Discovery of an Orally Efficacious MYC Inhibitor for Liver Cancer Using a GNMT-Based High-Throughput Screening System and Structure-Activity Relationship Analysis

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Discovery of an Orally Eﬃcacious MYC Inhibitor for Liver Cancer

Using a GNMT-Based High-Throughput Screening System and

Structure−Activity Relationship Analysis

Rajni Kant,^□Ming-Hui Yang,^□Chih-Hua Tseng,^□Chia-Hung Yen, Wei-You Li, Yu-Chang Tyan,

Marcelo Chen, Cherng-Chyi Tzeng, Wei-Cheng Chen, Kaiting You, Wen-Chieh Wang,

Yeh-Long Chen,* and Yi-Ming Arthur Chen *

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ABSTRACT: Glycine-N-methyl transferase (GNMT) downregula-

tion results in spontaneous hepatocellular carcinoma (HCC).

Overexpression of GNMT inhibits the proliferation of liver cancer

cell lines and prevents carcinogen-induced HCC, suggesting that

GNMT induction is a potential approach for anti-HCC therapy.

Herein, we used Huh7 GNMT promoter-driven screening to

identify a GNMT inducer. Compound K78 was identiﬁed and

validated for its induction of GNMT and inhibition of Huh7 cell

growth. Subsequently, we employed structure−activity relationship

analysis and found a potent GNMT inducer, K117. K117 inhibited

Huh7 cell growth in vitro and xenograft in vivo. Oral administration

of a dosage of K117 at 10 mpk (milligrams per kilogram) can inhibit

Huh7 xenograft in a manner equivalent to the eﬀect of sorafenib at a

dosage of 25 mpk. A mechanistic study revealed that K117 is an MYC inhibitor. Ectopic expression of MYC using CMV promoter

blocked K117-mediated MYC inhibition and GNMT induction. Overall, K117 is a potential lead compound for HCC- and MYC-

dependent cancers.

GNMT-based drug screening assay and identiﬁed a natural

compound, PGG, as a GNMT inducer. Our study demonstrated

the anti-HCC eﬀect of PGG in vitro and in vivo. Interestingly, our

study showed various known HCC drugs also induced GNMT

and provided a proof of concept that a GNMT-based assay has

potential to identify drugs for HCC.²²Due to the lack of

eﬀective treatment, this study started with the aim of identifying

novel small molecules for the treatment of HCC with a potency

the same as or higher than that of sorafenib. Using GNMT-based

high-throughput screening, we identiﬁed various small mole-

cules that induced GNMT. One of the GNMT inducers was

characterized for anti-HCC activity in vitro and in vivo.

INTRODUCTION

■

Hepatocellular carcinoma (HCC) is a major health burden with

an increasing global incidence.¹It is the second common cause

of cancer deaths, and treatment options are very scarce.²For

many years, there was only one FDA-approved drug (sorefenib)

for advanced HCC.³Sorafenib works only in selected

individuals with side eﬀects, and the price of this drug is very

high.⁴⁻⁶In addition, the pace of new drug development is very

slow and many compounds failed in clinical trials from 2007 to

2016.⁷Although in the past 2 years four new drugs showed

survival beneﬁts in patients with advanced HCC, the median

overall survival is still approximately 1 year.^8,9Therefore, ﬁnding

new drugs for HCC treatment to improve patient survival and

help to control disease progression is is urgently needed.¹⁰

GNMT is a tumor suppressor protein in HCC and has various

important functions in liver protection and homeostasis.¹¹⁻¹³

The level of expression of GNMT is very low in patients at risk of

HCC and is highly downregulated in HCC patients of various

etiologies.¹⁴⁻¹⁷Furthermore, GNMT knockout mice from

diﬀerent groups developed HCC spontaneously.^18,19GNMT

re-expression in HCC cell lines as well as transgenic mice

showed a beneﬁcial eﬀect and established GNMT as a target for

drug discovery.^20,21In previous studies, we established a

RESULTS

■

Small-Molecule Compound Library Screening for

GNMT Inducers. We used H7GPL cells described previously

Received: January 19, 2021

Published: June 16, 2021

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Figure 1. Identiﬁcation of K78 by high-throughput luciferase reporter screening. (A) Schematic outline of the high-throughput screening to identify

GNMT inducers. (B) Chemical structures of hits. (C) H7GPL cells were treated with the indicated concentrations of K78 for 24 h and harvested for

luciferase assay. Relative luciferase activity was calculated by normalizing luciferase activity to cell viability and presented as fold to control. Results are

means the standard deviation (n = 3). (D) Huh7 cells were treated with K78 (2.5 μM) for 24 h and harvested for mRNA and protein. GNMT mRNA

expression was assessed by qRT-PCR. Data were normalized to the respective amount of internal control and expressed as fold to solvent control. The

graph shows means the standard deviation (n = 3) (top). The bottom panel shows GNMT protein expression after K78 treatment (2.5 μM). β-Actin

expression was used as a loading control. ***P < 0.001; **P < 0.01 (Student’s t test).

to identify small molecules that induce GNMT expression.²²

The NHRI library, which represents a collection of 11920 small

molecules, was screened. In primary screening and conforma-

tional screening, we identiﬁed eight hits that induced GNMT

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Figure 2. K78 inhibits liver cancer cell growth in vitro and in vivo. (A) Huh7 cells were treated with various concentrations of K78 for 72 h. Cell

viabilities were determined by the Alamar blue assay. The percentages of viable cells normalized to the solvent-treated control were plotted. Results are

means the standard deviation (n = 3). (B) Huh7 cells were treated with the indicated concentrations of K78. Cell proliferation was evaluated on days

1, 2, 3, and 4 using the Alamar blue assay. Results are shown as means the standard deviation (n = 3). (C and D) Huh7 xenografts were developed in

NOD-SCID mice and treated with the indicated doses of K78, sorafenib, and solvent for 30 days. The mouse body weight was measured during the

course of treatment (C). Average tumor volume of each group 30 days after drug treatment (D). *P < 0.05; **P < 0.01 (Student’s t test).

promoter-driven luciferase expression by >1.8-fold compared

with solvent-treated control cells (schematic representation

shown in Figure 1A). Next, we assessed the cell viability of eight

hits by a MTS assay to evaluate whether some of the induction

eﬀects were caused by cytotoxicity. Of the eight eﬀective

compounds, ﬁve induced reporter gene expression at concen-

trations that were nontoxic or only slightly aﬀected cell viability

(Figure 1B). To conﬁrm the induction capacity of the selected

compounds, we performed a dose−response assay with these

ﬁve compounds. K78 {3-hydroxy-N′-[1-(pyridin-2-yl)-

ethylidene]-2-naphthohydrazide} showed a higher level of

dose-dependent induction of luciferase activity (Figure 1C

and Figure S1) than all other hits and was selected as a potential

candidate for induction of reporter gene expression. To conﬁrm

whether K78 enhances endogenous GNMT expression, we

examined GNMT mRNA and protein levels in Huh7 cells. As

shown in Figure 1D, K78 upregulated endogenous GNMT

expression at mRNA and protein levels in Huh7 cells.

ﬁrst evaluated the inhibitory eﬀect of K78 on the growth of

Huh7 cells. K78 suppressed the proliferation of Huh7 cells in a

dose-dependent manner after treatment for 72 h (Figure 2A).

Subsequently, exposure for 24, 48, 72, and 96 h to K78

signiﬁcantly inhibited the proliferation of Huh7 cells in a time-

dependent manner (Figure 2B). Next, we conducted studies on

a xenogeneic model using Huh7 cells. Tumor-bearing nude mice

were treated orally with a solvent, 25 mpk (milligrams per

kilogram) K78, 100 mpk K78, and 300 mpk K78. Sorafenib was

chosen as a comparison control because it is an FDA-approved

drug for HCC.²³There was no evidence of the dependence of

toxicity on body weight in any group (Figure 2C). As shown in

Figure 2D, K78 inhibits tumor growth in this model in

comparison with the solvent control. However, sorafenib has

the most signiﬁcant diﬀerence, and tumor reduction was

comparable to previous studies. Taken together, these results

provide strong evidence for the anti-HCC activity of K78 using

in vitro and in vivo models.

Anti-HCC Eﬀect of K78 In Vitro and In Vivo. To

Identiﬁcation of the More Potent Novel GNMT Inducer

determine the therapeutic potential of K78 against HCC, we

Compound K117 through a Structure−Activity Relation-

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Figure 3. SAR studies of K78 to identify more potent GNMT inducers. (A) Outline of the general synthesis of K78 derivatives. (B−D) H7GPL cells

were treated with the indicated concentrations of K78 derivatives for 24 h and harvested for the luciferase assay. Structures, molecular formulas, and

reporter activities for K78 derivatives after pyridine ring modiﬁcation (B), after methyl group modiﬁcation (C), and after naphthalene ring

modiﬁcation (D).

ship Study. Because the anti-HCC eﬀect of K78 was not

potent HCC inducers as potential drug candidates. We utilized

comparable to that of sorafenib, we intended to identify more

H7GPL cells and analyzed the eﬀect of structural changes in the

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Scheme 1. Synthesis of k-Series Compounds

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Scheme 1. continued

K78 molecule on its ability to enhance GNMT promoter-driven

luciferase reporter activity. The SAR analysis focuses on three

functional groups: the pyridine ring, the methyl group, and the

naphthalene ring of 3-hydroxy-N′-[1-(pyridin-2-yl)ethylidene]-

2-naphthohydrazide (Figure 3A). The analogues of K78 were

prepared according to the general procedure in the Experimental

Section, and a general synthetic sequence is illustrated in

Scheme 1.

