Discovery of selective and orally bioavailable protein kinase Cθ (PKCθ) inhibitors from a fragment hit

Article abstract of DOI:10.1021/jm500669m

Protein kinase Cθ (PKCθ) regulates a key step in the activation of T cells. On the basis of its mechanism of action, inhibition of this kinase is hypothesized to serve as an effective therapy for autoimmune diseases such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and psoriasis. Herein, the discovery of a small molecule PKCθ inhibitor is described, starting from a fragment hit 1 and advancing to compound 41 through the use of structure-based drug design. Compound 41 demonstrates excellent in vitro activity, good oral pharmacokinetics, and efficacy in both an acute in vivo mechanistic model and a chronic in vivo disease model but suffers from tolerability issues upon chronic dosing.

Full text of DOI:10.1021/jm500669m

Article
pubs.acs.org/jmc
Discovery of Selective and Orally Bioavailable Protein Kinase Cθ
(PKCθ) Inhibitors from a Fragment Hit
Dawn M. George,*,† Eric C. Breinlinger,† Michael Friedman,† Yang Zhang,∥ Jianfei Wang,∥
Maria Argiriadi,† Pratima Bansal-Pakala,† Martine Barth,⊥ David B. Duignan,† Prisca Honore,‡
QingYu Lang,§ Scott Mittelstadt,‡ Dominique Potin,⊥ Lian Rundell,† and Jeremy J. Edmunds†
†AbbVie Bioresearch Center, 381 Plantation Street, Worcester, Massachusetts 01605, United States
‡AbbVie Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, United States
§AbbVie China R&D Center, 5F, North Jin Chuang Building No. 1, 4560 Jinke Road, Pudong New District, Shanghai 201201, P. R.
China
∥WuXi AppTec (Shanghai) Co., Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, P. R. China
⊥Inventiva, 50 Rue de Dijon, 21121 Daix, France
S
* Supporting Information
ABSTRACT: Protein kinase Cθ (PKCθ) regulates a key step
in the activation of T cells. On the basis of its mechanism of
action, inhibition of this kinase is hypothesized to serve as an
effective therapy for autoimmune diseases such as rheumatoid
arthritis (RA), inflammatory bowel disease (IBD), and
psoriasis. Herein, the discovery of a small molecule PKCθ
inhibitor is described, starting from a fragment hit 1 and
advancing to compound 41 through the use of structure-based
drug design. Compound 41 demonstrates excellent in vitro
activity, good oral pharmacokinetics, and efficacy in both an
acute in vivo mechanistic model and a chronic in vivo disease model but suffers from tolerability issues upon chronic dosing.
regulatory T cell function. Furthermore, blockade of PKCθ
by a small molecule inhibitor rescued the function of Tregs
isolated from RA patients as well as protected these cells from
the inhibitory effects of TNFα, highlighting a potential role for
PKCθ inhibition in restoring immune regulation in chronic
autoimmune diseases such as RA. These complementary effects
(inhibition of T cell activation and enhancement of Treg
function) upon PKCθ inhibition are hypothesized to confer
superior efficacy in T cell-mediated diseases than either
mechanism alone. Given the pivotal and nonredundant role
that PKCθ plays in mediating T cell activation and the finding
that PKCθ inhibition also enhances Treg function, we
specifically targeted this isoform to develop an effective oral
therapy for multiple autoimmune and inflammatory disease
indications such as RA, IBD, and psoriasis.
INTRODUCTION
■
Multiple PKC isoforms exist, each with different functions
including roles in cellular signaling, proliferation, differentiation,
migration, survival, and death.1 Of the 12 known isoforms,
PKCθ and PKCα play fundamental roles in T cell biology.
While PKCα is required for T cell proliferation2 and its
inhibition could contribute to efficacy in inflammatory disease
models, inhibition of this isoform is also associated with
undesirable increases in cardiac contractility.3 PKCθ is highly
expressed in T cells and plays an essential role in interleukin-2
(IL-2) production, cellular expansion, and activation. PKCθ
mediates activation of T cells induced by the T cell receptor
and the CD28 co-stimulation pathways. On the basis of this
mechanism of action, inhibition of PKCθ is expected to block T
cell activation and lead to ablation of T cell responses. PKCθ-
deficient mice display defects in responses to antigen resulting
from defective activation of NF-κB, NFAT, and AP-1 following
T cell receptor engagement.4,5 PKCθ-deficient mice are
resistant to collagen-induced arthritis (CIA),6 experimental
autoimmune encephalomyelitis (EAE),7 and models of
asthma8,9 suggesting that PKCθ inhibition alone may be
sufficient to drive efficacy in chronic autoimmune and
inflammatory diseases.
Although PKCθ has been the focus of intense research over
the past 2 decades,11−22 no PKC inhibitor has successfully
navigated through clinical trials to market. Novartis’ 2
(sotrastaurin, AEB071, Figure 1)11 inhibits multiple PKC
isoforms (IC50: PKCθ 1.0 nM, PKCα 2.1 nM, PKCβ1 2.0 nM,
PKCδ 1.3 nM, PKCε 6.2 nM, PKCη 6.1 nM). Most recently 2
Special Issue: New Frontiers in Kinases
Received: April 29, 2014
Inhibition of PKCθ was recently demonstrated to enhance
suppressive function in regulatory T cells (Tregs),10 suggesting
that PKCθ may mediate a negative feedback signal on
© XXXX American Chemical Society
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has been shown to increase contractility;27 thus, we
hypothesized that improved selectivity over this isoform
would be an effective strategy to minimize potential
cardiovascular safety concerns. Starting from 19 μM (TR-
FRET data) fragment hit 1, structure-based drug design
investigating substitution at the 4-, 6-, and 7-positions led to
the identification of potent compounds that demonstrated
excellent PKCθ/α selectivity, good PK/ADME properties, and
efficacy in acute and chronic in vivo models.
CHEMISTRY
■
Compound 9 was synthesized as described in Scheme 1.
Cyclization of aminobromophenol 4 with 2-chloroacetyl
chloride provided benzo[1,4]oxazinone 5. Alkylation with
ethyl bromoacetate yielded intermediate 6. Treatment with
Lawesson reagent to 7 with subsequent cyclization provided
bromotriazinone 8. Further transformation through Suzuki
chemistry provided 7-substituted triazinone 9. Similar chem-
istry was applied to effect the syntheses of 4-, 6-, and 7-
substituted compounds 15a−w as outlined Scheme 2. Benzo-
[1,4]oxazinones 10 appropriately substituted on the 6- and 7-
positions were prepared from commercially available starting
materials (see Supporting Information). As described pre-
viously, alkylation yielded compound 11 and treatment with
Lawesson reagent afforded intermediate 12. From this
intermediate, two divergent routes were developed. In the
first route, reduction of the nitro group generated aniline 13.
