Discovery, Synthesis, and Evaluation of Oxynitidine Derivatives as Dual Inhibitors of DNA Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1), and Potential Antitumor Agents

Article abstract of DOI:10.1021/acs.jmedchem.8b00639

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme repairing DNA lesions resulting from stalled topoisomerase IB (TOP1)-DNA covalent complex. Inhibiting TDP1 in conjunction with TOP1 inhibitors can boost the action of the latter. Herein, we report the discovery of the natural product oxynitidine scaffold as a novel chemotype for the development of TOP1 and TDP1 inhibitors. Three kinds of analogues, benzophenanthridinone, dihydrobenzophenanthridine, and benzophenanthridine derivatives, were synthesized and evaluated for both TOP1 and TDP1 inhibition and cytotoxicity. Analogue 19a showed high TOP1 inhibition (+++) and induced the formation of cellular TOP1cc and DNA damage, resulting in cancer cells apoptosis at nanomolar concentration range. In vivo studies indicated that 19a exhibits antitumor efficiency in HCT116 xenograft model. 41a exhibited additional TDP1 inhibition with IC₅₀ value of 7 μM and synergistic effect with camptothecin in MCF-7 cells. This work will facilitate future efforts for the discovery of natural product-based TOP1 and TDP1 inhibitors.

Full text of DOI:10.1021/acs.jmedchem.8b00639

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Article
Discovery, synthesis and evaluation of oxynitidine derivatives
as dual inhibitors of DNA topoisomerase IB (TOP1) and tyrosyl-
DNA phosphodiesterase 1 (TDP1), and potential antitumor agents
Xiaoru Zhang, Hao-Wen Wang, Wen-Lin Tang, Yu Zhang, Hui Yang, De-Xuan Hu, Azhar Ravji,
Christophe Marchand, Evgeny Kiselev, Kwabena Ofori-Atta, Keli Agama, Yves Pommier, and Lin-Kun An
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00639 • Publication Date (Web): 18 Oct 2018
Downloaded from http://pubs.acs.org on October 21, 2018
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Discovery, synthesis and evaluation of oxynitidine
derivatives as dual inhibitors of DNA topoisomerase
IB (TOP1) and tyrosylꢀDNA phosphodiesterase 1
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(TDP1), and potential antitumor agents
†
,‡
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XiaoꢀRu Zhang, HaoꢀWen Wang, WenꢀLin Tang, Yu Zhang, Hui Yang, DeꢀXuan Hu,
‖
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Azhar Ravji, Christophe Marchand, Evgeny Kiselev, Kwabena OforiꢀAtta, Keli Agama, Yves
*,ǁ
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Pommier, LinꢀKun An
†
School of Pharmaceutical Sciences, Sun Yatꢀsen University, Guangzhou 510006, China
Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for
ǁ
Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
0892, United States
2
KEYWORDS: Topoisomerase, TyrosylꢀDNA phosphodiesterase, Inhibitor, DNA damage,
Antitumor, Benzophenanthridinone, Dihydrobenzophenanthridine, Benzophenanthridine.
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ABSTRACT. TyrosylꢀDNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme
repairing DNA lesions resulting from stalled topoisomerase IB (TOP1)ꢀDNA covalent complex.
Inhibiting TDP1 in conjunction with TOP1 inhibitors can boost the action of the latter. Herein,
we report the discovery of the natural product oxynitidine scaffold as a novel chemotype for the
development of TOP1 and TDP1 inhibitors. Three kinds of analogues, benzophenanthridinone,
dihydrobenzophenanthridine and benzophenanthridine derivatives were synthesized and
evaluated for both TOP1 and TDP1 inhibition, and cytotoxicity. Analogue 19a showed high
TOP1 inhibition (+++) and induced the formation of cellular TOP1cc and DNA damage resulting
in cancer cells apoptosis at nanomolar concentration range. In vivo studies indicated that 19a
exhibits antitumor efficiency in HCT116 xenograft model. 41a exhibited additional TDP1
inhibition with IC value of 7 ꢀM and synergistic effect with camptothecin in MCFꢀ7 cells. This
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work will facilitate future efforts for the discovery of natural productꢀbased TOP1 and TDP1
inhibitors.
INTRODUCTION
Topoisomerase IB (TOP1) is an essential nuclear enzyme controlling the topology of DNA in
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many cellular metabolic processes. To perform its functions, TOP1 cleaves one strand of DNA
by nucleophilic attack of its active site tyrosine on the DNA phosphodiester backbone (Figure
1
A), resulting in nicks in the target DNA as part of TOP1ꢀDNA covalent cleavage complexes
TOP1cc, Figure 1B). TOP1cc are transient intermediates under normal physiological
circumstances and reverse via 5’ꢀOH attack of the nicked strand on the TOP1ꢀDNA
(
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, 5
phosphotyrosyl group, thus restoring the intact DNA and releasing TOP1.
TOP1 inhibitors,
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such as camptothecin (1, Figure 2S, Supporting Information) bind at the interface of TOP1cc by
intercalating between the base pairs at the site of cleavage and forming key hydrogen bonds with
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TOP1. This stabilizes TOP1cc and prevents further religation of the broken DNA. When
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replication and transcription machineries encounter the trapped TOP1cc, DNA damage is
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generated, triggering cell death.
To date, four wellꢀknown camptothecin TOP1 inhibitors
(Figure 2S, Supporting Information) have been approved for clinical treatment of cancers,
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including irinotecan (2) and topotecan (3) approved by the FDA, 10ꢀhydroxycamptothecin (4,
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HCPT, in China) and belotecan (5, in South Korea).
Therefore, TOP1 is a validated target
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for the discovery of antitumor agents.
The DNA damage generated from TOP1cc can be repaired through several pathways,
including homologous recombination (BRCA1, BRCA2, CtIP, Mre11, Rad52), cell cycle
checkpoint signaling, XPF/ERCC1, Mre11, CtIP and tyrosylꢀDNA phosphodiesteraseꢀdependent
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pathways, etc.
TyrosylꢀDNA phosphodiesterase 1 (TDP1) is a member of the phospholipase
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D superfamily. It catalytically hydrolyzes the 3'ꢀphosphotyrosyl bond (Figure 1C) associated
with TOP1cc. TDP1 generates DNA breaks with 5'ꢀhydroxyl and 3'ꢀphosphate ends (Figure 1D)
for further DNA repair by the XRCCꢀ1ꢀdependent pathway, including polynucleotide kinase
phosphatase (PNKP), poly(ADPꢀribose) polymerase 1 (PARP1), DNA ligase III (Lig3) and
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DNA polymerase β (Polβ), finally resulting in resealed DNA (Figure 1E).
In this pathway,
TDP1ꢀcatalyzed hydrolysis of the 3'ꢀphosphotyrosyl bond is a key step for initiating the repair of
TOP1ꢀmediated DNA damage. In addition, TDP1 catalyzes hydrolysis of 3’ꢀblocking lesions
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generated by DNA oxidation and alkylation,
as well as 5'ꢀphosphotyrosyl bond involved in
topoisomerase IIꢀmediated DNA damage, implying a broader role of TDP1 in the maintenance of
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genomic stability.
The physiological importance of TDP1 is further emphasized by the
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discovery of a rare neurodegenerative disease called spinocerebellar ataxia with axonal
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neuropathy (SCAN1), in which TDP1 bears a H493R mutation in its active site.
Because TDP1ꢀdeficient cells are hypersensitive to TOP1 inhibitors, it has been suggested that
overexpression of TDP1 will confer resistance to TOP1ꢀmediated DNA damage, and therefore
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TDP1 inhibitors would have the ability to sensitize cancer cells to TOP1 inhibitors. Indeed,
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cells overexpressing TDP1 show resistance to camptothecin.
Conversely, TDP1 knockout
mice, human cells deficient in TDP1 and the SCAN1 mutation all show hypersensitivity to
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camptothecin,
and some TDP1 inhibitors show synergistic activity to camptothecin
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derivatives.
Together, these observations imply that TDP1 is a rational target for the
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development of novel antitumor agent.
Due to the unique physiological functions of TOP1 and TDP1, the discovery of their inhibitors
has attracted attention. As TOP1 inhibitors, camptothecin derivatives have been well investigated,
and four derivatives (2ꢀ5, Figure 2S) are clinically available, as well as several tumorꢀtargeted
derivatives currently in clinical trials. Despite their effectiveness in solid tumors, camptothecin
inhibitors suffer from wellꢀestablished limitations, including chemical instability under
physiological condition, poor solubility, bone marrow doseꢀlimiting toxicity, severe
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gastrointestinal toxicity, and drug effluxꢀmediated resistance. Because of these shortcomings,
much attention is directed toward the discovery of nonꢀcamptothecin inhibitors. To date, nonꢀ
camptothecin TOP1 inhibitors have been reported, and three chemotypes, including
indolocarbazoles, dibenzonaphthyridinone and indenoisoquinolines have been developed. The
indenoisoquinolines LMP744 (48, Figure 2S, Supporting Information), LMP400 (Indotecan) and
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LMP776 (Indimitecan) are in clinical trials.
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Compared to TOP1 inhibitors, the discovery of TDP1 inhibitors was initiated relatively
recently, and there is a limited number of chemotypes reported as TDP1 inhibitors, which leaves
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much room for development.
