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

Article abstract of DOI:10.1021/ml100304b

Kinome-wide selectivity profiling of a collection of 2-amino-pyrido[2,3-d] pyrimidines followed by cellular structure-activity relationship-guided optimization resulted in the identification of moderately potent and selective inhibitors of BMK1/ERK5 exemp

Full text of DOI:10.1021/ml100304b

LETTER
pubs.acs.org/acsmedchemlett
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
||
||
Xianming Deng,†, Qingkai Yang,‡, Nicholas Kwiatkowski,† Taebo Sim,† Ultan McDermott,§
Jeffrey E. Settleman,§ Jiing-Dwan Lee,*,‡ and Nathanael S. Gray*,†
†Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology,
Dana-Farber Cancer Institute, 250 Longwood Ave, SGM 628, Boston, Massachusetts 02115, United States
‡Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla,
California 92037, United States
§Center for Molecular Therapeutics, Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown,
Massachusetts 02129, United States
S Supporting Information
b
ABSTRACT: Kinome-wide selectivity profiling of a collection
of 2-amino-pyrido[2,3-d]pyrimidines followed by cellular struc-
ture-activity relationship-guided optimization resulted in the
identification of moderately potent and selective inhibitors of
BMK1/ERK5 exemplified by 11, 18, and 21. For example, 11
possesses a dissociation constant (Kd) for BMK1 of 19 nM, a
cellular IC50 for inhibiting epidermal growth factor induced
BMK1 autophosphorylation of 0.19 ( 0.04 μM, and an Ambit
KINOMEscan selectivity score (S5) of 0.035. Inhibitors 18 and
21 are also potent BMK1 inhibitors and possess favorable pharmacokinetic properties which enable their use as pharmacological
probes of BMK1-dependent phenomena as well as starting points for further optimization efforts.
KEYWORDS: MAPK, BMK1, ERK5
he mitogen-activated protein kinases (MAPKs) are crucial
components of signaling cascades that regulate numerous
T
physiological processes.1,2 Four MAPK pathways have been
identified thus far, including extracelluar-signal-regulated kinase
1/2 (ERK1/2), c-Jun-amino-terminal kinase (JNK), p38, and
BMK1.2 As a new member of the MAPKs, BMK1 is activated by
a wide variety of mitogens and stress stimuli.3-6 A recently
reported link between abnormal levels of BMK1 expression
and cancer has greatly increased interest in this signaling path-
way.7 Deregulation of the BMK1 pathway has been shown to be
associated with various malignant properties of human cancers
including increased metastatic potential of prostate cancer cells,8
sustained malignant growth of ErbB2 overexpressing mammary
carcinomas,9 and chemoresistance of breast cancer cells.10 Since
BMK1 was cloned in 1995, only two dual inhibitors of BMK1
and its upstream kinase MEK5, ((Z)-3-(((3-((dimethylamino)-
methyl)-phenyl)amino)-(phenyl)methylene)-2-oxoindoline-6-
carboxamide (BIX02188) and (Z)-3-(((3-((dimethylamino)-
methyl)phenyl)amino)(phenyl)-methylene)-N,N-dimethyl-2-
oxoindoline-6-carboxamide (BIX02189)), have been reported
(Figure 1).11 Therefore, the development of potent and selective
small molecule BMK1 inhibitors for use in investigating and
validating the BMK1 pathway as a target are urgently needed.
In this Letter, we report the design and synthesis of novel
compounds to explore the BMK1 structure-activity relationship
Figure 1. Dual inhibitors of BMK1 and MEK5.
(SAR) using a 2-amino pyrido[2,3-d]pyrimidine template. Itera-
tive rounds of medicinal chemistry and biological evaluation of
these compounds led to the discovery of 11, 18, and 21 as potent
and selective BMK1 inhibitors.
