Novel β-carboline-based indole-4,7-quinone derivatives as NAD(P)H: Quinone-oxidoreductase-1 inhibitor with potent antitumor activities by inducing reactive oxygen species, apoptosis, and DNA damage

Article abstract of DOI:10.1111/cbdd.13752

Eighteen new β-carboline-based indole-4,7-quinone derivatives (12a–i and 13a–i) were designed and synthesized, and their in vitro and in vivo antiproliferative activities were studied. Most of target compounds showed strong inhibition on three human tumor cells' proliferation. In particular, the most active compound 13g not only displayed more prominent antiproliferative activities than β-lapachone, a clinical antitumor candidate, but also exerted significant NAD(P)H: quinone-oxidoreductase-1 (NQO1) inhibitory activity and NQO1-dependent cytotoxicity in HT29 cells. Furthermore, 13g dose-dependently induced high ROS levels in HT29 cells, and selectively inhibited cancer cell but not non-tumor colon cell proliferation in vitro. Importantly, 13g promoted HT29 cell apoptosis and DNA damage by regulating relative apoptotic proteins and H2AX expression. Finally, 13g displayed significant growth inhibition of HT29 human colorectal adenocarcinoma xenograft in mice without overt toxicity.

Full text of DOI:10.1111/cbdd.13752

PROFESSOR BOHUA CHEN (Orcid ID : 0000-0002-1286-2586)
Article type
: Research Article
Novel β-carboline-based indole-4,7-quinone derivatives as NQO1 inhibitor with potent
antitumor activities by inducing ROS, apoptosis, and DNA damage
Yibing Guo,#a,b Liancheng Xu,#a,b Changchun Ling,a Tao Yang,a,b Wenjie Zheng,a Jin Lva, Qingsong Guoa* and
Bohua Chen a,b*
aResearch Center of Clinical Medical, Affiliated Hospital of Nantong University, Nantong 226001, People’s
Republic of China
bDepartment of Pharmacy, Affiliated Hospital of Nantong University, Nantong 226001, People’s Republic of
China.
Correspondence: chenbohua1966@126.com (Bohua Chen), ntgglj@163.com (Qingsong Guo).
#These authors contributed equally to this work.
Abstract
Eighteen new β-carboline-based indole-4,7-quinone derivatives (12a−i and 13a-i) were designed and
synthesized, and their in vitro and in vivo antiproliferative activities were studied. Most of target compounds
showed strong inhibition on three human tumor cells’ proliferation. In particular, the most active compound 13g
not only displayed more prominent antiproliferative activities than β-lapachone, a clinical antitumor candidate,
but also exerted significant NAD(P)H: quinone-oxidoreductase-1 (NQO1) inhibitory activity and
NQO1-dependent cytotoxicity in HT29 cells. Furthermore, 13g dose-dependently induced high ROS levels in
HT29 cells, and selectively inhibited cancer cell but not non-tumor colon cell proliferation in vitro. Importantly,
13g promoted HT29 cell apoptosis and DNA damage by regulating relative apoptotic proteins and H2AX
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10.1111/CBDD.13752
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expression. Finally, 13g displayed significant growth inhibition of HT29 human colorectal adenocarcinoma
xenograft in mice without overt toxicity.
KEYWORDS: Antitumor activity, β-Carboline, Indole-4,7-quinone derivatives, Quinone oxidoreductase 1
(NQO1), Apoptosis, Reactive oxygen species (ROS)
1. INTRODUCTION
Due to aberrant metabolic processes and abnormal proliferative growth phenotypes, malignant cells often
expose to a relatively high level of reactive oxygen species (ROS) compared with normal cells. Such
biochemical differences make cancer cells more susceptible to induced oxidative stress (Dharmaraja, 2017;
Gorrini & Li, 2013). However, the high ROS levels and excessive oxidative stress irreversibly cause lipids and
DNA damage, and ultimately induce apoptosis of cancer cells (Wang et al., 2017). Therefore, selectively
targeting cancer cells based on different redox states between tumor and normal cells is a feasible strategy
(Wang, 2017; Moloney, 2018), especially when it comes to targeting a redox adaptive mechanism that confers to
multiple solid tumors (Wang et al., 2017).
NAD(P)H quinone oxidoreductase 1 (NQO1), a 2-electron reductase responsible for the reduction of
quinones to hydroquinones (Oh et al., 2015), is often overexpressed in multiple solid tumors, including colon
cancer, nonsmall cell lung carcinoma, pancreatic cancer, and breast cancer, among others, where it is
constitutively overexpressed at levels of 50- to 100-fold greater than that in correlative normal tissues (Xu &
Lewis, 2017; Ma, 2015; Yang & Lin, 2014; Yang, 2019). Furthermore, the high NQO1 level usually associates
with metastasis, later clinical tumor stage, lower disease-free and overall survival (Yang, 2019; Ji & Butsri,
2017; Wakai, 2011). Therefore, NQO1 target is considered as an important redox-modulation strategy for tumor
treatment.
The indolequinone structure, one of an important and active quinone, shows a good interaction with NQO1
target and can be activated through a non-reversible and obligate double electron reduction (Xu, 2017; Jiho,
2019; Reigan, 2007). Some indolequinones and their derivatives are cytotoxic to cancer cell lines which
overexpress NQO1, and induce high-level ROS (Xu, 2017; Jiho, 2019; Sasaki, 2014; Reigan, 2007). Moreover,
according to structure-activity relationship (SAR) analysis of quinone derivatives, the amino substituent on
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quinone ring moiety favorably impacts binding to the active site of NQO1 and ROS induction in various cancer
cells (Jiho, 2019; Sasaki, 2014; Parkinson, 2013; Ling, 2018; Valderrama, 2009). Many quinone analogs bearing
alkyl amino substitutions have been reported to have improved antitumor activities against different tumor cell
lines, however, the antitumor activities of some are modest (Nishiyama & Salvador, 2017; Kumar, 2019;
Parkinson, 2015).
Natural products play a significant and irreplaceable role in the discovery of novel antitumor drugs. Hybrid,
designed to combine with part of natural products, offers a promising potential lead with an advantage to acquire
high diversity, inherent biological activity, and improvement of therapeutic efficacy (Bérubé, 2016; Ling, 2019;
Li, 2015). It is known that β-carbolines, including harmine, harmol and so on, are a kind of naturally obtained
indole alkaloids with a planar tricyclic pyrido [3,4-b] indole structure (Cao et al., 2007). Many β-carboline
analogues have been implicated in a range of diverse biological activities, including antitumor, antiviral,
antimicrobial, and antiparasitic activities, and have the most attracted attention in the treatment of cancer due to
their intercalation into DNA, inhibition of DYRK1A, CDK, topoisomerase, and monoamine oxidase, and
antiangiogenesis effect (Carvalho, 2017; Cao, 2007; Kamal, 2015; Miao & Ling, 2018). Some β-carboline
derivatives exerted antiproliferative activities by inducing ROS or/and apoptosis (Zhang & Sarkar, 2016)
In view of the above description, we used molecular hybridization strategy through incorporation of the
natural β-carboline fragment into indolequinone, and formed novel hybrids with enhanced anticancer
efficacy/potency to a broader range of cancer, even solid tumors. Therefore, we coupled different substituted
β-carboline fragments to the C5- or C6-position of indole-4,7-quinones through different alkyl diamine linkers to
design novel β-carboline-based indole-4,7-quinone derivatives, and compared the antiproliferative effects of
these hybrids in multiple cancer cells. In the present study, we report the synthesis of novel β-carboline-based
indole-4,7-quinone derivatives (12a‒i and 13a‒i, Scheme 1) as well as their evaluation of anti-tumor activities in
vitro and in vivo.