First, the importance of pyridine was investigated through the

synthesis of eight derivatives, compounds 2−9 (Figure 3B).

None of the eight derivatives induced GNMT promoter reporter

activity because it was similar to that of the control, as a total loss

of activity was observed for compounds in which pyridine

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Figure 4. K78 derivatives enhance GNMT expression. H7GPL cells were treated with the indicated concentrations of (A) K90, (B) K96, (C) K102,

and (D) K117 for 24 h and harvested for the luciferase assay. Relative luciferase activity was calculated by normalizing luciferase activity to cell viability

and presented as fold to solvent control. Results are means the standard deviation (n = 3). Huh7 cells were treated with the indicated concentrations

of K117 for 24 h and harvested for mRNA and protein. (E) GNMT mRNA expression was assessed by qRT-PCR. Data were normalized to the

respective amount of internal control and expressed as fold to solvent control. The graph shows means the standard deviation (n = 3). (F) GNMT

protein expression was assessed by immunoblot analysis. β-Actin expression was used as a loading control. The bottom panel shows quantiﬁcation of

GNMT signal intensities in the top panel. ***P < 0.001; **P < 0.01; *P < 0.05 (Student’s t test).

groups were modiﬁed or replaced with other groups. This

conﬁrmed the importance of pyridine in inducing GNMT

reporter activity. Then, the importance of the methyl group

along with the pyridine ring was investigated. For this, six

derivatives were synthesized [compounds 10−15 (Figure 3C)].

Similar to the previous results, all derivatives reduced the

activities to that of the control with the exception of compound

15, which contains a pyridine ring. However, the reporter

activity of compound 15 was also lower than that of K78 at

concentrations of 2.5 and 10 μM, implying that the modiﬁcation

of this methyl group along with the pyridine ring is not favorable

for the induction of GNMT reporter activity. Next, the inﬂuence

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Figure 5. K117 inhibits liver cancer cell growth in vitro and in vivo. (A) Huh7, HepG2, and Hep3B cells were treated with various concentrations of

K117 for 72 h. Cell viabilities were determined by the Alamar blue assay. The percentages of viable cells normalized to the solvent-treated control were

plotted. Results are means the standard deviation (n = 3). (B) The long-term eﬀect of K117 on Huh7 and HepG2 growth was determined by colony

formation of Huh7 and HepG2 cells after treatment for 10 days (representative image; n = 3). (C) Quantiﬁcation means the standard deviation (n =

3). (D) Growth curves of Huh7 tumors in nude mice treated with K117 (25 and 10 mpk), sorafenib (25 mpk), or solvent over a period of 4 weeks.

Error bars represent the standard error (n = 6). *P < 0.05 (Student’s t test). (E) Mouse body weights were measured during the course of treatment.

(F) Eﬀect of K117 on ALT and AST levels in K117-treated NOD-SCID mice. ***P < 0.001; **P < 0.01; *P < 0.05 (Student’s t test).

of the variation of the 3-hydroxynaphthalene ring was examined

by synthesis of 11 derivatives. Figure 3D presents the structures

and reporter activities of 11 derivatives containing a variety of

modiﬁcations at the naphthalene ring. Interestingly, when

naphthalene was replaced with benzene (as in compound 16),

the GNMT promoter activity increased at 2.5 μM. However,

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Figure 6. Eﬀect of GNMT-inducing compound K117 on Myc expression. (A) Huh7 and (B) HepG2 cells were treated with the indicated

concentrations of K117 for 24 h and subjected to immunoblot analysis for HDAC1 and HDAC2 protein expression. GADPH expression was used as a

loading control. (C) Myc mRNA expression in Huh7 cells treated with K78, K90, and K102 (5 μM) determined by qRT-PCR after treatment for 24 h.

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Figure 6. continued

Data presented as fold to solvent control. The graph shows means the standard deviation (n = 3). Eﬀect of the indicated doses of K117 on Myc

mRNA expression in (D) Huh7 and (E) HepG2 cells determined by qRT-PCR after treatment for 24 h. Data presented as fold to solvent control. The

graph shows means the standard deviation (n = 3). Alterations in the mRNA levels of MYC target genes (E) p21 and (G) p27 in Huh7 cells after

K117 treatment for 24 h detected by qRT-PCR. Data presented as fold to solvent control. The graph shows means the standard deviation (n = 3).

(H) MYC and GNMT protein expression in Huh7 xenograft tumor tissues (described in Figure 5D) determined by an immunoblot assay (n = 6 mice

from each group). β-Actin expression was used as a loading control. Huh7 cells were transfected with the pcDNA-MYC or control vector plasmid for 72

h, treated with K117 or solvent control, and then harvested for (I) RNA and (J) protein. mRNA and protein expression of MYC was determined by

qRT-PCR and an immunoblot assay. Data were normalized to the internal control and presented as fold to vector control. The graph shows means

the standard deviation (n = 3). β-Actin expression was used as a loading control. (K) Huh7 cells were transfected with the pcDNA-MYC or control

vector plasmid for 72 h, treated with K117 or solvent control, and then harvested for luciferase activity. Data were measured and represented as

described above. The graph shows means the standard deviation (n = 3). ***P < 0.001; **P < 0.01; *P < 0.05 (Student’s t test).

removal of one benzene ring from naphthalene along with

changes in the methyl group (compounds 17 and 18) reduced

the activity (at 2.5 μM). This result further conﬁrmed the

importance of the methyl group for GNMT promoter activity.

Because compound 16 showed reporter activity higher than that

of K78, we further studied the substitution eﬀect on the benzene

ring of compound 16. Compound 19 exhibited higher GNMT

reporter activity at 2.5 μM compared to that of compound 16 in

which an additional hydroxyl group was added to the benzene

ring at the ortho position of the carboxamide group. However,

this induction was eliminated in compound 20 in which the

additional hydroxyl group was added to benzene at the para

position. Similarly, when a hydroxyl group was added to benzene

at the para position (as in compound 21), the induction eﬀect in

compound 17 was eliminated. Interestingly, changing the

position of the hydroxyl group from ortho to meta (16 vs 22)

induced the reporter activity at 10 μM; however, the removal of

the μ-hydroxyl group resulted in a reduction in GNMT reporter

activity (22 vs 23) at 10 μM. Next, the replacement of the

hydroxyl group with a methoxy group (compound 24) resulted

in a reduction in GNMT promoter activity, whereas a

replacement of the amino group (compound 25) induced

activity higher than that of compound 16. Finally, inspired by

the structure of compound 25, we synthesized a more potent

compound K117 (compound 26), which induced GNMT

promoter activity higher than those of all identiﬁed compounds.

Collectively, our SAR study led to the identiﬁcation of a number

of novel compounds that exhibited GNMT promoter activity

higher than that of the known K78. Next we studied the dose

response of four derivatives [compounds 16 (K90), 19 (K96),

25 (K102), and 26 (K117)] which exhibited GNMT promoter

activities higher than that of K78.

All derivatives showed dose-dependent induction of GNMT

promoter activity with a biphasic bell-shaped dose−response

curve (Figure 4A−D). Moreover, K117, the most potent

inducer, was chosen for further studies and induced GNMT

mRNA as well as protein expression in Huh7 cells (Figure 4E,F).

K117 Inhibits Liver Cancer Cells In Vitro and In Vivo.

Next, we analyzed the eﬀect of K117 on cell proliferation in

diﬀerent liver cancer cell lines, including Huh7, Hep3B, HepG2,

and Mahlavu cells. Results showed that K117 inhibited

proliferation of all tested liver cancer cells in a dose-dependent

manner (Figure 5A and Figure S2 for the SRB cell viability

assay). Furthermore, K117 reduced the extent of colony

formation of Huh7 and HepG2 cells in a dose-dependent

manner (Figure 5B,C). To further evaluate the antitumor eﬀect

of K117 in vivo, Huh7 cells were injected subcutaneously into

nude mice as described in the Experimental Section. These mice

were then randomly grouped and treated with the solvent

control, 25 mpk K117, 10 mpk K117, and 25 mpk sorafenib as a

comparison control. As shown in Figure 5D and Figure S3, a

signiﬁcant tumor growth reduction was observed in the K117

treatment group when compared with the control group.

Moreover, K117 at smaller doses of 10 mpk showed the same

eﬀect as sorafenib at doses of 25 mpk. In addition, K117

treatment produced no severe adverse events as monitored by

animal survival, body weight, liver function tests, and organ

morphology. The change in mouse body weight, liver function,

and organ morphology were similar in all groups (Figure 5E,F

and Figure S4). In addition, K117 was more abundant in the

liver tissue of mice treated with a dose of 25 mpk than in mice

treated with a dose of 10 mpk, indicating the dose-dependent

availability of K117 in the liver of mice (Figures S5 −S514).

Altogether, these results provide strong evidence for the tumor

growth-inhibiting activity and tolerability of K117 in vitro and in

vivo.

Mechanism of K78-Series GNMT Induction in HCC.

