Cyclane-aminol 10-hydroxycamptothecin analogs as novel DNA topoisomerase I inhibitors induce apoptosis selectively in tumor cells

Article abstract of DOI:10.1097/CAD.0000000000000083

A novel series of cyclane-aminol 10-hydroxycamptothecin (HCPT) analogs was designed and synthesized through the Mannich reaction using HCPT as the lead compound, such as 10-hydroxyl-9-L-prolinol (+) methylcamptothecin (PRPT), 10-hydroxyl-9-(4′-hydroxy) piperidinylmethylcamptothecin (PPPT), and 10-hydroxy-9-(4′-hydroxyethyl)-piperazinylmethycamptothecin (QPPT). Three kinds of new cyclane-aminols were introduced into the structure of HCPT, which modified strong cytotoxic HCPT into cyclane-aminol HCPT analogs with moderate cytotoxicity and improved selectivity toward DNA topoisomerase I inhibition in tumor cells. Special metabolic pathways for cyclane-aminol HCPT analogs in rats were discovered, which differed from other HCPT analogs. Cyclane-aminol HCPT analogs can capture O₂ and cause an increase in intracellular hydrogen peroxide levels with selective induction of apoptosis in tumor cells rather than in normal peripheral blood mononuclear cells. Among them, PPPT has a much better druggability than topotecan (TPT) and has the potential to be developed into an antitumor agent.

Full text of DOI:10.1097/CAD.0000000000000083

614 Preclinical report
Cyclane-aminol 10-hydroxycamptothecin analogs as novel
DNA topoisomerase I inhibitors induce apoptosis selectively
in tumor cells
a
a
b
c
a
Shixuan Zhang , Tao Geng , Bo Jiang , Ge Song , Lisha Ha ,
b
a
a
a
Chenguang Sun , Yuan Qian , Qingyu Fan and Hongmin Guo
A novel series of cyclane-aminol 10-hydroxycamptothecin
HCPT) analogs was designed and synthesized through
Among them, PPPT has a much better druggability than
topotecan (TPT) and has the potential to be developed into
an antitumor agent. Anti-Cancer Drugs 25:614–623 ꢀc
2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
(
the Mannich reaction using HCPT as the lead compound,
such as 10-hydroxyl-9-L-prolinol (+) methylcamptothecin
0
(
PRPT), 10-hydroxyl-9-(4 -hydroxy)
Anti-Cancer Drugs 2014, 25:614–623
piperidinylmethylcamptothecin (PPPT), and 10-hydroxy-9-
0
Keywords: cyclane-aminol 10-hydroxycamptothecin analogs
(4 -hydroxyethyl)-piperazinylmethycamptothecin (QPPT).
1
0-hydroxycamptothecin, intracellular hydrogen peroxide, PF1
Three kinds of new cyclane-aminols were introduced into
the structure of HCPT, which modified strong cytotoxic
HCPT into cyclane-aminol HCPT analogs with moderate
cytotoxicity and improved selectivity toward DNA
topoisomerase I inhibitors
a
State Key Laboratory of Fine Chemicals, School of Pharmaceutical Science
b
and Technology, School of Environmental and Biological Science and
c
Technology, Dalian University of Technology, Dalian and Norman Bethune
College of Medicine, Jilin University, Changchun, China
topoisomerase I inhibition in tumor cells. Special metabolic
pathways for cyclane-aminol HCPT analogs in rats were
discovered, which differed from other HCPT analogs.
Correspondence to Shixuan Zhang, PharmD, State Key Laboratory of Fine
Chemicals, School of Pharmaceutical Science and Technology, Dalian University
of Technology, Linggong Road No. 2, Dalian 116024, China
–
Tel/fax: + 86 411 8498 6179; e-mail: zhangsx56@aliyun.com
Cyclane-aminol HCPT analogs can capture O2 and cause
an increase in intracellular hydrogen peroxide levels with
selective induction of apoptosis in tumor cells rather
than in normal peripheral blood mononuclear cells.
Received 20 September 2013 Revised form accepted 31 December 2013
Introduction
metabolite 7-ethyl-HCPT (SN-38) by enzymatic esters
in the body to take effect. Its bioavailability is very low,
most of it being excreted as the prototype drug (about
55%), and the toxicity of SN-38 is stronger than that of
HCPT [5]. In 1996, topotecan [TPT; 9-[(dimethyl-
amino) methyl]-10-hydroxycamptothecin; SmithKline
Beecham Corp., Philadelphia, Pennsylvania, USA] was
marketed in the USA (US5004758) [6]. It is a water-
soluble HCPT analog with excellent bioavailability. The
inhibitory activity of DNA topoisomerase I and toxicity
of TPT are both reduced compared with HCPT, but
TPT is still a strong cytotoxic drug. In 2004, belotecan
Camptothecin (CPT) analogs, as DNA topoisomerase I
inhibitors, have a clear mechanism of action, strong
antitumor activity, broad spectrum, good security and
reliability, and show no cross-resistance with other
antitumor drugs; however, they still cause vomiting,
diarrhea, bone marrow suppression and other serious
adverse reactions. This situation inspires medical experts
to modify the chemical structure of CPT, and the number
of analog patents for CPT have so far been more than
1
50 [1].
In 1985, Redinbo and colleagues [2,3] reported the
antitumor mechanism of CPT, which is the inhibition of
DNA topoisomerase I, leading to large-scale studies on
chemical structure modification and structure–activity
relationship. In 1998, they reported that DNA topo-
isomerase I, as a key enzyme, regulates the changes in
DNA space configuration dynamically and confirmed the
molecular docking mechanism of CPT and DNA topo-
isomerase I.
[
7-[2-(N-isopropylamino) ethyl]-camptothecin; CKD602;
Chong Kun Corp., Seoul, Korea] was marketed in Korea
US6265413131), and it entered phase III clinical trial in
(
the USA in 2011 [7]. It has no significant differences
in survival compared with TPT [8].
