Mechanism of forming trimer, self-assembling nano-particle and inhibiting tumor growth of small molecule CIPPCT

Article abstract of DOI:10.1039/c4md00158c

The mechanisms of small molecules forming nanospecies and the effect of the nanospecies of small molecules on their pharmacological actions remain to be elucidated. As one of our efforts here, a uPA inhibitor, (5aS,12S,14aS)-5,14-dioxo-12-(2-tryptophanylthreo-nylbenzylester-N-yl-ethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo[1,2:4,5]-pyrazino[1,2:1,6]pyrido[3,4-b]indole (CIPPCT) was presented. Energy-minimization, FT-MS and 2-D ROESY spectra defined CIPPCT taking -like conformation, and the intermolecular association drove CIPPCT to form a finger ring-like trimer. Images from transmission electron, scanning electron and atomic force microscopies consistently visualized that in aqueous solution at pH 6.7 and 10^-10 M concentration, CIPPCT generally assembled nanoparticles of 9-67 nm in diameter. Mesoscale simulation demonstrated that a nanoparticle 9.4 nm in diameter contained 350 trimers. In vivo CIPPCT dose-dependently inhibited tumor growth in S180 mice. An ELISA assay confirmed that CIPPCT concentration-dependently downregulated serum uPA. The nanoparticles of CIPPCT are capable of occurring in mouse plasma and adhering on HeLa cells, and nanosized CIPPCT directly correlates the downregulation of uPA with inhibition of tumor growth.

Full text of DOI:10.1039/c4md00158c

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Mechanism of forming trimer, self-assembling
nano-particle and inhibiting tumor growth of small
molecule CIPPCT†
Cite this: Med. Chem. Commun., 2014,
5, 1634
Fengxiang Du,a Xiaoyi Zhang,a Shan Li,a Yaonan Wang,a Meiqing Zheng,a Yuji Wang,a
a
a
a
ab
a
*
*
Shurui Zhao, Jianhui Wu, Lin Gui, Ming Zhao
and Shiqi Peng
The mechanisms of small molecules forming nanospecies and the effect of the nanospecies of small
molecules on their pharmacological actions remain to be elucidated. As one of our efforts here,
a
uPA inhibitor, (5aS,12S,14aS)-5,14-dioxo-12-(2-tryptophanylthreo-nylbenzylester-N-yl-ethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo[1,2:4,5]-pyrazino[1,2:1,6]pyrido[3,4-b]indole (CIPPCT)
was presented. Energy-minimization, FT-MS and 2-D ROESY spectra defined CIPPCT taking -like
conformation, and the intermolecular association drove CIPPCT to form a finger ring-like trimer. Images
from transmission electron, scanning electron and atomic force microscopies consistently visualized that
in aqueous solution at pH 6.7 and 10ꢀ10 M concentration, CIPPCT generally assembled nanoparticles of
9–67 nm in diameter. Mesoscale simulation demonstrated that a nanoparticle 9.4 nm in diameter
contained 350 trimers. In vivo CIPPCT dose-dependently inhibited tumor growth in S180 mice. An ELISA
assay confirmed that CIPPCT concentration-dependently downregulated serum uPA. The nanoparticles
of CIPPCT are capable of occurring in mouse plasma and adhering on HeLa cells, and nanosized CIPPCT
directly correlates the downregulation of uPA with inhibition of tumor growth.
Received 4th April 2014
Accepted 20th June 2014
DOI: 10.1039/c4md00158c
www.rsc.org/medchemcomm
In various physiological processes related to tissue remod-
eling, the plasminogen activator (PA) system is of importance.
Introduction
Biological nanosystems have been perspectively viewed.1–3 To
prepare the nanospecies and characterize the properties of the
nanomaterials, various technologies have been developed4–11 that
effectively promote the development of nanomedicine.12–15
Regarding nanomedicine, the progress of the nanotechnology in
the past decades mainly occurred in the preparation of drug
delivery systems, such as liposomes.16,17 Recently, the self-
assembly of single small molecules at the nanoscale,18,19 and the
effect of the self-assembly of some small molecules on their bio-
logical properties has attracted intense interest.20,21 Based on
these advances, some bioactive small molecules capable of self-
assembly have been used in the functionalization of metal
surfaces,22 in the craing of functional biological materials,23 and
in the increase of anti-osteoporosis activity.24 The physical and
chemical mechanisms of small molecules forming nano-species,
as well as the effect of the nanospecies of small molecules on their
pharmacological actions, remain to be elucidated.
The PA system consists of PAI-1/PAI-2 (the inhibitors of PA), a
urokinase-type plasminogen activator (uPA), the receptor of uPA
(uPAR) and the plasminogen (the substrate of uPAR).25
Regarding physiology, PAI-1 is a primary inhibitor of plasmin-
ogen activation in hemostasis and is one of the main regulators
of the brinolytic system.26 Abnormal expression of the PAI-1
gene induces different diseases, including brosis, obesity,
cancer and vascular disease.27,28 Moreover, regarding carcino-
genesis, upregulation of the PAI-1 gene results in the inhibition
of tumor progression and correlates with a less aggressive
phenotype of some cancers, presumably due to the inhibition of
the uPA activity.27,29,30 The elevated level of PAI-1 in several
primary malignant tumors, such as breast, gastric and ovarian
cancers, as well as adenocarcinomas of lung, is considered as
their marker.27,28,31 On the other hand, a high level of PAI-2 in a
tumour is associated with improved prognoses. In the presence
of PAI-1, PAI-2 inhibits uPA, whereas in the presence of vitro-
nectin, PAI-2 inhibits adherent cells. In inhibiting uPA, the
elevated level of PAI-2 in a tumour microenvironment outcom-
petes PAI-1.32
aCollege of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, P.R.
