Bifunctional Naphtho[2,3- d][1,2,3]triazole-4,9-dione Compounds Exhibit Antitumor Effects in Vitro and in Vivo by Inhibiting Dihydroorotate Dehydrogenase and Inducing Reactive Oxygen Species Production

Article abstract of DOI:10.1021/acs.jmedchem.0c00512

Human dihydroorotate dehydrogenase (hDHODH) is an attractive target for cancer therapy. Based on its crystal structure, we designed and synthesized a focused compound library containing the structural moiety of 1,4-benzoquinone, which possesses reactive oxygen species (ROS) induction capacity. Compound 3s with a naphtho[2,3-d][1,2,3]triazole-4,9-dione scaffold exhibited inhibitory activity against hDHODH. Further optimization led to compounds 11k and 11l, which inhibited hDHODH activity with IC50 values of 9 and 4.5 nM, respectively. Protein-ligand cocrystal structures clearly depicted hydrogen bond and hydrophobic interactions of 11k and 11l with hDHODH. Compounds 11k and 11l significantly inhibited leukemia cell and solid tumor cell proliferation and induced ROS production, mitochondrial dysfunction, apoptosis, and cell cycle arrest. Nanocrystallization of compound 11l displayed significant in vivo antitumor effects in the Raji xenograft model. Overall, this study provides a novel bifunctional compound 11l with hDHODH inhibition and ROS induction efficacy, which represents a promising anticancer lead worthy of further exploration.

Full text of DOI:10.1021/acs.jmedchem.0c00512

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Article
Bifunctional Naphtho[2,3-d][1,2,3]triazole-4,9-dione Compounds Exhibit
Antitumor Effects In Vitro and In Vivo by Inhibiting Dihydroorotate
Dehydrogenase and Inducing Reactive Oxygen Species Production
zeping zuo, Xiaocong Liu, Xinying Qian, Ting Zeng, Na Sang, Huan Liu, Yue Zhou,
Lei Tao, Xia Zhou, Na Su, Yamei Yu, Qiang Chen, Youfu Luo, and Ying-Lan Zhao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00512 • Publication Date (Web): 04 Jun 2020
Downloaded from pubs.acs.org on June 4, 2020
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Bifunctional Naphtho[2,3-d][1,2,3]triazole-4,9-dione Compounds Exhibit
Antitumor Effects In Vitro and In Vivo by Inhibiting Dihydroorotate
Dehydrogenase and Inducing Reactive Oxygen Species Production
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Zeping Zuo,a,# Xiaocong Liu,a,# Xinying Qian,a,# Ting Zeng,a,# Na Sang, Huan Liu,
a
a
a
a
a
c
a
a
a,*
Yue Zhou, Lei Tao, Xia Zhou, Na Su, Yamei Yu, Qiang Chen, Youfu Luo,
Yinglan Zhaoa,b,*
a
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West
China Medical School, Sichuan University, and Collaborative Innovation Center for
Biotherapy, Chengdu, 610041, China
b
West China School of Pharmacy, Sichuan University, Chengdu, 610041, China
c
Department of Pharmacy, West China Hospital, Sichuan University, Chengdu 610041,
China.
ABSTRACT
Human dihydroorotate dehydrogenase (hDHODH) is an attractive target for
cancer therapy. Based on its crystal structure, we designed and synthesized a focused
compound library containing the structural moiety of 1,4-benzoquinone which
possesses reactive oxygen species (ROS) induction capacity. Compound 3s with a
naphtho[2,3-d][1,2,3]triazole-4,9-dione scaffold exhibited inhibitory activity against
hDHODH. Further optimization led to compounds 11k and 11l which inhibited
hDHODH activity with IC values of 9 nM and 4.5 nM, respectively. Protein-ligand
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cocrystal structures clearly depicted hydrogen bond and hydrophobic interactions of
11k and 11l with hDHODH. Compounds 11k and 11l significantly inhibited leukemia
cells and solid tumor cells proliferation, induced ROS production, mitochondrial
dysfunction, apoptosis and cell cycle arrest. Nanocrystallization of compound 11l
displayed significant in vivo antitumor effects in Raji xenograft model. Overall, this
study provides a novel bifunctional compound 11l with hDHODH inhibition and ROS
induction efficacy which represents a promising anticancer lead worth further
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exploration.
INTRODUCTION
Human dihydroorotate dehydrogenase (hDHODH) is the fourth and rate-limiting
enzyme in the pyrimidine de novo synthesis pathway, which is located in the inner
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mitochondrial membrane . In addition to catalyzing the oxidation of dihydroorotate to
orotate with the participation of the cofactors flavin mononucleotide (FMN) and
ubiquinone, hDHODH reduces ubiquinone, linking the pyrimidine pathway to the
3
mitochondrial respiratory chain . Overexpression of hDHODH has been observed in
4,5
many cancers, including acute myeloid leukemia (AML) , colorectal cancer,
melanoma and pancreatic cancer, and is negatively associated with the poor survival
6–9
rate of cancer patients . Knockdown of hDHODH effectively inhibits cancer cell
proliferation, invasion and migration. The inhibition of hDHODH is a strategy for
overcoming the differentiation blockade in AML, and affects ATP depletion,
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endogenous ROS and the mediation of S-phase arrest in cancer cells . Additionally,
hDHODH inhibitors sensitize multiple cancers to chemotherapy. Triple-negative breast
cancer cells exhibited reduced chemotherapy resistance and were sensitized to
1
1
genotoxic chemotherapy agents after hDHODH inhibition . Hence, hDHODH is a
promising target with a vital role in the treatment of cancers.
Leflunomide and brequinar (BRQ) are two representative hDHODH inhibitors
(Figure 1A). Leflunomide, an FDA-approved drug for the treatment of rheumatoid
arthritis and psoriatic arthritis, was the first reported hDHODH inhibitor12,13. The
second reported hDHODH inhibitor, BRQ, was initially applied in therapy to prevent
organ transplant rejection and then regarded as an antitumor agent for multiple cancer
treatments14,15. The antitumor efficiency of leflunomide and BRQ was evaluated in
clinical trials against several malignancies, including melanoma and breast cancer.
However, no approval was obtained for their clinical use in cancer treatment because
of their limited efficiency and potential toxicity16–21. More recently, an increasing
number of new hDHODH inhibitors have been investigated for cancer treatment22,23. 4-
Quinoline carboxylic acids and 2-hydroxypyrazolo[1,5-a]pyridine hDHDOH inhibitors
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were investigated for the treatment of colorectal cancer and AML in preclinical studies
24,25.
ASLAN003 , BAY2402234 and AG-636 were explored to treat AML in Phase
26
27
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II clinical trials in 2017, leukemia in Phase I clinical trials in 2018 and lymphoma in
Phase I clinical trials in 2019, respectively (Figure 1A). BAY2402234 showed strong
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anti-myeloid malignancies with good ADME properties . However, all of the above-
mentioned hDHODH inhibitors have not yet achieved success as a marketed drug to
clinically treat cancers. Consequently, the discovery of diverse classes of hDHODH
inhibitors with beneficial properties is desirable.