The cytotoxicity of CPT, HCPT, and SN-38 is very high;
hence, these drugs kill tumor cells, with the risk of
serious injury to normal cells as well. Currently, the major
problem in developing CPT analogs is the continuous
pursuit toward improving their cytotoxicity, thereby
neglecting their selectivity toward tumor cells, causing
harm to normal cells, severe adverse reactions, and their
final ruling out in clinical studies. Therefore, novel drugs
with enhanced selectivity for tumor cells and causing
In 1994, irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piper-
idino]-carbonyloxycamptothecin, CPT-11; Kabushiki
Kaisha Yakult Honsha, Tokyo, Japan] was marketed in
Japan (US4604463) [4]. It is a water-soluble HCPT
analog as a prodrug and is hydrolyzed into the active
0
959-4973 ꢀc 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
DOI: 10.1097/CAD.0000000000000083
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Novel cyclane-aminol HCPT analogs Zhang et al. 615
+
478.1922 [M + H] (calculated 478.1920), C H N O .
reduced damage to normal cells are still to be developed,
for the purpose of achieving highly effective CPT analogs
with low toxicity.
2
6
27
3 6
–
1
IR (KBr, cm ): 3423, 2938, 1749, 1655(n C = O), 1594,
1
1506, 1469, 1384. H-NMR (DMSO-d , 400 MHz) d: 8.72
6
(
(
(
s, 1H, 7-CH), 7.98 (d, 1H, J = 9.2Hz, 12-CH), 7.52
In this paper, three kinds of new cyclane-aminols were
introduced into the structure of HCPT through the
Mannich reaction, the method used for synthesizing
TPT, to yield water-soluble cyclane-aminol HCPT
analogs. The selective induction of apoptosis in tumor
cells and druggability of the cyclane-aminol HCPT
analogs were evaluated.
s, 1H, 14-CH), 7.40 (d, 1H, J = 9.2 Hz, 11-CH), 6.51
s, 1H, 20-OH), 5.42 (s, 2H, Ar-CH -), 5.24 (s, 1H,
2
5
-CH ), 3.57 (s, 2H, C-17-H), 2.97B2.94 (m, 2H,
2
0
0
0
0
2
1
1
1
a-CH, 6 a-CH), 2.37B2.32 (m, 2H, 2 b-CH, 6 b-CH),
.91 (s, 3H, CH COOH), 1.86 (q, 2H, J = 7.3 Hz,
3
0
0
9-CH2), 1.79B1.74 (m, 2H, 3 b-CH, 5 b-CH),
.46B1.42 (m, 2H, 3 a-CH, 5 a-CH), 0.88 (t, 3H,
0
0
J = 7.3 Hz, 18-CH3).
Materials and methods
Proton nuclear magnetic resonance ( H-NMR) spectra
0
1
10-Hydroxy-9-(4 -hydroxyethyl)-piperazinylmethylcamp-
tothecin (QPPT): yellow solid, purity, 95.2% (by HPLC-
were recorded on
(
a Varian INOVA spectrometer
400 MHz; Varian Inc., Palo Alto, California, USA).
UV) and yield, 70.1%. HP-MS (API-ESI, positive) m/z:
+
07.269[M + H] (calculated 507.2267), C H N O .
5
Infrared (IR) spectra were obtained using KBr tablets
on a Nicolet 50 ꢁ FT-IR spectrophotometer (Thermo
Fisher Scientific Inc., Waltham, Massachusetts, USA).
Mass spectra were obtained on a HP 1100 LC-MSD
spectrometer (Hewlett-Packard Company, Palo Alto,
California, USA).
27 30
4
6
–
1
IR (KBr, cm ): 3423, 1746, 1655(n C = O), 1589, 1504,
1
1
463, 1384, 1338. H-NMR (DMSO-d , 400 MHz) d:
6
8.73 (s, 1H, 7-CH), 7.98 (d, 1H, J = 9.2 Hz, 12-CH), 7.42
(
d, 1H, J = 9.2 Hz, 11-CH), 7.26 (s, 1H, 14-CH), 6.51 (s,
1
3
2
H, 20-OH), 5.42 (s, 2H, Ar-CH -), 5.24 (s, 1H, 5-CH ),
2 2
.52 (s, 2H, 17-CH), 3.18B3.10 (m, 2H, 8 -CH ),
0
2
.67B2.60 (m, 10H, (N-CH2) ꢁ 5), 1.91 (s, 3H,
Synthesis of cyclane-aminol HCPT analogs and
CH COOH), 1.85 (q, 2H, J=7.3Hz, 19-CH ), 0.88 (t,
3
2
9-methylene-10-carbonyl-CPT
3H, J= 7.3 Hz, 18-CH3).
HCPT was dissolved in acetic acid, to which cyclane-
aminol and 37% formaldehyde solution were added
M376: yellow solid, purity, 96.8% (by HPLC-UV) and
yield, 92.1%. HP-MS (API-ESI, positive) m/z:
377.1073[M + H] (calculate 377.1071), C H N O .
(
4
1 : 4 : 4 mol/l). The reaction mixture was heated at
0–901C for about 0.2–2 h. The reaction solution was
+
2
1
16
2 5
–
1
poured into a large quantity of ether, and a cotton-like
solid separated out; this was filtered, washed, and
recrystallized using acetone to yield cyclane-aminol
HCPT analogs. The cyclane-aminol HCPT analogs can
be oxidized to 9-methylene-10-carbonyl-CPT (M376) by
NaOCl, but not by hydrogen peroxide (H O ). 10-
IR (KBr, cm ): 3384, 2974, 2876, 1744, 1655(n C = O),
1
1591, 1505, 1413. H-NMR (DMSO-d , 400 MHz)
6
d: 0.86 (3H, t, 8-CH ), 1.83 (2H, m, 19-CH ), 4.19, 4.23
3
2
(1H, d, 9-CH ), 4.46, 4.49 (1H, d, 9-CH ), 5.24 (2H, s,
5-CH ), 5.42 (2H, s, 17-CH ), 6.52 (20-OH), 7.26 (1H, s,
2
2
2
2
2
2
14-CH), 7.40, 7.42 (1H, d, 11-CH), 7.95, 7.97 (1H, d,
12-CH), 8.80 (1H, s, 7-CH).