Reductive amination produced substituted amine 14, and final
cyclization with hydrazine provided target triazinones 15. In the
alternative approach from 12, the synthetic sequence was
reversed to accommodate larger substituents R2 at the C4
position. Initial cyclization of 12 with hydrazine to give
triazinone 16 followed by nitro reduction generated aniline 17.
Functionalization via reductive amination or acylation delivered
compounds 15. The synthesis of 7-iPr substituted analogue
(21) was accomplished by later-stage incorporation of the C7
substituent as shown in Scheme 3. Thus, intermediate 15x was
brominated to provide 7-bromo analogue 18. Suzuki coupling
yielded isopropenyl intermediate 19 followed by hydrogenation
to give alkane 20. Subsequent Boc deprotection furnished the
desired C7-iPr compound 21. The C7-trifluoromethylated
analogues were prepared as described in Scheme 4. Starting
Figure 1. Structures of PKC inhibitors from Novartis and Vertex.
completed phase II clinical trials for psoriasis and trans-
plantation (kidney and liver); however, the compound
development was ultimately halted in 2012, as it “did not
show sufficient therapeutic benefit over standard of care”.23
A
transient increase in heart rate was observed in clinical trials
with 2, leading to speculation about the feasibility of developing
a safer approach through selective inhibition of one PKC
isoform instead of multiple PKC isoforms.24,25 In early 2013,
Vertex reported a PKCθ selective pyrazolopyridine 3
(“compound 27”, Figure 1)13 that demonstrated good
selectivity against other PKC family members (Ki: PKCθ 0.08
nM, PKCα 356 nM, PKCβI 15 nM, PKCβII 393 nM, PKCδ 16
nM, PKCε 1 nM, PKCγ 725 nM, PKCη 3.5 nM, PKCμ 2420
nM, PKCζ >5000 nM) and activity in an acute in vivo model of
staphylococcal enterotoxin B-stimulated IL-2 production. The
200× selectivity observed for PKCθ versus PKCδ was
hypothesized to potentially improve the overall safety profile
by mitigating the risk of B cell autoimmunity associated with
inhibition of the PKCδ isoform. However, no preclinical
cardiovascular or toxicology safety data were disclosed and no
advancement into the clinic has been reported. Rigel has also
announced the nomination of a dual PKCα/PKCθ clinical
candidate, R524,26 presenting data arguing that dual α/θ
inhibition is required for full efficacy in a model of
transplantation. No clinical trials with this compound have
been reported, and the structure has not been disclosed.
Our focus was to identify highly potent compounds with
selectivity for PKCθ over the PKCα isoform. PKCα is a
negative regulator of heart contractility, and inhibition of PKCα
a
Scheme 1
a
(i) 2-Chloroacetyl chloride, NaHCO3, DME, water, 80 °C; (ii) ethyl 2-bromoacetate, K2CO3, acetone, 56 °C; (iii) Lawesson reagent, toluene, 120
°C; (iv) NH2NH2−H2O, EtOH, rt; (v) potassium dimethylaminomethyltrifluoroborate, Pd(OAc)2, 2-(dicyclohexylphosphino)-2′,4′,6′-
triisopropylbiphenyl, Cs2CO3, dioxane, water, 100 °C.
B
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a
Scheme 2
a
(i) 2-Bromoalkyl acid ethyl ester derivative, K2CO3, solvent, heat; (ii) Lawesson reagent, toluene, 120 °C; (iii) NH2NH2−H2O, EtOH, 80 °C; (iv)
zinc powder, ammonium chloride, MeOH, rt; (v) ketone or aldehyde, AcOH, MeOH, heat, then NaBH3CN, heat; (vi) for examples with BOC
group; HCl, EtOAc, rt; (vii) carboxylic acid derivative, iPr2EtN, HATU, DMF, rt.
a
Scheme 3
a
(i) n-Bu4NBr3, DCM, MeOH, rt; (ii) 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane, K2CO3, Pd(dppf)2Cl2, water, dioxane, 80 °C; (iii)
Pd/C, MeOH, H2 (55 psi); (iv) TFA, DCM, rt.
from substituted aminophenol 22 (prepared as described in
Materials and Methods), cyclization with 2-chloroacetyl
chloride and alkylation to ester 23 followed by cyclization
with hydrazine afforded triazinone 24. Introduction of a SEM
protecting group (25), Buchwald−Hartwig reaction to generate
amine 26, and two-step deprotection gave triazinone 28.
Scheme 5 describes the synthetic approaches to access -O- and
-CH2- C6-linked analogues. Starting from common intermedi-
ate 25, conversion to the boronate ester followed by oxidation
with hydrogen peroxide generated phenol 29. Formation of
ether 30 and dual SEM and Boc deprotection afforded
triazinone 31. Alternatively, Heck reaction of intermediate 25
provided methylene azetidine 32 which underwent hydro-
genation to 33. Deprotection using TBAF followed by TFA
afforded triazinone 34.
A synthetic approach employing Suzuki chemistry similar to
that used to prepare C7-iPr compound 21 but omitting the
reduction step was utilized for the synthesis of C7-Ph analogues
37 and 38. The stereoisomers were separated via chiral SFC
purification as detailed in the Supporting Information. Similarly,
racemic compounds 15v and 28 were separated into the (R)
and (S) isomers (35 and 36; 39 and 40, respectively) by chiral
SFC (see Supporting Information for details). 3-Methylazeti-
dine analogue 41 was prepared using a route similar to that for
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a
Scheme 4
a
(i) 2-Chloroacetyl chloride, K2CO3, DMF, then ethyl 2-bromopropanoate, Cs2CO3, rt; (ii) Lawesson reagent, toluene, reflux then NH2NH2−H2O,
80 °C; (iii) NaH, SEMCl, THF, rt; (iv) tert-butyl 3-aminoazetidine-1-carboxylate, Pd(OAc)2, Cs2CO3, BINAP, toluene, reflux; (v) TBAF, THF, 80
°C; (vi) TFA, DCM, rt.