Among TDP1 inhibitors, sodium orthovanadate (7, Figure 2)
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was the first reported TDP1 inhibitor mimicking the DNA substrate in the transition state, but
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with inhibition only at millimolar concentration.
Recently, a few more potent inhibitors with
IC50 values at low micromolar or submicromolar concentrations have been reported, such as
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aminoglycoside
neomycin,
methylꢀ3,4ꢀdephostatin,
arylidenethioxothiazolicinone,
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thioxopyrimidinedione (CD00509), benzopentathiepine. A few derivatives of the clinicallyꢀ
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developed TOP1 inhibitors, the indenoisoquinolines have been reported to also inhibit TDP1.
The indenoisoquinoline 10 (Figure 2) showed consistent TDP1 inhibition with IC value of 1.52
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M. Recently, three chemotypes (Figure 2), 7ꢀhydroxycoumarin derivatives (11), usnic acid
enamines (8) and aminoadamantanes (9) were reported to inhibit TDP1 and synergize with
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camptothecin derivatives.
RESULTS AND DISCUSSION
Hit discovery for TOP1 inhibitor. Natural products are an important source for medicinal
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chemistry and drug development.
To find novel TOP1 inhibitors, we screened our inꢀhouse
natural product library containing more than 900 natural products using a TOP1ꢀmediated DNA
relaxation assay. Several chemotypes were found to inhibit TOP1, including meroterpenoid
derivatives and oxynitidine (6, Figure 1S, Supporting Information). The meroterpenoid
derivatives showed high TOP1 relaxing inhibition, but did not have the ability to trap and
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stabilize TOP1cc, and therefore could be classified as TOP1 catalytic inhibitors. By contrast,
TOP1ꢀmediated DNA cleavage assay indicated that 6 was able to induce the formation of
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TOP1cc in a doseꢀdependent manner (Figure 1S, Supporting Information). It is noteworthy that
the cleavage sites induced by 6 are different from that induced by camptothecin, and similar to
the indenoisoquinoline 48 (Figure 2S, Supporting Information). For example the cleavage sites
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7, 35 and 79 can be induced by 6 and 48, but not by camptothecin, implying that this novel
TOP1 inhibitor might target the genome at different sites and translate to different cellular effects
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compared with camptothecin.
It was reported that nitidine can intercalate into doubleꢀstranded DNA (dsDNA) and inhibit
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TOP1.
Because 6 is the 6ꢀoxo analogue of nitidine, a FRET melting assay using F10T (5'ꢀ
FAMꢀdTATAGCTATAHEGꢀTATAGCTATAꢀTAMRAꢀ3') as substrate was conducted to detect
the enhanced melting temperature (∆T ) and investigate the interaction of 6 with dsDNA.
m
Intercalation of small molecules into dsDNA stabilizes the DNA double helix structure, resulting
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in an increased melting temperature by about 5ꢀ8 °C. The FRET melting assay indicated that 6
showed low ∆T value (0.4 °C) compared to the intercalator 49 (Figure 2S, Supporting
m
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Information) with ∆T value of 5.8 °C (Table 1), implying that 6 seems not be a DNA
m
intercalator.
These results indicate that the oxynitidine scaffold is a novel chemotype for discovery and
development of new classes of TOP1 inhibitors.
Chemistry. To synthesize the benzophenanthridinone derivatives, the synthetic route was
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designed as shown in Scheme 1 according to the method reported by Korivi et al. The reaction
of 2ꢀbromoꢀ4,5ꢀdimethoxybenzaldehyde with 2ꢀhydroxyethylamine or 3ꢀhydroxypropanylamine
gave the imine intermediate 12a or 12b in 100% yield. To avoid oxidation during Swern
oxidation at step e (Scheme 1), the hydroxy groups of 12a and 12b were protected with
methoxymethyl (MOM) group to give intermediates 13a and 13b, which were directly used
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without purification to react with 14, prepared through Sonogashira coupling reaction from 5ꢀ
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bromobenzo[d][1,3]dioxole and 3ꢀbutynꢀ1ꢀol. Under Niꢀbased catalysis, the cyclization
reaction of 13a/b with 14 gave the intermediates 15a/b in 45ꢀ57% yield for two steps (from
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2a/b). The structures of 15a and 15b were confirmed using 1D and 2D NMR spectra and
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HRMS. Following the Swern oxidation of the hydroxyl group of 15a and 15b, cyclization
reaction under acid condition give the alcohol products 17a and 17b with simultaneous
deprotection of MOM group.
Following the chlorination of 17a/b with SOCl , the target benzophenanthridinone derivatives
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(19a/bꢀ25a/b) were obtained from the reaction of 18a/b with the corresponding amines.
The dihydrobenzophenanthridine derivatives (26a/bꢀ32a/b) were obtained from the reduction
of the corresponding benzophenanthridinone substrates (19a/b-25a/b) with LiAlH4.
The structures of all the benzophenanthridinone and dihydrobenzophenanthridine derivatives
were characterized through IR, NMR and HRMS spectra. The signals of the lactam carbonyl
ꢀ1
stretching vibration were between 1630 and 1642 cm . The structure of
dihydrobenzophenanthridine derivative 20b was also confirmed with Xꢀray single crystal
analysis (Figure 3).
The 6ꢀalkoxy substituted benzophenanthridinone derivatives (39a/bꢀ45a/b) were synthesized
as shown in Scheme 2. The isoquinolinone 34 was obtained from the nickelꢀcatalyzed cyclization
of 14 and 33, prepared from the reaction of 2ꢀbromoꢀ4,5ꢀdimethoxybenzaldehyde with 4ꢀ
methoxyphenylmethanamine. Following the Swern oxidation of 34, the key intermediate 36 was
prepared from the acidꢀcatalytic cyclization of 35, meanwhile, the pꢀmethoxybenzyl (PMB)
group was removed. As shown in Scheme 2, two synthetic pathways were designed for the target
products 39aꢀ45a (Scheme 2, steps e and f) and 39bꢀ45b (Scheme 2, steps g and h).
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Surprisingly, the reaction of 36 with 1,3ꢀdibromopropane under strong base condition (NaH)
mainly gave the product 38 with 3ꢀbromopropyl group attached to oxygen atom at 6ꢀposition in
86% yield (Scheme 2, step g), but not the product with 3ꢀbromopropyl group attached to the
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nitrogen atom of lactam similar to that reported in many publications.
The reaction of 38
with amines gave the corresponding benzophenanthridines 39bꢀ45b with 3ꢀalkylamino propoxy
substituents at 6ꢀposition.
The reaction of 36 with 1,2ꢀdichloroethane gave a mixture of intermediate 46 (Figure 4) and
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8a, which are inseparable through silica gel column chromatography. It was proposed that the
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ꢀchloroethyl group might firstly attach to the oxygen atom at 6ꢀposition to give 46. Compound
46 was not stable and would undergo a transformation according to the pathway proposed by
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Nguyen et al., in which 46 undergoes intramolecular cyclization and elimination of chlorine
atom to produce an intermediate 47 as shown in Figure 4. Nucleophilic attack of the chloride
anion to 47 gives the products 18a or 46, respectively. Therefore, the products 39aꢀ45a with 2ꢀ
alkylamino ethoxy group at 6ꢀposition were synthesized through the steps e and f of Scheme 2,
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in which 36 firstly was chlorinated with POCl to give intermediate 37, and then reaction of 37
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with 2ꢀhydroxy ethylamines under strong base condition gave the target products 39aꢀ45a.
In summary, three groups of oxynitidine derivatives, the benzophenanthridinone,
dihydrobenzophenanthridine and benzophenanthridine derivatives were synthesized and their
identity and purity assessed through HRMS, 1D and 2D NMR spectra, and HPLC method.
TOP1 inhibition. All synthesized compounds were evaluated for TOP1 inhibition through our
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TOP1ꢀmediated cleavage assay using a 3'ꢀ[ P]ꢀlabeled doubleꢀstranded DNA fragment as
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substrate. The compounds were tested at 100, 10, 1 and 0.1 ꢀM concentrations using
camptothecin 1 and indenoisoquinoline 48 as positive controls, and semiꢀquantitatively scored on
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the basis of visual inspection of the number and intensities of the DNA cleavage bands relative to
the TOP1 inhibition of 1 at 1 ꢀM concentration: 0, no inhibitory activity; +, between 20% and
50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% activity; ++++,
equal activity to 1 (Table 1). For the benzophenanthridinone derivatives 19a/bꢀ25a/b, both
alkylaminoethyl and alkylaminopropyl substituents attached to the nitrogen atom of lactam
increased the TOP1 inhibition except 23b with 3ꢀmorpholinopropyl group attached at 5ꢀposition,
which showed equal activity to that of parent molecule 6 (+/0, very minor activity less than 10%
TOP1 inhibition of 1). Bigger substituents at the 5ꢀposition decreased TOP1 inhibitory activity.
Most benzophenanthridinone derivatives showed limited TOP1 inhibitory activity of +, except
compounds 19a (+++) and 21a (++), with high and moderate TOP1 inhibitory activity,
respectively. The compounds 17a and 17b with a hydroxy terminus showed slightly increased
TOP1 inhibition of +. Compound 19a was the most potent among the three synthesized
chemotypes with TOP1 inhibition of +++. Figure 5A demonstrates that 19a exhibits cleavage
sites similar to 48 but not to 1. It was reported that the dibenzonaphthyridinone derivative, ARCꢀ
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from 1 and 48, implying that, although 19a and ARCꢀ111 are structurally similar, they trap
TOP1cc at different DNA sequences.
