Design and Synthesis of Novel Reactive Oxygen Species Inducers for the Treatment of Pancreatic Ductal Adenocarcinoma

Article abstract of DOI:10.1021/acs.jmedchem.7b01463

Altering redox homeostasis provides distinctive therapeutic opportunities for the treatment of pancreatic cancer. Quinazolinediones (QDs) are novel redox modulators that we previously showed to induce potent growth inhibition in pancreatic ductal adenocarcinoma (PDAC) cell lines. Our lead optimization campaign yielded QD325 as the most potent redox modulator candidate inducing substantial reactive oxygen species (ROS) in PDAC cells. Nascent RNA sequencing following treatments with the QD compounds revealed induction of stress responses in nucleus, endoplasmic reticulum, and mitochondria of pancreatic cancer cells. Furthermore, the QD compounds induced Nrf2-mediated oxidative stress and unfolded protein responses as demonstrated by dose-dependent increases in RNA synthesis of representative genes such as NQO1, HMOX1, DDIT3, and HSPA5. At higher concentrations, the QDs blocked mitochondrial function by inhibiting mtDNA transcription and downregulating the mtDNA-encoded OXPHOS enzymes. Importantly, treatments with QD325 were well tolerated in vivo and significantly delayed tumor growth in mice. Our study supports the development of QD325 as a new therapeutic in the treatment of PDAC.

Full text of DOI:10.1021/acs.jmedchem.7b01463

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Article
Design and Synthesis of Novel Reactive Oxygen Species
Inducers for the Treatment of Pancreatic Ductal Adenocarcinoma
Yuting Kuang, Mario Sechi, Salvatore Nurra, Mats Ljungman, and Nouri Neamati
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01463 • Publication Date (Web): 12 Jan 2018
Downloaded from http://pubs.acs.org on January 12, 2018
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(
Journal of Medicinal Chemistry)
Design and Synthesis of Novel Reactive Oxygen Species Inducers for the
Treatment of Pancreatic Ductal Adenocarcinoma
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†
,§
¶,*
¶
£
†,*
Yuting Kuang, Mario Sechi, Salvatore Nurra, Mats Ljungman, and Nouri Neamati
†
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, 1600 Huron
Parkway, Ann Arbor, MI 48109, United States
§
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of
Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, United States
¶
Department of Chemistry and Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari,
Italy
£
Department of Radiation Oncology, University of Michigan, 1600 Huron Parkway,
Ann Arbor, MI 48109, United States
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ABSTRACT: Altering redox homeostasis provides distinctive therapeutic opportunities for
the treatment of pancreatic cancer. Quinazolinediones (QDs) are novel redox modulators that we
previously showed to induce potent growth inhibition in pancreatic ductal adenocarcinoma
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(PDAC) cell lines. Our lead optimization campaign yielded QD325 as the most potent redox
modulator candidate inducing substantial reactive oxygen species (ROS) in PDAC cells. Nascent
RNA sequencing following treatments with the QD compounds revealed induction of stress
responses in nucleus, endoplasmic reticulum, and mitochondria of pancreatic cancer cells.
Furthermore, the QD compounds induced Nrf2-mediated oxidative stress and unfolded protein
responses as demonstrated by dose dependent increases in RNA synthesis of representative genes
such as NQO1, HMOX1, DDIT3 and HSPA5. At higher concentrations, the QDs blocked
mitochondrial function by inhibiting mtDNA transcription and downregulating the mtDNA-
encoded OXPHOS enzymes. Importantly, treatments with QD325 were well tolerated in vivo and
significantly delayed tumor growth in mice. Our study supports the development of QD325 as a
new therapeutic in the treatment of PDAC.
Keywords: Pancreatic Cancer, ROS, Next-generation Sequencing, Unfolded Protein Response,
Mitochondrial Synthesis
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INTRODUCTION
Pancreatic cancer is the fourth leading cause of cancer-related death in both genders in
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the United States, estimated to claim over 40,000 lives in 2017. Given its asymptomatic and
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metastatic nature, over 50% of pancreatic cancer cases are diagnosed at late stages, when the
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tumor has metastasized and is often unresectable. Therefore, treatment of pancreatic cancer is
largely dependent on systemic chemotherapy. Ever since its approval by the FDA in 1996,
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gemcitabine-based regimes have been the standard of care for pancreatic cancer. However,
limited by late-stage diagnosis and inherent/acquired resistance to available chemotherapy, the
overall five-year survival rate of pancreatic cancer is only 6.7%, one of the lowest among all
types of cancers. Recently, two combination regimens with modest clinical activity have been
added as an option for pancreatic cancer patients. The combination of nab-paclitaxel (albumin-
bound paclitaxel) with gemcitabine has increased median overall survival from 6.7 to 8.5
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months. The combination cocktail including oxaliplatin, irinotecan, fluorouracil and leucovorin
(FOLFIRINOX) was approved for the treatment of metastatic pancreatic cancer by improving
median overall survival from 6.8 months in the gemcitabine group to 11.1 months in the
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FOLFIRINOX group, but increased toxicity is the major concern for these new treatment
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-8
options. Therefore, novel therapeutics are urgently needed to enhance the survival and quality
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of life of patients with this devastating disease.
Modulation of redox homeostasis in cancer cells provides a new opportunity for tumor
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intervention.
Reactive oxygen species (ROS), natural byproducts from mitochondrial
respiration and other cellular processes, play important roles as second messengers in cell
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signaling. However, when present at high concentrations, ROS can be detrimental to cells by
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inducing oxidative damage to DNA, lipids and proteins. Cells eliminate excess intracellular
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ROS via expression of antioxidant genes regulated by the ROS-detoxifying machinery. In tumor
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cells, antioxidant enzymes are often induced as a result of elevated levels of intrinsic ROS.
G12D
Expression of mutant oncogenic Kras
, commonly present in pancreatic ductal
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adenocarcinoma (PDAC), keeps the master transcription factor NRF2 elevated at basal state to
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mount an antioxidant response.
A shift in the redox homeostasis is expected to make tumor
cells more susceptible to induced oxidative stress, overwhelming their adaptive antioxidant
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capacity and promoting ROS-mediated cell death.
Among various classes of small-molecule drugs tested in our laboratory to induce ROS,
the quinone containing compounds showed great promise due to their significant increase in
oxygen consumption rate in treated cells. Many quinones inhibit multiple biochemical assays due
to their redox, metal-chelation, color, and in some cases, reactivity towards nucleophiles through
Michael addition. Therefore, they are classified as pan assay inhibitors in high throughput
screening campaigns. However, over two-dozen drugs containing a quinone moiety have been
approved by the FDA or are under clinical investigations not only in oncology but also in other
diseases. For example, doxorubicin and dozen of its analogs, mitoxantrone, and mitomycin C are
some of the most commonly used FDA approved chemotherapeutic agents for numerous cancers
that contain a quinone group. In addition, there are many other agents that are approved for
various indications or are in late stage clinical development that broadly fall under the quinone
class of drugs (Chart 1).
Previously, we showed that the quinazolinedione QD232 (Chart 2) exerts ROS-mediated
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cytotoxicity in pancreatic cancer models.
In this study, we performed a lead optimization
campaign and identified QD325 as a lead compound for in-depth preclinical and mechanistic
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studies. Several major modifications on QD232 were made by varying the substituents at the
nitrogen atom on position 6 of the quinazoline-5,8-dione backbone.