Efforts to identify inhibitors of a new kinase target often begin
with a screen of archived inhibitors that were developed for previous
kinase projects.12 We initiated our chemistry effort by utilizing
a small library of compounds containing a pyrido[2,3-d]pyrimidine
template. The pyrido[2,3-d]pyrimidine core can be classified as a
privileged scaffold with respect to targeting the ATP-site of kinases.
Received: September 30, 2010
Accepted: December 28, 2010
Published: January 12, 2011
r
2011 American Chemical Society
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Examples of kinase inhibitors containing this core scaffold include
BI-2536, a selective polo-like kinase family (PLK1, PLK2, and
PLK3) inhibitor13,14 and 2-((3,5-difluoro-4-hydroxyphenyl)-
amino)-8-isopentyl-5,7-dimethyl-7,8-dihydropteridin-6(5H)-
one (BI-D1870), an inhibitor of RSK.15
Using information gleaned from these examples,13-15 we
designed the potential kinase inhibitor chemotypes I and II
by expanding the 6-membered pyridone ring of BI-2536 to a
7-membered lactam ring (Figure 2). An efficient four-step synthetic
route was developed to enable the synthesis of several compounds
(4, 5, 10, and 11) derived from scaffolds I and II. The synthesis of 4
and 5 is outlined in Scheme 1. First, 2,4-dichloro-5-nitropyrimidine
was reacted with (S)-methyl 2-(pyrrolidin-2-yl)acetate hydro-
chloride using diisopropylethyl amine as base at room
temperature to give the amination product 1 in moderate
yield. This reaction was followed by iron-mediated reduc-
tion of 1 and in situ cyclization in acetic acid at 50 ꢀC to
afford the 7-member lactam intermediate 2 in good yield.
Compound 5 was obtained via methylation of 2 and followed
by palladium mediated amination of 3 with 2-methoxy-4-(4-
methylpiperazin-1-yl)-benzenamine. Similarly, compound 4
was synthesized via palladium mediated amination of inter-
mediate 2.
Figure 2. Pyrido[2,3-d]pyrimidine scaffold expansion.
Scheme 1. Synthesis of 2-Amino-7a,8,9,10-tetrahydro-5H-pyrimido[5,4-b]pyrrolo [1,2-d][1,4]diazepin-6(7H)-one 4a
a Reagents and conditions: (a) DIEA, 2-PrOH, RT; (b) Fe/HOAc, 50 ꢀC; (c) MeI/NaH, DMA, 0 ꢀC; (d) X-Phos (9% mol), Pd2(dba)3 (6% mol),
K2CO3 (3.0 equiv), t-BuOH, 100 ꢀC.
Scheme 2. Synthesis of 2-Amino-5,11-dimethyl-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-onea
a Reagents and conditions: (a) DIEA, dioxane, 50 ꢀC; (b) Fe/HOAc, 50 ꢀC; (c) MeI/NaH, DMA, 0 ꢀC; (d) X-Phos (9% mol), Pd2(dba)3 (6% mol),
K2CO3 (3.0 equiv), t-BuOH, 100 ꢀC.
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Table 1. Kinase Hits for 10 and 11
Table 3. SAR of Substituents of Anthranilic Acid Moiety for
BMK1
compd ID
kinase hitsa
10
ABL1(F317I), BRK, C-FMS, DCAMKL1, DCAMKL2, ERK5,
FRK, GAK, KIT(L576P), MYLK4, TNK1, TNK2
11
DCAMKL1, DCAMKL2, ERK5, FRK, MNK2, PKD1, PKD2,
RSK4∼b, TAO1, TAO3, TNK1, TNK2, ULK1, ULK2
a Kinases that exhibited greater than 95% displacement from an im-
mobilized ligand by the tested compounds. Kinase designations are
those used by Ambit; Erk5 is another name for BMK1. RSK4∼b stands
for RPS6KA6(Kin.Dom.2-C-terminal).