China. E-mail: sqpeng@bjmu.edu.cn; mingzhao@bjmu.edu.cn; Fax: +86-10-8391-
1528; +86-10-8280-2482; Tel: +86-10-8391-1528; +86-10-8280-2482
bDepartment of Biomedical Science and Environmental Biology, Kaohsiung Medical
University, Kaohsiung, Taiwan
Depending on the interactions of uPA with endogenous PAI-
1, it exerts dual effects on tumor progression. In murine
models, a stable overexpression of uPA promotes the growth of
colon tumors that naturally express high PAI-1, but inhibits the
growth of renal tumors that naturally express low PAI-1.33 In a
† Electronic supplementary information (ESI) available: NMR spectra of CIPPCT
were included. See DOI: 10.1039/c4md00158c
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Scheme 1 Discovery course of CIPPCT.
variety of malignant tumours, the overexpression of uPA and/or the expression of uPA.38 Wider investigations are needed to
uPAR has been strongly correlated with poor prognosis. successfully discover relevant lead compounds.
Impairing the function of uPA and/or uPAR, or inhibiting the
The course of discovering CIPPCT is depicted in Scheme 1.
expression of uPA and/or uPAR, limits the metastatic potential The (5aS,12S,14aS)-isomers of (5aS,12S/R,14aS)-5,14-dioxo-12-
of many tumours.34 Clinical and experimental evidence proves (2-aminoacid-N-ylethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-
the signicance of uPA and/or uPAR for a number of solid 5H,14H-pyrolo[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]indoles
cancers, and the downregulation of uPA and/or uPAR is (DAPPP) were previously explored as inhibitors of urokinase.39
considered a potential strategy in cancer therapy.35,36
Of (5aS,12S,14aS)-DAPPP analogs, the 2-trptophan-N-ylethyl-1-yl
Perturbing the interaction between uPA and uPAR has derivative (CIPPC) more effectively antagonized the thrombo-
attracted some interest, and a few novel compounds have been lytic activity of urokinase, which provided a lead to a uPA
provided for inhibiting uPA. In a preliminary structure-activity inhibitor.40 Of Trp-Trp-AA-OBzl, Trp-Trp-Thr-OBzl possessed the
exploration, amiloride analogs that inhibited uPA were highest anti-tumor activity, which provided a possible modi-
synthesized.37 The N-nicotinonitrile derivatives that allegedly cation of CIPPC with Trp-Thr-OBzl.41 In the formation of
perturb the interaction between uPA and uPAR failed to affect nanoparticles WY3-56, a PAD4 inhibitor, the intermolecular
Scheme 2 Synthetic route of CICCPT.
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p–p interactions were found to be essential that provided an
effective approach in designing nanoscale self-assembly.42,43
Based on this information, a uPA inhibitor (5aS,12S,14aS)-5,14-
dioxo-12-(2-tryptophanylthreo-nylbenzylester-N-ylethyl-1-yl)-
1,2,3,5,5a,6,11,12,14,14a-decahy-dro-5H,14H-pyrolo[1,2:4,5]pyr-
azino[1,2:1,6]pyrido[3,4-b]indole (CIPPCT) was designed to
investigate the mechanisms of nanoparticle formation with FT-
MS and 2-D ROESY spectra, to visualize the feature of the
nanoparticles with transmission electron, scanning electron
and atomic force microscopies, and to correlate the bioactivity
with the nanoparticles with the in vitro and the in vivo assays.
Preparing (5aS,12S,14aS)- and (5aS,12R,14aS)-5,14-dioxo-12-
(2-tryptophan-benzylester-N-ylethyl-1-yl)-1,2,3,5,-5a,6,11,12,14,14a-
decahydro-5H,14H-pyrolo[1,2:4,5]pyrazino-[1,2:1,6]pyrido[3,4-b]-
indoles (3a and 3b). To a solution of 2.3 g (5aS,12S/R,14aS)-5,14-
dioxo-12-cabonylmethyl-1,2,3,5,5a,6,11, 12, 14,14a-deca-hydro-
5H,14H-pyrolo[1,2:4,5]pyrazino-[1,2:1,6]pyrido[3,4-b]indole (2,
6.8 mmol) in 10 mL DMF was added 2.4 g HCl$Trp-OBzl
(6.8 mmol), 1.5 mL Et3N and 6 g anhydrous Na2SO4. The reac-
tion mixture was stirred at room temperature for 3 h, added to a
solution of 1 g KBH4 (18.4 mmol) in 10 mL CH3OH, and stirred
for another 1 h. The reaction mixture was evaporated in vacuo,
and the residue was dissolved with 40 mL ethyl acetate and 20
mL deionized water. The two-phase solution was treated with 2
mL hydrochloric acid (5 M) to decompose the excess KBH4, and
adjusted to pH 9 with aqua ammonia NH3 (5 M). The ethyl
acetate phase was separated, and the aqueous phase was
extracted with ethyl acetate (3 ꢂ 40 mL). The combined ethyl
acetate phases were washed with 40 mL saturated aqueous
NaCl, dried with anhydrous Na2SO4, ltered and concentrated
in vacuo. The residue was then puried and separated on
column chromatography (CHCl3/CH3OH, 30 : 1) to give 1.3 g
(61%) 3a and 1.5 g (71%) 3b.
Experimental
Synthesis
To conveniently obtain CIPPCT, a 5-step reaction sequence of
Scheme 2 was used, which can be divided into steps 1–3 for
preparing 3a, and steps 4 and 5 forꢁpreparing CIPPCT.
(i) CH3OH, hydrochloric acid, 45 C; (ii) SOCl2, CH2Cl2, diiso-
propylamine; (iii) DMF,Et3N, anhydrous Na2SO4, KBH4, CH3OH
and (iv) DMF,DCC, anhydrous Na2SO4,N-methylmorpholine.
