Discovery of a Series of 5,11-Dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-ones as Selective PI3K-δ/γ Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00209

Dual inhibition of PI3K-δ and PI3K-γ is an established therapeutic strategy for treatment of hematological malignancies. Reported molecules targeting PI3K-δ/γ selectively are chemically similar and based upon isoquinolin-1(2H)-one or quinazolin-4(3H)-one scaffolds. Here we report a chemically distinct series of potent, selective PI3K-δ/γ inhibitors based on a 5,11-dihydro-6H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6-one scaffold with comparable biochemical potency and cellular effects on PI3K signaling. We envisage these molecules will provide useful leads for development of next-generation PI3K-δ/γ targeting therapeutics.

Full text of DOI:10.1021/acsmedchemlett.6b00209

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Letter
Discovery of a series of 5,11-dihydro-6H-benzo[e]pyrimido[5,4-
b][1,4]diazepin-6-ones as selective PI3K-#/# inhibitors
Fleur M. Ferguson, Jing Ni, Ting-hu Zhang, Bethany Tesar, Taebo Sim, Nam Doo
Kim, Xianming DENG, Jennifer R. Brown, Jean J Zhao, and Nathanael S Gray
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00209 • Publication Date (Web): 02 Aug 2016
Downloaded from http://pubs.acs.org on August 4, 2016
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Discovery of a series of 5,11ꢀdihydroꢀ6Hꢀbenzo[e]pyrimido[5,4ꢀ
b][1,4]diazepinꢀ6ꢀones as selective PI3Kꢀδ/γ inhibitors
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Fleur M. Ferguson1, Jing Ni1, Tinghu Zhang1, Bethany Tesar2, 3, Taebo Sim4,5, Nam Doo Kim6, Xianꢀ
ming Deng1, Jennifer R. Brown2, 3, Jean J. Zhao1, Nathanael S. Gray1*
1Department of Cancer Biology, DanaꢀFarber Cancer Institute, Boston, MA, USA. 2Department of Medical Oncology, Dana
Farber Cancer Institute, Boston, MA, USA. 3Department of Medicine, Harvard Medical School, Boston, MA, USA.
4Chemical Kinomics Research Center, Korea Institute of Science and Technology, Republic of Korea. 5KUꢀKIST Graduate
School of Converging Science and Technology, Korea University, Republic of Korea. 6DaeguꢀGyeongbuk Medical Innovaꢀ
tion Foundation, Republic of Korea
KEYWORDS: PI3Kꢀδ, PI3Kꢀγ, phosphatidylinositolꢀ4,5ꢀbisphosphate 3ꢀkinaseꢀdelta, p110ꢀδ , p110ꢀγ , kinase inhibitor
ABSTRACT: Dual inhibition of PI3Kꢀδ and PI3Kꢀγ is an established therapeutic strategy for treatment of hematological maligꢀ
nancies. Reported molecules targeting PI3Kꢀδ/γ selectively are chemically similar and based upon isoquinolinꢀ1(2H)ꢀone or
quinazolinꢀ4(3H)ꢀone scaffolds. Here we report a chemically distinct series of potent, selective PI3Kꢀδ/γ inhibitors based on a 5,11ꢀ
dihydroꢀ6Hꢀbenzo[e]pyrimido[5,4ꢀb][1,4]diazepinꢀ6ꢀone scaffold with comparable biochemical potency and cellular effects on
PI3K signaling. We envisage these molecules will provide useful leads for development of nextꢀgeneration PI3Kꢀδ/γ targeting therꢀ
apeutics.
