Novel octapeptide-DTX prodrugs targeting MMP-7 as effective agents for the treatment of colorectal cancer with lower systemic toxicity

Article abstract of DOI:10.1016/j.ejmech.2020.112194

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death around the world. The current treatments of CRC exhibited high occurrence rate of side effects. Docetaxel (DTX), an important drug widely used in cancer c

Full text of DOI:10.1016/j.ejmech.2020.112194

Journal Pre-proof
Novel octapeptide-DTX prodrugs targeting MMP-7 as effective agents for the
treatment of colorectal cancer with lower systemic toxicity
Zheng-Yu Liu, Mei-Lin Tang, Jinfeng Ning, Yun-Peng Hao, Lu Zhou, Xun Sun
PII:
S0223-5234(20)30161-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112194
EJMECH 112194
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 October 2019
Revised Date: 25 February 2020
Accepted Date: 25 February 2020
Please cite this article as: Z.-Y. Liu, M.-L. Tang, J. Ning, Y.-P. Hao, L. Zhou, X. Sun, Novel octapeptide-
DTX prodrugs targeting MMP-7 as effective agents for the treatment of colorectal cancer with lower
systemic toxicity, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112194.
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Graphic Abstract:
Ten MMP-7 activated octapeptide-DTX/4FDT prodrugs were synthesized and evaluated. The
preferred compound 9a possessed lower systemic toxicity and similar anti-CRC activity to its
parent drug DTX.

Novel Octapeptide-DTX Prodrugs targeting MMP-7 as Effective Agents for the
Treatment of Colorectal Cancer with Lower Systemic Toxicity
a
a,c
a
a
d
Zheng-Yu Liu , Mei-Lin Tang , Jinfeng Ning , Yun-Peng Hao , Lu Zhou , Xun
Sun a,b,
*
a
Department of Natural Medicine, School of Pharmacy, Fudan University, 826
Zhangheng Road, Shanghai 201203, China
b The Institutes of Integrative Medicine of Fudan University, 12 Wulumuqi Zhong
Road, Shanghai 200040, China
c
State Key Laboratory of Molecular Engineering and Institutes of Biomedical
Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China
d
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826
Zhangheng Road, Shanghai 201203, China
*Corresponding author. Tel./Fax: +86-21-51980101. E-mail address:
sunxunf@shmu.edu.cn (X. Sun).

Abstract
Colorectal cancer (CRC) is the third most common cancer and the fourth leading
cause of cancer death around the world. The current treatments of CRC exhibited high
occurrence rate of side effects. Docetaxel (DTX), an important drug widely used in
cancer chemotherapy, showed serious toxicity in CRC. Reducing toxicity of DTX
could be a feasible and promising way to achieve the new indication of DTX for CRC.
In this study, a series of MMP-7 activated octapeptide-DTX/4FDT prodrugs (6a-10a
and 6b-10b) were designed and synthesized based on the features of MMP-7 which is
highly expressed in CRC and could specially recognize octapeptides with specific
sequences. Among them, 9a and 9b, both possessing an octapeptide
Gly-Pro-Gln-Gly-Ile-Ala-Met-Gln moiety, were the most potent prodrugs.
Compounds 9a and 9b were also tested their release rate in HCT116 cell culture fluids
and tumor homogenate along with in vivo anti-CRC activity and systemic toxicity.
Since 9a showed better anti-CRC activity and lower systemic toxicity than 9b in CRC
tumor bearing mice, it was further evaluated for its acute toxicity, pharmacokinetics
and tissue distribution in comparison with its parent drug DTX. These results revealed
that 9a possessed good systemic stability, rapid release rate in CRC and reduced
systemic toxicity, while retaining similar anti-CRC activity to its parent drug DTX.
Thus, 9a, an MMP-7 polypeptide prodrug of DTX, has been identified as a promising
candidate for the treatment of CRC.
Keywords: CRC, prodrug, DTX, MMP-7, systemic toxicity.

1. Introduction
Colorectal cancer (CRC) is one of the most incident and fast-growing malignancies
around the world, with an estimated 1.4 million new cases and 693,900 deaths in 2012
[1]. It will increase by 60% to more than 2.2 million new cases and 1.1 million deaths
to 2030 [2]. Currently, the most commonly used drugs for CRC include
fluoropyrimidines, irinotecan, oxaliplatin, angiogenesis inhibitors, and EGFR
inhibitors [3-5]. However, the serious toxicity of these drugs, such as diarrhea,
myelosuppression, neutropenia and alopecia, limited their clinical application and
treatment efficacy.
Docetaxel (DTX, Figure 1), which induces cell-cycle arrest and apoptosis by
stablishing microtubules through binding to beta tubulin, is one of most important and
exciting drugs used in cancer chemotherapy. This agent has been approved to treat
many types of cancer in clinical, including breast carcinoma, prostate cancer,
non-small cell lung cancer, head & neck cancer and stomach cancer [6-8]. However,
even DTX shows some efficacy with about 20% of response rate in advanced CRC
[9], the high rate of serious toxicity hinders its application in CRC[10, 11].
Figure 1. Structures of DTX and 4FDT
Interestingly, DTX exhibited high antiproliferative activity among 50 CRC cell
lines in a high-throughput study [12-14]. In our previous work, we synthesized a
series of fluorinated DTX derivatives [15] and found the compound 4FDT (Figure 1)
also exhibited good antiproliferative activity in CRC with better metabolic stability
and water solubility than DTX. Nevertheless, 4FDT still had the high systemic
toxicity issue [16, 17]. Therefore, developing novel DTX and 4FDT analogs via

reducing systemic toxicity could be an achievable goal to identify promising
candidates for the treatment of CRC.
For decades, the prodrug strategy has been sought as the “magic bullet” in drug
discovery to increase the druggability by improving solubility, metabolic stability,
target specificity and reducing systemic toxicity [18, 19]. Some prodrugs of DTX had
been reported to reduce toxicity, such as chitosan-conjugated DTX prodrugs [20],
ester-linked DTX prodrugs [21-24], albumin-hitchhiking DTX prodrugs [25] and
lipophilic thioether-bridged oleate DTX prodrugs [26]. However, how to empower
DTX to specially exert antiproliferation function in tumor is far from being well
addressed. In an effort to solve this challenge, the strategy of prodrug that can be
activated by particular biomarker of CRC was applied in the present work [18, 19].
Reports have shown that MMP-7 specifically expresses in primary and metastatic
CRC, but not in adjacent normal tissues [27, 28]. As a zinc-containing endopeptidase,
MMP-7 can promote tumor growth, microenvironment development, metastasis and
invasion by degrading several components of the extracellular matrix (ECM) [29, 30].
More importantly, the key character of MMP-7 is that it can recognize octapeptides
with specific sequences and hydrolyze them at P4-P5 peptide bond [31], including
Gly-Pro-Met-Gly-Ile-Ala-Gly-Gln,
Gly-Pro-Gln-Gly-Leu-Ala-Gly-Gln,
Gly-Pro-Gln-Ala-Ile-Ala-Gly-Gln,
Gly-Pro-Gln-Gly-Ile-Ala-Met-Gln, and
Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln. This significant character of MMP-7 could be
incorporated into prodrug strategy that polypeptides with specific sequences were
used as carriers to confer special antiproliferative function on chemotherapy drugs in
CRC [32]. The polypeptide-prodrug strategy has successfully been used before, such
as on doxorubicin [33] and auristatin [34]. Furtherly, this tactics enables prodrugs to
own the strengths of facile introduction of chemical modifications, low
immunogenicity and high tumor affinity and specificity [32, 35].

Figure 2. Design strategy and structures of DTX and 4FDT prodrugs
Herein, we designed a series of MMP-7 activated octapeptide prodrugs of DTX and
4FDT (6a-10a and 6b-10b). These prodrugs consist of three subunits (Figure 2) with
octapeptide carrier, linker and parent drug. The octapeptide carrier could be
specifically recognized and hydrolyzed peptide bond between fourth and fifth peptide
by MMP-7 [31]. The bridge chain was Leu-PABC which could possess both the
function of self-degradation and linker of the octapeptide carrier with the
chemotherapy drug [36]. DTX or 4FDT was chosen as the parent drug, since both
already have demonstrated efficacy for anti-CRC activity. It was envisaged that
promising analogs with good anti-CRC activity and reduced side effects could be
achieved via transporting octapeptide-prodrug into CRC environment, being
recognized and hydrolyzed by the high-expressing MMP-7 and releasing the parent
drug by self-degradation of the linker.
2. Results and Discussion
2.1. Synthesis
The synthesis of prodrugs 6a-10a and 6b-10b was outlined in Scheme 1 by
applying a five-step sequence. Followed a previously reported method [35],

Fmoc-L-Leu (1) was reacted with p-aminobenzyl alcohol (PABC) and
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) to give Fmoc-Leu-PABC
(2) in 73% yield [36]. Compound 2 was then treated with bis(p-nitrophenyl) carbonate
(bis-PNP) under Hünig's base to form an activated ester 3 in 82% yield by
recrystallization. Intermediate 3 was coupled with DTX or 4FDT [14] catalyzed by
DMAP to afford their desired corresponding 4a or 4b in 50-56% yield. Removal of
Fmoc group of 4a and 4b to be 5a and 5b was problematic here mainly due to the
unstable feature of the free amine. When exposed longer reaction time, multiple
impurities were appeared which resulted in lower yield and tedious separation. After
tried several conditions (e.g. HCl/Et2O, Et2NH/CH3CN, etc.), it was found that the
reaction was completed in 30 min under piperidine/DMF condition to give crude 5a
or 5b in 80-90% yield. With 5a and 5b in hand, analogs of 6a-10a and 6b-10b were
prepared in 15-35% yield (2 steps) by reacting with different octapeptides (A1-A5) in
the presence of 1-hydroxybenzotriazole (HOBT), N,N'-diisopropylcarbodiimide (DIC)
and N-methylmorpholine (NMM) in DMF.

