Discovery of Pamiparib (BGB-290), a Potent and Selective Poly (ADP-ribose) Polymerase (PARP) Inhibitor in Clinical Development

Article abstract of DOI:10.1021/acs.jmedchem.0c01346

Poly (ADP-ribose) polymerase (PARP) plays a significant role in DNA repair responses; therefore, this enzyme is targeted by PARP inhibitors in cancer therapy. Here we have developed a number of fused tetra- or pentacyclic dihydrodiazepinoindolone derivatives with excellent PARP enzymatic and cellular PARylation inhibition activities. These efforts led to the identification of pamiparib (BGB-290, 139), which displays excellent PARP-1 and PARP-2 inhibition with IC50 of 1.3 and 0.9 nM, respectively. In a cellular PARylation assay, this compound inhibits PARP activity with IC50 = 0.2 nM. Cocrystal of pamiparib shows similar binding sites with PARP with other PARP inhibitors, but pamiparib is not a P-gp substrate and shows excellent drug metabolism and pharmacokinetics (DMPK) properties with significant brain penetration (17-19%, mice). The compound is currently being investigated in phase III clinical trials as a maintenance therapy in platinum-sensitive ovarian cancer and gastric cancer.

Full text of DOI:10.1021/acs.jmedchem.0c01346

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Drug Annotation
Discovery of Pamiparib (BGB-290), a Potent and Selective Poly (ADP-
ribose) Polymerase (PARP) Inhibitor in Clinical Development
Hexiang Wang, Bo Ren, Ye Liu, Beibei Jiang, Yin Guo, Min Wei, Lusong Luo, Xianzhao Kuang,
Ming Qiu, Lei Lv, Hong Xu, Ruipeng Qi, Huibin Yan, Dexu Xu, Zhiwei Wang, Chang-Xin Huo,
Yutong Zhu, Yuan Zhao, Yiyuan Wu, Zhen Qin, Dan Su, Tristin Tang, Fan Wang, Xuebing Sun,
Yingcai Feng, Hao Peng, Xing Wang, Yajuan Gao, Yong Liu, Wenfeng Gong, Fenglong Yu, Xuesong Liu,
Lai Wang, and Changyou Zhou*
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ABSTRACT: Poly (ADP-ribose) polymerase (PARP) plays a
significant role in DNA repair responses; therefore, this enzyme is
targeted by PARP inhibitors in cancer therapy. Here we have
developed a number of fused tetra- or pentacyclic dihydrodiazepin-
oindolone derivatives with excellent PARP enzymatic and cellular
PARylation inhibition activities. These efforts led to the
identification of pamiparib (BGB-290, 139), which displays
excellent PARP-1 and PARP-2 inhibition with IC50 of 1.3 and
0.9 nM, respectively. In a cellular PARylation assay, this compound
inhibits PARP activity with IC50 = 0.2 nM. Cocrystal of pamiparib
shows similar binding sites with PARP with other PARP inhibitors,
but pamiparib is not a P-gp substrate and shows excellent drug
metabolism and pharmacokinetics (DMPK) properties with
significant brain penetration (17−19%, mice). The compound is currently being investigated in phase III clinical trials as a
maintenance therapy in platinum-sensitive ovarian cancer and gastric cancer.
the release of nicotinamide.5 This results in the fast
INTRODUCTION
■
recruitment of DNA repair factors such as XRCC1 to the
UV light, free radicals, ionizing radiation, and chemo-
sites of DNA breaks, which is necessary to complete BER.6
therapeutic agents may cause DNA damage and ultimately
BRCA1 and BRCA2 are important tumor suppressors for
lead to cell death.1 DNA single-strand breaks (SSBs) are the
DNA DSB repair by HR, and mutations in these genes
most frequent type of damage and can be converted into
predispose humans to breast and other cancers.7 Inhibition of
potentially clastogenic and lethal DNA double-strand breaks
PARP-1 results in blockage of BER and persistent SSBs,
(DSBs). While SSBs are repaired by base-excision repair
leading to the development of DSBs. In normal cells, these
(BER), DSBs are more problematic with repair requiring a
DSBs can be repaired through HR but not in BRCA1- and/or
functional homologous recombination (HR) or nonhomolo-
BRCA2-deficient tumor cells. Accumulation of DSBs becomes
gous end joining system.2 These DNA repair pathways can also
highly toxic to tumor cells and results in synthetic lethality.8
be used by cancer cells to overcome DNA damage-based
Drug discovery efforts led to the development of four FDA-
cancer therapy and are a cause of anticancer drug resistance.3
approved PARP inhibitors (Figure 1), namely, olaparib
Poly (ADP-ribose) polymerases (PARPs) are a family of
(Merck/AstraZeneca),9 niraparib (GSK/Zai Lab),10 rucaparib
proteins that contain a conserved PARP catalytic domain.4 At
least 18 PARPs have been discovered so far, including the best
characterized proteins PARP-1, -2, -5A (TNKS-1), and -5B
(Clovis),11 and talazoparib (Pfizer/Medivation),12 all of which
are analogues of nicotinamide. Several other PARP inhibitors
(TNKS-2). PARP-1 is the most abundant member of the
Received: August 1, 2020
family and plays an important role in BER. It is activated by
binding to damaged DNA through its N-terminal zinc fingers,
and then its C-terminal catalytic domain catalyzes the transfer
of one or more ADP-ribose units from nicotinamide adenine
dinucleotide (NAD+) to target proteins (histones, top-
oisomerases, DNA polymerases, P53, and PARP itself) with
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In a phase I study as of January 2016, 40 patients (pts) had
been enrolled in 8 escalating cohorts. Linear dose-dependent
PK was observed with ∼2-fold accumulation at steady state.
Pamiparib induced significant PARylation inhibition in
peripheral blood mononuclear cell (PBMC) from the starting
dose level and achieved robust (>80%) and sustained
PARylation inhibition at steady state at doses of ≥10 mg
BID. Two complete and seven partial responses were
demonstrated in the 20 evaluable pts with ovarian cancer.19
Further clinical data will be published in due course.
Pamiparib was combined with others antitumors, such as
TMZ and tislelizumab, which are also in clinical trial in
advanced solid tumor. In a phase 1a/b (NCT02660034) study,
49 patients enrolled in the combination clinical study for
pamiparib and tislelizumab. As of January 2018, the
combination was generally well tolerated, and complete or
partial response was observed in 10 patients.20
These clinical data showed that pamiparib demonstrated a
favorable safety profile and promising antitumor activity in
advanced solid tumor.
Figure 1. FDA-approved PARP inhibitors.
are in late stage clinical development, such as veliparib
(AbbVie),13 and fluzoparib (Jiangsu Hansoh/Jiangsu Hen-
grui). These clinical PARP inhibitors mainly target BRCA-
mutated tumors or tumors bearing defects in other HR-related
genes and are used as a monotherapy and/or combination
therapy.14
RESULTS AND DISCUSSION
■
Structure−Activity Relationship. Most PARP inhibitors
developed to date are designed to mimic the natural substrate
NAD+, particularly the nicotinamide moiety. The amide of
nicotinamide binds to Ser904 and Gly863 through three key
hydrogen bonds. These compounds inhibit PARP by blocking
the active binding site. Most attempts to improve the affinity of
inhibitors have involved locking the carboxamide group into
the desired biologically active conformation.21−23 In this
report, four series of compounds were designed and
synthesized such that the primary amide was locked into an
active conformation by introducing a seven-membered
dihydrodiazepinone ring. Not all data are presented here,
and only compounds that are illustrative of the key SARs are
discussed. All the synthesized compounds were evaluated for
PARP-1 enzymatic inhibitory activity and cellular PARylation
inhibitory activity, using olaparib as a reference in both assays.
The IC50 and EC50 values obtained for the positive control
olaparib were comparable to previously reported values.24
Exploration of Fused Tetracyclic Dihydrodiazepinoin-
dolones (Series 1). In our initial efforts we attempted to lock
the primary amide through a seven-membered dihydrodiaze-
pinone ring instead of an intramolecular hydrogen bond, as is
the case for niraparib (Table 1).25 First, a tetracyclic
compound, 33, was synthesized and it showed good potency
in both enzymatic (IC50 = 10.2 nM) and cellular (EC50 = 3.6
nM) assays. Initial SAR studies of the effect of modifying the
phenyl ring in the indole scaffold showed that introduction of
fluoride at C2 (compound 34) resulted in enhanced enzymatic
Brain metastases develop in approximately 10−30% of all
cancers, affecting many patients. In one regional incidence
evaluation, brain metastatic lesions developed in approximately
10% of all cancers, with the highest incidence ascribed to lung
cancer (20%) followed by melanoma (7%) and breast cancer
(5%).15 PARP inhibitors with brain penetration are a
prerequisite in glioma tumors or other tumors with brain
metastasis.16 Many PARP inhibitors are being assessed in the
clinic, but only veliparib17 and E701618 are known to have the
brain penetration property; others like olaparib and rucaparib
are of limited brain penetration ability. Unfortunately, veliparib
is weaker than olaparib and rucaparib, which may be a
handicap in terms of clinical efficacy. A PARP inhibitor not
only with good potency and PK profile but also with good
brain penetration is also strongly desired to meet the highly
unmet medical needs for treating cancer patients with brain
metastasis.
In this article, we describe the synthesis and structure−
activity relationship (SAR) investigation of a novel series of
fused tetracyclic or pentacyclic dihydrodiazepinoindolone
derivatives, leading to the discovery of compound 139
(pamiparib, BGB-290), which now is under various phase II/
III clinical trials. This compound displays excellent potency
against both the PARP-1 and PARP-2 enzymes with IC50 = 1.3
and 0.9 nM, respectively, showing good selectivity over TNKS-
1 and TNKS-2 (IC50 = 230 and 140 nM, respectively). In a
whole cell assay, compound 139 inhibits PARP activity with
EC50 = 0.2 nM. This compound possesses an excellent
pharmacokinetic profile, almost 100% oral bioavailability and
significant brain penetration. It strongly inhibits proliferation of
cancer cell lines with BRCA mutations. Furthermore,
compound 139 exhibits strong antitumor activity in a
BRCA1 mutated xenograft model as a single agent and
exhibits combination efficacy with TMZ in a small cell lung
cancer xenograft model. In addition, compound 139 is not a P-
gp substrate and does not inhibit transporters; therefore, it may
have potential to help patients of drug resistance due to
upregulation of efflux pumps.
activity (IC50 = 6.0 nM) and similar cellular activity (EC50
=
5.5 nM); however, introduction of fluoride at C3 had
detrimental effects on both enzymatic and cellular activities
(compound 35: IC50 = 24 nM and EC50 = 15 nM). Therefore,
most subsequent SAR studies on the aliphatic A-ring of the
indole scaffold were focused on compounds with a fluoride or a
proton at C2. Monomethyl or gem-dimethyl substitutions at
C7 in the A-ring (compounds 36, 37, 38, and 39) resulted in
high activities similar to those of the corresponding parent
compounds, with IC50 values ranging from 4.9 nM to 9.1 nM
and EC50 values from 1.7 nM to 4.8 nM. gem-Dimethyl
substitution at C8 (compound 40) had a detrimental effect,
resulting in lower enzymatic and cellular potency (IC50 = 28.8
B
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Table 1. In Vitro Activity of Fused Tetracyclic Dihydrodiazepinoindolones 33−48
a
b
Data shown are mean values obtained from three independent experiments performed in triplicate. NT: not tested.
nM and EC50 = 45.5 nM). Good enzymatic and cellular
potencies were observed with isopropyl substitution at C7 of
the A-ring (compound 41: IC50 = 3.3 nM and EC50 = 2.4 nM).
Although phenyl substitution at C7 (compound 42) also
C
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Drug Annotation
Figure 2. Predicted binding pose of compounds 33 (A) and 56 (B) with PARP-1. Compounds 33, 56, and amino acid residues of PARP-1
interacting with compounds are shown as stick and colored in green, magenta, and orange, respectively. The molecular docking is done using Glide
based on the crystal structure of PARP-1 in complex with compound 139 (PDB code 7CMW). Hydrogen bonds and π−π stacking between
̈
compounds and protein are shown as blue and cyan dashed lines, respectively. The figure is generated using Schrodinger software (Maestro 12.3 in
̈
Schrodinger Suite 2020-1).
maintained good enzymatic potency (IC50 = 5.8 nM), the
cellular activity of this compound (EC50 = 12.4 nM) decreased,
probably due to poor cellular permeability. Both enzymatic and
cellular activities decreased with dimethylaminophenyl replace-
ment at C7 (compound 43: IC50 = 25.6 nM and EC50 = 26.6
nM). Interestingly, the seven-membered ring compounds 44
and 45, which were produced by further enlargement of the A-
ring, retained potencies similar to those of the six-membered
ring compounds 34 and 35.
Although most of the compounds in this series showed good
potencies in both enzymatic and cellular assays, they had rather
poor solubility properties and thus low exposure (oral, 38, 521
ng·h·mL−1, 5 mpk) in rat PK study. Introduction of polar
groups such as hydroxyl or carbonyl groups on the A-ring with
the intention of improving aqueous solubility led to the new
compounds 46−48. Unfortunately, these compounds had
much lower potencies in enzymatic and cellular assays
compared with those of the parent compound 38.
Exploration of N-Alkylated Tetrahydrodiazepino-
carbazolones (Series 2). The unsubstituted NH on the
indole ring of compound 33 is pointing out of the pocket and
thus has space in the front, which indicated that we can
introduce solubilizing groups with an N-substitution on the
indole ring (Figure 2). Table 2 shows the series of compounds
with an N-substitution on the indole ring. The simple methyl-
substituted analogues 51 and 52 retained good enzymatic
potency with IC50 = 15.8 nM and 15.2 nM, respectively,
activities (IC50 = 350.0 nM and EC50 = 1510.0 nM). Although
gem-dimethyl-substituted compound 52 showed a weaker
cellular potency than the parent compound 51, the
corresponding distal N,N-dimethyl compound 59 demonstra-
ted more than 2-fold higher biochemical potency (IC50 = 5.5
nM) and cellular potency (EC50 = 4.9 nM) than its analogue
53. A fluorine substitution at C2 of the phenyl ring in
compound 60 seemed tolerable and improved the cellular
potency further (IC50 = 6.5 nM and EC50 = 2.4 nM). Generally
speaking, good and even better biochemical and cellular
potencies were observed in gem-dimethyl compounds than in
nonsubstituted compounds when the distal nitrogen was
substituted by other groups, as illustrated by compounds 53,
54, 56, 57, 61, 62, 63, 64, and 65. The distal basic nitrogen
seemed to help improve potency; the reason is that it forms an
additional salt bridge with E988 (Figure 2B). Replacement of
the distal nitrogen with oxygen proved to be detrimental in
terms of biochemical potency, regardless of whether the
oxygen was in a linear alcohol (compound 66) or embedded in
a cyclic ether (compound 67). Quite surprisingly, the highest
cellular potency (EC50 = 0.5 nM) was obtained for the N-
monomethyl substituted compound 69.
Unfortunately, despite having high solubility, almost all the
compounds described above displayed significant inhibitory
activity in an hERG functional cellular assay. For example,
compounds 59, 60, 61, 63, and 64 showed 77%, 73%, 95%,
97%, and 92% inhibition of hERG activity at 20 μM,
respectively. But parent compound 38 only showed 32%
inhibition of hERG activity at 20 μM, which indicated that
strong basic substitutions may induce hERG inhibition in this
series. Consequently, these series of compounds were
abandoned, and more efforts were undertaken to reduce
hERG inhibitory activity for this scaffold.
though partial loss of cellular potency was observed (EC50
=
75.0 nM and 206.9 nM, respectively). Surprisingly, when N-
(dimethylamino)ethyl was introduced as a solubilizing group
in compound 53, not only was the good biochemical potency
of the enzyme maintained (IC50 = 15.0 nM) but also the
cellular potency (EC50 = 13.9 nM) was restored. Addition of a
slightly bigger diethyl substituent at the distal nitrogen in
compound 54 maintained high potency (IC50 = 15.6 nM, EC50
= 30.0 nM), while addition of a large group such as that in the
dibenzyl-substituted compound 55 dramatically decreased
enzymatic potency (IC50 = 800.0 nM) and cellular potency
(EC50 = 2010.0 nM). When the distal nitrogen atom was
embedded in a cycle, for example, in the pyrrolidine compound
56 and piperidine analogue 57, both enzymatic and cellular
potencies were improved (IC50 = 10.5 nM and 10.3 nM, EC50
= 33.0 nM and 23.0 nM, respectively). However, the more
polar morpholine analogue 58 demonstrated rather poor
Exploration of Tetrahydrotetraazacyclohepta-
fluorenones (Series 3). In order to improve aqueous
solubility and reduce hERG inhibition, a new series of
compounds derived from fused unsubstituted and substituted
piperidine rings were synthesized and tested. As shown in
Table 3, similar to the previous cyclohexane-fused compounds
36 and 42, N-unsubstituted (R2 = H) piperidine-fused
compounds 77 and 78 showed good biochemical activity
(IC50 = 15.7 nM and 8.5 nM, respectively) and single digit
PARylation potency (EC50 = 6.9 nM and 8.0 nM, respectively).
