Synthesis and Structure-Activity Relationships of DCLK1 Kinase Inhibitors Based on a 5,11-Dihydro-6 H-benzo[ e]pyrimido[5,4- b][1,4]diazepin-6-one Scaffold

Article abstract of DOI:10.1021/acs.jmedchem.0c00596

Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that is overexpressed in gastrointestinal cancers, including esophageal, gastric, colorectal, and pancreatic cancers. DCLK1 is also used as a marker of tuft cells, which regulate type II immunity in the gut. However, the substrates and functions of DCLK1 are understudied. We recently described the first selective DCLK1/2 inhibitor, DCLK1-IN-1, developed to aid the functional characterization of this important kinase. Here we describe the synthesis and structure-activity relationships of 5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one DCLK1 inhibitors, resulting in the identification of DCLK1-IN-1.

Full text of DOI:10.1021/acs.jmedchem.0c00596

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Article
Synthesis and structure activity relationships of
DCLK1 kinase inhibitors based on a 5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one scaffold
Fleur M. Ferguson, Yan Liu, Wayne Harshbarger, Ling Huang, Jinhua Wang,
Xianming Deng, Stephen Joseph Capuzzi, Eugene N. Muratov, Alexander
Tropsha, Senthil Muthuswamy, Kenneth Dale Westover, and Nathanael S Gray
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00596 • Publication Date (Web): 12 Jun 2020
Downloaded from pubs.acs.org on June 13, 2020
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Journal of Medicinal Chemistry
Synthesis and structure activity relationships of DCLK1
kinase inhibitors based on a 5,11-dihydro-6H-
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benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one scaffold
Fleur M. Ferguson1, Yan Liu2, Wayne Harshbarger2, 3, Ling Huang4, Jinhua Wang1, Xianming Deng1, 5,
Stephen J. Capuzzi6, Eugene N. Muratov6, Alexander Tropsha6, Senthil Muthuswamy4, 7, Kenneth D.
Westover2, Nathanael S. Gray1, *
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1Department of Cancer Biology, Dana-Farber Cancer Institute, Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, MA 02215.
Departments of Radiation Oncology and Biochemistry, The University of Texas Southwestern Medical Center at Dallas,
2
Dallas, TX 75390.
3 Current address GSK Vaccines, 14200 Shady Grove Rd, Rockville, MD 20850.
4 Cancer Research Institute and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, MA, 02215.
5 Current address, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
6 UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599.
7 Departments of Medicine and Pathology, Harvard Medical School, Boston, MA 02215.
KEYWORDS: DCLK1, DCLK2, doublecortin-like kinase, kinase inhibitor, pancreatic cancer.
ABSTRACT: Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that is overexpressed in gastrointestinal cancers,
including esophageal, gastric, colorectal and pancreatic cancers. DCLK1 is also used as a marker of tuft cells, which regulate Type
II immunity in the gut. However, the substrates and functions of DCLK1 are understudied. We recently described the first selective
DCLK1/ 2 inhibitor, DCLK1-IN-1, developed to aid the functional characterization of this important kinase. Here we describe the
synthesis and structure activity relationships of 5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one DCLK1 inhibitors,
resulting in the identification of DCLK1-IN-1.
INTRODUCTION
compounds makes them unsuitable for use as chemical probes
for investigating the role of DCLK1 in cancer. We sought to
Doublecortin-like kinase 1 (DCLK1) was originally identified
as a regulator of microtubule polymerization in migrating
neurons and for these functions, the kinase domain has been
shown to be largely dispensable. However, DCLK1 is also
expressed in gastrointestinal cancers, including pancreatic
cancer. In models of pancreatic ductal adenocarcinomas,
DCLK1 has been reported to be expressed downstream of K-
Ras activation, and as a putative K-RasG12D interacting protein.1
The 5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-
one scaffold has been utilized to develop inhibitors for several
6
develop
5,11-dihydro-6H-benzo[e]pyrimido[5,4-
b][1,4]diazepin-6-ones with improved selectivity for DCLK1,
by reducing affinity for ERK5, LRRK2, and the bromodomains
of BRD4.
kinases2, including TNK23, PI3K-δ/γ4, Aurora A/B/C5
,
LRRK27 and ERK58 9. The LRRK2 inhibitor LRRK2-IN-17 and
the ERK5 inhibitors XMD8-92 and XMD8-8510 display off-
target activity against DCLK1 in profiling experiments (Figure
1). Confirmation of this DCLK1 affinity in follow-up kinase
assays has been reported for LRRK2-IN-1 and XMD8-9211 12
.
Recently, these molecules have also been shown to inhibit the
bromodomains of BRD4, an important transcriptional regulator
and cancer target.9 13
LRRK2-IN-1 and XMD8-92 have been used to study the role
of DCLK1 kinase activity in colorectal and pancreatic
cancer11,12,1. However, the multi-targeted nature of these
Figure 1. 5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-
6-ones that inhibit DCLK1 kinase. Discovrx KINOMEscan assay
results for DCLK1 listed below. % control = percent of the control
sample signal remaining upon treatment with 10 μM of compound.
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Table 1. Exploration of the R1 position. DCLK1 IC50 values were determined using a MSA kinase assay and are reported as the
average of three technical replicates ± the standard error of the mean (SEM). ERK5 % inhibition was measured using the KiNativ
platform in cell lysates, at a concentration of 10 μM. LRRK2 IC50 values were determined using an ADAPTA kinase assay, with the
ATP concentration set at the KM, and are reported as the average of two technical replicates ± SEM.
We recently described the selective kinase inhibitor DCLK1-
IN-1 as a chemical tool to examine the role of DCLK1 kinase
activity. Here we present the structure activity relationships
leading to the development of this molecule and report its anti-
proliferative activity in K-Ras mutant patient derived organoid
cultures.
analog XMD12-1 displaying the most potent inhibition in the
series. However, complete removal or ortho substitution
resulted in a 3-fold loss of activity. As previous work has
demonstrated that an ortho methoxy substituent in this series
confers desirable selectivity against the targets Aurora A/B/C
and PI3K, relative to ortho-methyl analogs, ortho-methoxy was
considered to afford the optimal balance of potency and
selectivity.4, 5 Overall, XMD9-19 is 20 fold less potent than
XMD8-85, indicating that 4-N-methyl piperazine is preferred to
4-morpholine as the aromatic substituent (Table 1).
N-linked phenyl 4-sulfonamide R1 substituents were found to
be highly potent (XMD17-86), and ortho-methoxy substitution
well tolerated (FMF-03-047-1). However, S-linked phenyl 4-
sulfonamide R1 substituents demonstrated significantly reduced
affinity (XMD12-70-2).
Finally, when examining LRRK2-IN-1 analogs, shortened 2-
ring systems such as XMD11-52 at R1 displayed dramatically
improved potency for DCLK1, similar to the trend observed in
the XMD-8-85 analog series (Table 1).