Scheme 1. Synthesis of prodrugs 6a-10a and 6b-10b.
2.2. Biological Evaluation
2.2.1 In vitro cytotoxicity
All synthesized target compounds were evaluated for their in vitro cytotoxicity
against colon cancer cell lines HCT116 and SW620, normal colon cell line CCD18Co,
and normal kidney cell line HEK293 in comparison with their parent drugs (DTX or
4FDT) (Table 1). In this study, 5-FU was employed as a positive control. Results
revealed that the antiproliferative activity of all prodrugs were much higher than the
5-FU. Except 6b, most prodrugs showed good activity against HCT116 cell line.
Among them, 9b showed a slightly more potent than DTX (IC50 = 2.77 nM vs. 3.47
nM), though 4FDT was much more potent. For SW620 cell line, compound 9a had
the most potent activity in this series which was similar to that of DTX (IC50 = 30 nM
vs. 20 nM) and 9-fold more potent than that of 4FDT (IC50 = 280 nM). Except 6b, all
prodrugs showed higher antiproliferative activity against HCT116 than that of

SW620.
All prodrugs (6a-10a and 6b-10b) were significantly reduced toxicity compared to
their corresponding parent drugs DTX or 4FDT against normal cell line CCD18Co.
Most prodrugs also demonstrated weak toxicity against normal cell line HEK293.
Compound 9a and 9b, contained a common octapeptide carrier (octapeptide A4),
exhibited little or no toxicity effect against both normal cell line CCD18Co (IC50 >
200 µM) and HEK293 (IC50 > 86 µM). Particularly, 9a retained strong
antiproliferative activity against HCT116 and SW620 cell line (IC50 = 9.0 nM for
HCT116; IC50 = 30 nM for SW620). These data initially validated the strategy that
linking an octapeptide carrier to DTX or 4FDT could significantly reduce the toxicity
of parent drugs against normal cell lines.
Table 1. Cytotoxic activity of prodrugs against CRC and normal cell lines.
IC50 (µM)a
compound
HCT116b
26042.5 ± 335.2×10-3
3.5 ± 0.05×10-3
0.1 ± 0.01×10-3
9.8 ± 0.2×10-3
439.4 ± 24.3×10-3
7.2 ± 0.1×10-3
20.9 ± 1.3×10-3
11.3 ± 0.8×10-3
5.4 ± 0.1×10-3
9.1 ± 0.1×10-3
2.8 ± 0.1×10-3
5.2 ± 0.2×10-3
25.7 ± 0.6×10-3
SW620b
6.96 ± 0.07
0.02 ± 0.001
0.28 ± 0.013
1.01 ± 0.03
0.21 ± 0.01
1.14 ± 0.03
> 200
CCD18Cob
>200
HEK293b
79.32 ± 8.01
19.70 ± 1.31
0.36 ± 0.02
5-FU
DTX
4FDT
6a
116.54 ± 4.31
17.13 ± 0.74
> 200
15.24 ± 0.76
38.31 ± 1.71
11.02 ± 0.83
21.31 ± 0.52
106.00 ± 3.51
94.11 ± 2.73
136.83 ± 4.32
86.79 ± 2.71
165.34 ± 5.33
6.17 ± 0.34
6b
> 200
7a
> 200
7b
> 200
8a
> 200
> 200
8b
0.65 ± 0.02
0.03 ± 0.001
6.59 ± 0.73
21.92 ± 1.02
> 200
> 200
9a
> 200
9b
> 200
10a
10b
> 200
> 200
a: the concentration of drug to inhibit cell proliferation by 50% after 48 h or 72 h of treatment. b: cell
lines were treated for 48 h (HCT116, HEK293) or 72 h (SW620, CCD18Co) with different
concentrations of compound (10-10, 10-9, 10-8, 10-7,10-6, 10-5, and 10-4 mol/L).
2.2.2. In vitro stability and release rate
To investigate the stability of ten prodrugs, the half-life time in PBS and in plasma
of SD rat were tested (Table 2). After dissolving prodrugs in media, the unhydrolyzed

prodrugs were measured at set time points by RP-HPLC. The time needed for
prodrugs to reduce their concentration to half was calculated. Except 10a (t1/2 = 35.11
h) and 10b (t1/2 = 48.33 h), all other prodrugs showed a longer hydrolysis half-life
time in PBS (> 72 h). The high stability of these prodrugs in plasma of SD rat was
also observed. Except 8a and 10a, hydrolysis half-life time of all others reached to
around or more than 24 h. Overall, these results of in vitro stability indicated that 6a,
6b, 9a and 9b exhibited higher stability in both PBS and rat plasma.
Taken the results of their cytotoxic activity and stability in vitro together,
compounds 9a and 9b, both containing the carrier octapeptide A4, were chosen to
further evaluate their release rate in cell culture fluids and tumor homogenate of
HCT116. After incubating 9a or 9b in the culture mediate of HCT116 cells or in
0
HCT116 tumor homogenate at 37 C, the unhydrolyzed 9a or 9b and their
corresponding released drug DTX (as DTX*) or 4FDT (as 4FDT*) were measured by
RP-HPLC. Results showed that the percentage of release rate for 9a and 9b reached to
96.9% and 88.7% respectively during 4 h in HCT116 cell culture mediate; whilst in
HCT116 tumor homogenate, their percentage release rate reached to 96.3% and 98.7%
during 5 min respectively. These indicated that 9a and 9b can be rapidly released to
be their parent drugs in CRC environment while keeping stable in mock systemic
circulations of PBS and rat plasma.
As mentioned before, the hypothesis to design peptide-prodrugs was that these
prodrugs could keep stable in circulating system and then release parent drug in CRC
environment. The above results supported the feasibility of our rationale. The
preferred compounds 9a and 9b, stable in PBS and rat plasma but rapidly released
DTX* and 4FDT* in CRC circumstance, might have good antitumor activity to CRC
with lower systemic toxicity compared to their parent DTX and 4FDT. These results
inspired us to continue to explore their in vivo antitumor activity and systemic
toxicity.
Table 2. Stability of prodrugs in phosphate saline buffer (PBS) and rat plasma (37 0C).

a
T1/2
compound
PBS
Plasma
6a
6b
7a
＞ 72 h
＞ 7 2h
＞ 72 h
＞ 72 h
＞ 72 h
＞ 72 h
＞ 72 h
＞ 72 h
35.11 h
48.33 h
35.87 h
47.81 h
23.93 h
23.96 h
11.97 h
24.04 h
24.18 h
43.80 h
14.01 h
24.05 h
7b
8a
8b
9a
9b
10a
10b
a: At fixed time points (e.g. 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 10 h, 24 h, 48 h, 72 h),
equal samples were taken out and analyzed by RP-HPLC. See Experimental Section for details.
Figure 3. Release rate of 9a and 9b in the culture mediate of HCT116 cells or in HCT116 tumor
homogenate. (A) Concentration percentage of 9a and its released DTX (DTX*) in HCT116 cells
culture fluids at different time points. (B) Concentration percentage of 9b and its released 4FDT
(4FDT*) in HCT116 cells culture fluids at different time points. (C) Concentration percentage of 9a
and DTX* in HCT116 tumor homogenate at different time points. (D) Concentration percentage of 9b

