo activity compared to CPT-11 (Table 5). SN-38 was less active than 5e and CPT-11, showing an
inhibition rate of only 8.6%. No toxic death occurred after drug treatment, and the body weight losses
induced by 5e, CPT-11, and SN-38 at the given dose were similar.
Table 5. Tumor growth inhibitory activity of 5e in a human colon xenograft model in vivo (x ± SD).
Dose
Tumor Weight Inhibitory Rate Body Weight Change
Compound
(mg/kg)
(g/10g)
(%)
(g)
Vehicle
5e
0.35 ± 0.12
0.17 ± 0.05 *
0.17 ± 0.04 *
0.32 ± 0.12
+0.95
−2.72
−3.10
−1.45
60
60
60
51
51
Irinotecan
SN-38
8.6
*: p < 0.05, compared with vehicle.
2.6. Discussion
In order to overcome the drawbacks of the pharmacokinetic properties of irinotecan and find new
water soluble camptothecin prodrugs with improved activity and lower side effects, novel prodrugs of
SN-38 were designed and synthesized using amino acids or dipeptides as prodrug carriers to replace the
4-piperidinopiperidine moiety of irinotecan. Because of the poor enzymolysis stability of ester bonds
directly formed by the 10-OH moiety f SN-38 when reacting with α-COOH of amino acids, carbamate
linkages were used to conjugate the 10-OH of SN-38 and the α-NH2 of amino acids or dipeptides. Firstly,
the water solubility of the designed prodrugs was improved significantly by converting the free carboxyl
groups to their carboxylic salts. Next, we tested the chemical stability and plasma stability of these new
compounds in different pH conditions and in human plasma, respectively. The results suggested that the
new prodrugs could be stored in acidic solution or in solid state and converted to the active SN-38 in
physiological conditions or in human plasma easily, indicating the improved pharmacokinetics property
compared with that of Irinotecan. Both in vitro and in vivo biologic evaluation results showed the
predominant antitumor activity of these new compounds. Meanwhile, the compounds exhibited only
weak inhibitory activity against AChE, reducing the acute cholinergic diarrhea associated with
Irinotecan. These results suggest that these compounds could be used as leads for the further design and
development of novel anticancer camptothecin derivatives.
3. Experimental Section
3.1. General Information
Commercially available reagents were purchased from Alfa Aesar (Shanghai, China), and used as
supplied without further purification. Reactions were monitored by thin-layer chromatography
performed on silica gel GF254 pre-coated plate. Visualization was realized by UV light (365 nm).
Purification by flash chromatography was realized on silica gel 200–300 mesh. Proton nuclear magnetic
resonance (1H-NMR) spectra were recorded in DMSO-d6 at 400 MHz on a JNM-ECA-400 MHz instrument

Molecules 2014, 19
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(JEOL Ltd., Tokyo, Japan). Low Resolution Mass Spectroscopy (AB Sciex Pte. Ltd., Framingham, MA,
USA) was recorded on an API 150EX LC/MS system, with an electron ionization spray (ESI) technique.
3.2. Synthesis
10-OCO-(Gly-OBzl)-SN-38 (4a). H-Gly-OBzl•HCl (1.23 g, 6.12 mmol) was dissolved in a solution of
DCM (60 mL) and NaHCO3 (saturated aqueous solution 60 mL), and cooled down to 0 °C . After 10 min,
BTC (668 mg, 2.25 mmol) was added and the mixture was stirred for 20 min. The mixture was extracted
twice with DCM solution. The combined organic extract were dried over anhydrous sodium sulfate,
filtered, and evaporated to give the product as colorless oil without further purification. SN-38 (600 mg,
1.53 mmol) was dissolved in THF, and Et3N (618 mg, 6.12 mmol) was added. The mixture was set under
N2 atmosphere and stirred at room temperature for 30 min. Thereafter, the isocyanate mentioned above
was added and the reaction mixture was stirred at 40 °C overnight. The mixture was purified by silica
gel chromatography with DCM-acetone = 5:1 as eluent to give compound 4a (736 mg, 82.5% yield).