Specifically, mono/poly substitution on the phenyl ring with various functionalities
and/or bulky aromatic system was mainly explored to modulate lipophilicity and steric effects
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(
Chart 2). Furthermore, to evaluate a potential synergistic effect in terms of ROS modulation, we
sought to conjugate the quinazoline-5,8-dione scaffold to highly lipophilic
Disruption of mitochondrial-
a
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triphenylphosphonium cation for mitochondrial targeting.
mediated cell redox modulation represents a promising avenue for future therapy in cancer. Since
our compounds increase ROS production we tested their ability to interfere with mitochondrial
processes.
Our results show that QD325 caused selective inhibition of transcription of the
mitochondrial genome potentially by abrogating the mitochondrial D-loop, critical for mt-DNA
transcription. We propose that such targeting could be efficacious and should be further
explored as an innovative therapeutic approach to target cancers that heavily depend on
mitochondrial function. Importantly, QD325 was found to be well tolerated and showed tumor-
suppressing activities in a pancreatic cancer xenograft model.
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RESULTS
Chemistry. The synthesis of compounds QD324-338, 353-357 (Table 1) was carried out
using Bracher's methodology, according to our previously reported procedure with some
modifications. Scheme 1 illustrates the synthesis of the key synthone QD323 from the readily
available dimethoxybenzaldehyde (1). Nitration of compound 1 with concentrated nitric acid in
the presence of acetic anhydride under simple magnetic stirring afforded the 3,6-dimethoxy-2-
nitrobenzaldehyde (2) in good yield. This regioisomer was converted to the diformamido-
derivative 3 under gaseous HCl. Compound 3 was then cyclized to dimethoxyquinazoline 4 by
treatment with zinc powder and acetic acid. Final oxidation by cerium ammonium nitrate
resulted in the production of quinazoline-5,8-dione QD323. Regioselective addition of
appropriate aminobenzenes to 6 position of QD323 in the presence of Ce(III) ions gave QD324-
338, 353-357 (Schemes 2 and 3).
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Then, we wanted to obtain triphenylphosphonium-based model derivatives of compound QD331
and QD232. Compounds QD340 and QD359 were therefore generated by adapting a phosphine
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conjugation method, as we previously described.
The synthesis of triphenylphosphonium-
based compounds QD340 and QD359 is illustrated in Schemes 4 and 5. Initially, 3-
bromopropylamine hydrobromide (5) was reacted with triphenylphosphine in refluxing
acetonitrile for 16 hours, and the resulting triphenylphosphonium intermediate (6) was isolated
after treatment with n-hexane/diethyl ether/isopropanol. Next, the 4- and 3-aminobenzamido-
propyl-triphenylphosphonium bromides QD339 and QD358 were prepared by conjugating 6 with
4- or 3-aminobenzoic acid, respectively, via a standard coupling protocol using DIPEA, HBtU,
DMAP, in CH
2
Cl
2
. Finally, QD340 and QD359 were obtained by nucleophilic addition of
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appropriate triphenylphosphonium salts (QD339 or QD358) to QD323 in the presence of Ce(III),
according to the above-mentioned procedure.
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QD Compounds Inhibit Proliferation of Pancreatic Cancer Cells. We designed and
synthesized 25 new analogues of our previous lead compound, QD232, to better elucidate their
mechanisms of action. Nine of these novel analogues showed improved cytotoxicity compared to
QD232 in at least two PDAC cell lines in MTT assay (Table 1). QD325 was found to be the most
potent analogue with IC50 values <1 µM in the three PDAC cell lines (MIA PaCa-2, Panc-1, and
BxPC-3).
QD325 (as well as QD232) showed similar dose-dependent cytotoxicity in MIA PaCa-2
3
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and a gemcitabine-resistant derivative cell line MIA PaCa-2-GR (Table 2). In HPV16-E6E7
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immortalized pancreatic cell line HPDE, gemcitabine produced similar IC50 values as MIA
PaCa-2 cells, while the most potent QD325 compound showed a 3-fold higher sensitivity for
MIA PaCa-2 (Table 2).
Cytotoxicity of QD Compounds Correlates with Increased ROS Production.
Treatment with the QD compounds elicited significant ROS accumulation in MIA PaCa-2 cells
as shown in the H2DCFDA reactivity assay (Figures 1 and S2). To validate ROS induction as the
mechanism for cytotoxicity, the effect of QD compounds were evaluated in the presence and
absence of the antioxidant N-acetyl-cysteine (NAC). For the lead compound QD232 and the two
active analogues QD325 and QD326, we observed a time- and dose-dependent accumulation of
ROS (Figure 1A). While H
2
2
O treatment lead to an immediate conversion of H2DCFDA to
fluorescent DCF, treatment with the QD compounds resulted in a gradual induction of the
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fluorescent signal, which reached peak levels after 4-6 h, implying a progressive ROS
accumulation.
When cells were pretreated with 5 mM NAC, ROS induction by H
compounds was blocked (Figure 1B). In the MTT cell proliferation assay, NAC attenuated the
proliferation inhibitory effect of H , QD232, QD325 and QD326 (Figure 1C). Importantly, we
2
O
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and the QD
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did not observe any colloidal behavior or aggregation for QD compounds in our assays. These
results demonstrate that ROS accumulation is a major mechanism for cytotoxicity of the QD
compounds. However, NAC treatment did not completely block the effect of the QD compounds
and H
inhibition of cell proliferation. Testing many analogues in the presence of NAC and GSH at 1 to
mM we discovered that these antioxidants could protect toxicity anywhere from 10-80%
2
O2, suggesting these compounds may induce additional cellular effects responsible for the
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depending on the analogue. In most cases NAC addition never fully protects the cells even at
1000-fold excess of antioxidant. These studies suggest that the compounds not only produce
ROS but also bind to unique targets. It is also possible that the QD-NAC conjugate is partially
responsible for the ROS induction and cytotoxicity.
The faster induction of oxidative stress by the optimized compounds is perhaps due to
improved membrane permeability (predicted cLogP values for QD232 and QD325 are 1.99 and
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.89, respectively). We expected that better in vitro antiproliferative activity of QD325 should
translate to better in vivo efficacy. Therefore, we selected QD325 for extensive mechanistic and
in vivo studies.
In order to selectively target these compounds to the mitochondria, we synthesized two
derivatives containing a triphenylphosphine group. To our surprise, QD340 and QD359 did not
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show better ROS induction and cytotoxicity than QD325 and were not further explored. A
possible explanation of this unexpected behavior could be attributed to their redox properties,
since we previously showed that triphenylphosphoniums decreased cellular oxygen consumption
rate (OCR). Furthermore, the QD compounds without the triphenylphosphonium group cause a
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remarkable increase in cellular OCR.
The QD Compounds Induce Oxidative Stress and the Unfolded Protein Response.
We used the recently established bromouridine labeled RNA sequencing (Bru-seq) technique to
better characterize transcriptional effects of the QD compounds. Bru-seq captures nascent RNA
and provides information on ongoing transcription genome-wide without interference by pre-
36, 37
existing RNA.
We observed that the transcription signatures induced by 4-hour treatments
with QD232 or QD325 were similar according to Ingenuity Pathway Analysis (IPA) or Gene Set
Enrichment Analysis (GSEA) of the expressed genes (Figures 2 and S3-S6). This implies that the
two compounds have similar mechanisms of action. IPA profiling of all genes with >1.5-fold
change in expression upon treatment of MIA PaCa-2 cells with QD232 or QD325 identified the
“NRF2-mediated oxidative stress response” and “unfolded protein response” (UPR) as key
pathways activated by these compounds (Figure 2A).