Emerging studies have suggested that hDHODH depletion partially inhibits the
respiratory chain complex III, decreases the mitochondrial membrane potential, and
30
increases reactive oxygen species (ROS) generation . ROS are chemically reactive
molecules that have essential functions in living organisms. Excessive amounts of ROS
3
1
cause oxidative damage to the lipids, proteins and DNA of cancer cells . ROS
3
2
induction leads to the preferential killing and selective eradication of cancer cells .
Therefore, manipulating ROS levels by redox modulation is a way to selectively kill
cancer cells. Pharmacological use of ROS-inducing small molecules is considered an
effective strategy to combat against cancer.
It has been confirmed that compounds with a benzoquinone moiety can induce ROS
and apoptosis in cells. As shown in Figure 1B, the structural moiety of benzoquinone
is found to be included in many marketed anticancer drugs and investigational agents33–
37.
hDHODH. For example, decylubiquinone binds to the putative ubiquinone binding
Interestingly, ubiquinone derivatives are found to exhibit inhibitory activity against
3
8
pocket of hDHODH. DCL , another ubiquinone derivative, could interrupt the
oxidation of dihydroorotate with an IC value of 67 nM against hDHODH. Moreover,
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atovaquone, an antimalarial drug with a modest inhibitory effect against P. falciparum
DHODH (PfDHODH) in vitro (K = 27 μM) and weak inhibitory effect against
i
hDHODH (IC50 = 14.5 μM) contains benzoquinone fragments38,39. Thus, the
compounds with the structural moiety of benzoquinone might inhibit the activity of
hDHODH and induce the production of ROS.
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A
CF3
O
N
OH
O
O
F
O
O
H
N
O
O
Cl O
NH
N
N
O
N
OH
F
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F
Bayer-2402234, phase I
Decylubiquinone
ASLAN003, phase II
O
OH
F
O
Cl
O
O
F
F
F
OH
O
Cl
N
N
HO
O
H
O
N
F
Brequinar
DCL
Atovaquone
Leflunomide, approved
Cl
H
N
B
O
OH
OH
OH
O
O
HN
HN
OH
OH
O
OH
O
O
OH
NH2
O
HCl
O
O
O
OH
O
N
H
O
OH
Mitoxantrone
Napabucasin
Adriamycin
Figure 1. Chemical structures of reported hDHODH inhibitors and anticancer
agents with benzoquinone moiety. (A) Selected hDHODH inhibitors. (B) Anticancer
agents with ROS induction capability.
Based on the observations described above and the crystal structure of hDHDOH,
we designed a focused library of compounds containing the structural moiety of 1, 4-
benzoquinone to obtain active compounds with hDHODH inhibition and ROS-inducing
effects. In glide-docking experiments, we found that compounds with a naphtho[2,3-
d][1,2,3]triazole-4,9-dione scaffold possessed favorable binding mode to the protein
target (Figure 2A). The compound with a phenyl group substituted at the N-1 position
of the triazole ring occupied the proposed putative ubiquinone tunnel in hDHODH
(
PDB ID: 1D3G). Notably, it formed hydrogen bonds with the amino acid residues
R136 and Q47, which is similar to decylubiquinone (Figure 2B) and DCL (Figure 2C).
Furthermore, additional hydrophobic interactions were observed between the N-1
phenyl group of the described molecules and hDHODH. Thus, it is plausible that the
described molecules with hydrophobic substituents in the N-1 position of the triazole
ring may be bound to hDHODH and are promising agents for hDHODH inhibition.
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Figure 2. Chemical structures of designed hDHODH inhibitors and docking models
with hDHODH. (A) The designed naphtho[2,3-d][1,2,3]triazole-4,9-dione compound.
(
B) Docking models of a typical naphtho[2,3-d][1,2,3]triazole-4,9-dione compound
yellow) and decylubiquinone (cyan) with hDHODH. (C) Docking models of a typical
(
naphtho[2,3-d][1,2,3]triazole-4,9-dione compound (yellow) and DCL (green) with
hDHODH. Hydrogen bonds are shown as dashed lines. (D) Docking models of a typical
naphtho[2,3-d][1,2,3]triazole-4,9-dione compound (yellow) and atovaquone (pink)
with hDHODH. Hydrogen bonds are shown as dashed lines.
In the present study, we describe the design, synthesis, structure–activity
relationship (SAR) investigation, and X-ray crystallographic analysis of novel
bifunctional anticancer agents. The in vitro and in vivo biological assays (including
cell proliferation, cell cycle, apoptosis, uridine supplementation rescue experiment,
pharmacokinetics study, xenograft experiment and acute toxicity evaluation) were
performed to characterize the biological effects and action mechanisms of the designed
molecules. Our finding provides a novel structural class of hDHODH inhibitors with
ROS induction capability for cancer treatment.
RESULTS AND DISCUSSION
2
.1 Chemistry
To quickly access a panel of naphtho[2,3-d][1,2,3]triazole-4,9-dione compounds
for evaluating hDHODH inhibition, aryl azide building blocks with diverse structural
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features were synthesized. As illustrated in synthetic route A , the aryl azide
intermediates 2a–t were prepared from aryl boronic acids or aryl borates under copper-
catalyzed conditions. The final naphtho[2,3-d][1,2,3]triazole-4,9-dione compounds 3a–
t were made by condensation 2-hydroxynaphthalene-1,4-dione with different aryl
azides from the previous step. The Suzuki reaction was mainly employed to synthesize
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biphenyl azides with various substituent groups on either phenyl ring, which enabled
us to perform a more extensive SAR study (routes B and C)41, 42. Routes B and C differ
principally by the occurrence of such Suzuki couplings with a reactive functional group
R (-OH, -NH ) generated at a late and early stage, respectively. The advantage of route
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B is that R groups with sensitive moieties can be successfully added to produce
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biphenyl azides in high yields. However, route C was less tolerant of such hydrophilic
moieties and unable to obtain the desired compounds in a satisfactory manner. To
expand our SAR beyond the naphtho[2,3-d][1,2,3]triazole-4,9-dione limitations, we
developed routes D and E. Route D introduced diversity through the different
substituent 2-hydroxy naphthalene-1,4-dione via oxidation reaction with suitable
precursors, and route E enabled diversity through classic click chemistry between
different alkynyl and aryl azides. Double bond reduction was carried out by sodium
hydride to yield the corresponding alcohol (14j). From routes B–E, we obtained
compounds 7a–g, 11a–s, 14a–l, and 14k–l, respectively.