Encouraged by these results, several N-alkyl-substituted
D
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Table 2. In Vitro Activity of N-Alkylated Tetrahydrodiazepinocarbazolones 51−67 and 69
a
b
Data shown are mean values obtained from three independent experiments performed in triplicate. NT: not tested.
compounds were synthesized and evaluated. Assays of the
resulting compounds 97−101 illustrated that various N-
alkylations were tolerated and improved both enzymatic and
cellular potencies in comparison with the unsubstituted parent
compounds. A relatively big substitution was tolerable, as
illustrated in N-butylated compound 102, which demonstrated
excellent potency (IC50 = 3.8 nM and EC50 = 1.9 nM). At the
same time, comparable enzymatic and/or cellular activities
(single-digit nanomolar IC50 and/or EC50 values in most cases)
were obtained with N-acyl or sulfonyl substituents, such as in
compounds 103−118. As expected, hERG inhibition activity
was greatly reduced in compounds of this series. For example,
compounds 100, 103, and 116 showed weak hERG inhibition
levels of 39%, 12%, and 43% at 20 μM, respectively. But most
of compounds showed high clearance in mouse liver
microsomes (LM) study, for example, compound 97 (CL =
274.9 mL·min−1·kg−1), compound 100 (CL = 174.3 mL·
min−1·kg−1), and compound 103 (CL = 250 mL·min−1·kg−1).
N-Propyl-substituted compound 100 also displayed low
exposure (oral dose, 5 mpk, AUC0−24h: 141 ng·h·mL−1) with
high clearance (CL = 114 mL min−1 kg−1)and a modest oral
bioavailability (F = 19%) in rats PK study. Unfortunately, a
relatively high clearance was also observed in rats for N-
E
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prepared to block basic nitrogens, which are potential
metabolic hot spots, through introduction of steric hindrance.
Three compounds were prepared in this series (Table 4).
Table 3. In Vitro Activity of
Tetrahydrotetraazacycloheptafluorenones 77, 78, 97−107,
109, 110, and 114−118
Table 4. In Vitro Activity of Fused Pentacyclic
Tetrahydrotetraazacycloheptafluorenones 138, 139, and 146
a
Data shown are mean values obtained from three independent
experiments performed in triplicate.
Compound 138 displayed good PARP-1 enzymatic inhibitory
activity (IC50 = 8.2 nM) and excellent cellular PARylation
inhibitory activity (EC50 = 0.6 nM) comparable to that of
olaparib (EC50 = 0.4 nM) (Table 1). Introduction of fluoride
at C2 in compound 138 resulted in compound 139, which
demonstrated excellent PARP-1 enzymatic potency (IC50 = 5.1
nM) and the best PARylation activity (EC50 = 0.2 nM) among
all four series of compounds. Olaparib showed an EC50 of 0.4
nM in the same assay. In contrast, the pentacyclic regioisomer
compound 146 (racemate) displayed much weaker enzymatic
and cellular activity (IC50 = 73.0 nM and EC50 = 54.7 nM). As
hypothesized, the steric hindrance indeed greatly reduced
metabolic degradation; the clearance of 139 (CL = 12.9 mL·
min−1·kg−1) is lower than 97 (CL = 274.9 mL·min−1·kg−1) and
100 (CL = 174.3 mL·min−1·kg−1) in mouse liver microsomes
study; thus 139 demonstrated excellent bioavailability in mice
(98.7%) and rats (93.7%) even with 0.5% methylcellulose
(MC) as formulation. In addition, low clearances of 29.9 mL
min−1 kg−1 in mouse and 22.7 mL min−1 kg−1 in rat were also
observed (Table 8).
Structural Basis for Binding of 139 to PARP-1. To
rationalize the observed excellent enzymatic and cellular
activity shown by 139, an X-ray cocrystal structure of 139
within PARP-1 was solved (Figure 3). The key interactions of
the fused pentacyclic core with the PARP-1 active site are
highlighted. The amide of 139 forms key hydrogen bonds with
the hydroxyl group of Ser904 and the amide backbone of
Gly863, which are the same as with other PARP1 inhibitors,
such as niraparib and olaparib. Moreover, the indole ring is
engaged in π−π stacking with Phe897 and Tyr907.
a
Data shown are mean values obtained from three independent
b
experiments performed in triplicate. NT: not tested.
acylated compounds 103 and 116 (CL = 114 mL min−1 kg−1
and 71 mL min−1 kg−1, respectively).
Exploration of Fused Pentacyclic Tetrahydro-
tetraazacycloheptafluorenones (Series 4). To reduce
clearance in liver microsomes and improve pharmacokinetics
(PK) properties, several fused pentacyclic compounds were
PARP Isoform Profiling for Compound 139. Given its
excellent DMPK properties and potency in inhibiting PARP-1,
F
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tion.26 Compound 139 was tested for its potency in trapping
PARP on DNA. It showed potent DNA-trapping activity (with
an IC50 of 13 nM), similar to olaparib and rucaparib, and was
30-fold more potent than veliparib but 4-fold weaker than
talazoparib (Figure 4).
Figure 3. Cocrystal structure of PARP-1 in complex with compound
139 (PDB code 7CMW). PARP-1 protein is shown as ribbon and
colored in gray. Compound 139 and the surrounding amino acid
residues of PARP-1 are shown in stick form and are colored in green
and orange, respectively. Hydrogen bonds between 139 and protein
residues are shown as black dashed lines.
Figure 4. DNA trapping activity of PARP inhibitors.
the selectivity of compound 139 against several other PARP
family members was further profiled (Table 5). PARP-2 plays
Investigation of the in Vitro Cellular Potency of 139
in BRCA-Mutant Cells. Compound 139 was tested for the
ability to inhibit proliferation of several tumor cell lines
carrying BRCA1 or BRCA2 mutations. BRCA-mutated cell lines
were hypersensitive to PARP inhibition by compound 139 in
comparison with BRCA1- and BRCA2-proficient cells (Table
6). In MDA-MB-436 human breast cancer cells carrying
Table 5. Inhibitory Activity of 139 against PARP Isoforms
a
enzyme
IC50 (nM)
PARP-1
PARP-2
PARP-3
TNKS-1
TNKS-2
PARP-6
PARP-7
PARP-8
PARP-10
PARP-11
PARP-12
1.3
0.9
68
230
Table 6. Antiproliferation Activity of 139 in Tumor Cell
Lines
140
>100000
11000
8400
11000
2700
2400
cell line
mutation
EC50 (nM)
MDA-MB-436
UWB1.289
HCC1395
BRCA1
BRCA1
BRCA1/2
10.6
53.8
42.5
MDA-MB-231
SK-BR-3
>10000
>10000
a
Data shown are mean values obtained from three independent
experiments performed in triplicate.
BRCA1 mutations, 139 displayed IC50 = 10.6 nM, while in a
BRCA1 mutant human ovarian cancer cell line, UWB1.289,
139 showed IC50 = 53.8 nM. Due to BRCA1/2 deficiency,
HCC1395 cells are highly sensitive to PARP inhibition, and
compound 139 was found to be cytotoxic to this cell line with
IC50 = 42.5 nM. In contrast, breast cancer cell lines, such as
MDA-MB-231 and SK-BR-3, that do not have BRCA
mutations or HR deficiencies were resistant to 139 (IC50
values of >10 μM). These findings demonstrate that
compound 139 is selectively toxic to BRCA-mutated cancer
cells.
Evaluation of the Pharmacokinetic Profile of 139.
Compared with the compounds in series 3, compound 139
showed weak hERG inhibition (51% inhibition at 20 μM). It
also exhibited very weak inhibitory effects on the activities of
all six CYPs examined (CYP1A2, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, and CYP3A4) with all IC50 values of
>10 μM. Compound 139 possessed low clearance in human
and mouse liver microsomes and good permeability (Table 7).
Encouraged by this in vitro profile, the pharmacokinetic
properties of 139 were further evaluated in mice, rats, and
dogs. Table 8 shows the pharmacokinetic profiles of 139 in
an important role in DNA repair, and both PARP-1 and PARP-
2 are required for efficient BER. As expected from the close
homology between PARP-1 and PARP-2, compound 139 was
demonstrated to be a potent and selective inhibitor of both
PARP-1 and PARP-2 with IC50 = 1.3 and 0.9 nM, respectively.
It was 50-fold less effective against PARP-3. Moreover, it
displayed at least 100-fold higher selectivity for PARP-1 and
PARP-2 over TNKS-1 and TNKS-2 (IC50 = 230 and 140 nM,
respectively). Further evaluation of compound 139 revealed it
had minimal inhibition activity against PARP-6, PARP-7,
PARP-8, PARP-10, PARP-11, and PARP-12 with IC50 values of
>100000, 11000, 8400, 11000, 2700, and 2400 nM,
respectively. On the basis of these data, compound 139 may
serve as a potent inhibitor of PARP-1 and PARP-2 without
interfering with other catalytic PARP isoforms.
DNA Trapping Activity of Compound 139. PARP
inhibitors were reported to trap PARP enzymes on damaged
DNA. Trapped PARP-DNA complexes were found to be more
deleterious than SSBs caused by PARP inhibition, and the
cytotoxicity of different PARP inhibitors correlated better with
their trapping potency than their ability to inhibit PARyla-
G
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Table 7. In Vitro Properties of Compound 139
a
metabolic stability, CL (μL min−1 mg−1
)
plasma protein binding, fu (%)
HLM
7.9
MLM
12.9
RLM
7.2
DLM
9.5
human
4.3
mouse
15.0
rat
dog
Caco-2 Papp A−B (cm/s × 10−6) [B−A/A−B]
15.1
21.0
30.3 [1.2]
a
Human (H), mouse (M), rat (R), and dog (D) liver microsomes (LM).
Table 8. Rodent Pharmacokinetics of Compound 139
a
b
h
mouse
rat
dog
c
d
e
f
i
f
iv (1 mg/kg)
po (10 mg/kg)
iv (2 mg/kg)
po (5 mg/kg)
iv (1 mg/kg)
po (2 mg/kg)
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC0−inf (ng h/mL)
CL (mL kg−1 min−1
2713
0.583
3.16
861
1.00
5.5
502
0.417
3.62
1600
3.95
1757
29.9
5.65
5.3
2.07
1085
15.2
2.09
5521
1482
22.7
6.92
3469
)
Vdss (L/kg)
g
F
(%)
98.7
93.7
72.6
a
b
Data shown are mean values obtained from three ICR mice. Data shown are mean values obtained from three Sprague-Dawley rats.
c
d
e
f
g
Formulation: 5% DMA in water. Formulation: 0.5% MC. Formulation: 38% DMA in water. Formulation: 0.5% CMC-Na. Bioavailability.
h
i
Data shown are mean values obtained from three Beagle dogs. Formulation: 25% DMA in water.
mice, rats, and dogs following intravenous injection (iv) and
oral (po) administration. Excellent pharmacokinetic properties
were observed in both mice and rats, which confirmed the
ability of our SAR strategy to enhance PK. Olaparib showed
lower bioavailability (10%) when used with the same
formulation as 139 in the rat pk study.
used as a single agent, we treated female BALB/c nude mice
bearing established subcutaneous MDA-MB-436 breast cancer
xenografts harboring a BRCA1 mutation (Figure 5). In order to
Compound 139 was neither an inhibitor of P-gp at
concentrations up to 10.0 μM nor an inhibitor of OCT2 at
concentrations up to 50 μM. At 30 μM, the highest
concentration tested for BCRP, BGB-290 showed 43%
inhibition of BCRP activity. Compound 139 did not inhibit
BCRP for other tested concentrations lower than 30 μM. The
IC50 values of compound 139 were measured to be 19.4 μM,
19.4 μM, 47.8 μM, and 23.9 μM of OATP1B1, OATP1B3,
OAT1, and OAT3, respectively. The steady-state unbound
Cmax of 0.774 μM is significantly lower than the IC50 values.
Therefore, compound 139 is unlikely to be a clinically relevant
inhibitor of the intestinal efflux transporters P-gp and BCRP,
hepatic uptake transporters OATP1B1/1B3, and renal uptake
transporters OCT2 and OAT1/3. Compound 139 may have
potential to overcome drug resistances caused by upregulation
of cellular drug efflux pumps of other Parp inhibitors.
Figure 5. Antitumor activity of 139 in a BRCA1-mutated (MDA-MB-
436) mouse xenograft model. MDA-MB-436 tumor cells (5 × 106)
were implanted subcutaneously in female BALB/c nude mice. When
tumors reached a mean volume of approximately 130 mm3 in size,
mice were randomly allocated into groups and treated for 28 days as
indicated. Tumor volume was measured twice weekly. Data are
presented as mean tumor volume
standard error of the mean
(SEM) with nine animals in each group. p.o., oral gavage; BID × 28,
bis in die/twice per day for 28 days.
In addition, compound 139 showed the ability to cross the
blood−brain barrier in C57BL/6 mice (Table 9) with brain-to-
plasma ratios in the range of 17−19%. Brain penetration is a
highly desirable property for PARP inhibitors because it
enhances the efficacy of temozolomide (TMZ) in the
treatment of glioblastoma.
achieve similar free drug exposure and Cmax with clinic
approved dose of olaparib, based on published human PK,1
in vitro plasma protein binding cross species,2 and also internal
mouse PK data, two doses (50 mg/kg and 25 mg/kg, BID) of
olaparib were conducted for comparison. Olaparib and
compound 139 were both BID orally administrated for 28
consecutive days, and then the treatment was interrupted, and
the tumors were further monitored for an additional 2 months.
As shown in Figure 5, compound 139 induced dose-dependent
antitumor activity BRCA1 mutated xenografts. Consistently,
the data suggested that 139 can achieve similar or even more
durable in vivo efficacy with olaparib at an approximately 16-
fold lower dose (50/3.1, 25/1.6). No mortality or significant
body weight loss was observed during the study.
Evaluation of in Vivo Antitumor Activity of 139. To
assess the in vivo antitumor activity of compound 139 when
a
Table 9. Brain to Plasma Ratio in Mice
concentration
b
b
sample time (h) plasma (ng/mL)
brain (ng/g)
brain/plasma (%)
1
2
4
3527
1333
707
613
252
135
17.4
18.9
19.1
The ability of 139 to potentiate the effect of methylating
chemotherapeutic agent TMZ was also evaluated in a xenograft
tumor model. Female BALB/c nude mice bearing H209 small
a
b
Dose: po at 10 mg/kg (0.5% MC). Data shown are mean values
obtained from three C57BL/6 mice.
H
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Drug Annotation
cell lung cancer xenografted tumors were treated with
compound 139 (0.75 mg/kg, po) BID for 28 consecutive
days, TMZ (50 mg/kg, po) QD for 5 consecutive days at the
start of the experiment and then again 14 days later, or a
combination of 139 and TMZ. As shown in Figure 6, no
selenium dioxide gave a mixture of β-diketone 46 and
monoketone 47.30 Sodium borohydride reduction of 47
provided racemic alcohol 48.
Synthesis of N-Alkylated Tetrahydrodiazepino-
carbazolones 51−67 and 69 (Scheme 2). To avoid
competing alkylation of nitrogen, α,α-dimethylation of ketones
20 and 24 with iodomethane was performed prior to final ring
closure.31 Dihydrodiazepinones 51 and 52 were obtained from
ketoesters 49 and 50 by condensation with hydrazine hydrate.