Lastly, we explored the mechanism by which K78-series

derivatives enhance GNMT expression and inhibit liver cancer

cell proliferation. It has been recently shown that the MYC-

mediated repression and repression via histone deacetylation are

two independent mechanisms of GNMT suppression in HCC.²⁴

Therefore, ﬁrst, we tested the eﬀect of K117 on histone

deacetylase associated with transcriptional gene silencing, i.e.,

HDAC1 and HDAC2. As shown in panels A and B of Figure 6,

treatment with K117 did not aﬀect the protein expression of

HDAC1 and HDAC2 in Huh7 and HepG2 cells. Hence, the

K78 series induced GNMT expression by a diﬀerent

mechanism. Next, to determine whether K78-series compounds

exerted their eﬀects through MYC inhibition in HCC, we

examined the eﬀect of K78-series compounds (K78, K102, and

K90) on mRNA expression of MYC in Huh7 cells. As shown in

Figure 6C, all K78-series compounds (K78, K102, and K90)

inhibited MYC mRNA expression in Huh7 cells. We further

showed that K117 also suppressed MYC mRNA expression

dose-dependently in both Huh7 and HepG2 cells (Figure

6D,E). Moreover, we evaluated the eﬀect of K117 on MYC

function by analyzing the expression of MYC target genes after

K117 treatment. Consistent with MYC inhibition, K117

treatment increased the level of mRNA expression of p21 and

p27 in Huh7 cells (Figure 6F,G). Consistent with in vitro results,

the level of MYC protein expression was reduced in Huh7

xenograft tumors in K117-treated mice (Figure 6H and Figures

S15 and S16). These results demonstrated that K78-series

compounds are MYC inhibitors, which suppress MYC

expression in HCC cells. In our previous study, we have

shown that PGG inhibits MYC gene expression and enhances

the degradation of the MYC protein through proteasome- and

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calpain-independent mechanisms.²⁴To investigate this mech-

anism in K78-series compounds, we examined whether ectopic

expression of MYC rescued the K117-induced suppression of

MYC mRNA and protein in Huh7 cells. Interestingly, ectopic

expression of MYC could rescue K117-induced MYC mRNA

and protein suppression in Huh7 cells (Figure 6I,J and Figure

S17). In addition, promoter assay results showed that over-

expression of MYC restored K117-induced GNMT promoter

activity in H7GPL cells (Figure 6K). Collectively, K78-series

compounds suppressed MYC transcription and enhanced

GNMT expression.

responsible for this biphasic eﬀect of K117 remains to be

determined. It has been postulated that various mechanisms

resulted in a bell-shaped response of xenoestrogens: small

molecules that bind at diﬀerent targets oppose one another with

diﬀerent eﬃciencies, a single receptor might have two binding

sites that bind at diﬀerent doses and result in induction and

inhibition, and molecules at diﬀerent doses might modulate the

metabolism or other factors that result in diﬀerent eﬀects.²⁷The

bell-shaped dose−response curve is common in many antitumor

agents.²⁸Compounds such as RGD-mimetic integrin inhib-

itors,²⁹plasminogen activator-1 (PAI-1),³⁰HMG-CoA reduc-

tase inhibitors,^31,32bortezomib,³³TGF-β3,³⁴and TGF-β1³⁵

were reported to exhibit biphasic bell-shaped dose−response

curves. Collectively, K117 also showed a bell-shaped biphasic

dose−response curve. Thus, understanding the biphasic dose−

response curve and further study related to the mechanism of

MYC inhibition by K117 are necessary.

K78 and K117 both inhibited liver cancer cell growth in vitro

and in vivo. Importantly K117 inhibited in vivo tumor growth

and its inhibition capability is comparable to that of sorafenib.

The multitarget kinase inhibitor sorafenib is the ﬁrst-line

treatment for patients who have advanced HCC.⁷The recently

approved lenvatinib, regorafenib, cabozantinib, nivolumab, and

ramucirumab showed survival beneﬁts in patients who

progressed on sorafenib.^8,9However, while sorafenib prolonged

survival by 3.5 months, its survival beneﬁt was shorter (∼37.3%),

it was associated with severe side eﬀects, and patients developed

resistance to it.³⁶In addition, sorafenib treatment is expensive

and not cost-eﬀective for use in advanced HCC.³⁷⁻³⁹Notably, in

vivo data showed that when K117 was administrated at a smaller

dose of 10 mpk, the xenograft growth inhibition was comparable

with that of sorafenib at a dose of 25 mpk (Figure 5D).

Furthermore, the drug showed no adverse eﬀects on mouse

body weight or liver function (Figure 5E,F). Moreover, K117

also showed a growth inhibitory eﬀect on pancreatic and

prostate cancer cell lines (manuscript in preparation). Finally,

preclinical pharmacokinetics and pharmacodynamics studies are

required to conﬁrm the safety and to understand the metabolism

of K117. In addition, a larger dose of K117 (25 mpk) did not

inhibit xenograft tumor growth when compared to sorafenib,

whereas a smaller dose of 10 mpk did. Therefore, dose−

response studies using smaller doses to obtain the same eﬀect or

a better eﬀect may provide optimization and characterization of

the K117 in the treatment of HCC.

A recently published study demonstrated that GNMT is a

target gene of MYC.²⁴In addition, the GNMT inducer natural

compound PGG enhances GNMT expression through inhib-

ition of MYC. Interestingly, a mechanistic study of how these

drugs induced GNMT found that all identiﬁed hits were potent

MYC inhibitors (Figure 6C). K117 inhibits MYC expression in

Huh7 and HepG2 cells in vitro and xenograft tumors in vivo.

Furthermore, this inhibition of MYC by K117 is correlated with

the induction of the expression of its target genes, p21 and p27

(Figure 6F,G). As shown previously, PGG inhibited MYC

expression by targeting MYC protein stability through induction

of proteasome-independent MYC proteolysis.²⁴In contrast,

K117 inhibits MYC expression at the transcriptional level via

other mechanisms. Overexpression of MYC via an ectopic CMV

promoter rescued K117-induced MYC inhibition in Huh7 cells

(Figure 6I,J). Because the MYC inhibitory mechanism of PGG

employs mostly protein stability and that of K117 employs

transcriptional inhibition, we postulate that K117 might be a

more speciﬁc MYC inhibitor than PGG. Hence, further studies

DISCUSSION

■

HCC remains a deadly disease largely owing to the lack of

treatment options and makes the discovery of novel therapeutic

approaches an urgent need. In this study, we identiﬁed and

validated novel molecules to target HCC. GNMT is highly

downregulated in HCC with diverse etiologies and in patients

with acute and chronic liver disease.^{12,14,16,25,26}We have

previously demonstrated that GNMT induction is a promising

therapeutic approach in HCC and established a GNMT

reporter-based drug screening assay for the discovery of novel

compounds for the treatment of HCC.²²Here we identiﬁed a

number of novel compounds that enhance GNMT promoter

activity and validated the most potent GNMT inducer.

Subsequent SAR studies uncovered K117 as a potent inducer,

and its mechanism of GNMT induction was deﬁned. Moreover,

the identiﬁed compounds K78 and K117 showed potent anti-

HCC activity. This conﬁrmed the utility of this GNMT-based

drug screening for the discovery of small compounds with anti-

HCC activity.

K78 SAR analysis showed that the modiﬁcation in the

pyridine ring and methyl group of 3-hydroxy-N′-[(1E)-1-

(pyridin-2-yl)ethylidene]naphthalene-2-carbohydrazide re-

duced GNMT promoter activity. However, modiﬁcation of

the naphthalene ring induced GNMT promoter activity.

Therefore, SAR results suggested that the pyridine ring and

methyl group of 3-hydroxy-N′-[(1E)-1-(pyridin-2-yl)-

ethylidene]naphthalene-2-carbohydrazide contribute to

GNMT promoter induction. In addition, naphthalene ring

substitution is the key to inducing GNMT promoter activity.

Further research such as in molecular docking is needed to

understand how compounds (K78, K90, K96, and K117)

interact with the GNMT promoter and how to improve

compound activity

The results of this study demonstrated that all identiﬁed

derivatives induced GNMT promoter activity dose-dependently

with a biphasic bell-shaped dose−response curve (Figure 4A−

D). These dose-dependent responses were decreased at larger

doses. Thus, K117 also induced GNMT mRNA and protein

expression at a smaller dose with a reduced level of expression at

a larger dose (Figure 4E,F). Furthermore, these in vitro results

are in line with in vivo xenograft results. The in vivo antitumor

eﬃcacy of K117 at a smaller dose (10 mpk) is better than that at

the larger dose (25 mpk) (Figure 5D). Moreover, the smaller

dose of K117 (10 mpk) showed better inhibition of MYC

protein expression in xenograft tumors compared to that of the

larger dose (25 mpk) (Figure S16). In addition, the amount of

hepatic K117 in mice treated with 25 mpk K117 is larger than

that of hepatic K117 in mice treated with 10 mpk K117 (Figures

S5 −S514). These data further support the idea that the reduced

eﬃcacy of K117 at a larger dose is due to the bell-shaped

biphasic dose−response curve of K117. The mechanism

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Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum

(FBS) (HyClone, Logan, UT), ^L-glutamine (2 mM), streptomycin

(100 μg/mL), penicillin (100 units/mL), and non-essential amino

acids (0.1 mM). All cell lines were maintained at 37 °C in a humidiﬁed

CO₂incubator. H7GPL stable cells established by the lentiviral system

were grown in DMEM supplemented with 1 μg/mL puromycin.

Sorafenib was purchased from Sigma-Aldrich (St. Louis, MO).

Plasmids, transfection, and gene overexpression were described

previously.²⁴

comparing K117 with known Myc inhibitors are needed.

Nevertheless, these results will provide a rationale for further

investigation of K117 in MYC ampliﬁcation/overexpression

cancer models. Additionally, it has been shown that repression

via histone deacetylation is also an important mechanism of

GNMT suppression in HCC.²⁴The treatment with K117 did

not aﬀect the protein expression of HDAC1 and HDAC2

(Figure 6A,B). These data further demonstrated that K117

enhances GNMT only through MYC inhibition.

