A Novel Redox Modulator Induces a GPX4-Mediated Cell Death That Is Dependent on Iron and Reactive Oxygen Species

Article abstract of DOI:10.1021/acs.jmedchem.0c01016

Redox modulators have been developed as an attractive approach to treat cancer. Herein, we report the synthesis, identification, and biological evaluation of a quinazolinedione reactive oxygen species (ROS) inducer, QD394, with significant cytotoxicity in pancreatic cancer cells. QD394 shows a transcriptomic profile remarkably similar to napabucasin, a cancer stemness inhibitor. Both small molecules inhibit STAT3 phosphorylation, increase cellular ROS, and decrease the GSH/GSSG ratio. Moreover, QD394 causes an iron- and ROS-dependent, GPX4 mediated cell death, suggesting ferroptosis as a major mechanism. Importantly, QD394 decreases the expression of LRPPRC and PNPT1, two proteins involved in mitochondrial RNA catabolic processes and both negatively correlated with the overall survival of pancreatic cancer patients. Pharmacokinetics-guided lead optimization resulted in the derivative QD394-Me, which showed improved plasma stability and reduced toxicity in mice compared to QD394. Overall, QD394 and QD394-Me represent novel ROS-inducing drug-like compounds warranting further development for the treatment of pancreatic cancer.

Full text of DOI:10.1021/acs.jmedchem.0c01016

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Article
A Novel Redox Modulator Induces a GPX4-mediated Cell
Death That Is Dependent on Iron and Reactive Oxygen Species
Shuai Hu, Mario Sechi, Pankaj Kumar Singh, Lipeng Dai,
Sean McCann, Duxin Sun, Mats Ljungman, and Nouri Neamati
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01016 • Publication Date (Web): 18 Aug 2020
Downloaded from pubs.acs.org on August 18, 2020
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A Novel Redox Modulator Induces a GPX4-mediated Cell Death That Is Dependent on
Iron and Reactive Oxygen Species
Shuai Hu,a, b Mario Sechi,c, * Pankaj Kumar Singh, Lipeng Dai, Sean McCann, Duxin Sun,
c
d
a
d
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e
a, *
Mats Ljungman, Nouri Neamati
a
Departments of Medicinal Chemistry, College of Pharmacy, Rogel Cancer center, University of
Michigan, Ann Arbor, Michigan 48109, United States
b
Department of Computational Medicine and Bioinformatics, University of Michigan, Ann
Arbor, Michigan 48109, United States
c
Department of Chemistry and Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari,
Italy
d
Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann
Arbor, Michigan 48109, United States
e
Department of Radiation Oncology, Rogel Cancer Center and Center for RNA Biomedicine,
University of Michigan, Ann Arbor, Michigan 48109, United States
*
Corresponding authors: neamati@umich.edu and mario.sechi@uniss.it
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ABSTRACT
Redox modulators have been developed as an attractive approach to treat cancer. Herein, we
report on the synthesis, identification and biological evaluation of a quinazolinedione reactive
oxygen species (ROS) inducer, QD394, with significant cytotoxicity in pancreatic cancer cells.
QD394 shows a transcriptomic profile remarkably similar to napabucasin, a cancer stemness
inhibitor. Both small molecules inhibit STAT3 phosphorylation, increase cellular ROS and
decrease the GSH/GSSG ratio. Moreover, QD394 causes an iron- and ROS-dependent, GPX4
mediated cell death, suggesting ferroptosis as a major mechanism. Importantly, QD394 decreases
the expression of LRPPRC and PNPT1, two proteins involved in mitochondrial RNA catabolic
processes and both negatively correlated with the overall survival of pancreatic cancer patients.
Pharmacokinetic-guided lead optimization resulted in the derivative QD394-Me which showed
improved plasma stability and reduced toxicity in mice compared to QD394. Overall, QD394
and QD394-Me represent novel ROS-inducing drug-like compounds warranting further
development for the treatment of pancreatic cancer.
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INTRODUCTION
Pancreatic cancer is the fourth leading cause of cancer death in the United States, and its
incidence and mortality rates are continuing to increase worldwide.1, 2 It is predicted to be the
second leading cause of cancer death in the USA by 2030.2, 3 Pancreatic cancer is primarily
diagnosed at a late stage because early-stage symptoms are non-specific. The 5-year survival rate
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at the advanced stage is only around 3%. The current standard-of-care, FOLFIRINOX (folinic
acid, 5-fluorouracil (5-FU), irinotecan, and oxaliplatin), and gemcitabine together with nab-
paclitaxel show modest efficacy because of drug resistance and systemic toxicity.2, 4 Therefore, it
is imperative to find new improved therapies to treat pancreatic cancer.
Reprogramming energy metabolism and oxidative stress are two hallmarks of cancer.5-8
Therefore, treating cancer with small molecule reactive oxygen species (ROS) inducers may
selectively overwhelm cancer cells. Cellular redox homeostasis is closely related to ROS
production and elimination. Cancer cells have higher redox requirements from normal cells due
to their increased cell metabolism, dysfunctional mitochondria, scarce nutrients, and oxygen-
9
poor microenvironment. Higher intracellular ROS levels force cancer cells to develop robust
antioxidant systems to adapt to oxidative stress. At low or moderate levels, ROS serve as
signaling molecules to promote cell growth; while at high levels, ROS induce oxidation of lipids,
proteins, and DNA, ultimately leading to cell death. Several ROS inducers are in clinical trials to
evaluate efficacy and safety profiles in cancer.10-12 ARQ761, a beta-lapachone (Chart 1) prodrug
that reacts with NAD(P)H Quinone Dehydrogenase 1 (NQO1) to generate ROS, showed modest
10
efficacy in an open-label, dose-escalation Phase I study. Napabucasin (Chart 1) is an orally
administered cancer stemness inhibitor with a quinone scaffold and has been tested in Phase III
clinical trials in multiple cancers.11, 12 Napabucasin inhibits Signal Transducer and Activator of
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Transcription 3 (STAT3) signaling and increases ROS levels predominately by NQO1
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bioactivation. Therefore, inducing cellular ROS is a promising method to treat pancreatic
cancer.
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Previously, we developed a series of quinazolinedione (QD)‐based ROS inducers that
1
4
were tested for their utility in pre-clinical models of pancreatic cancer. In this study, we have
conducted a lead optimization on the most effective compound (i.e. QD325, Chart 1) to select
compounds inducing maximum ROS production. A series of structural modifications on
arylamino substituent at position 6 of the quinazoline-5,8-dione backbone were made to
modulate its physicochemical properties. A set of QD derivatives were synthesized (Scheme 1,
Chart 1), and the Me-N-piperazino analogue, i.e. QD394, was selected for mechanistic studies.
Herein, we show for the first time that this new prototype, QD394, displays highly
similar transcriptomic profiles with napabucasin, suggesting similar downstream signaling
effects. Combination with cell death signaling inhibitors reveals that QD394 induces ferroptosis
but not caspase-dependent apoptosis, necroptosis, or autophagy. We further demonstrate that
QD394 induces lipid peroxidation, iron-, ROS-, and glutathione peroxidase 4 (GPX4)-mediated
cell death, all of which are closely related to ferroptosis. Ferroptosis was first described in 2012
15
to represent an iron-dependent regulated cell death with increased lipid peroxidation. Intrinsic
1
6, 17
mechanisms of ferroptosis depend on the activity of GPX4 and lipid peroxidation.
At least
four classes of ferroptosis inducers have been developed, including (1) SLC7A11 inhibitor
(erastin), (2) GPX4 inhibitor (RSL3), (3) FIN56 (depleting GPX4 protein), and (4) FINO
2
16
(
indirectly inhibiting GPX4). Ferroptosis inducers show synergistic anti-tumor efficacy with
select chemotherapies18 and sensitize cancer cells to radiation.19 We also observed synergy
between QD394 and select FDA-approved drugs, suggesting that combination studies with redox
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modulators as ferroptosis inducers could be a viable approach to treat pancreatic cancer.