NRF2 is a transcription factor that is rapidly activated in response to oxidative and
electrophilic exposure and it promotes the transcription of genes encoding various detoxifying
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enzymes. Upon oxidative challenge, KEAP1 inactivation allows nascent NRF2 proteins to
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translocate to the nucleus,
and activate transcription of antioxidant genes containing AREs
(antioxidant response element) or MAREs (MAF recognition element) in cis-acting enhancer
elements.
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NQO1 and HMOX1 are two target genes of NRF2 that counteract the effects of oxidative
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stress.
NQO1 encodes the flavoprotein NAD(P)H:quinone oxidoreductase 1 that catalyzes a
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two-electron reduction of quinones to hydroquinones and exhibits chemo-protective effects.
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HMOX1 encodes heme oxygenase 1 (HO-1), whose antioxidant properties arise from
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degradation of the pro-oxidant heme and production of antioxidant bilirubin from biliverdin.
As revealed by Bru-seq, synthesis of NQO1 and HMOX1 RNAs was upregulated in a dose-
dependent manner by treatments with either QD232 or QD325 (Figure 2B).
The unfolded protein response (UPR) comprises three pathways regulated by the ER
trans-membrane proteins inositol-requiring enzyme 1a (IRE1a), activating transcription factor 6
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(ATF6), and protein kinase RNA-like endoplasmic reticulum kinase (PERK), respectively.
Misfolded proteins in the ER lumen trigger the competitive binding of the ER chaperone GRP78,
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leading to the activation of IRE1a, ATF6 and PERK and downstream responses to UPR.
Depending on the severity and duration of the ER stress, the UPR can function as a pro-survival
mechanism and restore homeostasis, or trigger apoptosis if the stress burden is beyond the
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capacity of this adaptive response.
DDIT3 and HSPA5 are genes that are induced as a result of UPR signaling. HSPA5
encodes GRP78, the key regulatory protein of ER stress. DDIT3 is a downstream target gene that
transcriptionally responds to all three arms of the UPR. As a transcription factor, the DDIT3 gene
product CHOP promotes apoptosis under prolong ER stress by inducing transcription of pro-
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apoptotic genes including PUMA and BIM.
Transcription of the two stress responsive genes
DDIT3 and HSPA5 is significantly increased by QD232 or QD325 treatment in a dose-dependent
manner (Figure 2C).
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Upregulation of mRNA synthesis detected by Bru-seq was found to also result in
increased protein levels of the major stress responsive genes. We observed time-dependent
increased protein levels of CHOP and GRP78 in MIA PaCa-2, Panc-1, and BxPC-3 cells treated
with the QD compounds (Figure 3A-C). For the oxidative stress responsive genes, HO-1 was
upregulated by QD treatments in MIA PaCa-2 and BxPC-3 cells, while no significant change
was detected in Panc-1 cells. Of note, the NQO1 gene is known to be deleted in Panc-1 cells, and
as expected, no transcription of the gene was observed in this cell line using Bru-seq. In MIA
PaCa-2 and BxPC-3, NQO1 showed high basal expression levels with no further induction
following treatment. These results suggest that MIA PaCa-2 and BxPC-3 cells have a high
intrinsic oxidative stress burden and that this may make them sensitive to exogenous agents
inducing oxidative stress. Indeed, IPA analysis showed significant activation of apoptosis
signaling at higher concentration of QD232 (3 times IC50) or QD325 (5 times IC50) after 4 h
treatment (Figure S3), suggesting that apoptosis is a mode of death following exposure to the QD
compounds.
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Previously, it was shown that ROS-inducing compounds such as phenethylisothiocyanate
and curcumin efficiently downregulate specificity protein (Sp) transcription factors Sp1, Sp3,
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and Sp4 in pancreatic cancer cells,
however, we did not observe selective downregulation of
these transcription factor genes in our Bru-seq studies and these factors were not in our top 100
affected genes.
The QD Compounds Inhibit Transcription of mtDNA from the D-loop. Mitochondria
play an important role in regulating redox homeostasis in mammalian cells. Deregulation of the
expression of mitochondrial genes can lead to interruption of the oxidative phosphorylation
pathway, resulting in the accumulation of ROS. Mitochondrial DNA (mtDNA) harbors 13 genes
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that encode proteins with important functions in the electron transport chain. The double-
stranded circular mitochondrial genome comprises the guanine-rich heavy strand and the
cytidine-rich light strand. Using Bru-seq, we observed significant inhibition of mtDNA
transcription after a 4-hour treatment with QD compounds at higher concentrations (Figure 4A;
Figure S7). Both compounds decreased COX III protein levels, which correlated to a reduced
rate of transcription of the gene (Figure 4B). These results strongly suggest that the QD
compounds disrupt mitochondrial function.
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The D-loop (displacement loop) is a noncoding area of the mtDNA genome composed of
a short three-stranded structure required for the regulation of initiation of mtDNA replication and
transcription. This region contains two promoters on the heavy strand HSP1 and HSP2, one on
the light strand, LSP, and also the mtDNA replication origin O
H
. The D-loop region has been
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reported to be frequently mutated in lung, hepatocellular, colorectal and cervical cancers
and
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these D-loop mutations are linked to a poor prognosis of the disease.
Using Bru-seq we found that the QD compounds inhibited the transcription of mtDNA
from both the heavy strand promoter HSP2 (top long arrow in Figure 4A) and the light strand
promoter LSP (bottom arrow), thus inhibiting the expression of mitochondrial genes that are
essential for mitochondrial oxidative phosphorylation. However, the activity of the heavy strand
promoter HSP1 (top short arrow, Figure 4A), which regulates transcription of 12s rRNA and 16s
rRNA, was less affected by the QDs. This pattern of transcriptional inhibition was not observed
following treatments with UV light, camptothecin or with 40 FDA approved and other novel
drugs developed in our laboratory (data not shown). Thus, the Bru-seq data showing selective
transcriptional inhibition of the mitochondrial genome suggest that this mechanism may be
linked to the toxicity induced by the QD232 and QD325 compounds.
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QD325 Delays Tumor Growth without Systemic Toxicity. In NOD/SCID mice, MIA
PaCa-2-derived xenograft showed significantly delayed growth following treatment with QD325
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(
5 mg/kg). On day 44 the xenografts treated with QD325 had only reached a size of 308 ± 72
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mm (p = 2.1E6) compared to 1291 ± 168 mm for the xenografts in the control group (Figure
5A).
No symptoms of gross toxicity such as weakness, weight loss or lethargy were observed
in any treatment group for the duration of the treatments (Figure 5B). H&E stained organ
sections did not reveal any major histopathological changes, further confirming the in vivo
tolerability of QD325 (Figure 5C). Following the 44-day treatment, two mice from each group
were used to evaluate efficacy and safety of QD325 at higher doses. While tumors in the control
group exhibited rapid growth, QD325 treatment was able to delay growth of the tumors without
any overt systemic toxicity observed even at doses as high as 20 mg/kg (Figure 6A-B).
In line with the tumor growth inhibition, QD325 treatment decreased the staining for the
proliferation marker Ki67 in tumor tissues, suggesting inhibition of cell proliferation (Figure 5D).
To further evaluate the mechanisms of action of QD325 in vivo, we examined the protein levels
of stress response markers in tumor lysates. NQO1, HO-1, CHOP and GRP78 protein levels
were significantly upregulated in QD325 treated tumors compared to tumors treated with vehicle,
further confirming induction of oxidative stress and UPR as major mechanisms of action for
QD325 in these pancreatic cancer models (Figure 5E).