Scheme 1. Synthesis of Naphtho[2,3-d][1,2,3]Triazole-4,9-Dione Analogs
Synthetic route A
O
OH
N
N
O
O
a
b
R1
or R1
R1 N3
B
B
N
OH
O
R1
1a-t
2a-t
3a-t
1
1
1
a, 2a, 3a : R = 2-cyanophenyl
1k, 2k, 3k : R = 6-chloropyridin-3-yl
1
1b, 2b, 3b : R = 3-nitrilephenyl
1
1
l, 2l, 3l : R = 2-chloropyridin-4-yl
1
1
1
c, 2c, 3c : R = 3-hydroxyphenyl
1m, 2m, 3m : R = pyridin-4-yl
1
1
1
1
d, 2d, 3d : R = 2-methoxyphenyl
1n, 2n, 3n : R = naphthalen-2-yl
1
1
e, 2e, 3e : R = 3-iodophenyl
1o, 2o, 3o : R = 2H-1,3-benzodioxol-5-yl
1
1
1
f, 2f, 3f : R = 3-aminophenyl
1p, 2p,
1q, 2q,
3
p : R = 1H-1,3-benzodiazol-2-yl
1
1
1g, 2g, 3g : R = 4-isopropylphenyl
4-(pyridin-3-yl)phenyl
3
q : R =
1
1
h, 2h, 3h : R =2-(trifluoromethyl)phenyl
r, 2r, 3r : R =
4-(pyridin-4-yl)phenyl
1
1
1
1
1
1
i, 2i, 3i : R = 4-fluoro-3-methylphenyl
1s, 2s, 3s : R = 1,1'-biphenyl]-4-yl
1
1
j, 2j, 3j : R = 4-methyl-3-(trifluoromethyl)phenyl
1t, 2t, 3t : R = 6-(morpholin-4-yl)pyridin-3-yl
Reagents and conditions. (a) Aryl boronic acid or borate, NaN , CuSO , MeOH, 80 °C,
3
4
overnight (45–78%). (b) 2-Hydroxynaphthalene-1,4-dione, DBU, CH CN, 80 °C, 3–4
3
h (34–64%).
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Synthetic route B
O
N3
N3
NH2
N
N
a
b
c
N
O
R2
Br
4
Br
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R2
6a-g
7
a-g
2
6e, 7e : R2= 4-aminophenyl-1-yl
6
a, 7a : R = pyrimidin-5-yl
2
6
6
b, 7b : R = 2-methylpyridin-4-yl
2
6
f, 7f : R = 3-aminophenyl-1-yl
2
c, 7c : R = 2-hydroxyphenyl-1-yl
2
6
g, 7g : R = 2-aminophenyl-1-yl
2
6d, 7d : R = 3-hydroxyphenyl-1-yl
Reagents and conditions. (a) (i) Tert-butylnitrite, THF, -20 °C to rt, 2 h. (ii) NaN3,
CH CN/H O (30–79%). (b) Aryl boronic acid or borate, Pd(dppf)Cl ·DCM, Cs CO or
3
2
2
2
3
K CO , 1,4-dioxane/H O, overnight (30–75%). (c) 2-Hydroxynaphthalene-1,4-dione,
2
3
2
DBU, CH CN, 80 °C, 3–4 h (34–64%).
3
Synthetic route C
N3
NH2
O
O
NH2
a
b
R3
c
N
N
R3
R3
N
R4
Br
a-s
R4
R3
8
9
9a-s
10a-s
R4
11a-s
3
4
3
4
a, 10a, 11a : R = H, R =2'-F
9k, 10k, 11k : R = 2, 6-difluoro, R = 3'-OCH3
3
4
3
4
9
b, 10b, 11b : R = H, R =3'-F
9l, 10l, 11l : R = 2, 6-difluoro, R = 2'-F
3
4
3
4
9
c, 10c, 11c : R = H, R =3'-COOCH
9m, 10m, 11m : R = 2, 6-difluoro, R = 3'-F
3
3
4
3
4
9n, 10n, 11n : R = 2, 6-difluoro, R = 4'-F
9
9
9
d, 10d, 11d : R = H, R =3', 4', 5'-OCH
3
3
4
3
4
9o, 10o, 11o : R = 2, 6-
difluoro, R = 3'-CH2OH
e, 10e, 11e : R = 2-OCH , R = H
3
3
4
9p, 10p,
3
4
f, 10f, 11f : R = H, R = 3'-OCH
11p : R = 2, 6-difluoro, R = 3'-CH OCH
9q, 10q, 11q : R = 2, 6-difluoro
3
2
3
3
4
3
4
9g, 10g, 11g : R = H, R = 3', 5'-bis(trifluoromethyl)
, R = 3'-CH2COOCH3
9r, 10r, 11r : R = 2, 6-difluoro, R4
3
3
4
9
h, 10h, 11h : R = 3-F, R = 4'-isopropyl
= 3'-N(CH3)2
3
4
3
4
9s, 10s, 11s : R = 2, 6-difluoro
, R = 2'-F, 3'-CH2OH
9
i, 10i, 11i : R = 3, 5-difluoro, R = 4'-isopropyl
3
4
9
j, 10j, 11j : R = 2, 6-difluoro, R = 4'-isopropyl
Reagents and conditions. (a) Aryl boronic acid or borate, Pd(dppf)Cl ·DCM, Cs CO
3
2
2
or K CO , 1,4-dioxane/H O, 100 °C, overnight (30–75%). (b) (i) Boron trifluoride
2
3
2
diethyl etherate, Tert-butylnitrite, THF, -20 °C to rt, 2 h. (ii) NaN , CH CN/H O (30–
3
3
2
79%). (c) 2-Hydroxynaphthalene-1,4-dione, DBU, CH CN, 80 °C, 3–4 h (34–64%).
3
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Synthetic route D
N3
NH2
NH2
a
b
N
N
R6
F
F
F
N
F
F
F
F
F
c
Br
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
6
R = 13 a-f, or substituted-ketone
F
F
O
O
OH
d
R5
R5
14 a-i
O
12 a-b,e-f
13 a-b,e-f
6
6
1
2a,13a, 14a : R = 6-methoxynaphthalene-1,4-dione-yl
12b,13b, 14b : R = 7-methoxynaphthalene-1,4-dione-yl
e
e
6
6
1
2c,13c, 14c : R = 6-hydroxynaphthalene-1,4-dione-yl
12d,13d, 14d : R = 7-hydroxynaphthalene-1,4-dione-yl
6
6
1
2e,13e, 14e : R = 6-fluoronaphthalene-1,4-dione-yl
12f,13f, 14f : R = 7-fluoronaphthalene-1,4-dione-yl
6
6
13g, 14g : R = 5,5-dimethylcyclohex-2-en-1-one-y
13h, 14h : R = cyclohex-2-en-1-one-yl
6
13i, 14i : R = 5-isopropylcyclohex-2-en-1-one-yl
O
O
OH
OH
N
N
N
f
N
N
N
F
F
F
F
F
F
1
4j
11l
Reagents and conditions. (a) Boronic acid or boronic acid ester, Pd(dppf)Cl ·DCM,
2
Cs CO /K CO , 1,4-dioxane/H O, 100 °C, overnight (30–75%). (b) (i) Boron
2
3
2
3
2
trifluoride diethyl etherate, Tert-Butylnitrite, THF, -20 °C to rt, 2 h, (ii) NaN3,
CH CN/H O (30–79%). (c) 2-Hydroxynaphthalene-1,4-dione, DBU, CH CN, 80 °C,
3
2
3
3
6
–4 h (34–64%). (d) Anhydrous tert-butanol, O , Potassium tert-butoxide, 3 h, rt, 52-
2
8%. (e) Boron tribromide, DCM, 0–5 °C, 64-67%. (f) Sodium hydride, MeOH, rt, 39%.