Most of the dihydrobenzophenanthridine derivatives, such as 26a/bꢀ31a/b and 32a showed
equal or slightly increased TOP1 inhibitory activity (+/0 or +) compared to the parent 6. Among
this series of derivatives, the most potent compound 32b with 3ꢀimidazolylpropyl group at the 5ꢀ
position, showed moderate TOP1cc inhibitory activity (scored ++). Surprisingly, most of
benzophenanthridine derivatives did not exhibit TOP1 inhibitory activity except compounds 40a
(+) and 43a (+/0) showing weak activity.
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Molecular modeling was performed to rationalize the molecular binding mode of the
benzophenanthridinone derivatives within the TOP1ꢀDNA complex. A hypothetical binding
model was built using inꢀsilico docking from the Xꢀray crystal of the TOP1ꢀDNAꢀligand ternary
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complex (PDB ID: 1K4T). Compounds were energyꢀminimized and docked into the binding
model. As shown in Figure 5D, the benzophenanthridinone scaffold of 19a intercalates in the
DNA break made by TOP1 and readily stacks with the +1 and ꢀ1 base pairs flanking the DNA
cleavage site. The Aꢀ and Bꢀring of 19a stack with the bases of nonꢀcleaved strand (C and A),
while the Cꢀ and Dꢀring stack with the scissile strand bases (G and T). In addition, the
dimethylaminoethyl substituent of 19a extends into the minor groove of the DNA. Also, a
hydrogen bond (2.9 Å) was observed between the lactam oxygen atom and Arg364, implying the
importance of hydrogen bond acceptor, which is consistent with the cytotoxicity of 19a against
the prostate cancer cells DU145ꢀRC0.1, resistant cells with a R364H mutation in TOP1. DU145ꢀ
RC0.1 cells showed high resistance to 19a (Table 4). The interaction between the two oxygen
atoms in the dioxole ring and Asn722 (3.7 Å) or Thr718 (3.6 Å), respectively, might also
contribute to the trapping of TOP1cc.
TDP1 inhibition. Because of the unique function of TDP1 to repair TOP1ꢀmediated DNA
damage, and the recently reported inhibition of TDP1 by the indenoisoquinoline TOP1
54ꢀ56
inhibitors,
TDP1 inhibition by the synthesized compounds were also screened through a
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fluorescence assay.
A quenched fluorescent singleꢀstranded oligonucleotide (5'ꢀFAMꢀ
AGGATCTAAAAGACTTꢀBHQꢀ3') was used as substrate. The compounds were tested at 100
M concentration. Twelve of them, 19a, 21b, 22b, 39a/b, 40a/b, 41a/b, 42b and 44a/b exhibited
ꢀ
TDP1 inhibition with the percentage inhibition ranging from 12% to 98% (Table 1). The most
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potent TOP1 inhibitor 19a (+++) showed low TDP1 inhibition (12% at 100 ꢀM). None of the
dihydrobenzophenanthridine derivatives exhibited TDP1 inhibition up to 100 ꢀM concentration.
Seven compounds 39a/b, 40a/b, 41a/b and 42b with notable TDP1 inhibition (> 50%) were
further tested to determine their IC50 values (Table 2), expressed as the concentration of
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compound that inhibits 50% of TDP1 activity. Compound 41a was the most potent with an IC50
value of 7.0 ± 1.4 ꢀM in a doseꢀdependent manner (Figure 6A).
To further establish their TDP1 inhibition, these seven active compounds 39a/b, 40a/b, 41a/b
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and 42b were further tested by a gelꢀbased assay, in which a singleꢀstranded 5'ꢀ Pꢀlabeled
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oligonucleotide N14Y containing 3'ꢀphosphotyrosyl group was used as substrate. Their IC50
values are summarized in Table 2. Compound 41a was again found the most potent TDP1
inhibitor (IC = 8.2 ꢀM). Representative TDP1 inhibition gels are shown in Figure 6C. The
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indenoisoquinoline 10 was used as the positive control. Five compounds 39a/b, 40b and 41a/b
showed full inhibition at the highest concentration (111 ꢀM) with progressive doseꢀdependency.
To obtain a molecular view of the drugꢀinduced TDP1 inhibition, a hypothetical binding model
was built by using inꢀsilico docking. The TDP1 catalytic region containing two lysine residues
(
Lys265 and Lys495) and two histidine residues (His263 and His493) was constructed as the
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binding site from the TDP1 Xꢀray crystal structure (PDB ID: 1RFF). The inhibitors were
docked into the binding site. The hypothetical structure pose of the topꢀranked 41a is shown in
Figure 6B. The polycyclic core of 41a lies along the DNA binding groove in the proximity of the
catalytically important Phe259 residue indicating a potential πꢀπ stacking stabilizing drug TDP1
complex (Figure 6B), while the pyrrolidinyl ethyl side chain is directed to the catalytic site of
TDP1 through a narrow channel (Figure 3S, Supporting Information). Two hypothetical
hydrogen bonds form between the nitrogen atom in the pyrrolidinyl group and the catalytic
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residues His493 (3.3 Å) and Asn283 (3.1 Å), implying the importance of the side chain to TDP1
inhibition. In addition, the 1,3ꢀdioxole ring is close to Glu538, and hydrogen bond forms
between the oxygen atom and the amide group of Glu358 with the distance of 3.7 Å, which
might also contribute to TDP1 inhibition.
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Interaction with DNA. To evaluate the interaction of the synthesized compounds with DNA,
FRET melting assays were performed using F10T as a selfꢀannealing substrate. The ∆T values
m
of the compounds are summarized in Table 1. The FRET melting assay indicated that none of the
synthesized compounds significantly increase the melting temperature of F10T at 2 ꢀM
concentration, and that 27b has the highest ∆T value of 1.9 °C. Potent TOP1 inhibitor 19a
m
demonstrated a low ∆T value of 0.5 °C, implying that the synthesized compounds could not
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stabilize the DNA double helix and increase the melting temperature by acting as classical DNA
intercalators.
The binding of classical DNA intercalators between the base pair of circular DNA also
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produce TOP1ꢀmediated unwinding effect, thereby limiting TOP1ꢀmediated DNA relaxation.
To further evaluate 19a, TOP1ꢀmediated unwinding assays in the presence of excess TOP1 were
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performed. As shown in Figure 5B, the DNA intercalator ethidium bromide (EB) showed a
distributed DNA ladder along with the increased tested concentration using supercoiled pBR322
DNA as substrate. On the contrary, 19a produced no TOP1ꢀmediated unwinding effect up to 9
ꢀM concentration. The assay using relaxed pBR322 DNA as substrate was also performed
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(
Figure 5C) and confirmed that 19a had no unwinding effect.
Thus, we conclude from the FRET and TOP1ꢀmediated unwinding assays, the specificity for
TOP1 of 19a and its lack of detectable binding to DNA outside of TOP1cc.
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Cytotoxicity assays. The cytotoxicity of the compounds was assessed by MTT assay against
five human tumor cell lines, including colon cancer (HCT116), leukemia (CCRFꢀCEM), prostate
cancer (DUꢀ145), nonꢀsmall cell lung cancer (A549) and hepatocarcinoma (Huh7) cell lines. The
cells were incubated with the compounds for 72 hours in a fiveꢀdose assay with concentrations
ranging from 0.01 to 100 ꢀM. After drug treatments, MTT solution was added to measure the
percentage of cellular growth. The GI values, defined as the concentrations of the compounds
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that result in 50% cell growth inhibition, are plotted and summarized in Table 1.
Compared to the parent 6, most benzophenanthridinone derivatives 17a/b and 19a/bꢀ25a/b
showed increased cytotoxicity against the five human cancer cell lines, which is consistent with
their TOP1 inhibition. Compound 19a with the highest TOP1 potency (+++) also showed the
highest cytotoxicity against these five cancer cell lines with GI50 values at nanomolar range
(
0.076 ꢀM for HCT116, 0.029 ꢀM for CCRFꢀCEM, 0.018 ꢀM for DU145, 0.79 ꢀM for A549
and 0.12 ꢀM for Huh7). Although compounds 20a and 22a with alkylaminoethyl substituents at
ꢀposition showed weak TOP1 inhibition of +, they showed significant cytotoxicity against
5
HCT116 (0.21 ꢀM for 20a, 0.16 ꢀM for 22a), CCRFꢀCEM (0.18 ꢀM for 20a, 0.32 ꢀM for 22a)
and DU145 (0.054 ꢀM for 20a, 0.96 ꢀM for 22a) cell lines. Compound 21a with TOP1
inhibition of ++ showed cytotoxicity against the CCRFꢀCEM (0.62 µM) and DU145 (0.19 µM)
cell lines with GI values at submicromolar range. Compared to the compounds with hydroxyl
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terminus (17a/b) on the side chain at 5ꢀposition, the compounds with alkylamino termini,
including dimethylamino (19a/b), diethylamino (20a/b), pyrrolidinyl (21a/b) and piperidinyl
(22a/b) groups, showed increased cytotoxicity against HCT116 cells, while the morpholinyl
(23a/b) and 4ꢀmethylpiperazinyl (24a/b) termini led to decreased cytotoxicity. Compounds
2
3a/b and 24b showed low cytotoxicity against A549 and Huh7 cell lines with GI values more
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than 100 µM. The compounds with shorter side chain at 5ꢀposition showed more cytotoxicity
than that with corresponding longer side chain against HCT116 cells, for example 17a vs. 17b
and 19-23a vs. 19-23b, exceptions are the compounds with 4ꢀmethylpiperazinyl (24a/b) and
imidazolyl (25a/b) termini.