Table 2. SAR of 2-Amino Moiety for BMK1
a Compounds were tested at a concentration of 0.5 μM. The data are
expressed as inhibition of kinase activity in the presence of the inhibitors
relative to DMSO control. b IC50s were calculated using densitometry of
phospho-BMK1 separatated by BMK1 by SDS gel electrophoresis using
6-point titration curves ( standard error of mean.
competition binding assay at a concentration of 10 μM.16,17
Given the generic nature of the pyrimidine-derived hinge inter-
acting motif, we were surprised to discover that these compounds
exhibited highly selective profiles (see the Supporting Informa-
tion for complete profiling data). Kinases that exhibited greater
than 95% displacement from an immobilized ligand by com-
pound 10 and 11 are listed in Table 1.
Because of our interest in the BMK1 signaling pathway, we
were drawn to the strong inhibition effect of 10 and 11 against
BMK1 with ambit scores of 0.5 and 0, respectively.17 Measure-
ment of dissociation constants revealed that compound 11 exhib-
ited much better affinity toward BMK1 with a measured disso-
ciation constant (Kd) of 19 nM, while 10 possessed a Kd of
670 nM. The other kinase targets potently inhibited by 11 were
DCAMKL1 (Kd = 1.2 nM), DCAMKL2 (Kd = 10 nM), GAK
(Kd = 450 nM), TNK1 (Kd = 29 nM), and TNK2 (Kd = 440 nM).
The KINOMEscan selectivity score (S5) calculated at a threshold
of 5% of the dimethyl sulfoxide (DMSO) control for 11 is
0.035 (14 out of 402 kinases) is comparable to that calculated
for BI-2356 (S5 = 0.025, 9 out of 353) which represents a high
level of selectivity.18 Next, we examined the inhibitory activ-
ity against cellular BMK1 by measuring the ability of the
a Compounds were tested at a concentration of 0.5 μM. The data are
expressed as inhibition of kinase activity in the presence of the inhibitors
relative to DMSO control. b IC50s were calculated using densitometry of
phospho-BMK1 separatated by BMK1 by SDS gel electrophoresis using
6-point titration curves ( standard error of mean.
Similarly, 10 and 11 were synthesized by using N-methyl
anthranilic ethyl ester 6 as starting material with higher yields
(Scheme 2).
These four compounds were screened against a diverse panel
of 402 kinases (Ambit KINOMEscan) using an in vitro ATP-site
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compounds to inhibit epidermal growth factor (EGF)-stimulated
autophosphorylation of BMK1 in HeLa cells. The cellular data
demonstrated that 11 (but not 10) significantly inhibited BMK1
autophosphorylation at a concentration of 1 μM. These cellular
results are consistent with the ambit biochemical data, which
reveals that 11 is a promising lead having the ability to inhibit
BMK1 both in vitro and in cells.
We next investigated the effects of modification to the N-sub-
stituent (R3) of the anthranilic acid and to the aryl ring (R4)
(Table 3). N-Substituents (R3) ranging from methyl, ethyl, iso-
propyl, to cyclopentyl are exemplified by 11, 23, 24, and 25, and all
maintained similar activity suggesting tolerance for further elabora-
tion at this position. There appears to be limited tolerance for
substitution on the aryl ring of anthranilic acid, as the 5-methyl,
4-fluoro, 5-chloro, and 4-chloro analogues (26, 27, 28, and 29
respectively) all exhibited a dramatic loss in activity. While these
analogues might suggest a limited tolerance for further elaboration of
the aryl ring, modifications at corresponding positions of other kinase
scaffolds has enabled access to an additional hydrophobic bind-
ing pocket as a result of a switch to the DFG-out conformation.20,21
Further investigation will be required to examine whether “type-II”
BMK1 inhibitors can be accessed through this approach.
The SAR exploration of the 2-amino-5,11-dimethyl-5H-
benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one scaffold re-
vealed that N-methyl substitution at lactam position (R1), the
2-methoxy group of 4-substituted aniline, and the absence of
substituents (R4 = H) on the aryl ring of anthranilic acid were key
structural features required to achieve potent cellular inhibitory
activity against BMK1. The two most active compounds 11 and
21 inhibited EGF induced BMK1 autophosphorylation with
submicromolar cellular IC50 values of 0.19 ( 0.04 and 0.13 (
0.03 μM, respectively.