Synthetic route E
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
R7
N3
N
N
NH2
NH2
Br
N
a
b
g
F
F
F
F
F
F
F
F
F
R7
F
F
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14k-l
7
7
1
4k ; R = acetophenone-yl
14l ; R = phenyl
Reagents and conditions. (a) Boronic acid or boronic acid ester, Pd(dppf)Cl ·DCM,
2
Cs CO /K CO , 1,4-dioxane/H O, 100 °C, overnight (30–75%). (b) (i) Boron
2
3
2
3
2
trifluoride diethyl etherate, Tert-Butylnitrite, THF, -20 °C to rt, 2 h. (ii) NaN3,
CH CN/H O (30–79%). (g) CuSO4 (0.02eq.), (+)-sodium L-ascorbate (0.1 eq) in 1:1
3
2
H O:t-BuOH, rt for 24 h 46% or 41%.
2
2
.2 Structure-Activity Relationship (SAR)
After visualization by in silico docking results, we firstly investigated the
hDHODH inhibition activity of twenty naphtho[2,3-d][1,2,3]triazole-4,9-dione
compounds with diverse N-1 substitutions of phenyl, pyridyl, naphthyl, benzimidazolyl
and biaryl structures (Scheme 1A). Lipophilic ligand efficiency (LipE) is believed to
be an important metric for prediction of absorption, distribution, metabolism, and
excretion (ADME) properties and BRQ is a lipophilic drug that has relatively low LipE.
Thus, in our medicinal chemistry campaign for finding novel hDHODH inhibitors as
potential anticancer agents against solid tumors, we hope to modulate the LipE values
of the compounds, while maintaining their hDHODH inhibitory effect. The results
showed that compound 3s with a diphenyl group at the N-1 position was the most potent
for hDHODH inhibition (IC = 19 nM), and it possessed an improved lipophilic ligand
5
0
efficiency (LipE = 2.69) compared with BRQ (1.9). Compound 3q with a 4-(4-(pyridin-
3-yl)phenyl)-group at the N-1 position of the naphtho[2,3-d][1,2,3]triazole-4,9-dione
scaffold had a better LipE value than compound 3s, but exhibited a decreased activity
against hDHODH with IC50 value of 422 nM. Compound 3g showed an enzyme
inhibition (IC = 560 nM) that was similar to compound 3q, but had a lower LipE value
50
than BRQ. Other compounds in this series did not exhibit inhibition more than 50%
against hDHODH activity at maximum test concentration (1000 nM). Collectively,
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these results indicated that compound with a diphenyl group at the N-1 position favors
hDHODH activity and improves lipophilic ligand efficiency, thus is worthy of further
optimization.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Physicochemical Properties and Biological Activity of Compounds 3a–t
and 7a–b
O
N
N
N
R
O
Compd
R
clogP
6.38
LipE
1.9
IC (nM)
50
BRQ
5.2
3
a
2.72
2.72
2.89
3.25
4.27
2.30
4.57
4.03
3.79
4.53
2.77
2.77
2.05
4.32
<3.28
<3.28
<3.11
<2.75
<1.73
<3.70
1.68
＞1000
CN
3
b
＞1000
＞1000
＞1000
＞1000
＞1000
560
NC
HO
3
c
3d
O
3
e
I
3
f
H2N
3
g
3
h
<1.97
<2.11
<1.47
<3.23
<3.23
<3.95
<1.68
＞1000
＞1000
＞1000
＞1000
＞1000
＞1000
＞1000
CF3
F
3i
3
j
F3C
Cl
3
k
N
N
3
l
Cl
N
3
m
3n
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
3
o
3.20
2.99
3.54
3.54
5.03
2.05
2.38
4.03
<2.80
<2.01
2.83
＞1000
＞1000
422
O
H
N
3
3
p
q
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
3r
<2.46
2.69
＞1000
19
3
s
O
N
3
t
<3.95
<3.62
<1.97
＞1000
＞1000
＞1000
N
N
7
a
N
N
7
b
Data for cLogP values were predicted using ChemBioDraw Professional 14 Software.
4
3
LipE was calculated as follows: LipE = -pIC (M) -cLogP .
50
Considering compound 3s with diphenyl skeleton exhibited potent inhibitory
activity against hDHODH and improved LipE value, we next designed and synthesized
twenty-four analogs by introducing diverse substituent groups at different positions of
the diphenyl skeleton to further enhance the enzyme activity of compounds. As shown
in Table 2, 50% of the synthesized analogs (12 out of 24) reached single-digit
nanomolar IC50 values. Four compounds (11d, 11g, 11h and 11i) displayed low
inhibitory effects with IC values over 1000 nM. These compounds seemed to have
5
0
substituent(s) that created high steric hindrances, such as the 3',4',5'-trimethoxy groups
linked to compound 11d, 3',5'-trifluoromethyl groups to 11g and 4'-isopropyl groups to
1
1h and 11i. Nevertheless, compound 11j, similarly bearing a 4'-isopropyl group,
showed potent activity with an IC value of 76 nM, which is much stronger than that
50
of compound 11i. The difference between compounds 11i and 11j is the location of two
fluorine atoms. Compound 11i is 2,6-difluorosubstitution, whereas compound 11j is
3,5-difluorosubstitution. The helices α1 and α2 in hDHODH form a slot in the so-called
hydrophobic patch, which narrows toward the proximal redox site (Figures 2B and 2C).
The size of the tunnel and substituent may afford undesirable clashes or limit the
degrees of conformational freedom. Compound 11i with 3,5-difluorosubstitution might
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results in a repulsive interaction with the hydrophobic patch, afford undesirable clashes
or limit the degrees of conformational freedom, whereas compound 11j with 2,6-
difluorosubstitution might not. Thus, 2,6-difluorosubstitution is favored for enzyme
inhibition activity over 3,5-difluorosubstitution. This observation prompted us to
synthesize more analogs (11k–s) with the favored 2, 6-difluorosubstitution. All of these
newly synthesized molecules displayed good inhibitory activity against hDHODH at
nanomolar or subnanomolar concentrations (Table 2). Similar to lead compound 3s,
most of the active compounds described herein have improved lipE values, except for
compound 11j. The hydrophobic property of the propyl group on compound 11j may
account for its low lipE value (0.37).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Physicochemical Properties and Biological Activity of Compounds 7c–g
and 11a–s
O
N
N
N
R
O
Compd
R
clogP
LipE
IC (nM)
5
0
BRQ
6.38
4.06
1.9
5.2
OH
7
c
2.58
230
21
HO
7d
4.36
3.80
3.80
3.80
5.17
5.17
5.00
4.33
3.32
3.80
4.7
H2N
7
e
25
H2N
7
f
3.1
7
g
3.89
3.17
2.08
1.26
<1.67
20
NH2
1
1a
11b
1c
1d
4.5
F
56
F
1
550
＞1000
O
O
O
O
1
O
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
1e
1f
4.58
4.95
3.80
2.05
4.1
O
1
100
O
F3C
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
11g
6.80
6.60
<-0.8
＞1000
F3C
1
1h
11i
1j
11k
1l
1m
11n
<-0.60
<-0.75
0.37
＞1000
＞1000
76
F
F
6.75
6.75
5.24
5.46
5.46
5.46
F
F
F
1
F
2.8
9.0
O
F
F
F
1
2.88
4.5
F
F
1
3.10
2.7
F
F
F
F
F
2.70
6.9
F
1
1o
4.28
5.12
4.64
3.67
1.2
1.6
F
F
OH
11p
F
O
F
F
11q
5.00
3.33
4.6
O
O
F
F
1
1r
5.49
4.43
3.81
4.49
0.5
1.2
N
F
F
F
11s
OH
Data for cLogP values were calculated using ChemBioDraw 14 trial. LipE was
4
3
calculated using LipE = -pIC (M) - cLogP .