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Among the dihydrobenzophenanthridine derivatives (26a/bꢀ32a/b), 26b with
diethylaminopropyl substituent at 5ꢀposition exhibited the highest cytotoxicity against HCT116
(
GI = 0.27 µM), CCRFꢀCEM (GI = 0.12 µM) and DU145 (GI = 0.081 µM) cell lines. The
50 50 50
other five dihydrobenzophenanthridine derivatives 26a, 28b, 29b and 30a/b showed high
cytotoxicity against CCRFꢀCEM cells with GI values at submicromolar concentration, while
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two dihydrobenzophenanthridine derivatives 28b and 29b showed high cytotoxicity against
HCT116 cells with GI values of 0.66 µM and 0.64 µM, respectively. 32b showed increased
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cytotoxicity against these five cancer cell lines compared to parent 6, which was consistent with
its TOP1 inhibition of ++. Contrary to the benzophenanthridinones, the
dihydrobenzophenanthridines with shorter side chain at 5ꢀposition showed lower cytotoxicity
against HCT116 cells than that corresponding compounds with longer side chain (for example
2
6a-30a vs. 26b-30b). Exceptions are the compounds with 4ꢀmethylpiperazinyl (31a/b) and
imidazolyl (32a/b) termini.
The benzophenanthridines (39a/bꢀ45a/b) with alkoxy substituents at the 6ꢀposition generally
showed decreased cytotoxicity compared to benzophenanthridinones (19a/bꢀ25a/b) with the
corresponding side chain at the 5ꢀposition. Although the benzophenanthridines 39a/b and 41a/b
did not show TOP1 inhibition at up to 100 ꢀM concentration, they had good TDP1 inhibitory
activity with IC values at low micromolar concentration, and showed good cytotoxicity against
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four cancer cell lines, including CCRFꢀCEM, DU145, A549 and Huh7 cells, with GI values
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between 1.29 and 8.95 ꢀM, suggesting additional cellular targets.
Compound 19a being the most potent inhibitor of TOP1 and the most cytotoxic, was selected
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and submitted to the National Cancer Institute (NCI, USA) for testing against the 60 tumor cell
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lines representing nine tissue types (NCIꢀ60).
According to the NCI established procedures,
the cells were incubated with 19a for 48 hours and stained with sulforhodamine B dye. Cell
growth inhibition (GI50 in Table 3) was calculated relative to cells without treatment. High
cytotoxicity was observed for 19a with a mean graph midpoint (MGM) for growth inhibition of
all cancer cell lines of 0.145 µM, and its GI50 values against HCT116, SR, NCIꢀH522 and
UACCꢀ62 cell lines were at nanomolar range (<100 nM).
Because the NCIꢀ60 database contains several thousand chemicals and anticancer drugs, which
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have been tested across the cancer cell line panel
(http://discover.nci.nih.gov/cellminer and
http://discover.nci.nih.gov/cellminercdb), we were able to perform a pattern comparison analysis
with 19a. The drugs with the highest correlation with 19a were all TOP1 inhibitors including
camptothecin and its derivatives as well as the indenoisoquinolines. Figure 7 shows the high
correlation between the cytotoxicity of 19a and camptothecin and the clinical indenoisoquinoline
LMP400.
The cytotoxicity of 19a was further evaluated against a panel of isogenic camptothecinꢀ and
doxorubicinꢀresistant cell lines using MTT assay (Table 4). The HCT116ꢀsiTOP1 subline was
developed by transfection of colon cancer parental cells HCT116 with short hairpin RNA vectors
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expressing siRNA for TOP1. Compared to the parental cell HCT116, HCT116ꢀsiTOP1 subline
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showed 8.3ꢀfold resistant to 1, of which TOP1 is the only known cellular target,
5.9ꢀfold to 19a, implying that TOP1 is a major cellular target of 19a.
and about
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The camptothecinꢀresistant prostate cancer DU145ꢀRC0.1 cells have a R364H mutation in
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TOP1 relative to the wildꢀtype parental DUꢀ145. The TOP1 with R364H mutation is
catalytically active, but leads to RC0.1 cells being resistant to 1 because the R364 residue is
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close to the catalytic tyrosine and can stabilize the open form of TOP1cc.
The DU145ꢀRC0.1
cells were highly resistant to 1 (225ꢀfold) and 19a (132ꢀfold), which is consistent with the
molecular modeling (Figure 5D) showing hydrogen bonding between R364 and 19a.
Pꢀglycoprotein (Pꢀgp) mediated drug efflux is generally responsible for classical multiple drug
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resistance. The chemotherapeutic agent doxorubicin (DOX) is a substrate of Pꢀgp, and both
breast cancer MCFꢀ7/ADR (77.8ꢀfold) and hepatocellular HepG2/ADR sublines (47.6ꢀfold),
which overexpress Pꢀgp, are highly resistant to doxorubicin (77.8ꢀ and 47.6ꢀfold, respectively;
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Table 4). Compound 19a appeared to be less a Pꢀgp substrate (Table 4) with a resistance ratio
of 2.8 (MCFꢀ7/ADR:MCFꢀ7) and 10.7 (HepG/ADR:HepG2), respectively.
Synergistic effects of 41a and 41b with camptothecin. The combined effects of the TDP1
inhibitors 41a and 41b were tested with camptothecin in MCFꢀ7 human breast cancer cells using
MTT assay. As shown in Figure 8A, after 96 hours incubation at 37 °C, the cytotoxicity of
camptothecin against MCFꢀ7 cells increased in the presence of 41a. At 2.5 ꢀM concentration,
almost 60% cells were killed by camptothecin itself and over 90% cells were killed when being
coꢀincubated with 41a at 5 ꢀM concentration, which implies that a supraꢀadditive effect of 41a
with camptothecin is consistent with TDP1 inhibition in cells. Similarly, 41b showed a
synergistic effect with camptothecin in MCFꢀ7 cells (Figure 8B).
Induction of cellular TOP1cc and DNA damage by 19a. The immunocomplex of enzyme to
DNA (ICE) assay was conducted to evaluate the induction of TOP1cc by 19a in HCT116 cancer
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cells. Figure 9A shows that 19a induces cellular TOP1cc in a doseꢀdependent manner, similar to
the positive control 1.
To assess the DNA damage in cancer cells induced by 19a, γH2AX foci were assessed by
immunofluorescence microscopy in human colon cancer HCT116 cells treated with 19a. After
incubation with 19a for 3 hours, HCT116 cells were stained with γH2AX antibodies. γH2AX
foci were induced by 19a at 1 ꢀM concentration (Figure 9C), consistent with DNA damage due
to the trapping of cellular TOP1cc by 19a.
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Cancer cell apoptosis induced by 19a. Flow cytometry assays were also conducted in
HCT116 cells to assess the induction of apoptosis by 19a. After being incubated with 19a for 24
hours, apoptotic HCT116 cells were detected. Figure 9B shows that 19a could significantly
induce HCT116 cells apoptosis in a doseꢀdependent manner. Approximately 60.9% (25.11%
early apoptotic cells and 35.8% late apoptotic cells) of the treated cells were scored as apoptotic
following 24 hours incubation with 19a at 2 ꢀM.
Pharmacokinetics of 19a. In vivo pharmacokinetic (PK) study of 19a was conducted in
SpragueꢀDawley (SD) rats. The SD rats were divided into two groups (n = 3) and treated by
intravenous injection (i.v.) at 1 mg/kg dose and intragastrical administration (i.g.) at 5 mg/kg
dose, respectively. Plasma samples were collected up to 24 hours postꢀdosing and the
concentration of 19a was measured. The PK parameters are summarized in Table 5. Following
i.v. administration, the AUC0→24h was 119 ± 10 h⋅ng/mL, and T1/2 was 0.855 ± 0.011 hour. After
oral administration, the Tmax, Cmax and AUC0→24h were 1.17 ± 0.76 hour, 23.5 ± 16 ng/mL and
8
9.7 ± 36 h⋅ng/mL, respectively. The bioavailability (F) was 15.5%.
Acute toxicity of 19a in vivo. To assess the toxicity of 19a, Kunming male mice were
randomly divided into six groups (n = 4) and administered with 19a by i.p. injection at single
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doses of 300, 240, 192, 154 and 123 mg/kg, respectively. The control group was injected with
sterile water. All mice survived after seven days of administration with 19a in the groups of 192
mg/kg dose (Figure 10A), 154 mg/kg dose (data not shown) and 123 mg/kg dose (data not
shown). Four mice died within five days in the 300 mg/kg group, and one mouse died within
seven days in the 240 mg/kg dose group (Figure 10A).