The preparation of the compounds are described in
following section; NMR spectra are available in the ESI.†
3a. FT-MS (m/z): 615.2830 [M]+; mp:175–176 ꢁC; 1HNMR (300
MHz, CDCl3): d ¼ 8.223 (s, 1H), 7.756–7.584 (dd, J ¼ 7.5 Hz, J ¼
7.5 Hz, 2H), 7.494–7.411 (m, 3H), 7.411(m, 3H), 7.386–7.317 (m,
5H), 7.155–7.149 (m, 3H), 7.05 (m, 1H), 6.084–6.057 (d, J ¼ 8, 1
Hz, 1H), 5.538–5.488 (q, J ¼ 4.8 Hz, 5.1 Hz, 1H), 5.216 (s, 2H),
4.174 (m, 1H), 4.106 (m, 1H), 4.004–3.988(d, J ¼ 4.8 Hz, 1H),
3.559–3.542 (m, 1H), 3.434–3.288(td, J ¼ 5.1 Hz, J ¼ 3.0 Hz, J ¼
3.0 Hz, 2H), 2.959–2.910 (m, 2H), 2.886–2.757 (m, 2H), 2.743–
2.655 (m, 1H), 2.466–2.376 (m, 1H), 2.318–2.198 (m, 2H), 2.098–
1.913 (m, 2H); 13C NMR (75 MHz, CDCl3): d ¼ 173.86, 169.48,
165.90, 165.83, 156.14, 136.34, 135.96, 135.71, 135.38, 133.96,
133.53, 129.60, 128.61, 128.34, 128.21, 126.99, 126.10, 125.66,
123.71, 122.59, 122.13, 121.39, 120.30, 120.12, 120.01, 119.41,
118.88, 118.17, 117.66, 115.44, 112.07, 111.83, 111.43, 111.23,
106.39, 105.70, 66.68, 62.21, 59.34, 59.19, 58.89, 57.40, 56.95,
50.28, 49.14, 46.24, 45.37, 44.46, 40.43, 35.44, 29.51, 28.64,
28.54, 23.24, 23.12, 21.41, 21.23, 10.00; IR (KBr): 3308, 2978,
2954, 2880, 1659, 1454, 1398, 1335, 1236, 1096, 745 cmꢀ1. In the
ROESY 2D NMR spectrum, a positive NOE signal between 5aS-H
and 12-H was observed.
Preparing
1-(2,2-dimethoxyethyl)-1,2,3,4-tetrahydrocarbo-
line-3-carboxylic acid methyl ester (1). A suspension of 5.0 g
L-tryptophan methyl ester (24.5 mmol) and 6.0 mL 1,1,3,3-tet-
ramethoxypropane (23.6 mmol) in 50 mL CH3OH was adjusted
to pH 2 with hydrochloric acid (5 M) and stirred at 45 ꢁC for
48 h. The reaction mixture was evaporated in vacuo to remove
the solvent, the residue was diluted with 50 mL water, and the
formed solution was extracted 3 times with 30 mL ethyl acetate.
Further, the extracts were combined and successively washed
with 10% aqueous Na2CO3 (3 ꢂ 30 mL) and saturated aqueous
NaCl (2 ꢂ 30 mL), then dried over anhydrous Na2SO4,
ltered and concentrated in vacuo to provide 1. ESI-MS (m/z):
319 [M + H]+.
Preparing
(5aS,12S/R,14aS)-5,14-dioxo-12-cabonylmethyl-
1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo[1,2:4,5]pyr-
azino[1,2:1,6]pyrido [3,4-b]indole (2). To 5.0 g CBz-L-Pro (14.8
mmol), 50 mL SOCl2 was added dropwise, and the reaction
mixture was heated for 5 h reux. The mixture was concentrated
in vacuo to remove excess SOCl2. and the residue was treated
with 30 mL ether to provide CBz-protected prolinyl chloride as
colorless powder, to which a solution of 3.2 g 1-(2,2-dimethox-
yethyl)-1,2,3,4-tetrahydrocarboline-3-carboxylic acid methyl
ester (1, 10 mmol) in 50 mL CH2Cl2 was added at 0 ꢁC and
stirred for 0.5 h. The reaction mixture was adjusted to pH 9 with
diisopropylamine and stirred at room temperature for 24 h. The
reaction mixture was concentrated in vacuo, the residue was
dissolved in 150 mL acetone, and the solution was treated with
200 mg p-TsOH. Aer stirring at 45 ꢁC for 1 h, the reaction
mixture was treated with 5 mL Et3N and concentrated in vacuo;
the residue was puried by column chromatography (CHCl3/
CH3OH, 40 : 1). The fraction of interest was evaporated in vacuo,
and the residue was recrystallized from acetone to provide 1.5 g
(29%) of the title compound. ESI-MS (m/z): 338 [M + H]+.
3b. FT-MS (m/z): 615.2845 [M]+; mp:105–106 ꢁC;1HNMR (300
MHz, CDCl3): d ¼ 11.087 (s, 1H), 10.677 (s, 1H), 7.458–7.404 (dd,
J ¼ 4.8 Hz, J ¼ 5.2 Hz, 2H), 7.295–7.250 (m, 7H), 7.075–6.935 (m,
4H), 6.068–6.040 (dd, J ¼ 1.8 Hz, J ¼ 1.8 Hz, 1H), 5.216–5.134(dt,
J ¼ 7.8 Hz, J ¼ 7.8 Hz, 2H), 4.765–4.734 (dd, J ¼ 2.7 Hz, J ¼ 2.4
Hz, 1H), 4.258–4.235 (d, J ¼ 6.9 Hz, 1H), 4.193–4.138(m, 2H),
3.806–3.790 (m, 1H), 3.428–3.388(dd, J ¼ 2.7 Hz, J ¼ 2.7 Hz, 2H),
3.333 (m, 4H) 3.012–2.982 (m, 2H), 2.875–2.727 (m, 1H), 2.407–
2.361(t, J ¼ 6.9 Hz, J ¼ 6.9 Hz, 1H), 2.226–2.209 (t, J ¼ 2.4 Hz, 2.7
Hz, 1H), 2.176–2.124 (m, 1H), 1.934–1.918 (m, 1H), 1.855–1.788
(m, 2H); 13C NMR (75 MHz, CDCl3): d ¼ 174.03, 165.51, 164.42,
136.66, 136.55, 136.29, 135.99, 133.66, 128.81, 128.29, 127.82,
127.13, 126.58, 121.68, 121.14, 119.23, 118.82, 118.26, 118.02,
111.61, 111.25, 106.00, 105.56, 66.04, 58.77, 53.54, 52.90, 46.90,
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46.40, 45.06, 29.93, 28.63, 25.22, 21.46; IR (KBr): 3251, 3233, 2974, 2928, 2884, 1742, 1649, 1520, 1454, 1402, 1339, 1196,
2957, 2930, 2857, 1659, 1454, 1398, 1150, 748 cmꢀ1. In the 1111, 745, 845 cmꢀ1
ROESY 2D NMR spectrum, no NOE signal between 5aS-H and
12-H was observed.