a
Scheme 5
a
(i) Bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, dioxane, 90 °C, then aqueous H2O2, dioxane, AcOH, rt; (ii) tert-butyl 3-((methylsulfonyl)-
oxy)azetidine-1-carboxylate, Cs2CO3, DMF, 80 °C; (iii) TFA, DCM, rt, then aqueous NH4OH, rt; (iv) 3-methyleneazetidine-1-carboxylic acid tert-
butyl ester, Et3N, CH3CN, tri(o-tolyl)phosphine, Pd(OAc)2, 110 °C; (v) Pd(OH)2/C, MeOH, H2 (50 psi); (vi) TBAF, THF, 80 °C, then TFA,
DCM, rt.
compound 39. The absolute configuration of 35 was confirmed
by small molecule X-ray (vide infra), and its isomer 36 was
assigned by comparison. The absolute configurations of 37, 38,
39, 40, and 41 were initially assigned based on their relative
activity, with the R isomer assumed as the more active isomer as
predicted by a superior fit in the PKCθ active site. Specific
rotation confirmed that the more active isomers had a negative
rotation. The absolute stereochemistry of 39 and 41 was later
confirmed by stereoselective syntheses prepared using the route
described in Scheme 4 starting from chiral 23 (synthesis
described in the Supporting Information).
a recurring challenge. A high-throughput screen (HTS) of our
corporate compound collection provided a dearth of compel-
ling starting points; hence, a complementary approach, via a
TR-FRET screen of a ∼250-member library of fragment-sized
kinase hinge binders, was conducted. From this effort, tricyclic
triazinone hit 1 (PKCθ TR-FRET IC50 = 19 μM, PKCθ
enzyme IC50 = 453 μM) was identified. Although the potency
was moderate, the favorable binding efficiency (BEI = 25 based
on TR-FRET data) and unique nature of the triazinone scaffold
obligated additional investigation. The synthesis of compound
1 was previously described. Two analogues were noted to have
moderate efficacy in a carrageenan-induced rat paw edema
model,28 but no subsequent findings on this chemotype have
been reported.
RESULTS AND DISCUSSION
■
PKCθ as a target has tantalized medicinal chemists with the
potential to block T cell signaling based on the key role of
PKCθ in regulating T cell activation. However, identification of
chemical matter with desirable activity, selectivity, and
physicochemical properties to enable in vivo dosing has been
As is frequently the case with fragment-based discovery, we
relied on crystallographic data for initial compound design. As
the result of two potential donor−acceptor pairings from the
triazinone ring (CO/NH or NH/N), modeling of
compound 1 against PKCθ suggested multiple potential
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binding modes. Only two solved X-ray structures in PKCθ have
been reported, one with staurosporine and one with a
maleimide compound: 1XJD at 2.00 Å29 and 2JED at 2.32
Å.30 Internal efforts have shown that PKCθ protein expression,
purification, and crystallization are less robust compared to
other kinase targets. As a result, PKCα has been utilized as a
reasonable crystallographic substitute in combination with
PKCθ homology models.11 In our experience, generation of
PKCθ and PKCα crystal structures requires significant
(minimally, IC50 < 1 μM) PKCθ or PKCα activity. Thus,
because of the weak binding affinity of 1 for PKCθ (IC50 = 19
μM) and PKCα (IC50 > 50 μM), efforts to generate a surrogate
crystal structure were pursued. Because of the ease of
crystallization, focal adhesion kinase (FAK) was selected as a
robust crystallographic surrogate. In spite of modest potency
for FAK (TR-FRET IC50 = 156 μM), we were able to obtain a
binding mode of 1 at high resolution in the kinase ATP binding
pocket region (Figure 2, 2.06 Å, PDB accession code 4Q9S).
Figure 3. Compound 9 model in PKCα compared to 2. Red surface =
acidic regions. Blue surface = basic regions. White surface = neutral
regions.
compound (9) did not deliver a desired improvement in
potency (PKCθ IC50 = 16 μM). A related approach, targeting
the PKCθ DFG Asp522 (Asp481 in PKCα) in a nearby region
in the protein by substitution off the C6-position of the scaffold
with an amino azetidine moiety (Figure 4, 15a), met with
Figure 2. Compound 1 crystal structure in FAK (PDB code 4Q9S).
The FAK−1 cocrystal structure clearly identified the N-NH
combination on the triazinone ring as the key hinge binding
element to FAK backbone residues Glu500 and Cys502
(Glu459 and Leu461 in PKCθ). On the basis of this
crystallographic information, we developed a PKCθ triazi-
none-bound model that maintained this binding mode and
predicted a water-mediated H-bond of the CO with back
pocket Thr442, a residue unique to the PKC family. This water
molecule was observed in internal PKCα structures with other
chemotypes (not shown). We hypothesized that, based on the
nonplanar conformation of this simple core combined with the
predicted interaction with unique, back pocket Thr442, broad
kinome selectivity with this chemotype would be achievable.
A structure-based design strategy was developed based on
the FAK binding mode. Thus, a modeling overlay of the FAK
compound 1 structure with a known structure of 2 in PKCα
(PDB accession code 3IW4)11 was utilized for design (Figure
3). An initial approach to drive potency was to target an
interaction with the acidic region of the protein with a basic
amine. Although several acidic residues line this cavity, initial
design focused on targeting Asp508 (Asp467 in PKCα), similar
to the interaction observed in the 2 crystal structure. Modeling
of a -CH2NMe2 moiety off the C7 position appeared to provide
the length and trajectory for optimal interaction; however, this
Figure 4. Model of compound 15a in PKCθ. Red surface = acidic
regions. White surface = neutral regions.
greater success (PKCθ IC50 = 1.3 μM). Comparator
compounds replacing the azetidine with a cyclobutyl (15b)
or oxetanyl (15c) group were significantly less potent (Table 1,
PKCθ IC50 = 38 or PKCθ IC50 > 50 μM, respectively), lending
credence to the hypothesis of an amine−Asp interaction.
Additional exploration of the C6 position with a variety of
amines, via amine or amide linkers, provided compounds 15d−
q (Table 1) with a range of potencies, none of which were
improved over aminoazetidine 15a. We conjectured that even
minor alterations in the trajectory of the amine toward the key
Asp interaction could result in a loss in potency.
To further improve the potency of these analogues, we
targeted a small “lipophilic pocket” proximal to residues Ala407
and Val394 above the plane of the core that could be accessed
by substitution adjacent to the carbonyl on the triazinone ring.