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Antitumor efficiency of 19a in vivo. Because 19a showed high TOP1 inhibition and cytotoxic
activity in vitro, its antitumor efficiency in vivo was assessed in a human colon cancer HCT116
xenograft nude mice model. Male nude mice were randomly divided into three groups (n = 6)
and administered with saline or with 19a at 10 mg/kg or 5 mg/kg dose by i.p. injection daily.
Administration of 19a significantly reduced tumor volume in a doseꢀdependent manner (Figure
1
0C). Meanwhile, the mice treated with 19a at 5 mg/kg dose showed no obvious body loss
compared to the saline control group. Body weight loss was observed in the 10 mg/kg group
(Figure 10B). Tumor weight inhibitions (TWI) by 19a were 59.6% (10 mg/kg, P < 0.01) and
3
2.7% (5 mg/kg) (Figure 10D). These results demonstrate the antitumor activity of 19a.
CONCLUSIONS
Here we report that the natural product oxynitidine scaffold represents a productive chemotype
for TOP1 and TDP1 inhibitors. In this work, three subgroups of oxynitidine derivatives
(benzophenanthridinone, dihydrobenzophenanthridine and benzophenanthridine derivatives)
were synthesized and evaluated as inhibitors of TOP1 and TDP1 and for cytotoxicity against
human cancer cell lines (HCT116, CCRFꢀCEM, DUꢀ145, A549 and Huh7 cell lines). Enzymeꢀ
and cellꢀbased assays show that the benzophenanthridinone derivative 19a with
dimethylaminoethyl substituent at the 5ꢀposition is potent at trapping TOP1cc (+++) and exhibits
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antiproliferative activity in human cancer cell lines at nanomolar concentration range GI values.
5
0
Compound 19a also shows consistent antiproliferative activity in the NCIꢀ60 cell lines with
MGM value of 0.145 ꢀM. Six benzophenanthridine derivatives 396a/b, 40b, 41a/b and 42b
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exhibit TDP1 inhibition with IC50 values between 7.0 and 24 ꢀM in biochemical assays.
Consistent with TDP1 inhibition, both 41a and 41b show synergy in combination with
camptothecin in MCFꢀ7 human breast cancer cells. Cellꢀbased assays demonstrated that 19a
induces the formation of cellular TOP1cc and DNA damage in HCT116 cells, promotes
apoptosis, and is a poor substrate of Pꢀgp, a drug efflux protein responsible for multiꢀdrug
resistance. The PK, acute toxicity and antitumor efficiency of 19a suggest its potential value for
further development.
EXPERIMENTAL SECTION
General Experiments. The major chemical reagents for synthesis were purchased from Alfa
Aesar, Sigma Aldrich Co. or Aladdin Reagent Database Inc (Shanghai), and were used without
further purification unless otherwise indicated. 4ꢀ(Benzo[d][1,3]dioxolꢀ5ꢀyl)butꢀ3ꢀynꢀ1ꢀol (14)
6
6
was prepared in our lab according to the Korivi's method. Chemical reaction courses were
monitored by silica gel GF254 thin layer chromatography (TLC). Melting points were determined
in open capillary tubes on a MPA100 Optimelt Automated Melting Point System without being
corrected. Nuclear magnetic resonance spectra were recorded on a Bruker AVANCE III 400
MHz spectrometer using tetramethylsilane as an internal reference. Mass spectra were analyzed
on an Agilent 6120 (Quadrupole LCꢀMS) mass spectrometer. The highꢀresolution mass spectra
were analyzed on a SHIMADZU LCMSꢀITꢀTOF mass spectrometer. All compounds tested for
biological activities were analyzed by HPLC and their purities were more than 95%. The
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analysis condition is: detection at 220 nm, 1.0 ml/min flow rate, a linear gradient of 50% to 15%
PBS buffer (pH 3) and 50% to 85% MeOH in 35 min.
All animals were obtained from Laboratory Animal Center of Sun Yatꢀsen University. All
procedures were approved by the Animal Ethics Committee of Sun Yatꢀsen University, in
accordance with National Institute of Health and Nutrition Guidelines for the Care and Use of
Laboratory Animals.
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General procedure for synthesis of Schiff's base 12a and 12b. The reaction solution of 6ꢀ
bromoveratraldehyde (9.8 g, 40 mmol) and amine (ethanolamine or 3ꢀaminopropanol, 42 mmol)
in methanol (200 mL) was stirred at room temperature for 12 h, and then concentrated under
reduced pressure. The residue was washed with petroleum ether (2 x 10 mL) to give a white
1
solid, yield 100%. The Schiff's base intermediates were pure as shown by their H NMR spectra
and were used for next synthesis without further purification.
1
Nꢀ(2ꢀhydroxylethyl)ꢀ6ꢀbromoveratraldimine (12a). H NMR (CDCl ) δ 8.58 (s, 1H), 7.54 (s,
3
1
H), 6.99 (s, 1H), 3.95ꢀ3.89 (m, 8H), 3.79 (t, J = 4.7 Hz, 2H).
1
Nꢀ(3ꢀhydroxylprapyl)ꢀ6ꢀbromoveratraldimine (12b). H NMR (CDCl ) δ 8.53 (s, 1H), 7.45 (s,
3
1
H), 7.00 (s, 1H), 3.91ꢀ3.86 (m, 8H), 3.82 (t, J = 6.2 Hz, 2H), 1.96 (quint, J = 6.0 Hz, 2H).
General procedure for the synthesis of 13a and 13b. Under nitrogen gas, the solution of NaH
(60%, 6.69 g, 167 mmol) in THF (100 mL) was stirred and cooled to 0 °C for 15 min, and then,
added dropwise to a solution of 12a (or 12b, 33 mmol) in THF (100 mL). The reaction solution
was stirred at 0 °C for 1 h. And then, chloromethyl methyl ether (15 mL, 198 mmol) was added
dropwise and stirred for 1 h. The reaction was quenched by the addition of ethanol (20 mL) at 0
°C. The reaction solution was concentrated under reduced pressure. The residue was dissolved in
chloroform (100 mL) and was washed with saturated saline (3 × 50 mL). The organic layer was
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dried (MgSO ) overnight and concentrated under reduced pressure to give crude intermediates
4
1
3a (or 13b), which was used immediately for next synthesis without further purification.
General procedure for synthesis of 15a and 15b. Under nitrogen gas, the reaction solution of
3a (or 13b, 2.7 mmol), 14 (520 mg, 2.73 mmol), Ni(cod) (40 mg, 0.14 mmol), P(oꢀTol) (80
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mg, 0.26 mmol) in freshly distilled MeCN (10 mL) was stirred at 80 °C for 3 h. Then
K [Fe(CN) ] (6.6 g, 20 mmol), CsOH solution (50% w/w in water, 3.6 mL, 40 mmol), MeOH
3
6
(20 mL) and water (20 mL) were added. The reaction mixture was stirred vigorously at 80 °C for
1
2 h. The reaction mixture was cooled and extracted with chloroform (3 × 50 mL). The
combined organic layers were dried (MgSO ) and concentrated under reduced pressure. The
4
residue was purified by silica gel column chromatography to give the target product.
3
ꢀ(Benzo[d][1,3]dioxolꢀ5ꢀyl)ꢀ4ꢀ(2ꢀhydroxyethyl)ꢀ6,7ꢀdimethoxyꢀ2ꢀ(2ꢀ
1
(methoxymethoxy)ethyl)isoquinolinꢀ1(2H)ꢀone (15a). White solid, yield 45% (from 12a). H
NMR (CDCl ) δ 7.89 (s, 1H), 7.11 (s, 1H), 6.91 (d, J = 7.7 Hz, 1H), 6.79 (s, 1H), 6.77 (d, J = 7.9
3
Hz, 1H), 6.05 (s, 2H), 4.48 (s, 2H), 4.07ꢀ3.95 (m, 8H), 3.77ꢀ3.62 (m, 4H), 3.20 (s, 3H), 2.80ꢀ2.66
1
3
(
m, 2H). C NMR (CDCl ) δ 161.4, 153.6, 149.1, 148.0, 147.9, 140.1, 131.8, 128.3, 123.7,
3
1
19.1, 111.5, 110.5, 108.4, 108.1, 103.9, 101.5, 96.1, 64.4, 62.3, 56.1, 56.1, 55.0, 45.6, 32.1.
+
ESIꢀMS m/z: 458.1 [M + H] . The structure was further confirmed with 2D NMR spectra.
3
ꢀ(Benzo[d][1,3]dioxolꢀ5ꢀyl)ꢀ4ꢀ(2ꢀhydroxyethyl)ꢀ6,7ꢀdimethoxyꢀ2ꢀ(3ꢀ
1
(methoxymethoxy)propyl)isoquinolinꢀ1(2H)ꢀone (15b). White solid, yield 57% (from 12b). H
NMR (CDCl ) δ 7.90 (s, 1H), 7.11 (s, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.79ꢀ6.74 (m, 2H), 6.07 (s,
3
2
H), 4.48 (s, 2H), 4.02 (s, 3H), 4.01 (s, 3H), 3.71 (t, J = 6.7 Hz, 2H), 3.44 (t, J = 6.3 Hz, 2H),
13
3.27 (s, 3H), 2.82ꢀ2.67 (m, 2H). C NMR (CDCl ) δ 161.3, 153.4, 149.1, 148.0, 139.9, 131.7,
3
1
28.4, 123.4, 111.3, 110.1, 108.5, 108.1, 103.7, 101.6, 96.0, 93.0, 91.5, 65.3, 62.4, 56.2, 56.1,
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5.1, 44.0, 32.0, 29.2. ESIꢀMS m/z: 472.2 [M + H] . The structure was further confirmed with
2
D NMR spectra.