.
Generation of energy-minimized conformation
Preparing
ylethyl-1-yl)-1,2,3,5,5a,6,11,12,14,14a-decahydro-5H,14H-pyrolo-
[1,2:4,5]pyrazino[1,2:1,6]pyrido[3,4-b]-indoles (CIPPC). To
(5aS,12S,14aS)-5,14-dioxo-12-(2-tryptophan-N-
CIPPCT was sketched in ChemDraw 10.0, converted to 3D
conformation in Chem3D 10.0 and then energy-minimized in
Discovery Studio 3.5 with an MMFF force eld. The energy-
minimized conformation was utilized as the starting confor-
mation for conformation generation. The energy-minimized
conformations of CIPPCT were sampled in the whole confor-
mational space via systematic search and BEST methods in
Discovery Studio 3.5. Both the systematic search and BEST
methods were practiced with SMART minimizer using a
CHARMM force eld. In addition, the energy threshold was set
to 20 kcal molꢀ1 at 300 K. The maximum number of minimi-
zation steps was set to 200 and the minimization RMS gradient
a
solution of 1.3 g (5aS,12S,14aS)-5,14-dioxo-12-(2-trypto-phan-
benzylester-nylethyl-1-yl)-1,2,3,5,-5a,6,11,12,14,14a-decahydro-
5H,14H-pyrolo-[1,2:4,5]pyrazino[1,2:1,6]-pyrido[3,4-b]indoles (3a,
2.1 mmol) in 5 mL methanol aqueous, NaOH (4 M) was added
dropwise to adjust the pH to 12, and the reaction mixture was
stirred at room temperature for 2 h. The reaction mixture was
adjusted to pH 5 with hydrochloric acid (2 M), evaporated
under vacuum, and the residue was puried on silica gel
column to provide 863 mg (90%) of CIPPC. ESI-MS (m/z): 526
1
[M + H]+; H NMR (400 MHz, DMSO), d(ppm) ¼ 11.27 (s, 1H),
˚
was set to 0.1 A. The maximum generated conformations were
10.86 (s, 1H), 7.54 (dd, J ¼ 7.6, J ¼ 7.6 Hz, 2H), 7.33 (m, 2H),
7.13 (s, 1H), 7.06 (s, 2H), 6.92 (t, J ¼ 7.2 Hz, 1H), 5.38 (s, 1H),
4.50 (s, 1H), 4.29 (m, 2H), 3.45–3.34 (m, 4H), 3.24 (s, 1H), 3.15
˚
set to 255 with a RMSD cutoff 0.2 A.
(m, 2H), 2.83 (m, 1H), 2.51 (s, 1H), 2.22 (s, 1H), 1.98–1.79 (m, Measuring two-dimensional ROESY spectra
5H). 13C NMR (100 MHz, DMSO), d(ppm) ¼ 171.11, 170.44,
The one-dimensional 1H NMR spectrum of 10 mg CIPPCT in 0.5
165.55, 136.34, 136.00, 133.98, 125.95, 124.11, 121.65, 118.64,
118.47, 118.19, 111, 71, 111.47, 109.49, 105.53, 101.53, 63.10,
62.17, 58.69, 56.32, 52.87, 49.42, 45.03, 43.18, 40.37, 40.16,
39.95, 36.27, 34.60, 28.21, 26.75, 22.93, 21.33. IR (KBr):
3390.86, 3269.34, 3113, 3057, 3014, 2954, 2933, 2877, 1640,
mL deuterated dimethyl sulfoxide (DMSO-d6) was measured on a
Bruker 800 MHz spectrometer. The probe temperature was
regulated to 298 K. Using a simple pulse-acquire sequence zg30,
1
the spectra were recorded. To ensure full relaxation of the H
resonances, typical acquisition parameters consisted of 64 K
points covering a sweep width of 16447 Hz, a pulse width (pw90)
of 8.63 ms and a total repetition time of 24 s were used. Before
Fourier transformation, the digital zero lling to 64 K and a
0.3 Hz exponential function were applied to FID. The resonance
at 2.5 ppm indicated an impurity (CD2HSOCD2H) in the residual
solvents, and tetramethylsilane (TMS) was used as internal
reference. Standard absorptive two-dimensional 1H–1H chemical
shi correlation spectra (COSY) were tested with the same spec-
trometer. Each spectrum consisted of a matrix of 2 K (F2) by 0.5 K
(F1) covering a sweep width of 9615.4 Hz. Before Fourier trans-
formation, the matrix was zero lled to 1 K by 1 K, and the
standard sinebell apodization functions were applied in both
dimensions. Two-dimensional ROESY experiments were carried
out in the phase-sensitive mode using the same spectrometer.
Each spectrum consisted of a matrix of 2 K (F2) by 1 K (F1) and
covered a sweep width of 9615.4 Hz. Spectra were obtained using
spin-lock mixing periods of 200 ms. Before Fourier trans-
formation, the matrix was zero lled to 1 K by 1 K, and qsine
apodization functions were applied in both dimensions.