Hydroxyl-9-L-prolinol (+) methylcamptothecin (PRPT;
3 mg) was added to 400 ml of NaOCl solution, and the
reaction solution was extracted with acetic ester and
5
Assay of solubility and ratio of ester/carboxylic acid in
the plasma by HPLC-UV
evaporated to dryness. The product was purified by
(methanol/EtOAc = 1 : 2;
column
CN2013100629663).
chromatography
A solution of 0.2 ml drug and PBS (1 mg/ml) was mixed
with 0.2 ml plasma, vortexed for 1 min, and incubated for
2 h at 371C; 3.6 ml of 1% formic acid methanol solution
was added; the solution was stirred for 1 min and
centrifuged at 1500 ext/min for 10 min. The supernatant
was subjected to HPLC-UV.
PRPT: yellow solid, purity 99.1% (by HPLC-UV) and yield
0.2%. HP-MS (API-ESI, positive) m/z: 478.1921[M +
H] (calculated 478.1920), C H N O . IR (KBr, cm ):
9
+
– 1
2
6
27
3 6
1
3
384, 2974, 2876, 1744, 1655, 1591, 1505. H-NMR
(
1
7
DMSO-d , 400 MHz) d: 8.84 (s, 1H, 7-CH), 8.07 (d,
6
The HPLC-UV system (Dalian Jiangshen Separation and
Technology Corp., Dalian, China) consisted of a Jiang-
shen pump (Dalian Jiangshen Separation and Technology
Corp.) and UV detector. Separations were performed
on a Kromasil ODS C18 reversed-phase column (5 mm,
H, J = 6.6 Hz, 12-CH), 7.52 (d, 1H, J = 6.6 Hz, 11-CH),
.28 (s, 1H, 14-CH), 6.53 (s, 1H, 20-OH), 5.43 (s, 2H,
0
Ar-CH -), 5.26 (s, 1H, 5-CH ), 3.76B3.32 (m, 2H, 6 -CH ),
2
2
2
0
3
2
1
4
.66 (s, 2H, 17-CH), 3.08B3.03 (m, 2H, 5 -CH ),
2
0
.07B2.02 (m, 2H, 3 -CH ), 1.88 (s, 3H, CH COOH),
2
3
4.6 mm internal diameter ꢁ 250 mm; AkzoNobel, Shanghai,
.86 (q, 2H, J= 7.0 Hz, 19-CH ), 1.71B1.69 (m, 2H,
2
China). The elution system for analytical chromatography
consisted of methanol–isopropyl alcohol–0.15% methane
sulfonic acid in water (50 : 1 : 49, v/v/v). The flow rate was
1 ml/min, and the injection volume of the sample solution
was 20 ml for all cases at ambient temperature. The UV
0
-CH ), 0.88 (t, 3H, J= 7.0 Hz, 18-CH ).
2
3
0
0-Hydroxyl-9-(4 -hydroxy)-piperidinylmethylcamptothe-
cin (PPPT): yellow solid, purity 96.7% (by HPLC-UV)
1
and yield, 86.8%. HP-MS (API-ESI, positive) m/z:
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

616 Anti-Cancer Drugs 2014, Vol 25 No 6
detector wavelength was set at 278 nm, and the data
were processed on a Zhida 2000 Chromatography Data
System (Zhejiang IT Information Engineering Co. Ltd,
Hangzhou, China). The theoretical plate number was
more than 3000. The elution system for preparative
chromatography consisted of a Vercopak Inertsil 10 ODS
C18 reversed-phase column (10 mm, 4.6 mm internal
diameter ꢁ 250 mm) and methanol–0.2% phosphoric acid
in water (45 : 55, v/v). The UV detector wavelength was
set at 268 nm.
100 mmol/l. PBS containing 0.1% DMSO served as the
control. The plates were incubated at 371C for 48 h; after
the medium was removed, 100 ml of 0.5 mg/ml MTT
solution diluted with PBS was added to the cells and the
plates were further incubated for 3 h. Thereafter, the
MTT solution was removed, 100 ml of DMSO was added
to each well to dissolve the formazan, and the plates were
read at 570 nm (630 nm as a reference) on a microplate
reader (Sunrise, TECAN, Grodig, Austria) according to
the Tim Mosmann method.
Analysis of extracellular and intracellular H O levels
Analysis of metabolites by LC–MS–MS
2
2
using a PF1 fluorescent probe
PF1 is an intracellular H O -selective fluorescent probe
synthesized by us. It has good permeability through the
cell membrane, stability, and selectivity, and does not
emit fluorescence. On entering cells, it can be oxidized
to fluorescein isothiocyanate (FITC), emitting yellow–
Rats received an oral dose of 25 mg/kg HCPT. Urine
samples were collected and frozen at – 151C to keep
them stable. A volume of 100 ml of urine sample was
mixed with 400 ml of methanol (including 0.1% methane-
sulfonic acid). Acidified methanol solution converted
carboxylated PRPT into its lactone form. After centrifu-
gation at 16 060g for 10 min, the supernatant was
subjected to liquid chromatography/tandem mass spec-
trometry (LC–MS–MS) analysis.
2
2
green fluorescence, by H O selectively (excitation
2
2
wavelength 488 nm, emission wavelength 525 nm) but
–
not by O2 or NaOCl. PF1 is currently the only truly
effective tool for assessment of intracellular H O level
2
induced by drugs [10,11].
