Styryl quinazolinones as potential inducers of myeloid differentiation via upregulation of C/EBPα

Article abstract of DOI:10.3390/molecules23081938

The CCAAT enhancer-binding protein α (C/EBPα) plays an important role in myeloid cell differentiation and in the enhancement of C/EBPα expression/activity, which can lead to granulocytic differentiation in acute myeloid leukemia (AML) cells. We found that styryl quinazolinones induce upregulation of C/EBPα expression, and thereby induce myeloid differentiation in human myeloid leukemia cell lines. We screened a series of active styryl quinazolinones and evaluated the structure–activity relationship (SAR) of these small molecules in inducing C/EBPα expression—thereby prompting the leukemic cells to differentiate. We observed that compound 78 causes differentiation at 3 μM concentration, while 1 induces differentiation at 10 μM concentration. We also observed an increase in the expression of neutrophil differentiation marker CD11b upon treatment with 78. Both the C/EBPα and C/EBPε levels were found to be upregulated by treatment with 78. These SAR findings are inspiration to develop further modified styryl quinazolinones, in the path of this novel differentiation therapy, which can contribute to the care of patients with AML.

Full text of DOI:10.3390/molecules23081938

molecules
Article
Styryl Quinazolinones as Potential Inducers of
Myeloid Differentiation via Upregulation of C/EBPα
Radhakrishnan Sridhar 1,†, Hisashi Takei 2,3,†, Riyaz Syed 4,5,†, Ikei S. Kobayashi 2,
Liu Bee Hui 1, Ahmed Kamal 4,6, Daniel G. Tenen 1,7,
*
and Susumu S. Kobayashi 2,7,8,
*
ID
ID
1
Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, Singapore;
csiradha@nus.edu.sg (R.S.); csilbh@nus.edu.sg (L.B.H.)
2
3
4
Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston,
MA 02215, USA; m14702054@gunma-u.ac.jp (H.T.); ikobayas@bidmc.harvard.edu (I.S.K.)
Department of Hematology, Gunma University Graduate School of Medicine, Maebashi,
Gunma 371-8511, Japan
Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007,
India; riyazsd@gmail.com (R.S.); ahmedkamal1915@gmail.com (A.K.)
5
6
7
8
Department of Chemistry, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad-500 085, India
School of Pharmaceutical Education and Research (SPER), Jamia Hamdard, New Delhi-110 062, India
Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02215, USA
Division of translational Genomics, Exploratory Oncology Research and Clinical Trial Center,
National Cancer Center, Kashiwa, Chiba 277-8577, Japan
*
Correspondences: daniel.tenen@nus.edu.sg (D.G.T.); sukobaya@east.ncc.go.jp (S.S.K.);
Tel.: +65-96587920 (D.G.T.); +81-4-7134-8786 (S.S.K.)
†
These authors contributed equally to this work.
Academic Editor: Takaomi Sanda
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 22 June 2018; Accepted: 31 July 2018; Published: 3 August 2018
Abstract: The CCAAT enhancer-binding protein
differentiation and in the enhancement of C/EBP
differentiation in acute myeloid leukemia (AML) cells. We found that styryl quinazolinones
induce upregulation of C/EBP expression, and thereby induce myeloid differentiation in
human myeloid leukemia cell lines. We screened a series of active styryl quinazolinones and
evaluated the structure–activity relationship (SAR) of these small molecules in inducing C/EBP
expression—thereby prompting the leukemic cells to differentiate. We observed that compound 78
causes differentiation at 3 M concentration, while induces differentiation at 10 M concentration.
We also observed an increase in the expression of neutrophil differentiation marker CD11b upon
treatment with 78. Both the C/EBP and C/EBP levels were found to be upregulated by treatment
α
(C/EBP
α) plays an important role in myeloid cell
α
expression/activity, which can lead to granulocytic
α
α
µ
1
µ
α
ε
with 78. These SAR findings are inspiration to develop further modified styryl quinazolinones, in the
path of this novel differentiation therapy, which can contribute to the care of patients with AML.
Keywords: myeloid differentiation; CCAAT/enhancer binding protein α
1. Introduction
Knowledge of the pathogenesis of acute myeloid leukemia (AML) has seen great progress due
to recent advances in genetic studies. However, the long-term survival of AML patients is still
unsatisfactory [
all AML subtypes. Currently, all-trans retinoic acid (ATRA) is the therapy of choice for the induction
of differentiation and the apoptosis of t(15;17)-positive acute promyelocytic leukemia (APL) cells [ ].