While the aminoazetidine moiety at C6 was held constant,
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Table 1. Structure−Activity Relationship for C6 Variants
a
Mean from n ≥ 2 experiments.
substitution at C4 was explored (Table 2), with initial
evaluation of compounds racemic at this position. Addition of
lipophilic substituents was undertaken, as modeling suggested
there was a hydrophobic region that could be accessed for
additional potency gain. Substituents such as CH3, F, iPr, and
CF3 were tolerated (Table 3) and maintained significant
selectivity for PKCθ over PKCα ranging from 17- to 69-fold.
These compounds were further profiled in a cellular assay
measuring the CD3/CD28-stimulated production of IL-2 from
human T-blasts (Table 3). In spite of poor permeability (in all
cases <1 × 10−6 cm/s (MDCK-MDR1 7.4 (pH): Papp from A to
B)), these secondary amines bearing a 7-substituent all
demonstrated good cellular activity (0.23−0.43 μM) and, in
general, the compounds had good stability in rat and human
microsomes. In the context of the C7-CF3 group, changes to
the linker at C6 were investigated. Replacement of the amino
linker with an ether (31) resulted in a 3-fold drop in cellular
potency. The methylene analogue (34) was tolerated but
presented no obvious potency advantage (Table 4).
Table 2. Structure−Activity Relationship for C4 Variants
a
IC50 (μM)
compd
R1
PKCθ
PKCα
α/θ
15a
15r
15s
15t
15u
H
1.3
7.1
4
Me
Et
0.23
1.0
9.4
41
5
5.4
cyPr
Ph
3.5
>50
>50
>14
>50
With promising analogues in hand, we next explored whether
the stereochemistry of the C4-methyl group was important for
activity (Table 5). Three pairs of analogues with varying groups
at C7 were separated. In each case, one enantiomer was more
potent toward PKCθ than the other, ranging from 7× to 16×.
The more potent isomer also showed improved selectivity for
PKCθ versus PKCα, up to >90× for compounds 35 and 39.
Stereochemistry had no effect on permeability or unbound
microsomal clearance. The (R) stereochemistry of the more
active isomer at C4 was later confirmed by small molecule X-
ray crystallography of C7-methyl derivative 35 (see Supporting
a
Mean from n ≥ 2 experiments.
a methyl group at C4 as in 15r provided a 5-fold boost in
potency compared to parent 15a and interestingly demon-
strated 41-fold selectivity over PKCα. The C4-Me group of
triazinone 15r fits optimally in a small lipophilic pocket (Figure
4), and therefore, larger substituents (Et, cyPr, Ph) at C4
resulted in reduced potency.
With enhanced substituents at C4 and C6 apparently
identified, subsequent exploration of the C7 position with
F
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Table 3. Structure−Activity Relationship for C7 Variants
a
IC50 (μM)
a
b
c
compd
R1
PKCθ
PKCα
α/θ
IC50 (μM), IL-2
permeability (10−6 cm/s)
CLint,u, hu/rat (L h−1 kg−1
)
15r
15v
15w
21
H
0.23
0.11
0.10
0.13
9.4
4.1
6.9
2.2
2.2
41
37
69
17
33
2.7
0.42
0.31
0.86
0.28
0.85
2.0/2.9
Me
F
0.43
0.31
0.34
<2.0/3.7
<1.6/3.0
<2.0/5.3
3.4/<4.0
iPr
CF3
28
0.067
0.23
a
b
c
Mean from n ≥ 2 experiments. MDCK-MDR1 7.4 (pH): Papp from A to B. In vivo predicted unbound CLint from human and rat microsome
stability studies.
Table 4. Structure−Activity Relationship for C6-Linker Variants
a
IC50 (μM)
a
b
c
compd
X
PKCθ
PKCα
α/θ
IC50 (μM), IL-2
permeability (10−6cm/s)
CLint,u, hu/rat (L h−1 kg−1
)
28
31
34
NH
O
0.067
0.19
2.2
3.0
1.6
33
16
27
0.23
0.91
0.85
3.4/<4.0
3.1/<4.4
<2.8/4.9
CH2
0.059
0.39
0.50
a
b
c
Mean from n ≥ 2 experiments. MDCK-MDR1 7.4 (pH): Papp from A to B. In vivo predicted unbound CLint from human and rat microsome
stability studies.
Table 5. Structure−Activity Relationship for C4 Stereoisomers
a
IC50 (μM)
a
b
c
compd
C4
R1
PKCθ
PKCα
α/θ
IC50 (μM), IL-2
permeability (10−6 cm/s)
CLint,u, hu/mo/rat (L h−1 kg−1
)
35
36
37
38
39
40
(R)
(S)
(R)
(S)
(R)
(S)
Me
Me
Ph
0.055
0.39
5.1
3.0
1.8
6.6
4.6
2.9
93
8
0.27
4.2
0.35
0.16
0.27
0.23
0.44
0.25
2.4/_/_
_/<4.4/_
0.049
0.76
37
9
0.12
2.4
5.4/28/12
6.6/25/<6.2
4.8/<8.3/<4.7
5.1/<7.2/7.1
Ph
CF3
CF3
0.049
0.32
94
9
0.22
1.0
a
b
c
Mean from n ≥ 2 experiments. MDCK-MDR1 7.4 (pH): Papp from A to B. In vivo predicted unbound CLint from human, mouse, and rat
microsome stability studies.
Information); a protein structure of 35 bound to PKCθ (2.6 Å,
PDB accession code 4Q9Z) confirmed the hypothesized
binding mode (Figure 5). The structure confirmed the N and
NH elements from the triazinone ring as the key hinge binder
elements with Glu459 and Leu461 of the protein backbone.
Two additional key contacts were observed, a water-mediated
interaction of the carbonyl group with back pocket Thr442 and
H-bonds between the azetidine NH with Asp522 and Asn509
of the protein. The (R)-stereochemistry positioned the methyl
group at C4 optimally to target a lipophilic pocket in that
region. Leu511 prevents an optimal fit for the (S)
configuration, thus explaining the steric preference for the
(R) enantiomer. Strikingly, residues within the C-terminal tail
proximal to the hinge (Pro658-Leu669) are completely ordered
in the X-ray structure and gives rise to a hydrophobic pocket
adjacent to the C7 extension. On the basis of the structure, we
hypothesized that the compelling selectivity for PKCθ versus
PKCα resulted from the optimal contacts of the triazinone to
regions of the Gly-rich loop, the C-terminal tail, and an ATP-
pocket Leu511 (which is a Met in PKCα). In general the
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anesthetized dog cardiovascular model, compound 39 rather
surprisingly did produce a dose-dependent increase in heart
contractility, coupled with decreases in systemic vascular
resistance and mean arterial pressure. However, upon treatment
with hexamethonium, an inhibitor of autonomic ganglionic
neurotransmission,31 the effect on contractility was mitigated
while the drop in blood pressure was exaggerated (Table 8),
suggesting that the increase in contractility observed in the dog
study was a compensatory effect driven by the drop in blood
pressure. The drop in blood pressure was related to a decrease
in vascular resistance suggesting that the hemodynamic changes
were driven by vasodilation. With no clear evidence that the
vasodilation was an on-target effect of PKCθ, we hypothesized
that this effect could potentially be screened out by an iterative
structure−activity relationship (SAR) approach.