Additionally, we performed proteomics analysis and observed a significant decrease in
LRPPRC and PNPT1 expression, two proteins involved in mitochondrial RNA catabolic
processes.20, 21 Expression of these two genes is negatively correlated with the survival of
pancreatic cancer patients in our analysis using TCGA PAAD dataset. Furthermore, a
pharmacokinetics (PK)-guided lead optimization campaign was conducted to develop a QD394
analog, QD394-Me, with improved PK properties.
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RESULTS
Chemistry. Eleven analogs of QD394 were synthesized to characterize their cytotoxicity in
pancreatic cancer cells. The target compounds QD385 to QD395 were synthesized by
regioselective coupling of quinazoline-5,8-dione 1 with appropriate amines in the presence of
Ce(III) ions (Scheme 1) under mild conditions. The key synthon 1 was prepared by following our
14
previously reported procedure (Scheme 2). Briefly, nitration of the dimethoxybenzaldehyde 2
with concentrated nitric acid in the presence of acetic anhydride under simple magnetic stirring
afforded the desired 3,6-dimethoxy-2-nitrobenzaldehyde (3) regioisomer, which was isolated in
good yield by flash chromatography. Next, 3 was converted to the diformamido-derivative 4 by
adding formamide, followed by exposure to gaseous HCl under controlled temperature. Then,
compound 4 was treated with zinc powder and acetic acid to obtain the dimethoxyquinazoline 5.
Final oxidation of 5 with cerium ammonium nitrate resulted in the production of 1, which was
used for final amino conjugation.
The QD394-Me (namely QD430) was synthesized starting from 2,5-dimethoxy-4-
methylbenzaldehyde by adapting the abovementioned synthetic route (Scheme 3), except for the
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first step. An ipso-nitration on the starting material occurred in the first step reaction, and the
1
,4-dimethoxy-2-methyl-5-nitrobenzene (6) was obtained as the main product, while the desired
nitrobenzaldehyde 7 was isolated in very low yields (~5%) after chromatographic separation.
Scale-up of this reaction was needed to reach the necessary amount of intermediate 7, which
resulted in increased consumption of the reagent that dramatically affected the overall yield of
this synthetic route. However, the 7-methyl-quinazoline-5,8-dione (10), bearing the methyl at the
position 7 on the quinazolinedione ring, was easily obtained after oxidation of 9, which was
prepared by cyclizing the diformamido-derivative 8 in the same reaction condition used for the
preparation of the nonmethylated analogue.
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In an attempt to expand the preparation of another QD394-Me isomer, where the methyl
substituent was placed at position 6 (Scheme 4), we setup an alternative way to prepare the
QD394 with improved overall yield. Specifically, the key methyldiketone 15 was obtained by a
two-step ring closure of the N-protected intermediate 12. The latter was prepared by reducing the
nitroderivative 6 with ammonium formate and Pd/C to give 11 quantitatively, followed by
protection of the amino functionality with ethylcloroacetate in THF. Compound 12 was then
reacted with hexamethylenetetramine (HMTA) in trifluoroacetic acid (TFA) to give the
2
2
dihydroquinazoline 13 after an intramolecular cyclization. Finally, oxidative dehydrogenation
by using potassium ferricyanide in aqueous ethanolic potassium hydroxide of derivative 13
afforded the synthon 15 in good yield. Instead of the expected QD394-Me isomer, coupling of 15
with 4-(4-methylpiperazin-1-yl)aniline in the presence of cerium ammonium nitrate resulted in
the nucleophilic substitution of the methyl group, which led to the isolation of QD394 (Scheme
4
), but with about ten-fold lesser overall yields than that obtained from the previous synthetic
route.
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a
Scheme 1. Preparation of QD385 to QD395
O
O
O
H
6
N
i
N
N
R
1
1
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+
H N
2
R
N
N
O
1
QD385-395
R
R
QD385
QD386
QD387
QD388
QD389
QD390
QD391
QD392
QD393
QD394
QD395
Cl
Cl
Cl
O
N
N
N
N
N
a
Reagents and conditions: (i) CeCl
3
Ÿ7H O, abs EtOH, rt, 1.5 h
2
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a
Scheme 2. Synthetic route for the preparation of the synthon 1 and its intermediates
O
OCH3
O
OCH3
OHCHN
OHCHN
OCH3
OCH3
O
O
i
N
ii
N
iii
iv
O N
O N
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OCH3
OCH3
OCH3
OCH3
2
3
4
5
1
a
Reagents and conditions: (i) conc. HNO3, (CH
3
CO)
2
O, 0 °C, 1.5 h; (ii) H
2
NCHO, HCl(g) from 40 to
8
0 °C, 1 h; (iii) glacial CH
3
COOH, Zn, 0 °C for 2 h, rt for 4 h; (iv) (NH
4
)
2
Ce(NO , CH CN/H O, 0 °C,
3
)
6
3
2
20 min. For synthetic details see in ref. 14.
a
Scheme 3. Synthetic Route for the Preparation of QD394-Me
O
O N
2
CH3
O
O
O
O
OHCHN
OHCHN
O
O
6 ( 80%)
~
H
i)
ii)
CH3
O
O
O N
CH3
2
2,5-dimethoxy-4-methylbenzaldehyde
O N
CH3
8
2
O
iii)
7
(
~
5%)
O
H
O
O
O
N
N
v)
iv)
N
N
N
CH3
N
N
CH3
N
CH3
O
N
O
QD394-Me
10
9
a
Reagents and conditions: (i) conc. HNO3, CH
iii) glacial CH
Ÿ7H O, abs EtOH, rt, 1.5 h.
2
Cl
2
, rt, 24 h; (ii) H
2
NCHO, HCl(g) from 40 to 80 °C, 1 h;
, CH CN/H O, 0 °C, 20 min.; (v)
(
3
COOH, Zn, 0 °C for 2 h, r.t. for 4 h; (iv) (NH
4
)
2
Ce(NO
3
)
6
3
2
CeCl
3
2
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Scheme 4. Alternative Synthetic Route for the Preparation of QD394
O
O
O
EtOOC
N
O
O
O N
H N
EtOOCHN
N
2
2
ii)
iii)
1
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0
i)
CH3
CH3
CH3
CH
3
O
O
O
6
11
12
13
iv)
O
O
O
H
N
O
N
vi)
N
N
N
v)
6
N
N
CH3
N
CH3
CH3
N
O
O
15
14
QD394-Me-iso
vi)
O
O
H
N
N
N
N
N
QD394
a
Reagents and conditions: Reagents and conditions: (i) HCOONH
HMTA, TFA; (iv) KOH, aqueous EtOH, K Fe(CN) ; (v) (NH
methylpiperazin-1-yl)aniline, CeCl Ÿ7H O, abs EtOH
4
; Pd-C; (ii) ClCO
2
Et, THF; (iii) (1)
O; (vi) 4-(4-
3
6
4
)
2
Ce(NO CN/H
3
)
6,CH
3
2
3
2
QD394 shows remarkable similarity to napabucasin and H
2
O in transcriptomic profiles.
2
Among 11 originally synthesized analogs (Table S1, S2, Scheme 1), QD394 had the highest
cytotoxicity in MIA PaCa-2 cells and similar cytotoxicity in PANC-1 and BxPC-3 cells (Table 1).
QD394 induced the highest level of intracellular ROS after 1 h compound treatment (Figure S1).