Gemcitabine is currently used as the standard of care for treatment of pancreatic cancer
patients. Unfortunately, inherent or acquired resistance to gemcitabine represents a major
challenge for successful treatment of this disease. Here we sought to explore the potential
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efficacy of administering QD325 in combination with gemcitabine. In mice studies, gemcitabine
is usually given at high doses (40-160 mg/kg) twice weekly. Considering its low tolerance in
NOD/SCID mice, we compared antitumor activity of two different gemcitabine treatment
schedules in a MIA PaCa-2 xenograft model in this mouse strain: 1) 15 mg/kg once a week for
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8 days; 2) 15 mg/kg twice a week for the first 15 days. Similar antitumor activity was achieved
by either schedule (Figure 6C). In both cases, gemcitabine was well tolerated and no weight loss
was observed (Figure 6D). Therefore, schedule 1 was used for comparison of efficacy with
QD325 at 5 mg/kg and the combination of gemcitabine and QD325. QD325 was given at 5
mg/kg five times a week and gemcitabine was given at 15 mg/kg once a week (Figure 6E). At
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the end of the 48-day treatment period, average tumor size was 1503 ± 189 mm for the control
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group, 387 ± 74 mm (p = 0.0049) for the gemcitabine group, 248 ± 72 mm (p = 0.0030) for
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the QD325 group, and 163 ± 83 mm (p = 0.0023) for the combination of gemcitabine and
QD325 group (Figure 6E). Single agent treatment with QD325 at 5 mg/kg showed similar anti-
tumor activity as gemcitabine. In this experiment, both gemcitabine and QD325 greatly inhibited
tumor growth as single agents. Importantly, the combination was well tolerated and no weight
loss was observed in any of the treatment groups, suggesting a reasonable safety profile of the
drug combination (Figure 6F).
DISCUSSION
Altered redox homeostasis has been observed in various types of cancers including
PDAC. ROS levels are increased as a result of elevated energy demands in cancer cells. To adapt
to changes in the microenvironment, cancer cells hijack the intracellular antioxidant machinery
to reach a new redox state that can facilitate their proliferation. Such dependency on an altered
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redox state provides novel therapeutic opportunities. Previously, we provided proof of concept
that generation of additional ROS using the tool compound QD232 can lead to the selective
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killing of cancer cells when they are challenged beyond their antioxidant capacity.
In order
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to expand upon our previous observations and to select a compound for future clinical
development, we performed a lead optimization campaign and identified QD325, which showed
a stronger induction of ROS and more potent cytotoxicity in PDAC cells (Figure 1).
As revealed by nascent RNA Bru-seq analysis, the NRF2-mediated oxidative stress
response is induced within 4 hours of treatment with the QD compounds (Figure 2). The
cytoprotective NRF2 signaling pathway has been found to be cancer preventive under certain
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circumstances but oncogenic under other conditions.
Activating mutations in NRF2 and
66
KEAP1 are often seen in cancers as an adaptation to elevated intrinsic ROS levels. In PDAC,
where such mutations are not very common, the redox state alteration can be supported through
G12D
other pathways. The Kras
mutation found in >90% of PDAC cases, activates the Nrf2
signaling through the MAPK pathway, thus modifying the antioxidant program of PDAC cells to
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promote tumor progression. This subtle balance is crucial for cancer cells as knockdown of
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NRF2 in PDAC cell lines results in decreased cell viability, showing dependency of cell
proliferation on the antioxidant pathway.
The NRF2 inhibitor, trig, can significantly sensitize PDAC cells to etoposide or TRAIL-
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induced apoptosis, and also enhances the antitumor response to etoposide in xenograft models.
However, trig was not cytotoxic as a single agent and the sensitization of cells was dependent on
the proteasome degradation pathway. An alternative strategy to target the altered redox balance
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in cancer cells would be to trigger ROS accumulation. ROS inducing-agents such as QD232,
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piperlongumine, and imexon have shown single agent anti-tumor activities in pancreatic
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cancer models. Induction of ROS by the QD compounds alters this delicate balance and cells
respond by upregulating the NRF2 pathway in an effort to counter the insult. However, excessive
ROS overwhelms the system to the point of no return leading to cell death. A phase I trial of
imexon and gemcitabine in patients with advanced pancreatic cancer demonstrated feasibility
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and antitumor responses encouraging clinical testing of more potent compounds such as
QD325. These results support the notion that manipulating redox homeostasis by inducing ROS
could be an efficacious therapeutic strategy.
Another significant pathway upregulated following 4 hours of QD treatment is the
unfolded protein response (UPR) (Figure 2). In the ER, where an oxidizing environment is
required for formation of disulfide bonds, the luminal redox state is under stringent regulation.
UPR is triggered when there is imbalance between protein-folding demands and protein folding
capacity, as well as any homeostatic perturbation that can cause protein misfolding, such as
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nutrition deprivation or redox insults. Furthermore, UPR serves as a sensitive cytoprotective
response to integrated stresses including oxidative stress. The activation of the NRF2-mediated
oxidative stress response and UPR both imply that QD232 and QD325 treatments induce
oxidative stress. While the activation of NRF2 protects cells against ROS, activation of UPR is
considered to be both an adaptive response and a major executor of cell death in response to
oxidative stress. UPR regulates cell survival on the basis of the severity and the duration of the
ER stress. The proapoptotic path of UPR is adopted through several mechanisms including
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induction of CHOP and its transcription targets to activate apoptosis. Upregulation of CHOP is
observed following treatment with the QD compounds (Figure 3), suggesting that ER-mediated
apoptosis is a major mechanism of cytotoxicity of the QD compounds.
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For the mitochondrial genome, we observed substantial inhibition of transcription from
the HSP2 and LSP promoters (Figure 4), with only modest change in the transcription from the
HSP1 promoter (Figure S6). This suggests selectivity in QD-mediated inhibition of transcription.
The displacement loop (D-loop) is a non-coding control region in the mitochondrial genome
where promoters for transcription of the H and L strands (HSP and LSP), as well as the origin of
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replication (O ) are located. There are two promoters for the H-strand, while transcripts from
H
HSP1 terminates right after the two rRNAs, transcripts from HSP2 cover the full strand and are
74, 75
subjected to subsequent processing into individual mRNA, tRNA and rRNA molecules.
The
selective transcriptional inhibition of two out of the three promoters implies that mechanisms
other than universal oxidative damaging contribute to such suppressed transcription, and might
serve as a pharmacologically induced model to further elucidate regulation of transcription from
the mitochondrial genome. We hypothesize that blockage of transcription initiation of selective
promoters in the D-loop is a unique feature of the mechanism of action of the QD compounds.
In our in vivo xenograft model for pancreatic cancer, QD325 showed substantial
antitumor efficacy with a favorable safety profile. Our original lead compound QD232
significantly delayed tumor growth at 20 mg/kg in a similar MIA PaCa-2 xenograft model as
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reported earlier, suppressing growth of tumor by 65% in the 31-day study. QD325, the most
potent compound in this lead optimization campaign, showed improved potency by achieving
comparable tumor inhibitory effect at a dose of only 5 mg/kg. QD325 did not show any toxicity
following repetitive treatments with doses as high as 20 mg/kg, suggesting that it is well
tolerated and that it has a favorable therapeutic window. We repeated the experiments with two
independent experiments under the same conditions (Figure 5A and Figure 6E), and observed
similar results (76% vs. 83% inhibition of tumor growth) for QD325-treated animals.