2 MATERIALS AND METHODS
2.1 Materials
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All chemical reagents were from Aladdin or Sigma-Aldrich and used without further purifications. 1H and 13
C
NMR spectra were obtained on a Bruker AV 400M spectrometer. HRMS was obtained on an Agilent
Technologies LC/MSD TOF. MS was obtained on a Mariner Mass Spectrum (ESI). The preparative
chromatogram was filled with silica gel (300–400 mesh, Aladdin) and detected by UV lamp at 254/365 nm.
L-tryptophan 1, different aldehydes 2a-c, and compound 7 were commercially available. Compounds 3-5, 8, and
9 were synthesized according to literature procedures (Jackson, 1995; Kamal, 2014). All target hybrids were of
purity (>95%) detected by HPLC.
2.2 Synthesis of target hybrids
2.2.1 General procedure for 6a-i
N-(2-aminoethyl)-1-methyl-9H-pyrido[3,4-b]indole-3-amide (6a)
To a solution of 5a (2.88g, 12 mmol) in 30 mL MeOH was added ethane-1,2-diamine 8 mL. The reaction
mixture was refluxed for 12 hours. Upon completion of the reaction, the solvent was evaporated. CH2Cl2 was
added and the mixture was washed by water for three times. The organic phase was dried by Na2SO4 and
evaporated under reduced pressure. The residue was isolated by column chromatography (CH2Cl2-CH3OH = 6:1,
v/v) to produce 6a a light yellow solid 2.6 g, yield: 82%. ESI-MS m/z = 269 [M+H]+.
N-(4-aminobutyl)-1-methyl-9H-β-carboline-3-amide (6b)
Compound 6b was synthesized in line with the preparation of 6a, except using butane-1,4-diamine instead of
ethane-1,2-diamine. ESI-MS m/z = 297 [M+H]+.
N-(6-aminohexyl)-1-methyl-9H-β-carboline -3-amide (6c)
Compound 6c was synthesized in line with the preparation of 6a, except using hexane-1,6-diamine instead of
ethane-1,2-diamine. ESI-MS m/z = 325 [M+H]+.
N-(2-aminoethyl)-1-(p-tolyl)-9H-β-carboline -3-amide (6d)
Compound 6d was synthesized in line with the preparation of 6a, except using 5b instead of 5a. ESI-MS m/z
= 345 [M+H]+.
N-(4-aminobutyl)-1-(p-tolyl)-9H-β-carboline -3-amide (6e)
Compound 6e was synthesized in line with the preparation of 6d, except using butane-1,4-diamine instead of
ethane-1,2-diamine. ESI-MS m/z = 373 [M+H]+.
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N-(6-aminohexyl)-1-(p-tolyl)-9H-β-carboline -3-amide (6f)
Compound 6f was synthesized in line with the preparation of 6d, except using hexane-1,6-diamine instead of
ethane-1,2-diamine. ESI-MS m/z = 401 [M+H]+.
N-(2-aminoethyl)-1-(4-methoxyphenyl)-9H-β-carboline -3-amide (6g)
Compound 6g was synthesized in line with the preparation of 6a, except using 5c instead of 5a. ESI-MS m/z =
361 [M+H]+.
N-(4-aminobutyl)-1-(4-methoxyphenyl)-9H-β-carboline -3-amide (6h)
Compound 6h was synthesized in line with the preparation of 6g, except using butane-1,4-diamine instead of
ethane-1,2-diamine. ESI-MS m/z = 389 [M+H]+.
N-(6-aminohexyl)-1-(4-methoxyphenyl)-9H-β-carboline -3-amide (6i)
Compound 6i was synthesized in line with the preparation of 6g, except using hexane-1,6-diamine instead of
ethane-1,2-diamine. ESI-MS m/z = 417 [M+H]+.
2.2.2 General synthetic procedure of 12a and 13a
To a solution of 1-tosyl-1H-indole-4,7-dione 9 (3.0 g, 10 mmol) in 50 mL methanol was added 6a (2.7 g, 10
mmol). The reaction solution was stirred at rt for 1h. After the reaction, NaOMe (1.1g, 20 mmol) was added into
the mixture solution, which was continuously stirred for 1h in ice bath. The residue was poured into 200 mL
water, and extracted by EtOAc (3 × 80 mL). The organic phase was combined and washed by brine, and
evaporated with vacuum and isolated by column chromatography to afford compounds 12a and 13a in yield of
33% and 24%, respectively.
N-(2-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)ethyl)-1-methyl-9H-β-carboline -3-amide (12a)
1
Analytical data for 12a: ESI-MS m/z = 414 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.86 (s, 1H, NH),
11.21 (s, 1H, NH), 8.72 (s, 1H, ArH), 8.27 (d, J = 7.8 Hz, 1H, ArH), 8.22 (m, 1H, NH), 7.88 (m, 1H, ArH), 7.71
(d, J = 7.8 Hz, 1H, ArH), 7.57 (s, 1H, NH), 7.54 – 7.48 (m, 1H, ArH), 7.21 (m, 1H, ArH), 5.91 (s, 1H, CH=),
3.57 (m, 2H, NCH2), 3.35 (t, J = 6.0 Hz, 2H, NCH2), 2.77 (s, 3H, CH3); 13C NMR (100MHz, DMSO-d6) δ
182.2, 179.8, 158.5, 153.1, 150.1, 145.6, 141.6, 135.6, 131.4, 130.0, 125.3, 123.1, 121.6, 120.8, 119.6, 119.0,
113.4, 112.6, 111.2, 106.7, 47.6, 43.0, 20.6. HRMS(ESI) calcd for C23H19N5O3 [M+H]+ 414.1521, found:
414.1532.
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N-(2-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)ethyl)-1-methyl-9H-β-carboline-3-amide (13a)
1
Analytical data for 13a: ESI-MS m/z = 414 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.94 (s, 1H, NH),
11.26 (s, 1H, NH), 8.70 (s, 1H, ArH), 8.34 (s, 1H, NH), 7.83 (s, 1H, ArH), 7.71 (d, J = 7.8 Hz, 1H, ArH), 7.64 (d,
J = 8.2 Hz, 1H, ArH), 7.59 (s, 1H, NH), 7.30 (m, 1H, ArH), 6.04 (m, 1H, ArH), 5.76 (s, 1H, CH=), 3.67 – 3.61
(m, 2H, NCH2), 3.41 (t, J = 6.0 Hz, 2H, NCH2), 2.85 (s, 3H, CH3); 13C NMR (100MHz, DMSO-d6) δ 182.3,
180.2, 158.5, 153.1, 150.2, 145.7, 141.6, 135.6, 131.4, 130.1, 125.3, 123.1, 121.6, 120.8, 119.7, 119.0, 113.4,
111.2, 108.5, 106.7, 47.8, 43.1, 20.6. HRMS(ESI) calcd for C23H19N5O3 [M+H]+ 414.1521, found: 414.1528.
2.2.3 General synthetic procedure of 12b and 13b
Compounds 12b and 13b were synthesized from 9 and 6b, based on the synthetic process of 12a and 13a in
yield of 31% and 22%, respectively.
N-(4-((4,7-dioxo-4,7-dihydro-1H-indol-5-yl)amino)butyl)-1-methyl-9H-β-carboline-3-amide (12b)
1
Analytical data for 12b: ESI-MS m/z = 442 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.91 (s, 1H, NH),
11.25 (s, 1H, NH), 8.64 (s, 1H, ArH), 8.24-8.32 (m, 2H, NH, ArH), 7.74 (d, J = 7.8 Hz, 1H, ArH), 7.63 (d, J =
8.2 Hz, 1H, ArH), 7.57-7.63 (m, 2H, NH, ArH), 7.28-7.37 (m, 2H, ArH), 5.77 (s, 1H, CH=), 3.40 (m, 2H,
NCH2), 3.26 (t, J = 6.0 Hz, 2H, NCH2), 2.82 (s, 3H, CH3), 1.65 (m, 4H, CH2-CH2); 13C NMR (100MHz,
DMSO-d6) δ 182.3, 180.2, 158.6, 153.4, 150.5, 145.8, 141.7, 135.9, 131.6, 130.1, 126.3, 123.4, 121.7, 120.6,
119.8, 119.1, 113.5, 112.6, 111.3, 105.9, 46.4, 42.7, 27.9, 27.6, 20.6. HRMS(ESI) calcd for C25H23N5O3 [M+H]+
442.1834, found: 442.1842.
