Suppression of Drug-Resistant Non-Small-Cell Lung Cancer with Inhibitors Targeting Minichromosomal Maintenance Protein

Article abstract of DOI:10.1021/acs.jmedchem.9b01783

Drug resistance has been a major threat in cancer therapies that necessitates the development of new strategies to overcome this problem. We report here a cell-based high-throughput screen of a library containing two-million molecules for the compounds that inhibit the proliferation of non-small-cell lung cancer (NSCLC). Through the process of phenotypic screening, target deconvolution, and structure-activity relationship (SAR) analysis, a compound of furanonaphthoquinone-based small molecule, AS4583, was identified that exhibited potent activity in tyrosine kinase inhibitor (TKI)-sensitive and TKI-resistant NSCLC cells (IC₅₀ = 77 nM) and in xenograft mice. The mechanistic studies revealed that AS4583 inhibited cell-cycle progression and reduced DNA replication by disrupting the formation of the minichromosomal maintenance protein (MCM) complex. Subsequent SAR study of AS4583 gave compound RJ-LC-07-48 which exhibited greater potency in drug-resistant NSCLC cells (IC₅₀ = 17 nM) and in mice with H1975 xenograft tumor.

Full text of DOI:10.1021/acs.jmedchem.9b01783

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Article
Suppression of Drug-Resistant Non-Small-Cell Lung Cancer
with Inhibitors Targeting Minichromosomal Maintenance Protein
Chia-Yi Lin, Hsin-Yi Wu, Yuan-Ling Hsu, Ting-Jen Rachel Cheng, Jyung-
Hurng Liu, Rou-Jie Huang, Tzu Hung Hsiao, Chia-Jen Wang, Pei-Fang Hung,
Albert Lan, Szu-Hua Pan, rj Chein, Chi-Huey Wong, and Pan-Chyr Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01783 • Publication Date (Web): 03 Mar 2020
Downloaded from pubs.acs.org on March 3, 2020
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Suppression of Drug-Resistant Non-Small-Cell Lung Cancer with
Inhibitors Targeting Minichromosomal Maintenance Protein
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Chia-Yi Lin,1, 2 Hsin-Yi Wu, Yuan-Ling Hsu, Ting-Jen Rachel Cheng, Jyung-Hurng Liu,
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6, 7, 8
Rou-
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1, 4, 12,
Jie Huang, Tzu Hung Hsiao, Chia-Jen Wang, Pei-Fang Hung, Albert Lan, Szu-Hua Pan,
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Rong-Jie Chein, * Chi-Huey Wong, and Pan-Chyr Yang
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Genome and Systems Biology Degree Program, National Taiwan University and Academia
Sinica, Taipei 100, Taiwan
2. Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
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Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan
Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National
Taiwan University, Taipei 100, Taiwan
5. The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
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Institute of Genomics and Bioinformatics, College of Life Sciences, National Chung Hsing
University, Taichung 402, Taiwan
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Agricultural Biotechnology Center, NCHU, Taichung 402, Taiwan
8. Rong Hsing Research Center for Translational Medicine, NCHU, Taichung 402, Taiwan
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Department of Chemistry, National Central University, Jhong-Li 320, Taiwan
0. Department of Medical Research, Taichung Veterans General Hospital, Taichung 407, Taiwan
11. Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital,
Taoyuan 333, Taiwan.
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2. Doctoral Degree Program of Translational Medicine, National Taiwan University, Taipei 100,
Taiwan
13. Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei
00, Taiwan
Corresponding authors
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KEYWORDS
Minichromosomal maintenance protein (MCM), DNA replication, cell cycle, and lung cancer
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ABSTRACT
Drug resistance has been a major threat in cancer therapies that necessitates the development
of new strategies to overcome this problem. We report here a cell-based high-throughput screen of a
library containing 2-million molecules for the compounds that inhibit the proliferation of non-small-
cell lung cancer (NSCLC). Through the process of phenotypic screening, target deconvolution and
structure-activity relationship (SAR) analysis, a compound of furanonaphthoquinone-based small
molecule, AS4583, was identified which exhibited potent activity in tyrosine kinase inhibitor (TKI)–
sensitive and TKI–resistant NSCLC cells (IC50 77 nM) and in xenograft mice. The mechanistic
studies revealed that AS4583 inhibited cell-cycle progression and reduced DNA replication by
disrupting the formation of the minichromosomal maintenance protein (MCM) complex. Subsequent
SAR study of AS4583 gave compound RJ-LC-07-48 which exhibited greater potency in drug-
resistant NSCLC cells (IC50 17nM) and in mice with H1975 xenograft tumor.
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INTRODUCTION
Lung cancer remains the leading cause of cancer-related death worldwide, and approximately
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.8 million new lung-cancer cases are diagnosed every year , the majority being non-small-cell lung
cancer (NSCLC). Despite advances in cancer treatment have been made over the past 20 years, less
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than 30% of lung-cancer patients respond to chemotherapy, and prognoses generally remain poor .
Although the development of specific tyrosine kinase inhibitors (TKIs) targeting the epidermal
growth factor receptor (EGFR) has improved patient outcomes, drug resistance has become a major
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concern. Therefore, it is essential to develop new anticancer agents with novel mode of action to
overcome the problem of drug resistance, especially of EGFR-TKI resistance.
Over the last years, target-based screens and cell-based screens have been the two
predominant strategies for drug discovery. With the growing knowledge of cancer biology, many
pharmaceutical companies have used target-based screens to develop therapeutic agents that
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specifically inhibit the key factors associated with cancer progression , e.g., the FDA-approved drugs
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gefitinib , erlotinib (targeting EGFR), crizotinib (targeting MET and ALK), imatinib (targeting
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ABL), temsirolimus (targeting mTOR) and dabrafenib (targeting BRAF). However, the problem
of drug resistance and the number of known targets that drive cancer progression remains limited,
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presenting a substantial challenge for target-based drug development . In order to identify new
targets for the development of anticancer agents, the strategy of cell-based phenotypic screens has
been used and several anticancer agents have been developed and approved by the FDA, e.g.,
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vorinostat , romidepsin , azacitidine , lenalidomide and pomalidomide .
Recently, the integration of high-throughput or high-content screening of large libraries
combined with computational data mining has been used to facilitate the drug discovery process,
including target identification and validation as well as the study of structure-activity relationship
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(SAR) and mechanisms of action . Herein, we present a successful hit-to-lead discovery of a novel
anticancer compound with high potency against both TKI-sensitive and TKI-resistant NSCLC using
cell-based, high-throughput screening combined with SAR study and in silico modeling and
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simulation. A high-throughput screen of lung-cancer cells with different EGFR mutation status was
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performed to identify compound AS4583, a furanonaphthoquinone-based small molecule, that
interferes with S-phase progression. In vitro analyses indicated that AS4583 interferes with cell-cycle
progression and DNA replication by disrupting the formation of the minichromosomal maintenance
protein (MCM) complex by promoting the proteasome degradation, especially of MCM2. Further
SAR study of AS4583 led to the identification of RJ-LC-07-48 with anticancer efficacy around 10-
fold higher than AS4583 against TKI-resistant NSCLC.