While ATRA treatment induces remission and constitutes a cure in nearly 70% of APL patients, it
1]. A block in differentiation is one of the characteristics of leukemia and is seen in
2
Molecules 2018, 23, 1938; doi:10.3390/molecules23081938
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1938
2 of 8
has no effect on other myeloid leukemias [
3]. Therefore, a novel differentiation therapy is sorely
needed. The CCAAT enhancer-binding protein
for the differentiation of cells in the liver, lung, adipose tissues, and bone marrow [
hematopoietic system, C/EBP is expressed in myeloid cells and is required for granulocytic and
monocytic differentiation [ ]. We have previously reported that C/EBP
and sufficient for neutrophil differentiation [ ]. Therefore, when C/EBP
α
(C/EBP
α
) is an essential transcription factor
4–7
]. In the
α
8
α
expression is necessary
9
α expression or function
is perturbed in myeloid progenitor cells, it can block differentiation and eventually lead to myeoid
leukemias. We demonstrated that C/EBP somatic mutations are detected in approximately 7%
of AML patients [10 11]. These mutations result in either the premature termination of C/EBP
α
,
α
translation or perturb DNA binding, thus supporting the notion that attenuated C/EBP
α activation is
associated with AML.
The quinazolinone scaffold (Figure 1) is widely represented among bioactive molecules [12].
For example, raltitrexed, a thymidylate synthase inhibitor, has been used for colorectal cancer and
malignant mesothelioma [13]. Ispinesib and tempostatin are in development for treatment of various
cancers [14
,15]. Several styryl quinazolinones specifically target methicillin-resistant staphylococcus
aureus infections [16
,
17]. Hamel et al. reported, in the L1210 leukemia model [18 20], 2-phenyl
–
quinazolinones and styryl quinazolinones to be inhibitors of tubulin polymerization and in vivo tumor
growth. Another report found styryl quinazolinones to cause the shortening of telomeres [21]. Based on
our hypothesis, that increased C/EBP
differentiation of leukemic cells, we screened several libraries of compounds for C/EBP
α
expression and/or activity in AML cells will stimulate the
induction
α
activity using a reporter assay [22]. We found that styryl quinazolinone analogue compound
1
(2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone increased C/EBP
α
activity, which in turn enhanced differentiation leading to growth arrest and the apoptosis of
leukemic cells.
A
NH2
O
O
N
O
N
O
N
H
HOOC
HOOC
Cl
Br
N
S
N
NH
N
NH
N
O
N
HO
Cl
O
Tempostatin
Raltitrexed
Ispinesib
B
C
O
N
O
O
O
Cl
Het Ar
N
NH
X
N
N
N
OH
OH
2-Styrylquinazolin-4(3H)ones
3-Heteroaryl 2-
Styrylquinazolin-4(3H)ones
Compound 1, (ICCB-280)
Figure 1. Quinazolinone compounds; (
development [13 15], ( ) Styryl quinazolinones explored as tubulin polymerization inhibitors [18
(C) Styryl quinazolinone 1, (ICCB-280) hit from small molecules C/EBPα inducer screens [22].
A
) quinazolinone drugs in the market and in early
–
B
–
20],
In this study, we screened a library of styryl quinazolinones to find more potent inducers of
C/EBP upregulation and differentiation. We also evaluated the structure-activity relationship (SAR)
α
of the styryl quinazolinones in terms of their ability to upregulate C/EBPα and to induce granulocytic
differentiation in human myeloid leukemia cells.

Molecules 2018, 23, 1938
3 of 8
2. Results and Discussion
We have earlier identified styryl quinazolinone
1
as a granulocytic differentiation inducer in
myeloid leukemia [21]. In order to arrive at more detailed SAR on this scaffold, we evaluated a
library of 80 styryl quinazolinones (Scheme 1A) using various approaches, namely the Wright-Giemsa
staining, to assess changes in morphology, nitroblue tetrazolium (NBT) reduction assay to evaluate
granulocyte function, and trypan-blue staining to detect apoptosis. Granulocyte differentiation was
determined (Table S1) from the Wright Giemsa staining and the NTB reduction assay.