QD394 (IC50, 0.64 ± 0. 04 µM) is slightly more cytotoxic and induces more ROS as compared to
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our previously described QD325 compound (IC50, 0.9 ± 0.2 µM) (Figure S2). To better
elucidate its mechanism of action, we performed Bru-seq, a nascent RNA-seq technique that uses
bromouridine to label newly synthesized RNAs.23 This method detects early changes in the
transcriptome caused by drugs and is useful in understanding their mechanisms of action and
toxicity. We discovered that the transcriptomic profile of QD394 was highly similar to that of
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napabucasin and H
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. The shrunkenLFC (shrunken log2FoldChange) of genes in QD394 Bru-
(r = 0.88)
seq samples significantly correlated with that of the napabucasin (r = 0.92) and H
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samples, indicating similar transcription regulation (Figure 1A). ShrunkenLFC reduces the effect
of low-expressing genes that result in over-inflated Log2FoldChange values. Gene Set
Enrichment Analysis (GSEA) revealed 82 upregulated and 51 downregulated enriched gene sets
in common between QD394 and napabucasin using the C2 curated gene sets from Molecular
Signatures (MSigDB) Database v7.0 with FDR below 0.001 (Figure 1B and 1C, Table S3). For
the Hallmark and KEGG pathway gene sets shared between QD394 and napabucasin, TNFA
signaling, UV response, and hypoxia hallmark gene sets were upregulated, and RNA degradation
KEGG gene set was downregulated (Figure 1D and 1E). TNF signaling and ROS are
interconnected in the regulation of cell homeostasis, survival, and death in inflammatory diseases
and cancers. ROS is closely related to TNF-related downstream signaling, such as NF-κB and
JNK activation, and TNF can induce antioxidant NRF2 pathway to protect cells from elevated
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ROS. UV radiation can cause DNA damage and corresponding cell death via oxidative stress.
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Moreover, cellular and mitochondrial ROS activate a series of transcription factors (TFs),
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which trigger the expression of a number of genes involved in the hypoxic response.
It is
interesting to note that QD394 affects RNA degradation in the KEGG pathway gene set, and this
is consistent with our proteomics study (see below).
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Since napabucasin is a STAT3 signaling inhibitor and exhibited a similar transcriptomic
profile as QD394, we sought to determine whether QD394 affected STAT3 signaling. Indeed,
QD394 inhibited the phosphorylation of STAT3 in MIA PaCa-2 and PANC-1 cells, and it also
increased the phosphorylation of H2AX, a DNA damage biomarker (Figure 1F).
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QD394 induces ROS-mediated cell death and decreases the GSH/GSSG ratio. As a redox
modulator, QD394 caused time-dependent cellular ROS accumulation within the first 4-hour of
treatment in MIA PaCa-2 cells (Figure 2A). The significant increase of ROS level could be
reduced in 30 minutes by the antioxidant N-acetylcysteine (NAC) (Figure 2B). Additionally,
NAC significantly increased the IC50 of QD394 from 0.53 μM to 1.11 μM (Figure 2C) and
rescued colony formation inhibition by QD394 (Figure 2D). Thus, the cytotoxicity of QD394 is
partially dependent on ROS induction.
Glutathione is a well-known cellular ROS scavenger. In the GSH/GSSG-Glo assay,
QD394 significantly decreased the GSH/GSSG ratio at both 2 h and 4 h treatment, similar to
napabucasin (Figure 2E). The decrease in ratio is due to reduced GSH level or increased GSSG
level or both. The reduction from QD394 treatment was caused by oxidation of GSH to GSSG,
rather than the direct decrease of GSH level (Figure S3). Buthionine sulfoximine (BSO) is an
inhibitor of GSH synthesis, and both napabucasin and BSO significantly lowered GSH level
(Figure 2E and S3).
QD394 causes iron-dependent and GPX4-mediated cell death. Since we observed that
QD394 modulated the GSH/GSSG ratio and its cytotoxicity was dependent on ROS, we
hypothesized that QD394 could induce ferroptosis. To determine the cell death mechanism, we
combined QD394 with a panel of signaling inhibitors (Table 2). We identified that deferoxamine
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DFO) and deferasirox, two iron chelators, prevented cell death caused by QD394, indicating
iron is a contributing factor for its cytotoxicity (Figure 3A and S4). Two known ferroptosis
inhibitors had different effects on cell death rescue. Liproxstatin-1 modestly rescued cell death
caused by QD394, while ferrostatin-1 did not. Z-VAD (a caspase-dependent apoptosis inhibitor)
and necrostatin-1 (a necroptosis inhibitor) did not rescue cell death, and chloroquine (an
autophagy inhibitor) slightly rescued cell death in the colony formation assay (Figure S4).
Interestingly, necrostatin-1 blocked the inhibition of colony formation caused by napabucasin,
indicating that cell death by napabucasin may proceed via necroptosis. The link between
napabucasin and necroptosis has not been previously reported. Moreover, these results
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demonstrate that QD394 and napabucasin have different cell death mechanisms.
The final step in ferroptosis is an increase in lipid peroxidation.15-18 We measured the
lipid ROS level in QD394-treated cells using the BODIPY C11 dye to validate QD394-induced
ferroptosis cell death. QD394 significantly increased lipid ROS in a dose-dependent manner
(Figure 3B). TBHP (an ROS inducer), RSL3 (a GPX4 inhibitor), and erastin (an SLC7A11
inhibitor) all increased lipid ROS. Beside iron and lipid peroxidation, GPX4 is another central
mediator of ferroptosis. GPX4 catalyzes the reduction of hydrogen peroxide, organic
hydroperoxides, and lipid hydroperoxides, and thereby protects cells against oxidative damage.15,
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Therefore, we investigated whether there is a connection between QD394 and GPX4. In the
cellular thermal shift assay (CETSA), QD394 destabilized GPX4 protein, indicated by the
reduction in its melting temperature (from 60.34 °C to 57.62 °C) in MIA PaCa-2 cells (Figure
3
C). Destabilization of GPX4 by QD394 was also observed in PANC-1 cells (Figure S5).
Binding to a protein generally stabilizes it, however, QD394 destabilized GPX4 and it may be
caused by an alternative binding mode or indirect interaction between them. Furthermore, the
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cytotoxicity of QD394 was significantly decreased in MIA PaCa-2 cells with GPX4 knocked
down using siRNA, further suggesting that QD394 regulates GPX4-mediated cell death (Figure
3D). Additionally, QD394 rescued the cytotoxicity of RSL3 in pancreatic cancer cells,
suggesting that QD394 targets GPX4 (Figure 3E). We hypothesized that QD394 and RSL3 may
work as competitive inhibitors to bind to the same site of GPX4, thus blocking both their activity
when they were combined at certain concentrations. In summary, QD394 induces an iron-
dependent and GPX4-mediated cell death, which is closely related to ROS induction.
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QD394 interferes with mitochondrial gene expression and RNA catabolic process. To
further investigate the Bru-seq results at the protein level, we performed proteomics and
measured the changes caused by QD394. QD394 increased the expression of 84 proteins and
decreased the expression of 146 proteins by at least 1.5-fold (FDR < 0.05) after 24 h treatment in
MIA PaCa-2 cells (n = 2). There are 17 upregulated and 35 downregulated genes/proteins in
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common between the proteomics and Bru-seq analysis (Figure 4A). In the STRING analysis,
the 17 proteins with increased expression were involved in response to endoplasmic reticulum
(ER) stress, the unfolded protein response, and the transcription from RNA polymerase II
promoter related to oxidative stress (Table S4). Furthermore, the 35 proteins with decreased
protein expression had a close connection with mitochondrial genes, mitochondrial translation
regulation, and cellular metabolic processes (Table S5, Figure 4B). QD394 reduced expression
of proteins involved in mitochondrial RNA catabolic processes, which is consistent with the Bru-
seq GSEA analysis showing that QD394 downregulated the KEGG RNA degradation gene set.