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RESULTS
Identification of AS4583 as an inhibitor of NSCLC cell proliferation
A high-throughput screening of 2 million compounds (each 10 µM) was initially carried out
to evaluate their ability to inhibit the proliferation of human H1975 NSCLC cells, which are resistant
to EGFR-TKIs and harbor an EGFR-TKI-sensitive mutation (L858R) and an acquired resistance
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mutation (T790M) . Approximately 6,800 hit compounds exhibited > 80% inhibition at 10 µM were
identified, which were further screened against other lung-cancer cells, including clinical isolates
(CL1-0 and CL1-5) as well as an ATCC cell line PC9 and its EGFR-TKI-derivative–resistant clone
PC9/IR. We then focused on compounds with potent activity against both TKI-sensitive and TKI-
resistant cells, such as PC9 and PC9/IR, respectively, to identify compounds with new modes of
action. After excluding established anticancer drugs, such as topotecan and doxorubicin analogs, a
total of 193 hits with >80% inhibition at 10 µM were identified and further assessed with respect to
their ability to inhibit the proliferation of both normal and cancer cells, especially inhibition of cell-
cycle progression. Among them, a furanonaphthoquinone-based small molecule, AS4583 (Figure 1A),
was found to inhibit the proliferation of both TKI-sensitive and TKI-resistant lung-cancer cells with
IC50 values of <100 nM and more than 10-fold higher potency for cancer cells than human normal
bronchial epithelial cells (NBE, Figure 1B and Table S1 and S2).
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Figure 1. AS4583 selected from high-throughput screening affects cell proliferation and inhibits
tumor growth in vivo. (A) Structure of AS4583. (B) Cytotoxicity assay for AS4583 with various normal
and tumor cell lines. Cells were treated with 0–10 μM AS4583 for 72 h, and survival was assessed with
the sulforhodamine B assay. (C) Tumor sizes and (D) TUNEL assay results from tumor xenograft models
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n=6 per group). H1975 cells (3  10 ) were subcutaneously injected into male nu/nu mice. Tumor size
was measured every 3 to 4 days. At day 27, the mice were sacrificed to examine tumor size as well as to
confirm histology (original magnification 400) and apoptosis status by hematoxylin and eosin (H&E)
and TUNEL staining. (E) Bar graph comparing percent tumor growth inhibition (%TGI) on the day of the
final measurement for the control group was compared across all treatments in vivo study
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AS4583 inhibits tumor growth in vivo
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To evaluate the antitumor efficacy of AS4583 in vivo, athymic nude mice bearing established
subcutaneous H1975 tumors were five days a week treated with 1 mg/kg or 4 mg/kg AS4583 (compared
with DMSO control) for 4 weeks (n=6 per group). The result showed that treatment with AS4583
significantly reduced H1975 xenograft tumor growth on post-treatment day 27 (average tumor size, 1,217
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± 516.6 mm for DMSO, 701.2 ± 196.5 mm for 1 mg/kg, and 518.0 ± 93.0 mm for 4 mg/kg, both p<0.05)
and induced apoptosis in the tumor tissues (Figure 1C and 1D). Both AS4583 treatment induced tumor
growth delay for a longer period of time when compared with DMSO controls. Compared to DMSO-
treated animals, AS4583 1 mg/kg and 4 mg/kg showed significant percent tumor growth inhibition (%
TGI) of 44 and 59, respectively (Figure 1E). To evaluate the potential cytotoxicity of AS4583, especially
in liver and kidney, the body weight of each mouse was measured and the serum was subjected to
biochemical analysis including aspartate transaminase, alanine transaminase, blood urea nitrogen, and
creatinine (Supplementary Figure 1). No significant difference was observed. These results indicated that
AS4583 could inhibit tumor growth without side effects.
AS4583 inhibits formation of the DNA replication fork and prolongs S phase in NSCLC cells
To examine the hypothesis that treatment of cells with AS4583 might disrupt cell cycle, we first
performed a flow cytometry analysis to examine the effect of this compound on cell-cycle progression.
H1975 cells treated with 140 nM AS4583 for 72 h were significantly arrested in the S phase (18% of the
treated vs. 2% of the control group) and the G2/M phase (38% of the treated vs. 17% of the control group)
(Table S3). Next, we investigated whether AS4583 inhibits cell proliferation by interfering with DNA
replication. The incorporation of bromodeoxyuridine (BrdU) into replicated DNA was monitored in
H1975 cells treated with DMSO (control) or AS4583 for 12, 24, or 48 h. The percentage of BrdU-positive
cells was lower in the treated group (at 24 h: 27.7 ± 3.9% vs. 44.5 ± 2.0% for the control, p=0.086; at 48
h: 16.0 ± 2.5% vs. 34.0 ± 5.6% for the control, p= 0.0411; Figure 2A and 2B).
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To further assess whether treatment with AS4583 interferes with DNA replication, we examined
the formation of replication forks by immunofluorescence staining of the replication marker, MCM2, in
both DMSO- and AS4583-treated H1975 cells released from G1 phase. Indeed, fewer replication forks
were found in the AS4583-treated group (48.8 ± 18.7) compared with the control (176.8 ± 8.7, p= 0.0007,
Figure 2C). Whether this decrease in the formation of replication forks could alter the distribution of cell-
cycle phases was then evaluated. H1975 cells were synchronized in G1/S phase using a double thymidine
block followed by release with DMSO and AS4583-containing medium up to 26 h. We observed that a
higher percentage of AS4583-treated cells (p= 0.0373) remained in the G1/S phase after 20 h (Figure 2D),
demonstrating that AS4583 increased the G1/S phase population, i.e., causing G1/S arrest. These results
revealed that AS4583 significantly inhibits replication-fork progression and hence prevents cell-cycle
progression.
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When further dissecting the potential apoptotic pathways that are activated by AS4583, the
expression levels of several specific apoptotic markers were detected by immunoblotting. As expected,
the cleaved form of three important regulatory proteins in the apoptotic pathway, caspase-3 and PARP-1,
appeared after AS4583 treatment. The appearance of cleaved caspase-8 implied that an intrinsic apoptotic
pathway was being activated by AS4583 treatment (Figure 2E).