and 4FDT* in HCT116 tumor homogenate at different time points.
2.2.3. In vivo efficacy and toxicity of compound 9a and 9b
To evaluate in vivo efficacy and toxicity of compound 9a and 9b, human CRC
cancer xenograft mouse models were established by subcutaneous inoculation of
HCT116 cells in the male BALB/C nude mice. After establishing solid tumor, the
mice were randomized into ten groups and each group assigned six mice. The mice
were injected intraperitoneally once every 7 days for two consecutive weeks and
tested groups received a different dose with 23.4 mg/kg of 9a (equivalent to DTX 10
mg/kg), or 11.7 mg/kg of 9a (equivalent to DTX 5 mg/kg), or 5.85 mg/kg of 9a
(equivalent to DTX 2.5 mg/kg), or 22.2 mg/kg of 9b (equivalent to 4FDT 10 mg/kg),
or 11.1 mg/kg of 9b (equivalent to 4FDT 5 mg/kg), or 5.55 mg/kg of 9b (equivalent
to 4FDT 2.5 mg/kg). The parent drug control groups received 5 mg/kg of DTX or 5
mg/kg of 4FDT and the positive control group received 5 mg/kg of 5-FU. The vehicle
control group received the solvent formula only. Tumor progression and weight of
mice were monitored once every other day. At the end of observation day, the mice
were scarified and the tumors were stripped and weighed.
As shown in Figure 4A, at the end of observation period (14th day), both tested
groups and the parent drug control groups exhibited statistically significant reduction
in tumor progression compared with the vehicle. There was no notable difference
between the positive control group of 5-FU (5 mg/kg) and vehicle control group in
tumor weight. Moreover, both 9a and 9b inhibited tumor growth in a dose-dependent
manner. At the same molar equivalent dose with 5 mg/kg DTX or 5 mg/kg 4FDT, the
tumor growth inhibition (TGI) of 9a (11.7 mg/kg), 9b (11.1 mg/kg), DTX (5 mg/kg)
and 4FDT (5 mg/kg) was 46.3%, 35.2%, 42.7% and 41.9% respectively (p < 0.05).
The TGI of 5-FU (5 mg/kg) was 11.4% (p > 0.05). At the same molar equivalent dose
with DTX (10 mg/kg) or 4FDT (10 mg/kg), groups of 9a (23.4 mg/kg) and 9b (22.2
mg/kg) exhibited enhanced inhibitory effect on the tumor growth with the TGI of 56.1%
(p < 0.01) and 46.3% (p < 0.05) respectively. At the same molar equivalent dose with
DTX (2.5 mg/kg) or 4FDT (2.5 mg/kg), tumor inhibition rate of groups 9a (5.85

mg/kg) and 9b (5.55 mg/kg) remained to reach 28.8% (p < 0.05) and 30.5% (p < 0.05)
respectively. These results indicated that 9a and 9b demonstrated similar in vivo
antitumor efficacy for CRC to their corresponding parent drugs DTX or 4FDT and
much more efficacious than 5-FU. These results were consistent with the conclusion
derived from in vitro cytotoxicity study.
At the end of observation period, animals treated with DTX (5 mg/kg), 9a (23.4
mg/kg), 9a (11.7 mg/kg), and 9a (5.85 mg/kg) exhibited body weight loss of 13.0%,
12.2%, 8.5% and 4.9% respectively. Similarly, mice treated with 4FDT (5 mg/kg), 9b
(22.2 mg/kg), 9b (11.1 mg/kg) and 9b (5.55 mg/kg) exhibited body weight loss of
9.5%, 8.7%, 8.5% and 4.6% respectively (Figure 4B and 4C). It was observed that 9a
and 9b had less effect on body weight of mice than their corresponding parent drugs
DTX or 4FDT at same molar dose (5 mg/kg) and even in high dose (10 mg/kg). This
indicated that prodrugs 9a or 9b might have lower systemic toxicity than DTX or
4FDT in vivo.
To further assess the systemic toxicity of prodrugs 9a and 9b, the heart and liver
tissues were collected from xenograft mice and stained by H&E at the end of
observation time. The systemic toxicity was compared among vehicle control group,
positive control group for 5-FU (5 mg/kg), groups for DTX (5 mg/kg) and 4FDT (5
mg/kg), 9a (11.73 mg/kg) and 9b (11.17 mg/kg). As shown in Figure 4D, DTX
caused concentrated nucleus and 4FDT caused tissue necrosis in liver, both which did
not appear on group of 9a and 9b. As shown in Figure 4E, disordered myocardial
fibers and inflammatory cell invasion in heart were significantly observed on DTX
and 4FDT, but not on 9a and 9b. This observation again indicated that prodrugs 9a or
9b may have lower systemic toxicity compared to DTX or 4FDT in vivo at the same
molar dose.

Figure 4. Inhibition of subcutaneous solid tumor growth in vivo by 9a, 9b, DTX, 4FDT and 5-FU.
(A) The weight of the excised tumor at the end of the observation period. (B) The mouse body weights
of groups 9a and DTX recorded during the observation period. (C) The mouse body weights of groups
9b and 4FDT recorded during the observation period. (D) Histological appearance of the liver (400×).
(E) Histological appearance of the heart (100×). Data are expressed as the mean ± SD (n = 6): (∗) p <
0.05 and (∗∗) p < 0.01 vs control. a: The same molar equivalent dose of DTX; b: the same molar
equivalent dose of 4FDT.
2.2.4. Acute toxicity in mice
Compound 9a was chosen for further evaluation of its acute toxicity in mice, since
it had better anti-CRC activity and lower systemic toxicity in vivo. Healthy Kunming
mice (weighing 20 ± 2 g) were randomly divided into five groups with equal of both
sexual and behavioral status. Death status of mice was continuously recorded for 14
days after intraperitoneally injected with different dose of tested compounds. Bliss
method was applied to calculate the value of LD50. Results showed that the LD50 of
prodrug 9a (245.25 mg/kg) was 3.3-fold higher than that of its parent drug DTX (74.1
mg/kg). When converted the LD50 value of 9a to the same molar equivalent dose of
DTX (104.3 mg/kg), it still showed 1.4-fold higher than that of parent drug DTX

(74.1 mg/kg). This confirmed that the safety margin of 9a was larger than its parent
drug DTX.
2.2.5. In vivo pharmacokinetics
Prodrug 9a and its parent drug DTX were administrated intravenously to Sprague
Dawley (SD) rats at a dose of 5 mg/kg respectively. The plasma concentrations of 9a,
DTX* (DTX released from 9a) and DTX were determined by LC-MS/MS. The
parameters of DTX group were calculated after converting DTX dose to the same
molar equivalent of 9a. The exposure level of 9a was ten-fold higher than that of DTX
and volume of distribution of 9a was two times lower than that of DTX (Table 3).
Moreover, 9a showed significantly larger mean residence time (MRT0-∞) and longer
half-life time than that of DTX, whereas the concentration of DTX* was much less
than that of the free parent drug DTX (Table 3, Figure 5). This meant that 9a could
keep primary type for long period in plasma and might contribute to decrease the
systemic toxicity induced by DTX*.
Table 3. The pharmacokinetic parameters of 9a, DTX*and DTX（iv, 5 mg/kg）
Compound 9a
Parameters
DTX i
9a
DTX*
247 ± 124
AUC0-t (µg/L*h)
AUC0-∞(µg/L*h)
MRT0-∞ (h)
t1/2 (h)
9069 ± 1786
9244 ± 1800
11.57 ± 2.76
12.81 ± 2.32
10.39 ± 2.47
0.56 ± 0.12
930± 139
867 ± 146
3.76 ± 0.39
5.75 ± 0.88
22.6 ± 2.76
2.76 ± 0.43
299 ± 103
9.04 ± 2.53
7.03 ± 1.76
206.32 ± 124.39
18.98 ± 6.90
Vz (L/kg)
CLz (L/h/kg)
Pharmacokinetic data were evaluated using a non-compartment model and showed as mean ± SD (n =
3). i: The parameters of DTX group were calculated after converting DTX dose to the same molar
equivalent of 9a.

Figure 5. The concentration-time profile after intravenous administration of 9a and DTX in SD rats (n
= 3).
2.2.6. In vivo distribution
Tissue distribution was evaluated in mice subcutaneously implanted with HCT116
tumors following administration of 9a and its parent drug DTX respectively. After
intravenous injection, concentrations of 9a, DTX* (DTX released from 9a) and the
free parent drug DTX in heart, liver, spleen, lung, kidney and tumor were measured
time course by LC-MS/MS. As shown in Figure 6, the concentration of free DTX
decreased rapidly in all tissues after 0.5 h, while the prodrug 9a kept relatively high
level for long time. Considering that 9a shows long half-life time (t1/2 for 9a was 12.8
h) and great AUC0-∞ (9069 µg/L*h) in plasma of SD rats in pharmacokinetics study,
9a was speculated more likely to be distributed in the blood of mice initially and
slowly distributed to various tissues and organs over time. As shown in Figure 6, the
concentration of DTX* was lower than that of 9a in heart, liver, spleen, lung, and
kidney at most time points, excepting in heart at 0.5 h and 4 h, and in lung and kidney
at 24 h. Moreover, the concentration of DTX* and 9a showed decreased trend in all
normal tissues since 8 h. However, in tumor, the concentration of DTX* was higher
than that of 9a from 8 h to 24 h and continued to increase over time, while 9a showed
decreased trend since 4 h. The different release rates of 9a in tumor and other tissues
indicated that the 9a may possess relatively higher stability in plasma and normal
tissues and more easily recognized and hydrolyzed in CRC tumor by MMP-7 to
release DTX. Thus, 9a may exhibit sustainable therapeutic efficacy for CRC and