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Gemcitabine is the standard of care treatment for pancreatic cancer patients. In our
studies, QD325 showed similar antitumor effect as gemcitabine, supporting the application of
QD325 as a novel therapeutic option with a distinct new mechanism, especially for patients with
gemcitabine resistance. Due to its remarkable ROS-induction in tumor cells, we expect QD325
to be less toxic than gemcitabine. In general, drug combinations demonstrate survival advantages
over single-agent therapy as long as they have manageable safety profiles. Our studies show that
combination with gemcitabine is tolerable without additional toxicity burden to mice. However,
to perform clinically relevant synergy studies both drugs need to be evaluated at optimal doses
and in additional cell lines and mice models. These findings warrant the development of QD325
as a novel treatment for PDAC as a single agent and in combination with standard of care drugs.
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CONCLUSIONS
In this study, we successfully optimized the anticancer ROS modulator QD232, leading
to the discovery of QD325 that shows significant ROS dependent anticancer activity in PDAC
models. Bru-seq analysis predicted cellular responses in the nucleus, ER, and mitochondrial for
the ROS-mediated cytotoxicity in pancreatic cancer cells, providing in-depth understanding of
the unique mechanisms of this active class of anti-cancer compounds. Significant antitumor
efficacy and the favorable safety profile of QD325 in vivo provides strong rationale for further
developing these QD drugs to target mitochondria as a novel approach to treat PDAC.
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EXPERIMENTAL SECTION
Chemistry
General methods
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The reference compound QD232 and the intermediate 6 were prepared as previously described
by us (see refs. 30 and 32, respectively). All solvents, including anhydrous solvents, and
chemicals, were purchased from Aldrich Co., Alfa Aesar, or Carlo Erba, 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. 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 phase. Melting points (mp) were determined using an
Electrothermal melting point or a Köfler apparatus and are uncorrected. Nuclear magnetic
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resonance ( H NMR, C NMR) spectra were recorded in CDCl
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or DMSO-d on 400 MHz
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Bruker Avance III. Chemical shifts (!) are reported in parts per million (ppm) downfield from
tetramethylsilane (TMS), used as an internal standard. Splitting patterns are designated as follow:
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.
2
Mass spectra were obtained on a Hewlett-Packard 5989 mass engine spectrometer, or a MALDI
micro MX (Waters, Micromass) equipped with a reflectron analyzer. Elemental analyses were
performed on a Perkin-Elmer Elemental Analyzer 2400-CHN at Laboratory of Microanalysis,
Department of Chemistry and Pharmacy, University of Sassari (Italy), and were within ±0.4% of
the theoretical values (Table S8), thus confirming "95% purity.
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General Method A: Preparation of Compounds QD324-327, 329, 331, 332, 334-336,
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38, 353-357. A solution of quinazoline-5,8-dione, cerium (III) chloride heptahydrate (CeCl
O, 1.1 eq.) and (3,4,5)-substituted aniline (1.1 eq.) in absolute ethanol 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 CH Cl . The organic layers were washed with water and
brine, dried over anhydrous Na SO and concentrated to dryness. Then, the crude product was
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7
8
9
0
2
2
2
4
purified by flash chromatography to give the expected product.
General Method B: Preparation of the Compounds QD328, 330, 333, 337. A solution
of quinazoline-5,8-dione, cerium (III) chloride heptahydrate (CeCl
3
K7 H O, 1.1 eq.) and (3,4,5)-
2
substituted aniline (1.1 eq.) in absolute ethanol was stirred at room temperature for 2-6 h. Next,
most of the ethanol was removed under vacuum, and water was added, followed by the
extraction with CH
2
Cl . The organic layers were dried over sodium sulfate (Na2SO4) and
2
concentrated to dryness. Then, the crude residue was treated with water, and the solid residue
that precipitated was filtered and triturated with petroleum ether to give the desired product.
3,6-Dimethoxy-2-nitrobenzaldehyde (2). Nitric acid (8.0 mL; 179.02 mmol), acetic
anhydride (8.0 mL, 84.24 mmol) and 2,5-dimethoxybenzaldehyde (1, 4.0 g, 24.07 mmol) were
added at 0 °C with stirring, respectively. After 1.5 h stirring, the mixture was poured onto 20 mL
ice/water. The resultant yellow solid was filtered, washed with cold water and then purified by
flash chromatography on silica gel using ethyl acetate-petroleum ether (1:1) to give first the
regioisomer 2,5-dimethoxy-4-nitrobenzaldehyde, and then (by further elution with only ethyl
acetate) the desired compound 2. Yield: 68%. Rf= 0.10 (ethyl acetate-petroleum ether 5:5); mp:
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67 °C. H-NMR 400 MHz (DMSO-d
6
): ! 10.25 (s, 1H), 7.70 (d, 1H), 7.48 (d, 1H), 3.95 (s, 3H),
): ! 10.39 (s, 1H), 7.30 (d, 1H), 7.12 (d, 1H), 3.97 (s,
1
3
.86 (s, 3H). H-NMR 400 MHz (CDCl
3
+
3H), 3.89 (s, 3H). MS: m/z 211 [M] .
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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3
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3
3
3
4
4
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4
5
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5
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5
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0
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7
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0
1
2
3
4
5
6
7
8
9
0
N,N’-[(3,6-dimethoxy-2-nitrophenyl)methanediyl)diformamide (3). A solution of 3,6-
dimethoxy-2-nitrobenzaldehyde (2, 11.90 g, 56.35 mmol) in formamide (66.5 eq., 150 mL),
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. Pale yellow colored
precipitate was formed, which was filtered, dried and triturated with ethyl acetate and petroleum
ether to yield the desired compound. Yield: 90%. Rf= 0.26 (dichloromethane-methanol 9.5:0.5);
1
mp: 255 °C. H-NMR 400 MHz (DMSO-d
6
): ! 8.67 (d, 2H), 7.92 (s, 2H), 7.28 (s, 2H), 6.77 (t,
+
1
H), 3.88 (s, 3H), 3.82 (s, 3H). MS: m/z 283 [M ].
5
,8-Dimethoxyquinazoline (4). Zinc powder (22.9 g) was added to a suspension of
N,N’-[(3,6-dimethoxy-2-nitrophenyl)methanediyl)]diformamide (3, 7.0 g, 24.71 mmol) in
triturated ice (92 g) and glacial acetic acid (32 mL), under constant magnetic stirring. The
reaction mixture was stirred for 2 h in 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 (120 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 in ethyl acetate, filtered, dried over anhydrous Na
2
SO , and
4
concentrated to dryness yielding the desired compound. Yield: 79%. Rf= 0.46 (dichloromethane-
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5
6
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methanol 9.5:0.5); mp: 106 °C. H-NMR 400 MHz (DMSO-d
6
): ! 9.64 (s, 1H), 9.28 (s, 1H), 7.39
+
(
d, 1H), 7.10 (d, 1H), 3.98 (s, 3H), 3.94 (s, 3H). MS: m/z 190 [M]
0
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2
3
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5
6
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9
0
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0
(
3-Aminopropyl)triphenylphosphonium bromide hydrobromide (6). To a 50 mL
round-bottom flask equipped with a magnetic stiring bar, triphenylphosphine (1.0 eq., 1.0 g, 3.82
mmol), 3-bromopropylamine hydrobromide (5, 1.0 eq., 0.84 g, 3.82 mmol), and acetonitrile (7
mL), were added. The resulting suspension was heated to reflux and the mixture was stirred for
1
6 h. The reaction was cooled to room temperature, then n-hexane was added and the resulting
solid was filtered, washed with n-hexane, dissolved in 100 mL isopropanol and precipitated with
cold diethyl ether, to give a white powder. Yield: 50%; Rf= 0.28 (dichlorometane-methanol 9:1);
1
mp: 200 °C. H-NMR 400 MHz (CDCl
3
): ! 7.95-7.92 (m, 3H), 7.84-7.74 (m, 15 H), 3.74 (m,
2
H), 3.00-2.98 (m, 2H), 1.85 (m, 2H).