5
0
To further explore the structure-activity relationship, we further synthesized a
series of derivatives by introduction of different substituents to the naphthyl ring of
compound 11l, or replacement of its benzoquinone scaffold with other groups. As
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shown in Table 3, most of these final molecules showed reduced activities compared
with 11l. However, compound 14j (with benzoquinone reduced to tetrahydroxyquinone)
maintained comparable potency against hDHODH. Nine compounds (14a–d, 14g–i and
1
1
4k–l) displayed a dramatic decrease of over 500 nM in their IC values. Compounds
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4e–f (IC = 98 or 152 nM) showed stronger activity than 14a–d. We deduced that the
5
0
fluorine atom fit well in the small pocket for compounds 14e–f. Conversely, compounds
4a–d (with the larger methoxyl or hydroxyl substituent) clashed with the protein.
Replacement of the benzoquinone scaffold of compound 11l with a cyclohexyl ketone
14g–i) or removal of its quinone structural fragment (14k–l) diminished hDHODH
1
(
inhibition.
Table 3. Physicochemical Properties and Biological Activity of Compounds 14a–i
R
F
F
F
Compd
R
clogP
6.38
LipE
1.9
IC (nM)
5
0
BRQ
5.2
4.5
1
1l
5.46
2.88
O
O
O
N
N
N
1
4a
5.40
5.40
5.24
5.24
5.61
5.61
<0.60
<0.60
<0.76
<0.76
<0.39
<0.39
>1000
>1000
>1000
>1000
98
O
N
N
1
4b
O
N
O
O
HO
N
N
1
4c
N
O
O
N
N
N
1
4d
HO
O
O
F
N
N
14e
14f
N
O
O
N
N
N
152
F
O
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
N
1
4g
N
5.28
4.24
5.69
<0.72
>1000
>1000
520
O
N
14h
N
<1.76
<0.31
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
1
4i
N
N
OH
OH
N
N
N
1
4j
3.20
5.52
2.10
5.1
O
N
N
N
1
4k
<0.48
＞1000
14l
N
N
6.33
<-0.33
＞1000
N
Data for cLogP values were calculated using ChemBioDraw 14 trial. LipE was
43
calculated using LipE = -pIC (M) - cLogP .
5
0
2
.3 Binding mode analysis through the crystal structure of hDHODH in complex
with compounds 11k, 11l, 11r and 11s
hDHODH is composed of a large C-terminal domain and a small N-terminal
domain, linked by an extended loop. The large C-terminal domain conceals the redox
4
4
site in which FMN and the dihydroorotate substrate bind . The N-terminal extension
consists of two helices, α1 and α2, harboring the binding site for the cofactor
ubiquinone. The helices α1 and α2 form a slot in the so-called hydrophobic patch with
the short α1 and α2 loop at the narrow end of the slot. This promotes the formation of
a tunnel that ends at the FMN cavity near the α1 and α2 loop. This tunnel narrows
toward the proximal redox site. A previous study suggested that ubiquinone
(
cosubstrate) was probably inserted into the tunnel and thus easily approached FMN for
3
the redox reaction . The crystal structures of hDHODH in complex with a brequinar
4
5
analog were recently published . To investigate the binding mode of naphtho[2,3-
d][1,2,3]triazole-4,9-dione-based inhibitors, the most potent compounds 11k, 11l, 11r
and 11s were selected for respective cocrystallization with hDHODH. The crystal
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structures were refined to 1.8 Å resolution, and the coordinates were deposited in the
Protein Data Bank as entries 6LP7, 6JME, 6LP8 and 6LP6, respectively. Details of the
data collection and refinement statistics are summarized in Table S1. The high
resolution and clear density map enabled us to unambiguously determine the position
and orientation of these inhibitors (Figure S1), thus revealing the detailed interactions
between them and hDHODH (Figures 3A, B, E, F). The binding sites of these
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compounds were clearly in the same pocket, along with the reported inhibitors24,25,46,47
,
again supporting the pocket as a rational target for the development of novel inhibitors.
All four inhibitors occupied the proposed putative ubiquinone tunnel, stabilized by a
substantial number of hydrophobic interactions with the side chains of hDHODH
residues lining the pocket (M43, L46, A55, L58, A59, F62, F98, M111, V143, L359,
Y356, T360 and P364) (Figures 3A–D). The biphenyl ring system occupied most of
the hydrophobic pocket. The naphtho[2,3-d][1,2,3]triazole-4,9-dione moiety occupied
the innermost part of the pocket near the redox site, including V143, L146, Y356,
L359, T360 and P364 (Figures 3A–D). The carbonyl and triazole rings of these
compounds formed hydrogen bonds with R136 (Figures 3E–H). Inhibitor 11l featured
a fluorobenzene ring that adopted two different orientations, which accounted for the
high potency of compound 11l. On the basis of the ligand omit map density (Figure
S1), the fluorine substituent was directed toward the α1 and α2 helices, and these two
conformations had the same occupancy. The methoxy oxygen of 11k pointed toward
the hydroxyl group of Y38 and the carbonyl group of L67 at distances of 3.7 Å and
4
.6 Å, respectively, too far to form a direct H-bond. In contrast to compound 11k, the
hydroxyl group of compound 11s formed hydrogen bonds with the side chain of Y38
and the main chain of L67 (Figure 3G). Compound 11r formed hydrophobic
interactions with Y38 and M111 via the dimethylamino group (Figure 3D). Moreover,
we carried out the binding mode assay of A771726, BRQ, Bay-2402234 and
compound 11l with hDHODH, and compared their complex structures (Figure S2).
The results showed that A771726, BRQ, Bay-2402234 and 11l all occupied the
ubiquinone tunnel, though the amino acid residues that interact with them were not
consistent. Hydrophobic interactions were also observed. These results supported the
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intrinsic plasticity of binding site with hDHODH . Thus, hDHODH may
accommodate a diverse range of inhibitors, rationalizing the strategy of designing
inhibitors with diverse scaffolds.