N-(4-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)butyl)-1-methyl-9H-β-carboline-3-amide (13b)
1
Analytical data for 13b: ESI-MS m/z = 442 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.91 (s, 1H, NH),
11.25 (s, 1H, NH), 8.63 (s, 1H, ArH), 8.33 (d, J = 7.8 Hz, 1H, ArH), 8.29-8.38 (m, 2H, NH, ArH), 7.71 (m, 1H,
ArH), 7.55 – 7.63 (m, 2H, NH, ArH), 7.27-7.35 (m, 2H, ArH), 5.84 (s, 1H, CH=), 3.39 (m, 2H, NCH2), 3.26 (m,
2H, NCH2), 2.82 (s, 3H, CH3), 1.43-1.62 (m, 4H, CH2-CH2); 13C NMR (100MHz, DMSO-d6) δ 182.1, 179.8,
158.5, 153.4, 150.5, 145.6, 141.7, 135.8, 131.6, 130.0, 126.3, 123.2, 121.5, 120.6, 119.7, 119.0, 113.5, 111.3,
108.7, 105.7, 46.5, 42.6, 27.9, 27.6, 20.6. HRMS(ESI) calcd for C25H23N5O3 [M+H]+ 442.1834, found:
442.1850.
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2.2.4 General synthetic procedure of 12c and 13c
Compounds 12c and 13c were synthesized from 9 and 6c, based on the synthetic process of 12a and 13a in
yield of 29% and 18%, respectively.
N-(6-((4,7-dioxo-4,7-dihydro-1H-indol-5-yl)amino)hexyl)-1-methyl-9H-β-carboline-3-amide (12c)
Analytical data for 12c: ESI-MS m/z = 470 [M+H]+; 1H NMR (400MHz, DMSO-d6) δ 11.90 (s, 1H, NH),
11.24 (s, 1H, NH), 8.69 (s, 1H, ArH), 8.31 – 8.37 (m, 2H, NH, ArH), 7.71 (m, 1H, ArH), 7.53–7.62 (m, 2H, NH,
ArH), 7.21 – 7.30 (m, 2H, ArH), 6.79 (m, 1H, ArH), 5.91 (s, 1H, CH=), 3.42 (m, 2H, NCH2), 3.23 (m, 2H,
NCH2), 2.84 (s, 3H, CH3), 1.42-1.61 (m, 8H, 4 × CH2); 13C NMR (100MHz, DMSO-d6) δ 182.3, 180.1, 158.5,
152.9, 150.0, 146.2, 141.8, 135.6, 131.2, 129.8, 125.3, 123.0, 121.5, 120.6, 119.8, 118.9, 113.4, 112.4, 111.2,
106.7, 46.2, 42.3, 30.5, 29.8, 27.6, 27.3, 20.6. HRMS(ESI) calcd for C27H27N5O3 [M+H]+ 470.2147, found:
470.2133.
N-(6-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)hexyl)-1-methyl-9H-β-carboline-3-amide (13c)
Analytical data for 13c: ESI-MS m/z = 470 [M+H]+; 1H NMR (400MHz, DMSO-d6) δ 11.90 (s, 1H, NH),
11.23 (s, 1H, NH), 8.65 (s, 1H, ArH), 8.29-8.36 (m, 2H, NH, ArH), 7.70 (d, J = 7.8 Hz, 1H, ArH), 7.54–7.60 (m,
2H, NH, ArH), 7.25-7.31 (m, 2H, ArH), 6.75 (m 1H, ArH), 5.86 (s, 1H, CH=), 3.40-3.36 (m, 2H, NCH2), 3.19
(m, 2H, NCH2), 2.83 (s, 3H, CH3), 1.39-1.60 (m, 8H, 4 × CH2); 13C NMR (100MHz, DMSO-d6) δ 182.2, 180.1,
158.4, 152.9, 150.0, 145.9, 141.8, 135.6, 131.1, 129.9, 125.3, 122.9, 121.5, 120.5, 119.6, 118.9, 113.3, 111.1,
108.4, 106.7, 46.1, 42.2, 30.5, 29.7, 27.5, 27.2, 20.6. HRMS(ESI) calcd for C27H27N5O3 [M+H]+ 470.2147,
found: 470.2156.
2.2.5 General synthetic procedure of 12d and 13d
Compounds 12d and 13d were synthesized from 9 and 6d, based on the synthetic process of 12a and 13a in
yield of 32% and 23%, respectively.
N-(2-((4,7-dioxo-4,7-dihydro-1H-indol-5-yl)amino)ethyl)-1-(p-tolyl)-9H-β-carboline-3-amide (12d)
1
Analytical data for 12d: ESI-MS m/z = 490 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.86 (s, 1H, NH),
11.21 (s, 1H, NH), 8.65 (s, 1H, ArH), 8.25 (m, 1H, NH), 8.05 (d, 1H, J = 7.8 Hz, ArH), 7.92 (d, 2H, J = 7.8 Hz,
ArH), 7.57-7.63 (m, 2H, NH, ArH), 7.35 (d, 2H, J = 7.8 Hz, ArH), 7.23 (m, 1H, ArH), 7.13 (m, 1H, ArH), 6.75
(m, 1H, ArH), 5.87 (s, 1H, CH=), 3.59 (m, 2H, NCH2), 3.38 (t, J = 6.0 Hz, 2H, NCH2), 2.39 (s, 3H, CH3); 13C
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NMR (100MHz, DMSO-d6) δ 182.0, 180.2, 161.5, 158.7, 151.5, 145.6, 141.2, 138.9, 132.3, 130.2, 129.8, 127.6,
124.5, 123.0, 122.6, 121.7, 121.1, 119.6, 113.4, 112.6, 111.2, 106.4, 102.3, 47.6, 43.1, 21.4. HRMS(ESI) calcd
for C29H23N5O3 [M+H]+ 490.1834, found: 490.1849.
N-(2-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)ethyl)-1-(p-tolyl)-9H-β-carboline-3-amide (13d)
1
Analytical data for 13d: ESI-MS m/z = 490 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.82 (s, 1H, NH),
11.16 (s, 1H, NH), 8.67 (s, 1H, ArH), 8.25 (m, 1H, NH), 8.04 (d, 1H, J = 7.8 Hz, ArH), 7.91 (d, 2H, J = 7.8 Hz,
ArH), 7.58-7.65 (m, 2H, NH, ArH), 7.32 (d, 2H, J = 7.8 Hz, ArH), 7.22 (m, 1H, ArH), 7.11 (m, 1H, ArH), 6.74
(m, 1H, ArH), 5.76 (s, 1H, CH=), 3.63 (m, 2H, NCH2), 3.42 (m, 2H, NCH2), 2.38 (s, 3H, CH3); 13C NMR
(100MHz, DMSO-d6) δ 182.1, 180.2, 161.4, 158.7, 151.5, 145.5, 141.1, 138.9, 132.3, 130.1, 129.8, 127.6, 124.5,
123.0, 122.5, 121.6, 121.1, 119.5, 113.4, 111.1, 108.6, 106.5, 102.3, 47.6, 43.1, 21.4. HRMS(ESI) calcd for
C29H23N5O3 [M+H]+ 490.1834, found: 490.1843.