Selective alkylation at the indole nitrogen was achieved by
treatment of compounds 33, 37, and 38 with K2CO3 and alkyl
halides in DMF, affording 53−65.32 Compound 37 was
alkylated with THP-protected 2-bromoethanol followed by
THP removal to afford compound 66. The reaction of 37 and
epichlorohydrin to generate 67 did not work well. This
problem was successfully overcome by changing the base from
K2CO3 to NaH. Tosylation of compound 66 led to
intermediate 68. A nitrogen atom was introduced by SN2
displacement of the tosyloxy group with methylamine to afford
compound 69.33
S y n t h e s i s o f T e t r a h y d r o t e t r a a z a c y c l o -
heptafluorenones 77, 78, 97−107, 109, 110, and 114−
118 (Scheme 3). Ketoesters 77 and 78 were prepared in a
similar fashion to compound 33. Condensation products 71
and 72 were synthesized from N-Cbz 3,5-piperidinedione.
Intramolecular Heck cyclization and removal of the Cbz group
by catalytic hydrogenolysis in ethyl acetate provided ketoesters
75 and 76, which were condensed with hydrazine hydrate to
provide 77 and 78. Ketoesters 79−96 were obtained through
alkylation of 75 and 76 via reductive amination with carbonyl
derivatives in the presence of NaBH3CN.34 Amide derivative
was obtained by acylation with acyl chloride using DIPEA in
DCM or with acid using HATU and DIPEA in DMF.35
Sulfonamide 96 was obtained by treatment with cyclo-
propanesulfonyl chloride at 0 °C. Finally, compounds 97−
114 were prepared using hydrazine hydrate. N-Boc depro-
tection under acidic conditions or N-Cbz deprotection under
hydrogen afforded the amines 115−118 in good yield.
Synthesis of Fused Pentacyclic Tetrahydro-
tetraazacycloheptafluorenones 138, 139, and 146
(Scheme 4). To prepare the chiral key intermediate 126, we
started from D-proline 119, which was reacted with chloral to
give 120 in good yield as a single diastereomer at C3, with the
CCl3 group exclusively in cis with the hydrogen at C7a.
Stereoselective alkylation of oxazolidinone 120 with iodo-
methane in THF at −78 °C in the presence of LDA provided
121 as a single cis isomer in moderate yield. Treatment with
thionyl chloride in methanol furnished 122 in enantiomerically
pure form as a hydrochloride salt.36 Protection of 122 as the
Cbz derivative 123 and reduction with DIBAL-H afforded the
aldehyde 124.37 Ohira−Bestmann reaction of the aldehyde
with the diazophosphonate 125 yielded the key intermediate
alkyne 126.38 Commercially available aminobenzoate 3 was
subjected to Sonogashira coupling with 126 in the presence of
Pd(PPh3)4 and CuI to afford 128 with a yield of 59%.39
Cyclization with zinc powder and dibromoethane in refluxing
ethanol for 8 h provided 130, although with a yield of only
28%.40 Subsequent removal of N-Cbz by catalytic hydro-
genolysis in methanol provided indole 132. For preparation of
5-fluoro analog 133, the steps were modified as follows:
Aniline precursor, 4, was first protected with TFAA to give
127. Sonogashira coupling and cyclization of anilide 129 in the
presence of ZnBr2 provided indole 131 with a yield of 51%.
Figure 6. Antitumor activity of 139 in combination with TMZ in an
H209 mouse xenograft model. H209 tumor cells (1 × 107) were
implanted subcutaneously in female BALB/c nude mice. When
tumors reached a mean volume of approximately 175 mm3 in size,
mice were randomly allocated into four groups and treated as
indicated. 139 was orally dosed BID for 28 consecutive days, and
TMZ was orally dosed QD for 5 consecutive days (days 1−5) and
then again on days 15−19. Tumor volume was measured twice
weekly. Data are presented as mean tumor volume standard error of
the mean (SEM) with eight animals in each group.
significant antitumor activity was observed when 139 was used
as a single agent, while a robust tumor shrinkage effect was
observed in both the TMZ single agent and combination
treatment groups. The PR rate in the combination group was
much higher (50%) than that in the TMZ single agent group
(13%). Tumor relapse in the TMZ group occurred on day 48,
while no tumor relapse in the combination group occurred
until day 69. The combination of 139 and TMZ was well
tolerated, with no significant body weight loss compared with
TMZ monotherapy.
CHEMISTRY
■
Synthesis of Fused Tetracyclic Dihydrodiazepino-
indolones 33−48 (Scheme 1). Substituted anilines 3 and
4 are commercially available, while 5 was prepared from 1 via
esterification and reduction of the nitro group with iron/acetic
acid.27 Substituted anilines 3−5 and commercially available β-
diketones were condensed by heating in acetic acid to give
enaminones 6−17. Pd-catalyzed intramolecular Heck cycliza-
tion of 6−17 in the presence of Pd(OAc)2 and tri(o-
tolyl)phosphine afforded tetrahydrocarbazolone derivatives
20, 21, and 23−32.28 Compound 22 could not be obtained
by this route as the aniline starting material was difficult to
source or prepare. We therefore chose another route, even
though the overall yield was low. Specifically, commercially
available aniline 18 was diazotized with sodium nitrite under
acidic conditions, and reduction with tin(II) chloride yielded
hydrazine hydrochloride 19. Next, condensation with 1,3-
cyclohexanedione, followed by microwave-assisted Fischer
indole synthesis, provided the tetrahydrocarbazolone ring
system.29 Condensation of ketoesters 20−32 with hydrazine
hydrate in refluxing methanol produced the desired dihy-
drodiazepinones 33−45 in good yield. Oxidation of 38 with
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Drug Annotation
a
Scheme 1. Synthesis of Fused Tetracyclic Dihydrodiazepinoindolones 33−48
a
Reagents and conditions: (i) MeOH, SOCl2, 0 °C to reflux, 8 h; (ii) EtOH, AcOH, Fe, reflux, 3.5 h; (iii) AcOH, appropriate β-diketones, 80 °C, 8
h; (iv) Pd(OAc)2, TEA, (CH3C6H4)3P, CH3CN, 100 °C, 20 h; (v) H2O, HCl, NaNO2, 0 °C, 45 min, then SnCl2, HCl, 0 °C to room temperature
(rt), 1 h; (vi) TFA, cyclohexane-1,3-dione, microwave, 150 °C, 100 min; (vii) MeOH, AcOH, NH2NH2·H2O, reflux, 8 h; (viii) SeO2, dioxane,
reflux, 40 h; (ix) MeOH, NaBH4, 0 °C, 0.5 h.
For this substrate, we found that N-Cbz, which was removed
during TFA protection, migrated from the indole to the
pyrrolidine ring. Removal of the TFA protective group with
NaBH4 furnished indole 133.41
CONCLUSIONS
■
The discovery and characterization of a novel PARP inhibitor,
139, have been described here. Investigation of tetracyclic
dihydrodiazepinoindolone derivatives allowed the discovery of
three distinct series of PARP inhibitors with good enzymatic
and cellular activity; however, the solubility, hERG inhibition
activity, and/or plasma clearance was suboptimal. The
introduction of another fused ring led to identification of
compound 139, which is a potent and selective PARP-1 and
PARP-2 inhibitor, displaying more than 50-fold selectivity over
other PARP isoforms. It exhibits double-digit nanomolar
antiproliferative activity against several BRCA -mutant tumor
cells and displays high selectivity over cell lines without BRCA
mutations or HR defects. The PK properties are excellent, with
high oral bioavailability in mice and rats. Furthermore, 139 is
able to cross the blood−brain barrier. It is efficacious both as a
single agent and in combination with TMZ in MDA-MB-436
and H209 mouse xenograft models, respectively.
With the two indole intermediates in hand, alkylation of
nitrogen with methyl bromoacetate and cyclization of the
resulting esters with methanesulfonic acid at 60 °C were
carried out, affording 136 and 137.42 The resulting ketoesters
were cyclized with hydrazine hydrate to provide 138 and 139.
The absolute configuration of compound 139 sesquihydrate
was determined by single crystal X-ray analysis, which
confirmed the absolute configuration as (R)-C10a. A unit
cell of the crystal and an ORTEP plot of the crystal structure
are shown in Figure 7.
Racemic dihydrodiazepinone 146 was prepared by alkylation
of α-methyl proline ester 140 with bromoacetone, followed by
KO-t-Bu-promoted cyclization, yielding cyclic β-diketone
142.43 Reaction between 142 and aniline 4 provided no
desired product. This issue was addressed by conversion of
142 to enol ether 143, followed by condensation with aniline 4
to give enaminoketone 144.44 Intramolecular Heck cyclization
gave ketoester 145. Condensation with hydrazine hydrate then
provided the desired product 146.
Many PARP inhibitors have demonstrated good efficacy via
synthetic lethality interactions on treatment of tumors with
BRCA1 or BRCA2 mutations.45,46 The biological and
pharmacological properties of 139 warranted its further
evaluation in clinical settings. On the basis of these
encouraging results, more than 10 clinical trials have been
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Drug Annotation
a
Scheme 2. Synthesis of N-Alkylated Tetrahydrodiazepinocarbazolones 51−67 and 69
a
Reagents and conditions: (i) CH3I, THF, t-BuOK, 0 °C to rt, 3 h; (ii) MeOH, AcOH, NH2NH2·H2O, reflux, 30 min; (iii) K2CO3, DMF,
appropriate alkyl halide, 0−70 °C, 4 h; (iv) K2CO3, DMF, 2-(2-bromoethoxy)tetrahydropyran, 70 °C, 12 h, then PTSA, MeOH, rt, 1 h; (v) NaH,
DMF, epichlorohydrin, N2, 0 °C to rt, 1 h; (vi) TsCl, DIPEA, DMAP, THF, rt, 2 h; (vii) MeNH2, sealed tube, 40 °C, 18 h.
i.d., 5 μm, Gemini NX- C18) at a flow rate of 20 mL/min and an
injection volume of 2 mL at room temperature with UV detection at
214 and 254 nm. HPLC spectrometer detector for purity analysis of
compound 139: Waters 2695 Empower 3 data handling software.
Detector: UV 254 nm. Mobile phase: A, pH 6.5 buffer; B, ACN.
Column: Waters XBridge C18, 4.6 mm × 150 mm, 5 μm. Gradient
method: flow rate 1.0 mL/min. Column temperature: 35 °C. Injection
volume:10 μL. HPLC spectrometer detector for purity analysis of
compounds except compound 139: instrument, Waters Acquity
UPLC H-Class. Column, BEH C18, 2.1 mm × 50 mm, 1.7 μm.
Column temperature, 40 °C. Injection volume, 2 μL. Detection
wavelength, UV 214, 254, 280 nm. Run time, 5.5 min. Flow rate, 0.8
mL/min. Diluent, CAN. Mobile phase A: H2O (0.05% TFA). Mobile
phase B: ACN (0.05% TFA). Gradient method.
Synthesis of Representative Key Examples. Methyl 2-
Bromo-3-((3-oxocyclohex-1-en-1-yl)amino)benzoate (6).
Methyl 3-amino-2-bromobenzoate 3 (2.39 g, 10.0 mmol) and
cyclohexane-1,3-dione (1.12 g, 10.0 mmol) were dissolved in 10
mL of acetic acid at 25 °C, under nitrogen. The mixture was stirred at
80 °C for 8 h. The resultant solid was purified by chromatography on
silica gel (elution with hexane/ethyl acetate) to afford 2.46 g (76%) of
methyl 2-bromo-3-((3-oxocyclohex-1-en-1-yl)amino)benzoate as a
tan foam. 1H NMR (CDCl3-d1) δ 7.53−7.55 (m, 2H), 7.37 (dd,
1H, J = 7.2, 8.4 Hz), 6.34 (br s, 1H), 5.57 (s, 1H), 3.95 (s, 3H),
2.56−2.59 (m, 2H), 2.40−2.42 (m, 2H), and 2.08−2.11 (m, 2H). MS
(ESI) m/e [M + 1]+ 324.0.
initiated using compound 139 as single or combination with
other drugs, such as single drug in participants with advanced
solid tumors (NCT03333915), in combination with TMZ in
participants with locally advanced or metastatic solid tumors
(NCT02660034), in combination with radiation and/or TMZ
in participants with newly diagnosed or recurrent glioblastoma
(NCT03150862), and a phase III clinical trial as a
maintenance therapy in platinum-sensitive ovarian cancer and
in gastric cancer (NCT03519230).47,48
EXPERIMENTAL SECTION
■
General. Efforts have been made to ensure accuracy with respect
to numbers used (for example, amounts, temperature, etc.), but some
experimental errors should be accounted for. All solvents and
chemicals used were reagent grade. Unless indicated otherwise, the
reactions described below were performed under a positive pressure
of nitrogen or argon or with a drying tube in anhydrous solvents; the
reaction flasks were fitted with rubber septa for the introduction of
substrates and reagents via syringe, and glassware was oven-dried and/
or heat dried. 1H NMR spectra were recorded on an Agilent
1
instrument operating at 400 or 600 MHz. H NMR spectra were
obtained using CDCl3, CD3OD, D2O, and DMSO-d6 as a solvent and
tetramethylsilane (0.00 ppm) or residual solvent (CDCl3, 7.25 ppm;
CD3OD, 3.31 ppm; D2O, 4.79 ppm; DMSO-d6, 2.50 ppm) as the
reference standard. Coupling constants, when given, are reported in
hertz (Hz). LC−MS spectrometer (Agilent 1260) detector: MWD
(190−400 nm). Mass detector: 6120 SQ. Mobile phase: A,
acetonitrile with 0.1% formic acid; B, water with 0.1% formic acid.
Column: Poroshell 120 EC-C18, 4.6 mm × 50 mm, 2.7 μm. Gradient
method: 5%−95% B in 1.5 min, 95% B for 0.5 min, 95%−5% B from
2.0 to 2.1 min, 5% B from 2.1 to 3.0 min. Flow rate: 1.8 mL/min.
Preparative HPLC was conducted on a column (150 mm × 21.2 mm
Methyl 4-Oxo-2,3, 4,9-tetrahydro-1H-carbazole-5-carboxy-
late (20). A mixture of methyl 2-bromo-3-(3-oxocyclohex-1-
enylamino)benzoate (compound 6; 0.97 g, 3.0 mmol), palladium
acetate (0.14 g, 0.6 mmol), tri-o-tolylphosphine (0.73 g, 2.4 mmol),
and triethylamine (0.38 g, 3.6 mmol) in acetonitrile (10 mL) was
heated in a sealed tube and flushed with nitrogen at 100 °C for 20 h.
The cooled reaction mixture was diluted with DCM (3 × 50 mL) and
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Drug Annotation
a
Scheme 3. Synthesis of Tetrahydrotetraazacycloheptafluorenones 77, 78, 97−107, 109, 110, and 114−118
a
Reagents and conditions: (i) AcOH, benzyl 3,5-dioxopiperidine-1-carboxylate, 70 °C, 8 h; (ii) Pd(OAc)2, TEA, (CH3C6H4)3P, CH3CN, 100 °C,
9 h; (iii) H2, Pd/C, EtOAc, rt, overnight; (iv) MeOH, AcOH, NH2NH2·H2O, reflux, 8 h; (v) appropriate aldehyde or acetone, NaBH3CN,
CH3OH, AcOH, rt, overnight; (vi) appropriate acyl chloride or cyclopropanesulfonyl chloride, DIPEA, DCM, 0 °C, 1 h; (vii) appropriate acid,
HATU, DIPEA, DMF, rt, 16 h; (viii) 4 M HCl/EtOAc, EtOAc, rt, 5 h; (ix) H2, Pd/C, MeOH, rt, overnight.
water (10 mL). The organic layer was separated, washed with water,
dried (Na2SO4), and concentrated. The remaining residue was
chromatographed on silica gel, eluted with gradient of 0%−100%
Hz), 2.77−2.79 (m, 2H), 2.35−2.37 (m, 2H), 1.92−1.93 (m, 2H).
MS (ESI) m/e [M + 1]+ 226.0. HPLC purity: 99.87% (254 nm).
8-Fluoro-2,3,5,10-tetrahydro[1,2]diazepino[3,4,5,6-def ]-
carbazol-6(1H)-one (34). Compound 34 was prepared from methyl
3-amino-2-bromo-5-fluorobenzoate (compound 4) according to the
procedures for compound 33. 1H NMR (DMSO-d6) δ 11.83 (s, 1H),
10.03 (s, 1H), 7.23 (dd, 1H, J = 10.8, 1.8 Hz), 7.12 (dd, 1H, J = 11.4,
1.8 Hz), 2.76−2.78 (m, 2H), 2.35−2.37 (m, 2H), 1.91−1.93 (m, 2H).
MS (ESI) m/e [M + 1]+ 244.0. HPLC purity: 99.69% (254 nm).