meanwhile reduce systemic toxicity for other tissues.
Figure 6. Tissue bio-distribution of 9a and DTX in mice bearing HCT116 xenograft at 0.5, 4, 8 and 24
h after intravenous injection (n = 5 for each group at each time point).
3. Conclusion
In present study, with the aim of reducing systemic toxicity of DTX or 4FDT and
developing their new indication for CRC, a series of MMP-7 activated
octapeptide-DTX/4FDT prodrugs were designed, synthesized and biologically
evaluated. Most of the synthesized prodrugs displayed comparable antitumor activity
to CRC cells (HCT116, SW620) and less cytotoxicity to normal cells (CCD18Co,
HEK293) when compared with their parent drugs. In addition, all prodrugs exhibited
good stability in PBS and plasma of rats. Among them, compound 9a and 9b, both
containing the same octapeptide carrier A4, were chosen to test their release rate in
CRC circumstance of cell culture fluids and tumor homogenate of HCT116. The fast
release rates of 9a and 9b in CRC circumstance and good stability in plasma
encouraged us to further study their systemic toxicity and anti-CRC activity in vivo.
At the same molar equivalent (5 mg/kg) as their parent drugs, 9a and 9b exhibited less

effect on body weight and lower toxicity injury on heart and liver of mice than DTX
and 4FDT. Also, 9a and 9b showed similar antitumor activity to DTX and 4FDT in a
subcutaneous solid tumor model of CRC. Prodrug 9a was further tested for its acute
toxicity, pharmacokinetics and bio-distribution. Acute toxicity experiment showed
that LD50 of 9a was increased 3.3-fold than its parent drug DTX which demonstrated
that 9a had a relatively larger safety margin than parent drug DTX. Pharmacokinetic
study indicated that 9a exhibits much higher distribution and longer half-life time in
plasma than DTX. The release rate of 9a in plasma was only 16.2% and the
concentration of DTX* (9a-released DTX) was much lower than that of free parent
drug DTX. In bio-distribution study, it was found the release rate of 9a was high in
tumor, but relatively low in other organs. In conclusion, the new prodrug 9a possessed
low systemic toxicity to normal tissues, while kept similar antitumor activity for CRC
to its parent drug DTX. These results demonstrated that these novel prodrugs of DTX
or 4FDT, conjugated an octapeptide by linker, could be feasible way to reduce
systemic toxicity of DTX or 4FDT and maintain good antitumor activity for CRC.
4. EXPERIMENTAL SECTION
4.1. Materials and physical measurements
Compound 4FDT was previously synthesized and published by our group [15].
Octapepetides were custom-made by Jie peptide Biological Technology Co., Ltd.
(Shanghai, China) with a purity ≥ 95%. Commercially available chemicals were
purchased from Aldrich and TCI Chemical companies. All solvents were purified and
dried in accordance with standard procedures unless otherwise indicated. Reactions
were monitored by TLC using Yantai (China) GF254 silica gel plates (5
×
10 cm) or
by HPLC using Waters e2695 with X-Bridge C18-column (5 µm, 3.0 × 50 mm).
Silica gel column chromatography was performed on silica gel (300-400 mesh) from
Yantai (China). Semi-preparative reversed phase liquid chromatography was
1
conducted on Combi Flash Rf from Teledyne Isco (USA). H NMR spectra were

13
recorded at Varian Mercury 400 (400 MHz) or Bruker Avance DPX400, C NMR
spectra were recorded at Bruker (151 MHz). Low-resolution Mass spectra data were
obtained using an Agilent 1946D LC/MSD (ESI) and high-resolution Mass spectra
data were performed on AB SCIEX TripleTOF™ 5600+.
4.2. Chemistry
4.2.1. Fmoc-Leu-PABC-PNP (3)
To a solution of N-ethoxycarbonyl-2-ethoxy-1,2- dihydroquinoline (EEDQ) (3.15 g,
2.59 mmol) and Fmoc-L-Leu (1, 4.15 g, 2.35 mmol) in anhydrous DCM (250 mL)
was added 4-aminobenzyl alcohol (PAB-OH) (1.55 g, 2.59 mmol). The mixture was
continuously stirred at room temperature for 24 h. The solvent was then evaporated
under reduced pressure and the resulting residue was purified by silica gel
chromatography (DCM/MeOH = 50 : 1 to 10 : 1) to afford 2 (1.00 g, 73%) as a
colorless powder. ESI-MS: m/z 459.2 [M+H]+, 481.2 [M+Na]+, HPLC analysis (λ =
220 nm) > 95% of peak area.
To a solution of 2 (4.00 g, 8.73 mmol) and bis(p-nitrophenyl)carbonate (bis-PNP)
(13.30 g, 43.65 mmol) in anhydrous N,N-dimethylformamide (DMF) (100 mL),
N,N-diisopropylethylamine (DIPEA) (4.33 mL, 26.19 mmol ) was added dropwise at
0
0 C. After stirring at ice-bath for 5 minutes, the reaction mixture was continuously
stirred at room temperature for approximately 6 h. Subsequently, water (300 mL) was
added and the mixture was extracted with ethyl acetate (3
×
200 mL). The combined
organic extracts were washed with brine (200 mL) and dried over Na2SO4. After
filtered, the solvent was concentrated and the resulting residue was purified roughly
by flash chromatography (petroleum ether/acetone = 7 : 1) to remove most of
impurities. Then, the concentrated residue was further recrystallized with ethyl
acetate/petroleum ether (V/ V = 1 : 1) to give compound 3 ( 4.46 g, 82%) as a
colorless powder. 1H NMR (400 MHz, CDCl3) δ ppm: 8.27 (d, J = 8.0 Hz, 2H), 7.76
(d, J = 8.0 Hz, 2H), 7.61-7.49 (m, 4H), 7.49-7.44 (m, 1H), 7.41-7.35 (m, 6H),
7.30-7.28 (m, 1H), 5.24 (s, 2H), 4.51-4.45 (m, 2H), 4.29 (s, 1H), 4.21 (dd, J = 8.0 Hz,
13
12.0 Hz, 1H), 1.80-1.68 (m, 2H), 1.04 (d, J = 4.0 Hz, 1H), 0.98-0.95 (m, 6H); C

NMR (100 MHz, CDCl3) δ ppm: 170.34, 155.53, 152.44, 145.43, 143.56, 141.36,
129.73, 127.83, 126.60, 125.47, 125.35, 124.89, 122.14, 121.78, 120.08, 70.60, 67.24,
47.17, 24.76, 22.93. ESI-MS: m/z 623.6 [M+H]+, 646.6 [M+Na]+; C35H33N3O8:
HRMS calcd. 646.2160 [M+Na]+, found 646.2162.
4.2.2. General procedure for the synthesis of 4a or 4b.
Compound 3 (535.95 mg, 0.86 mmol), 4-(dimethylamino)pyridine (DMAP)
(105.07 mg, 0.86 mmol) and appropriate DTX (or 4FDT ) (0.86 mmol) were
dissolved in anhydrous DCM (37 mL) under N2. The reaction was stirred at room
temperature for 48 h. Then, the organic solvent was concentrated under reduced
pressure, and the crude residue was purified by flash chromatography column
(DCM/MeOH = 100 : 1) to afford 4a (550 mg, 50%) as a colorless powder or 4b (550
mg, 54%) as a light yellow powder.
Fmoc-Leu-PABC-DTX (4a). Colorless powder. Yield: 50%. 1H NMR (400 MHz,
Acetone-d6) δ ppm: 9.43 (s, 1H), 8.12 (d, J = 7.6 Hz, 2H), 7.86 (d, J = 7.6 Hz, 2H),
7.76-7.64 (m, 5H), 7.58 (t, J = 7.6 Hz, 2H), 7.51 (d, J = 7.6 Hz, 2H), 7.46-7.35 (m,
6H), 7.34-7.25 (m, 3H), 7.04 (d, J = 9.6 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.11 (d, J =
8.4 Hz, 1H), 5.68 (d, J = 7.2 Hz, 1H), 5.40 (d, J = 9.2 Hz, 1H), 5.30 (d, J = 5.2 Hz,
1H), 5.25 (s, 1H), 5.19 (d, J = 11.80 Hz, 1H), 5.11 (d, J = 11.80 Hz, 1H), 4.97 (d, J =
8.8 Hz, 1H), 4.39-4.30 (m, 5H), 4.27-4.21 (m, 2H), 4.17 (s, 2H), 3.93 (d, J = 6.8 Hz,
1H), 3.68 (s, 1H), 2.83 (s, 1H), 2.45 (d, J = 17.6 Hz, 4H), 2.38-2.28 (m, 1H), 2.12 (d,
J = 8.4 Hz, 1H), 1.93-1.77 (m, 5H), 1.75-1.65 (m, 5H), 1.45- 1.25 (m, 20H), 1.17 (d, J
13
= 12.4 Hz, 6H), 0.96 (dd, J = 10.8, 6.6 Hz, 6H), 0.88 (t, J = 6.6 Hz, 1H). C NMR
(151 MHz, Acetone-d6) δ ppm: 209.96, 204.48 , 170.69, 169.18, 168.00, 165.15,
155.70, 154.72, 153.59, 143.60, 140.59, 138.83, 137.22, 137.10, 135.99, 132.56,
129.80, 129.74, 129.34, 128.72, 128.05, 127.88, 127.47, 127.01, 126.61, 126.42,
126.40, 124.62, 119.28, 118.86, 83.58, 80.24, 78.15, 77.27, 77.13, 75.30, 74.52, 73.67,
73.55, 71.43, 70.85, 70.74, 69.12, 65.66, 57.04, 54.34, 53.71, 46.55, 45.95, 42.63,
40.49, 36.10, 35.08, 31.10, 30.47, 30.16, 29.08, 28.8, 27.00, 25.50, 23.97, 21.94,
21.79, 21.62, 20.43, 19.88. ESI-MS: m/z 1292.4 [M+H]+, 1314.2 [M+Na]+;