1H-NMR δH (ppm): 0.87–0.91 (t, J = 7.2 Hz, 3H), 1.27–1.31 (t, J = 7.6 Hz, 3H), 1.84–1.91 (m, 2H),
3.15–3.20 (q, J = 7.6 Hz, 2H), 3.83–3.84 (d, J = 6.0 Hz, 2H), 5.30 (s, 2H), 5.43 (s, 2H), 6.55 (s, 1H),
7.31 (s, 1H), 7.61–7.64 (dd, J = 9.6 Hz, 2.4 Hz, 1H), 7.95 (d, J = 2.4 Hz, 1H), 8.16–8.18 (d, J = 9.6 Hz,
1H), 8.28–8.31 (t, J = 6.0 Hz, 1H), 12.68 (s, 1H). MS (ESI) m/z: 584.4 [M+H]+, 606.2 [M+Na]+.
10-OCO-(Gly-OH)-SN-38 (5a). Compound 4a (736 mg, 1.26 mmol) was dissolved in the solution of
THF and ethyl alcohol mixed at a ratio of 7:4. Afterwards, 10% Pd/C (294 mg) was added and the stirred
overnight under H2 atmosphere. The reaction mixture was filtered, the filtrate was evaporated
immediately and chromatographed on silica gel (DCM–acetone = 2:1 with 1% TFA) to give compound
5a as a yellow powder (446 mg, 71.7% yield). 1H-NMR δH (ppm): 0.87–0.91 (t, J = 7.2 Hz, 3H), 1.27–1.31
(t, J = 7.6 Hz, 3H), 1.84–1.91 (m, 2H), 3.15–3.20 (q, J = 7.6 Hz, 2H), 3.83–3.84 (d, J = 6.0 Hz, 2H),
5.30 (s, 2H), 5.43 (s, 2H), 6.55 (s, 1H), 7.31 (s, 1H), 7.61–7.64 (dd, J = 9.6 Hz, 2.4 Hz, 1H), 7.95 (d,
J = 2.4 Hz, 1H), 8.16–8.18 (d, J = 9.6 Hz, 1H), 8.28–8.31 (t, J = 6.0 Hz, 1H), 12.68 (s, 1H). MS(ESI) m/z:
494.3 [M+H]+, 516.3 [M+Na]+.
10-OCO-(Ala-OH)-SN-38 (5b). Compound 5b was synthesized by a similar procedure as 5a.
H-Ala-OBzl•TosOH (2.26 g, 6.43 mmol) and SN-38 (600 mg, 1.53 mmol) were employed to produced
4b (yellow powder, 862 mg, 94%), which was then hydrogenated to 5b with Pd/C, producing a yellow
powder (625 mg, 85.4%). δH (ppm): 0.87–0.90 (t, J = 7.2 Hz, 3H), 1.27–1.31 (t, J = 7.6 Hz, 3H),
1.38–1.41 (d, J = 7.6 Hz, 3H), 1.82–1.91 (m, 2H), 3.15–3.20 (q, J = 7.6 Hz, 2H), 4.12–4.15 (m, 1H),
5.31 (s, 2H), 5.44 (s, 2H), 6.55 (s, 1H), 7.31 (s, 1H), 7.58–7.63 (dd, J = 9.2 Hz, 2.4 Hz, 1H), 7.94–7.95
(d, J = 2.4 Hz, 1 H), 8.14–8.18 (d, J = 9.2 Hz, 1 H), 8.35–8.37 (t, J = 7.6 Hz, 1H), 12.79 (s, 1H). MS
(ESI) m/z: 508.4 [M+H]+.
10-OCO-(Val-OH)-SN-38 (5c). Compound 5c was synthesized by a similar procedure as 5a.
H-Val-OBzl•TosOH (1.94 g, 5.12 mmol) and SN-38 (500 mg, 1.28 mmol) were employed to produce
4c as a yellow powder (797 mg, 100%), then hydrogenated to 5c with Pd/C, producing a yellow powder
1
(271 mg, 39.7%). H-NMR δH (ppm): 0.87–0.90 (t, J = 7.2 Hz, 3 H), 0.98–1.01 (q, J = 3.2 Hz, 6 H),
1.28–1.32 (t, J = 7.6 Hz, 3H), 1.82–1.93 (m, 2H), 2.14–2.19 (m, 1H), 3.16–3.22 (q, J = 7.6 Hz, 2H),

Molecules 2014, 19
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3.96–4.00 (dd, J = 5.6 Hz, 8.4 Hz, 1H), 5.32 (s, 2H), 5.43 (s, 2H), 6.51 (s, 1H), 7.32 (s, 1H), 7.61–7.63
(dd, J = 2.4 Hz, 9.2 Hz, 1H), 7.95 (d, J = 2.4 Hz, 1H), 8.17–8.19 (d, J = 9.2 Hz, 1H), 8.22–8.44 (d,
J = 8.4 Hz, 1H), 12.72 (s, 1H). MS (ESI) m/z: 536.6 [M+H]+.