A
R2'
R2'
O
N
R3CHO
AcOH
H2N
(iii)
R2'
O
O
N
O
R1
R2
R1
R1
N
R1
Ac2O
140oC, 12 h
OH
NH2
O
N
R2
R3
80 to 100oC,
3 to 6h
AcOH,
Reflux, 6h
R2
N
(i)
(ii)
(iv)
1 to 80
B
Substitutions except 6-
fluoro are inactive or not
Phenyl ring R2
showing significant effect
ortho and para
R2'
O
N
substitutions found active
and there is preference for
non-polar substituents
R1
N
R2
R3
Hydrogen donors and furan ring
were active. 5-nitro furan more
promising than other rings screened
Quinazolinone
ring R1
Linker L
Phenyl or fuan or
heteroaryl ring R3
Preference for double
bond and trans isomer
Scheme 1. Styryl quinazolinones synthesis, structure, and activity.
(A
) General synthetic
protocol for Styryl Quinazolinone synthesis.
differentiation inducers.
(B) SAR of styryl quinazolinones as granulocytic
The library of styryl quinazolinones was structurally differentiated at three parts of the scaffold,
namely the phenyl rings at R2 and R3, and the quinazolinone ring R1 (Scheme 1B). Granulocyte
differentiation was affected by the substitution of the heteroaryl ring R1. With the exception of the
6-fluoro substitution, other groups such as the iodo derivative, 32, dimethoxy derivatives 20
54 55 58, and dichloro derivative 51 were generally not favored. There was a general preference for
hydrophobic groups at R2, such as (2-methoxy Ph), 25 (3-methoxy Ph), 62 (3,4,5-trimethoxy Ph),
, 42, 50,
,
,
1
66 (4-carboxy methyl Ph), 68, and 79 (4-fluoro Ph). On the other hand, polar OH groups were preferred
at R3. Replacing phenyl R3 with a furan ring, preferably substituted with 2-nitro, improved activity.
The stereochemistry of the ethenyl linker, connecting ring R3 to the quinazolinone scaffold, was critical
and should be trans.
These general SAR trends are illustrated in Figure 2 with specific examples. Compound
ortho-MeO at ring R2 and mono-OH at ring R3 was inactive, but activity was restored when ring
3 was substituted with two OH groups in . Interestingly, retaining this motif and moving the
methoxy on ring R2 from ortho to meta, decreased activity (12). Two other inactive analogs related
to were and 15. In , ring R2 was not substituted, and in 15, ring R3 was replaced with pyridyl.
2 with
1
1
7
7
These marked changes in activity suggests, firstly, that the substitution on both rings R2 and R3 is
important, and secondly, that there is a critical dependency between the two substitution patterns that

Molecules 2018, 23, 1938
4 of 8
remains to be determined. Activities of the compounds
1
,
12, and 25, provide further confirmation of
this inference. Keeping the seemingly favored ortho OMe on ring R2 of 25, while replacing the phenyl
ring R3 with furan, led to some loss in activity. Inserting a nitro substituent to the furan ring R3 of 25
resulted in potent analogues 66, 67, 78, and 79.
O
O
O
N
O
N
O
N
O
N
O
N
O
N
O
N
F
N
N
N
O
O
OH
OH
OH
OH
NO2
OH
Compound 78,
highly active
Compound 25,
slightly active
Compound 1,
active
Compound 12,
slightly active
Compound 2,
inactive
O
O
O
N
N
N
N
OH
OH
N
Compound 7,
inactive
Compound 15,
inactive
Figure 2. Schematic showing the potent compound 78 and the structure-based variation in
differentiation of HL-60.
Compound 78, with 6-fluoro substitution at the quinazolinone ring R1, and leaving ring R2
unsubstituted (seen in
from our screening efforts. Styryl quinazolinone 78 at 3
7
to be abolished activity), was found to be the most active analogue identified
M induced granulocytic differentiation in
µ
HL-60 cells, upon treatment for seven days, as characterized by a decreased nucleus-cytoplasm ratio,
the presence of granules, and nuclear segmentation (Figure 3A). Similar granulocytic differentiation
was observed with 10 µM of 1, whereas the negative control Dimethyl Sulfoxide (DMSO) showed no
characteristic differentiation. In the NBT reduction assay to detect the production of superoxide anion,
55% were positive in 78 treated cells, compared to 0–5% in DMSO treated control cells (Figure 3B).