For a preliminary evaluation of the antiproliferative activity of
these new BMK1 kinase inhibitors against cancer cell lines, 11
was profiled against a panel of 252 cancer cell lines which
represent diverse tumor types (see the Supporting Information
for a detailed list). Then the most active compound 21 was tested
in the subset of cancer cell lines that exhibited more than 50% of
cell growth inhibition at a concentration of 5.0 μM of 11
(Table 4). These selected cell lines were sensitive to the growth
inhibitory activity of 21 but to different extents. Most of the
selected cell lines exhibited EC50 values in the single-digit
micromolar range consistent with BMK1 signaling contributing
to proliferation of cancer cell lines but more potent compounds
are needed to establish what correlation exists between inhibition
of BMK1 autophosphorylation and antiproliferative activity.8-10
The pharmacokinetic properties of 18 and 21 were also
evaluated following intravenous and oral delivery in rats and
mice, respectively. These studies demonstrated that 18 and 21
exhibit favorable pharmacokinetic properties with a T1/2 of 2.0
Using 11 as a lead, the SAR for the 2-amino-5,11-dimethyl-
5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-ones was
explored, utilizing inhibition of BMK1 autophosphorylation in
HeLa cells as a readout. HeLa cells were serum starved overnight
followed by treatment with inhibitors for 1 h. Cells were then
stimulated with epidermal growth factor (EGF, 20 ng/mL)
for 17 min, and BMK1 activation was detected by mobility
retardation.19 We first explored the 2-position of this scaffold
by replacing the aniline substituent of 11 with different
2-methoxy-4-substituent anilines (Table 2). Introduction of
4-hydroxyl-piperidin-1-yl, piperazin-1-yl, and 4-(4-methylpi-
perazin-1-yl)piperidin-1-yl groups at the 4-position of the
2-methoxy aniline resulted in compounds 12, 14, and 15, respec-
tively. These modifications all yielded compounds possessing
similar activities (Table 2). Compounds 13 and 16 containing
substitutents with morpholino and 1-methylpiperidin-4-yl groups,
respectively, exhibited slight decreases in activity. Substitution of
the 2-methoxy group (11) with a methyl group (17) led to a sharp
decrease in activity, while the 2-ethoxy substituted compound 18
maintained similar activity. These results indicated that the
2-alkyloxy group contributes significantly to the overall potency
of this series of analogues. When 2-methoxy-4-(4-methylpiper-
azin-1-yl)-aniline was replaced with 4-sulfonamideaniline, the
resulting compound (19) was less effective. Substitution with
various amides at the 4-position resulted in the synthesis of
compounds 20-22 with 21 exhibiting the best activity within
this set of compounds.
Table 4. Antiproliferative Activity of 21 against a Diverse
Panel of Selected Cancer Cell Lines
cell line
organ
histology
EC50 (μM)a
PC-3
prostate
prostate
breast
adenocarcinoma
3.6
1.6
BPH-1
MCF7
benign prostate hyperplasia
adenocarcinoma
>10
>10
4.4
AU565
SK-N-AS
breast
adenocarcinoma
and 2.6 h, AUC of 903 and 7871 h ng/mL, and %F of 68.6 and
3
brain
neuroblastoma
86.6, respectively (Table 5).
NCI-H1299 lung:NSCLC nonsmall cell lung cancer
NCI-H522 lung:NSCLC nonsmall cell lung cancer
a The cells were treated with 21 at nine concentrations and incubated for
72 h. The data are expressed as the required compound concentration
for inhibiting cell growth at 50%.