We observed that LRPPRC and PNPT1 were involved in many of the biological processes
downregulated by QD394 (Table S5). LRPPRC (Leucine Rich Pentatricopeptide Repeat
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Containing) encodes a protein localized primarily to mitochondria, and it may regulate RNA
metabolism and transcription in both nuclei and mitochondria. PNPT1 (Polyribonucleotide
Nucleotidyltransferase 1) encodes a mitochondrial intermembrane protein in the polynucleotide
phosphorylase family comprised of phosphate-dependent 3’-to-5’ exoribonucleases implicated in
RNA processing and degradation. More importantly, we discovered that high expression of both
genes significantly correlated with reduced overall survival of pancreatic cancer patients in the
TCGA PAAD dataset (Figure 4C). In the 24 h proteomics experiment, QD394 reduced the
expression of these two proteins (FDR < 0.05); additionally, QD394 significantly decreased their
expression at 4 h (Figure 4D). Napabucasin also decreased the protein expression of these two
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genes at 4 h, while H
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QD394 and napabucasin to assess the effects on the expression of LRPPRC and PNPT1 (Figure
S6). NAC did not affect the downregulation of these two proteins caused by QD394 and
napabucasin; however, dicoumarol blocked the reduction, indicating that NQO1 is a potential
regulator in this process. Moreover, in PANC-1 cells, which have little intrinsic NQO1
expression, we did not observe the downregulation of LRPPRC and PNPT1 proteins by QD394
and napabucasin (Figure S6).
Besides these two mitochondrial proteins related to RNA processing, QD394 also
reduced expression of other mitochondrial proteins in the proteomics study, substantiating its
possible role in affecting mitochondrial function (Figure 4E). Overall, QD394 downregulated
mitochondrial proteins, including LRPPRC and PNPT1 in both Bru-seq and proteomics
experiments.
QD394 is synergistic with napabucasin and select FDA-approved drugs. ROS inducers
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showed synergism in the colony formation assay in both MIA PaCa-2 and PANC-1 cells (Figure
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A and S7), suggesting the potential development of combination studies between redox
modulator QD394 and cancer stemness inhibitors such as napabucasin. We next tested whether
QD394 would act in synergy with the ROS inducer arsenic trioxide (As ), which is approved
to treat acute promyelocytic leukemia. As has been shown to reduce protein expression of
Bcl-2, increase cellular ROS, induce permeability transition pore complex opening, and impair
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cell proliferation and apoptosis. QD394 sensitizes cells to As
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potentials between QD394 and different redox modulators (Figure 5B). Since QD394 causes
DNA damage, we assessed the synergy between QD394 and compounds regulating DNA repair.
Olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, is an FDA-approved drug to treat
breast, ovarian, and pancreatic cancer. Combination of QD394 and olaparib resulted in
synergistic effects in the colony formation assay (Figure 5C). The full colony formation assay
images and their synergism calculation using Highest Single Agent (HSA) and Bliss models in
the Combenefit software are shown in the Figure S8. Cumulatively, QD394 is synergistic with
cancer stemness inhibitors, other redox modulators, and PARP inhibitors.
QD394-Me shows improved plasma stability and reduced toxicity in mice as compared to
QD394. We determined the stability of QD394 in mouse liver microsomes and plasma. The half-
life of QD394 is over 60 minutes in mouse liver microsomes, yet only six minutes in mouse
plasma, indicating it has good microsomal stability but poor metabolic stability in plasma (Table
3). We then determined its potential metabolites in mouse plasma to better guide the design of
more metabolically stable derivatives (Figure 6A). M1 and M2 metabolites have hydroxyl
groups on the rings, and M3 and M4 metabolites are produced through a Michael addition. We
then synthesized QD394-Me with a methyl group to block Michael addition (Figure 6B). This
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improved the plasma stability of QD394 from six minutes to over two hours. Next, we calculated
the PK parameters of QD394 and QD394-Me in CD-1 mice following intraperitoneal injection
(IP, 10 mg/kg), oral administration (PO, 20 mg/kg), or intravenous (IV, 10 mg/kg) injection.
Plasma concentration of both QD394 and QD394-Me rapidly decreased within the first two
hours (Figure S9). Notably, two out of three mice administered with QD394 via IV injection died
likely due to systemic toxicity, while all three mice that received IV injections of QD394-Me
survived throughout the study (Table 3). Therefore, QD394-Me appears to be less toxic and more
tolerable in mice compared to QD394.
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To understand why the concentration of QD394-Me rapidly decreased in the plasma, we
assessed tissue distribution of QD394-Me administered via IV and PO in mice. After one-hour
treatment, colon had the highest concentration of QD394-Me, although this was relatively low
compared to the dosage (Colon: around 0.3 mg/kg, PO: 20 mg/kg, IV: 10 mg/kg) (Table S6).
Besides distribution to organs, we identified potential metabolites of QD394-Me. The major
metabolite of QD394-Me contains two hydroxyl groups on the right benzene or piperazine ring
(Figure S10). As early as 15 min, we observed the major metabolite in the plasma; however, both
the parent compound and metabolite were distributed and excreted quickly over time (Figure
S10).
On the basis of tissue distribution results, e.g. where colon tissue had much higher
concentration of QD394-Me than pancreas, we conducted a preliminary animal study in
immuno-competent Balb/c mice using mouse colon cancer cell line CT-26. Three out of five
mice displayed tumor shrinkage in the QD394-Me IV (10 mg/kg) and QD394 IP (20 mg/kg)
treatment groups. (Figure S11). A full assessment of in vivo efficacy studies in orthotopic models
of colon and pancreatic cancers will be published in due course.
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QD394-Me and QD394 share similar cell death mechanisms. QD394-Me and QD394 show
similar cytotoxicity in PANC-1 and BxPC-3 cells, while QD394-Me is less potent than QD394
in MIA PaCa-2 cells (Table 1). QD394-Me also significantly increased cellular ROS in MIA
PaCa-2 cells, and NAC blocked the induction of ROS (Figure 7A). Similar to QD394, QD394-
Me increased lipid ROS significantly in a dose-dependent manner (Figure 7B). QD394-Me
destabilized GPX4 protein in pancreatic cancer cells in the CETSA experiments (Figure 7C and
S4), and its cytotoxicity was significantly reduced in the GPX4-knockdown experiments (Figure
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D, Table S7). Moreover, QD394-Me significantly decreased protein expression of LRPPRC and
PNPT1 at 4 h, consistent with QD394 in MIA PaCa-2 cells (Figure 7E). Therefore, QD394-Me
shows a similar mechanism of action to QD394 in pancreatic cancer cells but has improved
plasma stability and less in vivo toxicity.
Bru-seq analysis of QD394-Me. We analyzed gene expression profile of QD394-Me in MIA
PaCa-2 using Bru-seq. QD394-Me induced hallmark gene sets related to TNF signaling and
hypoxia, which is similar to QD394. Additionally, QD394-Me increased unfolded protein
response, suggesting potential ER stress (Figure 8, Table S8). QD394-Me also induced ROS
pathway and JAK/STAT3 signaling hallmark gene sets, supporting its ROS-inducing capability
and STAT3-targeting effects (Table S8). KEGG pathways related to proteasome, ribosome and
spliceosome were enriched by QD394-Me, indicating its downstream effects on protein and gene
regulations (Figure 8, Table S9). There are no common downregulated enriched gene sets
between QD394 and QD394-Me Bru-seq samples.
DISCUSSION
ROS induction alters the activity of multiple transcription factors, including STAT3, NF-κB, and
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hypoxia-inducible factor-1α (HIF-1A). STAT3 signaling is downstream of MAPK, JAK, and
Src pathways. Redox modulation can inactivate these kinases leading to either activation or
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inhibition of STAT3 signaling, depending on cellular context and duration of ROS generation.
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TNFA signaling and hypoxia hallmark gene sets were upregulated by QD394 and napabucasin.