Optimization of R1 resulted in identification of potent DCLK1
inhibitors, however all lead compounds were also highly active
against off targets LRRK2 and ERK5. Therefore we examined
the effects of substitution of the R2 position (Table 2), which
had successfully been exploited to improve selectivity of
ERK5-IN-1 against LRRK2.10 As many of the analogs
identified in Table 1 displayed high biochemical potency for
RESULTS AND DISCUSSION
We developed a kinase peptide substrate mobility shift assay
(MSA) to determine the IC50 of the reported compounds for
DCLK1. The assay utilizes purified components including the
DCLK1 kinase domain (residues 364- 689) and measures the
conversion of peptide substrates, in this case a derivative of
PRAK (p38 Regulated Activated Protein Kinase), to a
phosphorylated form using microcapillary electrophoresis14. Of
the reported inhibitors, XMD8-85 is the most potent (11 nM),
and also display potent inhibition of ERK5 and LRRK2.
LRRK2-IN-1 inhibits DCLK1 to a lesser extent (186 nM), and
is significantly more potent against LRRK2 and ERK5. XMD8-
92 was found to be a weakly active DCLK1 inhibitor (716 nM)
(Table 1). We began by examining how variations in the R1
substituent affected the activity of these three molecules, along
with sulfonamide XMD17-86 identified in a screening effort
(Table 1). The N-methyl piperazine on XMD8-85 could be
substituted for oxygen without detriment to the activity.
However, removal of the N-methyl group, or extension with a
piperidine ring, led to reduced potency.
DCLK1, the concentration of ATP was increased from 15 μM
to 100 μM for testing future molecules, in order to increase the
stringency of the assay and allow discrimination between potent
analogs.
We next examined the SAR around XMD8-92. To optimize the
ortho-ethoxy substituent we explored the effects of reducing or
increasing its’ bulkiness. Potency improved as the ortho
substituent size was reduced, with the ortho methyl-substituted
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Having failed to improve the selectivity via substitution of R2
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or R3, we turned to exploration of the R4 group. DCLK1 and
LRRK2 possess a methionine gatekeeper residue (Met465 and
Met1947 respectively), unlike ERK5, which has a leucine
gatekeeper (Leu137). Examination of the ERK5-XMD8-9215
and the DCLK1-XMD8-85 co-crystal structures revealed that
the R4 methyl group is oriented towards the gatekeeper residue
(Supporting Figure 1). Therefore, we hypothesized that varying
the R4 group may lead to improved selectivity against ERK5.
Kinase
inhibitors
bearing
a
5,11-dihydro-6H-
9
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one scaffold have
recently been reported to possess off-target activity for the
bromodomains of BRD4.9 BRD4 is a prominent anti-cancer
target, and inhibition of BRD4 has pleiotropic effects16.
Therefore, we sought to engineer out the BRD4 activity which
would confound elucidation of DCLK1 dependent
pharmacology. The N-methyl amide of XMD8-85 has been
shown to bind in the acetyl lysine binding site of BRD4, and act
as a substrate mimetic, with the core of the scaffold engaging in
hydrophobic contacts.13 We hypothesized that changes to this
position would alter BRD4 binding and tested all R4 substituted
analogs using a BRD4 AlphaScreen assay, to assess the impact
of varying the N-methyl amide.
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We sought to both improve kinase selectivity and to remove
BRD4 activity from our DCLK1 inhibitors by varying the R4
position. We generated R4 variants incorporating the three most
potent R1 substituents, and kept R2 as methyl and R3 as
hydrogen (Table 4, and Ref 21 entries 1-5).
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Table 2. Exploration of the R2 position. DCLK1 IC50 values
were determined using a MSA kinase assay and are reported as
the average of three technical replicates ± SEM. ERK5 %
inhibition was measured using the KiNativ platform on cell
lysates, at a concentration of 10 μM. LRRK2 IC50 values were
determined using an ADAPTA kinase assay, with the ATP
concentration set at the KM, and are reported as the average of
two technical replicates ± SEM. ERK5 IC50 values were
assayed in a 32P kinase assay, as described previously8, and are
reported as the average of two technical replicates ± SEM.
XMD8-85 was used as a reference compound, and displayed a
1.5-fold decrease in affinity as a result of increasing the ATP
concentration (11 nM to 16 nM).
Increasing the bulk of the R2 substituent improved the
selectivity of the compounds against LRRK2, but not against
ERK5, consistent with reported SAR10. No significant
improvement in DCLK1 potency or selectivity was achieved by
variation of R2 (Table 2).
We next analyzed the effects of substitution of R3 (Table 3).
Substitution at the 4-position greatly reduced the potency for
DCLK1, ERK5 and LRRK2, likely due to steric interference
with the hinge binding interaction of the aminopyrimidine10.
Table 3. Exploration of the R3 substituent. DCLK1 IC50
values were determined using a MSA kinase assay and are
reported as the average of three technical replicates ± SEM.
ERK5 % inhibition was measured using the KiNativ platform
Previous work targeting PI3K-δ/γ4 and Aurora kinases A/B/C5
in cell lysates, at a concentration of 10 μM. LRRK2 IC50 values
were determined using an ADAPTA kinase assay, with the ATP
concentration set at the KM, and are reported as the average of
two technical replicates ± SEM. ERK5 IC50 values were
assayed in a 32P kinase assay, as described previously8, and are
reported as the average of two technical replicates ± SEM.
with this scaffold had shown that substitution at the 8-
5
position confers significant selectivity.4,
However, this
modification was also not well tolerated by DCLK1, and
compounds bearing an 8-methyl (XMD11-34) or 8-chloro
(XMD10-126) suffered more than
a 100-fold loss in
biochemical activity. Finally, substitution at the 9-position was
also not well tolerated by DCLK1, ERK5 or LRRK2 (Table 3).
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Table 4. Exploration of the R4 position. DCLK1 IC50 values were determined using a MSA kinase assay and are reported as the
average of three technical replicates ± SEM. LRRK2 IC50 values were determined using an ADAPTA kinase assay, with the ATP
concentration set at the KM, and are reported as the average of two technical replicates ± SEM. ERK5 IC50 values were assayed in
a 32P kinase assay, as described previously8, and are reported as the average of two technical replicates ± SEM. BRD4 IC50s were
measured using an AlphaScreen displacement assay, and are reported as the average of three technical replicates ±SEM.
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Removal of the R4 methyl of XMD8-85 (XMD8-87) was not
tolerated by DCLK1, ERK5 or BRD4, and led to a modest
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reduction in potency for LRRK2. Substitution of the R4 methyl
with an ethyl group was well tolerated by DCLK1 and LRRK2
in all cases (FMF-03-055-1, FMF-03-059-1, FMF-03-087-1).
However, these molecules displayed significant reduction in
affinity for ERK5 and BRD4. Based on this positive selectivity
data, R4 was further increased to an isopropyl group. This
resulted in greater than 100-fold losses in activity for DCLK1
compared to the R4 = methyl analogs.
Altering the polarity of the R4 group to reduce its acetyl-lysine
like properties, from a hydrophobic ethyl to an electronegative
[1,1,1]-trifluoroethyl group led to a modest reduction in
DCLK1 activity (5 – 25-fold), but a dramatic increase in
selectivity against ERK5, LRRK2 and BRD4. FMF-03-146-1
displayed a superior selectivity profile against ERK5, LRRK2
and BRD4, and was profiled against the kinome. KINOMEscan
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Table 5. Generation of a DCLK1 negative control molecule.