1465, 1303, 1130, 1103, 1008, 762, 702 cmꢀ1
Preparing (5aS,12S,14aS)-5,14-dioxo-12-(2-tryptophanyl-
.
threoninebenzylester-N-ylethyl-1-yl)-1,2,3,5,-5a,6,11,12,14,14a-
decahydro-5H,14H-pyrolo[1,2:4,5]pyrazino[1,2:1,6]pyrido-[3,4-b]-
indoles (CIPPCT). To a solution of 120 mg (0.23 mmol) CIPPC
and 77 mg (0.23 mmol) Thr-OBzl in 15 mL anhydrous DMF
100 mg (0.46 mmol), DCC and 30 mg anhydrous Na2SO4 were
added. The reaction mixture was adjusted to pH 9 with
N-methylmorpholine, and stirred at room temperature for 1 h.
The reaction mixture was evaporated under vacuum, the residue
was dissolved in 20 mL ethyl acetate, the solution was washed
with 10% aqueous Na2CO3 (15 mL ꢂ 3) and dried with anhy-
drous Na2SO4. Aer ltration the ltrate was evaporated under
vacuum, and the residue was puried on a silica gel column to
provide 129 mg (78%) CIPPCT as yellow powder. Mp 127–
20
ꢁ
128 C. [a]D ¼ ꢀ191 (c ¼ 0.6, CH2Cl2). FT-MS(m/z): 717.33161
[M + H]+. 1H NMR (300 MHz, CDCl3): d(ppm) ¼ 8.27 (s, 1H), 8.02
(t, J ¼ 5.4 Hz, 1H), 7.974 (d, J ¼ 7.8 Hz, 1H), 7.464 (m, 2H), 7.364
(m, 5H), 7.319 (d, J ¼ 2.1 Hz, 1H), 7.173 (m, 2H), 7.120 (m, 1H),
6.128 (s, 1H), 5.186 (s, 2H), 4.264 (d, J ¼ 6.6 Hz, 1H), 4.240 (m,
1H), 4.203 (d, J ¼ 6.6 Hz, 1H), 4.040 (d, J ¼ 4.8 Hz, 1H), 3.964 (m,
1H), 3.879 (dd, J ¼ 5.1 Hz, J ¼ 7.2 Hz, 1H), 3.768 (m, 1H), 3.630
Measuring FT-MS spectra
(m, 2H), 3.490 (m, 1H), 3.350 (m, J ¼ 5.1 Hz, 1H), 2.900 (m, 2H), ESI mass spectra of CIPPCT were measured on a solariX FT-ICR
2.511 (m, 2H), 2.467 (m, 2H), 2.241 (m, 1H), 2.005 (m, 3H), 1.241 mass spectrometer (Bruker Daltonics, Billerica, MA, USA) con-
(dd, J ¼ 4.2 Hz, J ¼ 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3): sisting of an ESI/MALDI dual ion source and a 9.4 T supercon-
d(ppm) ¼ 171.1, 170.7, 165.8, 162.6, 136.5, 135.6, 135.2, 133.8, ductive magnet. The measurements were performed with the
128.6, 128.5, 128.4, 127.6, 127.0, 125.8, 124.9, 122.5, 121.8, positive MALDI ion mode. The ion source was a Smart-beam-II
119.9, 119.8, 119.4, 117.9, 112.0, 111.4, 105.7, 68.2, 67.3, 65.3, laser (wavelength, 355 nm; focus setting, ‘medium’; repetition
60.7, 60.4, 59.3, 57.4, 48.0, 46.1, 45.2, 42.8, 36.5, 35.1, 31.4, 29.7, rate, 1000 Hz). The QCID mass was set to 2150.98172 m/z, and
29.6, 28.6, 23.3, 21.0, 20.8, 20.0, 14.2, 8.6. IR (KBr): 3387, 3325, the isolation window was 5 m/z. Data were collected using
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solariXcontrol soware. Spectral data were processed with Data 2,5-diphenyltetrazolium bromide] staining according to stan-
Analysis soware (Bruker Daltonics).
dard procedures. HL-60, HT-29, HeLa, SW480 and HepG2 cells
(5 ꢂ 103 cells per well) were grown in RPMI-1640 or DMEM
medium [containing 10% (v/v) fetal calf serum, 60 mg mLꢀ1
penicillin, and 100 mg mLꢀ1 streptomycin]. Stock solutions of
CIPPCT were prepared in DMSO and diluted with culture
medium to ꢁthe desired concentration. Cultures were propa-
gated at 37 C in a humidied atmosphere (with 5% CO2) for
24 h, and then CIPPCT was added (nal concentrations were 0,
1, 5 10, 25, 50 and 100 mM). Aer 48 h of treatment, MTT
solution was added (5 mg mLꢀ1; 25 mL per well), and cells were
incubated for an additional 4 h. Aer adding 100 mL of DMSO to
dissolve the MTT-formazan product, the optical density was
measured at 570 nm by a microplate reader (n ¼ 6).
Measuring TEM images
Shape and size measurements of CIPPCT nanospecies were
performed using transmission electron microscopy (TEM; JSM-
6360 LV, JEOL, Tokyo, Japan). An aqueous solution of CIPPCT
(pH 6.7, 10ꢀ10 M) was dripped onto a formvar-coated copper
grid, to which a drop of anhydrous ethanol was added to
promote the removal of water. The grid was allowed to dry
ꢁ
thoroughly in air and then was heated at 35 C for 24 h. The
copper grids were viewed under TEM. The shape and size
distributions of the nanospecies were determined by counting
>100 species in randomly selected regions on the copper grid.
All determinations were carried out on triplicate grids and at
80 kV (the electron beam accelerating voltage). Images were
recorded on an imaging plate (Gatan Bioscan Camera Model
1792, Pleasanton, CA, USA) with 20 eV energy windows at 6000–
400 000ꢂ and were digitally enlarged.