C72H81N3O19: HRMS calcd. 1314.5356 [M+Na]+, found 1314.5366.
1
Fmoc-Leu-PABC-4FDT (4b). Light yellow powder. Yield: 54%. H NMR (400
MHz, Acetone-d6) δ ppm: 9.46 (s, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 7.6 Hz,
2H), 7.79 (d, J = 9.6 Hz, 1H), 7.75-7.67 (m, 5H), 7.67-7.61 (m, 1H), 7.56-7.47 (m,
3H), 7.47-7.35 (m, 7H), 7.34-7.25 (m, 3H), 6.84 (d, J = 8.0 Hz, 1H), 6.10 (t, J = 9.2
Hz, 1H), 5.66 (d, J = 6.8 Hz, 1H), 5.40-5.34 (m, 1H), 5.32 (d, J = 5.6 Hz, 1H), 5.24
(brs, 1H), 5.20 (d, J = 12.0 Hz, 1H), 5.12 (d, J = 12.0 Hz, 1H), 4.98 (d, J = 9.2 Hz,
1H), 4.41-4.28 (m, 7H), 4.24 (dd, J = 6.8 Hz, 8.0 Hz, 1H), 4.20-4.12 (m, 2H), 3.92 (d,
J = 7.2 Hz, 1H), 3.79 (brs, 1H), 2.52-2.42 (m, 5H), 2.26 (dd, J = 15.6, 8.6 Hz, 2H),
2.02-1.95 (m, 1H), 1.90-1.75 (m, 7H), 1.73-1.63 (m, 6H), 1.62-1.50 (m, 7H),
13
1.44-1.34 (m, 1H), 1.34-1.24 (m, 5H), 1.17 (brs, 4H), 1.00-0.84 (m, 7H). C NMR
(151 MHz, Acetone-d6) δ ppm: 209.83, 170.74, 169.13, 167.96, 163.95, 161.90 (d, J
= 245.4 Hz), 155.72, 153.52, 153.07, 143.60, 143.45, 140.59, 138.86, 137.00, 136.51,
135.99, 132.11 (d, J = 7.4 Hz), 130.07 (J = 7.7 Hz), 129.69, 128.77, 128.18, 127.76,
127.01, 126.68, 126.42, 126.40, 125.35, 124.63, 124.62 (q, J = 282.1 Hz), 119.56 (d, J
= 21.4 Hz), 119.28, 118.89, 115.74 (d, J = 23.1 Hz), 83.57, 80.23, 77.13, 77.06, 75.19,
75.05, 73.64, 71.39, 70.81, 69.22, 65.68, 57.01, 54.81, 53.72, 46.55, 45.90, 42.60,
40.49, 36.05, 35.13, 25.46, 23.97, 21.94, 21.57, 20.44, 19.89, 18.30, 12.91, 8.82.
ESI-MS: m/z 1364.2 [M+H]+, 1386.2 [M+Na]+ ；C72H77F4N3O19: HRMS calcd.
1386.4980 [M+Na]+, found 1386.5001.
4.2.3. General procedure for the synthesis of 5a and 5b.
To solution of compound 4a or 4b (0.5 mmol) in anhydrous DMF (1 mL) was
0
added piperidine (5.00 equiv) at 0 C. The mixture reaction was stirred at room
temperature for half an hour. After removed DMF, the residue was diluted with DCM
(20 mL) and washed with brine (20 mL). The organic layer was concentrated next and
dried in vacuo to give crude 5a or 5b in 80%-90% purity which was used directly for
the step without further purification.
4.2.4. General procedure for the synthesis of peptide-prodrugs (6a-10a, 6b-10b).

To a solution of octapeptide (A1-A5) (0.04 mmol), 5a or 5b (0.04 mmol) and
1-hydroxybenzotriazole (HOBt) (0.12 mmol) in anhydrous DMF (4 mL),
0
4-methylmorpholine (0.16 mmol) was added dropwise under N2 at 0 C. After the
reaction mixture stirred at 0 0C for 15 min, N,N'-diisopropylcarbodiimide (DIC) (0.24
mmol) was added and the mixture was stirred at 5 0C for about 72 h to fully generated
peptide-prodrugs (6a-10a, 6b-10b). Subsequently, DMF was removed by vacuum
pump and the crude product was purified by reverse-phase preparative HPLC
chromatography with acetonitrile/water (V/V = 0% ~ 100% within 50 min, 1 mL/min)
as mobile phase to afford 6a-10a and 6b-10b. Purity analysis and reaction monitoring
of peptide-prodrugs were performed using HPLC with the UV detector set at 254 nm,
acetonitrile and water (V/V = 0 ~ 100% within 25 min) were used as mobile phase at
a flow-rate of 0.5 mL/min.
1
DTX-PABC-Leu-A1 (6a). Colorless powder. Yield: 17%. H NMR (400 MHz,
DMSO-d6) δ ppm: 9.95 (s, 1H), 8.16-8.05 (m, 4H), 7.99 (d, J = 8 Hz, 1H), 7.94 (d, J =
6.4 Hz, 3H), 7.90 (d, J = 8 Hz, 2H), 7.85 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 7.6 Hz, 3H),
7.67-7.63 (m, 3H), 7.43-7.39 (m, 6H), 7.37-7.28 (m, 7H), 7.16 (t, J = 7.2 Hz, 1H),
6.82 (brs, 1H), 5.77 (dd, J = 8.0 Hz, 12.0 Hz, 1H), 5.39 (d, J = 7.2 Hz, 1H), 5.14 (brs,
2H), 5.11-5.06 (m, 3H), 5.03 (d, J = 7.2 Hz, 2H), 4.97 (brs, 1H), 4.90 (d, J = 10.4 Hz,
1H), 4.46-4.39 (m, 3H), 4.31 (d, J = 8.0 Hz, 1H), 4.26-4.19 (m, 7H), 4.03-3.97 (m,
3H), 3.80-3.70 (m, 5H), 3.64 (d, J = 6.8 Hz, 2H), 3.51-3.43 (m, 1H), 2.27-2.21 (m,
5H), 2.15-2.08 (m, 2H), 1.93-1.83 (m, 4H), 1.72 (brs, 3H), 1.65-1.59 (m, 4H), 1.51
(brs, 6H), 1.33 (s, 9H), 1.26-1.19 (m, 6H), 0.96 (brs, 6H), 0.91 (d, J = 6.0 Hz, 3H),
0.88-0.84 (m, 6H), 0.83- 0.74 (m, 12H). 13C NMR (151 MHz, DMSO-d6) δ ppm:
209.14, 173.76, 172.48, 172.22, 171.46, 171.32, 171.11, 171.00, 170.69, 169.42,
168.81, 168.73, 168.61, 167.47, 165.13, 156.35, 156.24, 154.94, 153.62, 143.69,
140.54, 139.08, 137.04, 136.81, 135.64, 133.24, 129.88, 129.54, 129.40, 129.03,
128.49, 127.97, 127.45, 127.20, 126.91, 125.09, 119.93, 83.61, 80.14, 78.36, 77.51,
76.61, 75.23, 74.63, 73.57, 71.36, 70.57, 69.39, 65.60, 65.54, 59.52, 58.37, 56.83,
56.60, 56.47, 54.99, 52.21, 51.89, 50.99, 50.88, 48.35, 48.30, 46.45, 45.81, 45.74,