2.2.6 General synthetic procedure of 12e and 13e
Compounds 12e and 13e were synthesized from 9 and 6e, based on the synthetic process of 12a and 13a in
yield of 30% and 21%, respectively.
N-(4-((4,7-dioxo-4,7-dihydro-1H-indol-5-yl)amino)butyl)-1-(p-tolyl)-9H-β-carboline-3-amide (12e)
1
Analytical data for 12e: ESI-MS m/z = 518 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.92 (s, 1H, NH),
11.24 (s, 1H, NH), 8.59 (m, 2H, ArH, NH), 8.07 (d, 1H, J = 7.8 Hz, ArH), 7.95 (d, 2H, J = 7.8 Hz, ArH),
7.53-7.66 (m, 2H, NH, ArH), 7.33 (d, 2H, J = 7.8 Hz, ArH), 7.25 (m, 1H, ArH), 7.15 (m, 1H, ArH), 6.79 (m, 1H,
ArH), 5.90 (s, 1H, CH=), 3.45 (m, 2H, NCH2), 3.29 (t, J = 6.0 Hz, 2H, NCH2), 2.41 (s, 3H, CH3), 1.67 (m, 4H,
CH2-CH2); 13C NMR (100MHz, DMSO-d6) δ 181.9, 180.1, 161.3, 158.5, 151.3, 145.5, 141.2, 138.9, 132.3,
130.1, 129.7, 127.6, 124.5, 122.9, 122.5, 121.7, 121.0, 119.6, 113.4, 112.6, 111.0, 106.4, 102.3, 46.5, 42.7, 27.9,
27.5, 21.4. HRMS(ESI) calcd for C31H27N5O3 [M+H]+ 518.2147, found: 518.2161.
N-(4-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)butyl)-1-(p-tolyl)-9H-β-carboline-3-amide (13e)
1
Analytical data for 13e: ESI-MS m/z = 518 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.86 (s, 1H, NH),
11.19 (s, 1H, NH), 8.67 (s, 1H, ArH), 8.22 (m, 1H, NH), 8.03 (d, 1H, J = 7.8 Hz, ArH), 7.88 (d, 2H, J = 7.8 Hz,
ArH), 7.53-7.62 (m, 2H, NH, ArH), 7.29 (d, 2H, J = 7.8 Hz, ArH), 7.21 (m, 1H, ArH), 7.08 (m, 1H, ArH), 6.70
(m, 1H, ArH), 5.73 (s, 1H, CH=), 3.59 (m, 2H, NCH2), 3.40 (m, 2H, NCH2), 2.35 (s, 3H, CH3), 1.64 (m, 4H,
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CH2-CH2); 13C NMR (100MHz, DMSO-d6) δ 182.1, 179.9, 161.5, 158.7, 151.6, 145.5, 141.0, 138.7, 132.4,
130.1, 129.7, 127.6, 124.6, 123.1, 122.5, 121.7, 121.1, 119.5, 113.4, 111.2, 108.5, 106.6, 102.5, 46.4, 42.7, 27.9,
27.6, 21.4. HRMS(ESI) calcd for C31H27N5O3 [M+H]+ 518.2147, found: 518.2155.
2.2.7 General synthetic procedure of 12f and 13f
Compounds 12f and 13f were synthesized from 9 and 6f, based on the synthetic process of 12a and 13a in
yield of 26% and 19%, respectively.
N-(6-((4,7-dioxo-4,7-dihydro-1H-indol-5-yl)amino)hexyl)-1-(p-tolyl)-9H-β-carboline-3-amide (12f)
1
Analytical data for 12f: ESI-MS m/z = 546 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.87 (s, 1H, NH),
11.20 (s, 1H, NH), 8.69 (s, 1H, ArH), 8.26 (m, 1H, NH), 8.09 (m, 1H, ArH), 7.96 (d, 2H, J = 7.8 Hz, ArH),
7.55-7.71 (m, 2H, NH, ArH), 7.35 (d, 2H, J = 7.8 Hz, ArH), 7.28 (m, 1H, ArH), 7.16 (m, 1H, ArH), 6.76 (m, 1H,
ArH), 5.92 (s, 1H, CH=), 3.48 (m, 2H, NCH2), 3.35 (t, J = 6.0 Hz, 2H, NCH2), 2.41 (s, 3H, CH3), 1.46-1.69 (m,
8H, 4 ×CH2); 13C NMR (100MHz, DMSO-d6) δ 182.0, 180.2, 161.2, 158.6, 151.3, 145.6, 141.1, 138.8, 132.3,
130.1, 129.8, 127.6, 124.5, 123.1, 122.6, 121.7, 121.1, 119.6, 113.4, 112.4, 111.1, 106.4, 102.3, 46.2, 42.3, 30.6,
29.8, 27.6, 27.3, 21.4. HRMS(ESI) calcd for C33H31N5O3 [M+H]+ 546.2460, found: 546.2472.
N-(6-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)hexyl)-1-(p-tolyl)-9H-β-carboline-3-amide (13f)
Analytical data for 13f: ESI-MS m/z = 546 [M+H]+; 1H NMR (400MHz, DMSO-d6) δ 11.78 (s, 1H, NH), 11.13
(s, 1H, NH), 8.65 (s, 1H, ArH), 8.23 (m, 1H, NH), 8.04 (m, 1H, ArH), 7.92 (d, 2H, J = 7.8 Hz, ArH), 7.51-7.66
(m, 2H, NH, ArH), 7.33 (d, 2H, J = 7.8 Hz, ArH), 7.23 (m, 1H, ArH), 7.12 (m, 1H, ArH), 6.73 (m, 1H, ArH),
5.77 (s, 1H, CH=), 3.41 (m, 2H, NCH2), 3.32 (t, J = 6.0 Hz, 2H, NCH2), 2.36 (s, 3H, CH3), 1.43-1.64 (m, 8H, 4
×CH2); 13C NMR (100MHz, DMSO-d6) δ 182.0, 180.3, 161.1, 158.5, 151.2, 145.5, 141.0, 138.7, 132.1, 130.0,
129.7, 127.6, 124.5, 122.9, 122.4, 121.6, 121.0, 119.5, 113.2, 110.9, 108.6, 106.5, 102.3, 46.1, 42.3, 30.5, 29.8,
27.6, 27.2, 21.4. HRMS(ESI) calcd for C33H31N5O3 [M+H]+ 546.2460, found: 546.2451.
2.2.8 General synthetic procedure of 12g and 13g
Compounds 12g and 13g were synthesized from 9 and 6g, based on the synthetic process of 12a and 13a in
yield of 31% and 23%, respectively.
N-(2-((4,7-dioxo-4,7-dihydro-1H-indol-5-yl)amino)ethyl)-1-(4-methoxyphenyl)-9H-β-carboline-3-amide
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(12g)
1
Analytical data for 12g: ESI-MS m/z = 506 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.85 (s, 1H, NH),
11.12 (s, 1H, NH), 8.71 (s, 1H, ArH), 8.42-8.49 (m, 2H, ArH), 8.17-8.22 (m, 2H, NH, ArH), 7.75 (d, 2H, J = 7.8
Hz, ArH), 7.57-7.63 (m, 2H, NH, ArH), 7.22 (m, 1H, ArH), 7.10 (d, 2H, J = 7.8 Hz, ArH), 6.73 (m, 1H, ArH),
5.89 (s, 1H, CH=), 3.87 (s, 3H, OCH3), 3.63 (m, 2H, NCH2), 3.40 (t, J = 6.0 Hz, 2H, NCH2); 13C NMR
(100MHz, DMSO-d6) δ 182.1, 179.9, 161.8, 159.3, 152.3, 151.6, 141.5, 139.8, 136.1, 132.2, 128.5, 124.6, 123.5,
123.1, 121.9, 121.1, 115.2, 113.4, 112.6, 111.1, 106.9, 103.1, 47.6, 43.2, 41.7. HRMS(ESI) calcd for
C29H23N5O4 [M+H]+ 506.1784, found: 506.1777.