In vivo anti-tumor assay
Following the protocol review and approval by the ethics
committee of Capital Medical University, the investigations
were performed. The committee assures that the welfare of mice
was maintained in accordance with the requirements of the
animal welfare act. Male ICR mice (22 ꢃ 2 g, purchased from
Capital Medical University) were maintained at 21 ꢁC with a
natural day/night cycle in a conventional animal colony. Mice
were 10 weeks old at the beginning of the experiment. S180
ascites tumor cells were subcutaneously injected to form solid
tumors. To initiate subcutaneous tumors, cells obtained in
ascitic form from tumor-bearing mice were serially transplanted
once per week. Subcutaneous tumors were implanted under the
skin at the right armpit by injecting 0.2 mL of NS (normal
saline) containing 1 ꢂ 107 viable tumor cells. Twenty-four hours
aer implantation, mice were randomly divided into treatment,
positive control and vehicle control groups (7 per group). For
treatment groups CIPPCT in 0.2 mL of 0.3% carmellose sodium
(CMC-Na) was orally administered at 4.0, 0.8 and 0.16 mmol
kgꢀ1 per dose. For the positive control group, doxorubicin in NS
was intraperitoneally injected at 2 mmol kgꢀ1 per dose. For
vehicle control, 0.2 mL of CMC-Na (0.3%, negative control) was
orally administered. Oral administrations for treated groups or
for vehicle control or intraperitoneal injections for positive
control were administered every day for 7 days, and mice were
weighed daily. Twenty-four hours aer the last administration,
the mice were weighed and blood sampled from eyes; they were
then sacriced by ether anesthesia and dissected to immedi-
ately obtain and weigh the tumor and organ samples.
Measuring SEM images
The shape and size of lyophilized powders of CIPPCT (from a
solution of CIPPCT in ultrapure water) were measured by
scanning electron microscopy (SEM, JEM-1230, JEOL, Tokyo,
Japan) at 50 kV. The lyophilized powders were attached to a
copper plate with double-sided tape (Euromedex, Strasbourg,
France). The specimens were coated with 20 nm gold-palladium
using a Joel JFC-1600 Auto Fine Coater. The coater was operated
at 15 kV, 30 mA, and 200 mTorr (argon) for 60 s. The shape and
size distributions of the lyophilized powders of CIPPCT were
measured by examining >100 particles in randomly selected
regions on the SEM alloy. All measurements were performed on
triplicate copper plates. Images were recorded on an imaging
plate (Gatan Bioscan Camera Model1792) with 20 eV energy
windows at 100–10 000ꢂ and were digitally enlarged.
Measuring AFM images
Atomic force microscopy (AFM) images of contact mode were
recorded on a Nanoscope 3D AFM (Veeco Metrology, Santa Bar-
bara, CA, USA) under ambient conditions. Samples of CIPPCT in
water (pH 6.7, 10ꢀ10 M) were used for recording the images.
Theoretically predicting nanoparticle size
The mesoscale simulation soware was used to perform the
calculation and to predict how many trimers of CIPPCT can
construct the smallest nanoparticle revealed by TEM. Discover
module of the Materials Studio soware was used for the simu-
lation. CIPPCT molecule was built and optimized simply in the
Visualizer window. “Beads” were constructed from atomistic
simulations and placed at the center-of-mass of groups of atoms
corresponding to particular parts of the CIPPCT molecule.
uPA ELISA assay
uPA level in the serum of S180 mice treated with CIPPCT at 4, 0.8
and 0.15 mmol kgꢀ1 per dose was measured by enzyme immu-
noassay according to the manufacturer's instructions (mouse
uPA ELISA kit, Uscn Life Science Inc., Wuhan). In brief, 24 h aer
the last administration, mouse blood was collected in 3.8%
aqueous solution of sodium citrate from the eyes, kept at room
temperature for 30 min, centrifuged at 3000 rpm for 20 min; the
separated serum was kept at ꢀ20 ꢁC before use. In the ELISA
In vitro anti-proliferation assay
In vitro cell viability assays were carried out using 96-well assay, murine monoclonal antibody specic for uPA was coated
microtiter culture plates and MTT [3-(4,5-dimethylthiazol-2-yl)- on the 96-well microplate. Each standard and serum sample was
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added to the plate and incubated. Aer washing away unbound ions. The ion 717.33161 (bearing one positive charge) repre-
proteins, enzyme-linked antibody specic for uPA was added to sents the mass of a CIPPCT monomer (theoretical value:
the wells. Absorbency was measured at 492 nm, and the detection 716.3322) plus H. The ion 1433.66550 (bearing one positive
limit was 100 pg mLꢀ1. All samples were run in duplicate. The charge) represents the mass of a CIPPCT dimer (theoretical
variations of intra- and inter-assays were less than 10%.
value: 1432.6644) plus H. The ion 2149.96613 (bearing one
positive charge) represents the mass of a CIPPCT trimer (theo-
retical value: 2148.9966) plus H. Furthermore, the monomer ion
was characterized by the highest peak among the 3 ions. To
clarify the correlation of the momomer, the dimer and the
trimer, a qCID spectrum was measured and inserted into
Fig. 2a. The qCID spectrum demonstrated that the dimer and
the monomer were derived from the fragmentation of the
trimer under FT-MS conditions. Therefore, the spectra support
the interpretation that the intermolecular association leads to
trimerization and in aqueous CIPPCT exists as the trimer.