GNMT Enhancer Drug Screening Assay. The −1812/+14

GNMT promoter-driven luciferase reporter cell line H7GPL was

constructed in our previous study.²²The small-molecule compound

library that contained 11920 pure compounds was provided by the

National Health Research Institutes (NHRI), Taiwan. For primary

screening, H7GPL cells were seeded in 96-well plates at a density of 5 ×

10⁴cells/well. After 24 h, cells were treated with individual drugs at a

concentration of 10 μM in 1% dimethyl sulfoxide (DMSO) for 18 h. At

the end of the incubation, neolite reagent (PerkinElmer) was

distributed to cells (50 μL/well). The plates were placed in the dark

at room temperature (RT) for 10 min, and luminescence was measured

by Victor²V (PerkinElmer). The primary screening hits were retested

using 10 μM compound in 0.1% DMSO as the per primary screening

protocol in the conﬁrmation screening. The hits of the conﬁrmation

screen were sorted by a change of >1.8-fold and used for the cytotoxicity

assay. In the cytotoxicity assay, H7GPL-1 cells were seeded in 96-well

plates at a density of 3 × 10³cells/well overnight. Then, cells were

treated with 10 μM compound for 72 h. At the end of the incubation, for

a 96-well microtiter plate, 3 mL of reagent containing MTS

{tetrazolium compound [3-(4,5-dimethylthiozol-2-yl)-5-(3-carboxy-

methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (Prom-

ega, Madison, WI)} and PMS [phenazine methosulfate (Sigma)] in a

ratio of 20:1 was added and used as the detection reagent. The medium

in the well was removed, and the MTS mixed reagent was distributed to

cells (100 μL/well). The plates were incubated for 1.5 h at 37 °C in a

humidiﬁed 5% CO₂atmosphere, and the absorbance was then recorded

at 490 nm. The hits showing >90% of the cytotoxicity of the control

(100% of control means no cytotoxicity) were subjected to detailed

concentration−response testing using diﬀerent doses of the com-

pounds. Concentration−response testing was performed as described

previously.²²Top hits were selected for detailed anti-HCC studies.

Chemistry Methods. Melting points were determined on an

Electrothermal IA9100 melting point apparatus and are uncorrected. IR

spectra were recorded on a Bruker Corp. Alpha FT-IR spectropho-

tometer. Nuclear magnetic resonance (¹H and ¹³C) spectra were

recorded on a Varian-Unity-400 spectrometer. Chemical shifts were

expressed in parts per million (δ) with tetramethylsilane (TMS) as an

internal standard. Thin-layer chromatography was performed on silica

gel 60 F-254 plates purchased from E. Merck and Co. The elemental

analyses were performed in the Instrument Center of the National

Science Council at National Taiwan University using Heraeus CHN-O

Rapid EA, and all values are within 0.4% of the theoretical

compositions. The purities of all target compounds that were used

were ≥95%.

MYC protein encoded by MYC proto-oncogene is a

transcription factor that regulates various biological processes

such as cell growth, metabolism, diﬀerentiation, proliferation,

and apoptosis.⁴⁰⁻⁴³MYC is overexpressed or ampliﬁed in a wide

variety of cancers, including HCC, and contributes to tumor

initiation, progression, metastasis, poor prognosis, poor patient

survival, and drug resistance.^44,45Due to the crucial role of MYC

overexpression in cancer, it is an ideal and well-known target for

cancer therapy.⁴⁶⁻⁴⁸However, no MYC inhibitors have been

clinically approved after 40 years of MYC research.⁴⁹Direct

targeting of MYC remains challenging due to a lack of binding

pockets for small molecules, and most indirect MYC inhibitors

target MYC as various labels are still at diﬀerent stages of

development.⁵⁰⁻⁶¹We found K117 to be a novel MYC inhibitor

in liver cancer cells, and further validation and understanding of

the underlying mechanism of MYC inhibition are needed.

Here, using a GNMT-based assay, we identiﬁed several small

molecules that inhibit MYC expression to induce GNMT.

Furthermore, PGG, which was identiﬁed using this platform,

induced GNMT through MYC inhibition. Moreover, other

compounds identiﬁed through this platform also inhibit MYC to

induce GNMT. Surprisingly, somehow all GNMT inducer

compounds identiﬁed by our GNMT-based drug screening

platform established in our lab were potent MYC inhibitors.

Further studies of the characteristics of this platform and

possibilities of using this platform for identiﬁcation of MYC

inhibitors for cancer therapy are warranted

CONCLUSION

■

In summary, novel GNMT inducer compounds were identiﬁed

via high-throughput screening and SAR studies aimed at protein

GNMT. Interestingly, all identiﬁed hits were potent MYC

inhibitors and enhanced GNMT expression through MYC

inhibition. MYC is an important oncogene, and inhibition of

MYC is known as an approach to cancer therapy. Therefore, it

will be valuable to further understand the MYC inhibition

mechanism of this molecule and anticancer activities in MYC-

driven cancers. K117 showed potent anti-HCC activity in vitro

and in vivo when compared to sorafenib at a smaller dose.

Therefore, further clinical evaluation of K117 for the treatment

of HCC using physiologically more relevant cancer models such

as patient-derived xenograft, transgenic mouse models, and

chemically derived spontaneous mouse models of HCC is

recommended. GNMT was downregulated in HCC and in other

liver disorders such as cirrhosis, ﬁbrosis, NAFLD, and chronic

cholesteric liver disease.^11,62Our results showed that GNMT

upregulation could suppress tumor growth, and it would be

interesting to test whether GNMT enhancers could be applied

to other liver diseases.

General Procedure for the Preparation of Aroylhydrazide. A

mixture of ethyl 3-hydroxy-2-naphthoate, hydroxyl-substituted ethyl

benzoate, or ethyl 2-aminonicotinate (1.0 mmol) and hydrazine-

hydrate (80%, 3 mL) was heated under reﬂux in 20 mL of ethanol for 4

h. The mixture was cooled at room temperature after the reaction had

ceased. The precipitate was ﬁltered and washed with water. The crude

solid was crystallized in ethanol to aﬀord the appropriate aroylhy-

drazides.

General Procedure for the Preparation of K-Series Derivatives. A

solution of aroylhydrazide (1.0 mmol) in 20 mL of ethanol and the

appropriate aldehyde or ketone (1.0 mmol) were reﬂuxed for 4 h. Then,

the mixture was cooled at room temperature and ethanol evaporated.

The product was dried and crystallized with ethanol to aﬀord the

appropriate K-series derivatives.

EXPERIMENTAL SECTION

■

Cell Culture and Reagents. All cell lines were maintained as

described previously.²⁴In brief, human liver cancer cell lines Huh7,

HepG2, Hep3B, and Mahlavu were cultured in Dulbecco’s modiﬁed

3-Hydroxy-N′-[1-(pyridin-2-yl)ethylidene]-2-naphthohydrazide

(K78).⁶³⁻⁶⁷Compound K78 was obtained from 3-hydroxy-2-

naphthohydrazide and 2-acetylpyridine in 85% yield as a white solid.

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Mp: >200 °C. IR (KBr, cm⁻¹): 3058, 1645, 1554, 1218. ¹H NMR (400

MHz, DMSO-d₆): δ 11.85 (br s, 1H, OH), 11.70 (s, 1H, NH), 8.70 (s,

1H), 8.65−8.64 (m, 1H), 8.21−8.19 (m, 1H), 8.03−8.01 (m, 1H),

7.93−7.88 (m, 1H), 7.81−7.78 (m, 1H), 7.56−7.51 (m, 1H), 7.40−

7.35 (m, 3H), 2.49 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ

161.51, 154.84, 152.62, 152.50, 148.67, 136.67, 135.85, 132.56, 128.99,

128.38, 127.24, 125.73, 124.18, 123.94, 120.63, 120.47, 110.74, 11.87.

3-Hydroxy-N′-[1-(4-methoxyphenyl)ethylidene]-2-naphthohy-

drazide (K79). Compound K79 was obtained from 3-hydroxy-2-

naphthohydrazide and 4-methoxyacetophenone in 82% yield as a white

solid. Mp: 227−228 °C. IR (KBr, cm⁻¹): 3053, 1644, 1510, 1255. ¹H

NMR (400 MHz, DMSO-d₆): δ 11.68 (br s, 1H, OH), 11.47 (s, 1H,

NH), 8.65 (s, 1H), 8.00−7.98 (m, 1H), 7.86−7.85 (m, 2H), 7.78−7.77

(m, 1H), 7.53−7.51 (m, 1H), 7.39−7.36 (m, 2H), 7.03−7.01 (m, 2H),

3.82 (s, 3H, OCH₃), 2.35 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-

d₆): δ 161.44, 160.39, 152.77, 152.43, 135.69, 132.08, 130.28, 128.88,

128.22, 127.98 (2C), 127.18, 125.68, 123.85, 120.76, 113.75 (2C),

110.63, 55.23, 13.80. Anal. Calcd for C₂₀H₁₈N₂O₃: C, 71.84; H, 5.43; N,

8.38. Found: C, 71.76; H, 5.33; N, 8.38.

3-Hydroxy-N′-[1-(pyridin-3-yl)ethylidene]-2-naphthohydrazide

(K80). Compound K80 was obtained from 3-hydroxy-2-naphthohy-

drazide and 2-acetylpyridine in 81% yield as a white solid. Mp: 260−

261 °C. IR (KBr, cm⁻¹): 3056, 1672, 1562, 1343, 1223. ¹H NMR (400

MHz, DMSO-d₆): δ 11.78 (br s, 1H, OH), 11.64 (s, 1H, NH), 9.06 (s,

1H), 8.66−8.63 (m, 2H), 8.26−8.24 (m, 1H), 8.01−8.00 (m, 1H),

7.80−7.78 (m, 1H), 7.55−7.49 (m, 2H), 7.40−7.36 (m, 2H), 2.42 (s,

3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ 161.67, 152.67, 150.35,

150.11, 147.60, 135.80, 133.79, 133.54, 132.36, 128.96, 128.37, 127.21,

125.73, 123.94, 123.50, 120.72, 110.70, 13.80. Anal. Calcd for

C₁₈H₁₅N₃O₂: C, 70.81; H, 4.95; N, 13.76. Found: C, 70.72; H, 4.83;