9-Fluoro-2,3,5,10-tetrahydro[1,2]diazepino[3,4,5,6-def ]-
carbazol-6(1H)-one (35). Compound 35 was prepared from methyl
3-amino-4-fluorobenzoate (compound 18) according to the proce-
dures for compound 33. 1H NMR (DMSO-d6) δ 12.26 (s, 1H), 9.93
(s, 1H), 7.43 (dd, 1H, J = 8.4, 4.8 Hz), 6.96 (dd, 1H, J = 13.3, 8.4
Hz), 2.82−2.85 (m, 2H), 2.41−2.43 (m, 2H), 1.97−1.99 (m, 2H).
MS (ESI) m/e [M + 1]+ 244.0. HPLC purity: 97.59% (254 nm).
1
EtOAc in hexane to afford the title compound (0.61 g, 84%). H
NMR (CDCl3-dl) δ 9.47 (s, 1H), 7.36−7.40 (m, 2H), 7.22 (t, 1H, J =
7.8 Hz), 2.90−2.92 (m, 2H), 2.51−2.54 (m, 2H), 2.14−2.16 (m, 2H).
MS (ESI) m/e [M + 1]+ 244.0.
2,3,5,10-Tetrahydro[1,2]diazepino[3,4,5,6-def ]carbazol-
6(1H)-one (33). A solution of methyl 4-oxo-2,3,4,9-tetrahydro-1H-
carbazole-5-carboxylate (compound 20; 73 mg, 0.3 mmol), acetic acid
(0.15 mL, 2.6 mmol), and hydrazine hydrate (0.86 mL, 1.5 mmol) in
methanol (4 mL) was heated to reflux. After 8 h, the solid was
collected by hot filtration and washed with water, EtOAc, and DCM
1
to give the target compound (42 mg, 62%). H NMR (DMSO-d6) δ
11.70 (s, 1H), 9.79 (s, 1H), 7.36−7.38 (m, 2H), 7.05 (t, 1H, J = 7.8
L
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Drug Annotation
a
Scheme 4. Synthesis of Fused Pentacyclic Tetrahydrotetraazacycloheptafluorenones 138, 139, and 146
a
Reagents and conditions: (i) CCl3CHO, CHCl3, reflux, 6 h; (ii) LDA, THF, −78 °C, 0.5 h, then CH3I, −78 °C to rt, 1 h; (iii) SOCl2, MeOH, 0
°C to reflux, overnight; (iv) CbzCl, Na2CO3, THF, H2O, 0 °C to rt, overnight; (v) DCM, DIBAL-H, −78 °C, 2 h; (vi) K2CO3, MeOH, 125, 0 °C
to rt, overnight; (vii) TFAA, K2CO3, DCM, 0 °C to rt, overnight; (viii) Pd(PPh3)4 or Pd(PPh3)2Cl2, CuI, toluene or DMF, TEA or tetramethyl
guanidine, 126, 100 °C, 18 h; (ix) Zn, BrCH2CH2Br, EtOH, reflux, 8 h; (x) ZnBr2, toluene, 80 °C, 15 h; (xi) H2, Pd/C, MeOH, rt, 2 h; (xii)
MeOH, NaBH4, reflux, 4 h; (xiii) methyl bromoacetate, MeCN, DIPEA, rt, 20 h; (xiv) MeSO3H, 60 °C, 1 h; (xv) MeOH, AcOH, NH2NH2·H2O,
reflux, 5 h; (xvi) bromoacetone, DIPEA, MeCN, 80 °C, 3 h; (xvii) KO-t-Bu, THF, reflux, 4 h; (xviii) HC(OMe)3, MeOH, PTSA, reflux, 12 h; (xix)
4, PTSA, toluene, reflux, 4 h; (xx) Pd(OAc)2, TEA, (CH3C6H4)3P, CH3CN, reflux, 3 h.
Figure 7. X-ray crystal structure of 139 sesquihydrate: (A) unit cell of a single crystal; (B) ORTEP plot.
M
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Drug Annotation
8-Fluoro-2-methyl-2,3,5,10-tetrahydro[1,2]diazepino-
[3,4,5,6-def]carbazol-6(1H)-one (36). Compound 36 was pre-
pared from methyl 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 33. 1H NMR (DMSO-d6)
δ 12.1 (s, 1H), 10.0 (s, 1H), 7.26 (dd, 1H, J = 9.6, 2.4 Hz), 7.16 (dd,
1H, J = 10.2, 2.4 Hz), 2.87−2.91 (m, 1H), 2.40−2.43 (m, 2H), 2.15−
2.19 (m, 2H), 1.10 (d, 3H, J = 6.0 Hz). MS (ESI) m/e [M + 1]+
258.0. HPLC purity: 100% (254 nm).
2,2-Dimethyl-2,3,5,10-tetrahydro[1,2]diazepino[3,4,5,6-
def]carbazol-6(1H)-one (37). Compound 37 was prepared from
methyl 3-amino-2-bromobenzoate (compound 3) according to the
procedures for compound 33. 1H NMR (DMSO-d6) δ 11.69 (s, 1H),
9.84 (s, 1H), 7.42 (d, 2H, J = 7.8 Hz), 7.09 (t, 1H, J = 7.8 Hz), 2.67
(s, 2H), 2.23 (s, 2H), 1.05 (s, 6H). MS (ESI) m/e [M + 1]+ 254.0.
HPLC purity: 100% (254 nm).
8-Fluoro-2,2-dimethyl-2,3,5,10-tetrahydro[1,2]diazepino-
[3,4,5,6-def]carbazol-6(1H)-one (38). Compound 38 was pre-
pared from methyl 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 33. 1H NMR (DMSO-d6)
δ 11.84 (s, 1H), 10.08 (s, 1H), 7.29 (dd, 1H, J = 10.8, 1.8 Hz), 7.18
(dd, 1H, J = 11.4, 1.8 Hz), 2.63−2.67 (m, 2H), 2.25−2.26(m, 2H),
1.06 (s, 6H). MS (ESI) m/e [M + 1]+ 272.0. HPLC purity: 99.21%
(254 nm).
7-Fluoro-2,2-dimethyl-2,3,5,10-tetrahydro[1,2]diazepino-
[3,4,5,6-def]carbazol-6(1H)-one (39). Compound 39 was pre-
pared from methyl 3-amino-2-bromo-6-fluorobenzoate (compound
5) according to the procedures for compound 33. 1H NMR (DMSO-
d6) δ 11.75 (s, 1H), 9.60 (s, 1H), 7.41 (dd, 1H, J = 9.0, 3.0 Hz), 6.87
(dd, 1H, J = 12.0, 3.0 Hz), 2.65 (s, 2H), 2.21 (s, 2H), 1.05 (s, 6H).
MS (ESI) m/e [M + 1]+ 272.0. HPLC purity: 100% (254 nm).
8-Fluoro-3,3-dimethyl-2,3,5,10-tetrahydro[1,2]diazepino-
[3,4,5,6-def]carbazol-6(1H)-one (40). Compound 40 was pre-
pared from methyl 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 33. 1H NMR (DMSO-d6)
δ 11.7 (s, 1H), 10.1 (s, 1H), 7.26 (dd, 1H, J = 9.6, 2.4 Hz), 7.16 (dd,
1H, J = 10.2, 2.4 Hz), 2.81−2.83 (m, 2H), 1.74−1.81 (m, 2H), 1.16
(s, 6H). MS (ESI) m/e [M + 1]+ 272.0. HPLC purity: 99.34% (254
nm).
2-Fluoro-5,6,7,8-tetrahydro-4H-4,9,10-triazaindeno[2,1,7-
kla]heptalen-11(10H)-one (45). Compound 45 was prepared from
3-amino-2-bromo-5-fluorobenzoate (compound 4) according to the
procedures for compound 33. 1H NMR (DMSO-d6) δ 11.84 (s, 1H),
10.02 (s, 1H), 7.17−7.22 (m, 2H), 2.95−2.97 (m, 2H), 2.56−2.58
(m, 2H), 1.77−1.88 (m, 4H). MS (ESI) m/e [M + 1]+ 258.0. HPLC
purity: 95.54% (214 nm).
8-Fluoro-2,2-dimethyl-2,3-dihydro[1,2]diazepino[3,4,5,6-
def]carbazole-1,6(5H,10H)-dione (47). To a solution of 38 (0.5 g,
1.8 mmol) in anhydrous dioxane (25 mL) was added SeO2 (0.32 g,
2.7 mmol). The mixture was refluxed for 40 h and filtered through
Celite. The solid material was thoroughly washed with Et2O. The
filtrate was concentrated, and the residue was chromatographed to
give product (200 mg, yield 38%) as a solid. 1H NMR (DMSO-d6) δ
12.70 (s, 1H), 10.90 (s, 1H), 7.41 (dd, 1H, J = 10.2, 1.8 Hz), 7.09
(dd, 1H, J = 9.6, 1.8 Hz), 2.87 (s, 2H), 1.23 (s, 6H). MS (ESI) m/e
[M + 1]+ 286.0. HPLC purity: 98.51% (254 nm).
8-Fluoro-1-hydroxy-2,2-dimethyl-2,3,5,10-tetrahydro[1,2]-
diazepino[3,4,5,6-def]carbazol-6(1H)-one (48). To a solution of
8-fluoro-2,2-dimethyl-2,3-dihydro[1,2]diazepino[3,4,5,6-def ]-
carbazole-1,6(5H,10H)-dione (compound 47; 50 mg, 0.18 mmol) in
10 mL of MeOH was added NaBH4 (6.8 mg, 0.18 mmol) at 0 °C.
The mixture was stirred for additional 30 min. The solution was
poured into ice−water and extracted with EtOAc (5 mL × 3). The
organic layers were combined, washed with H2O (5 mL × 3) and
brine (5 mL × 3), dried over Na2SO4, and filtered. The filtrate was
concentrated, and the residue was chromatographed to give a crude
product, which was then purified by pre-HPLC to give the product (5
mg, 10%) as a yellow solid. 1H NMR (DMSO-d6) δ 11.9 (s, 1H), 10.1
(s, 1H), 7.23 (dd, 1H, J = 9.2, 2.0 Hz), 7.17 (dd, 1H, J = 10.8, 2.4
Hz), 5.68 (d, 1H, J = 6.0 Hz), 5.49 (d, 1H, J = 6.0 Hz), 2.31 (s, 2H),
1.00 (s, 3H), 0.88 (s, 3H). MS (ESI) m/e [M + 1]+ 288.0.
Methyl 9-Methyl-4-oxo-2,3,4,9-tetrahydro-1H-carbazole-5-
carboxylate (49). To a solution of methyl 4-oxo-2,3,4,9-tetrahy-
dro-1H-carbazole-5-carboxylate (compound 20; 0.27 g, 1 mmol) in
THF (5 mL) at 0 °C under N2 was added potassium tert-butoxide
(0.12 g, 1.05 mmol). The reaction mixture was stirred for 30 min
followed by the addition of methyl iodide (0.76 g, 5.0 mmol). After 3
h, the reaction mixture was concentrated to a residue and partitioned
between EtOAc (40 mL) and 1 N HCl (5 mL). The layers were
shaken and separated. The organic layer was washed with 1 N HCl (2
× 80 mL) and brine (2 × 10 mL), dried over Na2SO4, filtered, and
concentrated to give a solid (0.46 g, crude). The solid was used in the
next step without further purification. 1H NMR (DMSO-d6) δ 7.35−
7.39 (m, 2H), 7.29 (t, 1H, J = 7.2 Hz), 4.01 (s, 3H), 3.72 (s, 3H),
2.93−2.95 (m, 2H), 2.54−2.56 (m, 2H), 2.23−2.26 (m, 2H). MS
(ESI) m/e [M + 1]+ 258.0.
10-Methyl-2,3,5,10-tetrahydro[1,2]diazepino[3,4,5,6-def ]-
carbazol-6(1H)-one (51). Compound 51 was prepared from methyl
9-methyl-4-oxo-2,3,4,9-tetrahydro-1H-carbazole-5-carboxylate (com-
pound 49) according to the procedures for compound 33. 1H
NMR (DMSO-d6) δ 9.88 (s, 1H), 7.55 (d, 1H, J = 7.8 Hz,), 7.45 (d,
1H, J = 7.8 Hz,), 7.15 (t, 1H, J = 7.8 Hz), 3.70 (s, 3H), 2.77−2.79 (m,
2H), 2.35−2.37 (m, 2H), 1.92−1.93 (m, 2H). MS (ESI) m/e [M +
1]+ 240.0. HPLC purity: 95.3% (214 nm).
2,2,10-Trimethyl-2,3,5,10-tetrahydro[1,2]diazepino[3,4,5,6-
def]carbazol-6(1H)-one (52). Compound 52 was prepared from
methyl 2,2-dimethyl-4-oxo-2,3,4,9-tetrahydro-1H-carbazole-5-carbox-
ylate (compound 24) according to the procedures for compound 51.
1H NMR (DMSO-d6) δ 9.86 (s, 1H), 7.42−7.53 (m, 2H), 7.12 (t,
1H, J = 7.8 Hz), 3.66 (s, 3H), 2.67 (s, 2H), 2.20 (s, 2H), 1.04 (s, 6H).
MS (ESI) m/e [M + 1]+ 268.0. HPLC purity: 100% (254 nm).
10-(2-(Dimethylamino)ethyl)-2,3,5,10-tetrahydro[1,2]-
diazepino[3,4,5,6-def]carbazol-6(1H)-one (53). Compound 53
was prepared from 5,6,7,9-tetrahydro[1,2]diazepino[3,4,5,6-def ]-
carbazol-10(4H)-one (compound 33) according to the procedures
for compound 59. 1H NMR (DMSO-d6) δ 9.91 (s, 1H), 7.58 (d, 1H,
J = 8.4 Hz), 7.47 (d, 1H, J = 7.8 Hz), 7.16 (dd, 1H, J = 8.4, 7.8 Hz),
4.22−4.25 (m, 2H), 2.88−2.90 (m, 2H), 2.41−2.47 (m, 4H), 2.19 (s,
8-Fluoro-2-isopropyl-2,3,5,10-tetrahydro[1,2]diazepino-
[3,4,5,6-def]carbazol-6(1H)-one (41). Compound 41 was pre-
pared from methyl 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 33. 1H NMR (DMSO-d6)
δ 11.82 (s, 1H), 10.04 (s, 1H), 7.15−7.29 (m, 2H), 2.82−2.85 (m,
1H), 2.57−2.61 (m, 1H), 2.41−2.44 (m, 1H), 2.19−2.44 (m, 1H),
1.82−1.83 (m, 1H), 1.69−1.71 (m, 1H), 0.97 (d, 6H, J = 9.0 Hz). MS
(ESI) m/e [M + 1]+ 286.0. HPLC purity: 100% (254 nm).
8-Fluoro-2-phenyl-2,3,5,10-tetrahydro[1,2]diazepino-
[3,4,5,6-def]carbazol-6(1H)-one (42). Compound 42 was pre-
pared from methyl 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 33. 1H NMR (DMSO-d6)
δ 11.92 (s, 1H), 10.11 (s, 1H), 7.18−7.40 (m, 7H), 3.31−3.32 (m,
1H), 3.05−3.07 (m, 2H), 2.74−2.77 (m, 1H), 2.55−2.56 (m, 1H).
MS (ESI) m/e [M + 1]+ 320.0. HPLC purity: 98.89% (254 nm).
2-(4-(Dimethylamino)phenyl)-8-fluoro-2,3,5,10-tetrahydro-
[1,2]diazepino[3,4,5,6-def]carbazol-6(1H)-one (43). Compound
43 was prepared from methyl 3-amino-2-bromo-5-fluorobenzoate
1
(compound 4) according to the procedures for compound 33. H
NMR (DMSO-d6) δ 11.89 (s, 1H), 10.09 (s, 1H), 7.29 (dd, 1H, J =
9.0, 1.8 Hz), 7.17−7.19 (m, 3H), 6.69 (d, 2H, J = 8.4 Hz), 3.19−3.21
(m, 1H), 2.98−3.00 (m, 2H), 2.86 (s, 6H), 2.66−2.69 (m, 1H),
2.48−2.51 (m, 1H). MS (ESI) m/e [M + 1]+ 363.0. HPLC purity:
95.27% (254 nm).