therapy.6 Additionally Duvelisib has potent antiꢀinflammatory
INTRODUCTION
PI3Kꢀδ and PI3Kꢀγ are members of the Class I Type IA and
and joint protective effects in murine models of rheumatoid
arthritis.7 A Phase IIa exploratory clinical trial in mild allergic
Class
I
Type IB family of Phosphatidylinositolꢀ4,5ꢀ
asthma met several secondary endpoints demonstrating proofꢀ
ofꢀconcept that next generation PI3Kꢀδ/γ inhibitors may also
prove effective in this disease area.8
bisphosphate 3ꢀkinases (PI3Ks). Unlike the related PI3Kꢀα/ꢀβ
which are ubiquitously expressed, PI3Kꢀδ and PI3Kꢀγ are exꢀ
pressed primarily in leukocytes and perform a number of roles
in regulation of the immune system. PI3Kꢀδ has been shown to
be involved in Bꢀcell activation, proliferation, homing and
retention in lymphoid tissues, PI3Kꢀγ regulates Tꢀcell proliferꢀ
ation and cytokine production.1
PI3Kꢀδ and PI3Kꢀγ are the dominantly expressed PI3K
isoforms in Bꢀ and Tꢀcells respectively, where they are key
nodes in the PI3K/Akt/mTOR pathway. This pathway is misꢀ
regulated in a number of bloodꢀborne cancers including chronꢀ
ic lymphocytic leukemia (CLL), follicular lymphoma (FL) and
indolent nonꢀHodgkin’s lymphoma (iNHL).1
Figure 1: Structures of PI3Kꢀδ/γ selective inhibitors reported in
the literature and described in this work
PI3Kꢀδ signaling drives malignant Bꢀcell proliferation. Selecꢀ
tive inhibition of PI3Kꢀδ using small molecule inhibitor
Idelalisib has proven to be an effective treatment for CLL
when used in combination with rituximab, a chimeric monoꢀ
clonal antibody that targets the Bꢀlymphocyte antigen CD20.2
PI3Kꢀγ activation is key for inflammatory cell recruitment to
tumors, associated with angiogenesis and tumor growth, which
can be attenuated by knockdown or pharmacological inhibiꢀ
tion of PI3Kꢀγ.3,4 As these two kinases play distinct and comꢀ
plementary roles in immune function, dual inhibition of PI3Kꢀ
δ and PI3Kꢀγ is also an attractive strategy for broadly targeting
hematological malignancies. Inhibition of PI3Kꢀδ/γ is well
tolerated with mild, reversible side effects reported in the clinꢀ
ic.5 The dual inhibitor Duvelisib is currently in Phase III cliniꢀ
cal trials for CLL, FL and Phase II clinical trials for iNHL,
either alone, or in combination with monoclonal antibody
Currently reported selective dual inhibitors of PI3Kꢀδ/γ are
based upon isoquinolinꢀ1(2H)ꢀone or quinazolinꢀ4(3H)ꢀone
scaffolds (Figure 1).9,10, 11 Here we report a chemically distinct
series of potent, selective PI3Kꢀδ/γ inhibitors based on a 5,11ꢀ
dihydroꢀ6Hꢀbenzo[e]pyrimido[5,4ꢀb][1,4]diazepinꢀ6ꢀone scafꢀ
fold with comparable enzymatic potency and cellular effects
on PI3Kꢀδ signaling.
RESULTS AND DISCUSSION
Throughout the course of a screening campaign designed to
identify antiꢀleukemic compounds, we observed that comꢀ
pound 1 (FMFꢀ01ꢀ085ꢀ1) shows antiproliferative activity in Tꢀ
cell acute lymphocytic leukemia (TꢀALL) cell lines (IC50
MOLT4 cells = 33 nM, IC50 Jurkat cells = 166 nM). Subseꢀ
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quent kinome profiling revealed the primary targets of this δ over PI3Kꢀα and 270 fold selective over PI3Kꢀβ. The only
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compound are PI3Kꢀδ/γ (Table 1, Supporting Table 1, Supꢀ
porting Figure 1), leading us to explore the SAR of this series.
Compounds were synthesized according to Scheme 1. Analogs
from our initial screen lacking an arylꢀsulfonamide showed no
inhibitory effects on PI3Kꢀδ/γ (e.g. compound 19, FMFꢀ01ꢀ
086ꢀ2, Supporting Table 2), therefore we focused our synthetic
efforts on compounds containing this moiety. 12
offꢀtarget activity of concern is against Aurora kinases A and
B. Enzymatic testing revealed that Compound 1 has 30 fold
selectivity over Aurora A and 60 fold over Aurora B.
Scheme 1: Synthetic route for synthesis of 5,11ꢀdihydroꢀ
6Hꢀbenzo[e]pyrimido[5,4ꢀb][1,4]diazepinꢀ6ꢀones
We have previously reported that the 5,11ꢀdihydroꢀ6Hꢀ
benzo[e]pyrimido[5,4ꢀb][1,4]diazepinꢀ6ꢀone scaffold is capaꢀ
ble of binding to the ATP binding pocket of LRRK213,
ERK514, AuroraA/B kinases15 and to the acetylꢀlysine binding
pocket of the BRD4 bromodomains.16 However, methylation
of the phenyl ring in the tricyclic core is not tolerated by these
targets. Kinome profiling at 1 ꢁM compound concentration
revealed that 1 has excellent selectivity across the human kiꢀ
nome, with a selectivity score, S10 of 0.013. Importantly other
targets in the PI3K pathway such as Akt, DNAꢀPK, BTK and
mTOR are not inhibited (Supporting Table 1, Supporting Figꢀ
ure 1) and BRD4 activity is low (BRD4_1 IC50 = 6.0 ꢁM,
Supporting Table 3). The compound has some inhibitory efꢀ
fects on PIP5K2C (PIP4Kꢀγ), a lipid kinase with low levels of
activity in vitro. In our experience this level of inhibition corꢀ
responds to micromolar biochemical IC50.