Results and discussion
Energy-minimization and -like conformation
A
-like conformation of CIPPCT was generated in computer-
assisted molecular modeling. The systematic search method
produced 30 conformations, and the BEST method produced
203 conformations. The 233 generated conformations were
visually examined, and the conformation having the lowest free
energy was selected. This conformation has a ꢀ35.06 kcal molꢀ1
relative energy to the starting conformation. Regarding the
steric relationship between pyrolo[1,2:4,5]pyrazino[1,2:1,6]pyr-
ido[3,4-b]indole, tryptophan and benzyl moieties, CIPPCT had a
-like conformation, in which the distance from pyrrole-H of
pyrolo[1,2:4,5]pyrazino-[1,2:1,6]pyrido[3,4-b]indole moiety to
ROESY 2D NMR spectrum and trimerization manner
The pattern of the intermolecular association was identied
with the ROESY 2D NMR spectrum. In Fig. 2b, the ROESY-
related three cross-peaks are labeled with blue circles. Of the
three cross-peaks, the cross-peak a was assigned for the
interaction of the pyrrole-H in pyrolo[1,2:4,5]pyrazino-
[1,2:1,6]pyrido[3,4-b]indole moiety with the a-H of tryptophan
moiety and reected the steric relationship of two intra-
molecular H, according to the low energy conformation of
Fig. 1. The cross-peak b was assigned for the interaction of the
H of CH2 in benzyl moiety with the 13-H in pyrolo[1,2:4,5]-
pyrazino[1,2:1,6]pyrido[3,4-b]indole moiety; according to the
low energy conformation of Fig. 1, this interaction can only
result from two intermolecular H. The cross-peak c was
assigned for the interaction of the 19-H in pyrolo[1,2:4,5]-
pyrazino[1,2:1,6]pyrido-[3,4-b]indole moiety with the a-H of
threonine moiety; according to the low energy conformation
dened in Fig. 1, this interaction can also only result from two
intermolecular H. To t the requirements of cross-peaks b
and c, three CIPPCT molecules could associate in a manner
shown in Fig. 2c and form a nger ring-like species. The data
of NMR and MS together supported the interpretation that
CIPPCT existed as nger ring-like trimer.
˚
the a-H of tryptophan moiety was 2.298 A, which was reected
by cross-peak a in the ROESY 2D NMR spectrum (see Fig. 2b).
FT-MS spectroscopy and trimer
The intermolecular association of CIPPCT was explored with FT-
MS spectrum (Fig. 2a). This spectrum showed three interesting
Fig. 1
A
-like conformation, the energy-minimized conformation.
Fig. 2 (a) Ion peak in the FT-MS spectrum supporting trimerization of CIPPCT (bearing one positive charge, 2149.96613). The inset shows a qCID
spectrum, indicating that monomers and dimers result from fragmentation of the trimer under FT-MS conditions. (b) ROESY 2D NMR spectrum:
three cross-peaks indicating the intermolecular associations of CIPPCT (labeled with blue circles). (c) Finger ring-like trimer of CIPPCT, deduced
from the ROESY 2D NMR and FT-MS spectra, in which the distance of the hydrogens in the cross-peaks b and c are indicated.
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Fig. 3 TEM, SEM and AFM images, as well as mesoscale simulation software, assisted calculation of the nanoparticles of CIPPCT. (a) TEM images
of CIPPCT in pH 6.7 and 10ꢀ10 M aqueous solution. (b) SEM images of powders of CIPPCT precipitated from pH 6.7 and 10ꢀ10 M aqueous solution.
(c and d) AFM images of CIPPCT precipitated from pH 6.7 and 10ꢀ10 M aqueous solution. (e) Mesoscale simulation software-assisted calculation
of the structure of CIPPCT nanoparticles. CIPPCTs were divided into three beads. The green, yellow and pink beads represented pyrolo[1,2:4,5]-
pyrazino[1,2:1,6]pyrido[3,4-b]indole, tryptophan and threonine moieties, respectively.
approach to quantitatively estimate the number of the trimers
for an optimal nanoparticle.
TEM, SEM, AFM and nano-particles
The nano-structure was shown using TEM, SEM and AFM
images. The TEM image of Fig. 3a revealed that in an aqueous
solution of pH 6.7, and 10ꢀ10 M concentration CIPPCT formed
nanoparticles of 9–194 nm in diameter, and the diameter of
most particles was less than 125 nm. SEM images of Fig. 3b
revealed that the precipitates from the aqueous solution of pH
6.7 and 10ꢀ10 M concentration were the nanoparticles of 27–67
nm in diameter, and the diameter of most particles was less
than 47 nm. AFM images of Fig. 3c and d revealed that the
IC50 for inhibiting cancer cell proliferation
The cytotoxicity of CIPPCT to HL60, HT29, HeLa, SW480 and
HepG2 cells was examined using the MTT method, repre-
sented with IC50 values, and is shown in Fig. 4a. In the MTT
assays, the IC50 values in inhibiting HeLa and SW480 cells
were more than 100 mM, the IC50 value in inhibiting HepG2
cells was more than 60 mM, the IC50 value in inhibiting HT29
cells was more than 20 mM, and the IC50 value in inhibiting
HL60 cells was more than 10 mM. The relative higher IC50
values suggest CIPPCT had low cytotoxicity and should not be
a cytotoxic agent.
precipitates from an aqueous solution of pH 6.7 and 10ꢀ10
M
concentration were nanoparticles of 74–79 nm in diameter.
Therefore all images of TEM, SEM and AFM consistently visu-
alized that in 10ꢀ10 M aqueous solution CIPPCT existed as
nano-particles, while the spectra of FT-MS and ROESY 2D NMR
implied that the nano-particles were assembled by the trimers
of CIPPCT.