N-(2-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)ethyl)-1-(4-methoxyphenyl)-9H-β-carboline-3-amide
(13g)
1
Analytical data for 13g: ESI-MS m/z = 506 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.83 (s, 1H, NH),
11.09 (s, 1H, NH), 8.69 (s, 1H, ArH), 8.38-8.46 (m, 2H, NH, ArH), 8.24 (m, 1H, ArH), 7.74 (d, 2H, J = 7.8 Hz,
ArH), 7.55-7.63 (m, 2H, NH, ArH), 7.25 (m, 1H, ArH), 7.07 (d, 2H, J = 7.8 Hz, ArH), 6.74 (m, 1H, ArH), 5.79
(s, 1H, CH=), 3.85 (s, 3H, OCH3), 3.61 (m, 2H, NCH2), 3.39 (t, J = 6.0 Hz, 2H, NCH2); 13C NMR (100MHz,
DMSO-d6) δ 182.0, 179.6, 161.5, 159.1, 152.3, 151.6, 141.5, 139.6, 135.8, 132.1, 128.4, 124.6, 123.4, 123.0,
121.7, 121.1, 115.1, 113.4, 111.0, 108.6, 106.8, 103.1, 47.6, 43.1, 41.7. HRMS(ESI) calcd for C29H23N5O4
[M+H]+ 506.1784, found: 506.1793.
2.2.9 General synthetic procedure of 12h and 13h
Compounds 12h and 13h were synthesized from 9 and 6h, based on the synthetic process of 12a and 13a in
yield of 33% and 21%, respectively.
N-(4-((4,7-dioxo-4,7-dihydro-1H-indol-5-yl)amino)butyl)-1-(4-methoxyphenyl)-9H-β-carboline-3-amide
(12h)
1
Analytical data for 12h: ESI-MS m/z = 534 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.83 (s, 1H, NH),
11.05 (s, 1H, NH), 8.71 (s, 1H, ArH), 8.36-8.42 (m, 2H, ArH, NH), 8.19−8.25 (m, 2H, ArH), 7.89 (d, 2H, J = 7.8
Hz, ArH), 7.53-7.64 (m, 2H, NH, ArH), 7.46 (m, 1H, ArH), 7.07 (d, 2H, J = 7.8 Hz, ArH), 6.75 (m 1H, ArH),
5.83 (s, 1H, CH=), 3.87 (s, 3H, OCH3), 3.42 (m, 2H, NCH2), 3.25 (m, 2H, NCH2), 1.65 (m, 4H, CH2-CH2); 13
C
NMR (100MHz, DMSO-d6) δ 182.4, 180.1, 161.9, 159.3, 152.5, 151.6, 141.5, 140.1, 136.1, 132.3, 128.5, 124.6,
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123.5, 123.2, 122.2, 121.1, 115.2, 113.6, 112.8, 111.1, 107.1, 103.4, 46.4, 42.6, 41.7, 27.9, 27.5. HRMS(ESI)
calcd for C31H27N5O4 [M+H]+ 534.2097, found: 534.2109.
N-(4-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)butyl)-1-(4-methoxyphenyl)-9H-β-carboline-3-amide
(13h)
1
Analytical data for 13h: ESI-MS m/z = 534 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.84 (s, 1H, NH),
11.05 (s, 1H, NH), 8.71 (s, 1H, ArH), 8.39-8.43 (m, 2H, ArH, NH), 8.17−8.25 (m, 2H, ArH), 7.90 (d, 2H, J = 7.8
Hz, ArH), 7.55-7.63 (m, 2H, NH, ArH), 7.47-7.50 (m, 1H, ArH), 7.08 (d, 2H, J = 7.8 Hz, ArH), 6.75 (m 1H,
ArH), 5.77 (s, 1H, CH=), 3.88 (s, 3H, OCH3), 3.43 (m, 2H, NCH2), 3.27 (m, 2H, NCH2), 1.46-1.63 (m, 4H,
CH2-CH2); 13C NMR (100MHz, DMSO-d6) δ 182.0, 179.7, 161.5, 159.1, 152.4, 151.6, 141.5, 139.8, 136.0,
132.1, 128.4, 124.6, 123.4, 123.0, 122.0, 121.1, 115.1, 113.4, 111.0, 108.6, 106.7, 103.4, 46.5, 42.7, 41.7, 27.9,
27.6. HRMS(ESI) calcd for C31H27N5O4 [M+H]+ 534.2097, found: 534.2112.
2.2.10 General synthetic procedure of 12i and 13i
Compounds 12i and 13i were synthesized from 9 and 6i, based on the synthetic process of 12a and 13a in
yield of 28% and 19%, respectively.
N-(6-((4,7-dioxo-4,7-dihydro-1H-indol-5-yl)amino)hexyl)-1-(4-methoxyphenyl)-9H-β-carboline-3-amide
(12i)
1
Analytical data for 12i: ESI-MS m/z = 562 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.81 (s, 1H, NH),
11.03 (s, 1H, NH), 8.70 (s, 1H, ArH), 8.33-8.39 (m, 2H, ArH, NH), 8.17−8.25 (m, 2H, ArH), 7.86 (d, 2H, J = 7.8
Hz, ArH), 7.52-7.61 (m, 2H, NH, ArH), 7.46 (m, 1H, ArH), 7.07 (d, 2H, J = 7.8 Hz, ArH), 6.75 (m 1H, ArH),
5.86 (s, 1H, CH=), 3.89 (s, 3H, OCH3), 3.35 (m, 2H, NCH2), 3.20 (m, 2H, NCH2), 1.38-1.60 (m, 8H, 4 × CH2);
13C NMR (100MHz, DMSO-d6) δ 182.4, 180.1, 161.9, 159.3, 152.5, 151.6, 141.5, 140.0, 135.9, 132.2, 128.5,
124.6, 123.6, 123.1, 122.2, 121.1, 115.2, 113.6, 112.7, 111.1, 107.0, 103.5, 46.2, 42.3, 41.7, 30.5, 29.8, 27.6,
27.3. HRMS(ESI) calcd for C33H31N5O4 [M+H]+ 562.2410, found: 562.2421.
N-(6-((4,7-dioxo-4,7-dihydro-1H-indol-6-yl)amino)hexyl)-1-(4-methoxyphenyl)-9H-β-carboline -3-amide
(13i)
1
Analytical data for 13i: ESI-MS m/z = 562 [M+H]+; H NMR (400MHz, DMSO-d6) δ 11.83 (s, 1H, NH),
11.06 (s, 1H, NH), 8.72 (s, 1H, ArH), 8.37-8.43 (m, 2H, ArH, NH), 8.20-8.31 (m, 3H, ArH), 7.74 (m, 1H, ArH),
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7.53-7.61 (m, 2H, NH, ArH), 7.48 (m, 1H, ArH), 7.11 (d, 2H, J = 7.8 Hz, ArH), 6.79 (m 1H, ArH), 5.81 (s, 1H,
CH=), 3.88 (s, 3H, OCH3), 3.43 – 3.38 (m, 2H, NCH2), 3.21 (m, 2H, NCH2), 1.39-1.62 (m, 8H, 4 × CH2); 13C
NMR (100MHz, DMSO-d6) δ 182.0, 179.7, 161.5, 159.1, 152.3, 151.6, 141.3, 139.7, 136.0, 132.1, 128.5, 124.6,
123.5, 123.0, 121.9, 121.1, 115.2, 113.3, 111.1, 108.7, 106.9, 103.2, 46.3, 42.4, 41.7, 30.6, 29.8, 27.7, 27.3.