N, 13.73.

drazide and 2-acetylquinoline in 76% yield as a white solid. Mp: 273−

274 °C. IR (KBr, cm⁻¹): 3054, 1646, 1555, 1358, 1219. ¹H NMR (400

MHz, DMSO-d₆): δ 11.86−11.82 (m, 2H, OH and NH), 8.71 (s, 1H),

8.45−8.36 (m, 2H), 8.10−8.07 (m, 1H), 8.04−8.02 (m, 2H), 7.83−

7.79 (m, 2H), 7.67−7.63 (m, 1H), 7.56−7.52 (m, 1H), 7.41−7.37 (m,

2H), 2.63 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ 161.53,

154.90, 152.58, 152.31, 146.77, 136.32, 135.88, 132.67, 129.87, 129.13,

129.00, 128.42, 127.83 (2C), 127.24, 127.19, 125.72, 123.96, 120.58,

118.22, 110.76, 11.55. Anal. Calcd for C₂₂H₁₇N₃O₂: C, 74.35; H, 4.82;

N, 11.82. Found: C, 74.04; H, 4.59; N, 11.77.

3-Hydroxy-N′-(quinolin-2-ylmethylene)-2-naphthohydrazide

(K85). Compound K85 was obtained from 3-hydroxy-2-naphthohy-

drazide and 2-formylquinoline in 79% yield as a brown solid. Mp: 241−

242 °C. IR (KBr, cm⁻¹): 3050, 1651, 1529, 1215. ¹H NMR (400 MHz,

DMSO-d₆): δ 12.91 (br s, 1H, OH), 11.19 (br s, 1H, NH), 8.61 (s, 1H),

8.48−8.43 (m, 2H), 8.19−8.17 (m, 1H), 8.09−8.03 (m, 2H), 7.96−

7.93 (m, 1H), 7.84−7.77 (m, 2H), 7.68−7.65 (m, 1H), 7.55−7.51 (m,

1H), 7.39−7.35 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 163.97,

153.86, 148.31, 147.38, 136.83, 135.82, 130.40, 130.13, 128.90, 128.64,

128.21, 128.04, 127.96, 127.41, 126.81, 125.85, 125.75, 123.81, 121.18,

117.59, 110.48. Anal. Calcd for C₂₁H₁₅N₃O₂·0.25H₂O: C, 72.92; H,

4.53; N, 12.15. Found: C, 72.88; H, 4.33; N, 12.11.

3-Hydroxy-N′-(pyridin-2-ylmethylene)-2-naphthohydrazide

(K86).⁶⁹⁻⁷²Compound K86 was obtained from 3-hydroxy-2-

naphthohydrazide and pyridine-2-carbaldehyde in 71% yield as a

white solid. Mp: 222−224 °C. IR (KBr, cm⁻¹): 3257, 1686, 1537, 1462,

1199. ¹H NMR (400 MHz, DMSO-d₆): δ 12.18 (br s, 1H, OH), 11.16

(br s, 1H, NH), 8.65−8.63 (m, 1H), 8.49 (s, 1H), 8.44 (s, 1H), 8.05−

8.03 (m, 1H), 7.94−7.91 (m, 2H), 7.79−7.77 (m, 1H), 7.54−7.51 (m,

1H), 7.46−7.44 (m, 1H), 7.39−7.35 (m, 2H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 164.05, 153.88, 153.11, 149.56, 148.52, 136.92, 135.84,

130.26, 128.62, 128.22, 126.77, 125.83, 124.52, 123.79, 120.74, 120.10,

110.51. Anal. Calcd for C₁₇H₁₃N₃O₂·0.25H₂O: C, 69.01; H, 4.61; N,

14.21. Found: C, 68.83; H, 4.54; N, 13.98.

N′-[(1H-Pyrrol-2-yl)methylene]-3-hydroxy-2-naphthohydrazide

(K87).^73,74Compound K87 was obtained from 3-hydroxy-2-naph-

thohydrazide and pyrrole-2-carbaldehyde in 76% yield as a white solid.

Mp: 216−218 °C. IR (KBr, cm⁻¹): 3248, 1620, 1560, 1227. ¹H NMR

(400 MHz, DMSO-d₆): δ 11.76 (s, 1H, OH), 11.62 (s, 1H, NH), 11.48

(br s, 1H, NH), 8.47 (s, 1H), 8.32 (s, 1H), 7.92−7.90 (m, 1H), 7.78−

7.75 (m, 1H), 7.53−7.49 (m, 1H), 7.39−7.35 (m, 1H), 7.33 (s, 1H),

6.97−6.96 (m, 1H), 6.56−6.54 (m, 1H), 6.19−6.17 (m, 1H). ¹³C NMR

(100 MHz, DMSO-d₆): δ 163.51, 154.45, 141.72, 135.80, 129.82,

128.63, 128.16, 126.84, 126.74, 125.83, 123.76, 122.93, 119.90, 113.97,

110.58, 109.41. Anal. Calcd for C₁₆H₁₃N₃O₂: C, 68.81; H, 4.69; N,

15.05. Found: C, 68.61; H, 4.61; N, 14.96.

3-Hydroxy-N′-[1-(4-nitrophenyl)ethylidene]-2-naphthohydra-

zide (K81). Compound K81 was obtained from 3-hydroxy-2-

naphthohydrazide and 4-nitroacetophenone in 82% yield as a yellow

solid. Mp: 291−292 °C. IR (KBr, cm⁻¹): 3067, 1640, 1557, 1514, 1345,

1219. ¹H NMR (400 MHz, DMSO-d₆): δ 11.74 (br s, 1H, OH), 11.69

(s, 1H, NH), 8.65 (s, 1H), 8.32−8.31 (m, 2H), 8.15−8.14 (m, 2H),

8.01−8.00 (m, 1H), 7.79−7.78 (m, 1H), 7.54−7.52 (m, 1H), 7.39−

7.37 (m, 2H), 2.44 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ

161.67, 152.57, 149.95, 147.61, 144.02, 135.83, 132.46, 128.95, 128.39,

127.56 (2C), 127.20, 125.71, 123.94, 123.59 (2C), 120.65, 110.70,

13.78. Anal. Calcd for C₁₉H₁₅N₃O₄: C, 65.32; H, 4.33; N, 12.03. Found:

C, 65.12; H, 4.30; N, 11.93.

3-Hydroxy-N′-(1-phenylethylidene)-2-naphthohydrazide (K82).⁶⁸

Compound K82 was obtained from 3-hydroxy-2-naphthohydrazide

and acetophenone in 76% yield as a white solid. Mp: 250−252 °C. IR

1

(KBr, cm⁻¹): 3058, 1632, 1549, 1362, 1220. H NMR (400 MHz,

N′-(Furan-2-ylmethylene)-3-hydroxy-2-naphthohydrazide

(K88).⁷⁴Compound K88 was obtained from 3-hydroxy-2-naphthohy-

drazide and furfural in 63% yield as a brown solid. Mp: 210−211 °C. IR

DMSO-d₆): δ 11.71 (br s, 1H, OH), 11.55 (s, 1H, NH), 8.67 (s, 1H),

8.01−7.99 (m, 1H), 7.90−7.89 (m, 2H), 7.79−7.78 (m, 1H), 7.54−

7.52 (m, 1H), 7.48−7.45 (m, 3H), 7.39−7.37 (m, 2H), 2.39 (s, 3H,

CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ 161.57, 152.72, 152.41,

137.91, 135.74, 132.24, 129.40, 128.90, 128.37 (2C), 128.26, 127.20,

126.46 (2C), 125.69, 123.87, 120.74, 110.67, 13.92. Anal. Calcd for

C₁₉H₁₆N₂O₂: C, 74.98; H, 5.30; N, 9.20. Found: C, 74.89; H, 5.13; N,

9.22.

N′-[Di(pyridin-2-yl)methylene]-3-hydroxy-2-naphthohydrazide

(K83). Compound K83 was obtained from 3-hydroxy-2-naphthohy-

drazide and di(2-pyridyl) ketone in 81% yield as a yellow solid. Mp:

235−236 °C. IR (KBr, cm⁻¹): 3054, 1672, 1524, 1298, 1221. ¹H NMR

(400 MHz, DMSO-d₆): δ 13.94 (s, 1H, OH), 11.25 (br s, 1H, NH),

8.89−8.87 (m, 1H), 8.68 (s, 1H), 8.58−8.57 (m, 1H), 8.15−8.13 (m,

1H), 8.04−7.99 (m, 3H), 7.78−7.76 (m, 1H), 7.64−7.60 (m, 1H),

7.54−7.47 (m, 3H), 7.38−7.34 (m, 1H), 7.32 (s, 1H). ¹³C NMR (100

MHz, DMSO-d₆): δ 161.89, 155.41, 152.62, 151.19, 148.80, 148.53,

148.49, 137.24, 137.06, 136.00, 133.12, 129.07, 128.43, 127.14, 126.05,

125.64, 124.42, 123.99, 123.83, 122.83, 120.42, 110.49. Anal. Calcd for

C₂₂H₁₆N₄O₂: C, 71.73; H, 4.38; N, 15.21. Found: C, 71.60; H, 4.23; N,

15.16.