5,6,7,8-Tetrahydro-4H-4,9,10-triazaindeno[2,1,7-kla]-
heptalen-11(10H)-one (44). Compound 44 was prepared from
methyl 3-amino-2-bromobenzoate (compound 3) according to the
procedures for compound 33. 1H NMR (DMSO-d6) δ 11.74 (s, 1H),
9.79 (s, 1H), 7.46 (d, 1H, J = 7.8 Hz), 7.38 (d, 1H, J = 7.8 Hz), 7.07
(t, 1H, J = 7.8 Hz), 2.98−3.01 (m, 2H), 2.59−2.61 (m, 2H), 1.89−
1.94 (m, 2H), 1.70−1.84 (m, 2H). MS (ESI) m/e [M + 1]+ 240.0.
HPLC purity: 97.34% (254 nm).
N
https://dx.doi.org/10.1021/acs.jmedchem.0c01346
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
6H), 1.92−2.00 (m, 2H). MS (ESI) m/e [M + 1]+ 297.0. HPLC
purity: 99.9% (254 nm).
Compound 61 was prepared from 6,6-dimethyl-5,6,7,9-tetrahydro-
[1,2]diazepino[3,4,5,6-def ]carbazol-10(4H)-one (compound 37) ac-
cording to the procedures for compound 59. 1H NMR (DMSO-d6) δ
9.89 (s, 1H), 7.56 (d, 1H, J = 7.8 Hz), 7.46 (d, 1H, J = 7.8 Hz), 7.15
(t, 1H, J = 7.8 Hz), 4.15−4.17 (m, 2H), 2.76 (s, 2H), 2.62−2.64 (m,
2H), 2.43−2.46 (m, 4H), 2.25 (s, 2H), 1.07 (s, 6H), 0.81 (t, 6H, J =
7.2 Hz). MS (ESI) m/e [M + 1]+ 353.0. HPLC purity: 99.92% (254
nm).
10-(2-(Dibenzylamino)ethyl)-2,2-dimethyl-2,3,5,10-
tetrahydro[1,2]diazepino[3,4,5,6-def]carbazol-6(1H)-one (62).
Compound 62 was prepared from 6,6-dimethyl-5,6,7,9-tetrahydro-
[1,2]diazepino[3,4,5,6-def ]carbazol-10(4H)-one (compound 37) ac-
cording to the procedures for compound 59. 1H NMR (DMSO-d6) δ
9.87 (s, 1H), 7.40 (d, 1H, J = 7.2 Hz), 7.31 (d, 1H, J = 7.6 Hz), 7.13−
7.22 (m, 10 H), 7.00 (dd, 1H, J = 7.2, 7.6 Hz), 4.15−4.18 (m, 2H),
3.61 (br s, 4H), 2.56−2.59 (m, 2H), 2.24 (s, 2H), 2.11 (s, 2H), 0.87
(s, 6H). MS (ESI) m/e [M + 1]+ 477.0. HPLC purity: 98.80% (254
nm).
10-(2-(Diethylamino)ethyl)-2,3,5,10-tetrahydro[1,2]-
diazepino[3,4,5,6-def]carbazol-6(1H)-one (54). Compound 54
was prepared from 5,6,7,9-tetrahydro[1,2]diazepino[3,4,5,6-def ]-
carbazol-10(4H)-one (compound 33) according to the procedures
for compound 59. 1H NMR (DMSO-d6) δ 9.88 (s, 1H), 7.56 (d, 1H,
J = 7.2 Hz), 7.45 (d, 1H, J = 7.2 Hz), 7.15 (t, 1H, J = 7.2 Hz), 4.16−
4.18 (m, 2H), 2.89−2.91 (m 2H), 2.63−2.65 (m, 2H), 2.40−2.45 (m,
6H), 1.98−2.00 (m, 2H), 0.82 (t, 6H, J = 7.2 Hz). MS (ESI) m/e [M
+ 1]+ 325.0. HPLC purity: 99.55% (254 nm).
4-(2-(Dibenzylamino)ethyl)-5,6,7,9-tetrahydro[1,2]-
diazepino[3,4,5,6-def]carbazol-10(4H)-one (55). Compound 55
was prepared from 33 according to the procedures for compound 59.
1H NMR (DMSO-d6) δ 9.89 (s, 1H), 7.40 (d, 1H, J = 7.6 Hz), 7.29
(d, 1H, J = 7.6 Hz), 7.06−7.19 (m, 10H), 6.98 (t, 1H, J = 7.6 Hz),
4.18−4.21 (m, 2H), 3.55 (br s, 4H), 2.61−2.64 (m, 2H), 2.46−2.48
(m, 2H), 2.27−2.31 (m, 2H), 1.78−1.81 (m, 2H). MS (ESI) m/e [M
+ 1]+ 449.0. HPLC purity: 99.18% (254 nm).
2,2-Dimethyl-10-(2-(pyrrolidin-1-yl)ethyl)-2,3,5,10-
tetrahydro[1,2]diazepino[3,4,5,6-def]carbazol-6(1H)-one (63).
Compound 63 was prepared from 6,6-dimethyl-5,6,7,9-tetrahydro-
[1,2]diazepino[3,4,5,6-def ]carbazol-10(4H)-one (compound 37) ac-
cording to the procedures for compound 59. 1H NMR (DMSO-d6) δ
9.91 (s, 1H), 7.58 (d, 1H, J = 7.8 Hz), 7.47 (d, 1H, J = 7.2 Hz), 7.17
(dd, 1H, J = 7.2, 7.8 Hz), 4.23−4.26 (m, 2H), 2.69−2.74 (m 4H),
2.45 (br s, 4H), 2.25 (br s, 2H), 1.64−1.65 (m, 4H), 1.07 (s, 6H). MS
(ESI) m/e [M + 1]+ 351.0. HPLC purity: 100% (254 nm).
2,2-Dimethyl-10-(2-(piperidin-1-yl)ethyl)-2,3,5,10-
tetrahydro[1,2]diazepino[3,4,5,6-def]carbazol-6(1H)-one (64).
Compound 64 was prepared from 6,6-dimethyl-5,6,7,9-tetrahydro-
[1,2]diazepino[3,4,5,6-def ]carbazol-10(4H)-one (compound 37) ac-
cording to the procedures for compound 59. 1H NMR (DMSO-d6) δ
9.89 (s, 1H), 7.57 (d, 1H, J = 8.4 Hz), 7.47 (d, 1H, J = 7.8 Hz), 7.17
(dd, 1H, J = 8.4, 7.8 Hz), 4.20−4.22 (m, 2H), 2.89 (br s, 2H), 2.50
(br s, 2H), 2.35 (br s, 4H), 2.25 (br s, 2H), 1.35−1.46 (m, 6H), 1.07
(s, 6H). MS (ESI) m/e [M + 1]+ 365.0. HPLC purity: 100% (254
nm).
10-(2-(Pyrrolidin-1-yl)ethyl)-2,3,5,10-tetrahydro[1,2]-
diazepino[3,4,5,6-def]carbazol-6(1H)-one (56). Compound 56
was prepared from 5,6,7,9-tetrahydro[1,2]diazepino[3,4,5,6-def ]-
carbazol-10(4H)-one (compound 33) according to the procedures
for compound 59. 1H NMR (DMSO-d6) δ 9.91 (s, 1H), 7.57 (d, 1H,
J = 7.8 Hz), 7.46 (d, 1H, J = 7.8 Hz), 7.16 (t, 1H, J = 7.8 Hz), 4.25−
4.27 (m, 2H), 2.87−2.89 (m 2H), 2.72 (br s, 2H), 2.41−2.47 (m,
6H), 1.98−2.00 (m, 2H), 1.66 (br s, 4H). MS (ESI) m/e [M + 1]+
323.0.
10-(2-(Piperidin-1-yl)ethyl)-2,3,5,10-tetrahydro[1,2]-
diazepino[3,4,5,6-def]carbazol-6(1H)-one (57). Compound 57
was prepared from 5,6,7,9-tetrahydro[1,2]diazepino[3,4,5,6-def ]-
carbazol-10(4H)-one (compound 33) according to the procedures
for compound 59. 1H NMR (DMSO-d6) δ 9.89 (s, 1H), 7.57 (d, 1H,
J = 7.8 Hz), 7.46 (d, 1H, J = 7.8 Hz), 7.16 (t, 1H, J = 7.8 Hz), 4.23−
4.25 (m, 2H), 2.89−2.91 (m 2H), 2.51−2.55 (m, 2H), 2.36−2.43 (m,
6H), 1.98−2.00 (m, 2H), 1.36−1.45 (m, 6H). MS (ESI) m/e [M +
1]+ 337.0.
10-(2-Morpholinoethyl)-2,3,5,10-tetrahydro[1,2]diazepino-
[3,4,5,6-def]carbazol-6(1H)-one (58). Compound 58 was pre-
pared from 5,6,7,9-tetrahydro[1,2]diazepino[3,4,5,6-def ]carbazol-
10(4H)-one (compound 33) according to the procedures for
2,2-Dimethyl-10-(2-morpholinoethyl)-2,3,5,10-tetrahydro-
[1,2]diazepino[3,4,5,6-def]carbazol-6(1H)-one (65). Compound
65 was prepared from 6,6-dimethyl-5,6,7,9-tetrahydro[1,2]diazepino-
[3,4,5,6-def ]carbazol-10(4H)-one (compound 37) according to the
1
compound 59. H NMR (DMSO-d6) δ 9.90 (s, 1H), 7.59 (d, 1H, J
1
procedures for compound 59. H NMR (DMSO-d6) δ 9.91(s, 1H),
= 7.8 Hz), 7.47 (d, 1H, J = 7.8 Hz), 7.16 (t, 1H, J = 7.8 Hz), 4.25−
4.27 (m, 2H), 3.52−3.54 (m, 4H), 2.89−2.91 (m, 2H), 2.58−2.60
(m, 2H), 2.41−2.43 (m, 6H), 1.99−2.01 (m, 2H). MS (ESI) m/e [M
+ 1]+ 339.0.
7.59 (d, 1H, J = 8.4 Hz), 7.47 (d, 1H, J = 7.8 Hz), 7.16 (dd, 1H, J =
8.4, 7.8 Hz), 4.24−4.26 (m, 2H), 3.53−3.54 (m, 4H), 2.77 (br s, 2H),
2.57−2.59 (m, 2H), 2.42 (br s, 4H), 2.25 (br s, 2H), 1.08 (s, 6H). MS
(ESI) m/e [M + 1]+ 367.0. HPLC purity: 100% (254 nm).
10-(2-(Dimethylamino)ethyl)-2,2-dimethyl-2,3,5,10-
tetrahydro[1,2]diazepino[3,4,5,6-def]carbazol-6(1H)-one (59).
To a cooled solution (0 °C) of 37 (94 mg, 0.37 mmol) and DMF (10
mL) were slowly added K2CO3 (205 mg, 1.48 mmol) and then N,N-
dimethylamino-2-chloroethane (53 mg, 0.37 mmol). The resulting
solution was stirred at 70 °C for 4 h. The solution was allowed to
cool, and water was added (10 mL). The mixture was extracted with
ethyl acetate (2 × 20 mL). The organic layers were combined, dried
over Na2SO4, and filtered. The filtrate was concentrated, and the
residue was chromatographed to give the product (90 mg, 75%) as a
10-(2-Hydroxyethyl)-2,2-dimethyl-2,3,5,10-tetrahydro[1,2]-
diazepino[3,4,5,6-def]carbazol-6(1H)-one (66). To a solution of
37 (100 mg, 0.39 mmol) in dry DMF (8 mL) was added 2-(2-
bromoethoxy)tetrahydro-2H-pyran (194 mg, 1.17 mmol). K2CO3
(215 mg, 1.6 mmol) was added, and the mixture was heated at 70
°C for 12 h. Then water (100 mL) was added to the mixture, which
was then extracted with EtOAc (50 mL × 3). The organic layers were
combined, washed with brine, dried over Na2SO4, and concentrated
to provide a crude yellow oil. Then MeOH (15 mL) was added to the
residue, followed by the addition of PTSA·H2O (100 mg, 0.52 mmol),
and the mixture was stirred at room temperature for 1 h. Water (100
mL) was added to the mixture, and the mixture was extracted with
EtOAc (50 mL × 3). The organic layers were combined, washed with
saturated aqueous NaHCO3 and brine, dried over Na2SO4, and
concentrated. The residue was purified by chromatography on silica
gel (elution with hexane/ethyl acetate) to give 80 mg (69% yield) of
1
yellow solid. H NMR (DMSO-d6) δ 9.91 (s, 1H), 7.58 (d, 1H, J =
7.8 Hz), 7.47 (d, 1H, J = 7.8 Hz), 7.16 (t, 1H, J = 7.8 Hz), 4.23 (m,
2H), 2.74 (s, 2H), 2.51 (m, 2H), 2.25 (s, 2H), 2.18 (s, 6H), 1.07 (s,
6H). MS (ESI) m/e [M + 1]+ 325.0. HPLC purity: 95.42% (254 nm).
10-(2-(Dimethylamino)ethyl)-8-fluoro-2,2-dimethyl-
2,3,5,10-tetrahydro[1,2]diazepino[3,4,5,6-def ]carbazol-6(1H)-
one (60). Compound 60 was prepared from 2-fluoro-6,6-dimethyl-
5,6,7,9-tetrahydro[1,2]diazepino[3,4,5,6-def ]carbazol-10(4H)-one
1
66 as a yellow solid. H NMR (DMSO-d6) δ 9.89 (s, 1H), 7.58 (d,
1
1H, J = 8.4 Hz), 7.47 (d, 1H, J = 7.2 Hz), 7.15 (dd, 1H, J = 7.2, 8.4
Hz), 4.88 (t, 1H, J = 5.4 Hz), 4.21 (t, 2H, J = 5.4 Hz), 3.65−3.68 (m,
1H), 2.76 (s, 2H), 2.25 (s, 2H), 1.07 (s, 6H). MS (ESI) m/e [M + 1]+
298.1. HPLC purity: 96.68% (254 nm).
2,2-Dimethyl-10-(oxiran-2-ylmethyl)-2,3,5,10-tetrahydro-
[1,2]diazepino[3,4,5,6-def]carbazol-6(1H)-one (67). Compound
67 was prepared from 6,6-dimethyl-5,6,7,9-tetrahydro[1,2]diazepino-
(compound 38) according to the procedures for compound 59. H
NMR (DMSO-d6) δ 10.1 (s, 1H), 7.56 (dd, 1H, J = 9.6, 1.8 Hz), 7.21
(dd, 1H, J = 10.2, 1.8 Hz), 4.22 (m, 2H), 2.74 (s, 2H), 2.51 (m, 2H),
2.26 (s, 2H), 2.17 (s, 6H), 1.06 (s, 6H). MS (ESI) m/e [M + 1]+
343.0. HPLC purity: 95.61% (254 nm).
10-(2-(Diethylamino)ethyl)-2,2-dimethyl-2,3,5,10-
tetrahydro[1,2]diazepino[3,4,5,6-def]carbazol-6(1H)-one (61).
O
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Drug Annotation
[3,4,5,6-def]carbazol-10(4H)-one (compound 37) according to the
procedures for compound 59. H NMR (DMSO-d6) δ 9.96 (s, 1H),
prepared from 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 77. 1H NMR (DMSO-d6)
δ 11.9 (s, 1H), 10.1 (s, 1H), 7.36 (dd, 1H, J = 1.8, 9.6 Hz), 7.20 (dd,
1H, J = 1.8, 10.2 Hz), 3.75 (s, 2H), 3.24 (s, 2H), 2.44 (s, 3H). MS
(ESI) m/e [M + 1]+ 259.0. HPLC purity: 99.16% (254 nm).
2-Ethyl-6-fluoro-2,3,4,9-tetrahydro-2,4,9,10-tetraaza-
cyclohepta[def ]fluoren-8(1H)-one (98). Compound 98 was
prepared from 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 77. 1H NMR (DMSO-d6)
δ 11.8 (s, 1H), 10.1 (s, 1H), 7.35 (dd, 1H, J = 2.4, 9.6 Hz), 7.19 (dd,
1H, J = 2.4, 10.2 Hz), 3.80 (s, 2H), 3.30 (s, 2H), 2.60−2.64 (m, 2H),
1.09 (t, 3H, J = 7.2 Hz). MS (ESI) m/e [M + 1]+ 273.0. HPLC purity:
97.32% (254 nm).