10-OCO-(Glu-OH)-SN-38 (5d). Compound 5d was synthesized by a similar procedure as 5a.
H-Glu(OBzl)-OBzl•TosOH (2.55 g, 5.10 mmol) and SN-38 (500 mg, 1.28 mmol) were employed to
produce 4d (yellow powder, 1.03 g, 100%), which was then hydrogenated to 5d with Pd/C, producing a
yellow powder (368 mg, 51.0%). 1H-NMR δH (ppm): δ = 0.87–0.90 (t, J = 7.2 Hz, 3H), 1.28–1.31 (t,
J = 7.6 Hz, 3H), 1.86–1.91 (m, 2H), 2.09–2.12 (m, 2H), 2.48–2.51 (m, 2H), 3.18–3.20 (q, J = 6.8 Hz,
2H), 4.12–4.13 (m, 1H), 5.32 (s, 2H), 5.43 (s, 2H), 6.54 (s, 1H), 7.32 (s, 1H), 7.62–7.64 (d, J = 8.4 Hz,
1H), 7.96 (s, 1H), 8.15–8.17 (d, J = 8.0 Hz, 1H), 8.32–8.34 (d, J = 7.6 Hz, 1H), 12.58 (s, 2H). MS (ESI)
m/z: 566.4 [M+H]+, 588.4 [M+Na]+.
10-OCO-(Leu-OH)-SN-38 (5e). Compound 5e was synthesized by a similar procedure as 5a.
H-Leu-OBzl•TosOH (2.01 g, 5.10 mmol) and SN-38 (500 mg, 1.28 mmol) were employed to produce
4e (yellow powder, 600 mg, 73.6%), which was then hydrogenated to 5e with Pd/C, producing a yellow
powder (51 mg, 19.9%). 1H-NMR δH (ppm): 0.94–1.02 (m, 9 H), 1.33–1.36 (t, J = 7.6 Hz, 3H), 1.63–1.66
(m, 1H), 1.69–1.73 (m, 2H), 1.89–1.96 (m, 2H), 3.23–3.27 (q, J = 7.6 Hz, 2H), 4.12–4.14 (m, 1H), 5.38
(s, 2H), 5.50 (s, 2H), 6.61 (s, 1H), 7.37 (s, 1H), 7.65–7.68 (dd, J = 9.2 Hz, 2.8 Hz, 1H), 8.00–8.01 (d,
J = 2.4 Hz, 1H), 8.22–8.25 (d, J = 9.2 Hz, 1H), 8.38–8.40 (d, J = 8 Hz, 1H), 12.81 (s, 1H). MS (ESI)
m/z: 550.4 [M+H]+, 572.4 [M+Na]+.
10-OCO-(Ile-OH)-SN-38 (5f). Compound 5f was synthesized by a similar procedure as 5a.
H-Ile-OBzl•TosOH (2.01 g, 5.10 mmol) and SN-38 (500 mg, 1.28 mmol) were employed to produce 4f
(yellow powder, 757 mg, 92.8%), which was then hydrogenated to 5f with Pd/C, producing a yellow
1
powder (40 mg, 15.0%). H-NMR δH (ppm): 0.86–0.97 (m, 9 H), 1.27–1.33 (m, 4 H), 1.47–1.51 (m,
1H), 1.83–1.93 (m, 3H), 3.16–3.22 (q, J = 7.6 Hz, 2H), 4.00–4.02 (m, 1H), 5.32 (s, 2H), 5.44 (s, 2H),
6.55 (s, 1H), 7.32 (s, 1H), 7.61–7.64 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 7.95–7.96 (d, J = 2.4 Hz, 1H),
8.17–8.19 (d, J = 8.8 Hz, 1H), 8.27–8.29 (d, J = 8.4 Hz, 1H), 12.80 (s, 1H). MS (ESI) m/z: 550.3 [M+H]+,
572.4 [M+Na]+.