2
LC–MS–MS analysis was carried out in the electrospray
ionization (ESI) mode on a Triple Quad mass analyzer
Human peripheral blood mononuclear cells (PBMCs)
were isolated from heparinized venous blood of healthy
volunteers by Ficoll–Paque density gradient centrifuga-
tion. After washing with PBS twice, PBMCs were
cultured in RPMI 1640 medium with 10% fetal calf
(
Thermo Fisher Scientific Inc.). The LC portion
comprised an Agilent 1100 series LC system (Agilent
Technologies Inc., Santa Clara, California, USA). Separa-
tion was carried out on a Zorbax C18 reversed-phase
column (1.8 mm, 2.1 mm internal diameter ꢁ 100 mm).
The mobile phase consisted of methanol (A) and 0.2%
formic acid in water (B). The following gradient was
applied at a flow rate of 0.3 ml/min: 0–30 min, from 10% A
and 90% B to 80% A and 20% B; 30–40 min, from
serum at 371C in a humidified atmosphere of 5% CO2.
After incubation for 6 h on a plastic plate, nonadherent
cells were collected and used for cytotoxicity assay [12].
5
MCF-7 and BGC-823 cells and PBMCs (B2 ꢁ 10 ) were
8
0% A and 20% B to 100% A. The injection volume was 10 ml.
seeded in a 96-well black-transparent chamber; each
3
chamber contained 90 ml of solution (5 ꢁ 10 ). The
Analysis of effects on DNA topoisomerase I
sample was maintained at 371C in a 5% CO humidified
2
pUC19 DNA (0.5 mg) was incubated with or without
atmosphere for 24 h. Ten microliters of drug PBSs with
different gradients (drug referring to HCPTanalogs, TPT
and HCPT has been separately reserved as 1 mg/ml drug
DMSO solutions, and these solutions were further
diluted with PBS to get drug PBSs. Gradients: cyclane-
aminol HCPT analogs were diluted into 250, 125, 62.5,
1
0
3
.5 U topoisomerase I in a 20 ml solution containing 0.1,
.5, and 2.5 mmol/l HCPT or HCPT analogs at 371C for
0 min. Thereafter, the sample was separated by 1%
agarose gel electrophoresis, with a run time of 35 min.
The gel was stained with ethidium bromide solution (PB,
1
(
mg/ml) and then visualized under a UV transilluminator
Syngene, Westminster, Maryland, USA) [6,9].
3
0
1.25, 0 mmol/l, separately; TPT into 100, 50, 25, 12.5,
mmol/l; and HCPT into 50, 25, 12.5, 6.25 mmol/l) were
added, with each concentration located in four chambers
as repeats, and incubated for 2 h. Then 10 ml PF1
fluorescence probe solution (PF1 has been reserved as
1 mg/ml DMSO solution, and this solution was further
diluted with PBS into 100 mmol/l to obtain the PF1
fluorescence probe solution) was added to each chamber,
and the plate was shaken for 0.5 h at 371C. The
fluorescence intensity of H O in the cells under this
Anticancer activity in vitro by MTT
Cells from five human tumor cell lines, including gastric
adenocarcinoma cell line BGC-823, small cell lung cancer
cell line MCI-H1688, small cell lung cancer cell line
NCI-H446, liver cancer cell line SMMC-7721, and breast
cancer cell line MCF-7 cells (from Cell Bank of Shanghai
Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences) were each seeded in 96-well
2
2
condition was detected using a fluorescence assay
(ELISA; excitation wavelength 48 nm, emission wave-
length 525 nm). Extracellular and intracellular H O
3
microtiter plates (5 ꢁ 10 cells/well) and incubated at
3
PBS and added to the appropriate wells in four replicates,
71C for 24 h. HCPT analog solutions were diluted with
2
2
levels in the cells were calculated on the basis of a
standard curve. The cells were washed twice with 0.1 ml
with different final concentrations ranging from 0.01 to
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Novel cyclane-aminol HCPT analogs Zhang et al. 617
PBS, and intracellular H O fluorescence was detected
2
Scheme 1
2
under a fluorescence microscope.
X
Standard curve: in each chamber, 90 ml plasma culture
HO
HO
medium, 10 ml H O PBS containing different gradient
2
2
N
O
O
N
O
N
solutions (H O solution was diluted with PBS into 1000,
00, 250, 125, 62.5, 31.25, and 0 mmol/l solutions; each
RNH
N
2
2
5
HO
CH2O/HAc
HO
O
concentration was assigned to four chambers), and 10 ml
PF1 fluorescence probe solution were added; the mixture
was shaken for 0.5 h at 371C. The fluorescence intensity
O
O
HCPT
OH
CH CH OH
2 2
N
[optical density (OD)] was measured and the standard
curve was drawn. Linear regression equation: C (H O
2
2
2
CH OH
N
N
N
N
2
mmol/l) = 0.592 SQRT (OD) (R = 0.995, n = 6); the
linear relationship was good.
X=RN=
PRPT
PPPT
QPPT
TPT
HCPT analogs were prepared by the Mannich reaction. HCPT,
Apoptosis assay
0
1
0-hydroxycamptothecin; PPPT, 10-hydroxyl-9-(4 -hydroxy)
5
MCF-7 cells and PBMCs (B2 ꢁ 10 ) were seeded in a
piperidinylmethylcamptothecin; PRPT, 10-hydroxyl-9-L-prolinol (+)
0
3
methylcamptothecin; QPPT, 10-hydroxy-9-(4 -hydroxyethyl)-
9
6-well black-transparent chamber [90 ml in (5 ꢁ 10 )
piperazinylmethycamptothecin; TPT, topotecan.
each chamber]. The sample was maintained at 371C in a
% CO2 humidified atmosphere for 24 h. Then 10 ml
HCPT analog PBSs (HCPT analogs has been reserved as
mg/ml DMSO solutions, and these solutions were
5
1
further diluted with PBS into 125 mmol/l to get HCPT
analog PBSs) were added separately, with each concen-
tration located in four chambers as repeats, and incubated
for 24 h. Then 500 ml of Annexin V binding buffer was
added, and the plate was further incubated with 5 ml of
Annexin V-FITC at room temperature for 5 min in the
dark, followed by addition of 5 ml propidium iodide (PI)
solution and 1 min in the dark. Thereafter, the cells were
immediately subjected to flow cytometric analysis to
determine the extent of early apoptosis, late apoptosis,
and necrosis.