DCLK1 IC50 values were determined using a MSA kinase assay
and are reported as the average of three technical replicates ±
SEM.
profiling at 1 μM compound concentration against a panel of
In order to ensure additional kinase targets were not picked up
by the negative control, FMF-03-112-1 was also profiled by
KINOMEscan at 1 µM and found to be inactive against the
kinome.17 With selective and well characterized molecules in
hand, we sought to evaluate DCLK1 as a therapeutic target in
PDAC. We previously reported that DCLK1+ PDAC organoids
468 kinases revealed that this molecule displays exquisite
selectivity for DCLK1 and DCLK2.17
As the function of DCLK1 is poorly characterized, we sought
to develop a close structural analog of FMF-03-146-1 that could
be used as a negative control compound, in order to increase the
confidence that phenotypes observed in experiments with FMF-
03-146-1 are DCLK1-kinase dependent. Therefore, we
modified FMF-03-146-1 with R1 or R3 substituents that reduced
DCLK1 activity in this series (Table 4), resulting in the
identification of FMF-03-112-1.
derived from
a patient at various points throughout
chemotherapy and harboring a BRAF insertion mutation were
sensitive to DCLK1 inhibition.17
A
Panc286
Panc281
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
DMSO
0.01
Concentration (µM)
DCLK1-NEG DCLK1-IN-1
0.1
1.0
DMSO
0.01
Concentration (µM)
DCLK1-NEG DCLK1-IN-1
0.1
1.0
B
Panc286
Panc281
1.2
1.0
0.8
0.6
0.4
0.2
0
1.2
1.0
0.8
0.6
0.4
0.2
0
DMSO
0.01
Concentration (µM)
DCLK1-NEG DCLK1-IN-1
0.1
1.0
DMSO
0.01
Concentration (µM)
DCLK1-NEG DCLK1-IN-1
0.1
1.0
Figure 2. Biological characterization of DCLK1-IN-1 and DCLK1-NEG. A. Viability effects following organoid treatment with
compounds at the indicated concentrations for 4 d. B. Viability effects following organoid treatment with compounds at the indicated
concentrations for 12 d. Data in A, B, shown as the mean ± SEM (n = 3).
To explore the relationship between DCLK1 inhibition and
viability in PDAC organoids harboring K-Ras mutations, we
evaluated the effects of DCLK1-IN-1 and DCLK1-NEG on
organoids derived from samples taken from two unrelated
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PDAC patients using a previously reported method.18 The
mL) was heated at 50˚C for 6 hours. After the reaction was
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organoids were treated for 4 days or 12 days of (Figure 2 A,
B). These lines have multiple oncogenic mutations, including
K-RasG12D (Panc030) and K-RasG12V (Panc281).19 Here minimal
effects were observed after 4 d treatment, but both organoids
were sensitive over long term treatments, with between 50%
and > 90% loss of viability observed by 12 d at 1 µM compound
concentration. Minimal loss of viability with DCLK1-NEG was
observed, suggesting these effects are on target.
complete as monitored by thin layer chromatography (TLC),
the reaction solution was concentrated and the residue was
purified by silica-gel column chromatography with ethyl
acetate and hexane (1/20, v/v) to give the title compound (2.51
g, 93%). 1H NMR (600 MHz, CDCl3) δ 8.44 (s, 1H), 8.02 (d, J
= 7.2 Hz, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H),
7.22 (d, J = 7.8 Hz, 1H), 4.28-4.18 (m, 2H), 3.58 (s, 3H), 1.29
(t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 164.4,
160.8, 157.0, 155.2, 142.8, 134.1, 132.5, 128.9, 127.7, 61.6,
42.0, 14.0. MS (ESI) m/z 337 (M+H)+
9
CONCLUSIONS
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Here we present comprehensive structure activity relationships
for a series of DCLK1 inhibitors based on a 5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one scaffold. This
work culminated in the identification of DCLK1-IN-1, a
chemical probe for the DCLK1 kinase domain. As previously
reported, DCLK1-IN-1 has suitable pharmacokinetic properties
for use in mice (T1/2: 2.09 h, AUC: 1269.5 hr*ng mL-1, Cl: 26.26
mL min-1 kg-1) Using information garnered from the SAR study,
we also designed structurally related negative control
compound DCLK1-NEG, to be used in concert. Our previous
studies demonstrated that DCLK1 inhibition could reduce
viability in patient-derived PDAC organoids harboring an
activating BRAF insertion mutation. In this work, we further
the characterization of the role of DCLK1 in PDAC, this time
examining effects in patient-derived organoids harboring K-Ras
mutations, which were also sensitive to prolonged DCLK1
inhibition. We envisage these molecules will provide useful
leads for the investigation of the effects of DCLK1 in PDAC
and other DCLK1 expressing cancers.
2-chloro-11-methyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (3). A mixture
of compound 2 (2.35 g, 6.98 mmol) and iron power (3.9 g, 69.8
mmol) in acetic acid (100 mL) was heated at 50 ˚C for 9 hours.
After the reaction was complete as monitored by reverse phase
analytical
liquid-chromatography
electrospray
mass
spectrometry (LC-MS), the excess of iron was removed and the
mixture was concentrated in vacuo. The resulting residue was
poured into ice-water which resulted in a solid precipitate that
was collected by filtration, washed with water and air dried to
give the title compound (1.55 g, 85%). 1H NMR (600 MHz,
DMSO-d6) δ 10.44 (s, 1H), 8.14 (s, 1H), 7.72 (d, J = 4.8 Hz,
1H), 7.58 (s, 1H), 7.27 (d, J = 6.0 Hz, 1H), 7.21 (s, 1H), 3.33 (s,
3H). 13C NMR (150 MHz, DMSO-d6) δ 167.6, 161.4, 153.4,
149.7, 147.9, 134.2, 132.0, 125.9, 124.6, 124.3, 120.1, 37.2. MS
(ESI) m/z 261 (M+H)+
2-chloro-5,11-dimethyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (4). To a stirred
suspension of compound 3 (688 mg, 2.64 mmol) and MeI (0.25
mL, 4.0 mmol) in dimethyl acetamide (DMA, 40.0 mL) was
added NaH (360 mg, 60 % suspension in mineral oil) at -10 ˚C
and the reaction was gradually warmed to 0 ˚C. After the
reaction was complete as monitored by LC-MS, the solution
was poured into ice-water which resulted in a solid precipitate.
The precipitate was collected by filtration, washed with water
and air dried to give the title compound (563 mg, 77%). 1H
NMR (600 MHz, DMSO-d6) δ 8.57 (s, 1H), 7.68 (dd, J = 1.2,
7.2 Hz, 1H), 7.54-7.51 (m, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.20-
7.18 (m, 1H), 3.41 (s, 3H), 3.32 (s, 3H). 13C NMR (150 MHz,
DMSO-d6) δ 167.1, 163.8, 153.7, 153.4, 148.6, 133.5, 132.4,
128.7, 126.0, 124.6, 118.9, 38.1, 36.4. MS (ESI) m/z 275
(M+H)+.