HRMS(ESI) calcd for C33H31N5O4 [M+H]+ 562.2410, found: 562.2401.
2.3 Experimental procedures for biological evaluation
2.3.1 Cell lines and culture
Human colonic carcinoma cells (HT29), gastric carcinoma cells (HGC27), oral epithelial carcinoma cells
(KB), and human non-tumor colon cells (CCD841) were from Shanghai Institute of Cell Biology (SICB,
Shanghai, China). All of them were routinely put in DMEM/RMPI 1640 medium possessing 10% FBS
(Invitrogen), 100 units/ml penicillin, and 100 units/ml streptomycin at 37 °C in an atmosphere (humid, 5% CO2).
For subculture, cells were detached with 0.25% trypsin-EDTA (Ethylenediaminetetra-acetic acid) mixture, and
1×106 cells were seeded into new flasks.
2.3.2 MTT assay
The in vitro antiproliferative effects were assessed by the MTT method referring to previous literature (Ling,
et al. 2019). After washing the cells with PBS buffer, 0.5 mg/mL MTT solution was added and kept at 37 °C for
3 h. MTT medium was removed, and the OD value was detected at 570 nm using a BioTek Microplate
Absorbance Reader according to the protocol. The inhibitory rates were expressed as percentage. IC50 data were
then processed through corresponding software (GraphPadPrism Version 4.03).
2.3.3 NQO1 activity assay
The NQO1 activity was analyzed according to NQO1 assay kit (Abcam, MA, USA). HT29 cells were
dissolved in 1X extraction buffers at 0-4°C for 25 min, and centrifuged at 18,000 × g at 0-4°C for 25 min.
Supernatant was transferred to new tube and the concentration of protein was detected according to the Bio-Rad
protein assay. The reaction buffers with or without inhibitor were prepared, and OD value was detected at 442
nm every 20 seconds for 5 min through the Bio-Tek Synergy HT Multi-Mode Reader.
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2.3.4 Measurement of Intracellular ROS Generation
The tested cells (1 × 106 cells/well) were pre-treated with H2O (control) or 40 μM NAc for 2 h, followed by
treatment with an indicated concentration of compound 13g for 3, 6, 9 h, and the cells were stained with
dihydroethidium (DHE, Beyotime). The levels of intracellular ROS were measured as the fluorescent signals
using a fluorescence microplate reader (300 and 610 nm).
2.3.5 Flow cytometry assay
HT29 cells were cultured for 12h and incubated with 13g (2.0, 5.0 μM), β-lap (2.0 μM), or vehicle for 72 h.
The cells were stained with annexin-V APC and 7AAD for 15 min. Cell apoptotic rates were measured by flow
cytometric analysis (Calibur, BD, USA), using the annexin-V APC signal (ex633 nm, em660 nm) and 7AAD
signal (ex488 nm, em647 nm). The data were processed through WinList3D software 7.1, and the histogram was
draw through Excel2016.
2.3.6 Western blotting analysis
HT29 cells were placed in 6 cm wells to incubate for 24h, and then dealed with or without 13g, β-lap at
different dosages for 72h. The cells were subsequently harvested and lysed. After protein concentrations
quantification through a Bio-Rad protein test kit (Bio-Rad Laboratories Inc., Hercules, CA, USA),
corresponding cell lysates were lysed and severed through sodium dodecyl sulfatepolyacrylamide gel
electrophoresis (7.5% gel) and transformed to corresponding polyvinylidenedifluoride membranes. When
blocked by 5% fatfree milk for 2 h, the protein was respectively probed with anti-Bax, anti-Bcl-2,
anti-cleaved-caspase 3, anti-H2AX(S139ph), or anti-β-actin (Cell Signaling Technology, MA, USA). The bound
antibody was detected by peroxidase-conjugated second antibody for 1 h, and then conceived by
chemiluminescent reagent.
2.3.7 In vivo tumor xenograft model
Each female BALB/c nude mouse at 6~8-week old was cultured by 1×106 HT29 cells in the right flank.
After establishment of solid tumors at an average volume of 50-100 mm3, the animals were stochasticly treated
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by vehicle alone, compound 13g, or β-lap daily for 22 days. The tumor volumes and body weights of all animals
were surveyed every 3 days throughout the study. Tumor volumes were counted by a formula: tumor volume
(mm3) = W2 × L/2, where W = width and L = length (mm).
3 RESULTS AND DISCUSSION
3.1 Chemistry
The whole synthetic route of target hybrids 12a‒i and 13a‒i is depicted in Scheme 1. Treatment of
commercially available L-tryptophan 1 with different substituted aldehydes 2a-c produced the intermediates
3a-c via a Pictet-Spengler reaction. Compounds 3a-c were then esterified by SOCl2 in CH3OH to prepare methyl
esters 4a-c, which were further oxidized by catalysis of potassium permanganate in DMF to get compounds
5a-c. Intermediates 5a-c were next reacted with different alkyl diamines to produce monoamide derivatives 6a-i.
On the other hand, 4,7-dimethoxy-1H-indole 7 was reacted with 4-methylbenzenesulfonic anhydride to provide
4,7-dimethoxy-1-Ts-indole 8, which was then oxidized with cerium ammonium nitrate (CAN) in CH3CN to
obtain 1-Ts-indole-4,7-quinone 9. The key intermediate 9 was reacted with compounds 6a-i to produce
intermediates 10a-i and 11a-i. Finally, deprotection of compounds 10a-i and 11a-i afforded target compounds
12a-i and 13a-i. All synthetic compounds were isolated and purified using column chromatography, and their
1
molecule structures were characterized by H NMR, 13C NMR, MS, and HRMS. Each compound possessed
purity (>95%), and was detected by HPLC.
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O
O
COOH
NH2
O
OH
b
a
CHO
NH
NH
+
R
N
N
N
H
H
R
H
R
3a-c
2a-c
4a-c
1
O
O
N
NH2
O
N
n
d
H
c
2-5a
2-5b
2-5c
R = Me;
R = 4-MePh;
R = 4-MeOPh;
N
N
H
N
H
R
R
5a-c
6a-i
O
N
O
H
N
H
N
n
N
N
O
O
O
O
H
10a-i
Ts
R
R
O
e
f
g
O
N
H
N
N
H
N
Ts
Ts
O
O
O
N
7
8
9
H
n
N
N
H
O
11a-i
N
Ts
O
O
O
H
H
N
N
O
h
N
N
+
H
n
H
n
N
N
N
N
H
N
O
H
H
13a-i
12a-i
R
R
HN
12f
O
6 10-13a
12a
12b
12c
13a
: 33%, : 24%;
,
n = 1, R = Me,
n = 3, R = Me;
n = 5, R = Me;
6 10-13f
13f
: 19%;
,
n = 5, R = 4-MePh;
: 26%,
6 10-13b
13b
,
: 31%,
: 22%;
6 10-13g
12g
13g
,
n = 1, R = 4-MeOPh,
n = 3, R = 4-MeOPh,
n = 5, R = 4-MeOPh,
: 31%,
: 33%,
: 28%,
: 23%;
: 21%;
: 19%.
6 10-13c
13c
: 32%,
: 30%,
,
: 29%,
12d
12e
: 18%;
13d
: 23%;
13e
: 21%;
6 10-13h
12h
12i
13h
13i
,
6 10-13d
,
n = 1, R = 4-MePh,
n = 3, R = 4-MePh;
6 10-13i
,
6 10-13e
,
SCHEME 1 Reagents and conditions: (a) H+ or OH-, rt to reflux, 2-4h, 72-81% (b) SOCl2, MeOH, 0 °C, 1h, and then
reflux 4-6h, 85-92%; (c)KMnO4, DMF, rt, 2-3h, 71-80%; (d) different alkyl diamines, MeOH, reflux, 24h, 73-81%;
(e) Ts2O, NaH, THF, 0 °C 15 min, and then rt. 3h, 93%; (f) CAN, CH3CN, rt. 1h, 73%; (g) 6a-i, MeOH, rt, 2h; (h)
NaOMe, MeOH, 0°C, 1h, 18-33%.