1

(KBr, cm⁻¹): 3058, 1630, 1551, 1223. H NMR (400 MHz, DMSO-

d₆): δ 11.93 (s, 1H, OH), 11.28 (s, 1H, NH), 8.44 (s, 1H), 8.38 (s, 1H),

7.93−7.91 (m, 1H), 7.89 (d, 1H, J = 1.2 Hz), 7.78−7.76 (m, 1H),

7.53−7.50 (m, 1H), 7.38−7.33 (m, 2H), 6.98 (d, 1H, J = 2.4 Hz), 6.67

(dd, 1H, J = 2.4, 1.2 Hz). ¹³C NMR (100 MHz, DMSO-d₆): δ 163.75,

154.00, 149.29, 145.41, 138.21, 135.82, 130.17, 128.62, 128.20, 126.76,

125.82, 123.79, 120.33, 114.00, 112.26, 110.54. Anal. Calcd for

C₁₆H₁₂N₂O₃: C, 68.56; H, 4.32; N, 9.99. Found: C, 68.16; H, 4.20; N,

9.80.

3-Hydroxy-N′-(thiophen-2-ylmethylene)-2-naphthohydrazide

(K89).⁷⁴⁻⁷⁶Compound K89 was obtained from 3-hydroxy-2-

naphthohydrazide and thiophene-2-carbaldehyde in 75% yield as a

light yellow solid. Mp: 240−242 °C. IR (KBr, cm⁻¹): 3060, 1630, 1558,

1224. ¹H NMR (400 MHz, DMSO-d₆): δ 11.94 (s, 1H, OH), 11.27 (br

s, 1H, NH), 8.69 (s, 1H), 8.43 (s, 1H), 7.93−7.91 (m, 1H), 7.78−7.76

(m, 1H), 7.71 (d, 1H, J = 3.6 Hz), 7.53−7.50 (m, 2H), 7.38−7.36 (m,

1H), 7.33 (s, 1H), 7.17 (dd, 1H, J = 3.6, 2.4 Hz). ¹³C NMR (100 MHz,

DMSO-d₆): δ 163.61, 153.99, 143.58, 138.85, 135.80, 131.30, 130.12,

129.27, 128.61, 128.19, 127.92, 126.75, 125.82, 123.78, 120.38, 110.54.

Anal. Calcd for C₁₆H₁₂N₂O₂S: C, 64.85; H, 4.08; N, 9.45. Found: C,

64.84; H, 3.96; N, 9.51.

3-Hydroxy-N′-[1-(quinolin-2-yl)ethylidene]-2-naphthohydrazide

(K84). Compound K84 was obtained from 3-hydroxy-2-naphthohy-

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2-Hydroxy-N′-[1-(pyridin-2-yl)ethylidene]benzohydrazide

(K90).^77,78Compound K90 was obtained from 2-hydroxybenzohy-

drazide and 2-acetylpyridine in 84% yield as a white solid. Mp: 236−

238 °C. IR (KBr, cm⁻¹): 3044, 1639, 1548, 1462, 1232. ¹H NMR (400

MHz, DMSO-d₆): δ 11.81 (br s, 1H, OH), 11.48 (s, 1H, NH), 8.64−

8.63 (m, 1H), 8.17−8.15 (m, 1H), 8.05−8.03 (m, 1H), 7.91−7.87 (m,

2,6-Dihydroxy-N′-[1-(pyridin-2-yl)ethylidene]benzohydrazide

(K96). Compound K96 was obtained from 2,6-dihydroxybenzohy-

drazide and 2-acetylpyridine in 82% yield as a gray solid. Mp: 214−216

1

°C. IR (KBr, cm⁻¹): 3262, 1598, 1552, 1233. H NMR (400 MHz,

DMSO-d₆): δ 12.78 (br s, 2H, OH), 11.87 (s, 1H, NH), 8.63−8.62 (m,

1H), 8.15−8.13 (m, 1H), 7.90−7.86 (m, 1H), 7.45−7.42 (m, 1H), 7.26

(t, 1H, J = 8.0 Hz), 6.46 (d, 2H, J = 8.0 Hz), 2.43 (s, 3H, CH₃). ¹³

C

1H), 7.47−7.42 (m, 2H), 7.07−7.00 (m, 2H), 2.45 (s, 3H, CH₃). ¹³

C

NMR (100 MHz, DMSO-d₆): δ 166.83, 160.36 (2C), 154.45, 153.56,

148.74, 136.78, 134.34, 124.43, 120.55, 107.59 (2C), 102.12, 11.84.

Anal. Calcd for C₁₄H₁₃N₃O₃: C, 61.99; H, 4.83; N, 15.49. Found: C,

61.62; H, 5.04; N, 15.21.

NMR (100 MHz, DMSO-d₆): δ 161.92, 156.34, 154.86, 152.20, 148.65,

136.65, 133.54, 130.85, 124.12, 120.39, 119.82, 117.90, 116.89, 11.77.

Anal. Calcd for C₁₄H₁₃N₃O₂: C, 65.87; H, 5.13; N, 16.46. Found: C,

65.91; H, 5.18; N, 16.45.

2,4-Dihydroxy-N′-[1-(pyridin-2-yl)ethylidene]benzohydrazide

(K97). Compound K97 was obtained from 2,4-dihydroxybenzohy-

drazide and 2-acetylpyridine in 76% yield as a white solid. Mp: 245−

247 °C. IR (KBr, cm⁻¹): 3200, 1595, 1468, 1260. ¹H NMR (400 MHz,

DMSO-d₆): δ 11.72 (br s, 1H, OH), 11.27 (s, 1H, NH), 10.18 (br s, 1H,

OH), 8.61−8.59 (m, 1H), 8.13−8.11 (m, 1H), 7.88−7.84 (m, 2H),

7.42−7.39 (m, 1H), 6.45−6.41 (m, 2H), 2.40 (s, 3H, CH₃). ¹³C NMR

(100 MHz, DMSO-d₆): δ 162.26 (2C), 158.41, 155.03, 151.23, 148.65,

136.65, 132.47, 124.02, 120.35, 109.17, 108.23, 102.73, 11.70. Anal.

Calcd for C₁₄H₁₃N₃O₃·1.0H₂O: C, 58.12; H, 5.24; N, 14.53. Found: C,

58.42; H, 5.25; N, 14.56.

N′-[Di(pyridin-2-yl)methylene]-2,4-dihydroxybenzohydrazide

(K98). Compound K98 was obtained from 2,4-dihydroxybenzohy-

drazide and di(2-pyridyl)ketone in 75% yield as a light yellow solid. Mp:

278−280 °C. IR (KBr, cm⁻¹): 3362, 1639, 1259. ¹H NMR (400 MHz,

DMSO-d₆): δ 13.66 (s, 1H, OH), 11.18 (s, 1H, NH), 10.15 (s, 1H,

OH), 8.84−8.82 (m, 1H), 8.54−8.53 (m, 1H), 8.07−8.06 (m, 1H),

8.00−7.93 (m, 2H), 7.81 (d, 1H, J = 8.8 Hz), 7.59−7.55 (m, 1H),

7.47−7.43 (m, 2H), 6.40 (dd, 1H, J = 8.8, 2.0 Hz), 6.36 (d, 1H, J = 2.0

Hz). ¹³C NMR (100 MHz, DMSO-d₆): δ 162.59, 162.36, 158.44,

155.57, 151.37, 148.69, 148.44, 147.10, 137.20, 136.97, 132.48, 125.97,

124.24, 123.77, 122.67, 109.10, 108.21, 102.49. Anal. Calcd for

C₁₈H₁₄N₄O₃: C, 64.66; H, 4.22; N, 16.76. Found: C, 64.78; H, 4.22; N,

16.79.

N′-[Di(pyridin-2-yl)methylene]-2-hydroxybenzohydrazide

(K91).⁷⁹⁻⁸¹Compound K91 was obtained from 2-hydroxybenzohy-

drazide and di(2-pyridyl)ketone in 79% yield as a white solid. Mp:

179−181 °C. IR (KBr, cm⁻¹): 3057, 1657, 1595, 1378, 1225. ¹H NMR

(400 MHz, DMSO-d₆): δ 13.78 (s, 1H, OH), 11.26 (br s, 1H, NH),

8.86−8.84 (m, 1H), 8.57−8.55 (m, 1H), 8.11−8.09 (m, 1H), 8.02−

7.96 (m, 3H), 7.61−7.58 (m, 1H), 7.50−7.40 (m, 3H), 7.00−6.96 (m,

2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 162.31, 156.35, 155.43,

151.22, 148.73, 148.50, 148.06, 137.25, 137.04, 133.73, 131.09, 126.03,

124.37, 123.94, 122.76, 119.70, 117.74, 116.78. Anal. Calcd for

C₁₈H₁₄N₄O₂: C, 67.91; H, 4.43; N, 17.60. Found: C, 68.00; H, 4.41; N,

17.63.

2-Hydroxy-N′-(pyridin-2-ylmethylene)benzohydrazide (K92).^82,83

Compound K92 was obtained from 2-hydroxybenzohydrazide and

pyridine-2-carbaldehyde in 71% yield as a white solid. Mp: 223−225

1

°C. IR (KBr, cm⁻¹): 3048, 1629, 1544, 1462, 1230. H NMR (400

MHz, DMSO-d₆): δ 12.01 (s, 1H, OH), 11.65 (br s, 1H, NH), 8.64−

8.63 (m, 1H), 8.48 (s, 1H), 8.00−7.99 (m, 1H), 7.94−7.86 (m, 2H),

7.47−7.42 (m, 2H), 7.04−6.95 (m, 2H). ¹³C NMR (100 MHz, DMSO-

d₆): δ 164.99, 158.85, 153.07, 149.56, 148.71, 136.91, 133.90, 128.67,

124.49, 120.07, 118.99, 117.240, 116.18. Anal. Calcd for C₁₃H₁₁N₃O₂:

C, 64.72; H, 4.60; N, 17.42. Found: C, 64.66; H, 4.557; N, 17.38.