TNFA/TNF are important cytokines for maintaining cellular homeostasis and mediating the
activation of NF-κB, which in turn regulates downstream prosurvival genes, MAPK signaling,
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and cell death in cancers and inflammatory diseases.
The transcription factor HIF-1A is
37, 38
activated by hypoxic conditions and responds to accumulated ROS via NF-κB activation.
ROS induction by QD394 and napabucasin can trigger these signaling pathways. Napabucasin is
a cancer stemness inhibitor that reduces the proliferation, invasion, and stemness of cancer
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cells.
Napabucasin is bioactivated by NQO1 and generates ROS to induce DNA damage and
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cell death. In this study, we report that necrostatin-1 rescued napabucasin-mediated cell death,
suggesting that napabucasin induces necroptotic cell death in pancreatic cancer cells. However,
QD394 did not cause necroptosis, but iron-, ROS-dependent, and GPX4-mediated ferroptosis
(Figure 9).
Liproxstatin-1 inhibits ferroptosis induced by erastin, BSO and RSL3 in vitro, and it
4
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prevents ferroptosis and ROS accumulation in GPX4 knockout cells both in vitro and in vivo.
Ferrostatin-1 is a first-generation ferroptosis inhibitor that rescues cell death caused by erastin
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and RSL3.
Additionally, both compounds are effective radical-trapping antioxidants in the
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lipid bilayers. However, ferrostatin-1 did not prevent H
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ferroptosis is defined as a necrotic-like cell death. The fact that QD394 induces ROS may
explain why ferrostatin-1 did not rescue QD394-induced cell death. Although ferrostatin-1
blocked lipid peroxidation and ferroptosis, QD394 could cause other ROS-involved DNA
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damage and necrotic cell death (Figure 9). Therefore, we suggest that QD394 and QD394-Me
kill cells through a combination of different cell death pathways and ferroptosis is an essential
mechanism since GPX4 protein expression regulates the cytotoxicity of QD394 and QD394-Me.
Interestingly, we determined potential interactions between QD394, QD394-Me, and GPX4 in
the cells, but the detailed binding site requires further investigation.
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LRPPRC and PNPT1 are closely related to mitochondrial translation and RNA catabolic
2
0, 21, 44, 45
processes in different diseases.
LRPPRC deficiency alters mitochondrial electron
46
transport chain, mitochondrial permeability, and transmembrane ROS diffusion. PNPT1
regulates the correct maturation of mitochondrial ND6 transcripts and efficient mitochondrial
2
1, 45
RNA degradation.
Both proteins are involved in mitochondrial function and negatively
associated with the survival of pancreatic adenocarcinoma patients. Our redox modulators,
QD394 and QD394-Me, decreased the expression of these two proteins, which could be used as
potential pharmacodynamic biomarkers in pancreatic cancer (Figure 9). Meanwhile, further
understanding of the connection between overexpression of these two genes and pancreatic
cancer is needed and may lead to novel approaches to treat pancreatic cancer.
CONCLUSIONS
In this study, we developed a novel redox modulator QD394 and comprehensively studied its
mechanisms of action in pancreatic cancer cells. QD394 shares a highly similar transcriptomic
profile to napabucasin, inhibits STAT3 phosphorylation, increases ROS levels, and induces DNA
damage. However, while napabucasin causes necroptosis, QD394 induces an iron-, ROS-
dependent, and GPX4-mediated cell death. An essential cell death mechanism of QD394 is
ferroptosis, since QD394 increases lipid peroxidation, destabilizes GPX4 protein, and its
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cytotoxicity is dependent on GPX4. Bru-seq and proteomics analysis reveal that QD394
decreases both nascent transcripts and protein expression of LRPPRC and PNPT1. Furthermore,
QD394 acts in synergy with several FDA-approved drugs, suggesting its potential utility to
overcome drug resistance. A PK-guided optimization campaign resulted in QD394-Me with
improved mouse plasma stability and less systemic toxicity in mice.
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EXPERIMENTAL SECTION
Chemistry. General Methods. All solvents and other chemicals were obtained from Sigma-
Aldrich, Alfa Aesar, Carlo Erba (analytical or anhydrous grade) or Apollo Scientific Limited,
Oakwood Chemical, Combi Blocks, Enamine (for amines), and were used without further
purification). All reactions involving air- or moisture-sensitive compounds were performed
under a nitrogen atmosphere using oven-dried glassware and syringes to transfer solutions.
Melting points (Mp) were determined using an electrothermal melting point or a Köfler
apparatus and are uncorrected. Nuclear magnetic resonance (H H-NMR, 13C-NMR) spectra
1
were determined in CDCl or DMSO-d and were recorded on a 400 MHz Bruker Avance III.
3
6
Chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane (TMS)
and used as an internal standard. Splitting patterns are designated as follows: s, singlet; d,
doublet; t, triplet; q, quadruplet; m, multiplet; brs, broad singlet; dd, double doublet. The
assignment of exchangeable protons (OH and NH) was confirmed by the addition of D O. Mass
2
spectra were obtained on a Hewlett-Packard 5989 mass engine spectrometer, or a MALDI micro
MX (Waters, Micromass) equipped with a reflector analyzer. Analytical thin-layer
chromatography (TLC) was carried out on Merck silica gel F-254 plates. Flash chromatography
purifications were performed on Merck Silica gel 60 (230–400 mesh ASTM) as a stationary
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phase. Analytical HPLC for the lead compounds was carried out on an Agilent 1260 Infinity II
Quaternary pump (Agilent Technologies, Italia S.p.A.). Elemental analyses for tested compounds
were performed on a Perkin-Elmer 2400 spectrometer at Laboratorio di Microanalisi,
Dipartimento di Chimica, Università di Sassari (Italy), and were within ± 0.4% of the theoretical
values (Table S1), thus confirming ≥95% purity.
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General method for the preparation of the compounds QD385 - 395
A solution of quinazoline-5,8-dione (1), cerium (III) chloride heptahydrate (CeCl
3
Ÿ7H O, 1.1 eq.)
2
and substituted aniline (1.0 eq.) in absolute ethanol was stirred at room temperature for 1.5 h.
Next, after removing most of the ethanol under vacuum, the crude residue was diluted with water,
extracted with CH
(Na SO ) and concentrated to dryness. Then, the product was purified by flash chromatography
to give the desired compound.
-(Biphenyl-3-ylamino)quinazoline-5,8-dione [QD 385]
Quinazoline-5,8-dione (1, 0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
CeCl Ÿ7H O, 1.1 eq., 0.128 g, 0.343 mmol), 3-aminobiphenyl (1.0 eq., 0.053 g, 0.312 mmol),
2
Cl
2
and washed with water. The organic layers were dried over sodium sulfate
2
4
6
(
3
2
and absolute ethanol (6 mL). Flash chromatography (dichloromethane-methanol = 9.8:0.2) gave
compound QD385 as a violet powder. Yield: 20%. Rf: 0.41 (ether petroleum : ethyl acetate =
1
1
3:7). mp: 199-200 °C. H-NMR 400 MHz H-NMR (DMSO-d
6
): δ 9.63 (s, 1H), 9.43 (s, 1H),
13
7
.69-7.67 (m, 3H), 7.61-7.59 (d, 2H), 7.49-7.46 (t, 2H), 7.41-7.36 (t, 3H), 6.34 (s, 1H). C-NMR
(
DMSO-d
6-(4'-Chlorobiphenyl-4-ylamino)quinazoline-5,8-dione [QD 386]
Quinazoline-5,8-dione (1, 0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
6
): δ 163.78, 130.33, 129.03, 127.15, 125.61, 121.74, 105.15. MS: m/z 327 (M+).