General procedure for synthesis of 16a and 16b. To a solution of (COCl) (215 ꢁL, 2.5
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mmol) in freshly distilled CH Cl (5 mL), DMSO (365 ꢁL, 5 mmol) was added dropwise at ꢀ60
2
2
°
C and the reaction solution was stirred for 15 min. A solution of 15a (or 15b, 0.5 mmol) in
freshly distilled CH Cl (3 mL) was added dropwise and stirred at ꢀ60 °C for 30 min. And then,
2
2
Et N (1.5 mL, 10 mmol) was added dropwise. The reaction solution was brought to room
3
temperature and stirred for 2 h. The reaction was quenched by the addition of water (1 mL) at 0
°C. The reaction mixture was extracted with CH2Cl2 (3 x 20 mL). The organic layer was washed
with saturated saline (3 × 30 mL) and dried (MgSO ) overnight, and then concentrated under
4
reduced pressure. The residue was purified by silica gel column chromatography to give the
target product.
2
ꢀ(3ꢀ(Benzo[d][1,3]dioxolꢀ5ꢀyl)ꢀ6,7ꢀdimethoxyꢀ2ꢀ(2ꢀ(methoxymethoxy)ethyl)ꢀ1ꢀoxoꢀ1,2ꢀ
1
dihydroisoquinolinꢀ4ꢀyl)acetaldehyde (16a). White solid, yield 85%. H NMR (CDCl ) δ 9.57 (t,
3
J = 1.8 Hz, 1H), 7.90 (s, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.81ꢀ6.73 (m, 3H), 6.08 (s, 2H), 4.50 (s,
2
3
H), 4.09 (d, J = 6.8 Hz, 2H), 4.03 (s, 3H), 3.97 (s, 3H), 3.79ꢀ3.67 (m, 2H), 3.53ꢀ3.47 (m, 2H),
1
3
.21 (s, 3H). C NMR (CDCl ) δ 199.7, 161.6, 153.8, 149.3, 148.4, 148.2, 141.8, 131.6, 128.0,
3
123.7, 119.5, 110.2, 108.7, 108.3, 106.4, 103.4, 101.7, 96.2, 64.4, 56.2, 56.1, 55.1, 45.9, 44.6.
+
ESIꢀMS m/z: 455.2 [M + H] .
2
ꢀ(3ꢀ(Benzo[d][1,3]dioxolꢀ5ꢀyl)ꢀ6,7ꢀdimethoxyꢀ2ꢀ(3ꢀ(methoxymethoxy)propyl)ꢀ1ꢀoxoꢀ1,2ꢀ
1
dihydroisoquinolinꢀ4ꢀyl)acetaldehyde (16b). White solid, yield 87%. H NMR (CDCl ) δ 9.58 (s,
3
1H), 7.91 (d, J = 2.4 Hz, 1H), 6.96ꢀ6.90 (m, 1H), 6.80ꢀ6.74 (m, 3H), 6.08 (s, 2H), 4.71ꢀ4.60 (m,
2
H), 4.50 (s, 2H), 4.02 (s, 3H), 3.96 (s, 3H), 3.52ꢀ3.44 (m, 4H), 3.29 (s, 3H), 1.88ꢀ1.83 (m, 2H).
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C NMR (CDCl ) δ 199.6, 161.4, 153.7, 149.3, 148.4, 148.3, 141.5, 131.4, 128.0, 123.3, 119.7,
3
1
09.8, 108.8, 108.4, 106.3, 103.4, 101.7, 96.1, 65.3, 56.2, 56.1, 55.1, 44.5, 44.3, 29.2. ESIꢀMS
+
m/z: 470.2 [M + H] .
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General procedure for the synthesis of 17a and 17b. The reaction solution of 16a (or 16b, 0.5
mmol) and concentrated hydrochloric acid (0.4 mL) in MeOH (10 mL) was added to a 50 mL
roundꢀbottomed flask. The flask was sealed with a rubber stopper. The reaction solution was
stirred at room temperature overnight. And then, the formed precipitate was filtered and washed
with saturated sodium bicarbonate and water consecutively. The crude solid was dried and
purified by silica gel column chromatography to give the target product.
12ꢀ(2ꢀHydroxyethyl)ꢀ2,3ꢀdimethoxyꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
1
1
3(12H)ꢀone (17a). White solid, yield 97%, mp = 228.2ꢀ229.7 °C. H NMR (CDCl ) δ 8.00 (d, J
3
=
8.8 Hz, 1H), 7.91 (s, 1H), 7.59ꢀ7.57 (m, 2H), 7.25 (s, 1H), 7.19 (s, 1H), 6.11 (s, 2H), 5.08 (t, J
1
3
= 5.6 Hz, 1H), 4.54 (t, J = 4.2 Hz, 2H), 4.37ꢀ4.33 (m, 2H), 4.12 (s, 3H), 4.06 (s, 3H). C NMR
CDCl ) δ 165.8, 154.0, 149.8, 147.5, 147.5, 135.4, 131.9, 129.4, 123.8, 120.8, 118.7, 118.3,
(
3
1
17.3, 108.5, 105.0, 102.8, 102.0, 101.7, 64.0, 56.8, 56.3, 56.2. HRMS (ESI) m/z: 394.1276 [M +
+
+
H] , calcd for C H NO 394.1285. ESIꢀMS m/z: 394.1 [M + H] .
2
2
20
6
1
2ꢀ(3ꢀHydroxypropyl)ꢀ2,3ꢀdimethoxyꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
1
1
3(12H)ꢀone (17b). White solid, yield 96%, mp = 263.4ꢀ264.2 °C. H NMR (CDCl ) δ 7.98 (d, J
3
= 8.8 Hz, 1H), 7.92 (s, 1H), 7.60 (s, 1H), 7.59ꢀ7.55 (m, 2H), 7.19 (s, 1H), 6.12 (s, 2H), 4.72 (t, J
13
=
6.6 Hz, 2H), 4.12 (s, 3H), 4.07 (s, 3H), 3.54 (t, J = 6.6 Hz, 2H), 2.16ꢀ2.07 (m, 2H). C NMR
(CDCl ) δ 165.0, 153.7, 149.8, 147.6, 147.4, 135.1, 131.7, 129.0, 123.5, 121.1, 119.4, 118.4,
3
117.5, 108.9, 104.9, 102.8, 102.3, 101.6, 60.1, 56.2, 56.2, 48.3, 32.8. HRMS (ESI) m/z: 408.1457
+
+
[
M + H] , calcd for C H NO 408.1442. ESIꢀMS m/z: 408.1 [M + H] .
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General procedure for the synthesis of 18a and 18b. To a solution of 17a (or 17b, 0.5 mmol)
in freshly distilled chloroform (10 mL), SOCl (0.8 mL) was added dropwise through a syringe.
2
The reaction solution was stirred at room temperature for 1 h, and then quenched by the addition
of water (5 mL) at 0 °C. The precipitate was filtered and washed with saturated sodium
bicarbonate and water consecutively. The residue was purified by silica gel column
chromatography to give the target product.
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2ꢀ(2ꢀChloroethyl)ꢀ2,3ꢀdimethoxyꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ13(12H)ꢀ
1
one (18a). White solid, yield 94%, mp = 236.5ꢀ237.3 °C. H NMR (DMSO) δ 8.83 (d, J = 9.0
Hz, 1H), 8.31 (s, 1H), 8.26 (s, 1H), 8.16 (d, J = 9.0 Hz, 1H), 7.72 (s, 1H), 7.68 (s, 1H), 6.33 (s,
2
H), 5.76 (t, J = 9.0 Hz, 2H), 5.35 (t, J = 9.0 Hz, 2H), 4.20 (s, 3H), 4.04 (s, 3H). ESIꢀMS m/z:
+
4
12.1 (100%), 414.0 (33%) [M + H] .
12ꢀ(3ꢀChloropropyl)ꢀ2,3ꢀdimethoxyꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
1
1
3(12H)ꢀone (18b). White solid, yield 96%, mp = 235.5ꢀ236.1 °C. H NMR (CDCl ) δ 7.97 (d, J
3
=
8.8 Hz, 1H), 7.89 (s, 1H), 7.59ꢀ7.55 (m, 2H), 7.51 (s, 1H), 7.19 (s, 1H), 6.12 (s, 2H), 4.69 (t, J
= 6.8 Hz, 2H), 4.11 (s, 3H), 4.06 (s, 3H), 3.39 (t, J = 6.4 Hz, 2H), 2.43ꢀ2.33 (m, 2H). HRMS
+
(
ESI) m/z: 426.1090 [M + H] , calcd for C H NO Cl 426.1103. ESIꢀMS m/z: 426.1 (100%),
23
21
5
+
4
28.1 (33%) [M + H] .
General procedure for the synthesis of 19a/bꢀ25a/b. The solution of 18a (or 18b, 0.87 mmol),
NEt (870 mg, 8.7 mmol) and amines (in Pressure Vessel for dimethylamine, 8.7 mmol) in PhMe
3
(
20 mL) was stirred and heated under reflux for 3ꢀ6 h, and then, cooled to room temperature. The
solvent was evaporated under reduced pressure. The residue was purified by silica gel column
chromatography to give target product.