4.2
Inhibitor 18 has been used as a “tool” compound to investigate
the BMK1-mediated inhibition of the tumor suppressor activity
of the promyelocytic leukemia protein in tumor cells.22 The in vivo
efficacy of 18 was examined in a HeLa cell xenograft model.
The tumor-bearing mice were injected intraperitoneally with 18
0.95
Table 5. Pharmacokinetic Parameters of 18 and 21a
compd
route
dose (mg/kg)
Tmax (h)
Cmax (ng/mL)
AUC0-¥ (hr ng/mL)
T1/2 (hr)
CL (mL/min/kg)
Vss (L/kg)
F (%)
3
18
IV
1
2
0.08
0.50
439
247
658
903
2.0
3.4
25.5
37.0
3.4
PO
68.6
21
IV
1
907
2.62
18.4
5.36
PO
10
2.0
1287
7871
86.6
a IV = intravenous injection, PO = oral delivery, Tmax = time of maximum plasma concentration, Cmax = maximum plasma concentration, AUC = area
under the curve (measure of exposure), T1/2 = half-life, CL = plasma clearance, Vss = volume of distribution, F = oral bioavailability.
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(50 mg/kg) or vehicle solution twice a day for 28 days. The in vivo
experimental results showed that inhibition of BMK1 by 18 signifi-
cantly suppressed tumor growth by 95%, demonstrating the efficacy
and tolerability of BMK1-targeted cancer treatment in animals (for a
more comprehensive in vivo efficacy study, please see ref 22).
In conclusion, 11, 18, and 21 represent a new chemotype
exhibiting potent and highly selective BMK1 activities. They were
discovered using kinome-wide profiling followed by cellular BMK1-
guided SAR study. Given their excellent kinase selectivity, favorable
pharmacokinetic parameters, and efficacy in xenograft tumor mod-
els, the 2-amino-5,11-disubstituted-5H-benzo[e]pyrimido [5,4-b]-
[1,4]diazepin-6(11H)-ones may represent a privileged scaffold for
the development of therapeutic agents targeting BMK1.
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BMK1, big MAP kinase 1; DIEA, N,N-diisopropylethylamine;
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’ ASSOCIATED CONTENT
S
Supporting Information. Procedures and characteriza-
b
tion data for all compounds; procedures for biochemical assays
and cellular assay, ambit profiling data for 4, 5, 10, 11, 18, and BI-
2536, and cancer cell line profiling data for 11. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*(N.S.G.) Telephone: 617-582-8590. Fax: 617-582-8615. E-mail:
Nathanael_Gray@dfci.harvard.edu. (J.D.L.) Telephone: 858-784-
8703. Fax: 858-784-8343. E-mail: jdlee@scripps.edu.
Author Contributions
These authors contributed equally. N.S.G., X.D. and T.S.
designed the chemistry scaffold. X.D. and N.K. performed the
chemical synthesis and characterization. Q.Y., J.-D.L. and N.S.G.
designed the biological experimental research. Q.Y. performed
the biological experiment and analysis. U.M. and J.E.S. per-
formed the cancer cell lines profiling and analyzed the data.
X.D. and N.S.G co-wrote the paper. All authors read and edited
the manuscript.
Funding Sources
This work was supported by NIH Grant P41 GM079575-03
(N.S.G.), NIH Grants CA079871 and CA114059 (J.-D.L.),
funds from the Tobacco-Related Disease, Research Program of
the University of California, 19XT-0084 (J.-D.L.), and the Sanger
Research Centre (U.M.).
’ ACKNOWLEDGMENT
We wish to thank Life Technologies Corporation, SelectScreen
Kinase Profiling Service for performing enzymatic biochemical kinase
profiling, Ambit Bioscience for performing KINOMEscan profiling,
and SAI Advantium for performing pharmacokinetic studies.
(18) Ambit KINOMEscan selectivity score (S) is a quantitative
measure of compound selectivity. The selectivity score for primary
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ambit score with 5% Ctrl from single point data, S5 = (number of kinases
with %Ctrl e 5)/total number of kinases tested.
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