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Figure 2. Treatment of cells with AS4583 interrupts DNA replication, causes cell cycle arrest and
induces apoptosis. (A-B) AS4583 inhibits DNA replication in H1975 cells. H1975 cells were pulsed with
BrdU and analyzed by flow cytometry. A: representative DNA histograms from DMSO- and AS4583-treated
H1975 cells over time (three independent experiments). B: average percentage of cells in which BrdU was
incorporated. (C) AS4583 decreases the number of DNA replication forks. The replication forks were analyzed
by immunofluorescence staining for MCM2 (green) and DAPI (blue, for nuclei). Data were quantified by
counting clear, green spots using Image J software (n=5 cells per group; original magnification, 1000). (D)
G1/S phase is prolonged in AS4583-treated H1975 cells. A double thymidine block was performed, and the
data were collected at the indicated time points. (E) AS4583 induced intrinsic apoptosis pathway. H1975
cells were treated with AS4583 for 24, 48 or 72 h. The apoptotic marker protein expressions were detected
by immunoblotting.
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The decrease in MCM2 level could lead to cell death through silencing the expression of MCM2 via
lentivirus-based short hairpin RNAs
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As a significant reduction of MCM2 in AS4583 treated cells was observed in the DNA replication
fork study, we were curious about whether the decrease of MCM2 could directly interfere in the cell
survival. To test this hypothesis, we silenced the expression of MCM2 via lentivirus-based short hairpin
RNAs, and then, confirmed whether the breakdown of MCM2 accounts for AS4583-induced cell death.
The results showed that MCM2 knockdown resulted in a 33% inhibition of cell proliferation (p< 0.0001,
Figure 3A), and the efficiency of knockdown depended on the amount of short hairpin RNA (p< 0.0001,
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Figure 3B). These data are consistent with the observations reported by Liang and his colleagues .
Moreover, we also found that AS4583-induced cell death correlated positively with the expression of
MCM2 in different lung-cancer cell lines and skin fibroblast cell line (Figure 3C, 3D and Table S4). These
data implied that MCM complex might be involved in AS4583 –induced lung cancer cell death.
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Figure 3. The correlation between MCM2 level and cell death via lentivirus-based short hairpin RNAs
or AS4583 treatment. (A) Cell survival decreases after MCM2 knockdown. The cells were infected with the
indicated lentivirus in medium containing polybrene (8 µg/ml). At 24 h post-infection, the cells were
transferred to fresh medium and incubated for 48 h, then analyzed by immunoblotting and the sulforhodamine
B assay. (B)MCM2 knockdown decreases cell survival in a dose-dependent manner. The cells were infected
with the indicated lentivirus in medium containing polybrene (8 μg/ml). At 24 h post-infection, the cells were
incubated in fresh medium lacking lentivirus and polybrene for 48 h and analyzed with the MTS assay over
an additional 24 to 120 h. (C) Cellular levels of MCM2 were detected in different lung-cancer cells as assessed
with immunoblotting. (D) Quantification of MCM2 levels and IC50 values for each cell line were used to
calculate the correlation between the two cofactors in these different lung-cancer cell lines (IC50 versus MCM2,
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R = 0.5282).
High expression of the MCM complex is associated with poor clinical outcomes of NSCLC patients
To further validate whether the MCM complex is a potential therapeutic target for NSCLC, we
examined the cellular levels of MCM components by analyzing the gene expression data obtained from
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0
226 pathological stage I and II lung adenocarcinomas in the public database GSE31210 . The results
indicated that high expression of MCM2, MCM4, MCM5, or MCM6 is associated with poor overall
survival compared with the low-expression group (Figure 4). Thus, the components of the MCM complex
may serve as markers for high-risk patients and as potential therapeutic targets for the treatment of early
stage lung adenocarcinoma.
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Figure 4. Clinical significance and prognostic value of MCM in NSCLC. Kaplan-Meier analysis of MCM
expression and overall survival for 226 human lung adenocarcinoma samples, pathological stage I or II,
published as GSE31210. The p-values were obtained with a two-sided log-rank test.
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AS4583 promotes ubiquitination of MCM2–7 and their degradation in lung-cancer cells
The hetero-hexametric MCM2-7 protein complex has been identified as an important component
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for the precisely coordinated replication of the entire genome each time a cell divides . We next
determined whether AS4583 treatment could modulate the expression of MCM protein components. As
expected, the cellular levels of MCM2, MCM6, and MCM7 decreased significantly (Figure 5A) in an
AS4583 dose–dependent manner (Figure 5B) at 24 h after treatment, whereas the levels of MCM3, MCM4
and MCM5 decreased after 72 h (Figure 5A). This decrease in MCM expression appeared to be a
consequence at the protein level because the levels of the corresponding mRNAs did not change over the
course of AS4583 treatment (Supplementary Figure 2). In support of this idea, the observed decrease in
MCM2, MCM6, and MCM7 was abrogated after treatment with the proteasome inhibitor MG132 (Figure
5
C). Indeed, the MCM2 levels in H1975 cells were almost completely restored to the control level by
MG132, whereas partial restoration was observed for MCM6 and MCM7. Further experiments confirmed
that ubiquitination of MCM2 increased in the presence of AS4583 (Figure 5D).
To explore whether the AS4583-induced MCM protein degradation was due to interfere with the
DNA binding ability of MCM2, H1975 cells were synchronized using a double thymidine block followed
by release and subsequent treatment with DMSO (control) or AS4583 for 9 h. Then, the chromatin-bound
MCM2 was detected by immunoblotting. As shown in Figure 5E, temporal binding of MCM2 to
chromatin was observed in DMSO-treated cells. The bound MCM2 level decreased at 4.5 h after release
from the block and later returned to the pre-block level. In the AS4583-treated group, the dynamic binding
was not observed; instead, the level of MCM2 decreased significantly during the course of treatment.
Moreover, we also observed that AS4583 induced-MCM2 degradation could be restored upon treatment
of cells with the cullin inhibitor MLN4924 (Figure 5F), which is consistent with the fact that
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MCM2/3/5/6/7 have been identified as substrates of cullin-RING ligases . Collectively, all these suggest
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that MCM2 might be the therapeutic target of AS4583 treatment.
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Figure 5. AS4583 induces proteasome-mediated degradation of MCM2 after loss chromatin bound
ability. (A) Increased degradation of MCM2/6/7 upon treatment of H1975 cells with AS4583. AS4583-treated
cells were harvested at 24, 48, and 72 h, and the levels of proteins of the MCM complex were analyzed with
immunoblotting. (B) Dose-dependent degradation of MCM2/6/7 in different lung cancer cell lines. All cells
were treated with AS4583 for 24 h and then harvested. The levels of MCM2/6/7 were examined with
immunoblotting. (C) The proteasome inhibitor MG132 reverses the effects of AS4583 on the cellular levels
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of MCM2/6/7. H1975 cells were treated with AS4583 for 48 h, then incubated in the absence or presence of
MG132 (10 μM) for an additional 12 h, and then harvested. The levels of MCM2/6/7 were examined with
immunoblotting. (D) AS4583 induces ubiquitination of MCM2. The cells were transiently transfected with
pcDNA3-Myc-Ub and then incubated in the absence or presence of AS4583 (140 nM) and MG132 (10 μM).