42.71, 42.62, 42.08, 41.89, 40.58, 40.39, 40.34, 36.59, 36.48, 36.31, 34.50, 31.71,
31.27, 28.87, 27.93, 27.76, 26.28, 24.16, 24.10, 24.04, 23.99 22.90, 22.83,
22.29,21.89, 21.34, 21.04, 20.57, 17.54, 17.50, 15.09, 13.49, 10.86, 9.61. ESI-MS:
m/z 1015.4 [M/2+Na]+; C103H132N12O28: HRMS calcd. 2007.9166 [M+Na]+, found
2007.9246.
1
DTX-PABC-Leu-A1 (6b). Colorless powder. Yield: 22%. H NMR (400 MHz,
DMSO-d6) δ ppm: 10.00 (s, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.28–8.07 (m, 4H), 8.05–
7.93 (m, 3H), 7.90 (d, J = 8.0 Hz, 2H), 7.82 (d, J = 8.0 Hz, 2H), 7.75-7.69 (m, 3H),
7.67-7.60 (m, 2H), 7.49-7.37 (m, 7H), 7.35-7.27 (m, 5H), 7.19 (t, J = 8.0, 4.0 Hz, 1H),
6.82 (brs, 1H), 5.78 (dd, J = 8.0 Hz, 12.0 Hz, 1H), 5.38 (d, J = 4.0 Hz, 1H), 5.22-5.08
(m, 4H), 5.06-4.96 (m, 3H), 4.91 (d, J = 8.0 Hz, 1H), 4.54 (brs, 1H), 4.41 (brs, 1H),
4.34-4.17 (m, 7H), 4.16-4.08 (m, 6H), 4.07-3.95 (m, 3H), 3.90-3.49 (m, 5H),
3.19-3.15 (m, 14H), 2.89 (brs, 3H), 2.73 (brs, 3H), 2.24 (brs, 4H), 1.72 (brs, 5H),
1.57-1.49 (m, 10H), 1.23 (s, 5H), 0.98 (s, 5H), 0.91 (d, J = 8.0 Hz , 3H), 0.87 (brs,
5H), 0.80 (d, J = 8.0 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) δ ppm: 209.01,
173.77, 172.48, 172.23, 171.47, 171.11, 171.01, 170.70, 169.32, 168.80, 168.61,
167.47, 167.20, 163.95, 162.15, 160.99, 156.35, 153.56, 153.18, 143.69, 140.54,
139.09, 136.82, 136.36, 135.53, 132.16, 132.12, 130.89, 129.49, 129.02, 128.55,
128.22, 127.45, 127.20, 126.91, 125.60, 125.09, 120.29, 119.93, 119.06, 115.84,
83.57, 80.14, 78.52, 78.33, 77.26, 76.59, 75.15, 73.54, 71.34, 70.55, 69.48, 65.60,
65.54, 59.51, 56.80, 56.60, 56.46, 55.33, 52.20, 51.88, 50.98, 50.88, 48.43, 46.45,
45.76, 42.68, 42.62, 42.07, 40.58, 40.40, 40.33, 36.48, 36.27, 35.61, 34.49, 31.26,
30.62, 28.85, 27.75, 26.28, 24.15, 23.98, 22.89, 22.82, 22.24, 21.88, 21.33, 21.03,
20.56, 19.16, 19.11, 17.53, 17.49, 15.08, 13.46, 10.85, 9.58. ESI-MS: m/z 1029.4
[M/2+H]+; C103H128F4N12O28: HRMS calcd. 2079.8789 [M+Na]+, found 2079.8879.
1
DTX-PABC-Leu-A2 (7a). Colorless powder. Yield: 21%. H NMR (400 MHz,
DMSO-d6) δ ppm: 9.99 (s, 1H), 8.19 (d, J = 7.9 Hz, 1H), 8.13-7.09 (m, 3H),
8.05-7.92 (m, 6H), 7.86-7.79 (m, 1H), 7.75-7.69 (m, 1H), 7.65 (d, J = 7.2 Hz, 4H),
7.41 (t, J = 7.2 Hz, 2H), 7.38-7.31 (m, 4H), 7.29 (brs, 1H), 7.17 (t, J = 7.2 Hz 1H),

6.82 (brs, 1H), 5.78 (dd, J = 8.0 Hz, 12.0 Hz, 1H), 5.40 (d, J = 6.9 Hz, 1H), 5.15 (brs,
2H), 5.08 (d, J = 8.3 Hz, 2H), 5.03 (d, J = 7.3 Hz, 2H), 4.96 (s, 1H), 4.90 (d, J = 9.2
Hz, 1H), 4.5-4.37 (m, 3H), 4.33-4.17 (m, 5H), 4.12-4.96 (m, 4H), 3.95-3.89 (m, 1H),
3.87-3.78 (m, 2H), 3.78-3.67 (m, 4H), 3.64 (d, J = 6.4 Hz, 1H), 3.53 (brs, 1H), 2.24
(brs, 5H), 2.15-2.08 (m, 2H), 2.03 (brs, 4H), 1.86 (brs, 5H), 1.84 (brs, 2H), 1.72 (brs,
4H), 1.51 (brs, 6H), 1.34 (brs, 10H), 1.23 (d, J = 6.0 Hz, 6H), 0.98 (brs, 7H),
0.93-0.85 (m, 7H), 0.85-0.76 (m, 7H). 13C NMR (151 MHz, DMSO-d6) δ ppm:
209.13 , 173.76, 172.47, 171.60, 171.26, 171.11, 170.70, 169.42, 169.37, 169.17,
168.81, 168.72, 168.54, 168.44, 167.42, 167.15, 165.14, 154.94, 153.62, 139.07,
137.04, 136.81, 135.64, 133.25, 129.88, 129.55,129.40, 129.04, 128.49, 127.97,
127.20, 119.06, 83.61, 80.14, 78.36, 77.51, 76.62, 75.23, 74.63, 73.57, 71.36, 70.57,
69.40, 59.59, 58.54, 56.83, 56.61, 54.99, 52.21, 51.88, 51.76, 48.35, 46.58, 45.88,
45.81, 42.72, 42.07, 41.22, 40.40, 36.49, 31.43, 31.27, 29.47, 28.92, 27.76, 26.29,
24.22, 24.17, 24.06, 22.84, 22.30, 22.18, 21.19, 21.35, 20.57, 17.52, 15.10, 14.42,
13.49, 10.88, 9.61. ESI-MS: m/z 1843.8 [M+Na]+; C89H122N12O27S: HRMS calcd.
1845.8155 [M+Na]+, found 1845.8219.
1
DTX-PABC-Leu-A2 (7b). Colorless powder. Yield: 23%. H NMR (400 MHz,
DMSO-d6) δ ppm: 10.00 (s, 1H), 8.52 (d, J = 8.0Hz, 1H), 8.11 (d, J = 8.0 Hz, 2H),
8.06-7.93 (m, 3H), 7.82 (dd, J = 8.0, 4.0 Hz, 2H), 7.78-7.61 (m, 5H), 7.47-7.39 (m,
3H), 7.37-7.29 (m, 4H), 7.18 (t, J = 7.0 Hz, 1H), 6.82 (brs, 1H), 5.78 (dd, J = 8.0 Hz,
12.0 Hz, 1H), 5.38 (d, J = 8.0 Hz, 1H), 5.35-5.29 (m, 1H), 5.21-5.07 (m, 4H), 5.05 (d,
J = 8.0Hz, 1H), 5.01 (brs, 2H), 4.92 (d, J = 8.0 Hz, 1H), 4.54 (brs, 1H), 4.51-3.66 (m,
13H), 3.66-3.38 (m, 1H), 3.29-2.96 (m, 4H), 2.89 (brs, 1H), 2.73 (brs, 1H), 2.24 (brs,
3H), 2.15-2.08 (m, 2H), 2.03 (brs, 4H), 1.89-1.82 (m, 5H), 1.79-1.62 (m, 9H), 1.55
(brs, 3H), 1.54-1.48 (m, 7H), 1.23 (brs, 9H), 0.98 (brs, 6H), 0.91 (d, J = 8.0 Hz, 3H),
0.89-0.85 (m, 4H), 0.83-0.75 (m, 7H). 13C NMR (151 MHz, DMSO-d6) δ ppm:
209.10, 174.14, 173.77, 172.48, 171.61, 171.52, 171.23, 170.71, 170.65, 169.39,
169.33, 169.18, 168.81, 168.61, 168.55, 167.42, 167.16, 163.96, 162.61, 160.99,
153.56, 153.18, 139.09, 136.82, 136.36, 135.53, 132.16, 130.89, 129.49, 129.03,