Examination of the crystal structure suggested that additional
potency could be derived by incorporation of a small
substituent at the 3-position of the azetidine ring and this
modification was made in the context of the PK-favorable C7-
CF3 moiety. Compound 41 was highly selective across an in-
house panel of 81 kinases (see Supporting Information), with
only two kinases being <100× selective versus PKCθ (Clk2,
54x; Cdk8, 87x), and displayed a selectivity profile across PKC
isoforms similar to compound 39 (PKCθ versus: PKCα, 74×;
PKCβ, 26×; PKCδ, 14×; PKCε, 4×; PKCγ, 726×; PKCι,
213×; PKCζ, >1000×). Profiling across 80 receptors (CEREP;
see Supporting Information) at 10 μM revealed only two hits
with >50% inhibition: κ (KOP, agonist, 61%) and Ca2+ channel
(L, verapamil site, antagonist, 66%). No inhibition of the hERG
channel (IC50 > 30 μM) was observed. Because of good cellular
potency (EC50 = 0.12 μM), selectivity in kinome and CEREP
profiling, and in vitro ADME properties, compound 41 was
advanced to mouse and rat PK (Table 9). Compound 41 has
high oral exposure in mouse PK which enabled evaluation of
the compound in an acute in vivo model of inflammation
measuring concanavalin A (Con A) induced IL-2 production in
mice. The dosing regimen was designed to administer
compound so that Cmax would coincide with Con A
administration. Thus, a single oral dose of compound 41
given 2 h prior to Con A administration resulted in dose-
dependent inhibition of IL-2 production (Figure 7). Mean
terminal concentrations escalated linearly with dose: 0.037 μM
(1 mg/kg), 0.11 μM (3 mg/kg), 0.31 μM (10 mg/kg), 0.90 μM
(30 mg/kg), and 2.5 μM (100 mg/kg). 41 had moderate
plasma protein binding (fu = 0.27). On the basis of the
promising results in the acute model, compound 41 was
advanced into a chronic model of arthritis. The glucose 6-
phosphate isomerase (GPI) model in DBA/1 mice has been
reported in the literature.32,33 This model was appealing
because of its T cell-dependent mechanism and relatively short
duration (17 days). For our purposes, GPI was generated in-
house32 and the GPI arthritis model protocol was followed as
reported in the literature. With twice-daily oral dosing of
Figure 5. X-ray structure of compound 35 in PKCθ (PDB code
4Q9Z). Red surface = acidic regions.
compounds are highly selective across the kinome and over
PKC isoforms α, γ, ι, and ζ but are only moderately selective
against PKCβ, PKCε, and PKCδ (Table 6). While both the C7-
methyl and phenyl derivatives (35 and 37, respectively)
suffered from poor oral exposure in rat PK, the CF3 derivative
was differentiated by good overall characteristics in both rat and
mouse (Table 7).
In order to test our hypothesis that selectivity for PKCθ over
PKCα would reduce the chance of increased contractility,
compound 39 (PKCα/PKCθ = 94-fold) was profiled in an
anesthetized rat cardiovascular model measuring contractility,
heart rate, and mean arterial pressure (see Supporting
Information). When dosed over 30 min infusions at 3, 10,
and 30 mg/kg to deliver increasing plasma concentrations of
1.5, 4.8, and 19 μM, the CV profile was devoid of effects on left
ventricular dP/dt50 (Figure 6A), supporting our selectivity
hypothesis as a reasonable approach to mitigate changes in
contractility. This was in contrast to 2 which showed a
significant increase in dP/dt50 in the anesthetized rat (14% at
1.3 μM, 35% at 5.8 μM, and 76% at 9.7 μM). The CV profile of
compound 39 did show a significant drop in mean arterial
pressure (Figure 6B) and a moderate drop in heart rate (Figure
6C) starting at the end of the second infusion, possibly due to a
break in selectivity versus an off-target effect at higher
concentrations. CEREP profiling (see Supporting Information)
identified Ca2+ channel (L-verapamil site, antagonist, 51%
inhibition) as the only receptor inhibited by >50% at 10 μM,
and across an in-house panel of 81 kinases (see Supporting
Information), only three kinases were <100× selective versus
PKCθ (PKA, 58×; Cdk8, 80×; Clk2, 86×). Compound 39 did
not inhibit the hERG channel (IC50 > 30 μM). In an
a
Table 6. Selectivity of Compounds 35, 37, and 39 across PKC Isoforms
IC50 (μM)
PKCδ
compd
PKCθ
PKCα
PKCβ
PKCε
PKCγ
PKCι
PKCζ
b
a
35
37
39
0.054
0.049
0.045
5.1
1.8
4.6
0.52
0.75
0.33
0.47
−
0.46
23
−
0.86
−
8.5
b
0.087
1.5
13
b
0.14
7.8
3.9
33
a
b
Mean from n ≥ 2 experiments unless noted. Data from n = 1.
H
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a
Table 7. PK Data of Compounds 35, 37, and 39
compd
species
CLp (L h−1 kg−1
)
t1/2 (h)
Cmax (ng/mL)
AUC (ng·h/mL)
F (%)
35
37
39
39
rat
4.9
4.9
2.1
5.8
3.0
5.9
5.5
2.7
8.7
5.3
80
60
9.2
5.4
28
rat
43
rat
409
1730
mouse
342
85
a
Rat PK reported (iv bolus 1 mg/kg in 50% PEG400; po dose of 3 mg/kg in 0.5% HPMC and 0.02% Tween 80). Mouse PK reported (iv bolus 3
mg/kg in 50% PEG400; po dose of 10 mg/kg in 0.5% HPMC and 0.02% Tween 80). Data are reported as an average of three animals.
q = quartet, br = broad, m = multiplet), coupling constant (Hz),
integration. Analytical LCMS analyses were conducted using Shimadzu
LC-20AB pumps and a SPD-M20 PDA detector set at 220 and 254
nm with the MS detection performed with a MS-2010EV Micromass
Platform LC spectrometer in electrospray mode. Unless alternative
column and eluting conditions are detailed in the experiment, the
LCMS method used was as follows. Column: Ultimate XB-C18, 3 μm,
30 mm × 2.1 mm; mobile phase: acetonitrile (0.02% TFA) in water
(0.04% TFA), from 10% to 80% within 2 min. Flow rate: 1.2 mL/min.