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o
As some activity is present for PI3Kꢀα (and H1047L/Y muꢀ
tants) we measured PI3Kꢀα and PI3Kꢀβ IC50s to determine the
isoform selectivity. Compound 1 is 26 fold selective for PI3Kꢀ
Reaction conditions. i) DIEA, 1,4ꢀdioxane, 50 C; ii) Fe, AcOH,
50 oC; iii) NaH, MeI, DMF, 0 oC; iv) XPhos, Pd2(dba)3,
Cs2CO3,1,4ꢀdioxane, 95 oC
Table 1: SAR, isoform selectivity and Aurora kinase selectivity of PI3Kꢀδ/γ inhibitors
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a IC50s measured using ADAPTA assay format (ThermoFisher Scientific). b IC50s measured using Z’LYTE assay format (ThermoFisher
Scientific). IC50s plotted from the average of duplicate experiments. Errors are reported as ± 95% confidence interval.
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This prompted us to further investigate the factors conferring
selectivity to the series (Table 1). Meta substitution of the
aniline ring with an Nꢀsubstituted sulfonamide biases the poꢀ
tency of the compounds towards PI3Kꢀδ/γ (compounds 1, 3, 5,
7, 9, 10, 12). Conversely, the same substituents in the para
position improve the Aurora A/B potency and reduce the
PI3Kꢀδ/γ potency (compounds 2, 4, 6, 8, 11, 13).
(Figure 2A, Supporting Table 1). Additionally low BRD4 acꢀ
tivity was observed for all compounds (BRD4_1 IC50 = 18.8
ꢁM, 10.8 ꢁM respectively, Supporting Table 3).
In order to have a more direct comparison of potency to the
currently available clinical compounds we measured the IC50s
of Duvelisib and Idelalisib in the ADAPTA assay format (Figꢀ
ure 2B). In the PI3Kꢀδ assay, compounds 1, 9 and 12 are equiꢀ
potent to Idelalisib, whereas Duvelisib is more effective. In the
PI3Kꢀγ assay, compounds 1, 9 and 12 are comparable to
Duvelisib. Idelalisib, a PI3Kꢀδ specific inhibitor, is much less
potent against PI3Kꢀγ, as expected (Table 1, Figure 2B).
Compounds containing an unsubstituted sulfonamide nitrogen
are equipotent against PI3Kꢀδ/γ and AuroraA/B. (Compounds
14, 16, 17). It has been shown that orthoꢀsubstitution adjacent
to the hingeꢀbinding motif can remove AuroraA/B activity
from this scaffold.15 Orthoꢀmethylation of the aniline ring of
potent compound 16 to give compound 18 shows the expected
low AuroraA/B activity but also has dramatically reduced
PI3Kꢀδ/γ activity.
Encouraged by the potency of our inhibitors in comparison to
the current bestꢀinꢀclass molecules, we next explored the effect
of our compounds on PI3K signaling in isogenic HMEC cell
lines where PI3K signaling is driven exclusively by either CAꢀ
p110ꢀα, CAꢀp110ꢀβ or CAꢀp110ꢀδ under serum starved condiꢀ
tions, and compared them to Duvelisib and Idelalisib. Comꢀ
mensurate with their biochemical activities, Idelalisib, 9 and
12 show comparable inhibition of, and selectivity for, PI3Kꢀδ
signaling at 10 nM concentration, (Figure 2C). Duvelisib is the
most potent PI3Kꢀδ inhibitor, however it is less selective
against PI3Kꢀβ in a cellular context.