Dose-dependent inhibition for tumor growth
The in vivo anti-tumor activity of CIPPCT was assayed on an
S180 mouse model, represented by tumor weight, and are
Theoretical prediction and trimer number in nanoparticle
Mesoscale simulation predicted that the trimers of CIPPCT shown in Fig. 4b. The assays led CIPPCT to exhibit an orally
spontaneously formed nanoparticles and a nanoparticle of 9.4 dose-dependent inhibition and to having an low effective dose
nm in diameter contained 350 trimers (Fig. 3e). As seen in at 0.16 mmol kgꢀ1. With respect to 4 mmol kgꢀ1 oral dose,
Fig. 3a, the smallest nanoparticle had a diameter of 9.3 nm. The CIPPCT had the same efficacy as the 2 mmol kgꢀ1 of intra-
consistency of the theoretically predicted nanoparticle with the peritoneally injected doxorubicin, suggesting that an oral
experimentally tested nanoparticle emphasizes the utility of the administration of CIPPCT can offer desirable therapeutic
theoretical prediction. In addition, this prediction provided an effectiveness.
1640 | Med. Chem. Commun., 2014, 5, 1634–1643
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Fig. 4 CIPPCT possessed bioactivity on molecular, cell and animal models. (a) IC50 of CIPPCT in inhibiting cancer cell proliferation, n ¼ 6; (b) In
vivo CIPPCT dose-dependently inhibiting tumor growth, n ¼ 10; (c) In vivo CIPPCT dose-dependently decreasing serum uPA level, n ¼ 10; (d)
AFM image of mouse plasma alone; (e) AFM image of mouse plasma with CIPPCT; (f) fluorescence images demonstrating the time-dependent
adherence of CIPPCT nanoparticles onto the surface of HeLa cells.
were recorded. The images in Fig. 4e show that in mouse plasma,
the nanoparticles of CIPPCT are 99 nm in diameter, whereas the
mouse plasma gives no comparable nanoparticle (Fig. 4d).
uPA levels in serum of mice treated with CIPPCT
The anti-tumor mechanism of CIPPCT was monitored with
ELISA experiments; the uPA levels in the serum treated with 4.0,
0.8 and 0.16 mmol kgꢀ1 of oral CIPPCT are shown in Fig. 4c. In a
variety of malignant tumours, the overexpression of uPA has
been strongly correlated with poor prognosis. Experimental
evidence proved that the downregulation of uPA could be
considered a potential strategy in cancer therapy.11,12 CIPPCT
dose-dependently decreased uPA levels in the serum of mice,
reecting CIPPCT dose-dependently downregulated uPA
expression and suggested that by targeting uPA, CIPPCT
inhibited tumor growth.
Time-dependent adherence of CIPPCT nanoparticles onto the
surface of HeLa cells
With a 282 nm excitation wave, CIPPCT can emit blue uores-
cence at 418 nm, and this spectrum property was used to monitor
the nanoparticles. In this case, CIPPCT nanoparticles on the cell
surface were characterized by uorescence images (Fig. 4f). When
tumorigenic cells, HepG2, were treated with CIPPCT (10ꢀ10 M),
the blue uorescent nanoparticles of 100–135 nm in diameter
steadily increased as the treatment time lengthened from 15 min
to 240 min and especially as it went beyond 60 min.
AFM of CIPPCT nanoparticles in mouse plasma
To visualize the morphological features of CIPPCT in the blood,
an AFM test was performed on a Nanoscope 3D AFM (Veeco).
Conclusion
Using the contact mode, the images of mouse plasma alone In conclusion, with energy-minimization simulation, FT-MS
1
(negative control) and CIPPCT in mouse plasma (10ꢀ10 M, pH7.4) spectra, H NMR Noesy spectra, and images of TEM, SEM and
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AFM, the chemical and physical mechanism of CIPPCT-forming
nanoparticles could be claried. ELISA, anti-proliferation and
tumor growth assays provided evidence that downregulating
uPA was the pharmacological mechanism by which CIPPCT
inhibited tumor growth, beneted the increase of anti-tumor
activity and decreased cytotoxicity.
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Contribution of each author
The contribution of each author made to the manuscript
mechanism of forming trimer, self-assembling nanoparticle
and inhibiting tumor growth of small molecule CIPPCT is as
follows: chemical synthesis, in vivo anti-tumor assay and uPA
ELISA assay. Dr Fengxiang Du; mesoscale simulation and
gures: Dr Xiaoyi Zhang; in vitro anti-proliferation assay: Dr
Shan Li; MS, NMR and AFM data testing: Yaonan Wang,
Meiqing Zheng and Shurui Zhao; MS/NMR data collection and
interpretation: Dr Yuji Wang; literature search: Dr Jianhui Wu;
TEM and SEM measuring: Dr Lin Gui; experimental design: Dr
Ming Zhao; research guidance and paper writing: Dr Shiqi
Peng.
´
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H. Mendil-Jakani, S. Lyonnard and J. Jestin, Proton
conducting ionic liquid doped naon membranes: Nano-
structuration, transport properties and water sorption, J.
Phys. Chem. C, 2012, 116, 24413–24423.
11 R. H. Chou, S. C. Hsieh, Y. L. Yu, M. H. Huang, Y. C. Huang
and Y. H. Hsieh, Fisetin inhibits migration and invasion of
human cervical cancer cells by down-regulating urokinase
plasminogen activator expression through suppressing the
p38 MAPK-dependent NF-kB signaling pathway, PLoS One,
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12 A. H. Mekkawy, D. L. Morris and M. H. Pourgholami,
Urokinase plasminogen activator system as a potential
target for cancer therapy, Future Oncol., 2009, 5, 1487–
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Conflict of interest
The authors declare no competing nancial interest.
Acknowledgements
This work was supported by the Beijing area major laboratory of
peptide and small molecular drugs, the Project of Construction of
Innovative Teams and Teacher Career Development for Univer-
sities and Colleges Under Beijing Municipality, the Natural
Science Foundation (81373265 and 81270046), Beijing Natural
Science Foundation (7112016 and 7132032) and Beijing Munic-
ipal Science & Technology Commission (Z141100002114049).
13 B. Mishra, B. B. Patel and S. Tiwari, Colloidal nanocarriers: A
review on formulation technology, types and applications
toward targeted drug delivery, Nanomedicine, 2010, 6, 9–24.
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