In research, SN-38 was also used as a lead compound to
replace HCPT, but we did not obtain 9-[(dialkylamino)
methyl]-SN-38, although the reaction mixture was
refluxed for 5 h with acetic acid as a solvent. The reason
is that the ethyl group at C has large steric hindrance
7
that resists the Mannich reaction. Only dimethylamine
could react with SN-38, but the yield was low. As the
structure of cyclane-aminol is larger than that of
dimethylamine, cyclane-aminol could not react with
SN-38 [13].
Results
Structural design of cyclane-aminol HCPT analogs
Features of the structure–activity relationship of CPT
analogs that are essential include the 20-(S)-hydroxyl, the
pyridine moiety of the D-ring, the integrity of the lactone
moiety of the E ring, and the planarity of the five-
membered ring system. In general, modifications of the
C, D, and E rings of CPTresult in reduction of efficacy. In
contrast, modifications of positions 9, 10, and 11 of the A
ring and position 7 of the B ring are well tolerated and
attributed in many cases to potency enhancement, both
in vitro and in vivo [1].
Solubility and ratio of ester/carboxylic acid in the
plasma
One of the major problems associated with CPT is its
poor solubility. Solubility is an important evaluation
when designing HCPT analogs, as shown in Table 1.
In particular, the solubility of PRPT and PPPT in PBS
was 1.39 and 0.84 mg/ml, respectively, which was more
than 68-fold and five-fold increased compared with that
of the lead compound HCPT, and more than 41-fold and
two-fold increased compared with that of the control drug
TPT. The solubility of both PPPT and PRPT in octanol
was 0.45 mg/ml, which is about 21-fold and three-fold
increased compared with that of HCPT and TPT. The
results indicate that improved solubility is associated
with the promotion of drug transportation and enhanced
bioavailability.
To reduce the cytotoxicity of HCPT, cyclane-aminol was
introduced into the structure of HCPT, which modified it
into water-soluble cyclane-aminol HCPT analogs through
the Mannich reaction (Scheme 1). The molecular
structures of these analogs are well established by
modern spectrometry, such as IR, MS, and H-NMR.
The signal of C –H, whose chemical shift in HCPT is at
d = 6.60, disappeared when various cyclane-aminols were
1
A major limitation for the clinical use of CPT analogs
springs from the chemical equilibrium between the CPT
lactone form and the E ring-opened carboxylate form
(Scheme 2). When the ratio of lactone/carboxylate
increases, CPT analogs gain access into the cells easily
and do not bind DNA topoisomerase I, leading to a
9
introduced at the C site by the Mannich reaction, and
the signal of Ar-CH –N appeared at about d = 4.17–4.72
9
2
1
in the H-NMR spectrum of these analogs.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

618 Anti-Cancer Drugs 2014, Vol 25 No 6
decrease in selectivity in tumor cells. When the ratio of
lactone/carboxylate decreases, CPT analogs can readily
bind DNA topoisomerase I, but do not have easy access
into cells, leading to an increase in tumor cell selectivity.
To improve the selectivity in tumor cells and reduce the
impact on normal cells, an appropriate ratio of lactone/
carboxylate is required, and it is also a major evaluation
for designing HCPT analogs, as shown in Table 1. In
particular, the lactone form of PPPT in the plasma was
appropriate at 86%. The appropriate ratio of lactone/
carboxylate in the plasma is associated with binding DNA
topoisomerase I exclusively [1,14].
Antiproliferative activity
Strongly cytotoxic drugs could kill tumor cells and
simultaneously damage normal cells, therefore causing a
significant number of side effects. Drugs with medium
cytotoxicity could kill tumor cells, whereas they are
Table 2 Inhibition of DNA topoisomerase I by 10-
hydroxycamptothecin analogs (n = 4)
Compounds
IC50 (mmol/l)
HCPT
TPT
PRPT
PPPT
QPPT
1.0
3.2
0.3
0.3
0.4
Inhibition of DNA topoisomerase I
HCPT, 10-hydroxycamptothecin; IC, inhibitory concentration; PPPT, 10-hydroxyl-
DNA topoisomerase I is an important enzyme that is
present in all living organisms and participates in many
cellular metabolic processes relevant to separation of
DNA strands such as replication, transcription, recombi-
nation, and repair. Data on the inhibitory activity of
cyclane-aminol HCPT analogs, HCPT, and TPT on
topoisomerase I are shown in Table 2. In particular, the
inhibitory activity of PPPTwas increased by about three-
fold and 10-fold, respectively, compared with that of the
lead compound, HCPT, and the control drug, TPT, as
shown in Fig. 1. These results indicate that the inhibitory
activity of cyclane-aminol HCPT analogs is stronger than
that of HCPT and TPT, and the increase in inhibitory
activity toward DNA topoisomerase I is associated
exclusively with induction of apoptosis in the prolifera-
tion phase in malignant rather than in normal cells [6].
0
9
-(4 -hydroxy) piperidinylmethylcamptothecin; PRPT, 10-hydroxyl-9-L-prolinol (+)
0
methylcamptothecin; QPPT, 10-hydroxy-9-(4 -hydroxyethyl)-piperazinylmethy-
camptothecin; TPT, topotecan.