After 12 h, the cells were harvested, and MCM2 was precipitated with an MCM2-specific antibody.
Ubiquitination of MCM2 was detected with immunoblotting. IgG served as the antibody negative control for
the immunoprecipitation (IP) experiment. (E) Treatment with AS4583 decreases the level of the chromatin-
bound form of MCM2. A double thymidine block was performed, and the data were collected at the indicated
time points. (F)MCM2 protein levels were recovered upon treatment of H1975 cells with MLN4924. Cells
were treated with DMSO or AS4583 for 24 h, and then co-treated with different dose of MLN4924. After 6 h,
the cells were harvested for detection of MCM2.
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AS4583 binds the N-terminal portion of MCM2
Our results thus far raised an important question: does AS4583 bind directly to MCM2? To address
this issue, we incubated H1975 lysates with AS4583-conjugated magnetic beads (Figure 6A) for 24 h at
4
°C, and the results showed that more MCM2 was pulled down by the mag-beads-AS4583 compared
with the mag-beads-control (Figure 6B). In addition, we also performed an in silico modeling and
simulation to provide structural insights into the binding of AS4583 to MCM2 (Supplementary Results
and Supplementary Figure 3-5). We modelled the MCM2 structure based on the crystal structure of an
archaeal MCM from Sulfolobus solfataricus (ssoMCM, PDB: 3F9V). Although the amino acid sequence
identity of MCM2 to ssoMCM is only 38%, the confidence estimate of the fold recognition server is 100%,
suggesting this template used for the homology modeling is highly reliable. The final structure of MCM2
was optimized by molecular dynamics procedure, and the validation results revealed that our model
structure is of good quality for further studies. The protein-ligand docking program iGEMDOCK
predicted that the binding site for AS4583 is in the N-terminal domain of MCM2. This site resides within
a cavity surrounded by domains A, B and C (Figure 6C and D). The solvent-accessible portion of the
binding site is close to the interface between MCM2 and MCM6 in the MCM hexamer (Figure 6E). The
B domain of MCM6 may rotate and insert into this site, thereby promoting the interaction with MCM2.
The predicted binding energy is –8.8 kcal/mol for AS4583 in complex with MCM2 (Figure 6F); and
residues from the three domains, including His282 and Gln257 in domain A, Gln341 in domain B, and
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Asn442 in domain C, are involved in the AS4583 interaction. In particular, Gln341 contributes
substantially to the intermolecular hydrogen-bond formation.
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To validate the in silico docking results concerning Gln341, we constructed the plasmids p3xFlag-
MCM2-WT and p3xFlag-MCM2-Q341A for overexpression in H1975 cells. The western blotting
analysis of MCM2-WT or MCM2-Q341A cells shows that Flag-tagged protein expressed. The magnetic-
bead pulldown assay revealed significantly less binding of MCM2-Q341A to mag-beads-AS4583
compared with the binding of the control, Flag-tagged MCM2-WT (Figure 6G). Next, we investigated
that if MCM2 MCM2-Q341A can serve as a resistant clone by testing IC50 of AS4583 and ubiquitination
of MCM2. The IC50 of AS4583 is 71nM for MCM2-WT cells and 150nM for MCM2-Q341A cells
(Supplementary Figure 6A). We also performed the immunoprecipitation assay to pull down Flag-tagged
MCM2-WT or -Q341A with AS4583 treatment for 24 hours and then detected the polyubiquitinated
species of MCM2 by immunoblotting. In accordance with the previous result, treatment with AS4583
significantly enhanced the ubiquitination of MCM2-WT, but reduced ubiquitination of MCM2-Q341A in
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93T cells (Supplementary Figure 6B). These data indicate that Q341A weakened the cytotoxicity and
proteasomal-dependent ubiquitination of AS4583. This result supports the critical contribution of Gln341
for binding of AS4583 to MCM2.
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IP
A
B
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O
O
H
N
N
O
OH
H
O
12
Mag-beads-Control
O
O
O
O
H
N
N
O
N
N
N
H
H
H
O
O
12
N
N
O
MCM2
Mag-beads-AS4583
C
D
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E
F
G
IP: Mag-beads-
Input
Control
AS4583
Flag-MCM2
Flag
IB
β-actin
Figure 6. The interaction between AS4583 and MCM2. (A) Structure of mag-beads-control and mag-beads-
AS4583. (B) AS4583 binds MCM2. H1975 cell lysates were incubated with magnetic beads-control or
magnetic beads-AS4583 overnight, and the bound MCM2 proteins were detected with immunoblotting. (C)
Domain organization of MCM2. N-C, N to C linker; α/β, α/β domain of the AAA+ ATPase core structure;
α/β-α, linker between subdomains in the ATPase core; α, α domain; WH, winged-helix domain. The domain
annotation is based on the structure of Sulfolobus solfataricus (ssoMCM, PDB: 3F9V). (D) Model for MCM2
complexed with AS4583. Domains and linkers have the same coloring as depicted in panel c. Helices are
depicted as cylinders and β-strands as arrows. The AS4583 structure is shown as a space-filling model (pink,
carbon; blue, nitrogen; red, oxygen). (E) Model of AS4583 in the MCM hexamer. Protomers (MCM2–7) are
colored differently and labeled. AS4583 is shown as red spheres. (F) The AS4583 binding pocket. The domains
have the same coloring as in panel c. The residues involved in AS4583 binding are shown as stick structures.
The black dashed lines represent polar contacts between amino acid residues and AS4583. (G) Mag-beads-
AS4583 pulled down less MCM2 Q341A than MCM2 wild type. Immunoprecipitation (IP) was performed by
incubating lysates of cells that overexpressed FLAG, FLAG-MCM2 wild-type, or the Q341A
mutant with mag-beads-control or -AS4583.
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Cell-based SAR study of AS4583 reveals essential molecular functionality of AS4583 that
mediates the inhibition of H1975 cell proliferation
To identify the structural components of AS4583 that are essential for its ability to inhibit
the proliferation of lung-cancer cells, a preliminary SAR study was carried out with modifications
of the X, R1 and R2 groups of AS4583 in Table 1 and the length (n) of the side chain on the furan
ring. The ability of each analog to inhibit H1975 cell proliferation was evaluated using the
1
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sulforhodamine B assay. As shown in Table 1, analogs with different amide side-chain lengths
1
(
compound 1, n=0, and compound 2, n=2) and R group sizes (compound 3-5) suppressed H1975
cell proliferation with IC50 of <1.0 μM. The IC50 value of the pyrrole analog 6 was 100-fold higher
than that of AS4583, and benzofuran 11 had little or no effect up to 10 μM. These results suggested
that both the furan oxygen and benzoquinone moiety are essential for AS4583 activity.
Replacement of the imidazole group at the end of the amide chain of AS4583 with morpholine
(compound 8) did not affect the IC50 value (80 nM), and compound 10, with a benzyl tail end, was
unable to suppress H1975 cell proliferation even at a dose of 10 μM. These results indicated that
the tertiary nitrogen of the amide chain is essential for the anti-proliferation activity of AS4583.