128.55, 127.21, 125.61, 120.43, 120.29, 119.07, 115.84, 115.69, 83.53, 80.14, 77.26,
76.59, 75.15, 73.55, 71.34, 70.55, 69.48, 59.59, 56.80, 56.61, 52.20, 51.87, 51.75,
48.35, 45.88, 45.77, 42.68, 41.86, 31.42, 31.26, 29.46, 28.91, 28.84, 28.65, 27.75,
26.29, 24.21, 24.15, 24.05, 22.82, 22.24, 22.17, 21.34, 20.56, 19.16, 19.12, 17.50,
15.09, 14.41, 13.46, 10.86, 9.58. ESI-MS: m/z 948.6 [M/2+H]+; C89H118F4N12O27S:
HRMS calcd. 1917.7778 [M+Na]+, found 1917.7831.
1
DTX-PABC-Leu-A3 (8a). Colorless powder. Yield: 18%. H NMR (400 MHz,
DMSO-d6) δ ppm: 9.97 (s, 1H), 8.22-8.10 (m, 4H), 8.07-7.95 (m, 6H), 7.79-7.61 (m,
7H), 7.47-7.27 (m, 8H), 6.81 (brs, 2H), 5.86-5.72 (m, 1H), 5.45-5.36 (m, 1H),
5.17-5.01 (m, 6H), 5.00-4.88 (m, 2H), 4.44 (brs, 2H), 4.26 (s, 4H), 4.18-4.11 (m, 2H),
4.07-3.98 (m, 3H), 3.90 (brs, 2H), 3.74 (brs, 3H), 3.58-3.47 (m,2H), 2.29-2.20(m, 4H),
2.17-2.06 (m, 5H), 1.91-1.79 (m, 8H), 1.76-1.68 (m, 6H), 1.67-1.61 (m, 3H), 1.52 (s,
5H), 1.33 (brs, 9H), 1.24 (brs, 9H), 1.01-0.95 (m, 6H), 0.92-0.87 (m, 6H), 0.83-0.77
13
(m, 6H). C NMR (151 MHz, DMSO-d6) δ ppm: 209.13, 173.89, 173.75，173.53,
173.49, 172.56, 172.41, 172.07, 171.90, 171.71, 171.37, 171.13, 170.99, 170.66,
170.62, 170.76, 169.40, 168.88, 168.73, 167.68, 167.03, 165.13, 154.94, 153.62,
139.07, 137.04, 136.81, 135.64, 133.24, 129.88, 129.55, 129.40, 129.03, 128.49,
127.97, 127.20, 119.06, 83.61, 80.14, 78.36, 77.51, 76.62, 75.23, 74.63, 73.57, 71.36,
70.57, 69.40, 59.86, 58.53, 56.77, 54.99, 52.44, 52.31, 51.90, 48.46, 48.29, 47.90,
45.89, 45.81, 42.72, 42.12, 41.26, 40.38, 40.29, 36.31, 36.25, 34.50, 31.29, 28.88,
27.94, 27.68, 27.02, 26.29, 24.23, 24.15, 24.10, 22.85, 22.30, 22.19, 21.33, 20.57,
17.89, 17.69, 17.54, 15.13, 13.49, 10.85, 9.61. ESI-MS: m/z 940.2 [M/2+Na]+;
C90H123N13O28: HRMS calcd. 1856.8493 [M+Na]+, found 1856.8486.
1
DTX-PABC-Leu-A3 (8b). Colorless powder. Yield: 21%. H NMR (400 MHz,
DMSO-d6, rotamers) δ ppm: 9.97 (d, J = 12.0 Hz, 1H), 8.52 (d, J = 12.0 Hz, 1H),
8.23-7.97 (m, 12.1 Hz, 7H), 7.82 (d, J = 8.0 Hz, 2H), 7.76 (d, J = 8.0 Hz, 1H),
7.74-7.68 (m, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.45-7.38 (m, 3H), 7.37-7.25 (m, 5H),
7.18 (d, J = 4.0 Hz, 1H), 6.81 (d, J = 8.0 Hz, 2H), 5.77 (dd, J = 8.0, 12.0 Hz, 1H),
5.38 (d, J = 8.0 Hz, 1H), 5.22-4.95 (m, 7H), 4.91 (d, J = 8.0 Hz, 1H), 4.54 (brs, 1H),

4.40 (brs, 1H), 4.34-4.18 (m, 4H), 4.12 (brs, 7H), 4.01 (brs, 2H), 3.90 (brs, 1H), 3.73
(brs, 1H), 3.66-3.46 (m, 1H), 3.19-3.16 (m, 5H), 2.88 (brs, 1H), 2.72 (brs, 1H),
2.34-2.17 (m, 5H), 2.16-2.05 (m, 5H), 1.91-1.82 (m, 6H), 1.76-1.69 (m, 6H),
1.56-1.48 (m, 10H), 1.28-1.15 (m, 7H), 1.01-0.95 (m, 5H), 0.93-0.84 (m, 6H),
13
0.84-0.73 (m, 7H). C NMR (151 MHz, DMSO-d6) δ ppm: 209.02, 173.91, 173.77,
173.46, 173.07, 172.57, 172.08, 171.91, 171.72, 171.39, 171.14, 171.00, 170.93,
170.77, 169.42, 169.33, 168.88, 168.61, 167.69, 163.96, 162.62, 160.99, 153.56,
153.18, 139.08, 136.82, 136.36, 135.53, 132.16, 130,85, 129.49, 129.02, 128.55,
128.22, 127.21, 125.60, 120.43, 119.07, 83.58, 80.14, 77.26, 76.59, 75.12, 73.55,
71.34, 70.55, 69.48, 59.86, 56.80, 55.33, 52.44, 52.30, 51.89, 48.43, 48.29, 45.89,
45.77, 42.68, 42.11, 41.25, 36.24, 31.34, 31.28, 28.87, 27.67, 27.01, 26.28, 24.22,
24.14, 24.09, 22.83, 22.24, 22.17, 21.31, 20.65, 19.16, 19.12, 17.67, 17.52, 15.12,
13.46, 10.83, 9.58. ESI-MS: m/z 954.0 [M/2+H]+; C90H119F4N13O28: HRMS calcd.
1928.8116[M+Na]+, found 1928.8183.
1
DTX-PABC-Leu-A4 (9a). Colorless powder. Yield: 24%. H NMR (400 MHz,
DMSO-d6, rotamers) δ ppm: 10.03 (s, 1H), 8.29-8.15 (m, 3H), 8.12-7.91 (m, 8H),
7.84-7.61 (m, 6H), 7.47-7.16 (m, 8H), 6.80 (s, 2H), 5.90-5.68 (m, 1H), 5.40 (d, J =
4.0 Hz, 1H), 5.21-4.84 (m, 8H), 4.55-4.37 (m, 3H), 4.35-4.14 (m, 6H), 4.07-3.96 (m,
3H), 3.89-3.54 (m, 8H), 2.29-2.19 (m, 4H), 2.18-2.07 (m, 5H), 2.06-2.00 (m, 3H),
1.95-1.82 (m, 9H), 1.77-1.63 (m, 9H), 1.56-1.45 (m, 6H), 1.34 (brs, 9H), 1.27-1.16 (m,
13
6H), 1.03-0.95 (m, 6H), 0.93-0.85(m, 6H), 0.83-0.75 (m, 6H). C NMR (151 MHz,
DMSO-d6) δ ppm: 209.13, 173.84, 173.64, 173.45, 172.28, 172.10, 171.66, 171.51,
171.44, 171.32, 171.08, 171.02, 170.75, 170.57, 169.51, 169.42, 168.73, 168.62,
168.33, 167.66, 167.04, 165.13, 154.94, 153.62, 139.09, 137.04, 136.81, 135.64,
133.24, 129.88, 129.55，129.39, 129.08, 128.49, 129.04, 128.49, 127.97, 127.20,
119.05, 83.61, 80.14, 78.36, 77.51, 76.62, 75.23, 74.63, 73.57, 71.36, 70.57, 69.40,
59.82, 58.56, 56.83, 56.70, 54.99, 52.52, 52.30, 52.03, 51.82, 48.33, 46.56, 45.88,
45.81, 42.72, 42.00, 41.31, 40.53, 36.49, 36.31, 34.50, 31.69, 31.29, 29.20, 28.81,
27.94, 27.48, 27.16, 26.29, 24.26, 24.17, 24.10, 22.88, 22.30, 22.19, 21.90, 21.27,

20.57, 17.42, 17.31, 15.05, 14.40, 13.49, 10.86, 9.61. ESI-MS: m/z 969.8 [M/2+Na]+;
C92H127N13O28S: HRMS calcd. 1916.8526 [M+Na]+, found 1916.8535.
1
DTX-PABC-Leu-A4 (9b). Colorless powder. Yield: 25%. H NMR (400 MHz,
DMSO-d6, rotamers) δ ppm: 10.01 (d, J = 20.8 Hz, 1H), 8.47 (d, J = 8.4 Hz, 1H), 8.14
(d, J =6.4 Hz, 2H), 8.08-7.98 (m, 3H), 7.95-7.91 (m, 2H), 7.78 (q, J = 16.4, 12.4 Hz,
2H), 7.71-7.66 (m, 1H), 7.66-7.57 (m, 4H), 7.37 (t, J = 7.6 Hz, 2H), 7.32 (d, J = 7.6
Hz, 2H), 7.29 (d, J = 8.6 Hz, 2H), 7.24 (s, 2H), 7.14 (t, J = 8.0 Hz, 1H), 6.76 (brs, 2H),
5.74 (dd, J = 8.0 Hz, 12.0 Hz, 1H), 5.34 (d, J = 8.0 Hz, 1H), 5.17-5.07 (m, 4H), 5.04
(d, J = 8.0 Hz, 1H), 5.01-4.96 (m, 2H), 4.87 (d, J = 8.0 Hz, 1H), 4.43-4.34 (m, 1H),
4.39 (s, 1H), 4.32-4.11 (m, 6H), 4.07-3.97 (m, 3H), 3.86 (dd, J = 16.0, 8.0 Hz, 1H),
3.80-3.64 (m, 2H), 3.62-3.42 (m, 2H), 3.34 (brs, 1H), 2.85 (brs, 3H), 2.69 (brs, 3H),
2.43 (brs, 2H), 2.20 (brs, 3H), 2.16-2.03 (m, 4H), 1.98 (s, 3H), 1.95-1.88 (m, 3H),
1.87-1.84 (m, 4H), 1.76-1.69 (m, 4H), 1.68 (brs, 3H), 1.65-1.57 (m, 4H), 1.55 (brs,
3H), 1.52-1.48 (m, 6H), 1.21-1.14 (m, 6H), 0.94 (s, 6H), 0.87 (d, J = 6.2 Hz, 3H),
0.83 (d, J = 6.2 Hz, 3H), 0.83-0.76 (m, 6H). 13C NMR (151 MHz, DMSO-d6) δ ppm:
209.00, 173.84, 173.65, 173.46, 172.28, 171.66, 171.51, 171.07, 171.01, 170.74,
169.52, 169.32, 168.60, 167.66, 163.94, 162.15, 160.98, 153.55, 139.09, 136.81,
136.36, 135.52, 132.16, 132.11, 130.89, 130.83, 129.48, 129.02, 128.54, 128.21,
127.20, 125.60, 120.42, 120.28, 119.05, 115.69, 83.58, 80.14, 77.26, 76.59, 75.15,
73.54, 71.34, 70.55, 69.48, 59.82, 58.55, 56.80, 55.33, 52.51, 52.35, 52.28, 52.02,
51.81, 48.32, 45.88, 45.77, 42.68, 41.99, 41.30, 40.53, 36.49, 36.28, 35.61, 34.50,
31.69, 31.64, 31.28, 30.61, 29.19, 28.80, 28.65, 28.51, 28.40, 27.58, 27.54, 27.47,
27.15, 26.38, 26.29, 24.26, 24.17, 24.09, 22.87, 22.24, 22.18, 21.89, 21.26, 20.56,
19.17, 19.12, 17.41, 17.30, 15.04, 14.39, 13.47, 10.84, 9.58. ESI-MS: m/z 984.2
[M/2+H]+; C92H123F4N13O28S: HRMS calcd. 1988.8150 [M+Na]+, found 1988.8169.
1
DTX-PABC-Leu-A5 (10a). Colorless powder. Yield: 24%. H NMR (400 MHz,
DMSO-d6) δ ppm: 9.96 (s, 1H), 8.14 (d, J = 28.0Hz, 5), 8.03 (s, 1H), 7.95 (d, J = 21.6
Hz, 5H), 7.68 (dd, J =24.4, 6.4 Hz, 5H), 7.41 (d, J =6.0 Hz, 2H), 7.31 (d, J = 26.4 Hz,
6H), 7.17 (brs, 1H), 6.81 (d, J = 14.8 Hz, 2H), 5.78 (dd, J = 8.0 Hz, 12.0 Hz, 1H),