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Figure 3. Interaction between hDHODH and compounds 11k (yellow PDB 6LP7),
11l (green PDB 6JME), 11s (cyan PDB 6LP6) and 11r (blue PDB 6LP8). Residues
that interacted with the inhibitors are labeled and shown as stick representation. (A)–
(D) showed the hydrophobic interactions, and (E)–(H) showed the hydrogen bonds (red
dashed lines).
2
.4 In vitro antiproliferative activity of compounds against human cancer cell lines
The proliferation inhibitory effect of the compounds against human cancer cell
lines was measured using an MTT assay. BRQ was used as a positive control. The
results showed that the cell proliferation inhibitory effects of the compounds were
largely related to their solubility and effect on the enzyme inhibitory activity (Table 4).
For instance, compounds without any substituent at the diphenyl skeleton (3s), or with
hydroxyl at meta (7d), amino at para (7e) or ortho (7g) of biphenyl terminal ring which
showed moderate inhibition activity against hDHODH (IC50 = 19-25 nM) only
exhibited weaker anti-proliferative effects with IC values of 0.2-9.04 μM in several
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cancer cells and > 10 μM in most of cancer cells. Introduction the amino at meta (7f),
fluorine at ortho (11a), methoxyl at meta (11e) of biphenyl intermediate ring
significantly increased inhibitory potency against hDHODH, whereas only slightly
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improved the antiproliferative efficacy compared with compounds 7e and 7g. The poor
membrane permeability or the solubility values might be responsible for the moderate
cell proliferation inhibition of compounds 7f, 11a and 11e.
Notably, compounds 11k and 11l showed superior antiproliferative activities to
BRQ. Compound 11k exhibited potent anti-proliferative effects against human
melanoma A375 cells and human colorectal cancer HCT116 cells with IC values of
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.04 and 0.60 μM, respectively, and considerable potency against human B-cell
lymphoma Raji cells (IC = 0.48 μM), human chronic myelogenous leukemia K562
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cells (IC = 0.34 μM) and human B-cell lymphoma Nalmawa cells (IC = 1.04 μM).
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50
Compound 11l displayed more potent activity against A375 and HCT-116, with IC50
values of 0.02 and 0.29 μM, respectively, and considerable potency against Raji (IC50
=
0.16 μM), K562 (IC = 0.11 μM) and Nalmawa (IC = 0.99 μM). These data were
50 50
consistent with the potent inhibition activity against hDHODH (IC = 4.5-9 nM) and
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better solubility (21.4-33.8 μg/ml in PBS) of compounds 11k and 11l. On the other
hand, compounds with fluorine at para (11n), 3'-F (11m), 3'-F (11o), 3'-CH OCH
2
3
(11p), 3'-CH COOCH (11q), 3'-N(CH ) (11r), 2'-F, 3'-CH OH (11s) of biphenyl
2
3
3 2
2
terminal ring showed more potent hDHODH activity, but lower cellular bioactivities
compared with compounds 11k and 11l. Their relatively low inhibitory activity against
cancer cells could be attributed to its poor membrane permeability, as observed in the
predicted QPPCaco-2 values by Schrodinger_Suites or the poor solubility. Overall,
compounds 11k and 11l predominantly exhibited proliferation inhibition against cancer
cells, which deserves further investigation.
Table 4. Antiproliferation Activity of Compounds against Human Ccancer Cell
Lines
a
solubility
MTT Assay (IC )
5
0
Compd QPPCaco-2b
(µg/mL)
A375
0.59
5.83
HCT116 Hela A549 MCF-7 Raji
K562
0.54
>10
Nalmawa
>10
BRQ
328.45
464.29
75.17
nd
4.12
4.23
>10
>10
4.10
>10
>10
>10
2.29
4.61
3
s
1.87
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d
104.75
121.01
120.92
166.98
464.23
561.15
465.41
465.18
465.26
465.24
142.23
465.19
141.42
432.69
142.50
765.20
nd
>10
1.90
0.25
1.93
2.98
0.01
0.04
0.02
0.19
1.58
0.03
0.18
0.42
0.02
0.03
0.16
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>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.02
>10
>10
>10
>10
>10
>10
>10
>10
2.25
1.65
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
9.61
>10
>10
>10
>10
>10
>10
>10
>10
9.04
>10
1.36
3.84
2.23
0.79
0.48
0.16
1.51
>10
3.31
1.23
3.76
2.15
>10
>10
7.09
0.24
>10
2.16
1.87
0.21
0.34
0.11
>10
>10
>10
>10
2.41
>10
>10
>10
>10
>10
>10
5.05
2.79
>10
1.04
0.99
>10
>10
>10
>10
>10
>10
>10
>10
7
e
nd
7
f
<10.0
nd
0.88
2.30
3.33
0.18
0.60
0.29
0.55
2.25
2.21
1.13
1.61
0.41
0.15
0.58
7
g
0
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1
1a
1e
1k
1l
1m
1n
<10.0
15.4
21.4
33.8
<10.0
nd
1
1
1
1
1
1
1o
1p
1q
14.6
<10.0
<10.0
<10.0
17.6
<10.0
1
1
1
1r
1s
14j
1
>10
a
b
IC , the mean value of triplicate measurements. Data for QPPCaco-2 values were
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calculated by Schrodinger_Suites.
2.5. Uridine supplementation rescues cell proliferation inhibition induced by
compounds 11k and 11l
As the inhibition of hDHODH activity reduces the de novo pyrimidine nucleotide
synthesis that is vital for cancer cell proliferation, supplementation of exogenous
uridine in cancer cells could rescue the proliferation inhibitory effects of hDHODH
inhibitors. To evaluate whether the hDHODH inhibition by compounds 11k and 11l is
mainly responsible for their antiproliferative effect, we carried out uridine
supplementation rescue experiments in Raji cells. The results showed that uridine
supplementation significantly rescued the cell proliferation inhibition induced by
compounds 11k and 11l (Figure 4). Interestingly, the rescue effect was more obvious
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after compound 11l treatment than 11k treatment, which is consistent with the stronger
antiproliferation and hDHODH inhibitory activity of compound 11l. Besides hDHODH,
other enzymes such as carbamoyl phosphate synthetase Ⅱ, aspartate transcarbamoylase
and orotate phosphoribosyl transferase (OPRT) are involved in de novo pyrimidine
biosynthesis. The inhibition of above enzymes may also result in cell proliferation
rescue by uridine supplementation. We performed glide-docking experiments and
found that both compound 11l and BRQ can’t bind to OPRT (PDB ID :4HKP) which
is the only enzymes with crystal structure, suggesting the compound 11l might not bind
to OPRT.
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In conclusion, these data demonstrated that compound 11k and 11l inhibited
cancer cells proliferation by inhibiting hDHODH activity.