Quinazoline-5,8-dione (QD323). A solution of 5,8-dimethoxyquinazoline (4, 0.35 g,
1
.84 mmol) in (7:3) acetonitrile:water (10 mL) was cooled at 0 °C in an ice bath and a solution
of ceric ammonium nitrate (2.7 eq., 2.72 g, 4.97 mmol) in (9:1) acetonitrile:water (10 mL) was
added dropwise. The reaction mixture was stirred for 20 minutes, then poured into ice/water and
extracted (8-10 times) with CH
2
Cl . The organic layer was washed (5-6 times) with water, dried
2
over anhydrous Na
2
SO
4
and concentrated to dryness to give a brown powder. Yield: 69%. Rf=
1
0.62 (dichloromethane-methanol 9.5:0.5); mp: > 320 °C. H-NMR 400 MHz (DMSO-d
6
): ! 9.69
13
(
s, 1H), 9.43 (s, 1H), 7.28 (d, 1H), 7.18 (d, 1H). C-NMR 400 MHz (DMSO-d
6
): ! 184.07,
+
1
82.88, 162.08, 156.27, 152.61, 139.46, 137.74, 124.61. MS: m/z 160 [M]
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Journal of Medicinal Chemistry
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-((4-Phenoxyphenyl)amino)quinazoline-5,8-dione (QD324). Quinazoline-5,8-dione
(
QD323, 0.10 g, 0.62 mmol), cerium (III) chloride heptahydrate (CeCl
3
K7 H
2
O, 1.1 eq., 0.26 g,
0
.69 mmol), 4-phenoxyaniline (1.1 eq., 0.13 g, 0.69 mmol), and absolute ethanol (11 mL). Flash
1
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4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
chromatography (ethyl acetate-petroleum ether 6:4) gave compound QD324 as a violet powder.
1
Yield: 65%. Rf= 0.30 (ethyl acetate-petroleum ether 6:4); mp: 169-171 °C. H-NMR 400 MHz
(
CDCl
3
): ! 9.66 (s, 1H), 9.49 (s, 1H), 7.51 (s, 1H), 7.39 (t, 2H), 7.25 (d, 2H), 7.20-7.15 (m, 1H),
13
7
1
.09-7.04 (m, 4H), 6.53 (s, 1H). C-NMR 400 MHz (CDCl
3
): ! 180.65, 180.39, 163.76, 156.48,
56.31, 154.40, 145.15, 130.90, 130.01, 125.25, 124.04, 123.34, 119.76, 119.28, 104.65. MS:
+
m/z 343 [M]
6
-([1,1'-Biphenyl]-4-ylamino)quinazoline-5,8-dione (QD325). Quinazoline-5,8-dione
(QD323, 0.16 g, 1.01 mmol), cerium (III) chloride heptahydrate (CeCl
3
K7 H O, 1.1 eq., 0.41 g,
2
1
.11 mmol), 4-aminobiphenyl (1.1 eq., 0.19 g, 1.11 mmol), and absolute ethanol (19 mL). Flash
chromatography (ethyl acetate-petroleum ether 7:3) gave compound QD325 as a violet powder.
1
Yield: 58%. Rf= 0.48 (ethyl acetate-petroleum ether 8:2); mp: 230 °C. H-NMR 400 MHz
(
CDCl
3
): ! 9.68 (s, 1H), 9.51 (s, 1H), 7.68 (d, 2H), 7.65 (s, 1H), 7.60 (d, 2H), 7.48 (t, 2H), 7.42-
1
3
7
.36 (m, 3H), 6.73 (s, 1H). C-NMR 400 MHz (CDCl ): ! 180.67, 180.52, 163.78, 156.39,
3
154.32, 144.33, 139.71, 135.44, 128.99, 128.53, 127.53, 127.00, 123.26, 105.20. MS: m/z 327
+
[
M]
6-((3,4,5-Trimethoxyphenyl)amino)quinazoline-5,8-dione (QD326). Quinazoline-5,8-
dione (QD323, 0.07 g, 0.44 mmol), cerium (III) chloride heptahydrate (CeCl K7 H O, 1.1 eq.,
3
2
0.18 g, 0.48 mmol), 3,4,5-trimethoxyaniline (1.1 eq., 0.09 g, 0.48 mmol), and absolute ethanol (8
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2
2
2
2
2
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4
4
4
4
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5
5
5
5
5
5
5
5
6
mL). Flash chromatography (ethyl acetate-petroleum ether from 7:3 to 8:2) gave compound
QD326 as a violet powder. Yield: 88%. Rf= 0.18 (ethyl acetate-petroleum ether 8:2); mp: 161-
1
162 °C. H-NMR 400 MHz (CDCl
3
): ! 9.67 (s, 1H), 9.49 (s, 1H), 7.51 (s, 1H), 6.59 (s, 1H), 6.50
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
s, 2H), 3.88 (s, 9H). C-NMR 400 MHz (CDCl
3
): ! 180.61, 180.40, 163.77, 156.33, 154.15,
+
1
44.90, 136.96, 131.87, 123.30, 105.00, 101.20, 61.07, 56.39. MS: m/z 341 [M]
6
-((4-(Trifluoromethoxy)phenyl)amino)quinazoline-5,8-dione (QD327). Quinazoline-
5
,8-dione (QD323, 0.05 g, 0.31 mmol), cerium (III) chloride heptahydrate (CeCl K7 H O, 1.1
3
2
eq., 0.13 g, 0.34 mmol), 4-(trifluoromethoxy)aniline (1.1 eq., 0.046 mL, 0.34 mmol), and
absolute ethanol (6 mL). Flash chromatography (dichloromethane-methanol 9.7:0.3) gave
compound QD327 as a dark red powder. Yield: 67%. Rf= 0.53 (dichloromethane-methanol
1
9.5:0.5); mp: 114 °C. H-NMR 400 MHz (CDCl
3
): ! 9.68 (s, 1H), 9.51 (s, 1H), 7.55 (s, 1H), 7.33
+
(s, 4H), 6.58 (s, 1H). MS: m/z 335 [M]
4-((5,8-Dioxo-5,8-dihydroquinazolin-6-yl)amino)benzenesulfonamide
Quinazoline-5,8-dione (QD323, 0.05 g, 0.31 mmol), cerium (III) chloride heptahydrate (CeCl
O, 1.1 eq., 0.13 g, 0.34 mmol), sulfanilamide (1.1 eq., 0.06 g, 0.34 mmol), and absolute
(QD328).
3
K
7
H
2
ethanol (6 mL). The precipitate was filtered and triturated with petroleum ether gave compound
QD328 as a red powder. Yield: 59%. Rf= 0.16 (ethyl acetate-petroleum ether 8:2); mp: >320 °C.