10-OCO-(L-Asp-OH)-SN-38 (5g). Compound 5g was synthesized by a similar procedure as 5a.
H-L-Asp(OBzl)-OBzl•TosOH (2.48 g, 5.10 mmol) and SN-38 (500 mg, 1.28 mmol) were employed to
produce 4g (yellow powder, 920 mg, 98.6%), which was then hydrogenated to 5g with Pd/C, producing
1
a yellow powder (521 mg, 75.0%). H-NMR δH (ppm): 0.86–0.90 (t, J = 7.2 Hz, 3H), 1.27–1.31 (t,
J = 7.6 Hz, 3H), 1.81–1.92 (m, 2H), 2.65–2.72 (q, J = 7.6 Hz, 16.8 Hz, 1H), 2.80–2.85 (q, J = 5.8 Hz,
16.8 Hz, 1H), 3.16–3.22 (q, J = 7.6 Hz, 2H), 4.40–4.44 (m, 1H), 5.34 (s, 2H), 5.44 (s, 2H), 6.56 (s, 1H),
7.32 (s, 1H), 7.36–7.40 (m, 10H), 7.61–7.63 (dd, J = 9.2 Hz, 2.4 Hz, 1H), 7.96–7.97 (d, J = 2.4 Hz, 1H),
8.18–8.20 (d, J = 9.2 Hz, 1H), 8.34–8.36 (dd, J = 8.4 Hz, 1H), 12.89 (s, 2H). MS (ESI) m/z: 552.3
[M+H]+, 574.5 [M+Na]+.

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10-OCO-(D-Asp-OH)-SN-38 (5h). Compound 5h was synthesized by a similar procedure as 5a.
H-D-Asp(OBzl)-OBzl•TosOH (2.48 g, 5.10 mmol) and SN-38 (500 mg, 1.28 mmol) were employed to
produce 4h (yellow powder, 810 mg, 86.8%), which was then hydrogenated to 5h with Pd/C, producing
1
a yellow powder (422 mg, 70.0%). H-NMR δH (ppm): 0.86–0.89 (t, J = 7.2 Hz, 3H), 1.27–1.30 (t,
J = 7.6 Hz, 3H), 1.84–1.90 (m, 2H), 2.71–2.73 (q, 1H), 2.80–2.82 (q, 1H), 3.16–3.18 (q, J = 7.6 Hz,
2H), 4.44–4.47 (m, 1H), 5.31 (s, 2H), 5.43 (s, 2H), 6.54 (s, 1H), 7.31 (s, 1H), 7.59–7.62 (dd, J = 9.2 Hz,
2.4 Hz, 1H), 7.94–7.95 (d, J = 2.4 Hz, 1H), 8.16–8.18 (d, J = 9.2 Hz, 1H), 8.34–8.36 (d, J = 8.4 Hz,
1H), 12.80–12.83 (s, 2H). MS (ESI) m/z: 552.3 [M+H]+, 574.5 [M+Na]+.
10-OCO-(D-Asp-Gly-OBzl)-SN-38 (6a). Under the ice-bath conditions, 5a (446 mg, 0.90 mmol) was
dissolved in THF (100 mL), followed by addition of HOBt (224 mg, 1.80 mmol) and DIEA (582 mg,
4.50 mmol) in sequence. The mixture was placed under a N2 atmosphere and stirred for 20 min, and then
H-D-Asp (OBzl)-OBzl•TosOH (485.6 mg, 0.99 mmol), and DCC (206 mg, 0.99 mmol) were added.
Thereafter, the reaction mixture was stirred at room temperature for 24 h. The mixture was purified by
silica gel chromatography with DCM–acetone = 3:1 as eluent to give compound 6a ( 241 mg, 34%).
1H-NMR δH (ppm): 0.87–0.90 (t, J = 7.2 Hz, 3H), 1.28–1.32 (t, J = 7.6 Hz, 3H), 1.86–1.89 (m, 2H),
2.88–2.89 (q, 1H), 2.95–2.96 (q, 1H), 3.14–3.15 (q, J = 7.6 Hz, 2H), 3.84–3.89 (d, J = 5.6 Hz, 2H),
4.84–4.86 (q, 1H), 4.55–4.59 (q, 1H), 5.13 (s, 2H), 5.21 (s, 2H), 5.27 (s, 2H), 5.45 (s, 2H), 6.57 (s, 1H),
7.35 (s, 11H), 7.63–7.65(dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.94 (d, J = 2.0 Hz, 1H), 8.14–8.16 (d, J = 8.4 Hz,
1H), 8.25 (s, 1H), 8.66–8.68 (d, J = 8.0 Hz, 1H). MS (ESI) m/z: 789.7 [M+H]+, 811.7 [M+Na]+.