3.2 In vitro antiproliferative activities and NQO1-dependency evaluation
Novel β-carboline-based indole-4,7-quinone derivatives (12a‒i and 13a‒i) were first tested for their
antiproliferative activities against three cancer cells, including human colonic carcinoma cells (HT29), human
gastric carcinoma cells (HGC27), and human oral epithelial carcinoma cells (KB) in MTT assays. β-Lapachone
(β-lap), a known NQO1 inhibitor in multiple clinical trials (Kim et al., 2018), was used as a positive control. The
IC50 data (the effective concentration that inhibiting 50% of tumor cell proliferation) of 12a‒i and 13a‒i against
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three human tumor cells are depicted in Table 1. Most of tested compounds exhibited potent antiproliferative
activities with IC50’s in the single-digit micromole level in three cells. Interestingly, most of the compounds
showed slightly stronger cytotoxicities against HT29 cells than HGC27 and KB cells. Importantly, the
antiproliferative activities of 13g (IC50 = 1.87-2.49 µM) in three cancer cells were 2-to-3 folds more potent than
those of β-lap (IC50 = 2.91-4.13 µM), 7-to-8 folds more potent than those of indolequinone moiety (compound
9), and 17-to-25 folds more potent than those of harmine (IC50 = 42.6-52.1 µM). The pharmacophoric group in
the active compound 13g may be originated from two fragments indolequinone moiety and β-carboline portion.
We also tested the antiproliferative activities of two fragments, and found that indolequinone moiety (compound
9) showed more potent activities than β-carboline portion (harmine).
TABLE 1 IC50 data of hybrids 12a-i and 13a-i against three human cancer cell lines.
In vitro antiproliferative activity (IC50a, μM)
Compd.
R
n
HT29
HGC27
4.13 ± 0.51
42.6 ± 5.34
18.6 ± 1.63
7.52 ± 0.84
11.8 ± 1.36
>12.5
KB
β-lap
Harmine
9
/
/
/
2.91 ± 0.24
47.5 ± 3.89
15.2 ± 1.34
8.63 ± 0.77
10.5 ± 1.38
>12.5
3.63 ± 0.55
52.1 ± 4.69
NDb
/
/
/
12a
12b
12c
Me
1
3
5
1
3
5
1
3
5
1
3
5
10.1 ± 1.14
>12.5
Me
Me
>12.5
12d
12e
4-MePh
4-MePh
4-MePh
4-MeOPh
4-MeOPh
4-MeOPh
Me
4.77 ± 0.52
7.86 ± 0.59
11.7 ± 1.21
3.86 ± 0.53
6.35 ± 0.72
11.2 ± 1.34
4.96 ± 0.58
7.37 ± 0.86
>12.5
5.06 ± 0.63
7.58 ± 0.67
>12.5
5.89 ± 0.75
10.7 ± 0.92
>12.5
12f
12g
12h
12i
4.09 ± 0.60
7.67 ± 0.58
>12.5
4.74 ± 0.63
9.85 ± 1.10
>12.5
13a
13b
13c
5.71 ± 0.64
8.02 ± 0.69
>12.5
7.02 ± 0.66
8.87 ± 0.96
>12.5
Me
Me
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13d
13e
13f
13g
13h
13i
4-MePh
4-MePh
1
3
5
1
3
5
2.39 ± 0.36
3.75 ± 0.44
9.46 ± 1.12
1.87 ± 0.25
2.80 ± 0.22
9.12 ± 1.07
3.05 ± 0.29
3.68 ± 0.53
8.71 ± 0.94
2.49 ± 0.28
3.15 ± 0.49
9.50 ± 1.15
3.51 ± 0.43
5.90 ± 0.72
11.5 ± 1.34
2.27 ± 0.32
3.78 ± 0.35
10.8 ± 1.27
4-MePh
4-MeOPh
4-MeOPh
4-MeOPh
a The IC50 data (means ± SD) were from three separated experiments. bNot detected.
13g
80
13g + 40 μM DIC
70
13g + 40 μM NAc
60
50
40
30
20
10
0
0.5
1
2
5
10
Concentrations (μM)
FIGURE 1 Cytotoxicity of 13g toward HT29 cancer cells with or without the NQO inhibitor (DIC, 40 μM) and
the free radical scavenger (NAc, 40 μM).
We next detected the antiproliferative effects of the most potent compound 13g in the presence of NQO1
inhibitor dicoumarol (DIC) or free radical scavenger N-acetylcysteine (NAc) in human colon cancer HT29 cell
lines. As shown in Figure 1, the antiproliferative activities of 13g were NQO1-specific, science the cell
inhibitory rates in HT29 cells were marked decreased by addition of DIC or NAc. The cytotoxic IC50 value of
13g toward HT29 cells was increased from 1.87 μM (13g only) to higher than 5 μM (after addition of DIC or
NAc). These results implies that the antiproliferative activities of 13g may be influenced at least in part by their
NQO1-dependent bio-reductive activation.
3.3 NQO1 inhibitory effects
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According to the prominent antiproliferative activity of 12a-i and 13a-i against three human cancer cells, we
further tested six active compounds (12d, 12g, 13d, 13e, 13g, and 13h) for NQO1 inhibitory activities in vitro.
As depicted in Table 2, all 6 compounds exhibited high NQO1 inhibitory potencies in HT29 cell lines. Most of
these activities are in accordance with their antiproliferative activities. In particular, compounds 13d and 13g
exhibited significant NQO1 inhibitory activities with IC50 data of 1.5 μM and 1.2 μM, which were more potent
than β-lapachone and other target compounds. Therefore, NQO1 depletion by treatment with
β-carboline/indole-4,7-quinone hybrids might result in decreased cell proliferation.
TABLE 2 Effects of 12d, 12g, 13d, 13e, 13g, and 13h on NQO1 activity in HT29 cancer cell lines.
Compound
β-lap
12d
IC50a (µM)
1.7 ± 0.2
3.8 ± 0.5
4.1 ± 0.4
1.5 ± 0.2
2.2 ± 0.3
1.2 ± 0.1
2.1 ± 0.2
12g
13d
13e
13g
13h
a The IC50 data (means ± SD) were from three separated experiments.
80
CCD841
70
60
50
40
30
20
10
0
HT29
0.5
1
2
5
10
Concentrations of 13g (μM)
FIGURE 2. Inhibitory selectivity of 13g on the growth of HT29 and CCD841 cells. Each cell was incubated with the different
concentrations of 13g for 72h. Cell inhibitory activity was detected by the MTT assay. Data (means ± SD) of the inhibition (%)
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are from three separated experiments.
3.4 The inhibitory selectivity of 13g between tumor cells and normal cells
Based on the significant NQO1 target and tumor cell inhibitory activities of 13g in vitro, the cell inhibitory
selectivity analysis was further evaluated by determining the inhibitory potencies of 13g on the proliferation of
both colon cancer cells HT29 and normal epithelial cells CCD841. Compound 13g had little inhibitory activity
against non-tumor CCD841 cells under an increased dose, whereas the same doses exerted significant inhibition
on HT29 cells growth (Figure 2). These findings confirm that 13g might have selectivity in antiproliferative
activity to cancer cells but not normal cells. The possible reason may be that tumor cells often expose to a
relatively high level of ROS compared with normal cells, and 13g as an NQO inhibitor also augmented high
levels of intracellular ROS in HT29 cells. Therefore, the accumulation of high levels ROS in HT29 cells may
make cancer cells more selective to induce cell apoptosis or death.