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2ꢀ(2ꢀ(Dimethylamino)ethyl)ꢀ2,3ꢀdimethoxyꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
ꢀ1
1
3(12H)ꢀone (19a). White solid, yield 65%, mp =136.4ꢀ138.0 °C. IR (KBr, cm ), 1632, 1611,
1
1590. H NMR (CDCl ) δ 7.97 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.66 (s, 1H), 7.57ꢀ7.54 (m, 2H),
3
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
7
.18 (s, 1H), 6.10 (s, 2H), 4.66 (t, J = 7.0 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 2.78 (t, J = 7.0 Hz,
1
3
2
H), 2.21 (s, 6H). C NMR (CDCl ) δ 164.7, 153.6, 149.7, 147.5, 147.3, 135.5, 131.7, 128.9,
3
123.3, 121.3, 119.6, 118.3, 117.3, 108.8, 104.8, 102.9, 102.3, 101.5, 57.6, 56.2, 56.1, 50.1, 45.5.
+
HRMS (ESI) m/z: 421.1761 [M + H] , calcd for C H N O 421.1758.
2
4
25
2
5
1
2ꢀ(3ꢀ(Dimethylamino)propyl)ꢀ2,3ꢀdimethoxyꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀ
ꢀ
c]phenanthridinꢀ13(12H)ꢀone (19b). White solid, yield 70%, mp = 173.2ꢀ174.3 °C. IR (KBr, cm
1
1
)
, 1612 (sh.), 1582. H NMR (CDCl ) δ 7.97 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.58ꢀ7.53 (m,
3
2
H), 7.48 (s, 1H), 7.19 (s, 1H), 6.13 (s, 2H), 4.55 (t, J = 7.2 Hz, 2H), 4.12 (s, 3H), 4.07 (s, 3H),
1
3
2.51 (t, J = 7.2 Hz, 2H), 2.34 (s, 6H), 2.18 (quint, J = 7.2 Hz, 2H). C NMR (CDCl ) δ 164.7,
3
1
1
53.6, 149.6, 147.5, 147.4, 135.3, 131.7, 129.0, 123.3, 121.0, 119.5, 118.3, 117.4, 108.8, 104.8,
+
02.9, 102.0, 101.6, 56.2, 56.1, 55.5, 49.7, 43.5, 25.1. HRMS (ESI) m/z: 435.1919 [M + H] ,
calcd for C H N O 435.1914.
25 27
2
5
1
2ꢀ(2ꢀ(Diethylamino)ethyl)ꢀ2,3ꢀdimethoxyꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
ꢀ1
1
3(12H)ꢀone (20a). White powder, yield 86%, mp = 129.1ꢀ129.5 °C. IR (KBr, cm ), 1640,
1
1612, 1592. H NMR (CDCl ) δ 7.96 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.70 (s, 1H), 7.59ꢀ7.52
3
(
m, 2H), 7.17 (s, 1H), 6.10 (s, 2H), 4.64 (t, J = 6.4 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 2.82 (t, J =
13
6
.4 Hz, 2H), 2.39 (q, J = 7.2 Hz, 4H), 0.83 (t, J = 7.2 Hz, 6H). C NMR (CDCl ) δ 165.0, 153.7,
3
1
49.8, 147.6, 147.5, 135.8, 131.8, 129.2, 123.3, 121.3, 119.9, 118.5, 117.6, 109.0, 104.9, 103.0,
+
102.8, 101.7, 56.4, 56.3, 51.6, 50.6, 47.5, 12.0. HRMS (ESI) m/z: 449.2078 [M + H] , calcd for
C H N O 449.2071.
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2ꢀ(3ꢀ(Diethylamino)propyl)ꢀ2,3ꢀdimethoxyꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
ꢀ1
1
3(12H)ꢀone (20b). White solid, yield 60%, mp = 106.5ꢀ108.1 °C. IR (KBr, cm ), 1637, 1613,
1
1581. H NMR (CDCl ) δ 7.99 (d, J = 8.8 Hz, 1H), 7.92 (s, 1H), 7.59 (s, 1H), 7.57 (d, J = 8.8 Hz,
3
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
H), 7.53 (s, 1H), 7.20 (s, 1H), 6.12 (s, 2H), 4.56 (t, J = 7.0 Hz, 2H), 4.13 (s, 3H), 4.07 (s, 3H),
1
3
.63ꢀ2.43 (m, 6H), 2.22ꢀ2.10 (m, 2H), 0.98 (t, J = 6.5 Hz, 6H). C NMR (CDCl ) δ 164.7, 153.6,
3
149.7, 147.6, 147.4, 135.4, 131.7, 129.0, 123.4, 121.2, 119.5, 118.4, 117.3, 108.7, 104.9, 102.8,
+
1
02.1, 101.6, 56.3, 56.2, 50.1, 49.4, 46.6, 25.3, 10.8. HRMS (ESI) m/z: 463.2233 [M + H] , calcd
+
for C H N O 463.2227. ESIꢀMS m/z: 463.2 [M + H] . The structure was also confirmed with
2
7
31
2
5
and single crystal analysis. Compound 20b was crystallized from dichloromethane as white
cubic crystals. Data collections for 20b were performed at 103 K on a Xcalibur Nova
diffractometer, using Cu Kα radiation (λ = 1.54184 Å). The determination of crystal class and
unit cell parameters was carried out by CrysAlisPro. The raw frame data was processed using
CrysAlisPro (Rigaku Oxford Diffraction) to yield the reflection data file. The structure of 20b
2
was solved by use of SHELXTL (Bruker, 2005) program. Refinement was performed on F
anisotropically for all the nonꢀhydrogen atoms by the fullꢀmatrix leastꢀsquares method. The
hydrogen atoms were placed at the calculated positions and were included in the structure
calculation without further refinement of the parameters. Molecular formula = C H N O ,
2
7
32
2
4
molecular mass = 449.57, monoclinic, a = 9.41991(12) Å, b = 10.11440(12) Å, c = 23.7285(4)
3
Å, β = 91.6653(13)°, V = 2259.82(5) Å , T = 103 K, space group P2 /c (no. 14), Z = 4, ꢀ (Cu Kα)
1
ꢀ1
=
0.710 mm , 10230 reflections measured, 4283 unique (R = 0.0458) which were used in all
int
2
calculations. The final R (reflections) = 0.0500, wR (reflections) = 0.1495. Crystallographic
data for compound 20b has been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication number CCDC 1579803. Copies of the data can be obtained, free of
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charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44(0)1223ꢀ
3
36033 or eꢀmail: deposit@ccdc.cam.ac.uk].
2,3ꢀDimethoxyꢀ12ꢀ(2ꢀ(pyrrolidinꢀ1ꢀyl)ethyl)ꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
ꢀ
1
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
1
1
3(12H)ꢀone (21a). White solid, yield 47%, mp =209.2ꢀ210.3 °C. IR (KBr, cm ), 1639, 1610,
1
594. H NMR (CDCl ) δ 7.93ꢀ7.88 (m, 2H), 7.65 (s, 1H), 7.52 (s, 1H), 7.49 (d, J = 8.8 Hz, 1H),
3
7.12 (s, 1H), 6.08 (s, 2H), 4.66 (t, J = 7.2 Hz, 2H), 4.08 (s, 3H), 4.05 (s, 3H), 2.91 (t, J = 7.2 Hz,
1
3
2
1
H), 2.52ꢀ 2.42 (m, 4H), 1.73ꢀ1.61 (m, 4H). C NMR (CDCl ) δ 164.7, 153.5, 149.6, 147.5,
3
47.3, 135.4, 131.6, 129.0, 123.2, 121.1, 119.6, 118.2, 117.3, 108.8, 104.7, 102.8, 102.6, 101.5,
+
56.2, 56.1, 54.3, 54.1, 51.0, 23.6. HRMS (ESI) m/z: 447.1916 [M + H] , calcd for C H N O
2
6
27
2
5
+
4
47.1914. ESIꢀMS m/z: 447.2 [M + H] .
,3ꢀDimethoxyꢀ12ꢀ(3ꢀ(pyrrolidinꢀ1ꢀyl)propyl)ꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀ
c]phenanthridinꢀ13(12H)ꢀone (21b). White solid, yield 65%, mp = 205.8ꢀ207.4 °C. IR (KBr, cm
2
ꢀ
1
1
)
, 1640, 1612, 1596. H NMR (CDCl ) δ 7.95 (d, J = 8.8 Hz, 1H), 7.88 (s, 1H), 7.56 (s, 1H),
3
7
.54 (d, J = 8.8 Hz, 1H), 7.48 (s, 1H), 7.16 (s, 1H), 6.10 (s, 2H), 4.57 (t, J = 7.2 Hz, 2H), 4.10 (s,
H), 4.05 (s, 3H), 2.59 (s, br. 4H), 2.52 (t, J = 7.2 Hz, 2H), 2.19 (quint, J = 7.2 Hz, 2H), 1.77 (s,
3
1
3
br. 4H). C NMR (CDCl ) δ 164.7, 153.6, 149.7, 147.6, 147.4, 135.3, 131.7, 129.0, 123.4, 121.2,
3
1
19.5, 118.3, 117.3, 108.8, 104.9, 102.9, 102.1, 101.6, 56.2, 56.2, 53.8, 53.3, 49.9, 27.4, 23.4.