Covalent inhibitors have been reported for PI3Kꢀα that target a
nonꢀconserved cysteine unique to this isoform.17 Examination
of Xꢀray crystal structures showed there are no accessible cysꢀ
teine residues proximal to the ATP binding pocket in PI3Kꢀδ
and PI3Kꢀγ (PDB IDs: 4XE0, 4EZJ). As Compound 1 conꢀ
tains an acrylamide, we used LCMS/MS experiments with
purified PI3Kꢀδ protein to confirm that 1 does not covalentlyꢀ
label the protein. Therefore we sought to remove this reactive
functionality whilst maintaining onꢀtarget potency and kinome
selectivity. Gratifyingly compound 9 (FMFꢀ02ꢀ109ꢀ1) and
compound 12 (FMFꢀ02ꢀ063ꢀ1) both maintained potent inhibiꢀ
tion of PI3Kꢀδ/γ and showed comparable selectivity for PI3Kꢀ
δ against PI3Kꢀα (40 fold, 25 fold) and improved selectivity
against PI3Kꢀβ (360 fold, 2300 fold), Aurora A (65ꢀfold, 70ꢀ
fold) and Aurora B (71ꢀfold, 71ꢀfold). Kinome profiling reꢀ
vealed that compounds 9 and 12 also maintain an excellent
selectivity profile with S10 of 0.010 and 0.008 respectively
To rationalize the isozyme selectivity we docked 9 and 12 into
PI3Kꢀδ (PDB: 4XE0). It is known that exploitation of a selecꢀ
tivity pocket formed by rearrangement of a methionine residue
in the ATP binding pockets of PI3Kꢀδ and PI3Kꢀγ can imꢀ
prove isozyme selectivity. This has been achieved to date by
the design of molecules that adopt a propellorꢀlike conforꢀ
mation, such as Idelalisib and Duvelisib.18 In the docking
models of 9 and 12 in complex with PI3Kꢀδ the butterflyꢀlike
conformation of the benzodiazipinone scaffold causes the tolyl
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A
Cmpd 9
Cmpd 12
B
Compound IC50 (nM) a IC50 (nM) a
1
2
3
4
5
6
ID
9
PI3Kꢀδ
PI3Kꢀγ
1.7 ± 1.5
11 ± 4.1
12
2.1 ± 0.68
2.0 ± 0.54
< 0.5
6.5 ± 1.5
64 ± 30
Idelalisib
Duvelisib
2.9 ± 0.17
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PI3Kꢀδ/γ
PI3Kꢀδ/γ
C
CAꢀp110α
CAꢀp110β
Du
CAꢀp110δ
Du
12
9
Du
Id
12
9
Id
12
9
Id
pAKT308
pAKT473
AKT
pS6RP
S6RP
αꢀTubulin
Figure 2: A Kinomeꢀwide selectivity profile of compounds 9 and 12. B Comparison of biochemical IC50 values of compounds 9, 12,
Duvelisib and Idelalisib in PI3Kꢀδ and PI3Kꢀγ ADAPTA assays. C Effects of inhibitors 9 and 12 on AKT and S6RP phosphorylation in
isogenic HMEC lines expressing CAꢀp110α, CAꢀp110β or CAꢀp110δ treated with the indicated compounds at 0.01 ꢀM, 0.1 ꢀM, or 1 ꢀM
for 1h. Du, Duvelisib; Id, Idelalisib.
group to occupy the selectivity pocket formed by Met 752,
while still allowing the pyrimidineꢀhinge contact to occur. The
Hꢀbond of the sulfonamide to Thr 833 may explain the preferꢀ
ence for this functional group and the requirement for 3ꢀ
substitution vs 4ꢀsubstitution of the aniline ring (Figure 3,
Supporting Figure 3, Table 1).
ed a doseꢀdependent reduction in viability (Table 2, Supportꢀ
ing Figure 2).
Table 2: Activity of PI3Kꢀδ/γ inhibitors in cell viability
assays
Lys 779
Ile 825
Trp 760
Tyr 813
Met 752
a patient derived primary cells. IC50s plotted from average of three
replicates.
Val 828
The series of compounds described in this work represent a novel
class of PI3Kꢀδ/γ inhibitors. We were able to develop potent,
selective molecules with cellular activity and drugꢀlike properties
in the absence of structural information. We envisage these moleꢀ
cules will provide useful leads for development of nextꢀgeneration
PI3Kꢀδ/γ targeting therapeutics. Investigation into the binding
mode of these molecules by Xꢀray crystallography may yield
rationale for development of molecules with superior PI3Kꢀδ/γ
selectivity using structureꢀbased design.
Met 900
Thr 833
Figure 3: Docking model of 12 bound to PI3Kꢀδ.