Wavelength: 220 nm. Analytical SFC-MS analyses were conducted
using Mettler pumps and an Agilent G1315B detector set at 190−370
nm with the MS detection performed with Agilent 6110 following
detailed column and eluting conditions below. Preparative reverse
phase (RP) HPLC was performed using a Gilson 322 pump, a Gilson
156 UV detector set at 220 and 254 nm, and Gilson GX-281 liquid
handler following detailed column and eluting conditions below.
Normal-phase silica gel preparative purification was performed using
an automated Combiflash companion from ISCO using prepacked
silica gel cartridges supplied by Santai Technologies Inc. and Agela Inc.
SFC separation was performed using Berger SFC Analytix following
detailed column and eluting conditions below. Microwave reactions
were performed using Biotage INITIATOR60EXP microwave syn-
thesis system. Qualitative ( ) optical rotation data were collected
using a PDR-chiral advanced laser in-line polarimeter, model
ALP2002. Analytical TLC was performed using SGF254 thin layer
chromatography plates (0.2−0.25 mm) supplied by Combinol Reagent
(Yantai) Co., Ltd. Preparative TLC was performed using SGF254 thin
layer chromatography plates (0.4−0.5 mm) supplied by Yucheng
Chemical (Shanghai) Co., Ltd. The purity of final products was ≥95%
as established by analytical liquid chromatography using a PDA
compound 41 from day 1 to day 17, dose-dependent inhibition
of paw swelling was observed (Figure 8). Compound 41 gave
full inhibition at the 30 mg/kg dose, comparable to treatment
with anti-CD4 (rat anti-mouse GKI5.4 ra IgG2b/k, generated
internally) which is known to block T cell activation34 and
partial (62%) inhibition at the 10 mg/kg dose. Compound
exposure escalated in a linear fashion on day last (mean AUC0−t
of 4.5 μg·h/mL at 10 mg/kg and 15.1 μg·h/mL at 30 mg/kg).
Terminal unbound plasma concentrations confirmed com-
pound coverage over the unbound cellular EC80 at the 30 mg/
kg dose and partial coverage at the 10 mg/kg dose. However,
with b.i.d. dosing at 100 mg/kg for >10 days, the compound
was not tolerated and the animals had to be sacrificed. No cause
of the tolerability issue was apparent upon necropsy, and no
obvious explanation for the observed toxicity from off-target
receptors or kinases was apparent. Thus, in spite of the good in
vitro activity and in vivo efficacy of compound 41, we felt that a
∼3× margin based on dose was unacceptable, and advancement
of this compound was halted. In an effort to increase the overall
safety margin, efforts were focused on improving the potency of
this chemotype while maintaining good oral exposure in order
to reduce the high exposure required for efficacy while
minimizing Cmax/Cmin. These results will be described in a
subsequent communication.
CONCLUSION
■
The crystal structure of fragment hit 1 in surrogate kinase FAK
enabled the structure-based design and optimization of the
triazinone chemotype leading to the discovery of analogues
with good selectivity for PKCα versus PKCθ and moderate
cellular activity. When occupation of lipophilic pockets at C6
and C7 is targeted, a sequence of analogues with improved
cellular activity emerged; however, these suffered from
suboptimal PK. Incorporation of a C7-CF3 moiety dramatically
improved oral exposure while maintaining good in vitro activity,
and both 39 and 41 demonstrated dose-dependent inhibition of
IL-2 in an acute in vivo model. The most promising compound
(41) effectively reduced paw swelling in a mouse GPI model of
arthritis but required high exposures for efficacy and was poorly
tolerated at doses only marginally higher than efficacious doses.
Further optimization studies to identify compounds with
reduced efficacious exposures will be presented in the next
paper in this series.
1
detector at 220 nm and confirmed by H NMR.
6-(Azetidin-3-ylamino)-4-methyl-7-trifluoromethyl-2,10-di-
hydro-9-oxa-1,2,4a-triazaphenanthren-3-one Ditrifluoroacetic
Acid (28). Step A. To a 0 °C mixture of 4-bromo-3-(trifluoromethyl)-
phenol (Apollo, 10.8 g, 44.8 mmol) in AcOH (40 mL) was added
concentrated sulfuric acid (1.5 mL) followed by fuming nitric acid (5.2
g), and the reaction mixture was stirred for 30 min. Additional
concentrated sulfuric acid (9 mL) was added. The temperature was
allowed to rise to ambient temperature, and the reaction mixture was
stirred for 3 h at room temperature. The mixture was poured into ice−
water (500 mL), and the aqueous solution was extracted with EtOAc
(3 × 50 mL). The combined organic phase was washed with brine (50
mL), dried over anhydrous Na2SO4, and filtered. The filtrate was
evaporated in vacuo, and the residue was purified by column
chromatography on silica gel (eluting with 10−20% EtOAc in
petroleum ether) to give 4-bromo-2-nitro-5-(trifluoromethyl)phenol
1
(4.7 g, 36%) as a yellow oil. H NMR (DMSO-d6, 400 MHz): δ 12.0
(br s, 1H), 8.31 (s, 1H), 7.54 (s, 1H). TLC: Rf = 0.4 (eluting with 50%
DCM in petroleum ether).
MATERIALS AND METHODS
Step B. To a mixture of zinc powder (16.23 g, 248 mmol) and
ammonium chloride (13.28 g, 248 mmol) in MeOH (80 mL) was
added 4-bromo-2-nitro-5-(trifluoromethyl)phenol (7.1 g, 24.8 mmol),
and the reaction mixture was stirred overnight at ambient temperature.