However, other potent analogues, 66
quinazolinone substitution, the derivatives 67
towards inducing differentiation compared to 78.
,
67, and 79, lost activities at 3
µM (0–5%). Even with 6-fluoro
,
70 72 75 79, and 80 were found to be less active
,
,
,
Figure 3. Styryl quinazolinone derivatives induce granulocytic differentiation and are determined by
) Wright-Giemsa staining for morphology, and ( ) NBT assay for function. HL-60 cells were treated
with 0.1% DMSO, 10 µM compound 1, or 3 µM compound 78 for seven days. Bars = 50 µm.
(
A
B

Molecules 2018, 23, 1938
5 of 8
Additionally, compound 78 increased the expression of the neutrophil differentiation surface
marker CD11b (Figure 4A). Similar results were obtained using MOLM-14 cells, which were established
from a patient with acute monocytic leukemia (M5a) (Supplementary Figure S1). We have reported
that
1 and granulocyte-colony stimulating factor (G-CSF) synergistically enhance granulocytic
differentiation in HL-60 cells [21]. As expected, G-CSF enhanced the expression of CD11b, which was
induced by compound 78 (31.7% vs. 42.2%, Figure 3A). These results suggest that 78 can upregulate
G-CSF receptors, and render leukemic cells more sensitive to G-CSF stimulation.
Figure 4. Compound 78 induces the expression of the neutrophil differentiation surface marker CD11b
and upregulation of CEBPA and its downstream targets: (
A
) CD11b positive cells were evaluated by
flow cytometry analysis; and (B) expression of CEBPA and CEBPE was determined by quantitative real
time PCR analysis.
Further, we examined the gene expression levels of the C/EBP
gene (CEBPE) [21]—one of the downstream target genes of C/EBP
with 78, mRNA expression levels of CEBPA and CEBPE were increased in a time-dependent manner
(Figure 4B). We observed that 78 could induce the increase of C/EBP (2.5-fold) and C/EBP (10.3-fold)
α
gene (CEBPA) and the C/EBP
ε
α
. When HL-60 cells were treated
α
ε
by day seven. These results further demonstrate that 78 induces granulocytic differentiation in
leukemia cell lines, which is mediated through upregulation of C/EBPα.
3. Conclusions
In this study we have identified the derivative 78 as a potent inducer of granulocytic differentiation
in HL-60 cells—apart from 1. The improvement in potency may be mainly due to the introduction of
the 5-nitro furan group at ring R3. This modification not only improves the granulocytic differentiation
ability of the styryl quinazolinones, but also replaces the Pan Assay Interference compounds (PAINS)
susceptible moiety 3,4-dihydroxy phenyl group in compound 1. The other factor influencing the
case of 78 can be the fluoro substitution of the quinazolinone scaffold. Overall, we have identified
a new and improved inducer for myeloid differentiation in HL-60 cells by suitably modifying the
3,4-dihydroxy phenyl part of 1, with the 5-nitrofuran group, which can be further developed as a

Molecules 2018, 23, 1938
6 of 8
potential drug targeting inducer of C/EBP
α
expression for leukemia treatment. To the best of our
knowledge, this is the first report describing the structure activity relations of styryl quinazolinones
relating to the induction of C/EBP expression, and during the investigation we found a lead,
like molecule 78, with more promising activity (at 3 M concentration) than our earlier lead, compound
. We strongly believe that repurposing styryl quinazolnines, for inducing C/EBP expression in AML,
α
µ
1
α
would rejuvenate further research on these types of small molecules.
4. Materials and Methods
Reagents: All-trans retinoic acid (ATRA) was purchased from Sigma-Aldrich (R2625,
St Louis, MO, USA). Compounds
Facility at Harvard Medical School.
1
to 60 were purchased from the ICCB-Longwood Screening
Compounds 61 to 80 were obtained from Indian
Institute of Chemical Technology and the synthesis of these compounds were reported
earlier [20]. 2-[E-2-(3,4-dihydroxyphenyl), vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone,
compound 1, and 6-fluoro-2-[(E)-2-(5-nitrofuran-2-yl)ethenyl]-3-phenylquinazolin-4(3H)-one (78) were
resynthesized (Scheme 1) at the National University of Singapore. Stock solutions for the compounds
◦
were solved in DMSO and stored at
−
20 C. These compounds were diluted in a fresh complete
medium before the experiment, and the final concentration of DMSO was 0.1%. Human G-SCF was
purchased from Miltenyl Biotec (130-096-345, Bergisch Gladbach, Germany). Human G-CSF was
prepared in 0.1% bovine serum albumin/PBS, and stored at −20 ◦C.