Figure 4. Uridine supplementation rescues Raji cell proliferation inhibition
induced by compounds 11k and 11l. Raji cells were treated with 11k, 11l or BRQ
with or without uridine (U, 500 μM). Data are shown as the mean ± SD. *p < 0.05; **p
<
0.01; ***p < 0.001.
2
.6. Compounds 11k and 11l induce ROS production and decrease mitochondrial
membrane potential
As we aimed to develop an hDHODH inhibitor with ROS inducing efficiency, we
examined whether compounds 11k and 11l could induce the elevation of ROS levels in
the Raji cells. The results showed that compounds 11k or 11l treatment upregulated the
ROS levels in Raji cells in a concentration-dependent manner (Figure 5A). Compared
with compounds 11k or 11l, the classic hDHODH inhibitor BRQ did not increase the
ROS level at a concentration of 0.008 μM, and showed less increase at a concentration
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of 0.04 μM. These findings suggested that compounds 11k and 11l induced more
obvious ROS production than the classic hDHODH inhibitors. Moreover, we found that
compounds 11k or 11l induced ROS production at a very low concentration (8 nM),
which was similar to that of hDHODH inhibition (IC = 4.5-9 nM). This suggested
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that compounds 11k or 11l directly induced ROS production, but was not dependent on
hDHODH inhibition. We found that compounds 11k and 11l exhibited stronger
proliferation inhibitory effect than BRQ, and potent ROS induction effect of
compounds 11k and 11l might be responsible for this observation. Moreover, we found
that BAY-2402234 which has predominant anti-myeloid malignancies potency and
high oral bioavailability didn’t show inhibitory ability against solid tumor cells. BAY-
2
402234 is a high specific hDHODH inhibitor, whereas compound 11l could inhibit
hDHODH activity and induce ROS production as same time, which make it different
from BAY-2402234.
As mentioned, both hDHODH inhibition and ROS induction can cause
mitochondrial dysfunction and exhibit anti-proliferative effects in tumor cells. Loss of
mitochondrial membrane potential (ΔΨm) is a character of mitochondrial dysfunction.
Thus, the ΔΨm alteration was detected using the mitochondria-specific and voltage-
dependent dye tetramethyl rhodamine methyl ester (TMRM). The results show that
ΔΨm significantly decreased in a concentration-dependent manner when Raji cells
were treated with compound 11k or 11l (Figure 5B). Compared with compound 11k or
1
1l, BRQ exhibited less influence on the mitochondrial membrane potential of Raji
cells.
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Figure 5. Compounds 11k and 11l induce ROS production and decrease the
mitochondrial membrane potential. (A) Raji cells were treated with compounds 11k,
1
1l and BRQ. The ROS level was detected using dihydroethidium. (B) The Raji cells
were treated with compounds 11l, 11k, BRQ, CCCP (positive control). Mitochondrial
dysfunction was detected using TMRM. Data are shown as the mean ± SD. *p < 0.05;
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*
*p < 0.01; ***p < 0.001.
2
.7. Compounds 11k and 11l induce cell cycle S-phase arrest and apoptosis
As the inhibition of hDHODH activity could suppress the formation of pyrimidine
nucleotides needed for DNA synthesis, we investigated the influence of compounds
1k and 11l on the Raji cell cycle by flow cytometry. The results indicated that
compounds 11k and 11l significantly induced cell cycle arrest in the S-phase (Figure
). Compared with 52% of S-phase cells in the control group, compound 11k at a
1
6
concentration of 0.5 μM increased the amount of S-phase cells to 77.98%. Compound
11l at concentrations of 0.031 μM and 0.125 μM increased the amount of S-phase cells
to 76.62% and 86.31%, respectively, suggesting that compound 11l profoundly induced
S-phase cell arrest. As a positive control, BRQ showed a weaker effect than 11k or 11l.
Then, we further examined the effect of treatment with compounds 11k and 11l on
apoptosis induction using Annexin V/PI staining assays. We found that compounds 11k
and 11l induced apoptosis of Raji cells (Figure 7). Compound 11k treatment exhibited
an apoptosis rate of 8.51% at a concentration of 1 μM, and compound 11l induced an
apoptosis rate of 10.14% at concentrations of 0.125 μM. BRQ (1 μM) induced 7.53%
apoptosis of Raji cells. These data demonstrated that compounds 11k and 11l could
induce Raji cell apoptosis, which is consistent with the loss of ΔΨm induced by these
two compounds. Taken together, our data demonstrated that compounds 11k and 11l
effectively induced S-phase arrest and apoptosis in Raji cells.
The studies above showed that compound 11l exhibited profound efficacy against
hDHODH activity and cancer cell proliferation in vitro. Therefore, we selected
compound 11l for further biological function studies, including pharmacokinetic
character, antitumor effect and safety profile in vivo.
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Figure 6. Compounds 11k and 11l induce S-phase cell cycle arrest in Raji cells.
Raji cells were treated with various concentrations of compounds 11k, 11l or BRQ for
2
4 h. Cells were stained using PI. Data are shown as the mean ± SD.
Figure 7. Compounds 11k and 11l induce apoptosis of Raji cells. Raji cells were
treated with various concentrations of 11k, 11l or BRQ for 48 h. Cells were stained
using an Annexin V/FITC Apoptosis Detection Kit. Data are shown as the mean ± SD.
*
P < 0.05; ***P < 0.001; ****P < 0.0001.
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.8. Pharmacokinetic character of compound 11l
.8.1 Nanocrystalline preparation of compound 11l
Our study showed that the lipE and clogP of compound 11l are 2.88 and 5.46,
respectively, which suggested that the solubility of compound 11l might not be
adequate. To better understand the physical property of compound 11l, we detected its
solubility, and the result showed that its solubility in PBS is 33.8 μg/mL (Table S5).
As oral administration is the main administration route for small-molecule
anticancer drugs, the solubility of compound 11l might affect its in vivo
pharmacokinetic character which is an important part of druggability. Nanotechnology
has played a key role in drug delivery and disease treatment owing to its targeted-
delivery effect to deal with the problems of low solubility and poor bioavailability of
poorly soluble drugs. Therefore, we focused on screening nanocrystalline materials to
improve the solubility of compound 11l and obtain better in vivo pharmacokinetic
character. Nanocrystalline samples of compound 11l (11l-NC) were prepared using a
microfluidizer (AH100D) as the homogenization device. Compound 11l (100 mg) was
dispersed in a 20 ml aqueous solution of various stabilizers separately (CMC-Na (200
mg), F68 (20 mg) and CA (200 mg) or Tween 20 (240 mg), PEG-4000 (600 mg), CA
(100 mg), SDP (50 mg), DSP (100 mg) and NaOH (56.8 mg)) in different
concentrations. Samples for characterization were collected after pre-dissolving, as
well as after 2, 6, 10, 13, 16 and 20 min of homogenization. The mean particle size (z-
average) and polydispersity index (PDI) are summarized in (Table S2). The size and
PDI of formulations 1 and 2 were 532.4 or 494.3 nm and 0.142 or 0.256 mV after 20
min, respectively. The results were in line with the requirements of nanocrystalline
preparation. Storage stability tests were then performed to assess the effect of
temperature or time on the stability of formulations 1 and 2. The results show that the
PDI and size variation of formulation 1 were less than those of formulation 2,
suggesting that formulation 1 is more stable (Table S3 and S4). Moreover, the
formulation 1 resulted in a significant solubility improvement in aqueous buffer form
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
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2
3
4
5
6
7
8
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0
1
2
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6
7
8
9
0
3
3.8 to 91.8 μg/ml (Table S5).