1
H-NMR 400 MHz (DMSO-d ): ! 9.72 (s, 1H), 9.64 (s, 1H), 9.44 (s, 1H), 7.88 (d, 2H), 7.60 (d,
6
1
3
2H), 7.38 (s, 2H), 6.46 (s, 1H). C-NMR 400 MHz (DMSO-d
6
): ! 180.50, 180.32, 162.58,
1
55.78, 153.46, 145.33, 140.91, 140.20, 127.38, 127.05, 124.19, 123.17, 112.38, 105.11. MS:
+
m/z 331 [M+1]
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Journal of Medicinal Chemistry
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-((4-(Hydroxymethyl)phenyl)amino)quinazoline-5,8-dione (QD329). Quinazoline-
5,8-dione (QD323, 0.05 g, 0.31 mmol), cerium (III) chloride heptahydrate (CeCl
3
K7 H O, 1.1
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
eq., 0.13 g, 0.34 mmol), 4-aminobenzyl alcohol (1.1 eq., 0.04 g, 0.34 mmol), and absolute
ethanol (6 mL). Flash chromatography (dichloromethane-methanol 9.7:0.3) gave compound
QD329 as a brown-red powder. Yield: 27%. Rf= 0.30 (dichloromethane-methanol 9.5:0.5); mp:
1
2
03 °C. H-NMR 400 MHz (CDCl
3
): ! 9.67 (s, 1H), 9.50 (s, 1H), 7.59 (s, 1H), 7.47 (d, 2H), 7.29
+
(
d, 2H), 6.64 (s, 1H), 4.75 (s, 2H). MS: m/z 303 [M+Na]
4
-((5,8-Dioxo-5,8-dihydroquinazolin-6-yl)amino)benzamide (QD330). Quinazoline-
5
,8-dione (QD323, 0.05 g, 0.31 mmol), cerium (III) chloride heptahydrate (CeCl K7 H O, 1.1
3
2
eq., 0.13 g, 0.34 mmol), 4-aminobenzamide (1.1 eq., 0.05 g, 0.34 mmol), and absolute ethanol (6
mL). The precipitate was filtered and triturated with petroleum ether gave compound QD330 as
1
a brown powder. Yield: 24%. Rf= 0.58 (dichloromethane-methanol 9.5:0.5); mp: > 320 °C. H-
NMR 400 MHz (DMSO-d
6
): ! 9.65 (s, 1H), 9.63 (s, 1H), 9.43 (s, 1H), 7.95 (d, 2H), 7.49 (d, 2H),
+
7
.38 (s, 2H), 6.42 (s, 1H). MS: m/z 295 [M+1]
Methyl
Quinazoline-5,8-dione (QD323, 0.06 g, 0.37 mmol), cerium (III) chloride heptahydrate (CeCl
7 H O, 1.1 eq., 0.15 g, 0.41 mmol), methyl 4-aminobenzoate (1.1 eq., 0.06 g, 0.41 mmol), and
4-((5,8-dioxo-5,8-dihydroquinazolin-6-yl)amino)benzoate
(QD331).
3
K
2
absolute ethanol (7.2 mL). Flash chromatography (ethyl acetate-petroleum ether from 6:4 to 7:3)
gave compound QD331 as a red powder. Yield: 42%. Rf= 0.35 (ethyl acetate-petroleum ether
1
7:3); mp: 226-230 °C. H-NMR 400 MHz (CDCl
3
): ! 9.69 (s, 1H), 9.52 (s, 1H), 8.14 (d, 2H),
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2
2
2
2
2
2
2
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3
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3
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3
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3
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4
4
4
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5
5
5
5
5
5
5
6
13
7
.72 (s, 1H), 7.36 (d, 2H), 6.81 (s, 1H), 3.95 (s, 3H). C-NMR 400 MHz (CDCl
3
): ! 180.75,
1
80.40, 165.96, 163.85, 156.57, 153.96, 143.34, 140.64, 131.51, 127.69, 123.27, 121.66, 121.55,
+
106.35, 52.35. MS: m/z 309 [M]
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
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0
Ethyl
Quinazoline-5,8-dione (QD323, 0.13 g, 0.81 mmol), cerium (III) chloride heptahydrate (CeCl
O, 1.1 eq., 0.33 g, 0.89 mmol), ethyl 4-aminobenzoate (1.1 eq., 0.15 g, 0.89 mmol), and
4-((5,8-dioxo-5,8-dihydroquinazolin-6-yl)amino)benzoate
(QD332).
3
K
7
H
2
absolute ethanol (16 mL). Flash chromatography (ethyl acetate-petroleum ether 6.5:3.5) gave
compound QD332 as a red powder. Yield: 39%. Rf= 0.36 (ethyl acetate-petroleum ether 7:3);
1
mp: 206-207 °C. H-NMR 400 MHz (CDCl
3
): ! 9.69 (s, 1H), 9.52 (s, 1H), 8.14 (d, 2H), 7.72 (s,
1
3
1
H), 7.36 (d, 2H), 6.80 (s, 1H), 4.43-4.38 (q, 2H), 1.42 (t, 3H). C-NMR 400 MHz (CDCl ): !
3
180.74, 180.42, 165.49, 163.85, 156.56, 153.98, 143.38, 140.53, 131.47, 128.08, 123.27, 121.65,
+
1
06.31, 61.31, 14.34. MS: m/z 323 [M]
(3-((5,8-Dioxo-5,8-dihydroquinazolin-6-yl)amino)phenyl)boronic acid (QD333).
Quinazoline-5,8-dione (QD323, 0.05 g, 0.31 mmol), cerium (III) chloride heptahydrate (CeCl
3
K
7
H
2
O, 1.1 eq., 0.13 g, 0.34 mmol), 3-aminophenylboronic acid (1.1 eq., 0.05 g, 0.34 mmol), and
absolute ethanol (6 mL). The precipitate was filtered and triturated with petroleum ether gave
compound QD333 as a red powder. Yield: 48%. Rf= 0.37 (dichloromethane-methanol 9.5:0.5);
1
mp: 208-210 °C. H-NMR 400 MHz (DMSO-d
6
): ! 9.61 (s, 1H), 9.52 (s, 1H), 9.41 (s, 1H), 8.20
+
(s, 2H), 7.77-7.70 (m, 2H), 7.43 (m, 2H), 6.23 (s, 1H). MS: m/z 318 [M+Na]
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Journal of Medicinal Chemistry
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-((4'-Fluoro-[1,1'-biphenyl]-4-yl)amino)quinazoline-5,8-dione
(QD334).
Quinazoline-5,8-dione (QD323, 0.05 g, 0.31 mmol), cerium (III) chloride heptahydrate (CeCl
O, 1.1 eq., 0.13 g, 0.34 mmol), 4-amino-4'-fluorobiphenyl (1.1 eq., 0.06 g, 0.34 mmol), and
3
K
7
H
2
1
1
1
1
1
1
1
1
1
1
2
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2
2
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5
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5
5
5
6
0
1
2
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5
6
7
8
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1
2
3
4
5
6
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9
0
1
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
absolute ethanol (6 mL). Flash chromatography (ethyl acetate-petroleum ether from 7:3 to 8:2)
gave compound QD334 as a violet powder. Yield: 29%. Rf= 0.32 (ethyl acetate-petroleum ether
1
7
7
1
:3); mp: 285-289 °C. H-NMR 400 MHz (CDCl
3
): ! 9.68 (s, 1H), 9.51 (s, 1H), 7.63 (d, 2H),
13
.55 (t, 2H), 7.53 (s, 1H), 7.37 (d, 2H), 7.16 (t, 2H), 6.72 (s, 1H). C-NMR 400 MHz (CDCl
3
): !