Cells: HL-60 (CCL-240) cells were purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). MOLM-14 cells were kindly provided by Dr. Gary Gililand. These cell lines were
maintained in RPMI 1640 with 10% fetal bovine serum at 37 ◦C with 5% CO2.
Morphological Analysis and Nitro Blue Tetrazolium Reduction Assay: HL-60 cells were treated
with 0.1% DMSO, 10
µM of 1, and 3 µM of 78 for 7 days and stained with Wright-Giemsa. Maximum
cell concentrations were adjusted lower than 100,000 /mL. These treated HL-60 cells were incubated
with 1.0 mg/mL nitroblue tetrazolium/PBS, and 0.36 µM Phorbol-12-myristate-13-acetate (PMA) at
37 ◦C for 30–40 min and stained with safranin O. Morphological differentiation and apoptosis were
evaluated as − (0–5%), + (5–30%), and ++ (more than 30%), as previously described [21].
Flow Cytometry: HL-60 cells were treated with 0.1% DMSO, 10 µM of 1, and 3 µM of 78 with or
without 60 ng/mL of human G-CSF for 7 days and were stained with APC anti-mouse/human CD11b
antibody from Biolegend (101211, San Diego, CA), and was analyzed by LSRII flow (BD Biosciences,
Franklin Lakes, NJ, USA). We used FlowJo (FLOWJO, LLC, Ashland, OR, USA) to analyze the data.
Quantitative Real-Time PCR (QT-PCR): Total RNA was isolated from HL-60 cells using
RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized using
SuperScript II (Thermo Fisher Scientific, Waltham, MA, USA). The mRNA levels of genes were
measured by QT-PCR using the Rotor Gene 6000 sequence detection system (Qiagen, Valencia, CA,
USA) with iTaq Universal SYBR Green Supermix (BIO-RAD, Hercules, CA, USA). The expression
levels of the glyceraldehyde 3-phosohate dehydrogenase (GAPDH) were used for normalization.
Reactions were performed in technical triplicates. The primers for qPCR, human C/EBP
α sense
50-TGTGCCTTGGAAATGCAAAC-30, antisense 50-CGGGAAGGAGGCAGGAAA-30, human C/EBP
ε
sense 50-GCGTTCTCAAGGCCCCTT-30, antisense 50-GGGAGGGCGCCTTCAG-30, human GAPDH
sense 50-CCACATCGCTCAGACACCAT-30, and antisense 50-CCAGGCGCCCAATACG-30.
Statistical Analysis: Differences between the experimental groups were tested with Student’s
t-test. p-values of less than 0.05 were considered statistically significant.
Supplementary Materials: The following are available online, Table S1: Styryl quinazolinone derivatives
evaluated in this study. Figure S1: Compound 78 induces morphological changes in MOLM-14 cells.
Author Contributions: R.S. (Radhakrishnan Sridhar), D.G.T. and S.S.K. conceived and designed the experiments;
R.S. (Radhakrishnan Sridhar), H.T., I.S.K., and R.S. (Riyaz Syed) performed the experiments; R.S. (Radhakrishnan
Sridhar), H.T., I.S.K., L.B.H. and S.S.K. analyzed the data; A.K. contributed reagents; D.G.T. and S.S.K. supervised

Molecules 2018, 23, 1938
7 of 8
the study; R.S. (Radhakrishnan Sridhar) and S.S.K. wrote the paper. All authors read and approved the
final manuscript.