5.39 (d, J = 6.0 Hz, 1H), 5.15 (brs, 3H), 5.09-4.98 (m, 3H), 4.96 (brs, 1H), 4.90 (d, J
= 9.2 Hz, 1H), 4.47-4.37 (m, 2H), 4.35-4.12 (m, 5H), 4.10-3.89 (m, 4H), 3.88-3.77 (m,
1H), 3.72 (brs, 3H), 3.67-3.60 (m, 1H), 3.59-3.46 (m, 2H), 3.28 (brs, 1H), 2.24 (brs,
3H), 2.15-2.02 (m, 5H), 1.94-1.88 (m, 2H), 1.86 (brs, 3H), 1.85-1.73 (m, 3H), 1.68
-1.53 (m, 6H), 1.51 (brs, 3H), 1.47-1.42 (m, 2H), 1.33 (brs, 9H), 1.25-1.19 (m, 4H),
0.98 (brs, 6H), 0.92-0.82 (m, 13H). 13C NMR (151 MHz, DMSO-d6) δ ppm: 209.14 ,
173.89, 173.82, 173.79, 173.52, 172.63, 171.97, 171.89, 171.67, 171.51, 171.44,
171.35, 171.14, 171.01, 169.52, 169.44, 168.92, 168.74, 168.64, 168.48, 167.69,
167.06, 165.15, 154.95, 153.63, 139.07, 137.04, 136.81, 135.6, 133.26, 129.88,
129.55, 129.40, 129.04, 128.50, 127.98, 127.20, 119.07, 83.62, 80.14, 78.38, 77.52,
76.62, 75.23, 74.63, 73.58, 71.37, 70.58, 69.41, 59.79, 58.58, 56.83, 54.99, 54.48,
52.27, 51.09, 50.79, 48.49, 46.56, 45.89, 45.81, 42.72, 42.13, 41.92, 41.30, 40.38,
36.31, 34.50, 31.29, 28.79, 27.94, 27.68, 27.54, 27.13, 26.29, 24.25, 24.16, 23.90,
22.92, 22.85, 22.30, 22.18, 21.90, 21.33, 20.57,17.50, 17.45, 13.49, 9.61. ESI-MS:
m/z 1842.8 [M+Na]+; C89H121N13O28: HRMS calcd. 1842.8336 [M+Na]+, found
1842.8347.
1
DTX-PABC-Leu-A5 (10b). Colorless powder. Yield: 22%. H NMR (400 MHz,
DMSO-d6) δ ppm: 9.97 (s, 1H), 8.52 (d, J = 8 Hz, 1H), 8.17 (d, J = 8.0 Hz, 2H), 8.11
(dd, J = 6.0, 4.0 Hz, 3H), 8.07-8.01 (m, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.91 (dd, J =
8.0, 4.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.78-7.58 (m, 5H), 7.43-7.28 (m, 9H), 7.18
(t, J = 8.0 Hz, 1H), 6.81 (d, J = 16 Hz, 2H), 5.78 (dd, J = 8.0 Hz, 12.0 Hz, 1H), 5.38
(d, J = 8.0 Hz, 1H), 5.21-5.08 (m, 4H), 5.07-4.96 (m, 3H), 4.92 (d, J = 8.0 Hz, 1H),
4.54 (s, 1H), 4.51-3.85 (m, 10H), 3.84-3.68 (m, 4H), 3.64 (d, J = 8.0 Hz, 1H),
3.60-3.35 (m, 2H), 3.34 (brs,1H), 2.36-2.19 (m, 4H), 2.17-1.99 (m, 5H), 1.98-1.82 (m,
7H), 1.80-1.57 (m, 10H), 1.55 (brs, 3H), 1.54-1.48 (m, 7H), 1.48-1.31 (m, 4H),
1.26-1.18 (m, 3H), 0.98 (brs, 5H), 0.93-0.89 (m, 3H), 0.89-0.85(m, 6H), 0.94-0.77 (m,
4H). 13C NMR (151 MHz, DMSO-d6) δ ppm: 209.01, 173.87, 173.70, 173.49, 172.60,
171.87, 171.64, 171.49, 171.12, 171.05, 170.96, 169.48, 169.33, 168.89, 168.61,
167.67, 167.05, 163.95, 162.61, 160.99, 153.56, 153.18, 139.09, 136.82, 136.37,

135.53, 132.17, 130.89, 129.48, 129.03, 128.55, 128.22, 127.21, 125.61, 124.02,
120.43, 120.29, 119.06, 115.84, 115.69, 83.58, 80.14, 78.52, 78.33, 77.26, 76.59,
75.15, 73.55, 71.34, 70.55, 69.48, 59.78, 56.80, 55.33, 52.47, 51.88, 48.47, 45.88,
45.77, 42.68, 41.29, 36.28, 34.50, 31.63, 31.28, 28,78, 27.69, 27.54, 27.13, 26.29,
24.25, 24.15, 23.89, 22.92, 22.83, 22.24, 22.18, 21.89, 21.33, 20.56, 19.16, 19.12,
17.46, 13.47, 9.58. ESI-MS: m/z 947.2 [M/2+H]+; C89H117F4N13O28: HRMS calcd.
1914.7959 [M+Na]+, found 1914.7963.
4.3. Biological evaluation procedures
4.3.1. Cell lines and cultures
HCT116 (human colon carcinoma cell line), SW620 (human colon carcinoma cell
line), CCD18Co (human colon cell line) and HEK293 (human embryonic kidney 293)
were purchased from American Type Culture Collection (ATCC) by commercial
suppliers. DMEM and McCoy's 5A were obtained from NanJing KeyGen Biotech Co.,
Ltd. Fetal bovine serum (FBS) and Trypsine-EDTA (0.25%) were purchased from
Gibco (Invitrogen, USA). All these cell lines were cultured as recommended: SW620
and HEK293 were cultured in DMEM with 10% fetal bovine serum, CCD18Co was
cultured in DMEM with 15% FBS, HCT116 was cultured in McCoy's 5A with 10%
FBS. All cells were incubated at 37℃ in a humidified atmosphere containing 5%
CO2.
4.3.2. Cell proliferation assay
Cell proliferation assays were conducted to measure and compare the cytotoxicity
and selectivity of these prodrugs and their parent drug DTX or 4FDT. Cells were
seeded onto a 96-well plate at a density of 5000 cells per well and precultured for 1
day. Stock solutions (0.1 mol/L) of test compounds were prepared freshly by
dissolving in DMSO. An increased concentration (10-10, 10-9, 10-8, 10-7,10-6, 10-5 and
10-4 mol/L) and a control (blank) were applied to the cells in sixplicate for 48h
o
(HCT116 and HEK293) or 72 h (SW620 and CCD18Co) at 37 C in 5% CO2
atmosphere. Cell cytotoxicity was evaluated by MTT assay. MTT solution (25 µL, 5
mg/mL in PBS) was added to the culture media. After incubating at 37 oC in the dark