6-Fluoro-2-propyl-2,3,4,9-tetrahydro-2,4,9,10-tetraaza-
cyclohepta[def ]fluoren-8(1H)-one (99). Compound 99 was
prepared from 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 77. 1H NMR (DMSO-d6)
δ 11.8 (s, 1H), 10.1 (s, 1H), 7.36 (dd, 1H, J = 1.8, 9.0 Hz), 7.19 (dd,
1H, J = 1.8, 10.2 Hz), 3.80 (s, 2H), 3.30 (s, 2H), 2.50−2.54 (m, 2H),
1.50−1.54 (m, 2H), 0.89 (t, 3H, J = 7.8 Hz). MS (ESI) m/e [M + 1]+
287.0. HPLC purity: 98.46% (254 nm).
1
7.65 (d, 1H, J = 8.4 Hz), 7.49 (d, 1H, J = 7.8 Hz), 7.18 (dd, 1H, J =
8.4, 7.8 Hz), 4.60−4.63 (m, 1H), 4.21−4.24 (m, 1H), 3.27−3.29 (m,
1H), 2.76−2.77 (m, 1H), 2.75 (s, 2H), 2.46−2.48 (m, 1H), 2.26 (s,
2H), 0.92 (s, 6H). MS (ESI) m/e [M + 1]+ 310.0. HPLC purity:
98.04% (254 nm).
Benzyl 3-((2-Bromo-3-(methoxycarbonyl)phenyl)amino)-5-
oxo-5,6-dihydropyridine-1(2H)-carboxylate (71). Compound 3
(0.25 g, 1.1 mmol) and benzyl 3,5-dioxopiperidine-1-carboxylate
(compound 70; 0.13 g, 0.55 mmol) were dissolved in 10 mL of acetic
acid at 25 °C, under nitrogen. The mixture was stirred for 8 h at 70
°C. The resultant solid was purified by chromatography on silica gel
(elution with hexane/ethyl acetate) to afford 0.13 g (51%) of 71 as a
tan foam. 1H NMR (CDCl3-d1) δ 7.53−7.58 (m, 3H), 7.42−7.48 (m,
5H), 5.56 (s, 1H), 5.16 (s, 2H), 4.46 (s, 2H), 4.13 (s, 2H), 3.93 (s,
3H). MS (ESI) m/e [M + 1]+ 459.0.
2-Benzyl 5-Methyl 4-oxo-3,4-dihydro-1H-pyrido[3,4-b]-
indole-2,5(9H)-dicarboxylate (73). A mixture of 71 (0.13 g, 0.28
mmol), palladium acetate (0.013 g, 0.06 mmol), tri-o-tolylphosphine
(0.72 g, 0.19 mmol), and triethylamine (0.36 g, 0.36 mmol) in
acetonitrile (2 mL) was heated in a sealed tubule flushed with
nitrogen at 100 °C for 9 h. The cooled reaction mixture was diluted
with DCM (3 × 50 mL) and water (10 mL). The organic layer was
separated, washed with water, dried (Na2SO4), and concentrated. The
remaining residue was chromatographed on silica gel, eluted with a
gradient (0%−100%) of EtOAc in hexane to give the title compound
(0.076 g, 72%). 1H NMR (CDCl3-d1) δ 9.62 (s, 1H), 7.24−7.50 (m,
8H), 5.18 (s, 2H), 4.88 (s, 2H), 4.27 (s, 2H), 3.98 (s, 3H). MS (ESI)
m/e [M + 1]+ 379.0.
2-Isopropyl-2,3,4,9-tetrahydro-2,4,9,10-tetraazacyclohepta-
[def]fluoren-8(1H)-one (100). Compound 100 was prepared from
methyl 3-amino-2-bromobenzoate (compound 3) according to the
1
procedures for compound 77. H NMR (DMSO-d6) δ 11.7 (s, 1H),
9.83 (s, 1H), 7.43 (d, 1H, J = 8.4 Hz), 7.38 (d, 1H, J = 7.6 Hz), 7.08
(dd, 1H, J = 8.4, 7.6 Hz), 3.78 (s, 2H), 3.26 (s, 2H), 2.93−2.96 (m,
1H), 1.04 (d, 6H, J = 6.4 Hz). MS (ESI) m/e [M + 1]+ 269.0. HPLC
purity: 97.12% (254 nm).
6-Fluoro-2-isopropyl-2,3,4,9-tetrahydro-2,4,9,10-tetraaza-
cyclohepta[def]fluoren-8(1H)-one (101). Compound 101 was
prepared from 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 77. 1H NMR (DMSO-d6)
δ 11.8 (s, 1H), 10.1 (s, 1H), 7.35 (dd, 1H, J = 2.4, 9.6 Hz), 7.18 (dd,
1H, J = 2.4, 10.2 Hz), 3.82 (s, 2H), 3.32 (s, 2H), 2.98−3.00 (m, 1H),
1.09 (d, 6H, J = 7.2 Hz). MS (ESI) m/e [M + 1]+ 287.0. HPLC
purity: 99.09% (254 nm).
2-Butyl-6-fluoro-2,3,4,9-tetrahydro-2,4,9,10-tetraaza-
cyclohepta[def]fluoren-8(1H)-one (102). Compound 102 was
prepared from 3-amino-2-bromo-5-fluorobenzoate (compound 4)
according to the procedures for compound 77. 1H NMR (DMSO-d6)
δ 11.8 (s, 1H), 10.1 (s, 1H), 7.36 (dd, 1H, J = 2.4, 9.6 Hz), 7.19 (dd,
1H, J = 2.4, 10.2 Hz), 3.80 (s, 2H), 3.30 (s, 2H), 2.54−2.57 (m, 2H),
1.47−1.50 (m, 2H), 1.30−1.34 (m, 2H), 0.91 (t, 3H, J = 7.2 Hz). MS
(ESI) m/e [M + 1]+ 301.0. HPLC purity: 98.50% (254 nm).
2-(Cyclopropanecarbonyl)-2,3,4,9-tetrahydro-2,4,9,10-
tetraazacyclohepta[def ]fluoren-8(1H)-one (103). Compound
103 was prepared from methyl 2-(cyclopropanecarbonyl)-4-oxo-
2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-5-carboxylate (compound
85) according to the procedures for compound 77. 1H NMR
(DMSO-d6) δ 11.9 (s, 1H), 10.0 (s, 1H), 7.47−7.53 (m, 2H), 7.17
(dd, 1H, J = 7.2, 7.8 Hz), 4.89 (s, 2H), 4.35 (s, 2H), 2.08−2.11 (m,
1H), 0.78−0.79 (m, 4H). MS (ESI) m/e [M + 1]+ 295.0. HPLC
purity: 99.77% (254 nm).
2,3,4,9-Tetrahydro-2,4,9,10-tetraazacyclohepta[def ]-
fluoren-8(1H)-one (77). A mixture of compound 73 (34 mg, 0.1
mmol) and palladium (10%) on carbon (10 mg) in 10 mL of EtOAc
was stirred at room temperature under a balloon of hydrogen
overnight. The mixture was then filtered through a pad of Celite. The
catalyst cake was washed with EtOAc. The filtrate was concentrated to
afford the intermediate 75. A solution of compound 75, acetic acid
(0.08 mL, 1.4 mmol), and hydrazine hydrate (0.47 mL, 0.8 mmol) in
methanol (3 mL) was heated at reflux. After 8 h, the solid was
collected by hot filtration and washed with water, EtOAc, and
1
dichloromethane to give the target compound (9.6 mg, 47%). H
NMR (DMSO-d6) δ 11.7 (s, 1H), 9.86 (s, 1H), 7.40−7.45 (m, 2H),
7.09 (t, 1H, J = 8.0 Hz), 3.94 (s, 2H), 3.41 (s, 2H). MS (ESI) m/e [M
+ 1]+ 227.0.
6-Fluoro-2,3,4,9-tetrahydro-2,4,9,10-tetraazacyclohepta-
[def ]fluoren-8(1H)-one (78). Compound 78 was prepared from 3-
amino-2-bromo-5-fluorobenzoate (compound 4) according to the
1
procedures for compound 77. H NMR (DMSO-d6) δ 11.8 (s, 1H),
10.04 (s, 1H), 7.29 (dd, 1H, J = 10.0, 1.6 Hz), 7.11 (dd, 1H, J = 10.4,
1.6 Hz), 3.93 (s, 2H), 3.45 (s, 2H). MS (ESI) m/e [M + 1]+ 245.0.
HPLC purity: 98.23% (254 nm).
Methyl 2-(Cyclopropanecarbonyl)-4-oxo-2,3,4,9-tetrahy-
dro-1H-pyrido[3,4-b]indole-5-carboxylate (85). To a solution
of methyl 4-oxo-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-5-carbox-
ylate (compound 75; 0.21 g, 0.82 mmol) and cyclopropanecarbonyl
chloride (0.074 mL, 0.82 mmol) in CH2Cl2 (10 mL) was added
DIPEA (0.143 mL) at 0 °C, and the mixture was stirred at 0 °C for 1
h. Then the solvent was evaporated to give the crude product, which
was purified by pre-TLC to give the title compound (170 mg, 63%).
MS (ESI) m/e [M + 1]+ 313.0.
Methyl 2-(2-(((Benzyloxy)carbonyl)amino)-2-methylpropa-
noyl)-4-oxo-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-5-car-
boxylate (93). . A solution of HATU (86 mg, 0.23 mmol) in DMF
(2 mL) was added to a mixture of 75 (36 mg, 0.15 mmol), 2-
(((benzyloxy)carbonyl)amino)-2-methylpropanoic acid (21 mg, 0.09
mmol), diisopropylethylamine (58 mg, 0.45 mmol), and DMF (8
mL), and the resultant mixture was stirred at ambient temperature for
16 h. The DMF was evaporated to give 93, which was used in the next
step without further purification.
2-(Cyclopropanecarbonyl)-6-fluoro-2,3,4,9-tetrahydro-
2,4,9,10-tetraazacyclohepta[def ]fluoren-8(1H)-one (104).
Compound 104 was prepared from methyl 7-fluoro-4-oxo-2,3,4,9-
tetrahydro-1H-pyrido[3,4-b]indole-5-carboxylate (compound 76)
1
according to the procedures for compound 103. H NMR (DMSO-
d6) δ 9.64 (s, 1H), 6.80−7.08 (m, 2H), 4.95 (s, 1H), 4.74 (s, 1H),
4.40 (s, 1H), 4.20 (s, 1H), 2.03−2.07 (m, 1H), 0.75 (s, 4H). MS
(ESI) m/e [M + 1]+ 313.0.
6-Fluoro-2-pivaloyl-2,3,4,9-tetrahydro-2,4,9,10-tetraaza-
cyclohepta[def]fluoren-8(1H)-one (110). Compound 110 was
prepared from 76 according to the procedures for compound 103. 1H
NMR (DMSO-d6) δ 11.9 (s, 1H), 10.2 (s, 1H), 7.45 (dd, 1H, J = 2.4,
9.6 Hz), 7.22 (dd, 1H, J = 2.4, 10.2 Hz), 4.92 (s, 2H), 4.42 (s, 2H),
1.24 (s, 9H). MS (ESI) m/e [M + 1]+ 329.0. HPLC purity: 96.91%
(254 nm).
6-Fluoro-2-methyl-2,3,4,9-tetrahydro-2,4,9,10-tetraaza-
cyclohepta[def ]fluoren-8(1H)-one (97). Compound 97 was
Benzyl (2-Methyl-1-oxo-1-(8-oxo-8,9-dihydro-2,4,9,10-
tetraazacyclohepta[def ]fluoren-2(1H,3H,4H)-yl)propan-2-yl)-
P
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Drug Annotation
carbamate (111). Compound 111 was prepared from 93 according
to the procedures for compound 103. H NMR (DMSO-d6) δ 11.8
concentrated in vacuo. The residue was purified by flash column
chromatography (5%, ethyl acetate−hexane) to give 123 as a colorless
1
1
(s, 1H), 10.2 (s, 1H), 7.18−7.55 (m, 8H), 4.82−4.91 (m, 4H), 4.43−
4.55 (m, 2H), 1.25 (s, 6H). MS (ESI) m/e [M + 1]+ 446.0.
2-(2-Amino-2-methylpropanoyl)-2,3,4,9-tetrahydro-
2,4,9,10-tetraazacyclohepta[def ]fluoren-8(1H)-one (116).
Compound 116 was prepared from 111 according to the procedures
for compound 75. 1H NMR (DMSO-d6) δ 11.8 (s, 1H), 9.99 (s, 1H),
7.48 (d, 1H, J = 8.0 Hz), 7.43 (d, 1H, J = 7.2 Hz), 7.13 (dd, 1H, J =
8.0, 7.2 Hz), 5.29−5.31 (m, 2H), 4.69−4.75 (m, 2H), 1.26 (s, 6H).
MS (ESI) m/e [M + 1]+ 312.0.
oil (78.2 g, 97%). H NMR (CDCl3-d1, rotamer) δ 7.37−7.26 (m,
5H), 5.20−5.01 (m, 2H), 3.71, 3.4 (s, 3H), 3.66−3.58 (m, 2H),
2.21−2.16 (m, 1H), 1.97−1.86 (m, 3H), 1.61, 1.54 (s, 3H).
(R)-Benzyl 2-Formyl-2-methylpyrrolidine-1-carboxylate
(124). To a solution of 123 (55.2 g, 0.20 mol) in 1 L of DCM,
220 mL of DIBAH (1 N in toluene) was added dropwise at −78 °C
over 0.5 h under an atmosphere of nitrogen. The mixture was stirred
for 2 h at −78 °C and quenched by 1 L of aq NH4Cl. The organic
layer was separated. The water layer was extracted with CH2Cl2 (3 ×
600 mL). The combined organic layers were dried with Na2SO4,
filtered, and concentrated in vacuo. The residue was used in the next
step without further purification.
2-(2-Amino-2-methylpropanoyl)-6-fluoro-2,3,4,9-tetrahy-
dro-2,4,9,10-tetraazacyclohepta[def]fluoren-8(1H)-one (117).
Compound 117 was prepared from 76 according to the procedures
1
for compound 116. H NMR (DMSO-d6) δ 12.1 (s, 1H), 10.2 (s,
(R)-Benzyl 2-Ethynyl-2-methylpyrrolidine-1-carboxylate
(126). To a solution of 124 and 100 g of K2CO3 in 500 mL of
MeOH, 60 g of diethyl 1-diazo-2-oxo-2-phenylethylphosphonate
(0.21 mol) was added dropwise at 0 °C over 10 min. The mixture was
allowed to warm to room temperature and stirred at room
temperature overnight. The mixture was concentrated in vacuo. The
residue was poured into 500 mL of aq NaHCO3 and extracted with
CH2Cl2 (3 × 500 mL). The combined organic layers were dried with
Na2SO4, filtered, and concentrated in vacuo. The residue was purified
by flash column chromatography (5%, ethyl acetate−hexane) to give
1H), 7.40 (dd, 1H, J = 1.8, 10.2 Hz), 7.20 (dd, 1H, J = 1.8, 10.2 Hz),
5.25 (s, 2H), 4.75 (s, 1H), 1.32 (s, 6H). MS (ESI) m/e [M + 1]+
330.0.
(3S,7aR)-3-(Trichloromethyl)-tetrahydropyrrolo[1,2-c]-
oxazol-1(3H)-one (120). To a solution of (R)-pyrrolidine-2-
carboxylic acid (90 g, 0.78 mol) in 500 mL of chloroform was
added 2,2,2-trichloroacetaldehyde (172.5 g, 1.17 mol) at room
temperature. The mixture was heated to reflux and refluxed for 6 h
with a reverse Dean−Stock trap. The solution was washed with water
(2 × 150 mL), and the washes were extracted with chloroform (200
mL). The combined organic layers were dried with Na2SO4 and
filtered, and the solvent was removed in vacuo to afford a light brown
solid. The crude product was recrystallized from ethanol (350 mL) to
1
126 as a colorless oil (15.2 g, 31%, yield for two steps). H NMR
(CDCl3-d1, rotamer) δ 7.43−7.26 (m, 5H), 5.21, 5.15 (s, 2H), 3.62−
3.61, 3.46−3.45 (m, 2H), 2.36−2.28 (m, 2H), 2.02−1.95 (m, 2H),
1.87−1.83 (m, 1H), 1.72−1.62 (m, 3H).