Funding: R.S. (Radhakrishnan Sridhar), was supported by Cancer Science Institute of Singapore. This research
is supported by the Singapore Ministry of Health’s National Medical Research Council under its Singapore
Translational Research (STaR) Investigator Award, and by the National Research Foundation Singapore and
the Singapore Ministry of Education under its Research Centres of Excellence initiative. S.S.K was supported
by National Institution of Health (R21CA178301 and R01CA169259), American Cancer Society (RSG-13-047),
and Harvard Stem Cell Institute Blood Program (DP-0110-12-00). R.S. (Riyaz Syed) thanks CSIR-HRDG for the
award of CSIR-SRAship (13(8906-A)/2017-pool) and also acknowledges CSIR, New Delhi, for financial support
under the 12th Five Year plan project “Affordable Cancer Therapeutics (ACT)” (CSC0301). D.G.T was supported
by the National Institution of Health (P01 CA66996).
Acknowledgments: Authors thank Go Mei Lin for her helpful discussions during preparation of the manuscript
and for correcting the manuscript. Authors thank Brian W. Dymock for his valuable comments and helpful
discussions during the course of this work.
Conflicts of Interest: S.S.K. has previously received consulting fees from Pfizer and Ono Pharmaceutical.
The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation
of data; in the writing of the manuscript, and in the decision to publish the results. Other authors declare no
conflicts of interest.
References
1.
Ferrara, F.; Palmieri, S.; Mele, G. Prognostic factors and therapeutic options for relapsed or refractory acute
myeloid leukemia. Haematologica 2004, 89, 998–1008. [PubMed]
2.
Zhou, G.B.; Zhang, J.; Wang, Z.Y.; Chen, S.J.; Chen, Z. Treatment of acute promyelocytic leukaemia
with all-trans retinoic acid and arsenic trioxide: A paradigm of synergistic molecular targeting therapy.
Philos. Trans. R. Soc. B 2007, 362, 959–971. [CrossRef] [PubMed]
3.
4.
5.
6.
7.
8.
Nowak, D.; Stewart, D.; Koeffler, H.P. Differentiation therapy of leukemia: 3 decades of development. Blood
2009, 113, 3655–3665. [CrossRef] [PubMed]
Post, S.M.; Kantarjian, H.; Quintás-Cardama, A. Biology of adult myelocytic leukemia and myelodysplasia.
In The Molecular Basis of Cancer, 4th ed.; Elsevier-Saunders: Philadelphia, PA, USA, 2015; pp. 421–432.
Bernlohr, D.A.; Simpson, M.A. Biochemistry of lipids, lipoproteins and membranes. In New Comprehensive
Biochemistry; Elsevier: Waltham, MA, USA, 1996; pp. 257–281.
Mcdonald, P.P. Transcriptional Regulation in Neutrophils: Teaching Old Cells New Tricks. Adv. Immun. 2004
,
82, 1–48. [PubMed]
Morgan, E.T. Regulation of Drug-Metabolizing Enzymes and Drug Metabolism by Inflammatory Responses.
Drug Metab. Dis. 2017, 21–58.
Koleva, R.I.; Ficarro, S.B.; Radomska, H.S.; Carrasco-Alfonso, M.J.; Alberta, J.A.; Webber, J.T.; Luckey, C.J.;
Marcucci, G.; Tenen, D.G.; Marto, J.A. C/EBPα and DEK coordinately regulate myeloid differentiation. Blood
2012, 119, 4878–4888. [CrossRef] [PubMed]
9.
Zhang, D.E.; Zhang, P.; Wang, N.D.; Hetherington, C.J.; Darlington, G.J.; Tenen, D.G. Absence of
granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding
protein α-deficient mice. Proc. Natl. Acad. Sci. USA 1997, 94, 569–574. [CrossRef] [PubMed]
10. Pabst, T.; Mueller, B.U.; Zhang, P.; Radomska, H.S.; Narravula, S.; Schnittger, S.; Behre, G.; Hiddemann, W.;
Tenen, D.G. Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha
(C/EBPalpha), in acute myeloid leukemia. Nat. Genet. 2001, 27, 263–270. [CrossRef] [PubMed]
11. Koschmieder, S.; Halmos, B.; Levantini, E.; Tenen, D.G. Dysregulation of the C/EBPα Differentiation Pathway
in Human Cancer. J. Clin. Oncol. 2009, 27, 619–628. [CrossRef] [PubMed]
12. Jafari, E.; Khajouei, M.R.; Hassanzadeh, F.; Hakimelahi, G.H.; Khodarahmi, G.A. Quinazolinone and
quinazoline derivatives: Recent structures with potent antimicrobial and cytotoxic activities. Res. Pharm. Sci.