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(
CeCl
3
Ÿ7H
2
O, 1.1 eq., 0.128 g, 0.343 mmol), 4-amino-4’-chlorobiphenyl (1.0 eq., 0.064 g, 0.312
mmol), and absolute ethanol (6 mL). Flash chromatography (ethyl acetate-petroleum ether = 5:5)
gave compound QD386 as a violet powder. Yield: 95%. Rf: 0.20 (ethyl acetate-petroleum ether
0
1
2
3
4
5
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9
0
1
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4
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9
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
=
7:3). mp: 292-293 °C. H-NMR (DMSO-d
6
): δ 9.62 (s, 2H), 9.42 (s, 1H), 7.79-7.77 (d, 2H),
1
3
7
.76-7.74 (d, 2H), 7.55-7.53 (d, 2H), 7.50-7.48 (d, 2H), 6.35 (s, 1H). C-NMR (DMSO-d ): δ
6
180.52, 180.03, 162.59, 155.67, 153.74, 145.89, 138.07, 137.42, 135.77, 132.34, 128.91, 128.26,
27.53, 124.06, 104.06. MS: m/z 361 (M+).
1
6
-(3',5’-Dichlorobiphenyl-4-ylamino)quinazoline-5,8-dione [QD 387]
Quinazoline-5,8-dione (1, 0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
(CeCl Ÿ7H O, 1.1 eq., 0.128 g, 0.343 mmol), 4-amino-3’,5’-dichlorobiphenyl (1.0 eq., 0.074 g,
.312 mmol), and absolute ethanol (6 mL). Flash chromatography (ethyl acetate-petroleum ether
3
2
0
=
5:5) gave compound QD387 as a violet powder. Yield: 95%. Rf: 0.50 (ethyl acetate-petroleum
1
ether = 7:3). mp: 260-261 °C. H-NMR (DMSO-d
6
): δ 9.63 (s, 2H), 9.43 (s, 1H), 7.87-7.85 (d,
13
2
1
H), 7.79 (s, 2H), 7.61 (s, 1H), 7.52-7.50 (d, 2H), 6.38 (s, 1H). C-NMR (DMSO-d ): δ 180.47,
6
80.15, 162.60, 155.70, 153.68, 145.75, 142.80, 138.28, 134.68, 133.99, 128.02, 125.20, 123.91,
104.34. MS: m/z 395 (M+).
-(4-(Piperidin-1-yl)phenylamino)quinazoline-5,8-dione [QD 388]
Quinazoline-5,8-dione (1, 0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
CeCl Ÿ7H O, 1.1 eq., 0.128 g, 0.343 mmol), 4-(1-piperidinyl)aniline (1.0 eq., 0.055 g, 0.312
6
(
3
2
mmol), and absolute ethanol (6 mL). Flash chromatography (ethyl acetate-petroleum ether = 7:3)
gave compound QD388 as a violet powder. Yield: 66%. Rf: 0.29 (ethyl acetate-petroleum ether
1
=
7:3). mp: 209 °C. H-NMR (DMSO-d
6
): δ 9.60 (s, 1H), 9.38 (s, 1H), 7.45 (s, 1H), 7.22-7.20 (d,
1
3
2H), 7.02-7.00 (d, 2H), 6.13 (s, 1H), 3.16 (s, 4H), 1.63-1.56 (d, 6H). C-NMR (DMSO-d ): δ
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80.65, 179.30, 162.59, 155.51, 154.08, 149.52, 146.47, 127.71, 124.93, 124.12, 115.91, 102.59,
9.23, 25.09, 23.83. MS: m/z 334 (M+).
6-(4-Morpholinophenylamino)quinazoline-5,8-dione [QD 389]
Quinazoline-5,8-dione (1, 0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
CeCl Ÿ7H O, 1.1 eq., 0.128 g, 0.343 mmol), 4-(4-morpholinyl)aniline (1.0 eq., 0.056 g, 0.312
1
1
1
1
1
1
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0
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8
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3
4
5
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7
8
9
0
(
3
2
mmol), and absolute ethanol (6 mL). Flash chromatography (ethyl acetate-petroleum ether = 7:3)
gave compound QD389 as a violet powder. Yield: 88%. Rf: 0.10 (ethyl acetate-petroleum ether
1
=
7:3). mp: 226-227 °C. H-NMR (DMSO-d
6
): δ 9.60 (s, 1H), 9.47 (s, 1H, H-N), 9.39 (s, 1H),
1
3
7
d
4
.26-7.24 (d, 2H), 7.05-7.03 (d, 2H), 6.14 (s, 1H), 3.76 (s, 4H), 3.15 (s, 4H). C-NMR (DMSO-
6
): δ 180.62, 179.41, 162.59, 155.53, 149.03, 146.49, 128.56, 124.97, 115.40, 102.70, 66.00,
8.18. MS: m/z 336 (M+).
6
-(9H-Fluoren-4-ylamino)quinazoline-5,8-dione [QD390]
Quinazoline-5,8-dione (1, 0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
CeCl Ÿ7H O, 1.1 eq., 0.128 g, 0.343 mmol), 1-aminofluorene (1.0 eq., 0.057 g, 0.312 mmol),
(
3
2
and absolute ethanol (6 mL). Flash chromatography (ethyl acetate-petroleum ether = 7:3) gave
compound QD390 as a violet powder. Yield: 69%. Rf: 0.46 (ethyl acetate-petroleum ether = 7:3).
1
mp: 217 °C. H-NMR (CDCl
3
): δ 9.68 (s, 1H), 9.53 (s, 1H), 7.57-7.55 (d, 2H), 7.52-7.49 (t, 1H),
1
3
7
.45-7.41 (t, 1H), 7.38-7.35 (t, 1H), 7.31-7.29 (d, 1H), 6.43 (s, 1H), 3.86 (s, 2H). C-NMR
(CDCl
3
): δ 163.78, 156.33, 128.79, 127.74, 127.25, 125.18, 121.70, 120.42, 119.04, 105.53. MS:
m/z 339 (M+).
6
-(9H-Fluoren-2-ylamino)quinazoline-5,8-dione [QD 391]
Quinazoline-5,8-dione (1, 0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
(CeCl
3
Ÿ7H
2
O, 1.1 eq., 0.128 g, 0.343 mmol), 2-aminofluorene (1.0 eq., 0.057 g, 0.312 mmol),
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and absolute ethanol (6 mL). Flash chromatography (ethyl acetate-petroleum ether = 5:5) gave
compound QD391 as a violet powder. Yield: 92%. Rf: 0.14 (ethyl acetate-petroleum ether = 7:3).
1
mp: 236-237 °C. H-NMR (DMSO-d
6
): δ 9.62 (s, 2H), 9.43 (s, 1H), 8.00-7.97 (d, 1H), 7.92-7.91
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
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7
8
9
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8
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13
(
d, 1H), 7.62-7.60 (d, 2H), 7.43-7.39 (t, 2H), 7.35-7.32 (t, 1H), 6.35 (s, 1H), 4.00 (s, 1H). C-
NMR (DMSO-d ): δ 155.64, 153.82, 146.13, 144.27, 143.19, 140.42, 138.78, 136.25, 126.84,
6
125.13, 124.14, 122.77, 120.67, 120.54, 119.99, 103.74, 65.70. MS: m/z 339 (M+).