EXPERIMENTAL SECTION
General Procedures. Unless otherwise noted, reagents and
solvents were obtained from commercial suppliers and were
used without further purification. 1H NMR spectra were
recorded on 500 MHz Bruker Avance III spectrometer, and
chemical shifts are reported in parts per million (ppm, δ)
downfield from tetramethylsilane (TMS). Coupling constants
(J) are reported in Hz. Spin multiplicities are described as s
(singlet), br (broad singlet), d (doublet), t (triplet), q (quartet),
and m (multiplet). Mass spectra were obtained on a Waters
Acquity UPLC. Preparative HPLC was performed on a Waters
Sunfire C18 column (19 mm × 50 mm, 5 μM) using a gradient
of 15−95% methanol in water containing 0.05% trifluoroacetic
acid (TFA) over 22 min (28 min run time) at a flow rate of 20
mL/min. Assayed compounds were isolated and tested as TFA
salts. Purities of assayed compounds were in all cases greater
than 95%, as determined by reverse-phase HPLC analysis.
2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-
5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-
b][1,4]diazepin-6-one (XMD8-85-1). A mixture of 4 (26 mg,
0.1 mmol), 2-methoxy-4-(4-methylpiperizin-1-yl)benzamine
(22 mg, 0.1 mmol), X-Phos (4.3 mg), Pd2(dba)3 (5.5 mg) and
K2CO3 (41.5 mg, 0.3 mmol) in 1.2 mL of t-BuOH was heated
at 100 °C in a seal tube for 4 h. Then the reaction was filtered
through celite and eluted with dichloromethane. The
dichloromethane was removed in vacuo and the resulting crude
product was purified by reverse-phase prep-HPLC using a
water (0.05% TFA)/acetonitrile (0.05% TFA) gradient to afford
the title compound 10 as TFA salt (20 mg, yield: 36%). 1H
NMR (600 MHz, CD3OD) δ 7.83 (dd, J = 1.2, 7.8 Hz, 1H), 7.71
(s, 1H), 7.62-7.59 (m, 2H), 7.30 (d, J = 8.4 Hz, 1H), 7.26 (t, J =
7.2 Hz, 1H), 6.75 (d, J = 1.8 Hz, 1H), 6.66 (d, J = 7.2 Hz, 1H),
3.92-3.87 (m, 5H), 3.66-3.60 (m, 2H), 3.49 (s, 3H), 3.30-3.24
(m, 2H), 3.14-3.08 (m, 2H), 2.97 (s, 3H). MS (ESI) m/z 446
Scheme 1
ethyl
yl)(methyl)amino)benzoate (2)
(methylamino)benzoate (1.44
2-((2-chloro-5-nitropyrimidin-4-
mixture of ethyl 2-
g, 8.0 mmol),
A
diisopropylethylamine (DIEA) (2.8 mL, 16.0 mmol) and 2,4-
dichloro-5-nitropyrimidine (2.30g, 12.0 mmol) in dioxane (40
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Journal of Medicinal Chemistry
MeOH in DCM) to give the title compound (167 mg, 29 %). 1H
(M+H)+.
1
NMR (500 MHz, DMSO-d6) δ 8.58 (s, 1H), 7.69 (dd, J = 7.8,
1.7 Hz, 1H), 7.52 (ddd, J = 8.3, 7.3, 1.7 Hz, 1H), 7.27 (dd, J =
8.5, 1.1 Hz, 1H), 7.21 (td, J = 7.5, 1.0 Hz, 1H), 4.52 (hept, J =
6.8 Hz, 1H), 3.35 (s, 3H), 1.80 – 0.85 (m, 6H). 13C NMR (126
MHz, DMSO-d6) δ 167.49, 166.18, 154.39, 148.79, 133.11,
132.29, 126.90, 125.90, 124.56, 118.46, 54.22, 36.01. MS (ESI)
m/z 304 (M+H)+
2
Scheme 2
3
4
5
6
7
8
9
All other compounds were prepared from intermediates 4, 5, 6
or 7 in an analogous fashion to XMD8-85.
2-chloro-11-methyl-5-(2,2,2-trifluoroethyl)-5,11-dihydro-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one
(5).
5-ethyl-2-((4-(4-hydroxypiperidin-1-yl)-2-
Compound 3 (500mg, 1.9 mmol), Cs2CO3 (1.3 g, 4.0 mmol) and
trifluoroethyl iodide ( 250 μL, 2.6 mmol) were dissolved in
DMA (5 mL) and heated to 70 °C for 2 h. The solvent was
evaporated and the residue purified by silica column
chromatography (0 – 5% MeOH in DCM) to give the title
compound (116 mg, 18 %). 1H NMR (500 MHz, DMSO-d6) δ
8.84 (s, 1H), 7.73 (dd, J = 7.8, 1.7 Hz, 1H), 7.59 (ddd, J = 8.4,
7.3, 1.7 Hz, 1H), 7.34 (dd, J = 8.4, 0.9 Hz, 1H), 7.26 (ddd, J =
8.0, 7.3, 1.0 Hz, 1H), 5.04 (br s, 2H), 3.38 (s, 3H). 13C NMR
(126 MHz, DMSO-d6) δ 167.79, 165.40, 155.37, 149.08,
134.04, 132.42, 126.15, 125.90, 125.17, 124.92, 123.66,
119.15, 48.61 (q, J = 32.5 Hz), 36.30. MS (ESI) m/z 343
(M+H)+
methoxyphenyl)amino)-11-methyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (XMD9-22). 1H
NMR (500 MHz, DMSO-d6) δ 8.33 (s, 1H), 7.94 (s, 1H), 7.72
(d, J = 8.8 Hz, 1H), 7.63 (dd, J = 7.7, 1.8 Hz, 1H), 7.50 – 7.45
(m, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.16 (t, J = 7.4 Hz, 1H), 6.61
(d, J = 2.6 Hz, 1H), 6.49 (dd, J = 8.8, 2.6 Hz, 1H), 4.15 – 4.01
(m, 1H), 3.80 (s, 3H), 3.62 (tq, J = 8.5, 4.4 Hz, 1H), 3.55 – 3.46
(m, 2H), 3.35 (s, 2H), 3.27 (s, 3H), 1.83 (dq, J = 12.4, 4.0 Hz,
2H), 1.50 (dtd, J = 13.0, 9.5, 3.8 Hz, 2H), 1.15 (t, J = 7.1 Hz,
3H). MS (ESI) m/z 475 (M+H)+
5-ethyl-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)-11-methyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (FMF-03-055-
1). 1H NMR (500 MHz, DMSO-d6) δ 9.75 (s, 1H), 8.35 (s, 1H),
7.81 (d, J = 8.7 Hz, 1H), 7.64 (dd, J = 7.7, 1.7 Hz, 1H), 7.49
(ddd, J = 8.7, 7.2, 1.7 Hz, 1H), 7.24 – 7.13 (m, 2H), 6.72 (d, J
= 2.6 Hz, 1H), 6.57 (dd, J = 8.8, 2.6 Hz, 1H), 4.20 – 3.92 (m,
1H), 3.89 (s, 2H), 3.83 (s, 3H), 3.54 (d, J = 12.1 Hz, 2H), 3.28
(s, 3H), 3.18 (q, J = 10.9 Hz, 2H), 2.94 (t, J = 12.6 Hz, 2H), 2.89
(d, J = 3.4 Hz, 3H), 1.16 (t, J = 7.1 Hz, 3H), .MS (ESI) m/z 474