1
form 120 as a white solid (135.0 g, 71%). H NMR (CDCl3-d1) δ
Methyl 2-Bromo-5-fluoro-3-(2,2,2-trifluoroacetamido)-
benzoate (127). To a solution of 4 (25.0 g, 100 mmol) and
K2CO3 (42.0 g, 302 mmol) in DCM (250 mL) was added 2,2,2-
trifluoroacetic anhydride (249.0 g, 1.197 mol) at 0 °C under a
nitrogen atmosphere. The mixture was stirred at room temperature
overnight. The reaction mixture was diluted with DCM, washed with
H2O (200 mL × 2) and saturated aq NaHCO3 (200 mL × 2), dried
over anhydrous Na2SO4, and concentrated to give 34.0 g (98%) of
5.17 (s, 1H), 4.13 (m, 1H), 3.43 (m, 1H), 3.13 (m, 1H), 2.23 (m,
1H), 2.13 (m, 1H), 1.90 (m, 1H), 1.72 (m, 1H).
(3S,7aR)-7a-Methyl-3-(trichloromethyl)tetrahydropyrrolo-
[1,2-c]oxazol-1(3H)-one (121). To a solution of diisopropylamine
(83.9 g, 0.83 mol) in 1 L of tetrahydrofuran 332 mL of n-butyllithium
(2.5 M, 0.83 mol) was added dropwise at −30 °C under an
atmosphere of nitrogen. The mixture was stirred for 0.5 h at −30 °C.
The solution was added dropwise to a solution of (3S,7aR)-3-
(trichloromethyl)tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one (135.0 g,
0.55 mol) in 500 mL of tetrahydrofuran at −78 °C over 0.5 h. The
mixture was stirred at −78 °C for 0.5 h, and then iodomethane (234.1
g, 1.65 mol) was added dropwise over 0.5 h at −78 °C. The mixture
was stirred at −78 °C for 1 h and allowed to warm to room
temperature. Water (750 mL) was added, and the solution was
extracted with chloroform (3 × 1 L). The combined organic extracts
were dried with Na2SO4, filtered, and evaporated to dryness in vacuo.
The residue was purified by flash column chromatography (10%, ethyl
acetate−hexane) to give 121 as a white solid (80.1 g, 56%). 1H NMR
(CDCl3-d1) δ 4.99 (s, 1H), 3.43−3.36 (m, 1H), 3.22−3.16 (m, 1H),
2.25−2.18 (m, 1H), 2.02−1.93(m, 1H), 1.91−1.69 (m, 2H), 1.56 (s,
3H).
1
127 as a white solid. H NMR (CDCl3-d1) δ 8.87 (s, 1H), 8.36 (d,
1H, J = 6.4 Hz), 7.43 (d, 1H, J = 5.2 Hz), 3.98 (s, 3H).
(R)-Benzyl 2-((4-Fluoro-2-(methoxycarbonyl)-6-
(2,2,2trifluoroacetamido)phenyl)ethynyl)-2-methylpyrroli-
dine-1-carboxylate (129). A mixture of 127 (27.52 g, 80 mmol),
Pd(PPh3)2Cl2 (2.8 g, 4 mmol), 126 (19.44 g, 80 mmol), copper(I)
iodide (764 mg, 4 mmol), and tetramethyl guanidine (27.6 g, 240
mmol) in DMF (200 mL) was heated at 80 °C under nitrogen
protection system for 16 h. The cooled reaction mixture was diluted
with EtOAc (3 × 200 mL) and water (800 mL). The organic layer
was separated, washed with water (2 × 200 mL), dried (Na2SO4), and
concentrated. The remaining residue was chromatographed on silica
gel, eluted with a gradient of 0−30% EtOAc in hexanes to give 129
1
(21 g, 53%) as a white solid. H NMR (DMSO-d6) δ 11.01 (s, 1H),
7.64−7.77 (m, 1H), 7.36 (m, 5H), 7.19−7.31 (m, 1H), 5.04−5.12
(m, 2H), 3.85 (s, 3H), 3.44−3.47 (m, 2H), 2.0−2.29 (m, 2H), 1.90−
1.97 (m, 2H), 1.69 (s, 3H). MS (ESI) m/e [M + 1]+ 507.0.
(R)-Methyl 2-Methylpyrrolidine-2-carboxylate Hydrochlor-
ide (122). To a solution of (3S,7aR)-7a-methyl-3-(trichloromethyl)-
tetrahydropyrrolo[1,2-c]oxazol-1(3H)-one (compound 121; 80.1 g,
0.31 mol) in 500 mL of methanol 100 mL of thionyl chloride was
added dropwise at 0 °C over 1 h. The mixture was allowed to warm to
room temperature and then heated to reflux overnight. The mixture
was concentrated in vacuo. The residue was poured into 300 mL of
dry THF, shaken, and filtered to give 122 as a white solid (52.1 g,
(R)-Methyl 2-(1-(Benzyloxycarbonyl)-2-methylpyrrolidin-2-
yl)-1H-indole-4-carboxylate (130). To a suspension of Pd(PPh3)4
(1.72 g, 1.5 mmol) and CuI (0.29 g, 1.5 mmol) in 54 mL of toluene
were added 3 (2.3 g, 10 mmol), 126 (3.0 g, 12 mmol), and TEA (7
mL, 50 mmol). The mixture was stirred for 18 h at 100 °C under a
nitrogen atmosphere. After cooling, water (20 mL) was added. The
mixture was extracted with EtOAc (3 × 20 mL), and the combined
organic layers were washed with brine (20 mL) and dried over
MgSO4. The mixture was filtered, and the filtrate was evaporated to
dryness. The residue was purified by column chromatography on
silica gel using CH2Cl2 as the eluent to provide 2.31 g of (R)-benzyl 2-
((2-amino-6-(methoxycarbonyl)phenyl)ethynyl)-2-methylpyrroli-
dine-1-carboxylate (compound 128). To a refluxing solution of 128
(1.1 g, 2.8 mmol) and dibromoethane (5.21 g, 2.8 mmol) in ethanol
(20 mL) was added zinc powder (1.43 g, 22 mmol) in one portion.
After refluxing for 8 h, the reaction mixture was filtered, and the
filtrate was concentrated to 3 mL and poured into water (15 mL).
1
93%). H NMR (DMSO-d6) δ 10.18 (s, 1H), 9.43 (s, 1H), 3.78 (s,
3H), 3.35−3.23 (m, 2H), 2.28−2.20 (m, 1H), 2.07−1.89 (m, 2H),
1.56 (s, 3H).
(R)-1-Benzyl 2-Methyl 2-methylpyrrolidine-1,2-dicarboxy-
late (123). To a solution of (R)-methyl 2-methylpyrrolidine-2-
carboxylate hydrochloride (compound 122; 52.1 g, 0.29 mol) and
Na2CO3 (81 g, 0.76 mol) in 500 mL of THF and 500 mL of water, a
solution of benzyl chloroformate (54.4 g, 0.32 mol) in 200 mL of
THF was added dropwise at 0 °C over 1 h. The mixture was allowed
to warm to room temperature and stirred at room temperature
overnight. The mixture was extracted with ethyl acetate (3 × 1 L).
The combined organic layer was dried with Na2SO4, filtered, and
Q
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Drug Annotation
The reaction mixture was extracted with EtOAc (20 mL × 3). The
combined extracts were dried over Na2SO4, filtered, and evaporated to
give a pale brown oil, which was chromatographed over silica gel
(elution with hexanes:ethyl acetate = 5:1) to give 130 (307 mg, 28%).
1H NMR (CDCl3-d1) δ 10.3 (s, 1H), 7.83 (d, 1H, J = 7.8 Hz), 7.53
(d, 1H, J = 7.8 Hz), 7.29−7.35 (m, 5H), 7.16 (t, 1H, J = 7.8 Hz), 6.96
(s, 1H), 5.15 (s, 2H), 3.95 (s, 3H), 3.56−3.59 (m, 2H), 2.83−2.85
(m, 1H), 2.03−2.07 (m, 2H), 1.84−1.93 (m, 4H). MS (ESI) m/e [M
+ 1]+ 393.0.
(R)-Methyl 11b-Methyl-6-oxo-2,3,5,6,11,11b-hexahydro-
1H-indolizino[8,7-b]indole-7-carboxylate (136). In a 25 mL
flask, 134 (146 mg, 0.44 mmol) was treated with anhydrous MeSO3H
(10 mL). The flask was fitted with a reflux condenser and heated to
60 °C for 1 h. Then, the reaction mixture was cooled in an ice bath
and diluted with distilled water (2.0 mL). The pH of the solution was
increased to ∼10 by the addition of saturated aq NaHCO3. The
reaction mixture was then extracted with EtOAc (3 × 20 mL), and the
organic extracts were washed with brine (20 mL), dried over Na2SO4,
filtered, and concentrated. The residue was purified by flash
chromatography (20% to 60% EtOAc/hexanes) to give 136 (58
mg, 44%). MS (ESI) m/e [M + 1]+ 299.0.
(R)-Methyl 9-Fluoro-11b-methyl-6-oxo-2,3,5,6,11,11b-hexa-
hydro-1H-indolizino[8,7-b]indole-7-carboxylate (137). In a 25
mL flask, 135 (100 mg, 0.29 mmol) was treated with anhydrous
MeSO3H (6 mL). The flask was fitted with a reflux condenser and
heated at 60 °C for 1 h. Then, the reaction mixture was cooled in an
ice bath and diluted with distilled water (6.0 mL). The pH of the
solution was increased to ∼10 by the addition of saturated aq
NaHCO3. The reaction mixture was then extracted with EtOAc (3 ×
5 mL). The organic extracts were combined and washed with brine (1
× 5 mL), dried over Na2SO4, filtered, and concentrated. The residue
was purified by preparative TLC to give 137 (30 mg, 33%). 1H NMR
(CDCl3-d1) δ 7.14−7.24 (m, 2H), 4.03 (s, 3H), 3.81−3.84 (m, 1H),
3.57−3.59 (m, 1H), 3.22−3.24 (m, 1H), 2.92−2.94 (m, 1H), 2.39−
2.40 (m, 1H), 2.16−2.17 (m, 1H), 1.93−1.94 (m, 1H), 1.63 (s, 3H),
1.56−1.57 (m, 1H). MS (ESI) m/e [M + 1]+ 317.0.
(R)-10a-Methyl-7,8,9,10,10a,11-hexahydro-5,6,7a,11-
tetraazacyclohepta[def ]cyclopenta[a]fluoren-4(5H)-one
(138). A solution of 136 (58 mg, 0.19 mmol), acetic acid (0.4 mL),
and hydrazine hydrate (0.2 mL) in methanol (10 mL) was heated at
reflux. After 7 h, the reaction was cooled and water (5 mL) was added.
The mixture was extracted with EtOAc (3 × 5 mL), and the
combined organic layers were washed with brine (10 mL) and dried
over MgSO4. The mixture was filtered and evaporated to dryness, and
the residue was purified by pre-TLC using CH2Cl2 as the eluent to
give 40 mg (74%) of 138. 1H NMR (DMSO-d6) δ 12.9 (s, 1H), 10.6
(s, 1H), 7.50−7.52 (m, 2H), 7.12 (t, 1H, J = 7.8 Hz), 3.24−3.26 (m,
1H), 2.91 (d, 1H, J = 18.4 Hz), 2.37−2.38 (m, 1H), 2.30−2.32 (m,
1H), 2.20−2.21 (m, 1H), 1.95−1.96 (m, 1H), 1.41−1.43 (m, 1H),
1.34 (s, 3H), 1.18−1.19 (m 1H). MS (ESI) m/e [M + 1]+ 281.0.
HPLC purity: 96.93% (254 nm).
(R)-Methyl 6-fFuoro-2-(2-methyl-1-(2,2,2-trifluoroacetyl)-
pyrrolidin-2-yl)-1H-indole-4-carboxylate (131). To a solution
of 129 (5.0 g, 10 mmol) in toluene was added zinc(II) bromide
(11.25 g, 50 mmol) at room temperature. The reaction mixture was
heated at 80 °C with a nitrogen protection system for 15 h. The
solvent was removed under reduced pressure, and the residue was
treated with DCM (500 mL) and water (800 mL). The organic layer
was separated, washed with water (2 × 200 mL), dried (Na2SO4), and
concentrated. The remaining residue was chromatographed on silica
gel, eluted with a gradient of 0−50% EtOAc in hexanes to give 131
1
(1.9 g, 51%) as a yellow solid. H NMR (CDCl3-d1) δ 9.97 (s, 1H),
7.62 (d, 1H, J = 10.2 Hz), 7.27 (d, 1H, J = 9.6 Hz), 7.05 (d, 1H, J =
1.2 Hz), 3.98 (s, 3H), 3.86−3.88 (m,2H), 2.91−2.96 (m, 1H), 2.25−
2.28 (m, 1H), 2.12−2.16 (m, 2H), 1.99 (s, 3H). MS (ESI) m/e [M +
1]+ 507.0.
(R)-Methyl 2-(2-Methylpyrrolidin-2-yl)-1H-indole-4-carbox-
ylate (132). A stirred mixture of 130 (307 mg, 0.78 mol), methanol
(10 mL), and 10% palladium on carbon (50 mg) was mixed under a
balloon-pressure of hydrogen at room temperature. After 2 h, the
mixture was filtered through Celite and the filtrate was concentrated
to give 132 (190 mg, 94%). 1H NMR (CDCl3-d1) δ 10.9 (s, 1H), 7.86
(d, 1H, J = 7.8 Hz), 7.63 (d, 1H, J = 7.8 Hz), 7.24 (t, 1H, J = 7.8 Hz),
7.14 (s, 1H), 3.96 (s, 3H), 3.40−3.43 (m, 1H), 3.12−3.15 (m, 1H),
2.78−2.81 (m, 1H), 2.23−2.26 (m, 3H), 1.94 (s, 3H). MS (ESI) m/e
[M + 1]+ 259.0.
(R)-Methyl 6-Fluoro-2-(2-methylpyrrolidin-2-yl)-1H-indole-
4-carboxylate (133). To a solution of 131 (1.0 g, 1.9 mmol) in
MeOH (20 mL) was added NaBH4 (706 mg, 11.4 mmol) at room
temperature. The reaction mixture was refluxed for 4 h with a
nitrogen protection system. The solvent was removed under reduced
pressure. The residue was dissolved in DCM (200 mL), and the
mixture was washed with water (200 mL) and brine (200 mL), dried
over Na2SO4, and concentrated to give 133 (727 mg, 98%) as a
1
yellow oil. H NMR (CD3OD-d4) δ 7.50 (dd, 1H, J = 10.2, 2.4 Hz),
(R)-2-Fluoro-10a-methyl-7,8,9,10,10a,11-hexahydro-
5,6,7a,11-tetraazacyclohepta[def ]cyclopenta[a]fluoren-4(5H)-
one (139). A solution of 137 (90 mg, 0.28 mmol), acetic acid (0.54
g, 9 mmol), and hydrazine hydrate (0.28 g, 5.6 mmol) in methanol
(30 mL) was heated at reflux. After 5 h, the reaction was cooled and
water (5 mL) was added. The mixture was extracted with EtOAc (3 ×
5 mL). The combined organic layers were washed with brine (10 mL)
and dried over MgSO4. The mixture was filtered, and the filtrate was
evaporated to dryness. The residue was purified by pre-TLC using
DCM as eluent to give 80 mg (94%) of 139. 1H NMR (DMSO-d6) δ
11.9 (s, 1H), 10.2 (s, 1H), 7.30 (d, 1H, J = 9.6 Hz), 7.20 (d, 1H, J =
10.2 Hz), 3.76 (d, 1H, J = 16.4 Hz), 3.34 (d, 1H, J = 16.4 Hz), 2.99−
3.02 (m, 1H), 2.54−2.58 (m, 1H), 2.35−2.40 (m, 1H), 1.90−1.94
(m, 1H), 1.73−1.75 (m, 1H), 1.48 (s, 3H), 1.43−1.45 (m, 1H). MS
(ESI) m/e [M + 1]+ 299.0. HPLC purity: 100% (254 nm); ee >99%.
Process Chemistry of Scaling Up Compound 139. Com-
pound 139 was scaled up to multikilograms per batch through a
modified synthetic process from the above medicinal chemistry route,
and the process chemistry will be published in due course.