Notably, compound 9, with a pyrrolidine moiety instead of an imidazole ring, had the best activity
with IC50 24 nM, while the activity was diminished with pyrrolyl side chain (7, IC50 918 nM).
According to the SAR study (Table 1), the furan oxygen is important for AS4583 activity. This
finding is coherent with our predicted binding model (Figure 6F), because the furan oxygen forms
a hydrogen bond with His282 sidechain. The size of R1 moiety is also closely related to AS4583
activity. In our predicted binding model, the R1 methyl moiety is surrounded by Ile281, Val283
and Leu256, and therefore a much bulky moiety cannot fit into this pocket. In addition, the
replacement of R1 by a proton would reduce the van der Waals force for the AS4583 binding.
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1
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1
1
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2
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2
2
3
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5
6
Table 1. SAR study of AS4583
O
X
R1
O
N
H
R2
n
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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9
0
1
2
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8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound n R1
R2
X
O
O
IC50 (nM)
77
AS4583
1
1 Me
0 Me
280
2
2 Me
O
250
3
4
1
Et
1 iPr
Ph
O
O
288
260
5
6
1
O
826
1 Me
NH
8727
7
8
1 Me
1 Me
O
O
918
80
9
1 Me
2 Me
O
O
24
10
>10µM
O
N
N
>10 µM
N
O
H
11
Synthesis of a more potent lead compound, RJ-LC-07-48
Although compound 9 was the most potent inhibitor of H1975 cell viability in the
aforementioned analysis (IC50 24 nM), it also exhibited toxicity toward skin fibroblast cells (Hs68,
IC50 77 nM). To further improve the potency of AS4583 and reduce its toxicity, a new analog, RJ-
LC-07-48 was synthesized from 7-fluoro-1-tetralone (12). As shown in Scheme 1, 7-fluoro-1-
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tetralone was regioselectively oxidized to 2-hydroxynaphthoquinone (13) under an oxygen
atmosphere. A three-components reaction23 was then applied to transfer compound 13 to a
phosphorus zwitterion 14. A Wittig reaction between 14 and acetyl chloride provided
furanonaphthoquinone 15, which was then hydrolyzed to acid 16 and then linked with the
imidazole side chain to complete the synthesis. Fortunately, the survival assay showed that RJ-
LC-07-48 had a better activity in the inhibition of H1975 cell viability (Supplementary Figure 7A,
1
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0
IC50 17nM). In addition, treatment with RJ-LC-07-48 markedly reduced the growth of H1975
xenograft tumors in mice compared with the control group: average tumor size, 546.2 ± 95.2 mm3
3
3
for DMSO/control, 128.2 ± 50.5 mm for 1 mg/kg RJ-LC-07-48, and 109.5 ± 23.1 mm for 4
mg/kg RJ-LC-07-48 on day 24; p< 0.05, n=6 per group, Supplementary Figure 7G). All these
results suggested that RJ-LC-07-48 is a potential lead compound for treatment of lung cancer.
SCHEME 1. Stereoselective synthesis of RJ-LC-07-48
Reagents and conditions: a) O
2
atmosphere, room temperature (r.t.), 50 min, 90%; b) CHOCO
2
Et, TFA,
PBu , THF, 0–64 °C, 3.5 h, 63%; c) acetyl chloride, Et
3
3
N, THF, 0 °C to r.t., 15 min, 77%; d) 3 M
NaOH, THF: H
r.t., 6 h, 64%.
2
O (4:1), 0 °C to r.t., 5 h, 97%; e) HBTU, DMAP, 1-(3-aminopropyl) imidazole, DMF,
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DISCUSSION
Through the high-throughput cell-based screening and SAR study, we have identified a
furanonaphthoquinone-based small molecule, AS4583, which can induce the proteasome
degradation of MCM2–7 and ultimately contribute to the death of NSCLC cells. The SAR study
of AS4583 further identified the furanonaphthoquinone moiety and the tertiary nitrogen at the end
of the side chain as crucial components for the inhibition of tumor growth. Through computer-
simulated docking, we found that AS4583 could bind to MCM2 by forming a hydrogen bond with
residue Q341, and this result was supported by immunoprecipitation experiments using anti-
MCM2. Finally, a more potent compound RJ-LC-07-48 was synthesized with better antitumor
effect than AS4583 in vivo.
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We found that AS4583 contributes to the induction of ubiquitinated-MCM2, leading to
degradation of MCM2 by the proteasome. MCM2 degradation may further interfere with the
formation of the MCM complex, cause inhibition of DNA replication, and prolong the duration of
S phase in cancer cells. MCM2 degradation may ultimately promote apoptosis in vitro and inhibit
tumor growth in vivo. Since MCM2–7 must be assembled as a hexamer or tetramer before nuclear
import19, 21, the degradation of ubiquitinated MCM2 in proteasomes may interfere with the
formation of the MCM complex, perturb its import into the nucleus, and inhibit chromatin loading.
The docking analysis revealed that AS4583 does not occupy the ATP binding site in the
C-terminal ATPase domain of MCM2 but rather binds to the cavity in the N-terminal domain.
Indeed, the N-terminal portion of MCM2 has been shown to be important for the hexamer
24
formation, DNA binding, and enzyme regulation . Mutational and biochemical analyses carried
out by others have further suggested that a conformational change in the N-terminal portion
(especially the movement of domain A) is required for helicase function25, 26. The binding of
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AS4583 to MCM2 may alter the rigidity of the N-terminal portion and hinder the movement of
domain A. In addition, the bound AS4583 molecule could interfere with the interaction between
MCM6 and MCM2 and therefore perturb hexamer assembly and helicase function. Based on these
findings, we propose a paradigm for AS4583–induced apoptosis. Cells typically progress through
the cell cycle using the MCM2–7 helicases to unwind the DNA double helix for DNA replication.
In cancer cells, however, the MCM2–7 complex is highly regulated through the G0-G1 to S phase.
Once this complex is disrupted, the cancer cells will undergo apoptosis. As important components
in the process of DNA replication for malignant proliferation, MCM2–7 represent a potential class
of drug targets for lung-cancer treatment in the near future.
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This is the first report to show that a small molecule can induce MCM2 ubiquitination and
suppress the expression of MCM2. Although previous studies reported that norcantharidin induced
2
7
the cleavage of MCM2 at the micro-molar level and that MCM2 is a therapeutic target of
2
8
Trichostatin A in colon cancer cells , their mechanisms of action remain unclear and need
clarification. In addition, high expression levels of MCM complex proteins are associated with
poor survival of patients with NSCLC. In summary, our results demonstrate that the MCM
complex can serve as a potential target for lung-cancer treatment, and this work paves the way for
development of new cancer agents to tackle the problem of drug resistance.