3.5 Compound 13g leads to an increase ROS level
Next, we assessed whether the antiproliferative effect of 13g on colon tumor cells involved ROS increase.
HT29 cells were pre-treated with H2O (control) or 40 μM NAc for 2 h, followed by treatment with an indicated
concentration of compound 13g for 3, 6, 9 h, respectively. Intracellular ROS levels were determined by
fluorescent spectrometry. Treatment with 13g remarkably increased the levels of intracellular ROS in HT29 cells
and this induction was concentration-dependent (Figure 3). However, this ROS induction could be reversed by
addition of the free radical scavenger NAc. These findings indicated that compound 13g led to an increasing
oxidative stress in HT29 cells, thus resulting in significant cytotoxicity to tumor cells.
4
3.5
3
13g 2.0 μM
13g 5.0 μM
13g 5.0 μM + NAc
2.5
2
1.5
1
0
3
6
9
12
Time (h)
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FIGURE 3 Compound 13g selectively induced ROS accumulation in HT29 cells. HT29 cells were pre-treated with
H2O (control) or 40 μM NAc for 2 h, followed by treatment with an indicated concentration of compound 13g for 3, 6,
9 h, respectively. Intracellular ROS levels were stained with dihydroethidium (DHE) and measured by a fluorescent
spectrometry. Data are the means of individual groups of cells (relative to the control group) from three separate
experiments.
3.6 Compound 13g promotes apoptosis in HT29 cells
To investigate whether the antiproliferative effects of 13g against colonic tumor cells are accompanied by
enhanced cell apoptosis, the cell apoptotic percentages were analyzed by flow cytometric assay. HT29 cells were
then incubated with increasing concentrations of 13g or β-lap for 72h. As depicted in Figure 4, the apoptotic
rates of 13g (45.3% and 58.9% for 2.0 and 5.0 µM, respectively) in annexin V+ HT29 cells gradually increased
with concentrations, which were more potent than the positive control group β-lap (37.6% for 2.0 µM). These
results indicated that the treatment with 13g could dose-dependently promote HT29 cell apoptosis.
FIGURE 4 Compound 13g induces colon cancer cell apoptosis. (A) HT29 cells were treated with the different concentrations of
13g or β-lap for 72h, and stained with FITC-Annexin V and PI through flow cytometric analysis. (B) The apoptotic rates were
quantitatively analyzed, and data (means ± SD) were collected from three separated experiments. *P< 0.01 vs control.
3.7 Compound 13g regulates apoptosis and DNA damage related proteins
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Proteins of the Bcl-2 family, particularly Bcl-2 and Bax, are involved in multiple apoptotic pathways.
Caspase-3 is closely associated with apoptosis. DNA damage is one of the molecular events associated with cell
apoptosis. Histone H2AX, a variant form of histone H2A, is required for checkpoint-mediated DNA repair
following double-stranded DNA breaks. To further investigate the ability of 13g to induce apoptosis and DNA
damage in the colon cancer cells, the expression level of apoptotic proteins (Bcl-2, Bax, and cleaved-caspase 3)
and DNA damage marker H2AX, in response to 13g treatment, was determined. HT29 cells were incubated with
13g for 72h, and the cells were lysed and subjected to analysis in western blot, using β-lap as a positive control.
It was observed that 13g dose-dependently augmented the pro-apoptotic Bax levels but diminished the
anti-apoptotic Bcl-2 levels (Figure 5). Importantly, 13g also resulted in more significant accumulation of cleaved
caspase-3 than both the control and β-lap-treated group as shown in Figure 5. In addition, H2AXS139ph levels
in 13g treatment groups were remarkably higher than that in β-lap. Taken together, these results confirmed that
the incubation with 13g could induce apoptosis and DNA damage in HT29 cells.
FIGURE 5 (A) The levels of apoptosis related proteins and DNA damage protein in HT29 cells upon with 13g or β-lap in WB
assay. (B) The relative levels of each apoptosis protein were quantitatively analyzed compared to the internal reference β-actin, and
the related data (means ± SD) were from three independent assays. *P < 0.01 vs vehicle control.
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1800
1600
1400
1200
1000
800
600
400
200
0
Control
13g 50 µmol/L
13g 100 µmol/L
β-lap 50 µmol/L
1
4
7
10
13
16
19
22
Days
FIGURE 6 The antitumor effect of 13g on HT29 xenograft in nude mice. After establishing xenograft mice model, tumor volume
changes in tumor-bearing mice administrated with saline control, 13g, or β-lap were surveyed during 21 days.
3.8 In vivo antitumor efficacy of 13g
In order to assess the in vivo antitumor efficacy of 13g, four BALB/c nude mice groups were subcutaneously
injected with HT29 cells. After establishing xenograft mice model, these tumor-bearing mice were
intraperitoneally injected with saline, 13g, and β-lap every day for 21 days, respectively. The body mass and
tumor size of nude mice were surveyed every three days during the 21 days. As shown in Figure 6, a sustainable
tumor growth was observed with the saline treated group. In contrast, the intraperitoneal treatment of 13g
apparently diminished the volumes of xenograft tumors. Notably, 13g produced greater tumor reduction at 100
μmol/L with the tumor weight (0.56 ± 0.07 g), which were reduced by 67.4% (w/w) compared to the vehicle
treated controls (1.72 ± 0.36 g). Furthermore, 13g also exhibited slightly more potent anti-tumor activity than the
β-lap treatment, where the tumor weight was 0.92 ± 0.18 g at 50 μmol/L. Together, these data clearly confirm
that 13g possessed remarkable inhibitory potency of the tumor growth in vivo.
3.9 Structure-Activity Relationships (SARs)
Analysis of SARs revealed that the linker length of target compounds 12a−i and 13a-i influences the
antitumor activities. The antitumor potency of hybrids declines with extending the linker. For example,
compounds linked with ethanediamine (n = 1) displayed better antitumor effects than those with butanediamine
(n = 3) or hexanediamine (n = 5). Carbolines with a 4-methoxyphenyl group showed more potent activities than
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the other two classes containing either methyl or 4-methylphenyl. The potency order of substituents is
4-methoxyphenyl > 4-methylphenyl > methyl. These findings indicate that a suitable substituent of the
1-position is preferred for antiproliferative activities of target compounds. In addition, C6-substituted
amino-indole-4,7-quinones 13a-i in general displayed slightly stronger antiproliferative activity than their
isomers
C5-substituted
amino-
indole-4,7-quinones
12a-i,
suggesting
the
C6-substituted
amino-indole-4,7-quinones is preferred for antitumor efficacy.
4 CONCLUSION
In summary, we have designed and prepared 18 new β-carboline-based indole-4,7-quinone derivatives
(12a−i and 13a-i), and evaluated their in vitro and in vivo antitumor activities. Most of these hybrids possessed
low micromolar proliferative inhibitory activity against three human tumor cells. The most active compound 13g
showed stronger antiproliferative activity than β-lap in three cancer cells. More importantly, 13g exerted
significant NQO1 inhibitory effects better than β-lap in HT29 cells. Furthermore, 13g induced higher levels of
ROS in dose-dependent manner in HT29 cells, and selectively inhibited tumor cell but not normal colon cell
growth in vitro. In addition, 13g exhibited a significant induction of cell apoptosis and DNA damage through
regulating apoptotic relative proteins and H2AX expression. Finally, 13g possessed satisfactory growth
inhibitory potency upon cancer cell xenograft in vivo. These results indicate that these new β-carboline-based
indole-4,7-quinone derivatives described here may be a potential candidate for the discovery of anti-colon tumor
drugs.
ACKNOWLEDGMENTS
The authors sincerely thank the fund support by Project of Jiangsu Pharmaceutical Association (A2017016),
Science and technology project of Nantong City (MS12018077, MS12018058, and JC2018125).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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