Fig. 1
M
1
2
3
4
5
6
Table 1 Solubility and ratios of lactone/carboxylate in the plasma
of 10-hydroxycamptothecin analogs
Compounds
Lactone/carboxylate
Octylalcohol (mg/ml)
PBS (mg/ml)
Fluorescence images of DNA topoisomerase I inhibition by PPPT from
agarose gel electrophoresis using EB (lane M, 1 kb DNA marker; lane
HCPT
TPT
PRPT
PPPT
QPPT
90/10
82/18
80/20
86/14
74/26
0.02
0.12
0.45
0.45
0.28
0.02
0.23
1.39
0.84
0.19
1
, 0.5 mg pUC19; lane 2, 0.5 mg pUC19 incubated with 1.5 U of Topo I
at 371C for 30 min; lanes 3–6, 0.5 mg pUC19 incubated with 1.5 U
of Topo I in the presence of 0, 0.1, 0.5, 2.5 mmol/l PPPT or 2.5 mmol/l
HCPT, at 371C for 30 min). EB, ethidium bromide; HCPT, 10-hydroxy-
0
camptothecin; H
2
2
O , hydrogen peroxide; PPPT, 10-hydroxyl-9-
HCPT, 10-hydroxycamptothecin; PPPT, 10-hydroxyl-9-(4 -hydroxy) piperidinyl-
methylcamptothecin; PRPT, 10-hydroxyl-9-L-prolinol (+) methylcamptothecin;
QPPT, 10-hydroxy-9-(4 -hydroxyethyl)-piperazinylmethycamptothecin; TPT, topotecan.
0
(4 -hydroxy) piperidinylmethylcamptothecin; Topo I, topoisomerase I.
0
Scheme 2
R9 R7
R9 R7
R9 R7
HO
HO
HO-Ph723-DNA Topo I
HO
N
O
O
N
O
O
O
N
N
HO
N
H O
N
HO
3
OH
OH
OH
HO
Ph723-DNA Topo I
O
O
O
ester
Binding DNA topoisomerase I at 723 site tyrosine
carboxylic acid
HCPT analogs binding to DNA topoisomerase I at tyrosine located at the 723 site. HCPT, 10-hydroxycamptothecin; Topo I, topoisomerase I.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Novel cyclane-aminol HCPT analogs Zhang et al. 619
Table 3 The antiproliferative activity of 10-hydroxycamptothecin
analogs in vitro (n = 4)
pathway is totally different from that of other marketed
drugs of HCPT analogs.
IC50 (mmol/l)
HCPT
TPT
PRPT
PPPT
QPPT
NCI-H1688
NCI-H446
SMMC-7721
BGC-823
MCF-7
4.0
2.1
2.5
0.8
1.0
2.1
0.94
8.2
2.5
6.3
16.4
11.4
9.0
17.1
4.3
23.8
6.5
25.6
17.0
30.0
20.0
35.3
25.6
5.66
Regulating effect on intracellular ROS
To confirm that cyclane-aminol HCPT analogs could
11.0
25.0
25.0
16.5
7.06
15.0
22.0
20.0
17.5
5.37
–
capture O2 and produce H O , an aqueous solution of
2
2
1% cyclane-aminol HCPT analogs was oxidized by
reactive oxygen species (ROS) dissolved in PBS; the
oxidation product was detected by LC-MS and the
generated H O using PF1. Oxidation product M376 by
Average
±
RSD
3.95
HCPT, 10-hydroxycamptothecin; IC, inhibitory concentration; PPPT, 10-hydroxyl-
2
2
0
9
-(4 -hydroxy) piperidinylmethylcamptothecin; PRPT, 10-hydroxyl-9-L-prolinol (+)
0
H O oxidation was not detected. Oxidation product
2 2
methylcamptothecin; QPPT, 10-hydroxy-9-(4 -hydroxyethyl)-piperazinylmethy-
camptothecin; TPT, topotecan.
–
M376 and H O (E1 = 0.68 eV) by light irradiation (O
)
2
2
2
oxidation were detected. Oxidation product M376 and
NaCl by NaOCl (E1 = 1.49 eV) oxidation were detected
but none by H O oxidation. These results indicate that
tolerated by normal cells. The antiproliferative activity in
terms of inhibitory concentration (IC ), as obtained
2
2
5
0
–
cyclane-aminol HCPT analogs can capture O2 by
generating H O and NaOCl by generating NaCl.
using the MTT assay, on BGC-823, MCI-H1688, NCI-
H446, SMMC-7721, and MCF-7 cell lines in vitro are
shown in Table 3. The IC50 values of HCPT, TPT, PRPT,
PPPT, and QPPTwere 2.1, 9.0, 16.5, 17.5, and 25.6mmol/l,
respectively. These results indicate that PPPTand PRPT
have medium cytotoxicity, which was reduced by eight-
fold compared with that of the lead compound HCPTand
two-fold compared with that of TPT. These results
indicate that the antiproliferative activity of PPPT and
PRPT was medium and that they have the ability to kill
tumor cells while being tolerated by normal cells.
2
2
Causing elevation in the intracellular level of H
Results of the fluorescence assays of H O in MCF-7 and
2 2
O
2
2
BGC-823 cells, and PBMCs incubated with HCPT
analogs for 2 h using the PF1 fluorescence probe are
shown in Fig. 2.
When MCF-7 cells were incubated with cyclane-aminol
HCPT analogs, extracellular and intracellular H O levels
showed a remarkable concentration-depended increase,
2
2
as in Fig. 2. A concentration of 20 mmol/l H O is
2
2
Major metabolic pathways
generated by treatment of MCF-7 cells with 12.5 mmol/l
PPPT, with induction of apoptosis rather than necrosis.
The fluorescence microscopy images, using the PF1
marker, of intracellular H O in MCF-7 cells incubated
with PPPTand HCPT for 2 h are presented in Fig. 3. The
ratio of apoptotic cells was increased to 26.2% and
necrotic cells to 0.2% in MCF-7 cells incubated with
Metabolic profiling assays revealed the presence of
metabolites in rat urine on LC-MS following oral
administration of HCPT analogs at 25 mg/kg [2]. The
results are shown in Scheme 3. The detection of
metabolites was summarized to depict the proposed
metabolic pathways associated.