-(4'-Ethylbiphenyl-4-ylamino)quinazoline-5,8-dione [QD 392]
Quinazoline-5,8-dione (1, 0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
CeCl Ÿ7H O, 1.1 eq., 0.128 g, 0.343 mmol), 4-amino-4’-ethylbiphenyl (1.0 eq., 0.061 g, 0.312
6
(
3
2
mmol), and absolute ethanol (6 mL). Flash chromatography (ethyl acetate-petroleum ether = 5:5)
gave compound QD392 as a violet powder. Yield: 77%. Rf: 0.19 (ether petroleum : ethyl acetate
1
=
5:5). mp: 247 °C. H-NMR 400 MHz (DMSO-d
6
): δ 9.63 (s, 2H), 9.43 (s, 1H), 7.76-7.74 (d,
2H), 7.64-7.62 (d, 2H), 7.49-7.47 (d, 2H), 7.33-7.31 (d, 2H), 6.36 (s, 1H), 2.69-2.63 (m, 2H),
13
1
1
.24-1.21 (t, 3H). C-NMR 100MHz (DMSO-d ): δ 185.78, 185.24, 167.73, 161.00, 159.01,
6
51.19, 148.40, 142.53, 141.91, 133.63, 132.54, 131.68, 129.33, 109.12, 33.02, 20.77. MS: m/z
355 (M+).
-(4-Cyclohexylphenylamino)quinazoline-5,8-dione [QD 393]
Quinazoline-5,8-dione (1, 0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
CeCl Ÿ7H O, 1.1 eq., 0.128 g, 0.343 mmol), 4-cyclohexylaniline (1.0 eq., 0.055 g, 0.312 mmol),
6
(
3
2
and absolute ethanol (6 mL). Flash chromatography (ethyl acetate-petroleum ether = 5:5) gave
compound QD393 as a violet powder. Yield 53%. Rf: 0.24 (ether petroleum : ethyl acetate =
1
5
:5). mp: 234 °C. H-NMR (DMSO-d
6
): δ 9.61 (s, 1H), 9.53 (s, 1H), 9.40 (s, 1H), 7.34-7.28 (m,
1
3
4H), 6.21 (s, 1H), 1.82-1.80 (m, 5H), 1.73-1.70 (d, 1H), 1.47-1.33 (m, 5H). C-NMR (DMSO-
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d
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): δ 180.60, 162.58, 155.59, 153.85, 145.32, 127.53, 123.91, 43.26, 38.87, 26.29, 25.53. MS:
m/z 333 (M+).
6-(4-(4-Methylpiperazin-1-yl)phenylamino)quinazoline-5,8-dione [QD 394]
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
5
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9
0
Quinazoline-5,8-dione (1,
CeCl Ÿ7H O, 1.1 eq., 0.128 g, 0.343 mmol), 4-(4-methylpiperazin-1-yl)aniline (1.0 eq., 0.060 g,
0.312 mmol), and absolute ethanol (6 mL). Flash chromatography (dicloromethane:methanol =
0.050 g, 0.312 mmol), cerium (III) chloride heptahydrate
(
3
2
8
:2) gave compound QD394 as a violet powder. Yield: 70%. Rf: 0.30 (dicloromethane:methanol
1
=
8:2). mp: 204-205 °C. H-NMR (DMSO-d
6
): δ 9.60 (s, 1H), 9.46 (s, 1H), 9.39 (s, 1H), 7.24-
7
.22 (d, 2H), 7.04-7.01 (d, 2H), 6.13 (s, 1H), 3.19-3.17 (m, 4H), 2.47 (s, 4H), 2.23 (s, 3H). MS:
m/z 349 (M+).
6
-(4-(Pyridin-2-yl)phenylamino)quinazoline-5,8-dione [QD 395]
Quinazoline-5,8-dione (1, 0.075 g, 0.468 mmol), cerium (III) chloride heptahydrate
(CeCl Ÿ7H O, 1.1 eq., 0.192 g, 0.515 mmol), 4-(2-pyridil)aniline (1.0 eq., 0.080 g, 0.468 mmol),
3
2
and absolute ethanol (9 mL). Flash chromatography (ether petroleum : ethyl acetate = 5:5) gave
compound QD395 as a violet powder. Yield: 54%. Rf: 0.16 (ether petroleum : ethyl acetate =
1
2:8). mp: 239 °C. H-NMR (DMSO-d
6
): δ 9.67(s, 1H), 9.63 (s, 1H), 9.44 (s, 1H), 8.69-8.65 (d,
1
H), 8.21-8.18 (d, 2H), 8.01-7.99 (d, 1H), 7.92-7.88 (m, 1H), 7.56-7.53 (dd, 2H), (m, 1H), 6.43
(
s, 1H). MS: m/z 328 (M+).
2
,5-Dimethoxy-4-methylnitrobenzene [6]
1
Yield: 80%. Rf = 0.90 (ethyl acetate-petroleum ether 3:7). mp: 116 °C. H NMR 400 MHz
13
(CDCl ): δ 10.29 (s, 1H), 6.93 (s, 1H), 3.95 (s, 3H), 3.87 (s, 3H), 2.42 (s, 3H). C NMR 400
3
MHz (CDCl
3
): δ 150.98, 147.69, 136.77, 135.25, 116.46, 107.05, 57.10, 56.00, 16.83. MS: m/z
198 [M+H]+.
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3
,6-Dimethoxy-4-methyl-2-nitrobenzaldehyde [7]
To a solution of 3,6-dimethoxy-4-methyl benzaldehyde (1 eq., 4.0 g, 24.07 mmol) in CH
mL), conc. HNO (4.54 eq., 6 mL) in CH Cl (25 mL) was added at room temperature under
stirring. After 24 h, the obtained clear solution was cautiously poured onto aqueous Na SO . The
organic layer was separated and dried over Na SO . The obtained crude product was then
2
Cl (50
2
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9
0
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9
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2
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purified by flash chromatography on silica gel using ethyl acetate-petroleum ether (2.5:7.5) to
give first the major side product, 2,5-dimethoxy-4-methylnitrobenzene (6), and then (by further
elution with only ethyl acetate) the desired compound (7). Yield: 5%. Rf = 0.60 (ethyl acetate-
1
petroleum ether 3:7). mp: 85 °C. H NMR 400 MHz (CDCl
3
): δ 10.29 (s, 1H), 6.93 (s, 1H), 3.95
): δ 185.46, 157.60, 143.47,
1
3
(s, 3H), 3.87 (s, 3H), 2.42 (s, 3H). C NMR 400 MHz (CDCl
3
42.28, 117.58, 115.54, 114.09, 62.90, 56.63, 17.47. MS: m/z 226 [M+H]+.
1
N,N
′
-[(3,6-Dimethoxy-4-methyl-2-nitrophenyl)methanediyl)diformamide [8]
A solution of 3,6-dimethoxy-4-methyl-2-nitrobenzaldehyde (7, 1.1 g, 4.87 mmol) in formamide
66.5 eq., 13 mL, 325 mmol), heated at 40°C, was exposed to dry HCl gas (1 h) until the
(
temperature reached 80°C. Then, the solution was cooled to room temperature, and water/ice was
added. A pale brownish yellow colored precipitate was formed, which was filtered, dried and
triturated with ethyl acetate and petroleum ether to yield the desired compound. Yield: 70%. Rf =
1
0
.16 (dichloromethane-methanol 9.5:0.5). mp: 238 °C. H NMR 400 MHz (DMSO-d
6
): δ 8.65 (d,
1
3
2H), 7.91 (s, 2H), 7.21 (s, 1H), 6.72 (t, 1H), 3.89 (s, 3H), 3.71 (s, 3H), 2.50 (s, 3H). C NMR
4
00 MHz (DMSO- d
6
): δ 160.14, 152.82, 141.76, 134.61, 119.71, 116.23, 116.09, 62.31, 56.54,
1
5.89. MS: m/z 298 [M+H]+.
5,8-Dimethoxy-7-methylquinazoline [9]
Zinc powder (15 eq., 1.4 g, 22 mmol,) was added to a suspension of N,N′-[(3,6-Dimethoxy-4-
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methyl-2-nitrophenyl)methanediyl)diformamide (8, 0.45 g, 1.5 mmol) in triturated ice and
glacial acetic acid (2 mL), under constant magnetic stirring. The reaction mixture was stirred for
2 h in an ice bath and for 4 h at room temperature. After filtration of the reaction mixture through
filter paper, the resulting solution was dropped on cooled 50% NaOH (5 mL), and the yellow
colored suspension thus formed was left without stirring for 1 h. Next, the suspension was
filtered and the solid dried (at 30−40 °C) to give a yellow powder. The solid was then solubilized
1
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0
in ethyl acetate, filtered, dried over anhydrous Na
2
SO
4
, and concentrated to dryness yielding the
1
desired compound. Yield: 50%. Rf = 0.30 (ethyl acetate-petroleum ether 3:7). mp: 132 °C. H
NMR 400 MHz (CDCl
3
): δ 9.65 (s, 1H), 9.31 (s, 1H), 6.72 (s, 1H), 4.02 (s, 3H), 4.00 (s, 3H),
1
3
2.51 (s, 3H). C NMR 400 MHz (CDCl ): δ 155.68, 155.27, 151.35, 146.12, 144.83, 136.39,
3
17.04, 108.33, 61.81, 55.86, 17.21. MS: m/z 205 [M+H]+.