The reaction mixture was filtered, and the filtrate was evaporated in
vacuo. The residue was partitioned between EtOAc (100 mL) and
water (300 mL). The organic phase was separated and washed with
brine (30 mL), dried over Na2SO4, filtered, and concentrated in vacuo
to give 2-amino-4-bromo-5-(trifluoromethyl)phenol (compound 22,
5.9 g, 93%) as a black oil, which was used in the next step without
■
Chemistry. All reagents and anhydrous solvents were of the
highest grade available and were purchased from commercial sources
and were used without further purification or distillation unless
otherwise noted. All NMR spectra were recorded on Bruker Avance III
400 MHz FT-NMR spectrometer with 5 mm BBO(F) probe or a
Varian 400 MHz, 1H/19F/31P/13C 5 mm PFG 4Nuc probe instrument.
1H chemical shifts are reported in δ values in ppm with MeOH-d3,
DMSO-d6, or CDCl3 as the reference standard. Data are reported as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet,
I
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Table 8. Anesthetized Dog Cardiovascular Profile for
Compound 39 with and without Hexamethonium
Change from Baseline Compared to Vehicle (n = 3)
39 with
hexamethonium,
plasma concn
39, plasma
concn (μM)
(μM)
2.0
7.7
1.7
6.9
mean arterial pressure (mmHg)
−3
−4
−12
−7
1
3
3
dP/dtmax (%)
34
Table 9. Profile for Compound 41
a
IC50 (μM)
c
CLint,u
,
a
b
IC50 (μM), permeability
hu/mo/rat
PKCθ PKCα α/θ
IL-2
(10−6 cm/s)
1.5
(L h−1 kg−1
)
0.023
1.7
74
0.12
<2.0/<5.7/<3.3
d
PK
CLp
t1/2
(h)
Cmax
(ng/mL)
species
(L h−1 kg−1
)
AUC (ng·h/mL) F (%)
rat
2.7
1.5
4.7
3.1
160
846
782
74
mouse
9260
108
a
b
Mean from n ≥ 2 experiments. MDCK-MDR1 7.4 (pH): Papp from
c
A to B. In vivo predicted unbound CLint from human, mouse, and rat
microsome stability studies. Rat PK reported (iv bolus 1 mg/kg in
d
50% PEG400; po dose of 3 mg/kg in 0.5% HPMC and 0.02% Tween
80). Mouse PK reported (iv bolus 3 mg/kg in 50% PEG400; po dose
of 10 mg/kg in 0.5% HPMC and 0.02% Tween 80). Data are reported
as an average of three animals.
Figure 6. Anesthetized rat cardiovascular profile for compound 39.
Shown are the percentage change in baseline dP/dt50 (A), change in
mean arterial blood pressure (B), and change in heart rate (C)
following three 30 min infusions of vehicle and compound 39 at 3, 10,
and 30 mg/kg in anesthetized rats.
Figure 7. Acute in vivo efficacy data (mouse Con A) with compound
41. Mice are predosed orally with compound 41 or 30 mg/kg
cyclosporin A (CsA), 2 h prior to challenge. Two h postchallenge,
serum is analyzed for murine IL-2 levels. Statistical significance was
determined by a one-way ANOVA using a Dunnett’s post-test.
1
further purification. H NMR (DMSO-d6, 400 MHz): δ 9.89 (br s
1H), 6.98 (s, 1H), 6.92 (s, 1H), 5.53 (br s, 2H). LCMS: tR = 1.096
the reaction mixture was stirred for an additional 3 h at ambient
temperature. The reaction mixture was poured into water (150 mL),
and the aqueous mixture was extracted with EtOAc (3 × 50 mL). The
combined organic phase was washed with brine (30 mL) and dried
over Na2SO4, filtered, and the filtrate was evaporated in vacuo. The
residue was purified by column chromatography on silica gel (eluting
with 10−17% EtOAc in petroleum ether) to give ethyl 2-(6-bromo-3-
oxo-7-(trifluoromethyl)-2H-benzo[b][1,4]oxazin-4(3H)-yl)-
min. MS m/z: 256, 258 [M + H]+.
Step C. To a mixture of 2-amino-4-bromo-5-(trifluoromethyl)-
phenol (5.9 g, 23.05 mmol) and K2CO3 (9.55 g, 69.01 mmol) in DMF
(80 mL) was added 2-chloroacetyl chloride (2.86 g, 25.3 mmol)
dropwise at room temperature, and the reaction mixture was stirred for
16 h at ambient temperature. Cs2CO3 (7.51 g, 23.05 mmol) and ethyl
2-bromopropanoate (6.26 g, 34.6 mmol) were added sequentially, and
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Journal of Medicinal Chemistry
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carboxylate (1.39 g, 8.09 mmol), diacetoxypalladium (0.27 g, 1.21
mmol), Cs2CO3 (1.32 g, 4.05 mmol), and BINAP (1.0 g, 1.62 mmol)
in toluene (10 mL) was heated at reflux for 2 h. The reaction mixture
was cooled to ambient temperature and filtered. The filtrate was
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (eluting with 20% EtOAc in petroleum
ether) to give 3-[4-methyl-3-oxo-7-trifluoromethyl-2-(2-
trimethylsilanylethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-
phenanthren-6-ylamino]azetidine-1-carboxylic acid tert-butyl ester
(compound 26, 2.1 g, 89%) as a pale yellow solid. 1H NMR
(CDCl3, 400 MHz): δ 7.14 (s, 1H), 5.86 (s, 1H), 5.10 (q, J = 9.2 Hz,
2H), 4.63 (m, 2H), 4.51 (m, 2H), 4.33 (m, 2H), 4.18 (m, 1H), 3.77
(m, 2H), 3.65 (t, J = 8.4 Hz, 2H), 1.47 (d, J = 6.4 Hz, 3H), 1.45 (s,
9H), 0.99 (t, J = 8.4 Hz, 2H), 0.00 (s, 9H). LCMS: tR = 1.503 min. MS
m/z: 608 [M + Na]+.
Step G. To a mixture of 3-[4-methyl-3-oxo-7-trifluoromethyl-2-(2-
trimethylsilanylethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-
phenanthren-6-ylamino]azetidine-1-carboxylic acid tert-butyl ester
(0.37 g, 0.632 mmol) in THF (4 mL) was added tetrabutylammonium
fluoride (1 M in THF, 2.53 mL, 2.53 mmol). The reaction mixture was
heated at 80 °C overnight and then cooled to ambient temperature,
quenched by the addition of water (10 mL), and extracted with EtOAc
(3 × 10 mL). The combined organic phase was washed with brine (2
× 10 mL) and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (eluting with 7−50% EtOAc in
petroleum ether) to give 3-(4-methyl-3-oxo-7-trifluoromethyl-2,3,4,10-
tetrahydro-9-oxa-1,2,4a-triazaphenanthren-6-ylamino)azetidine-1-car-
boxylic acid tert-butyl ester (compound 27, 0.19 g, 66%) as a white
solid. LCMS: tR = 1.203 min. MS m/z: 478 [M + Na]+. TLC: Rf = 0.6
(eluting with 33% EtOAc in petroleum ether).