CONCLUSION
The use of high-throughput drug screens for various lung-cancer cells and of in silico
docking prediction will provide effective means for identifying small molecules that target the
MCM complex, which is important for developing new cancer therapeutics. Our results
demonstrate that proteins of the MCM complex can serve as potential targets for lung-cancer
treatment, and our SAR analysis paves the way for design of potent lead compounds.
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EXPERIMENTAL SECTION
Synthesis and characterization of chemical compounds
Unless stated otherwise, reactions were performed in flame-dried glassware under a
positive pressure of nitrogen. Commercial grade reagents and solvents were used without further
0
1
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purification. CH
2
Cl
2
, CH CN and THF were purified by PS-MD-5 solvent purification system
3
(Innovative Technology, Inc). Magnetic bead was purchased from TAN Beads, Taiwan. The
progress of all the reactions were monitored by TLC, using TLC glass plates pre-coated with silica
®
gel 60 F254 (Merck). Flash column chromatography was performed with silica gel Geduran Si 60
(Merck). IR spectra were recorded with Thermo Nicolet iS-5 FT-IR spectrophotometer, νmax in
-1
1
13
cm . H and C NMR spectra were recorded with Bruker AV-III 400 MHz or Bruker AV-400
MHz spectrometers and chemical shifts were measured in δ (ppm) with residual solvent peaks as
1
13
internal standards (CDCl
3
, δ 7.24 ppm in H NMR, δ 77.0 ppm in C NMR; CD OD, δ 3.31 ppm
3
1
13
in H NMR, δ 49.0 ppm in C NMR). Coupling constants J, measured in Hz. HR FAB (LR FAB)
and HR EI (LR EI)-mass spectra were recorded on a JMS-700 double focusing mass spectrometer
(JEOL, Tokyo, Japan) with a resolution of 8000 (3000) (5 % valley definition) and HR (LR) ESI
®
(Electrospray)-mass spectra were recorded using dual ionization ESCi (ESI/APCi) source options,
Waters LCT premier XE (Waters Corp., Manchester, UK). Melting points were recorded on Büchi
M-565 apparatus. Purity of all compounds for biology test was found to be >98% as determined
by HPLC (Shimadzu LCMS-2020 with Unitary C18 columns).
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SCHEME 2. Synthesis of AS4583.
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Ethyl 2-methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylate (17)
A mixture of 2, 3-dichloro-1, 4-naphthoquione (500 mg, 2.20 mmol), K CO (760 mg, 5.51
2
3
mmol) and ethyl acetoacetate (0.31 mL, 2.42 mmol) in MeCN (30 mL) was stirred at reflux
temperature for 6 hr. After completion, the reaction mixture was diluted with EtOAc and filtered
®
through Celite , filtrate was evaporated in vacuo to yield the crude product. The residue was
purified by flash column chromatography (EtOAc/hexanes, 1/4 to 2/3) to give 17 as a yellow solid
o
1
(
305 mg, 48 % yield). R
CDCl ) δ 8.42 – 7.99 (m, 2H), 7.98 – 7.53 (m, 2H), 4.43 (q, J = 7.2 Hz, 2H), 2.70 (s, 3H), 1.43 (t,
J = 7.1 Hz, 3H); C NMR (100 MHz, CDCl
33.6, 131.5, 128.2, 127.4, 126.5, 113.8, 61.5, 14.2, 14.2.
f
(30 % EtOAc/hexanes) 0.52; Mp 149.3 – 159.9 C; H NMR (400 MHz,
3
1
3
3
) δ 178.7, 173.5, 164.4, 162.0, 151.3, 134.1, 133.7,
1
2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid (18)
To a stirred solution of 17 (378 mg, 1.33 mmol) in THF/H O (0.2 M, 7/3 v/v) was added LiOH
167 mg, 3.99 mmol) at room temperature. After stirred at room temperature for 3 hr, the reaction
mixture was quenched with 1N HCl (adjust to pH ~ 1, aqueous layer will show transparent),
extracted with EtOAc (15 mL x 3), dried over Na SO , and concentrated in vacuo. The residue
was purified by flash column chromatography (MeOH/CH Cl , 0/100 to 1/9) to give 18 as a yellow
2
(
2
4
2
2
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solid (324 mg, 95 % yield). R
f
(5 % MeOH/CH
2
Cl ) 0.35; IR (neat) 3425, 3022, 2975, 2920, 2712,
2
−1 1
1
744, 1673, 1581, 1413, 1217, 1192, 995, 721 cm ; H NMR (400 MHz, CDCl
3
) δ 8.25 (td, J =
7.4, 1.2 Hz, 2H), 7.84 (dtd, J = 18.8, 7.5, 1.3 Hz, 2H), 2.88 (s, 3H); C NMR (100 MHz, CDCl
δ 184.9, 172.4, 169.0, 160.9, 150.9, 135.8, 134.5, 131.8, 131.7, 128.0, 127.5, 126.0, 112.8, 14.5.
1
3
3
)
0
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N-(3-(1H-Imidazol-1-yl) propyl)-2-methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-
carboxamide (AS4583)
To a stirred solution of 18 (256.2 mg, 1.0 mmol), DMAP (12.2 mg, 0.1 mmol) and HBTU (455
mg, 1.2 mmol) in DMF (5 mL) was added 1-(3-Aminopropyl) imidazole (180 μL, 1.5 mmol) at
room temperature. The solvent was removed by vacuo after the reaction mixture was stirred at
room temperature for 1 day. Then the crude was washed by H
), dried over Na SO , and concentrated in vacuo. The residue was purified by flash column
chromatography (MeOH/CH Cl , 0/100 to 1/49) to give AS4583 as a yellow solid (240 mg, 66 %
yield). R (5 % MeOH/CH Cl ) 0.28; IR (neat) 3285, 3095, 2923, 2848, 1650, 1582, 1213, 991,
44, 715 cm ; H NMR (400 MHz, CDCl ) δ 9.79 (s, 1H), 8.21 (qd, J = 4.0, 1.7 Hz, 2H), 7.83 –
.76 (m, 2H), 7.59 (s, 1H), 7.07 (s, 1H), 7.00 (s, 1H), 4.11 (t, J = 7.0 Hz, 2H), 3.46 (dd, J = 12.5,
2
O, extracted with CH
2
Cl
2
(10 mL x
3
2
4
2
2
f
2
2
−
1 1
8
7
6
1
1
3
1
3
.2 Hz, 2H), 2.87 (s, 3H), 2.16 (quint, J = 6.8 Hz, 2H); C NMR (100 MHz, CDCl
3
) δ 183.3,
72.9, 166.5, 161.5, 151.1, 137.2, 134.8, 134.2, 132.7, 131.4, 129.3, 127.8, 126.9, 125.9, 118.9,
+
+
15.1, 44.5, 36.2, 30.9, 14.9; HRMS (ESI , TOF) calcd for C20
H
18
N
3
O [M + H] 364.1297, found
4
364.1289.