10-OCO-(D-Asp-Gly-OH)-SN-38 (7a). Compound 6a (241 mg, 0.31 mmol) was dissolved in a solution
of THF and ethyl alcohol mixed at the ratio of 7:4. Afterwards, 10% Pd/C (96.4 mg) was added and the
mixture was stirred overnight under a H2 atmosphere. The reaction mixture was filtered, the filtrate was
evaporated immediately and was chromatographed on silica gel (DCM–acetone = 2:1 with 1% TFA) to
give compound 7a (95 mg, 51% yield), as a yellow powder. 1H-NMR δH (ppm): 0.87–0.90 (t, J = 7.2 Hz,
3H), 1.28–1.32 (t, J = 7.6 Hz, 3H), 1.86–1.89 (m, 2H), 2.64–2.66 (q, 1H), 2.70–2.73(q, 1H), 3.18–3.20
(q, J = 7.6 Hz, 2H), 3.78–3.80 (d, J = 5.6 Hz, 2H), 4.59–4.62(q, 1H), 5.33 (s, 2H), 5.44 (s, 2H), 6.52 (s,
1H), 7.33 (s, 1H), 7.64–7.66 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 7.96 (d, J = 2.0 Hz, 1H), 8.13 (s, 1H),
8.17–8.19 (d, J = 8.8 Hz, 1H), 8.32–8.34 (d, J = 8.0 Hz, 1H), 12.64 (s, 2H). MS (ESI) m/z: 609.4 [M+H]+,
631.4 [M+Na]+.
10-OCO-(L-Asp-Gly-OH)-SN-38 (7b). Compound 7b was synthesized by a similar procedure as 7a.
H-L-Asp(OBzl)-OBzl•TosOH (336 mg, 0.69 mmol) and compound 4a (311 mg, 0.63 mmol) were
employed to produce 6b (yellow powder, 240 mg, 48.3%), which was then hydrogenated to 7b with
Pd/C, producing a yellow powder (115 mg, 72.7%). 1H-NMR δH (ppm): 0.86–0.90 (t, J = 7.2 Hz, 3H),
1.27–1.31 (t, J = 7.6 Hz, 3H), 1.81–1.92 (m, 2H), 2.60–2.65 (q, 1H), 2.69–2.73 (q, 1H), 3.18–3.20 (q,
J = 7.6 Hz, 2H), 3.78–3.79 (d, J = 6.0 Hz, 2H), 4.55–4.60 (q, 1H), 5.34 (s, 2H), 5.44 (s, 2H), 6.56 (s,
1H), 7.32 (s, 1H), 7.64–7.67 (dd, J = 9.2 Hz, 2.4 Hz, 1H), 7.96–7.97 (d, J = 2.4 Hz, 1H), 8.17–8.20 (d,
J = 9.2 Hz, 1H), 8.21–8.24 (t, J = 6.0 Hz, 1H), 8.33–8.35 (d, J = 7.6 Hz, 1H), 12.79 (s, 2H). MS (ESI)
m/z: 609.3 [M+H]+, 631.4 [M+Na]+.

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10-OCO-(Glu-Gly-OH)-SN-38 (7c). Compound 7c was synthesized by a similar procedure as 7a.
H-L-Glu(OBzl)-OBzl•HCL (382 mg, 1.05 mmol) and compound 4a (471 mg, 0.96 mmol) were
employed to produce 6c (yellow powder, 440 mg, 57.5%), which was then hydrogenated to 7c with
Pd/C, producing a yellow powder (230 mg, 72.8%). 1H-NMR δH (ppm): 0.86–0.90 (t, J = 7.2 Hz, 3H),
1.27–1.31 (t, J = 7.6 Hz, 3H), 1.83–2.00 (m, 4H), 2.29–2.31(m, 2H), 3.17–3.19 (q, J = 7.6 Hz, 2H),
3.79–3.82 (t, J = 5.6 Hz, 2H), 4.29–4.30 (m, 1H), 5.32 (s, 2H), 5.44 (s, 2H), 6.56 (s, 1H), 7.32 (s, 1H),
7.64–7.66 (dd, J = 9.2 Hz, 2.4 Hz, 1H), 7.96 (d, J = 2.4 Hz, 1H), 8.14–8.19 (m, 2H), 8.31 (d, 1H), 12.55
(s, 2H). MS (ESI) m/z: 623.4 [M+H]+, 645.4 [M+Na]+.