N′-[1-(Furan-2-yl)ethylidene]-3-hydroxy-2-naphthohydrazide

(K93).^70,84Compound K93 was obtained from 3-hydroxy-2-naph-

thohydrazide and 2-acetylfuran in 81% yield as a yellow solid. Mp:

211−213 °C. IR (KBr, cm⁻¹): 3109, 1650, 1223. ¹H NMR (400 MHz,

DMSO-d₆): δ 11.77 (br s, 1H, OH), 11.48 (s, 1H, NH), 8.64 (s, 1H),

8.00−7.97 (m, 1H), 7.84 (d, 1H, J = 1.6 Hz), 7.78−7.76 (m, 1H),

7.53−7.49 (m, 1H), 7.38−7.33 (m, 2H), 7.02 (d, 1H, J = 3.6 Hz), 6.63

(dd, 1H, J = 3.6, 1.6 Hz), 2.30 (s, 3H, CH₃). ¹³C NMR (100 MHz,

DMSO-d₆): δ 161.23, 152.64, 151.61, 144.77, 144.12, 135.74, 132.31,

128.93, 128.30, 127.21, 125.70, 123.90, 120.66, 112.05, 111.82, 110.66,

13.32. Anal. Calcd for C₁₇H₁₄N₂O₃·0.1H₂O: C, 68.95; H, 4.84; N, 9.46.

Found: C, 68.90; H, 4.80; N, 9.40.

3-Hydroxy-N′-[1-(pyridin-2-yl)ethylidene]benzohydrazide (K99).

Compound K99 was obtained from 3-hydroxybenzohydrazide and 2-

acetylpyridine in 72% yield as a white solid. Mp: 237−238 °C. IR (KBr,

1

cm⁻¹): 3181, 1652, 1529, 1295. H NMR (400 MHz, DMSO-d₆): δ

10.78 (br s, 1H, OH), 9.76 (br s, 1H, NH), 8.62−8.60 (m, 1H), 7.87−

7.84 (m, 1H), 7.43−7.40 (m, 1H), 7.32−7.27 (m, 4H), 6.99−6.96 (m,

1H), 2.44 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ 157.22,

155.12, 148.60, 147.76, 138.78, 136.57, 135.25, 129.35, 124.78, 124.11,

120.33, 118.53, 114.86, 12.46. Anal. Calcd for C₁₄H₁₃N₃O₂: C, 65.87;

H, 5.13; N, 16.46. Found: C, 66.06; H, 5.09; N, 16.48.

N′-[1-(Pyridin-2-yl)ethylidene]benzohydrazide (K100).^85,86Com-

pound K100 was obtained from benzohydrazide and 2-acetylpyridine in

78% yield as a white solid. Mp: 281−283 °C. IR (KBr, cm⁻¹): 3183,

1658, 1539, 1282. ¹H NMR (400 MHz, DMSO-d₆): δ 10.87 (br s, 1H,

NH), 8.62−8.61 (m, 1H), 8.17−8.11 (m, 1H), 7.95−7.81 (m, 3H),

7.64−7.50 (m, 3H), 7.42−7.38 (m, 1H, pyridine), 2.47 (s, 3H, CH₃).

¹³C NMR (100 MHz, DMSO-d₆): δ 155.10, 148.62, 147.80, 138.13,

3-Hydroxy-N′-[1-(thiophen-2-yl)ethylidene]-2-naphthohydra-

zide (K94). Compound K94 was obtained from 3-hydroxy-2-

naphthohydrazide and 2-acetylthiophene in 76% yield as a white

solid. Mp: 220−224 °C. IR (KBr, cm⁻¹): 3055, 1635, 1567, 1220. ¹H

NMR (400 MHz, DMSO-d₆): δ 11.71 (br s, 1H, OH), 11.47 (s, 1H,

NH), 8.62 (s, 1H), 7.99−7.97 (m, 1H), 7.78−7.76 (m, 1H), 7.63 (d,

1H, J = 4.8 Hz), 7.55 (d, 1H, J = 4.0 Hz), 7.53−7.49 (m, 1H), 7.38−

136.61, 133.93, 132.05, 131.60, 128.28, 127.06, 124.84, 124.14, 120.35,

12.56. Anal. Calcd for C₁₄H₁₃N₃O·0.1H₂O: C, 69.74; H, 5.48; N, 17.56.

Found: C, 69.58; H, 5.40; N, 17.56.

7.43 (m, 2H), 7.12 (dd, 1H, J = 4.8, 4.0 Hz), 2.40 (s, 3H, CH₃). ¹³

C

NMR (100 MHz, DMSO-d₆): δ 161.30, 152.73, 149.17, 143.03, 135.75,

132.11, 129.08, 128.92, 128.35, 128.30, 127.63, 127.18, 125.72, 123.91,

120.72, 110.66, 14.30. Anal. Calcd for C₁₇H₁₄N₂O₂S: C, 65.79; H, 4.55;

N, 9.03. Found: C, 65.79; H, 4.51; N, 9.04.

2-Methoxy-N′-[1-(pyridin-2-yl)ethylidene]benzohydrazide

(K101). Compound K101 was obtained from 2-methoxybenzohydra-

zide and 2-acetylpyridine in 81% yield as a white solid. Mp: 184−186

1

°C. IR (KBr, cm⁻¹): 3307, 1670, 1538, 1240. H NMR (400 MHz,

N′-[1-(1H-Pyrrol-2-yl)ethylidene]-3-hydroxy-2-naphthohydra-

zide (K95). Compound K95 was obtained from 3-hydroxy-2-

naphthohydrazide and 2-acetylpyrrole in 77% yield as a brown solid.

DMSO-d₆): δ 11.05 (s, 1H, NH), 8.63−8.62 (m, 1H), 8.14−8.12 (m,

1H), 7.96−7.85 (m, 2H), 7.60−7.55 (m, 1H), 7.44−7.41 (m, 1H),

7.26−7.24 (m, 1H), 7.15−7.12 (m, 1H), 4.01 (s, 3H, OCH₃), 2.44 (s,

3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ 161.39, 157.19, 154.89,

152.29, 148.66, 136.65, 133.28, 131.04, 124.14, 121.25, 121.05, 120.38,

112.38, 56.50, 11.59. Anal. Calcd for C₁₅H₁₅N₃O₂: C, 66.90; H, 5.61; N,

15.60. Found: C, 66.95; H, 5.62; N, 15.60.

2-Amino-N′-[1-(pyridin-2-yl)ethylidene]benzohydrazide

(K102).^87,88Compound K102 was obtained from 2-aminobenzohy-

drazide and 2-acetylpyridine in 73% yield as a green solid. Mp: 167−

169 °C. IR (KBr, cm⁻¹): 3430, 3315, 1645, 1248. ¹H NMR (400 MHz,

1

Mp: 266 °C dec. IR (KBr, cm⁻¹): 3413, 1630, 1447, 1248. H NMR

(400 MHz, DMSO-d₆): δ 10.46 (s, 1H, NH), 8.13 (s, 1H), 7.93−7.91

(m, 1H), 7.84−7.81 (m, 1H), 7.55−7.51 (m, 1H), 7.39−7.35 (m, 2H),

7.23 (dd, 1H, J = 2.8, 1.2 Hz), 7.04 (dd, 1H, J = 3.6, 1.2 Hz), 6.95 (dd,

1H, J = 3.6, 2.8 Hz), 2.69 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-

d₆): δ 152.90, 151.90, 144.84, 135.43, 131.31, 128.32, 127.65, 127.28,

126.00, 123.67, 123.63, 121.67, 115.93, 115.66, 109.76, 104.84, 18.62.

Anal. Calcd for C₁₇H₁₅N₃O₂: C, 69.61; H, 5.15; N, 14.33. Found: C,

69.98; H, 4.75; N, 14.46.

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Journal of Medicinal Chemistry

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Article

DMSO-d₆): δ 10.62 (br s, 1H, NH), 8.61−8.60 (m, 1H), 8.09−8.06

(m, 1H), 7.87−7.82 (m, 1H), 7.60−7.57 (m, 1H), 7.42−7.39 (m, 1H),

7.24−7.19 (m, 1H), 6.79−6.77 (m, 1H), 6.62−6.58 (m, 1H), 6.22 (br s,

2H, NH₂), 2.45 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ

155.26, 149.69, 148.60, 147.70, 136.57, 132.57, 132.21, 129.32, 124.01,

120.28, 116.29, 114.84, 114.53, 12.36. Anal. Calcd for C₁₄H₁₄N₄O: C,

66.13; H, 5.55; N, 22.03. Found: C, 66.13; H, 5.55; N, 21.84.

(GraphPad, La Jolla, CA). Data were considered statistically signiﬁcant

at the P < 0.05 level.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00093.