2
2
1
2.5 mmol/l PPPT for 24 h, on the basis of the Annexin
The major metabolic pathway of cyclane-aminol HCPT
–
analogs involves capturing oxygen free radicals (O2 ) in
V-FITC–PI fluorescence assay (Fig. 4).
the cells and producing H O . Of the total amount of
2
When BGC-823 were incubated with cyclane-aminol
HCPT analogs, extracellular and intracellular H O levels
2
PPPT, 20% was oxidized into M376 and 80% remained as
the prototype drug. QPPT was completely (100%)
oxidized into 9-methylene-10-carbonyl-CPT, and there
was no prototype drug excretion. The major metabolites
of PRPT were M396, sulfated metabolites, and de-
oxidized metabolites, methylated metabolites, and less
than 30% prototype drug. These results indicate that
metabolism of PPPTwas appropriate, QPPTwas too fast,
and PRPT was complex. Of the TPT, 15% was
metabolized into other metabolites and 85% was excreted
as the prototype drug. CPT-11 was mostly excreted in the
form of the prototype drug (55%), whereas some of it was
hydrolyzed into the active metabolite SN-38 by carbox-
ylesterase in vivo, and the rest was oxidized [5].
2
2
showed a remarkable concentration-depended increase at
low concentrations; however, they showed a concentra-
tion-depended decrease at high concentrations (Fig. 2).
A concentration of 25 mmol/l H O is generated on
2
2
treatment of BGC-823 cells with 6.25 mmol/l PPPT, with
induction of apoptosis rather than necrosis.
When PBMCs were incubated with cyclane-aminol
HCPT analogs, extracellular and intracellular H O levels
showed a remarkable concentration-depended increase
2
2
(
Fig. 2). A concentration of 25 mmol/l H O is generated
2
2
on treating PBMCs with 12.5 mmol/l PPPT or PRPT,
without induction of either apoptosis or necrosis. The
fluorescence microscopy images, using the PF1 marker, of
intracellular H O in PBMCs incubated with PPPT and
These results indicate that cyclane-aminol HCPTanalogs
–
can capture O2 and produce H O in rats, which is
2
2
HCPT for 2 h are shown in Fig. 3. The microscopy images
of PBMCs incubated with 12.5 mmol/l PPPT compared
with 2.5 mmol/l HCPT for 24 h are shown in Fig. 3.
2
2
associated with an elevation in H O levels in MCF-7 and
2
2
BGC-823 cells, and in PBMCs. This special metabolic
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620 Anti-Cancer Drugs 2014, Vol 25 No 6
Scheme 3
HO
HO
HO
HO
HO SO
3
HO SO
3
N
O
N
O
O
N
O
O
N
N
N
H
H C
OH
3
H C
3
HO
HO
OH
HO
H O
3
3
O
O
O
M396 100%
M506 blood
OH
M490 11%
O3−
2H
OH
OH
N
N
N
HO SO
HO
HO SO
3
3
N
N
O
O
2H
N
O
O
2H
N
O
N
N
H
H COC
3
HO
HO
HO
HO
HO
O
O
O
O
M617 18%
PRPTM477 28%
M575 35%
HO
HO
N
N
H O
2
2
N
H O
2
2
OH
O
OH
O2−
N
N
O
O
N
N
O
O
O2−
N
O
O
N
OH
OH
O
OH
O
O
PPPT (80%)
M376 (20%,100%)
QPPT (0%)
N
HO
HO
N
N
O
N
N
O
N
O
N
6H
H
4H
OH
OH
OH
OH
OH
OH
O
OH
O
O
O
M386 4%
TPT M421 87%
M366 2%
4H
N
O
N
N
H
N
O
N
OH
OH
OH
O
M358 4%
M301 3%
0
Metabolic pathways of PPPT, QPPT, PRPT, and TPT in rat. PPPT, 10-hydroxyl-9-(4 -hydroxy) piperidinylmethylcamptothecin; PRPT, 10-hydroxyl-9-L-
0
prolinol (+) methylcamptothecin; QPPT, 10-hydroxy-9-(4 -hydroxyethyl)-piperazinylmethycamptothecin; TPT, topotecan.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Novel cyclane-aminol HCPT analogs Zhang et al. 621
Fig. 2
50
40
30
20
10
0
5
4
3
0
0
0
50
40
30
20
10
0
20
10
0
0
5
10 15 20 25
mol/l
0
5
10 15 20 25
mol/l
0
5
10 15 20 25
mol/l
μ
μ
μ
PRPT
PPPT
TPT
HCPT
Assay of extracellular and intracellular H
O
2 2
levels in MCF-7 and BGC-823 cells, and in PBMCs, which were incubated with HCPT analogs for
2
1
h in the presence of PF1 (PRPR or PPPT at 3.125–25 mmol/l, TPT at 1.25–10 mmol/l, HCPT at 0.625–5 mmol/l, n = 4, UV488 nm, ꢁ 32). HCPT,
0
2 2
0-hydroxycamptothecin; H O , hydrogen peroxide; PBMCs, peripheral blood mononuclear cells; PPPT, 10-hydroxyl-9-(4 -hydroxy)
piperidinylmethylcamptothecin; PRPT, 10-hydroxyl-9-L-prolinol (+) methylcamptothecin; TPT, topotecan.
Fig. 3
2
00
200
200
2
00
200
200
MCF-7 12.5μmol/l PPPT 2h
MCF-7 0.625μmol/l HCPT 2h
PBMCs 12.5μmol/l PPPT 2h
PBMCs 2.5μmol/l HCPT 2h
PBMCs (control) 24h
PBMCs 12.5μmol/l PPPT 24h
PBMCs 2.5μmol/l HCPT 24h
Microscopy images of intracellular H
2
O
2
in MCF-7 tumor cells and PBMCs that were incubated with PPPT and HCPT.
0
2 2
HCPT, 10-hydroxycamptothecin; H O , hydrogen peroxide; PBMCs, peripheral blood mononuclear cells; PPPT, 10-hydroxyl-9-(4 -hydroxy)
piperidinylmethylcamptothecin.