N-(2-(1H-imidazol-1-yl)ethyl)-2-methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b]furan-3-
carboxamide (1)
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To a stirred solution of 18 (0.17 mmol), DMAP (21 mg, 0.17 mmol) and HBTU (97 mg,
0
.26 mmol) in DMF (0.85 mL) was added 2-Imidazol-1yl-ethylamine (47 mg, 0.43 mmol) at room
temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room
temperature for 1 day. Then the crude was washed by H O, extracted with CH Cl (10 mL x 3),
dried over Na SO , and concentrated in vacuo. The residue was purified by flash column
chromatography (MeOH/CH
Rf (5 % MeOH/CH Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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3
3
3
3
3
4
4
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4
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5
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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
2
2
2
2
4
2
Cl
2
, 0/100 to 1/49) to give 1 as a yellow solid (42 mg, 70 % yield).
1
2
2
) 0.28; H NMR (400 MHz, CD
3
OD) δ 8.27 – 8.14 (m, 3H), 7.92 – 7.81
(
m, 2H), 7.43 (s, 1H), 7.22 (s, 1H), 4.41 (t, J = 6.0 Hz, 1H), 3.86 (t, J = 6.0 Hz, 1H), 2.77 (s, 3H);
13C NMR (100 MHz, CD
OD) δ 184.0, 174.2, 167.1, 163.7, 152.6, 138.8, 135.9, 135.4, 134.2,
3
+
132.9, 129.3, 128.6, 127.5, 127.2, 120.8, 115.8, 47.2, 41.3 14.7; HRMS (ESI , TOF) calcd for
+
C
19
H
16
N
3
O
4
[M + H] 350.1141, found 350.1135. Sample for biology test was purified by
preparative HPLC with HPLC purity >99%.
N-(4-(1H-imidazol-1-yl)butyl)-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-
carboxamide (2)
To a stirred solution of 18 (95 mg, 0.37 mmol), DMAP (45 mg, 0.37 mmol) and HBTU
(
154 mg, 0.41 mmol) in DMF (1.9 mL) was added 4-(1H-imidazol-1-yl) butan-1-amine (77 mg,
.56 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture
was stirred at room temperature for 18 hr. Then the crude was washed by H O, extracted with
CH Cl (10 mL x 3), dried over Na SO , and concentrated in vacuo. The residue was purified by
flash column chromatography (MeOH/CH
0 % yield). Rf (5 % MeOH/CH Cl
0
2
2
2
2
4
2
Cl
2
, 0/100 to 1/49) to give 2 as a yellow solid (97 mg,
1
7
2
2
) 0.25; mp 158.6 – 164.1 °C; H NMR (400 MHz, CDCl
3
) δ
9.70 (s, 1H), 8.18 (d, J = 7.5 Hz, 2H), 7.83 – 7.75 (m, 2H), 7.49 (s, 1H), 7.03 (s, 1H), 6.93 (s, 1H),
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2
2
2
2
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2
.01 (t, J = 7.1 Hz, 2H), 3.47 (dd, J = 12.6, 6.4 Hz, 2H), 2.86 (s, 3H), 1.94 (dt, J = 15.1, 7.4 Hz,
1
3
H), 1.69 (dt, J = 14.2, 6.9 Hz, 2H); C NMR (100 MHz, CDCl ) δ 183.1, 172.8, 166.4, 161.3,
3
151.0, 137.0, 134.7, 134.1, 132.7, 131.4, 129.3, 127.6, 126.7, 125.9, 118.7, 115.2, 46.6, 38.5, 28.5,
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
+
+
2
6.4, 14.9; HRMS (ESI , TOF) calcd for C21
H N
20 3
O [M + H] 378.1454, found 378.1448. Sample
4
for biology test was purified by preparative HPLC with HPLC purity >99%.
N-(3-(1H-imidazol-1-yl)propyl)-2-ethyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-
carboxamide (3)
To a stirred solution of 2-ethyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxylic
acid (92 mg, 0.34 mmol), DMAP (42 mg, 0.34 mmol) and HBTU (142 mg, 0.37 mmol) in DMF
(
1.7 mL) was added 1-(3-Aminopropyl)imidazole (49 μL, 0.41 mmol) at room temperature. The
solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 1
day. Then the crude was washed by H O, extracted with CH Cl (10 mL x 3), dried over Na SO
and concentrated in vacuo. The residue was purified by flash column chromatography
2
2
2
2
4
,
(MeOH/CH
2
Cl
2
, 0/100 to 1/19) to give 3 as a yellow solid (116 mg, 90 % yield). Rf (5 %
1
MeOH/CH
2
Cl
2
) 0.31; H NMR (400 MHz, CDCl
3
) δ 9.82 (s, 1H), 8.26 – 8.18 (m, 2H), 7.84 – 7.74
(m, 2H), 7.66 (s, 1H), 7.08 (s, 1H), 7.02 (s, 1H), 4.12 (t, J = 7.0 Hz, 2H), 3.46 (dd, J = 12.5, 6.2
13
Hz, 2H), 3.35 (q, J = 7.5 Hz, 2H), 2.24 – 2.10 (m, 2H), 1.36 (t, J = 7.5 Hz, 3H); C NMR (100
MHz, CDCl
3
) δ 183.5, 173.0, 171.2, 161.5, 151.3, 137.3, 134.8, 134.2, 132.8, 131.6, 129.5, 127.8,
+
126.9, 118.9, 114.4, 44.6, 36.3, 31.0, 22.1, 12.0; HRMS (MALDI , TOF) calcd for C21
H
20
N O
3 4
+
[M + H] 378.1448, found 378.1457. Sample for biology test was purified by preparative HPLC
with HPLC purity >99%.
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N-(3-(1H-imidazol-1-yl)propyl)-2-isopropyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-
carboxamide (4)
To a stirred solution of 2-isopropyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-
carboxylic acid (41.8 mg, 0.15 mmol), DMAP (18 mg, 0.15 mmol) and HBTU (61 mg, 0.16 mmol)
in DMF (0.74 mL) was added 1-(3-Aminopropyl)imidazole (21 μL, 0.18 mmol) at room
temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room
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
temperature for 1 day. Then the crude was washed by H
dried over Na SO , and concentrated in vacuo. The residue was purified by flash column
chromatography (MeOH/CH
Rf (5 % MeOH/CH Cl
2
O, extracted with CH
2
Cl
2
(10 mL x 3),
2
4
2
Cl
2
, 1/199 to 1/19) to give 4 as a yellow solid (52 mg, 90 % yield).