■

sı

2-Amino-N′-[1-(pyridin-2-yl)ethylidene]nicotinohydrazide

(K117). Compound K117 was obtained from 2-aminonicotinohydra-

zide and 2-acetylpyridine in 74% yield as a white solid. Mp: 197−199

Small molecules identiﬁed after the analysis of the drug

screen assays (Figure S1), eﬀect of K117 on cell viability

(by the SRB assay) of liver cancer cells (Figure S2), eﬀect

of K117 on tumor growth in Huh7 xenograft nude mice

(Figure S3), eﬀect of K117 on organ morphology in

K117-treated NOD-SCID mice (Figure S4), HPLC

chromatograms obtained from the analysis of K177 in

liver tissue extracts of mice that received K117 (Figures

S5−S14), quantiﬁcation of MYC and GNMT signal

intensities from the Figure 6H immunoblot (Figure S15),

eﬀect of K117 on MYC protein expression in vivo (Figure

S16), quantiﬁcation of MYC signal intensities from the

1

°C. IR (KBr, cm⁻¹): 3425, 1658, 1572, 1524, 1463, 1248. H NMR

(400 MHz, DMSO-d₆): δ 8.61−8.60 (m, 1H), 8.13 (dd, 1H, J = 4.8, 1.6

Hz), 8.05 (br s, 1H), 7.97 (d, 1H, J = 8.0 Hz), 7.86−7.82 (m, 1H),

7.43−7.39 (m, 1H), 6.84 (s, 2H, NH₂), 6.67−6.64 (dd, 1H, J = 8.0, 4.8

Hz), 2.45 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ 165.56,

158.69, 155.12, 153.90, 151.53, 148.64, 138.01, 136.61, 124.14, 120.34,

111.41, 109.51, 12.55. Anal. Calcd for C₁₃H₁₃N₅O: C, 61.17; H, 5.13;

N, 27.43. Found: C, 61.23; H, 5.09; N, 27.41.

Cell Viability and Colony Formation Assay. The Alamar blue

assay (AbD serotec, Raleigh, NC) and the sulforhodamine B (SRB)

assay were used to evaluate cell proliferation and cell viability as

described previously.^89,90In brief, Huh7, HepG, Hep3B, and Mahlavu

cells were seeded in 96-well plates and exposed to various

concentrations of the drugs. Alamar Blue was added and further

incubated for 4 h to assess the eﬀect of the tested drugs. For the colony

formation assay, cells (1 × 10⁴cells/well) were seeded overnight in six-

well plates and exposed to speciﬁed concentrations of drugs. After

incubation for 10 days at 37 °C in a 5% CO₂atmosphere, surviving

colonies were analyzed through the crystal violet staining method as

previously described.²²OpenCFU colony counting software (http://

opencfu.sourceforge) was used for colony quantiﬁcation.

Immunoblotting. Cells or the xenograft tumors lysate was

prepared by using a radio-immunoprecipitation assay (RIPA) buﬀer

containing phosphatase inhibitors (10 mM Na₃VO₄, 1 mM NaF, and 5

mM NaPP_i) and a protease inhibitor cocktail (Roche Molecular

Biochemicals). The protein concentrations in the supernatant were

quantiﬁed by the Bio-Rad Protein Assay (Bio-Rad), and immunoblot-

ting was carried out as described previously.⁹⁰Antibodies anti-MYC

(D84C12, Cell Signaling Technology), anti-GNMT (YMAC-Biotech),

and anti-β-actin (AC-15, Sigma-Aldrich) were used according to the

description in the manufacturer’s instructions.

Quantitative Real-Time PCR (qRT-PCR). Total RNA from cell

lines and xenograft tumors was isolated by using Trizol reagent (Life

Technologies) according to the manufacturer’s instructions. Comple-

mentary DNA was produced using the SuperScript II RNase H-Reverse

Transcriptase Kit (Life Technologies). PCR ampliﬁcation was

performed on the Applied Biosystems Step-One-plus real-time PCR

system (Applied Biosystems, Foster City, CA) as described previously

using the LightCycler First Start DNA Master SYBR Green I reagent

(Roche Diagnostics, Basel, Switzerland).²²The normalization of real-

time RT-PCR data was performed using the TBP mRNA level as a

standard to calculate relative expression. The primers are described in

Table S1.

1

Figure 6J immunoblot (Figure S17), H and ¹³C NMR

spectra of the compounds (Figures S18−S67), rapid-scan

FT-IR spectra of the compounds (Figures S68−S93),

oﬃcial reports of the elemental analyses (Figures S94−

S96), HPLC purity reports (Figures S97−S100), and a list

of QPCR primers (Table S1) (PDF)

Molecular formula SMILE strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Yi-Ming Arthur Chen − Graduate Institute of Biomedical and

Pharmaceutical Science, Fu Jen Catholic University, New

Taipei City 24205, Taiwan; Email: 150110@mail.fju.edu.tw

Yeh-Long Chen − Drug Development and Value Creation

Research Center and Department of Medicinal and Applied

Chemistry, College of Life Science, Kaohsiung Medical

University, Kaohsiung 80708, Taiwan; Email: yeloch@

kmu.edu.tw

Authors

Rajni Kant − Graduate Institute of Biomedical and

Pharmaceutical Science, Fu Jen Catholic University, New

Taipei City 24205, Taiwan; orcid.org/0000-0003-4120-

9700

Ming-Hui Yang − Department of Medical Education and

Research, Kaohsiung Veterans General Hospital, Kaohsiung

81362, Taiwan

Chih-Hua Tseng − School of Pharmacy, College of Pharmacy,

Kaohsiung Medical University, Kaohsiung 807, Taiwan; Drug

Development and Value Creation Research Center, Kaohsiung

Medical University, Kaohsiung 80708, Taiwan

Chia-Hung Yen − Graduate Institute of Natural Products,

College of Pharmacy and Research Center for Natural Products

and Drug Development, Kaohsiung Medical University,

Kaohsiung 80708, Taiwan; orcid.org/0000-0002-6939-

5528

Xenograft Assay. The animal experiments were reviewed,

approved, and performed in accordance with relevant guidelines

provided by the Institutional Animal Care and Use Committee of

Kaohsiung Medical University. The xenograft model was prepared as

described previously.²⁴In brief, ﬁve- to six-week-old female Balb/c

nude mice were injected with Huh7 cells (1 × 10⁶) subcutaneously in

the right ﬂank. Once a palpable tumor was detected, mice were

randomly grouped and treated with 300, 100, and 25 mpk K78 and the

drug vehicle orally every day for 30 days. For K117, doses 10 and 25

mpk were used. For the sorafenib control group, a dose of 25 mpk was

used. Tumor growth was monitored throughout the experiment by

using a Vernier caliper. Measurements of the length (L) and width (W)

of the tumor were used for tumor volume (TV) calculation via the

formula TV = (L × W²)/2. Compounds K78 and K117 were dissolved

in 0.5% carboxymethyl cellulose sodium salt.

Wei-You Li − Graduate Institute of Biomedical and

Pharmaceutical Science, Fu Jen Catholic University, New

Taipei City 24205, Taiwan

Yu-Chang Tyan − Department of Medical Imaging and

Radiological Sciences, Kaohsiung Medical University,

Kaohsiung 80708, Taiwan

Marcelo Chen − Department of Urology, Mackay Memorial

Hospital, Taipei 10449, Taiwan

Statistical Analysis. P values were calculated using the unpaired

two-tailed Student’s t tests with MS excel and GraphPad Prism 5.0

9006

https://doi.org/10.1021/acs.jmedchem.1c00093

J. Med. Chem. 2021, 64, 8992−9009

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Cherng-Chyi Tzeng − Department of Medicinal and Applied

Chemistry, College of Life Science, Kaohsiung Medical

University, Kaohsiung 80708, Taiwan

Wei-Cheng Chen − Graduate Institute of Biomedical and

Pharmaceutical Science, Fu Jen Catholic University, New

Taipei City 24205, Taiwan

Kaiting You − Graduate Institute of Biomedical and

Pharmaceutical Science, Fu Jen Catholic University, New

Taipei City 24205, Taiwan

Wen-Chieh Wang − Institute of Biotechnology and

Pharmaceutical Research, National Health Research Institutes,

Zhunan, Miaoli County 35053, Taiwan

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00093

Author Contributions

^□R.K., M.-H.Y., and C.-H.T. contributed equally to this work.

Funding

This work was supported by the Program to Upgrade the R&D

Capabilities of Private Universities (MOST105-2632-B-037-

001), the Taiwan Protein Project (AS-KPQ-105-TPP), the

National Health Research Institutes (NHRI-104A1-PDCO-

0315162), the Kaohsiung Medical University Aim for the Top

Universities (KMU-TP105E08, KMU-TP105E14, and KMU-

TP104H05), the Aim for the Top 500 Universities (KMU-

DT105004), the KMU Global Networking Talent Plan (105

KMUOR02 and 105 KMUOR05), Kaohsiung Medical Uni-

versity Research Foundation Grants S107006 and NSYSUK-

MU107-P015, The University of Cologne-Kaohsiung Medical

University Alliance (CKA) Research Fund (KMU-SH000151),

the Ministry of Health and Welfare, Taiwan (MOHW104-TDL-

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(VGHKS109-181), and the Research Center for Environmental

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Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

■

The authors thank Dr. Jen-Shin Song of the National Health

Research Institutes of Taiwan for drug screening and for the

drug library. The authors also acknowledge Dr. Shih-Ping Liu

(China Medical University) for his generous gift of plasmid

pCDNA-MYC and the staﬀ from the Center of Infectious

Disease and Cancer Research (CICAR) of Kaohsiung Medical

University for technical assistance.

DEDICATION

■

This work is dedicated to Dr. Pao-Huei Chen, founding

chairman of the Taiwan Liver Cancer Medical Association, in

memory of his contribution to the prevention and treatment of

liver cancer.

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ABBREVIATIONS USED

■

HCC, hepatocellular carcinoma; GNMT, glycine-N-methyl

transferase; PGG, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyrano-

side

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Discovery of an Orally Efficacious MYC Inhibitor for Liver Cancer Using a GNMT-Based High-Throughput Screening System and Structure-Activity Relationship Analysis

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