(M+H)+
Scheme 3
2-chloro-5-ethyl-11-methyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (6). Compound
3 (500mg, 1.9 mmol), Cs2CO3 (1.3 g, 4.0 mmol) and ethyl
iodide ( 210 μL, 2.6 mmol) were dissolved in DMA (5 mL) and
heated to 70 °C for 2 h. The solvent was evaporated and the
residue purified by silica column chromatography (0 – 5%
MeOH in DCM) to give the title compound (280 mg, 51 %). 1H
NMR (500 MHz, DMSO-d6) δ 8.65 (s, 1H), 7.66 (dd, J = 7.8,
1.7 Hz, 1H), 7.53 (ddd, J = 8.4, 7.3, 1.7 Hz, 1H), 7.27 (dd, J =
8.4, 1.0 Hz, 1H), 7.22 (td, J = 7.5, 1.0 Hz, 1H), 4.10 (q, J = 5.2
Hz, 2H), 3.18 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H). 13C NMR (126
MHz, DMSO-d6) δ 167.06, 164.74, 153.97, 153.61, 148.71,
133.30, 132.08, 127.49, 126.71, 124.70, 118.82, 46.09, 36.31,
13.82. MS (ESI) m/z 290 (M+H)+
2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-
11-methyl-5-(2,2,2-trifluoroethyl)-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (FMF-03-146-
1). 1H NMR (500 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.48 (s, 1H),
8.18 (s, 1H), 7.78 – 7.67 (m, 2H), 7.57 – 7.50 (m, 1H), 7.30 –
7.25 (m, 1H), 7.20 (td, J = 7.6, 1.0 Hz, 1H), 6.71 (d, J = 2.6 Hz,
1H), 6.61 – 6.53 (m, 1H), 3.86 (d, J = 4.4 Hz, 1H), 3.83 (s, 2H),
3.54 (d, J = 12.0 Hz, 2H), 3.29 (s, 2H), 3.23 – 3.12 (m, 2H),
2.95 (t, J = 12.5 Hz, 2H), 2.88 (s, 3H). MS (ESI) m/z 528
(M+H)+
5-isopropyl-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)-11-methyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (FMF-03-149-
1). 1H NMR (500 MHz, DMSO-d6) δ 9.71 (s, 1H), 8.25 (s, 1H),
8.11 (s, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.66 (dd, J = 7.7, 1.7 Hz,
1H), 7.46 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H), 7.27 – 7.12 (m, 2H),
6.71 (d, J = 2.6 Hz, 1H), 6.57 (dd, J = 8.8, 2.6 Hz, 1H), 4.60
(hept, J = 6.8 Hz, 1H), 3.82 (s, 6H), 3.54 (d, J = 12.2 Hz, 2H),
3.28 (s, 3H), 3.17 (dd, J = 12.8, 9.2 Hz, 2H), 2.94 (t, J = 12.7
Hz, 2H), 2.88 (d, J = 3.3 Hz, 3H), 1.49 – 1.36 (m, 3H), 1.08 (d,
J = 6.8 Hz, 3H). MS (ESI) m/z 488 (M+H)+
Scheme 4
2-chloro-5-isopropyl-11-methyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (7). Compound
3 (500mg, 1.9 mmol), Cs2CO3 (1.3 g, 4.0 mmol) and isopropyl
iodide (250 μL, 2.2 mmol) were dissolved in DMA (5 mL) and
heated to 70 °C for 2 h. The solvent was evaporated and the
residue purified by silica column chromatography (0 – 5%
4-((5-ethyl-11-methyl-6-oxo-6,11-dihydro-5H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-
methoxy-N-(1-methylpiperidin-4-yl)benzamide (FMF-03-
059-1). 1H NMR (500 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.49 (s,
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Journal of Medicinal Chemistry
Page 8 of 12
1H), 8.36 (dd, J = 8.0, 5.3 Hz, 2H), 8.23 – 8.16 (m, 1H), 7.66 2.3 Hz, 1H), 4.02 (s, 2H), 3.82 (s, 3H), 3.31 (s, 3H), 2.98 (s,
1
2
3
4
5
6
7
8
(dd, J = 7.8, 1.7 Hz, 1H), 7.60 – 7.54 (m, 1H), 7.54 – 7.46 (m,
2H), 7.25 (dt, J = 8.4, 1.9 Hz, 1H), 7.19 (td, J = 7.5, 1.1 Hz,
1H), 4.61 (br s, 2H), 4.11 – 3.98 (m, 1H), 3.95 (d, J = 3.8 Hz,
3H), 3.49 (d, J = 12.2 Hz, 2H), 3.36 (s, 3H), 3.17 – 3.05 (m,
2H), 2.79 (d, J = 4.6 Hz, 3H), 2.08 – 1.98 (m, 2H), 1.82 – 1.69
(m, 2H), 1.18 (t, J = 7.0 Hz, 3H). MS (ESI) m/z 516 (M+H)+
3H), 1.16 (t, J = 7.1 Hz, 3H).
MS (ESI) m/z 469 (M+H)+
N-(3-methoxy-4-((11-methyl-6-oxo-5-(2,2,2-trifluoroethyl)-
6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-
yl)amino)phenyl)methanesulfonamide (FMF-03-147-1). H
1
NMR (500 MHz, DMSO-d6) δ 8.65 (s, 1H), 7.72 (dd, J = 7.8,
1.7 Hz, 1H), 7.60 – 7.54 (m, 1H), 7.32 (dd, J = 8.5, 1.0 Hz, 1H),
7.23 (td, J = 7.5, 0.9 Hz, 1H), 6.80 (s, 1H), 6.70 (d, J = 1.3 Hz,
2H), 3.75 (s, 3H), 3.65 (s, 2H), 3.29 (s, 3H), 3.17 (s, 3H). MS
(ESI) m/z 523 (M+H)+
2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-
11-methyl-5-(2,2,2-trifluoroethyl)-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (FMF-03-146-
1). 1H NMR (500 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.48 (s, 1H),
8.18 (s, 1H), 7.78 – 7.67 (m, 2H), 7.57 – 7.50 (m, 1H), 7.30 –
7.25 (m, 1H), 7.20 (td, J = 7.6, 1.0 Hz, 1H), 6.71 (d, J = 2.6 Hz,
1H), 6.61 – 6.53 (m, 1H), 3.86 (d, J = 4.4 Hz, 1H), 3.83 (s, 2H),
3.54 (d, J = 12.0 Hz, 2H), 3.29 (s, 2H), 3.23 – 3.12 (m, 2H),
2.95 (t, J = 12.5 Hz, 2H), 2.88 (s, 3H). MS (ESI) m/z 528
(M+H)+
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-(4-((5-isopropyl-11-methyl-6-oxo-6,11-dihydro-5H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-
methoxyphenyl)methanesulfonamide (FMF-03-150-2)
1H NMR (500 MHz, DMSO-d6) δ 9.57 (s, 1H), 8.30 (s, 1H),
8.16 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.66 (dd, J = 7.8, 1.7 Hz,
1H), 7.50 – 7.43 (m, 1H), 7.25 – 7.21 (m, 2H), 7.16 (td, J = 7.5,
1.0 Hz, 1H), 6.91 (d, J = 2.3 Hz, 1H), 6.83 (dd, J = 8.6, 2.3 Hz,
1H), 4.55 (dq, J = 38.7, 6.8 Hz, 1H), 3.81 (s, 3H), 3.30 (s, 3H),
2.98 (s, 3H), 1.44 (d, J = 5.6 Hz, 4H), 1.09 (d, J = 6.6 Hz, 4H).