10-OCO-(D-Asp-Ala-OH)-SN-38 (7d). Compound 7d was synthesized by a similar procedure as 7a.
H-D-Asp(OBzl)-OBzl•TosOH (652 mg, 1.34 mmol) and compound 4b (619 mg, 1.22 mmol) were
employed to produce 6d (yellow powder, 700 mg, 71.3%), which was then hydrogenated to 7d with
1
Pd/C, producing a yellow powder (90 mg, 40.0%). H-NMR δH (ppm): 0.87–0.91 (t, J = 7.2 Hz, 3H),
1.22–1.34 (m, 6H), 1.86 (m, 2H), 3.19–3.26 (m, 2H), 3.59 (m, 2H), 4.19 (m, 1H), 4.52–4.59 (q, 1H),
5.33 (s, 2H), 5.43 (s, 2H), 6.55 (s, 1H), 7.31 (s, 1H), 7.63 (dd, J = 9.2 Hz, 2.8 Hz, 1H), 7.95 (d, J = 2.8 Hz,
1H), 8.16–8.31 (m, 3H), 12.71 (s, 2H). MS (ESI) m/z: 623.4 [M+H]+, 645.3 [M+Na]+.
10-OCO-(L-Asp-Ala-OH)-SN-38 (7e). Compound 7e was synthesized by a similar procedure as 7a.
H-L-Asp(OBzl)-OBzl•TosOH (392 mg, 0.77 mmol) and compound 4b (392 mg, 0.77 mmol) were
employed to produce 6e (yellow powder, 400 mg, 64.6%), which was then hydrogenated to 7e with
Pd/C, producing a yellow powder (124 mg, 42.6%). 1H-NMR δH (ppm): 0.87–0.91 (t, J = 7.2 Hz, 3H),
1.29–1.31 (t, J = 7.6 Hz, 3H), 1.33–1.35 (d, J = 7.6 Hz, 3H), 1.82–1.93 (m, 2H), 2.62–2.67 (q, 1H),
2.70–2.76 (q, 1H), 3.15–3.20 (q, J = 7.6 Hz, 2H), 4.19–4.24 (m, 1H), 4.58–4.60 (m, 1H), 5.31 (s, 2H),
5.44 (s, 2H), 6.52 (s, 1H), 7.32 (s, 1H), 7.62–7.65 (dd, J = 9.2 Hz, 2.4 Hz, 1H), 7.94–7.95 (d, J = 2.4 Hz,
1H), 8.14–8.20 (m, 2H), 8.27–8.29 (d, J = 8.0 Hz, 1H), 12.64 (s, 2H). MS (ESI) m/z: 623.4 [M+H]+,
645.5 [M+Na]+.
10-OCO-(Glu-Ala-OH)-SN-38 (7f). Compound 7f was synthesized by a similar procedure as 7a.
H-L-Glu(OBzl)-OBzl•HCl (493 mg, 1.36 mmol) and compound 4b (625 mg, 1.23 mmol) were employed
to produce 6f (yellow powder, 690 mg, 69.0%), which was then hydrogenated to 7f with Pd/C, producing
1
a yellow powder (260 mg, 50.3%). H-NMR δH (ppm): 0.87–0.91 (t, J = 7.2 Hz, 3H), 1.29–1.31 (t,
J = 7.6 Hz, 3H), 1.33–1.35 (d, J = 7.2 Hz, 3H), 1.82–1.91 (m, 2H), 2.01–2.03 (m, 2H), 2.29–2.34 (m,
2H), 3.15–3.20 (q, J = 7.6 Hz, 2H), 4.19–4.22 (t, J = 7.2 Hz, 1H), 4.27–4.29 (m, 1H), 5.31 (s, 2H), 5.44
(s, 2H), 6.51 (s, 1H), 7.32 (s, 1H), 7.62–7.65 (dd, J = 9.2 Hz, 2.4 Hz, 1H), 7.94 (d, J = 2.4 Hz, 1H),
8.14–8.18 (m, 2H), 8.24–8.26 (d, J = 8.0 Hz, 1H), 12.42 (s, 2H). MS (ESI) m/z: 637.3 [M+H]+,
659.4 [M+Na]+.