2016, 11, 1–14. [PubMed]
13. Cunningham, D.; Zalcberg, J.; Maroun, J.; James, R.; Clarke, S.; Maughan, T.S.; Vincent, M.; Schulz, J.;
Barón, M.G.; Facchini, T. Efficacy, tolerability and management of raltitrexed (Tomudex™) monotherapy in
patients with advanced colorectal cancer: A review of phase II/III trials. Eur. J. Cancer 2002, 38, 478–486.
[CrossRef]

Molecules 2018, 23, 1938
8 of 8
14. Penna, L.S.; Henriques, L.A.P.; Bonatto, D. Anti-mitotic agents: Are they emerging molecules for cancer
treatment? Pharm. Ther. 2017, 173, 67–82. [CrossRef] [PubMed]
15. De Jonge, M.J.A.; Dumez, H.; Verweij, J.; Yarkoni, C.; Snyder, D.; Lacombe, D.; Marréaud, S.; Yamaguchi, T.;
Punt, C.J.A.; Van Oosterom, A. Phase I and pharmacokinetic study of halofuginone, an oral quinazolinone
derivative in patients with advanced solid tumours. Eur. J. Cancer 2006, 42, 1768–1774. [CrossRef] [PubMed]
16. Bouley, R.;
Kumarasiri, M.;
Peng, Z.;
Otero, L.H.;
Song, W.;
Suckow, M.A.;
Schroeder, V.A.; Wolter, W.R.; Lastochkin, E.; Antunes, N.T.; et al.
Discovery of Antibiotic
(E)-3-(3-Carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one. J. Am. Chem. Soc. 2015, 137, 1738–1741.
[CrossRef] [PubMed]
17. Bouley, R.; Ding, D.; Peng, Z.; Bastian, M.; Lastochkin, E.; Song, W.; Suckow, M.A.; Schroeder, V.A.;
Wolter, W.R.; Mobashery, S.; et al. Structure–Activity Relationship for the 4(3H)-Quinazolinone Antibacterials.
J. Med. Chem. 2016, 59, 5011–5021. [CrossRef] [PubMed]
18. Raffa, D.; Edler, M.C.; Daidone, G.; Maggio, B.; Merickech, M.; Plescia, S.; Schillaci, D.; Bai, R.; Hamel, E.
Synthesis, cytotoxicity, and inhibitory effects on tubulin polymerization of a new 3-heterocyclo substituted
2-styrylquinazolinones. Eur. J. Med. Chem. 2004, 39, 299–304. [CrossRef] [PubMed]
19. Hour, M.J.; Huang, L.J.; Kuo, S.C.; Xia, Y.; Bastow, K.; Nakanishi, Y.; Hamel, E.; Lee, K.H. 6-Alkylamino- and
2,3-Dihydro-3¢-methoxy-2-phenyl-4-quinazolinones and Related Compounds: Their Synthesis, Cytotoxicity,
and Inhibition of Tubulin Polymerization. J. Med. Chem. 2000, 43, 4479–4487. [CrossRef] [PubMed]
20. Jiang, J.B.; Hesson, D.P.; Dusak, B.A.; Dexter, D.L.; Kang, G.J.; Hamel, E. Synthesis and Biological Evaluation
of 2-Styrylquinazolin-4(3H)-ones, a New Class of Antimitotic Anticancer Agents Which Inhibit Tubulin
Polymerization. J. Med. Chem. 1990, 33, 1721–1728. [CrossRef] [PubMed]
21. Kamal, A.; Sultana, F.; Ramaiah, M.J.; Srikanth, Y.V.V.; Viswanath, A.; Bharathi, E.V.; Nayak, R.;
Pushpavalli, S.N.C.V.L.; Srinivas, C.; Pal-Bhadra, M. 3-Diarylethyne quinazolinones: A new class of
senescence inducers. Med. Chem. Commun. 2013, 4, 575–581. [CrossRef]
22. Radomska, H.S.; Jernigan, F.; Nakayama, S.; Jorge, S.E.; Sun, L.; Tenen, D.G.; Kobayashi, S.S. A Cell-Based
High-Throughput Screening for Inducers of Myeloid Differentiation. J. Biomol. Screen. 2015, 20, 1150–1159.
[CrossRef] [PubMed]
Sample Availability: Samples of the compound 78 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).