MS (ESI) m/z 483 (M+H)+
3-methoxy-4-((11-methyl-6-oxo-5-(2,2,2-trifluoroethyl)-
6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-
yl)amino)-N-(1-methylpiperidin-4-yl)benzamide (FMF-03-
148-1). 1H NMR (500 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.63 (s,
1H), 8.38 (d, J = 7.5 Hz, 1H), 8.34 (d, J = 10.0 Hz, 1H), 8.29
(d, J = 8.4 Hz, 1H), 7.71 (dd, J = 7.7, 1.7 Hz, 1H), 7.56 (ddt, J
= 10.1, 8.7, 3.3 Hz, 2H), 7.53 – 7.48 (m, 1H), 7.30 (dd, J = 8.2,
3.0 Hz, 1H), 7.25 – 7.20 (m, 1H), 5.27 (s, 1H), 4.62 (s, 1H),
4.17 – 3.97 (m, 1H), 3.94 (d, J = 3.7 Hz, 3H), 3.48 (s, 2H), 3.37
(d, J = 2.8 Hz, 3H), 3.18 – 3.05 (m, 2H), 2.79 (d, J = 4.6 Hz,
3H), 2.08 – 2.01 (m, 2H), 1.78 (qd, J = 13.7, 3.9 Hz, 2H). MS
(ESI) m/z 570 (M+H)+
5-ethyl-2-((2-methoxy-4-(4-(pyrrolidin-1-yl)piperidine-1-
carbonyl)phenyl)amino)-11-methyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (FMF-03-058-
2). 1H NMR (500 MHz, DMSO-d6) δ 9.78 (s, 1H), 8.47 (s, 1H),
8.29 (d, J = 8.2 Hz, 1H), 8.20 (s, 1H), 7.66 (dd, J = 7.7, 1.7 Hz,
1H), 7.50 (ddd, J = 8.7, 7.3, 1.8 Hz, 1H), 7.25 (dd, J = 8.4, 1.0
Hz, 1H), 7.19 (td, J = 7.5, 1.0 Hz, 1H), 7.09 – 7.00 (m, 2H),
4.05 (s, 2H), 3.91 (s, 3H), 3.53 (s, 2H), 3.47 – 3.37 (m, 1H),
3.35 (s, 3H), 3.16 – 3.05 (m, 2H), 2.96 (s, 1H), 2.55 (s, 2H),
2.09 (s, 2H), 2.06 – 1.92 (m, 2H), 1.93 – 1.78 (m, 2H), 1.63 –
1.49 (m, 1H), 1.17 (t, J = 7.1 Hz, 3H). MS (ESI) m/z 556
(M+H)+
4-((5-isopropyl-11-methyl-6-oxo-6,11-dihydro-5H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-
methoxy-N-(1-methylpiperidin-4-yl)benzamide (FMF-03-
151-1). 1H NMR (500 MHz, DMSO-d6) δ 9.44 (s, 1H), 8.39 (s,
1H), 8.33 (d, J = 8.4 Hz, 1H), 8.26 (s, 1H), 7.68 (dd, J = 7.7, 1.8
Hz, 1H), 7.57 (dd, J = 8.5, 1.9 Hz, 1H), 7.52 – 7.46 (m, 2H),
7.24 (dt, J = 8.7, 1.9 Hz, 1H), 7.18 (td, J = 7.5, 1.0 Hz, 1H), 4.59
(p, J = 6.8 Hz, 1H), 4.20 – 3.98 (m, 1H), 3.94 (d, J = 3.9 Hz,
3H), 3.49 (d, J = 12.2 Hz, 2H), 3.36 (s, 3H), 3.17 – 3.06 (m,
2H), 2.79 (d, J = 4.6 Hz, 3H), 2.09 – 1.99 (m, 2H), 1.78 (qd, J
= 13.6, 3.9 Hz, 2H), 1.47 (s, 3H), 1.11 (s, 3H). MS (ESI) m/z
530 (M+H)+
N-(4-((5-ethyl-11-methyl-6-oxo-6,11-dihydro-5H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-2-
yl)amino)phenyl)methanesulfonamide (FMF-03-083-1). H
1
NMR (500 MHz, DMSO-d6) δ 9.66 (s, 1H), 9.44 (s, 1H), 8.43
(s, 1H), 7.74 – 7.68 (m, 2H), 7.64 (dd, J = 7.7, 1.7 Hz, 1H), 7.48
(ddd, J = 8.8, 7.2, 1.7 Hz, 1H), 7.26 (dd, J = 8.4, 1.0 Hz, 1H),
7.16 (d, J = 8.8 Hz, 3H), 4.15 – 3.59 (m, 2H), 3.35 (s, 3H), 2.93
(s, 3H), 1.17 (t, J = 7.0 Hz, 3H). MS (ESI) m/z 439 (M+H)+
N-(4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-
methoxyphenyl)methanesulfonamide (FMF-03-047-1). 1H
NMR (500 MHz, DMSO-d6) δ 9.57 (s, 1H), 8.36 (s, 1H), 8.16
(s, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.69 (dd, J = 7.8, 1.7 Hz, 1H),
7.51 (ddd, J = 8.7, 7.2, 1.8 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H),
7.18 (t, J = 7.5 Hz, 1H), 6.91 (d, J = 2.3 Hz, 1H), 6.84 (dd, J =
8.6, 2.3 Hz, 1H), 3.82 (s, 3H), 3.39 (s, 3H), 3.32 (s, 4H), 2.98
(s, 3H). MS (ESI) m/z 455 (M+H)+
5-ethyl-2-((2-methoxy-4-(2-(4-methylpiperazin-1-
yl)ethoxy)phenyl)amino)-11-methyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (FMF-03-086-
1). 1H NMR (500 MHz, DMSO-d6) δ 8.35 (s, 1H), 8.14 (s, 1H),
7.79 (d, J = 8.7 Hz, 1H), 7.64 (dd, J = 7.7, 1.7 Hz, 1H), 7.48
(ddd, J = 8.7, 7.2, 1.8 Hz, 1H), 7.25 – 7.12 (m, 2H), 6.66 (d, J
= 2.7 Hz, 1H), 6.58 (dd, J = 8.8, 2.7 Hz, 1H), 4.21 (t, J = 5.2
Hz, 2H), 4.05 (s, 2H), 3.81 (s, 3H), 3.41 (s, 4H), 3.27 (s, 3H),
3.17 (s, 4H), 2.82 (s, 3H), 1.16 (t, J = 7.1 Hz, 3H). MS (ESI)
m/z 518 (M+H)+
N-(4-((5-ethyl-11-methyl-6-oxo-6,11-dihydro-5H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-2-yl)amino)-3-
methoxyphenyl)methanesulfonamide (FMF-03-087-1). 1H
NMR (500 MHz, DMSO-d6) δ 9.57 (s, 1H), 8.39 (s, 1H), 8.16
(s, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.64 (dd, J = 7.7, 1.7 Hz, 1H),
7.53 – 7.44 (m, 1H), 7.24 (dd, J = 8.4, 0.9 Hz, 1H), 7.17 (td, J
= 7.5, 1.0 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 6.84 (dd, J = 8.6,
4-((5-ethyl-11-methyl-6-oxo-6,11-dihydro-5H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-2-
yl)amino)benzenesulfonamide (FMF-03-088-1/-2). H NMR
(500 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.51 (s, 1H), 7.95 – 7.88
(m, 2H), 7.79 – 7.72 (m, 2H), 7.66 (dd, J = 7.8, 1.7 Hz, 1H),