1
2
2
) 0.32; H NMR (400 MHz, CDCl
3
) δ 9.83 (s, 1H), 8.20 (td, J = 5.3, 1.8
Hz, 2H), 7.83 – 7.73 (m, 2H), 7.72 (s, 1H), 7.08 (s, 1H), 7.05 (s, 1H), 4.34 – 4.27 (m, 1H), 4.14 (t,
J = 7.0 Hz, 2H), 3.46 (dd, J = 12.4, 6.2 Hz, 2H), 2.17 (quint, J = 6.8 Hz, 2H), 1.37 (s, 3H), 1.36 (s,
1
3
3H); C NMR (100 MHz, CDCl
3
) δ 183.6, 174.1, 172.9, 161.6, 151.3, 134.8, 134.2, 132.8, 131.6,
+
1
28.7, 127.7, 126.8, 125.9, 113.5, 44.8, 36.2, 30.9, 27.7, 20.4; HRMS (ESI , TOF) calcd for
+
C
22
H
22
N
3
O [M + H] 392.1610, found 392.1605. Sample for biology test was purified by
4
preparative HPLC with HPLC purity >98%.
N-(3-(1H-imidazol-1-yl)propyl)-4,9-dioxo-2-phenyl-4,9-dihydronaphtho[2,3-b]furan-3-
carboxamide (5)
To a stirred solution of 4,9-dioxo-2-phenyl-4,9-dihydronaphtho[2,3-b]furan-3-carboxylic
acid (50.9 mg, 0.16 mmol), DMAP (19.5 mg, 0.16 mmol) and HBTU (73 mg, 0.19 mmol) in DMF
(0.8 mL) was added 1-(3-Aminopropyl)imidazole (29 μL, 0.24 mmol) at room temperature. The
solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 1
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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
day. Then the crude was washed by H
2
O, extracted with CH
2
Cl
2
(10 mL x 3), dried over Na
2
SO ,
4
and concentrated in vacuo. The residue was purified by flash column chromatography
(MeOH/CH
MeOH/CH
2
Cl
2
, 0/100 to 1/19) to give 5 as a yellow solid (38 mg, 56 % yield). Rf (5 %
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
1
2
Cl
2
) 0.32; H NMR (400 MHz, CDCl
3
) δ 9.22 (s, 1H), 8.26 – 8.22 (m, 1H), 8.12 – 8.10
(m, 1H), 7.92 – 7.89 (m, 2H), 7.84 – 7.80 (m, 2H), 7.62 (s, 1H), 7.50 – 7.44 (m, 3H), 7.06 (s, 1H),
7.01 (s, 1H), 4.14 (t, J = 7.0 Hz, 2H), 3.48 (dd, J = 12.5, 6.3 Hz, 2H), 2.18 (quint, J = 6.8 Hz, 2H);
+
+
HRMS (ESI , TOF) calcd for C25
H
20
N
3
O [M + H] 426.1454, found 426.1451. Sample for
4
biology test was purified by preparative HPLC with HPLC purity >99%.
N-(3-(1H-imidazol-1-yl)propyl)-2-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indole-3-
carboxamide (6)
To a stirred solution of 2-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indole-3-carboxylic
acid (23 mg, 0.09 mmol), DMAP (17 mg, 0.14 mmol) and HBTU (51 mg, 0.14 mmol) in DMF
(0.45 mL) was added 1-(3-Aminopropyl)imidazole (13 μL, 0.11 mmol) at 0 °C. The solvent was
removed by vacuo after the reaction mixture was stirred at room temperature for 2 days. The
residue was purified by flash column chromatography to give 6 as a yellow solid (9.1 mg, 28 %
1
yield). Rf (5 % MeOH/CH
2
Cl
2
) 0.20; H NMR (400 MHz, CDCl
3
) δ 10.33 (t, J = 5.4 Hz, 1H),
8
1
.24 – 8.10 (m, 1H), 8.10 – 7.98 (m, 1H), 7.75 – 7.63 (m, 2H), 7.60 (s, 1H), 7.00 (s, 1H), 6.99 (s,
H), 4.10 (t, J = 7.1 Hz, 2H), 3.47 (q, J = 6.1 Hz, 2H), 2.67 (s, 3H), 2.17 – 2.05 (m, 2H); HRMS
+
+
(ESI , TOF) calcd for C20
H
19
N
4
3
O [M + H] 363.1457, found 363.1454. Sample for biology test
was purified by preparative HPLC with HPLC purity >99%.
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Page 29 of 67
Journal of Medicinal Chemistry
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N-(3-(1H-pyrrol-1-yl)propyl)-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-
carboxamide (7)
To a stirred solution of 18 (100 mg, 0.39 mmol), DMAP (47 mg, 0.39 mmol) and HBTU
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
(
163 mg, 0.43 mmol) in DMF (1.95 mL) was added 3-(1H-pyrrol-1-yl)propan-1-amine (58 mg,
.47 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture
was stirred at room temperature for 1 day. Then the crude was washed by H O, extracted with
CH Cl (10 mL x 3), dried over Na SO , and concentrated in vacuo. The residue was purified by
flash column chromatography (MeOH/CH Cl , 0/100 to 1/49) to give 7 as a yellow solid (67 mg,
7 % yield). Rf (CH Cl ) 0.07; Mp 143.5 – 150.7 C; H NMR (400 MHz, CDCl
0
2
2
2
2
4
2
2
o
1
4
2
2
3
) δ 9.73 (s, 1H),
8.26 – 8.13 (m, 2H), 7.84 – 7.71 (m, 2H), 6.74 (t, J = 2.1 Hz, 2H), 6.14 (t, J = 2.1 Hz, 2H), 4.05 (t,
13
J = 6.9 Hz, 2H), 3.42 (dd, J = 12.3, 6.6 Hz, 2H), 2.86 (s, 3H), 2.14 (quint, J = 6.8 Hz, 2H); C
NMR (100 MHz, CDCl
3
) δ 183.1, 172.9, 166.5, 161.3, 151.0, 134.7, 134.1, 132.8, 131.5, 127.7,
+
126.8, 126.0, 120.6, 115.3, 108.2, 47.0, 36.6, 31.2, 14.9; HRMS (MALDI , TOF) calcd for
+
C
21
H
19
N
2
O
4
[M + H] 363.1339, found 363.1325. Sample for biology test was purified by
preparative HPLC with HPLC purity >99%.
2
-Methyl-N-(3-morpholinopropyl)-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-
carboxamide (8)
To a stirred solution of 18 (23.8 mg, 0.093 mmol), DMAP (17 mg, 0.14 mmol) and HBTU
(54 mg, 0.14 mmol) in DMF (0.47 mL) was added 3-morpholinopropylamine (20.4 μL, 0.14 mmol)
at 0 °C. The solvent was removed by vacuo after the reaction mixture was stirred at room
temperature for 2 days. The residue was purified by flash column chromatography (MeOH/CH
2
Cl ,
2
1
0/100 to 1/9) to give 8 as a yellow solid. H NMR (400 MHz, CDCl
3
) δ 9.69 (s, 1H), 8.26 – 8.14
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