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2
.8.2. Compounds 11l and 11l-NC exhibit good physiological stability
The physiological stability of compounds 11l and 11l-NC were evaluated in
buffers with pH ranging from 1.0 to 9.0. The results show that the concentration
decrease of compounds 11l and 11l-NC was between 1.6% and 9% in buffer with pH
0
1
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9
0
1
–7.4, and approximately 15% in buffer with pH 9, which suggested that both
compounds exhibited good stability in buffers with pH values ranging from 1 to 7.4
Table S6). However, the concentration decrease of BRQ was between 6.3% and 12.1%
(
in buffer with pH 1–7.4, and 75.4% in buffer with pH 9. This result suggests that
compounds 11l and 11l-NC were more stable than BRQ (Table S6).
2
.8.3 In Vivo evaluation of oral bioavailability
The oral bioavailability of compound 11l and its nanocrystalline preparation (11l-
NC) were assessed on Sprague-Dawley (SD) rats after their oral (po) or intravenous
(iv) administration. Pharmacokinetic (PK) parameters are summarized in Table 5.
Compared with compound 11l, oral administration of 11l-NC obtained a longer half-
life (T , h) (8.83 vs 12.47) and a larger area under the concentration-time curve
1
/2
.
(AUC0–∞, μg/L h) (4879 vs 141075). The oral maximum plasma concentration (Cmax)
of 11l-NC was 6369 μg/L, which was larger than compound 11l. Importantly, 11l-NC
exhibited an improved oral bioavailability of 25.16%, which is 2.6-fold larger than that
of compound 11l.
Table 5. Pharmacokinetic Parameters of Compounds 11l and 11l-NC after Oral
or Intravenous Administration
AUC(
AUC
CLz/F
T1/2
(h)
Tmax
(h)
Cmax
(μg/L)
oral F
(%)
0–t)
.
(0–∞)
.
Route
(μg/L h) (μg/L h) (L/h/kg)
iv
po
iv
8414
8417
0.62
5.47
0.08
0.18
3.89
8.83
0.08
10
9450
279
1
1l
4879
6825
9.66
95721
142632
215279
12.01 0.08
12.47 27
76941
6369
1
1l-NC
po 141075
25.16
Expressed as mean, n = 6.
2
.8.4. Microsome stability of compounds 11l and 11l-NC
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The microsome stabilities of 11l and 11l-NC were evaluated using in vitro liver
microsome preparations from human, rat and mouse. The results show the clearances
of 11l-NC and 11l were 1.03 vs 2.63, 1.53 vs 4.21 and 1.52 vs 2.28 mL/min/kg in
human, rat and mouse liver microsomes, respectively (Table S7). The parameter
analysis showed that the T of 11l-NC and 11l was 63 vs 32, 54 vs 19.8 and 50 vs 37
1
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9
0
1
/2
min in human, rat and mouse liver microsomes, respectively (Table S7). These data
indicated that 11l-NC exhibited an encouraging level of microsomal stability compared
with compound 11l.
2
.9. Compounds 11l and 11l-NC inhibit Raji tumor growth in vivo
The in vivo antitumor efficacy of compounds 11l and 11l-NC was evaluated with
mouse subcutaneous xenograft models. Nude mice carrying Raji xenograft tumors were
orally administered compound 11l (30 mg/kg), 11l-NC (40, 80 mg/kg) and BRQ (10
mg/kg). Tumor volumes and body weights were measured every 3 days. The results
show that compound 11l inhibited tumor growth with a growth inhibition rate of 42%
(Figure S3). Compared with compound 11l, 11l-NC exhibited a more obvious
inhibition of tumor growth without decrease of body weight. The inhibition rate for 40
and 80 mg/kg 11l-NC were 52% and 68%, respectively (Figure 8). In contrast, although
BRQ treatment significantly reduced tumor growth with an inhibition rate of 74%, BRQ
treatment induced obvious body weight loss and liver damage (Figure 8, S4). Moreover,
no obviously adverse effects were observed during treatment with compounds 11l and
11l-NC, such as toxic death, dermatitis, or body weight loss. Raji cell lines are human
Burkitt’s lymphoma (BL) cell lines which are intensively used in BL research. BL is a
rare, highly aggressive subtype of non-Hodgkin lymphoma. Treatment options for
relapsed and refractory BL remain limited. Thus, novel therapy strategy for BL
4
9
treatment will be helpful for BL patients . Collectively, 11l-NC significantly inhibited
tumor growth in vivo and exhibited greater safety than BRQ.
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0
Figure 8. 11l-NC inhibits Raji tumor growth in vivo. Oral administration of 11l-NC
-1
-1
-1
(
40 or 80 mg kg d ) and BRQ (10 mg kg ) were initiated when the Raji tumor volume
3
reached 100–200 mm (six mice per group), 6 days per week for 3 weeks. (A)
Representative images of tumor. (B) Tumor weight. (C) Tumor volume. (D) Mice body
weight. The data are shown as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001;
*
***P < 0.0001.
2
.10. Safety profile of compounds 11l and 11l-NC in vivo
An acute toxicity assay was performed to detect the maximum tolerated dose
(MTD), which was used to determine the highest dosage of compounds 11l and 11l-
NC that does not cause obvious side effects. 11l-NC had a higher MTD value (800
mg/kg) compared with 11l (400 mg/kg). No significant differences of blood
biochemical parameters (including ALT, AST, TP, ALB, BILT, ALP, GLU, BUN,
CREA, UA, CHO1, TG, CK, LDH, HDL and LDLC) were observed after compounds
11l and 11l-NC treatment (Figure 9). Consistent with previous reports, BRQ exhibited
dose-dependent toxicity as measured by body weight loss in the treated mice. At the 30
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mg/kg dose, this body weight loss was large enough to require termination of that arm
of the study50-52
.
The high safety property and inhibitory against hDHODH of compound 11l are
quite encouraging. The hDHODH inhibitors could be used for treatment of rheumatoid
1
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arthritis and psoriatic arthritis besides malignant tumor . Moreover, hDHODH
inhibitor exhibited inhibitory activity against PfDHODH. For example, A771726, a
hDHODH inhibitor (IC = 261 nM) could inhibit PfDHODH activity with an IC
5
0
50
54
value of 191 μM . These studiessuggested that compounds with hDHODH inhibitory
activity may inhibit the activity PfDHODH though the efficacy is weak54-56. Thus, the
series described in our work may be used for treatment of rheumatoid arthritis, psoriatic
arthritis and growth of P. falciparum and further studies are needed.
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