7.53 – 7.47 (m, 1H), 7.27 (dd, J = 8.4, 1.0 Hz, 1H), 7.21 – 7.15
1
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b][1,4]diazepin-6-one (XMD10-47). 1H NMR (600 MHz,
(m, 3H), 3.53 (s, 2H), 3.39 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H). MS
(ESI) m/z 425 (M+H)+
MeOD-d4) 7.88 (d, J = 9.0 Hz, 1H), 7.76 (dd, J = 8.4 Hz, 1.2
Hz, 1H), 7.53 (m, 1H), 7.26 (m, 2H), 6.76 (d, J = 2.4 Hz, 1H),
6.67 (dd, J = 9.0 Hz, 2.4 Hz, 1H), 3.90 (s, 3H), 3.86 (m, 2H),
3.63 (m, 2H), 3.46 (s, 3H), 3.31 (s, 3H), 3.29 (m, 2H), 3.09 (m,
2H), 2.98 (s, 3H), 2.42 (s, 3H). MS (ESI) m/z 474 (M+H)+
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2
3
4
5
N-(2-(dimethylamino)ethyl)-4-((5-ethyl-11-methyl-6-oxo-
6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-2-
yl)amino)-3-methoxybenzamide (FMF-03-061-1). H NMR
1
(500 MHz, DMSO-d6) δ 9.41 (s, 1H), 8.64 (t, J = 5.7 Hz, 1H),
8.50 (s, 1H), 8.38 (d, J = 8.5 Hz, 1H), 8.23 (s, 1H), 7.66 (dd, J
= 7.7, 1.7 Hz, 1H), 7.57 (dd, J = 8.5, 1.9 Hz, 1H), 7.53 (d, J =
2.0 Hz, 1H), 7.50 (ddd, J = 8.8, 7.2, 1.7 Hz, 1H), 7.25 (dd, J =
8.4, 1.0 Hz, 1H), 7.19 (td, J = 7.5, 1.0 Hz, 1H), 4.09 (s, 2H),
3.94 (s, 3H), 3.62 (q, J = 5.9 Hz, 2H), 3.36 (s, 3H), 3.28 (q, J =
5.9 Hz, 2H), 2.87 (d, J = 4.7 Hz, 6H), 1.18 (t, J = 7.0 Hz, 3H).
MS (ESI) m/z 490 (M+H)+
6
7
8
9
11-benzyl-2-((2-methoxy-4-(4-methylpiperazin-1-
yl)phenyl)amino)-5-methyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (XMD11-32).
1H NMR (600 MHz, CDCl3) 8.14 (s, 1H), 8.08 (d, J = 8.4 Hz,
1H), 7.80 (dd, J = 7.2 Hz, 1.2 Hz, 1H), 7.39 (d, J = 7.2 Hz, 2H),
7.31 (m, 2H), 7.25 (m, 2H), 7.17 (m, 1H), 7.08 (m, 2H), 6.59
(dd, J = 8.4 Hz, 3.0 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 5.12 (br,
2H), 3.86 (s, 3H), 3.54 (s, 3H), 3.22 (m, 4H), 2.65 (m, 4H), 2.40
(s, 3H). MS (ESI) m/z 536 (M+H)+
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2-((4-(3-(dimethylamino)pyrrolidine-1-carbonyl)-2-
methoxyphenyl)amino)-5-ethyl-11-methyl-5,11-dihydro-
6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (FMF-03-
2-((2-methoxy-5-(4-(4-methylpiperazin-1-yl)piperidine-1-
carbonyl)phenyl)amino)-5,11-dimethyl-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (XMD11-56).
1H NMR (600 MHz, MeOD-d4) 8.42 (d, J = 1.8 Hz, 1H), 8.27
(s, 1H), 7.77 (dd, J = 7.8 Hz, 1.8 Hz, 1H), 7.53 (m, 1H), 7.26
(d, J = 8.4 Hz, 1H), 7.21 (m, 2H), 7.12 (d, J = 8.4 Hz, 1H), 4.69
(s, 2H), 4.02 (s, 2H), 3.98 (s, 3H), 3.52 (s, 4H), 3.48 (s, 3H),
3.46 (s, 3H), 3.41 (s, 4H), 2.93 (s, 3H), 2.12 (s, 2H), 1.68 (s,
2H). MS (ESI) m/z 571 (M+H)+
1
067-1). H NMR (500 MHz, DMSO-d6) δ 10.17 (s, 1H), 8.48
(s, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.22 (s, 1H), 7.66 (dd, J = 7.7,
1.7 Hz, 1H), 7.50 (ddd, J = 8.7, 7.3, 1.7 Hz, 1H), 7.27 – 7.15
(m, 4H), 3.95 (s, 2H), 3.92 (s, 3H), 3.75 – 3.66 (m, 2H), 3.61 (s,
2H), 3.35 (s, 3H), 2.85 (s, 6H), 2.40 – 2.26 (m, 1H), 2.12 (t, J =
10.6 Hz, 1H), 1.17 (t, J = 7.0 Hz, 3H). MS (ESI) m/z 516
(M+H)+
2-((2-isopropoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-
11-methyl-5-(2,2,2-trifluoroethyl)-5,11-dihydro-6H-
2-((4-(4-hydroxypiperidin-1-yl)-2-methylphenyl)amino)-
5,11-dimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-
b][1,4]diazepin-6-one (XMD12-68). 1H NMR (600 MHz,
DMSO-d6) 8.52 (s, 1H), 8.21 (s, 1H), 7.64 (dd, J = 7.8 Hz, 1.8
Hz, 1H), 7.46 (m, 1H), 7.18 (d, J = 8.4 Hz, 2.0 Hz, 2H), 7.13
(m, 1H), 6.76 (s, 1H), 6.71 (m, 1H), 4.63 (d, J = 3.0 Hz, 1H),
3.59 (m, 1H), 3.46 (m, 2H), 3.34 (s, 3H), 3.18 (s, 3H), 2.77 (m,
2H), 2.12 (s, 3H), 1.79 (m, 2H), 1.47 (m, 2H). MS (ESI) m/z
445 (M+H)+
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (FMF-04-077-
1). 1H NMR (500 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.50 (s, 1H),
8.02 (s, 1H), 7.84 (d, J = 8.8 Hz, 1H), 7.70 (dd, J = 7.7, 1.7 Hz,
1H), 7.58 – 7.50 (m, 1H), 7.27 (dd, J = 8.5, 1.0 Hz, 1H), 7.20
(td, J = 7.5, 1.0 Hz, 1H), 6.73 (d, J = 2.6 Hz, 1H), 6.57 (dd, J =
8.8, 2.6 Hz, 1H), 5.29 (d, J = 37.9 Hz, 1H), 4.68 (h, J = 6.1 Hz,
1H), 4.55 (s, 1H), 3.81 (d, J = 13.1 Hz, 2H), 3.30 (s, 3H), 3.17
(t, J = 5.5 Hz, 2H), 2.94 (t, J = 12.5 Hz, 2H), 2.90 – 2.85 (m,
3H), 1.25 (d, J = 6.0 Hz, 7H). MS (ESI) m/z 556 (M+H)+
N-(4-((5,11-dimethyl-6-oxo-6,11-dihydro-5H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-2-
2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-
8,11-dimethyl-5-(2,2,2-trifluoroethyl)-5,11-dihydro-6H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one (FMF-04-112-