1
7
-methylquinazoline-5,8-dione [10]
A solution of 5,8-Dimethoxy-7-methylquinazoline (9, 0.15 g, 0.75 mmol) in (7:3)
acetonitrile:water (5 mL) was cooled at 0 °C in an ice bath, and a solution of ceric ammonium
nitrate (3 eq., 1.2 g, 2.1 mmol) in (9:1) acetonitrile:water (5 mL) was added dropwise. The
reaction mixture was stirred for 20 min, then poured into ice/water and extracted (8−10 times)
with dichloromethane. The organic layer was washed (5−6 times) with water, dried over
anhydrous Na
2
SO
4
and concentrated to dryness to give a brown powder. Yield: 90%. Rf = 0.52
1
(
ethyl acetate-petroleum ether 3:7). mp: 118 °C. H NMR 400 MHz (CDCl
3
): δ 9.68 (s, 1H), 9.51
1
3
(s, 1H), 6.97 (s, 1H), 2.30 (s, 3H). C NMR 400 MHz (CDCl
3
): δ 183.29, 182.88, 162.84,
57.23, 152.51, 149.59, 135.12, 124.24, 16.81. MS: m/z 175 [M+H]+.
1
7
-Methyl-6-((4-(4-methylpiperazin-1-yl)phenyl)amino)quinazoline-5,8-dione [QD394-Me]
A solution of 7-methylquinazoline-5,8-dione (10, 0.050 g, 0.312 mmol), cerium (III) chloride
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heptahydrate (CeCl
3
Ÿ7H
2
O, 1.1 eq., 0.128 g, 0.343 mmol) and 4-(4-methylpiperazin-1-yl)aniline
(1.0 eq., 0.060 g, 0.312 mmol) in absolute ethanol (6 ml) was stirred at room temperature for 1−2
h. Next, most of the ethanol was removed under vacuum, and water was added, followed by the
extraction with dichloromethane. The organic layers were washed with water and brine, dried
0
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over anhydrous Na
2
SO , and concentrated to dryness. Then, the crude product was purified by
4
flash chromatography to give the expected product. Yield: 90%. Rf = 0.30 (dichloromethane-
1
methanol 9.5:0.5); mp: 142 °C. H NMR 400 MHz (DMSO-d
6
): δ 9.57 (s, 1H), 9.33 (s, 1H), 8.85
1
3
(s, 1H), 7.00 (d, 1H), 6.91 (d, 1H), 3.14 (m, 4H), 2.50 (m, 4H), 2.27 (s, 3H), 1.62 (s, 3H). C
NMR 400 MHz (DMSO-d ): δ 180.92, 180.54, 162.35, 155.21, 153.86, 147.78, 143.71, 131.55,
6
124.33, 123.77, 117.20, 115.11, 54.40, 47.94, 45.42, 13.22. MS: m/z 364 [M+H]+.
-methyl-2,5-dimethoxyaniline [11]
4
To a stirred suspension of 2,5-dimethoxy-4-methylnitrobenzene (6, 1 g, 5.07 mmol) and 10% Pd-
C (0.4 g) in dry methanol (20 ml), ammonium formate (4.5 eq., 1.5 g, 23.79 mmol) was added in
a single portion. The resulting mixture was stirred at room temperature for 40 min., the catalyst
was removed by filtration through a celite pad and washed with dry methanol. The filtrate was
evaporated either under reduced or at normal pressure. The resulting residue was extracted with
dichloromethane /water. Yield: 70%. Rf = 0.20 (ethyl acetate-petroleum ether 3:7). mp: 255 °C.
1H NMR 400 MHz (DMSO-d
H).
): δ 6.61 (s, 1H), 6.35 (s, 1H), 3.79 (s, 3H), 3.74 (s, 3H), 2.13 (s,
6
3
Ethyl (2,5-dimethoxy-4-methylphenyl)carbamate [12]
To a solution of 4-methyl-2,5-dimethoxyaniline (11, 0.5 g, 2.99 mmol) in tetrahydrofuran (30
mL), ethyl chloroformate (0.65 g, 0.57 mL, 2 eq., 6 mmol) was added. Further trimethylamine (1
mL, 2 eq., 6 mmol) was added into the reaction mixture and stirred at room temperature for 1
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hour. Reaction mixture was concentrated and extracted in ethyl acetate/water to obtain the
1
product. Yield: 60%. Rf = 0.10 (ethyl acetate-petroleum ether 3:7). mp: 106 °C. H NMR 400
MHz (CDCl
3
): δ 7.76 (s, 1H), 7.15 (s, 1H), 6.66 (s, 1H), 4.20 (q, 2H), 3.81 (s, 3H), 3.80 (s, 3H),
1
1
1
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.17 (s, 3H), 1.32 (t, 3H).
Ethyl-5,8-dimethoxy-6-methylquinazoline-1-(2H)-carboxylate [13]
A mixture of N-protected N-protected 4-methyl-2,5-dimethoxyaniline (12, 0.2 g, 0.84 mmol) and
hexamethylenetetramine (HMTA, 9 eq., 0.84 g, 7.2 mmol) in in trifluoroacetic acid (TFA, 4 mL)
was refluxed for 1 hour. After cooling, the mixture was diluted with cold water, neutralized with
NaHCO and extracted with ethyl acetate. The organic phase was evaporated and residue was
3
treated with cyclohexane. The undissolved solid was filtered off and the solution was evaporated
under reduced pressure to give product. Yield: 50%. Rf = 0.45 (ethyl acetate-petroleum ether
1
3
:7); mp: > 320 °C. H NMR 400 MHz (CDCl
3
): 8.58 (s, 1H), 6.83 (d, 2H), 6.56 (s, 1H), 4.14 (q,
2H), 3.94 (s, 3H), 3.84 (s, 3H), 2.30 (s, 3H), 1.28 (t, 3H).
,8-Dimethoxy-6-methylquinazoline [14]
5
A solution of 10% KOH in water/EtOH in 1/1 ratio (25 mL), quinazoline carboxylate (13, 0.55 g,
1.97 mmol) was added and refluxed for 1 hour. After cooling, potassium ferricyanide (45 eq.,
3
.24 g, 89.12 mmol) was added and mixture was refluxed for 4 hour. After cooling, the mixture
was diluted with water, extracted with dichloromethane, and the organic phase was evaporated
under reduced pressure. The residue was dissolved in the minimum amount of methanol and
dilute with water to obtain pure product. Yield: 35%. Rf = 0.30 (ethyl acetate-petroleum ether
1
3
:7). mp: > 320 °C. H NMR 400 MHz (CDCl
3
): δ 9.59 (s, 1H), 9.31 (s, 1H), 7.04 (s, 1H), 4.06
(s, 3H), 3.92 (s, 3H), 2.49 (s, 3H).
6-Methyl quinazoline-5,8-dione [15]
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