3.3. Cytotoxicity Study
The in vitro antitumor activity was evaluated on two human cancer cell lines with the MTT method.
SGC-7901 is a human gastric adenocarcinoma cancer cell line and HeLa is a human cervical carcinoma
cell line. Cells were provided by the Jilin Province Tumor Institute. Cells were maintained in IMDM
medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL

Molecules 2014, 19
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streptomycin, at 37 °C in a humidified atmosphere containing 5% CO2. The human cancer lines SGC-7901
and HELA at a concentration of 2.5 × 104 μg·mL−1 were incubated in a 96-well microtiter plate (100 μL
in every well) for 24 h (37 °C , 5%CO2). Then the cells were incubated continuously for 72 h following
various concentrations of test drugs were added. The concentrations are 50 μM, 10 μM, 2 μM, 0.4 μM,
0.08 μM, 0.016 μM, 0.0032 μM, respectively. After the 100 μL 0.05% MTT solution was added to each
well, the plate was incubated for a further 4 h before removal of medium, the media removed. Formazan
crystals were dissolved with 100 μL DMSO, then the absorbance was detected at 490 nm.
3.4. Acetylcholinesterase Inhibition Assay
The AChE inhibition by all compounds was studied according to a modification of the method of
Ellman et al. [15]. The activity was assayed with AChE from electric eel in the presence or absence of
the test compounds in phosphate buffer (pH7.3). After the incubation at 37 °C for 1 min, 0.38 mM
5,5-dithiobisnitrobenzoic acid and 0.56 mM ATCh were added to the buffer. The changes in absorbance
were read at 412 nm, per 15 s intervals for 3 min by a spectrophotometer (UV-2500, Shimadzu Corp.,
Kyoto, Japan).
3.5. Stability Test
The stability of all compounds was investigated in PBS and human plasma by HPLC using a C18
analytical column. The mobile phase was composed of water and acetonitrile at a ratio of 70:30,
respectively, containing 1% trifluoroacetic acid. The amount of the compound and its metabolite SN-38
were flowed at the rate of 0.8 mL·min−1 and detected by the UV wavelength of 370 nm. Stability test in
PBS: each compound was dissolved in PBS (pH 7.4 and pH 4.6), incubated at 37 °C and analyzed at 1,
3, 6, 12 h. Stability test in plasma: The test compounds were dissolved in DMSO and diluted to 1 mM
by 0.9% physiological saline. Forty μL of each solution was added into plasma with a final concentration
of 0.1 mM. The mixture was incubated in a 37 °C water bath following the vigorous vortex mixing for
15 s. Samples were analyzed at 1, 3, 6, 12 h. 400 μL of cold acetonitrile (containing 1% trifluoroacetic
acid) was added to each sample, vortex-mixed for 15 s and centrifuged at 3500 rpm for 15 min at 4 °C .
4. Conclusions
In summary, 14 novel SN-38 prodrugs were prepared by conjugating amino acids or dipeptides to the
10-hydroxy group of SN-38 via a carbamate linkage. These prodrugs were found to have much greater
antitumor activities than SN-38. The synthesized compounds completely regenerated SN-38 in pH 7.4
buffer or in human plasma, so higher antitumor activity and less interpatient pharmacokinetic variability
than with irinotecan treatment are expected. The ability of these prodrugs to inhibit AChE activity was
also significantly reduced; thus, the administration of these compounds should cause less severe side
effects owing to their diminished inhibition of AChE.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/12/19718/s1.

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Acknowledgments
This work was supported by a National Science and Technology Major Project of China grant
(2012ZX09301003-001-004).
Author Contributions
B.Z., W.S. and X.H. conceived the the project, M.Z. and M.L. designed the experiments and executed
the chemical synthesis. D.W., H.Y. and M.Z. performed the biological assays. M.L., M.Z. and P.Z. wrote
the paper and S.F and Y.Y. edited English language. All authors discussed the results and commented
on the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).