1). 1H NMR (500 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.45 (s, 1H),
8.15 (s, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 2.2 Hz, 1H),
7.34 (dd, J = 8.6, 2.3 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.71 (d,
J = 2.6 Hz, 1H), 6.57 (dd, J = 8.8, 2.6 Hz, 1H), 5.23 (s, 1H),
4.54 (s, 1H), 4.07 – 3.84 (m, 2H), 3.81 (s, 3H), 3.59 – 3.47 (m,
3H), 3.20 (d, J = 22.5 Hz, 2H), 2.95 (t, J = 12.8 Hz, 2H), 2.88
(s, 4H), 2.28 (s, 3H). MS (ESI) m/z 542 (M+H)+
yl)amino)phenyl)methanesulfonamide (XMD17-86). 1H
NMR (600 MHz, DMSO-d6) 9.62 (s, 1H), 9.42 (s, 1H), 8.37 (s,
1H), 7.68 (m, 3H), 7.48 (m, 1H), 7.25 (d, J = 8.4 Hz, 1H), 7.14
(m, 3H), 3.80 (s, 3H), 3.34 (s, 3H), 2.91 (s, 3H). MS (ESI) m/z
425 (M+H)+
ASSOCIATED CONTENT
AUTHOR INFORMATION
Corresponding Author
* Nathanael S. Gray, Nathanael_Gray@dfci.harvard.edu
Author Contributions
All authors have given approval to the final version of the
manuscript.
methyl
2-(4-(3-methoxy-4-((11-methyl-6-oxo-5-(2,2,2-
trifluoroethyl)-6,11-dihydro-5H-benzo[e]pyrimido[5,4-
b][1,4]diazepin-2-yl)amino)phenyl)piperazin-1-yl)acetate
1
(FMF-04-117-1). H NMR (500 MHz, DMSO-d6) δ 8.47 (s,
1H), 8.21 (s, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.70 (dd, J = 7.8, 1.7
Hz, 1H), 7.54 (ddd, J = 8.4, 7.3, 1.7 Hz, 1H), 7.27 (dd, J = 8.4,
1.1 Hz, 1H), 7.21 (td, J = 7.5, 1.0 Hz, 1H), 6.71 (d, J = 2.6 Hz,
1H), 6.57 (dd, J = 8.8, 2.6 Hz, 1H), 5.28 (s, 1H), 4.56 (s, 1H),
4.35 (s, 3H), 3.81 (s, 3H), 3.77 (d, J = 2.3 Hz, 2H), 3.73 – 3.61
(m, 4H), 3.29 (s, 3H), 3.12 (d, J = 15.9 Hz, 2H), 3.05 – 2.92 (m,
1H). MS (ESI) m/z 586 (M+H)+
Funding Sources
2017 AACR-Bayer Innovation and Discovery grant no. 17-80-44-
GRAY (N.S.G.), DF/HCC GI SPORE Developmental Research
Project Award P50CA127003 (N.S.G.) and Hale Center for
Pancreatic Research (N.S.G.). American Cancer Society Award
132205-RSG-18-039-01-DMC (K.D.W.), Welch Foundation Grant
I1829 (K.D.W.) and NIH grant 5U01CA207160 (S.J.C., E.N.M.,
A.T.).
2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-
4,5,11-trimethyl-5,11-dihydro-6H-benzo[e]pyrimido[5,4-
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8.
Deng, X.; Yang, Q.; Kwiatkowski, N.; Sim, T.;
Conflict of Interest
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McDermott, U.; Settleman, J. E.; Lee, J. D.; Gray, N. S.,
Discovery of a Benzo[e]pyrimido-[5,4-b][1,4]diazepin-6(11H)-
one as a Potent and Selective Inhibitor of Big MAP Kinase 1. ACS
Med Chem Lett 2011, 2 (3), 195-200.
Nathanael Gray is a founder, science advisory board
member (SAB) and equity holder in Gatekeeper, Syros,
Petra, C4, B2S and Soltego (board member). The Gray lab
receives or has received research funding from Novartis,
Takeda, Astellas, Taiho, Jansen, Kinogen, Her2llc,
Deerfield and Sanofi. Ken Westover is a science advisory
board member of Vibliome. The Westover lab receives or
has received research funding from Astellas and Revolution
Medicines.
9.
Lin, E. C.; Amantea, C. M.; Nomanbhoy, T. K.;
Weissig, H.; Ishiyama, J.; Hu, Y.; Sidique, S.; Li, B.; Kozarich,
J. W.; Rosenblum, J. S., ERK5 Kinase Activity is Dispensable for
Cellular Immune Response and Proliferation. Proc Natl Acad Sci
U S A 2016, 113 (42), 11865-11870.
10.
Deng, X.; Elkins, J. M.; Zhang, J.; Yang, Q.; Erazo,
9
T.; Gomez, N.; Choi, H. G.; Wang, J.; Dzamko, N.; Lee, J. D.;
Sim, T.; Kim, N.; Alessi, D. R.; Lizcano, J. M.; Knapp, S.; Gray,
N. S., Structural Determinants for ERK5 (MAPK7) and Leucine
Rich Repeat Kinase 2 Activities of Benzo[e]pyrimido-[5,4-
b]diazepine-6(11H)-ones. Eur J Med Chem 2013, 70, 758-767.
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ABBREVIATIONS
DCLK1, doublecortin-like kinase 1. SEM, standard error of the
mean. MSA, mobility shift assay.
11.
Weygant, N.; Qu, D.; Berry, W. L.; May, R.;
Supporting Information
Chandrakesan, P.; Owen, D. B.; Sureban, S. M.; Ali, N.;
Janknecht, R.; Houchen, C. W., Small Molecule Kinase Inhibitor
LRRK2-IN-1 Demonstrates Potent Activity Against Colorectal and
Pancreatic Cancer Through Inhibition of Doublecortin-like Kinase
1. Mol Cancer 2014, 13, 103.
The Supporting Information is available free of charge on the ACS
Publications website. Experimental protocols, compound
characterization, molecular formula strings, supporting figures
(PDF)
12.
Sureban, S. M.; May, R.; Weygant, N.; Qu, D.;
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