The apoptotic cell ratio was increased to 1.8% and necrotic
cell ratio to 2.0% in PBMCs incubated with 12.5mmol/l
PPPT for 24 h, on the basis of the Annexin V-FITC–PI
fluorescence assay (Fig. 4). These results indicate that
HCPT can induce apoptosis and necrosis in PBMCs, but
PPPT does not induce apoptosis or necrosis in PBMCs.
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622 Anti-Cancer Drugs 2014, Vol 25 No 6
Fig. 4
05
0.2%
3.6%
5.3%
105
2.0%
0.5%
1.3%
1
1
1
1
7
20.9%
96.2%
04
03
02
104
103
102
Q1
Q3
Q2
Q4
Q1
QQ 33
Q2
Q4
102
103
FITC-A
PBMCs/PPPT
104
105
1
02
103
FITC-A
104
105
MCF-7/PPPT
Annexin V-FITC–PI assay of apoptosis in MCF-7 cells, compared with PBMCs, that were incubated with PPPT for 24 h (PPPT 12.5 mM).
FITC, fluorescein isothiocyanate; HCPT, 10-hydroxycamptothecin; PBMCs, peripheral blood mononuclear cells; PI, propidium iodide;
0
PPPT, 10-hydroxyl-9-(4 -hydroxy) piperidinylmethylcamptothecin.
When MCF-7 and BGC-823 cells, and PBMCs were
incubated with 10–5 mmol/l TPT or 5–2.5 mmol/l HCPT,
the elevation in intracellular H O levels did not increase.
H O in tumor cells may be catalytically decomposed
2 2
primarily by CAT into water and oxygen. As CAT activity
in tumor cells is low, excess H O may not be catalytically
2
2
2
2
HCPT (2.5 mmol/l) can induce apoptosis and necrosis in
MCF-7 and BGC-823 cells, and in PBMCs in 24 h.
decomposed into water and oxygen. Thus, relative to
normal cells with the same H O levels, tumor cells are
more prone to apoptosis due to the cytotoxicity of H O .
2
2
2
2
Cyclane-aminol HCPT analogs can cause elevations in
H O levels in MCF-7 and BGC-823 cells, along with
Excess H O is critical, as in tumor cells the levels reach
2
2
2
2
the threshold of toxicity. The mechanisms of redox state
are different in normal cells and in tumor cells. The excess
intracellular H O in normal cells may oxidize glutathione
induction of apoptosis rather than necrosis. Cyclane-
aminol HCPTanalogs can cause elevations in H O levels
2
2
2
2
in PBMCs, and neither apoptosis nor necrosis, which is
associated with a regulating effect on intracellular ROS,
such as antioxidant activity on NaOCl. TPTor HCPTcan
induce apoptosis and necrosis rather than cause an
elevation in intracellular H O levels in MCF-7 and
to oxidized glutathione. The excess intracellular H O in
2
2
tumor cells may cause oxidization at specific kinases with
an active site of cysteine. Whereas targeted oxidation of
the CDC25 cysteine active site contributes to deactiva-
tion of CDKs/cyclins and leads to cell proliferation
inhibition, targeted oxidation of the cysteine active site
of caspases contributes to activation of the apoptosis
pathway, and targeted oxidation of p53 cysteine activation
site-175 contributes to cell proliferation inhibition and
selective induction of cancer cell apoptosis [15,16]. Thus,
any agent that increases H O levels slows down cancer
2
2
BGC-823 cells, and PBMCs. These results indicate that
cyclane-aminol HCPT analogs can cause elevations in
intracellular H O levels and can selectively induce
2
2
apoptosis in tumor cells rather than in normal PBMCs.
Discussion
The effect of ROS on cell proliferation depends on their
2
2
cell proliferation and leads to cell apoptosis. Conversely,
any agent that decreases intracellular H O2 levels
nature, their origin, and their intracellular level. The
lifetime of intracellular H O is longer than that of other
2
2
2
enhances cancer cell growth. Therefore, drugs that
specifically increase intracellular H O levels in cancer
cells can selectively induce cancer cell apoptosis [17].
ROS. Hence, H O could be an important messenger in
2
2
2
2
–
cell signaling pathways. In normal cells, 95% of O2 may
be catalytically decomposed into H O by superoxide
2
2
dismutase, which constitutes the first natural anti-ROS
defense in the body. In normal cells, excess of H O may
Because of the differential regulation of the redox status
in tumor cells and in normal cells, tumor cell proliferation
is inhibited and apoptosis is induced selectively by
cyclane-aminol HCPT analogs; however, with little
2
2
cause catalytic oxidation of glutathione to oxidized
glutathione by glutathione enzyme. Therefore, H O in
2
2
normal cells is maintained at a very low concentration,
with the cells in a nonproliferative state. In tumor cells,
levels of superoxide dismutase and catalase (CAT) were
lower than those in normal cells, leading to higher levels of
influence on the function of normal cells. Cyclane-aminol
–
HCPT analogs can capture O
intracellular H
and cause elevation in
levels with selective induction of
2
O
2
2
apoptosis in MCF-7 and BGC-823 cells, rather than
in normal PBMCs. The results suggest that elevation in
–
O2 in tumor cells, which promotes cell proliferation.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

Novel cyclane-aminol HCPT analogs Zhang et al. 623
intracellular H O levels may cause oxidization at specific
2
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2
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8
Lee JH, Lee JM, Kim JK, Kim JK, Ahn SK, Lee SJ, et al. Antitumor activity of
In this research, cyclane-aminol methylene was intro-
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series of water-soluble cyclane-aminol HCPT analogs
with medium cytotoxicity were synthesized, and the
selectivity of DNA topoisomerase I inhibition, compared
with TPT, was improved, which were associated with
apoptosis of tumor cells in the proliferation phase. We
first discovered that cyclane-aminol HCPT analogs had a
special metabolic pathway, which is different from that of
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–
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Acknowledgements
Conflicts of interest
There are no conflicts of interest.
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