80.64, 180.53, 163.97, 163.79, 161.51, 156.39, 154.30, 144.33, 138.70, 135.98, 135.47, 128.65,
+
128.39, 123.33, 116.03, 115.82, 105.21. MS: m/z 345 [M]
6
-((4'-Ethyl-[1,1'-biphenyl]-4-yl)amino)quinazoline-5,8-dione (QD335). Quinazoline-
5,8-dione (QD323, 0.05 g, 0.34 mmol), cerium (III) chloride heptahydrate (CeCl K7 H O, 1.1
3
2
eq., 0.14 g, 0.37 mmol), 4-amino-4'-ethylbiphenyl (1.1 eq., 0.07 g, 0.37 mmol), and absolute
ethanol (6.5 mL). Flash chromatography (ethyl acetate-petroleum ether from 6:4 to 7:3) gave
compound QD335 as a red-violet powder. Yield: 60%. Rf= 0.36 (ethyl acetate-petroleum ether
1
7
:3); mp: 232 °C. H-NMR 400 MHz (CDCl
3
): ! 9.66 (s, 1H), 9.49 (s, 1H), 7.68 (s, 1H), 7.66 (d,
1
3
2
H), 7.51 (d, 2H), 7.34 (d, 2H), 7.30 (d, 2H), 6.71 (s, 1H), 2.74-2.68 (q, 2H), 1.29 (t, 3H). C-
): ! 180.67, 180.48, 163.73, 156.35, 154.34, 144.35, 144.09, 139.64,
37.03, 135.15, 128.53, 128.46, 126.89, 123.35, 123.24, 105.11, 28.54, 15.55. MS: m/z 356
NMR 400 MHz (CDCl
3
1
+
[M+1]
6
-((4'-Methoxy[1,1'-biphenyl]-4-yl)amino)quinazoline-5,8-dione
(QD336).
3
Quinazoline-5,8-dione (QD323, 0.05 g, 0.34 mmol), cerium (III) chloride heptahydrate (CeCl K
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2
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5
5
5
5
5
5
6
7
H
2
O, 1.1 eq., 0.14 g, 0.37 mmol), 4'-methoxy-biphenyl-4-ylamine (1.1 eq., 0.07 g, 0.37 mmol),
and absolute ethanol (6.5 mL). Flash chromatography (ethyl acetate-petroleum ether from 7:3 to
10:0) gave compound QD336 as a dark violet powder. Yield: 69%. Rf= 0.29 (ethyl acetate-
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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8
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2
3
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0
petroleum ether 7:3); mp: 270-272 °C. H-NMR 400 MHz (CDCl
3
): ! 9.67 (s, 1H), 9.51 (s, 1H),
7
.63 (d, 2H), 7.61 (s, 1H), 7.53 (d, 2H), 7.32 (d, 2H), 7.01 (d, 2H), 6.71 (s, 1H), 3.87 (s, 3H).
1
3
C-NMR 400 MHz (CDCl
3
): ! 180.67, 180.47, 163.77, 159.58, 156.36, 154.37, 144.38, 139.38,
+
1
34.81, 132.20, 128.05, 128.01, 123.29, 123.19, 114.44, 105.09, 55.40. MS: m/z 357 [M]
6
-((4'-Amino-[1,1'-biphenyl]-4-yl)amino)quinazoline-5,8-dione
Quinazoline-5,8-dione (QD323, 0.05 g, 0.34 mmol), cerium (III) chloride heptahydrate (CeCl
O, 1.1 eq., 0.14 g, 0.37 mmol), benzidine (1.1 eq., 0.07 g, 0.37 mmol), and absolute ethanol
(QD337).
3
K
7
H
2
(6.5 mL). The precipitate was filtered and triturated with petroleum ether gave compound
QD337 as a dark violet powder. Yield: 16%. Rf= 0.71 (dichlorometane-methanol 9.5:0.5); mp:
1
>
320 °C. H-NMR 400 MHz (DMSO-d
6
): ! 9.66 (s, 1H), 9.63 (s, 1H), 9.44 (s, 1H), 7.83 (d, 2H),
7.64 (d, 1H), 7.53 (d, 2H), 7.40 (d, 2H), 6.65 (d, 1H), 6.40 (s, 1H), 5.27 (s, 2H). MS: m/z 342
+
[M]
6
-((2-Fluoro-4'-methyl-[1,1'-biphenyl]-4-yl)amino)quinazoline-5,8-dione (QD338).
Quinazoline-5,8-dione (QD323, 0.06 g, 0.35 mmol), cerium (III) chloride heptahydrate (CeCl
O, 1.1 eq., 0.14 g, 0.38 mmol), 2-fluoro-4'-methyl-biphenyl-4-ylamine (1.1 eq., 0.08 g, 0.38
3
K
7
H
2
mmol), and absolute ethanol (6.7 mL). Flash chromatography (ethyl acetate-petroleum ether,
from 6:4 to 8:2) gave compound QD338 as a violet powder. Yield: 60%. Rf= 0.26 (ethyl acetate-
1
petroleum ether 7:3); mp: 282-283 °C. H-NMR 400 MHz (CDCl
3
): ! 9.69 (s, 1H), 9.52 (s, 1H),
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Journal of Medicinal Chemistry
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.62 (s, 1H), 7.52 (t, 1H), 7.45 (d, 2H), 7.29 (d, 2H), 7.14 (t, 2H), 6.75 (s, 1H), 2.42 (s, 3H). C-
NMR 400 MHz (CDCl
3
): ! 180.61, 180.47, 163.83, 156.49, 143.93, 138.14, 131.79, 129.40,
+
128.89, 128.71, 123.31, 118.67, 110.85, 110.58, 105.75, 21.24. MS: m/z 359 [M]
1
1
1
1
1
1
1
1
1
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2
2
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2
2
2
2
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0
(
3-(4-Aminobenzamido)propyl)triphenylphosphonium bromide (QD339). To a solution of 4-
aminobenzoic acid (1.0 eq., 0.076 g, 0.55 mmol) in CH Cl (12 mL), N,N-diisopropylethylamine
DIPEA, 5 eq. 0.48 mL, 2.75 mmol), and HBtU (1.0 eq., 0.206 g, 0.55 mmol) were added. The
reaction mixture was stirred for 15 minutes, and (3-aminopropyl)triphenylphosphonium bromide
6, 3 eq. 0.80 g, 1.66 mmol) and DMAP (0.04 eq., 2.7 µg, 0.02 mmol) were added. The resulting
mixture was stirred at room temperature for 5 h, filtered, washed with CH Cl , and concentrated
2
2
(
(
2
2
to dryness. The crude product was purified by flash chromatography on silica gel using
dichloromethane-isopropanol (9.5:0.5) to give QD339 as a beige powder. Yield: 70%; Rf= 0.27
1
(dichlorometane-methanol 9:1); mp: 203-205 °C. H-NMR 400 MHz (CDCl
3
): ! 8.91 (t, 1H),
8
.10 (d, 2H), 7.77-7.72 (m, 9H), 7.61-7.58 (m, 6H), 6.70 (d, 2H), 3.94-3.90 (m, 2H), 3.72-3.71
(m, 2H), 1.95 (m, 2H). MS: m/z 439.
(
3-(4-((5,8-Dioxo-5,8-dihydroquinazolin-6-yl)amino)benzamido)propyl)
triphenylphosphonium bromide (QD340). A solution of quinazoline-5,8-dione (5, 1.0 eq., 0.04
g, 0.25 mmol), cerium (III) chloride heptahydrate (CeCl K7 H O, 1.1 eq., 0.102 g, 0.27 mmol)
and (3-(4-aminobenzamido)propyl)triphenylphosphonium bromide (QD339, 1.1 eq., 0.143 g,
.27 mmol) in absolute ethanol (5 mL) was stirred at room temperature for 2 h. Then, most of the
ethanol was removed under vacuum, and water was added, followed by the extraction with
3
2
0
CH
2
Cl
2
. The organic layers were washed with water, brine, dried over anhydrous sodium
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