Determination of the Absolute Configuration for 139
Sesquihydrate. A single crystal was grown from isopropanol and
water via vapor diffusion. X-ray intensity data from a yellow rod-like
crystal were collected at 293 K. All measurements were made on a
Bruker APEX DUO diffractometer with graphite monochromated Cu
Kα radiation (λ = 1.541 78 Å) and a CCD detector. A polarized light
microscopic image was captured at room temperature. Direct
methods structure solution, difference Fourier calculations, and full-
7.32 (d, 1H, J = 9.0, 2.4 Hz), 6.93 (s, 1H), 3.97 (s, 3H), 3.03−3.12
(m, 2H), 2.27−2.32 (m, 1H), 1.88−1.98 (m, 3H), 1.60 (s, 3H). MS
(ESI) m/e [M + 1]+ 276.0.
(R)-Methyl 2-(1-(2-Methoxy-2-oxoethyl)-2-methylpyrroli-
din-2-yl)-1H-indole-4-carboxylate (134). Compound 132 (190
mg, 0.74 mmol) was dissolved in CH3CN (25 mL). Methyl
bromoacetate (250 mg, 1.6 mmol) and DIPEA (350 mg, 2.7
mmol) were then added. The reaction mixture was stirred at room
temperature for about 20 h. The reaction mixture was then diluted
with CH2Cl2 (15 mL) and washed with water three times. The
organic layer was dried with MgSO4 and concentrated to give 146 mg
(60%) of 134. 1H NMR (CDCl3-d1) δ 7.85 (d, 1H, J = 7.8 Hz), 7.13−
7.29 (m, 3H), 4.92 (s, 2H), 3.95 (s, 3H), 3.70 (s, 3H), 3.56−3.62 (m,
2H), 1.95−2.06 (m, 4H), 1.94 (s, 3H). MS (ESI) m/e [M + 1]+
331.0.
(R)-Methyl 6-Fluoro-2-(1-(2-methoxy-2-oxoethyl)-2-methyl-
pyrrolidin-2-yl)-1H-indole-4-carboxylate (135). To a stirred
mixture of 133 (1.0 g, 1.27 mmol), CH3CN (50 mL), and methyl
bromoacetate (0.58 g, 3.82 mmol) was added DIPEA (0.82 g, 6.35
mmol). The reaction mixture was stirred at room temperature for
about 20 h. The reaction mixture was then diluted with CH2Cl2 (15
mL) and washed with water three times. The organic layer was dried
with MgSO4 and concentrated to give 0.85 g (67%) of 135. 1H NMR
(CD3OD-d4) δ 7.47 (dd, 1H, J = 2.4, 12.0 Hz), 7.27 (dd, 1H, J = 2.4,
9.0 Hz), 6.89 (s,1H), 3.95 (s, 3H), 3.66−3.68 (m, 1H), 3.64 (s, 3H),
3.16−3.17 (m, 2H), 2.72−2.75 (m, 1H), 1.88−2.02 (m, 4H), 1.44 (s,
3H). MS (ESI) m/e [M + 1]+ 349.0.
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matrix least-squares refinement against F2 were performed with
SHELXTL.
PARylation Assay. HeLa cells were seeded into a 96-well plate
with a clear bottom and black wall at an initial concentration of 5000
cells/well in culture medium (100 μL of DMEM containing 10% FBS,
0.1 mg/mL penicillin−streptomycin, and 2 mM L-glutamine). The
plates were incubated for 4 h at 37 °C under a 5% CO2 atmosphere
and then serial dilutions of compounds (0.01 nM yp 10 μM final
concentration range in 0.1% DMSO/culture medium) over eight
points. The plate was then incubated for 18 h at 37 °C in 5% CO2.
Then DNA damage was induced by the addition of 60 μL of H2O2
solution in PBS (final concentration 200 μM). As a negative control,
untreated cells were used. The plate was kept at 37 °C for 5 min.
Then the medium was gently removed by plate inversion, and the
cells were fixed by addition of ice-cold MeOH (100 μL/well) and
kept at −20 °C for 20 min. After removal of the fixative by plate
inversion and washing 10 times with PBS (120 μL), the detection
buffer (50 μL/well, containing PBS, Tween (0.1%), and BSA (1 mg/
mL)) together with a primary PAR mAb (Alexis ALX-804-220,
1:2000), a secondary anti-mouse Alexa Fluor 488 antibody
(MolecularProbes A11029, 1:2000), and the nuclear dye DAPI
(Molecular Probes D3571, 150 nM) were added. Following overnight
incubation at 4 °C in the dark, removal of the solution, and washing
six times with PBS (120 μL), the plate was read on an ArrayScan VTI
(ThermoFisher). PAR polymers were detected by monitoring
Alexa488 at XF100_485_20, with an exposure time of 0.05 s, and
nuclei were identified by tracking DAPI with XF100_386_23, with an
exposure time of 0.01 s. The mean of total intensity of cells was
calculated by measuring the average of total intensity of nuclei over
the total number of DAPI-labeled nuclei. The EC50 was determined
based on the residual enzyme activity in the presence of increasing
PARP inhibitor concentrations.
Proliferation Assay. The growth-inhibitory activity of compound
139 was determined using the CellTiter-Glo luminescent cell viability
assay (Promega). The number of cells seeded per well of a 96-well
plate was optimized for each cell line to ensure logarithmic growth
over the 7-day treatment period. Cells were left to attach for 16 h, and
cells were treated in duplicate with a 10-point dilution series.
Following a 7-day exposure to the compound, a volume of CellTiter-
Glo reagent equal to the volume of cell culture medium present in
each well was added. The mixture was mixed on an orbital shaker for
2 min to allow cell lysis, followed by 10 min of incubation at room
temperature to allow development and stabilization of the
luminescent signal, which corresponded to a quantity of ATP and
thus the quantity of metabolically active cells. Luminescent signal was
measured using a PHERAstar FS reader (BMG Labtech). Cell
viability was expressed relative to the mock treatment control.
Pharmacokinetic Analysis. The pharmacokinetic profile of
compound 139 was investigated in mice and rats. The protein
precipitation method was used to process mouse and rat plasma
samples before loading into a 96-well plate. Mouse brain samples were
homogenized with 3 volumes of saline followed by protein
precipitation with acetonitrile. After vortexing and centrifugation,
supernatants were transferred and diluted with water before being
injected into an LC−MS/MS instrument for analysis. Sample analysis
was performed using a Shimadzu LC-20A chromatography system
with a Phenomenex Synergi MRX-RP C18 5 cm column. The mobile
phase consisted of mixtures of acetonitrile and 0.1% formic acid in
water and was run in gradient mode at a flow rate of 0.4 mL/min.
Mass spectra were detected with an AB Sciex 4000 triple quadruple
equipped with an ESI source. Internal standards were used to track
the response of analytes in samples. The limit of detection was 2 ng/
mL. Pharmacokinetic analysis was performed with WinNonlin
software (version 5.2.1) using a noncompartmental analysis method.
In Vivo Pharmacology. All animal studies were conducted in
accordance with the Institutional Animal Care and Use Committee
(IACUC) of BeiGene. MDA-MB-436 tumor cells (5 × 106)
suspended in cold PBS and Matrigel (BD, catalog no. 354234,
PBS:Matrigel = 1:1) or H209 tumor cells (1 × 107) suspended in cold
PBS were implanted subcutaneously in the right flank of six 8-week-
old female BALB/c nude mice. After implantation, tumor volume was
measured twice weekly in two dimensions using a caliper and
Protein Preparation, Crystallization, and Structure Deter-
mination. The sequence encoding the catalytic domain (662−1011)
of human PARP-1 was cloned into the pET28a vector and
transformed into BL21(DE3) cells. Protein expression was induced
with 0.2 mM IPTG at 16 °C for 18 h. Protein was purified following a
standard Ni-affinity protocol and desalted into a buffer containing 50
mM Tris, pH 7.5, 250 mM NaCl for thrombin digestion to remove
the His tag. Ion exchange and gel filtration chromatography were used
to further purify the protein. Protein was concentrated to about 30
mg/mL in storage buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1.5
mM DTT) and then flash-frozen with liquid nitrogen and stored at
−80 °C.
Compound 139 was dissolved in 100% DMSO and incubated with
the PARP-1 catalytic domain protein solution at a 5:1 molar ratio at rt
for 1 h. Crystals grew from vapor-diffusion hanging-drops under the
following conditions: 2.2 M DL-malic acid, pH 7.0, 0.1 M Bis-Tris
propane, pH 7.0. The crystals were cryoprotected in mother liquor
supplemented with 20% glycerol and flash-frozen in liquid nitrogen.
Diffraction data were collected using the BL17U1 at the Shanghai
Synchrotron Radiation Facility and were processed with HKL-2000.49
The phase was solved with the program PHASER using a reported
PARP-1 catalytic domain structure (PDB code 4HHY) as the
molecular replacement search model.50,51 Phenix.refine was used to
perform rigid body, TLS, and restrained refinement against X-ray
data, followed by manual adjustment in COOT and further
refinement in Phenix.refine.52,53
PARP-1 Enzyme Assay. The PARP-1 enzyme assay was
conducted using a method modified from the PARP homogeneous
inhibition assay kit (Trevigen). The assays were composed of 1 ng/μL
PARP-1, 20 ng/μL activated DNA (Sigma), and 500 nM NAD in a
reaction buffer containing 100 mM Tris-HCl, pH 8.0, 100 mM NaCl,
and 20 mM MgCl2. PARP-1 was preincubated with different
concentrations of compounds for 30 min at room temperature
followed by the addition of NAD to initiate the auto-PARylation
reaction. The remaining NAD was detected by incubation with a
cycling assay solution containing 1% ethanol, 25 μM resazurin, 0.30
U/mL alcohol dehydrogenase, and 0.25 U/mL diaphorase. The
concentration of NAD is proportional to the fluorescence signal at Ex
540 nm/Em 590 nm. The IC50 values were calculated based on
residual enzyme activity (the rate of NAD decrease) in the presence
of increasing concentrations of compounds.
PARP Isoform Enzyme Assay. PARP isoform enzyme assays
were conducted using the commercial chemiluminescent assay kit
(BPS Biosciences) following the manufacturer’s protocols. Briefly,
histones were first coated in a high binding plate, then incubated with
PARP isoforms and increasing concentrations of compound 139 for
30 min. Then, substrate solution containing NAD and biotinylated
NAD with activated DNA (for PARP-1, PARP-2, PARP-3, PARP-6,
PARP-7, PARP-8, PARP-10, PARP-11, and PARP-12) or without
activated DNA (for TNKS-1 and TNKS-2) was added to the wells.
The biotinylated PARylation product was measured by adding
streptavidin-HRP and HRP substrates, which produce chemilumi-
nescence. The IC50 values were calculated based on residual enzyme
activity in the presence of increasing concentrations of compound
139.
DNA Trapping Activity. Compound 139, olaparib, and veliparib
were tested for DNA trapping activity using a fluorescence
polarization (FP) method.40 PARP1 enzyme and a serial dilution of
compounds were added to a 384 well plate. 5′-Alexa Fluor 488-
labeled DNA containing a nick and a 5′-dRP at the nicked site was
added, and the plate was incubated for 0.5 h at room temperature.
NAD was added to the wells to initiate the PARylation reactions.
PARylation reduced the FP signal by freeing the DNA from PARP1.
After reaction for 1 h at room temperature, FP values were measured
on a PHERAstar FS plate reader (BMG LABTECH). The EC50 values
were calculated based on inhibition of FP signal changes as a function
of increasing concentrations of compounds.
S
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Drug Annotation
calculated using the formula V = 0.5(ab2), where a and b are the long
and short diameters of the tumor, respectively. When tumors reached
a mean volume of approximately 100−200 mm3 in size, mice were
randomly allocated into groups and orally administered at a volume of
10 mL/kg with test articles as indicated. Olaparib was formulated in
PBS containing 10% dimethyl sulfoxide (DMSO) and 10% 2-
hydroxypropyl-b-cyclodextrin (HP-β-CD)] as reported, while TMZ
and compound 139 were formulated in 0.5% (w/v) methylcellulose
(MC). Data are presented as mean tumor volume standard error of
the mean.
Chang-Xin Huo − Department of Medicinal Chemistry,
BeiGene (Beijing) Co., Ltd., Beijing 102206, P. R. China
Yutong Zhu − Department of Discovery Biology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Yuan Zhao − Department of Discovery Biology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Yiyuan Wu − Department of Discovery Biology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Zhen Qin − Department of Discovery Biology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Dan Su − Department of DMPK, BeiGene (Beijing) Co., Ltd.,
Beijing 102206, P. R. China
Tristin Tang − Department of in Vivo Pharmacology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Fan Wang − Department of DMPK, BeiGene (Beijing) Co.,
Ltd., Beijing 102206, P. R. China
Xuebing Sun − Department of Biochemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Yingcai Feng − Department of Biochemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Hao Peng − Department of Biochemistry, BeiGene (Beijing)
Co., Ltd., Beijing 102206, P. R. China
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01346.
■
sı
1H NMR spectra and HPLC spectra of representative
key compounds (PDF)
Accession Codes
Coordinates and structure factors have been deposited at the
Protein Data Bank with accession code 7CMW for compound
139. Authors will release the atomic coordinates and
experimental data upon article publication.
Xing Wang − Department of in Vivo Pharmacology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Yajuan Gao − Department of in Vivo Pharmacology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Yong Liu − Department of in Vivo Pharmacology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Wenfeng Gong − Department of in Vivo Pharmacology,
BeiGene (Beijing) Co., Ltd., Beijing 102206, P. R. China
Fenglong Yu − Department of in Vivo Pharmacology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Xuesong Liu − Department of Biochemistry, BeiGene (Beijing)
Co., Ltd., Beijing 102206, P. R. China
AUTHOR INFORMATION
Corresponding Author
Changyou Zhou − Department of Medicinal Chemistry,
BeiGene (Beijing) Co., Ltd., Beijing 102206, P. R. China;
orcid.org/0000-0001-5214-8291; Phone: 86-10-
58958101; Email: changyou.zhou@beigene.com; Fax: 86-
10-58958088
■
Authors
Hexiang Wang − Department of Medicinal Chemistry,
BeiGene (Beijing) Co., Ltd., Beijing 102206, P. R. China
Bo Ren − Department of Medicinal Chemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Ye Liu − Department of Biochemistry, BeiGene (Beijing) Co.,
Ltd., Beijing 102206, P. R. China
Lai Wang − Department of in Vivo Pharmacology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01346
Beibei Jiang − Department of in Vivo Pharmacology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Yin Guo − Department of Discovery Biology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Min Wei − Department of Biochemistry, BeiGene (Beijing)
Co., Ltd., Beijing 102206, P. R. China
Notes
The authors declare no competing financial interest.
All authors are currently employees of BeiGene except Dexu
Xu, Yiyuan Wu, Zhen Qin, and Hao Peng, who were
employees of BeiGene at the time of their contribution to
this work.
Lusong Luo − Department of Discovery Biology, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Xianzhao Kuang − Department of Medicinal Chemistry,
BeiGene (Beijing) Co., Ltd., Beijing 102206, P. R. China
Ming Qiu − Department of Medicinal Chemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Lei Lv − Department of Medicinal Chemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Hong Xu − Department of Medicinal Chemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Ruipeng Qi − Department of Medicinal Chemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Huibin Yan − Department of Medicinal Chemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Dexu Xu − Department of Medicinal Chemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China
Zhiwei Wang − Department of Medicinal Chemistry, BeiGene
(Beijing) Co., Ltd., Beijing 102206, P. R. China;
orcid.org/0000-0001-8792-9573
ACKNOWLEDGMENTS
■
We gratefully acknowledge the contribution to this work of all
past and present members of the PARP team. We also thank
Joerg Deerberg for help in reviewing the manuscript, and we
thank all members of the compound management team, Yanli
Wang and Hongxia Liu and their team (analytical group) and
Xiaofeng Luo (NMR), for their supporting roles.
ABBREVIATIONS USED
■
CL, plasma clearance; Cmax, maximum plasma concentration;
F, oral bioavailability; HR, homologous recombination; NAD+,
nicotinamide adenine dinucleotide; PARP, poly (ADP-ribose)
polymerase; SSB, single-strand break; T1/2, terminal half-life;
TMZ, temozolomide; TNKS, tankyrase; Vdss, volume of
distribution at steady state; iv, intravenous injection; MTD,
T
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