ASSOCIATED CONTENT
Supporting Information
Finally we treated leukaemia cell lines and patient derived
primary CLL cells to examine the effects of PI3Kꢀδ/γ inhibiꢀ
tion on cell viability. Compounds 9 and 12 were comparably
potent to Duvelisib and more potent than Idelalisib in the testꢀ
ed cell lines. In the CLL cells Idelalisib showed little effect,
consistent with previously published reports19, as did
Duvelisib and compound 12, wheras compound 9 demonstratꢀ
The Supporting Information is available free of charge on the
ACS Publications website. Biochemical and cellular assay protoꢀ
cols, compound synthesis and characterization, supporting figures
and supporting tables.
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ACS Medicinal Chemistry Letters
9.
Winkler, D. G.; Faia, K. L.; DiNitto, J. P.; Ali, J. A.;
AUTHOR INFORMATION
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3
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White, K. F.; Brophy, E. E.; Pink, M. M.; Proctor, J. L.; Lussier,
J.; Martin, C. M.; Hoyt, J. G.; Tillotson, B.; Murphy, E. L.; Lim,
A. R.; Thomas, B. D.; Macdougall, J. R.; Ren, P.; Liu, Y.; Li, L.
S.; Jessen, K. A.; Fritz, C. C.; Dunbar, J. L.; Porter, J. R.;
Rommel, C.; Palombella, V. J.; Changelian, P. S.; Kutok, J. L.,
PI3Kꢀdelta and PI3Kꢀgamma inhibition by IPIꢀ145 abrogates
immune responses and suppresses activity in autoimmune and
inflammatory disease models. Chem. Biol. 2013, 20 (11), 1364ꢀ
74.
Corresponding Author
* Email: nathanael_gray@dfci.harvard.edu.
Author Contributions
All authors have given approval to the final version of the manuꢀ
script.
Conflict of Interest Disclosure
9
10.
Ikeda, H.; Hideshima, T.; Fulciniti, M.; Perrone, G.;
The authors declare no competing financial interest.
Miura, N.; Yasui, H.; Okawa, Y.; Kiziltepe, T.; Santo, L.; Vallet,
S.; Cristea, D.; Calabrese, E.; Gorgun, G.; Raje, N. S.;
Richardson, P.; Munshi, N. C.; Lannutti, B. J.; Puri, K. D.; Giese,
N. A.; Anderson, K. C., PI3K/p110{delta} is a novel therapeutic
target in multiple myeloma. Blood 2010, 116 (9), 1460ꢀ8.
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Current Address
Xianming Deng; State Key Laboratory of Cellular Stress Biology,
Innovation Center for Cell Signaling Network, School of Life
Sciences, Xiamen University, Xiamen, Fujian 361102, China
11.
Williams, O.; Houseman, B. T.; Kunkel, E. J.;
Aizenstein, B.; Hoffman, R.; Knight, Z. A.; Shokat, K. M.,
Discovery of dual inhibitors of the immune cell PI3Ks p110δ and
p110γ: a prototype for new antiꢀinflammatory drugs. Chem. Biol.
2010, 17 (2), 123ꢀ134.
ABBREVIATIONS
PI3K, phosphatidylinositolꢀ4,5ꢀbisphosphate 3ꢀkinase. CLL,
chronic lymphocytic leukemia. FL, follicular lymphoma. iNHL,
indolent nonꢀHodgkin’s lymphoma. TꢀALL, Tꢀcell acute lymphoꢀ
cytic leukemia. Akt, Protein kinase B. BTK, Bruton's tyrosine
kinase. mTOR, mechanistic target of rapamycin. DNAꢀPK, DNAꢀ
dependent protein kinase. BRD4, Bromodomainꢀcontaining proꢀ
tein 4. PLK1, poloꢀlike kinase 1. LRRK2, leucineꢀrich repeat
kinase 2. ERK5, extracellularꢀsignalꢀregulated kinase 5. JAK2,
Janus Kinase 2
12.
Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant,
P.; Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.;
Davis, M. I.; Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.;
Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.;
Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P., A
quantitative analysis of kinase inhibitor selectivity. Nat. Biotech.
2008, 26 (1), 127ꢀ132.
13.
Deng, X.; Dzamko, N.; Prescott, A.; Davies, P.; Liu, Q.;
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Discovery of a series of 5,11ꢀdihydroꢀ6Hꢀbenzo[e]pyrimido[5,4ꢀb][1,4]diazepinꢀ6ꢀones as selective PI3Kꢀδ/γ inhibitors
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Fleur M. Ferguson1, Jing Ni1, Tinghu Zhang1, Bethany Tesar2, 3, Taebo Sim4,5, Nam Doo Kim6, Xianming Deng1, Jennifer R.
Brown2, 3, Jean J. Zhao1, Nathanael S. Gray1*
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