+
HRMS (ESI) m/z: 461.2067 [M + H] , calcd for C H N O 461.2071.
2
7
29
2
5
2,3ꢀDimethoxyꢀ12ꢀ(2ꢀ(piperidinꢀ1ꢀyl)ethyl)ꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
ꢀ1
1
3(12H)ꢀone (22a). White solid, yield 44%, mp = 178.3ꢀ180.1 °C. IR (KBr, cm ), 1642, 1612,
1
1
596. H NMR (CDCl ) δ 7.98 (d, J = 8.8 Hz, 1H), 7.93 (s, 1H), 7.74 (s, 1H), 7.58 (s, 1H), 7.56
3
(d, J = 8.8 Hz, 1H), 7.19 (s, 1H), 6.12 (s, 2H), 4.69 (t, J = 6.6 Hz, 2H), 4.12 (s, 3H), 4.07 (s, 3H),
13
2
.69 (t, J = 6.6 Hz, 2H), 2.34ꢀ2.23 (m, 4H), 1.40ꢀ1.29 (m, 6H). C NMR (CDCl ) δ 165.0, 153.5,
3
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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
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5
5
5
5
5
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1
49.5, 147.5, 147.3, 135.6, 131.6, 129.1, 123.2, 121.2, 119.7, 118.2, 117.5, 108.9, 104.7, 102.8,
+
02.7, 101.5, 57.2, 56.2, 56.1, 54.6, 49.8, 25.8, 24.2. HRMS (ESI) m/z: 461.2076 [M + H] , calcd
+
for C H N O 461.2071. ESIꢀMS m/z: 461.2 [M + H] .
2
7
29
2
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
,3ꢀDimethoxyꢀ12ꢀ(3ꢀ(piperidinꢀ1ꢀyl)propyl)ꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
ꢀ1
1
3(12H)ꢀone (22b). White solid, yield 60%, mp =110.6ꢀ112.3 °C. IR (KBr, cm ), 1639, 1612,
1
1592. H NMR (CDCl ) δ 7.98 (d, J = 8.7 Hz, 1H), 7.91 (s, 1H), 7.60ꢀ7.55 (m, 2H), 7.48 (s, 1H),
3
7
2
.19 (s, 1H), 6.10 (s, 2H), 4.55 (d, J = 7.2 Hz, 2H), 4.12 (s, 3H), 4.06 (s, 3H), 2.50 (d, J = 7.2 Hz,
1
3
H), 2.34ꢀ2.13 (s, 6H), 1.51ꢀ1.30 (m, 6H). C NMR (CDCl ) δ 164.6, 153.7, 149.7, 147.6,
3
147.5, 135.1, 131.7, 128.9, 123.5, 121.0, 119.4, 118.3, 117.4, 108.7, 104.9, 102.9, 102.0, 101.6,
+
5
6.2, 56.1, 55.7, 53.9, 50.1, 25.4, 25.2, 23.9. HRMS (ESI) m/z: 475.2227 [M + H] , calcd for
C H N O 475.2227.
28
31
2
5
2
,3ꢀDimethoxyꢀ12ꢀ(2ꢀmorpholinoethyl)ꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
ꢀ1
1
3(12H)ꢀone (23a). White solid, yield 48%, mp = 220.5ꢀ221.1 °C. IR (KBr, cm ), 1639, 1611,
1
1
594. H NMR (CDCl ) δ 7.93 (d, J = 8.7 Hz, 1H), 7.88 (s, 1H), 7.64 (s, 1H), 7.54 (s, 1H), 7.51
3
(d, J = 8.7 Hz, 1H), 7.14 (s, 1H), 6.09 (s, 2H), 4.66 (t, J = 6.2 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H),
1
3
3
1
1
.34 (s, br. 4H), 2.67 (t, J = 6.2 Hz, 2H), 2.21 (s, br. 4H). C NMR (CDCl ) δ 165.2, 153.5,
3
49.6, 147.5, 147.4, 135.6, 131.5, 129.0, 123.2, 121.2, 119.7, 118.3, 117.6, 108.9, 104.8, 102.8,
+
02.5, 101.6, 66.9, 56.6, 56.2, 56.2, 53.4, 49.3. HRMS (ESI) m/z: 463.1875 [M + H] , calcd for
+
C H N O 463.1864. ESIꢀMS m/z: 463.2 [M + H] .
26
27
2
6
2
,3ꢀDimethoxyꢀ12ꢀ(3ꢀmorpholinopropyl)ꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀc]phenanthridinꢀ
ꢀ1
1
3(12H)ꢀone (23b). White solid, yield 60%, mp = 203.1ꢀ205.0 °C. IR (KBr, cm ), 1639, 1610,
1
1595. H NMR (CDCl ) δ 7.94 (d, J = 8.5 Hz, 1H), 7.90 (s, 1H), 7.58ꢀ7.48 (m, 3H), 7.16 (s, 1H),
3
6
.10 (s, 2H), 4.63 (t, J = 6.2 Hz, 2H), 4.10 (s, 3H), 4.05 (s, 3H), 3.42 (s, br. 4H), 2.12 (s, br. 4H),
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2
.08ꢀ1.96 (m, 4H). C NMR (CDCl ) δ 164.6, 153.5, 149.7, 147.5, 147.2, 135.9, 131.6, 128.9,
3
1
23.0, 121.1, 119.7, 118.3, 117.2, 108.8, 104.8, 102.8, 102.4, 101.5, 66.9, 56.2, 56.2, 55.3, 53.3,
+
49.8, 25.3. HRMS (ESI) m/z: 477.2032 [M + H] , calcd for C H N O 477.2020.
2
7
29
2
6
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
2
,3ꢀDimethoxyꢀ12ꢀ(2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)ethyl)ꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀ
ꢀ
c]phenanthridinꢀ13(12H)ꢀone (24a). White solid, yield 42%, mp = 207.5ꢀ208.3 °C. IR (KBr, cm
1
1
), 1635, 1613, 1590. H NMR (CDCl ) δ 7.93 (d, J = 8.8 Hz, 1H), 7.89 (s, 1H), 7.61 (s, 1H),
3
7
.54 (s, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.15 (s, 1H), 6.10 (s, 2H), 4.67 (t, J = 6.8 Hz, 2H), 4.10 (s,
H), 4.05 (s, 3H), 2.67 (t, J = 6.8 Hz, 2H), 2.64ꢀ2.56 (m, 2H), 2.38ꢀ2.32 (m, 4H), 2.23ꢀ2.16 (m,
3
1
3
5H). C NMR (CDCl ) δ 165.2, 153.5, 149.6, 147.5, 147.4, 135.5, 131.5, 129.0, 123.2, 121.2,
3
1
19.8, 118.2, 117.6, 109.0, 104.8, 102.8, 102.4, 101.5, 56.2, 56.2, 55.9, 54.7, 52.3, 49.5, 45.5.
+
HRMS (ESI) m/z: 476.2177 [M + H] , calcd for C H N O 476.2180. ESIꢀMS m/z: 476.2 [M +
27
30
3
5
+
H] .
2,3ꢀdimethoxyꢀ12ꢀ(3ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)propyl)ꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀ
ꢀ
c]phenanthridinꢀ13(12H)ꢀone (24b). White solid, yield 41%, mp = 170.9ꢀ172.8 °C. IR (KBr, cm
1
1
)
, 1631, 1614, 1590. H NMR (CDCl ) δ 7.96 (d, J = 8.7 Hz, 1H), 7.91 (s, 1H), 7.56 (s, 1H),
3
7
3
1
.54 (d, J = 8.7 Hz, 1H), 7.52 (s, 1H), 7.18 (s, 1H), 6.11 (s, 2H), 4.61 (t, J = 6.8 Hz, 2H), 4.10 (s,
13
H), 4.05 (s, 3H), 2.33ꢀ2.08 (m, 13H), 1.99 (t, J = 6.8 Hz, 2H). C NMR (CDCl ) δ 164.7, 153.5,
3
49.7, 147.5, 147.2, 135.8, 131.6, 128.9, 123.2, 121.4, 119.7, 118.3, 117.3, 108.8, 104.8, 102.8,
+
102.5, 101.5, 56.3, 56.2, 54.9, 52.5, 49.9, 45.8, 29.7, 25.7. HRMS (ESI) m/z: 490.2334 [M + H] ,
+
calcd for C H N O 490.2336. ESIꢀMS m/z: 490.2 [M + H] .
2
8
32
3
5
1
2ꢀ(2ꢀ(1Hꢀimidazolꢀ1ꢀyl)ethyl)ꢀ2,3ꢀdimethoxyꢀ[1,3]dioxolo[4',5':4,5]benzo[1,2ꢀ
c]phenanthridinꢀ13(12H)ꢀone (25a). White powder, yield 88%, mp = 254.2ꢀ255.6 °C. IR (KBr,
ꢀ1
1
cm ), 1630, 1609, 1588. H NMR (CDCl ) δ 7.92ꢀ7.86 (m, 2H), 7.55ꢀ7.50 (m, 2H), 7.32 (s, 1H),
3
ACS Paragon Plus Environment
29