Figure 8. Chronic in vivo efficacy data (mouse GPI) with compound
41. GPI immunized mice were dosed starting on day 0 with compound
41 or with vehicle orally twice a day. Anti-CD4 antibody was dosed
interperitoneally twice per week starting on day 0. Statistical
significance was determined by a two-way ANOVA using a Bonferroni
post-test.
1
propanoate (compound 23, 4.2 g, 46%) as a yellow oil. H NMR
(CDCl3, 400 MHz): δ 7.35 (s 1H), 7.12 (s, 1H), 5.26 (q, J = 7.2 Hz, 1
H), 4.68 (d, J = 15.4 Hz, 1H), 4.62 (d, J = 15.4 Hz, 1H), 4.24 (q, J =
7.2 Hz, 2H), 1.65 (d, J = 7.2 Hz, 3 H), 1.24 (t, J = 7.2 Hz, 3H). TLC:
Rf = 0.5 (eluting with 25% EtOAc in petroleum ether).
Step H. A mixture of 3-(4-methyl-3-oxo-7-trifluoromethyl-2,3,4,10-
tetrahydro-9-oxa-1,2,4a-triazaphenanthren-6-ylamino)azetidine-1-car-
boxylic acid tert-butyl ester (0.012 g, 0.029 mmol) in dichloromethane
(0.9 mL) and trifluoroacetic acid (0.3 mL) was stirred for 1 h at
ambient temperature. The solvent was removed in vacuo, and the
residue was purified by preparative HPLC (column, Phenomenex
Synergi C18 150 mm × 25 mm, 4 μm; MeCN in water (0.075% TFA),
from 0% to 30% in 10 min; flow rate, 25 mL/min; wavelength, 220/
254 nm; column temperature, 30 °C) to give 6-(azetidin-3-ylamino)-4-
methyl-7-trifluoromethyl-2,10-dihydro-9-oxa-1,2,4a-triazaphenanth-
ren-3-one ditrifluoroacetic acid (compound 28, 0.0105 g, 62%) as a
Step D. A mixture of ethyl 2-(6-bromo-3-oxo-7-(trifluoromethyl)-
2H-benzo[b][1,4]oxazin-4(3H)-yl)propanoate (1.30 g, 3.28 mmol)
and Lawesson reagent (1.46 g, 3.61 mmol) in toluene (10 mL) was
heated at reflux for 1 h. The reaction mixture was cooled to ambient
temperature. Hydrazine hydrate (0.84 g, 26.3 mmol) was added, and
the reaction mixture was heated at 80 °C for 1 h. The reaction mixture
was cooled to ambient temperature, and the solvent was evaporated in
vacuo. The residue was purified by column chromatography on silica
gel (eluting with 16−33% EtOAc in petroleum ether) to give 6-bromo-
4-methyl-7-trifluoromethyl-2,10-dihydro-9-oxa-1,2,4a-triazaphenanth-
1
white solid. H NMR (MeOH-d6, 400 MHz): δ 7.17 (s, 1H), 6.26 (s,
1H), 4.67−4.71 (m, 2H), 4.46−4.62 (m, 4H), 4.10−4.14 (m, 2H),
1.47 (d, J = 6.8 Hz, 3H). LCMS (column, Ultimate XB-C18, 3 μm, 30
mm × 2.1 mm; mobile phase, acetonitrile (0.02% TFA) in water
(0.04% TFA), from 0% to 60% within 4 min; flow rate, 1.2 mL/min;
wavelength, 220 nm), tR = 1.916 min. MS m/z: 356 [M + H]+.
6-(Azetidin-3-ylamino)-4(R)-methyl-7-trifluoromethyl-2,10-
dihydro-9-oxa-1,2,4a-triazaphenanthren-3-one Trifluoroacetic
Acid (39) and 6-(Azetidin-3-ylamino)-4(S)-methyl-7-trifluoro-
methyl-2,10-dihydro-9-oxa-1,2,4a-triazaphenanthren-3-one
Trifluoroacetic Acid (40). Step A. To a solution of 3-[4-methyl-3-
oxo-7-trifluoromethyl-2-(2-trimethylsilanylethoxymethyl)-2,3,4,10-tet-
rahydro-9-oxa-1,2,4a-triazaphenanthren-6-ylamino]azetidine-1-carbox-
ylic acid tert-butyl ester (compound 26, 1.6 g, 2.73 mmol) in THF (5
mL) was added a solution of TBAF (1 M in THF, 4.1 mL, 4.1 mmol).
The reaction mixture was heated at reflux for 3 d, then cooled to
ambient temperature. The solvent was removed in vacuo, and the
residue was purified by chiral SFC separation (column, Chiralpak AD
250 mm × 30 mm i.d., 5 μm; mobile phase, supercritical CO2/EtOH =
65/35; flow rate, 55 mL/min; wavelength, 220 nm) to give 3-(4(S)-
methyl-3-oxo-7-trifluoromethyl-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triaza-
phenanthren-6-ylamino)azetidine-1-carboxylic acid tert-butyl ester
(enantiomer 1, SFC (column, Chiralpak AD-H 250 mm × 4.6 mm
i.d., 5 μm; mobile phase, ethanol (0.05% DEA) in supercritical CO2
from 5% to 40%; flow rate, 2.35 mL/min; wavelength, 220 nm), tR =
8.78 min, 0.280 g, 22%) and 3-(4(R)-methyl-3-oxo-7-trifluoromethyl-
2,3,4,10-tetrahydro-9-oxa-1,2,4a-triazaphenanthren-6-ylamino)-
azetidine-1-carboxylic acid tert-butyl ester (enantiomer 2, SFC
(column, Chiralpak AD-H 250 mm × 4.6 mm i.d., 5 μm; mobile
1
ren-3-one (compound 24, 0.75 g, 63%) as a white solid. H NMR
(DMSO-d6, 400 MHz): δ 10.92 (s, 1H), 7.66 (s, 1H), 7.34 (s, 1H),
4.88 (q, J = 6.8 Hz, 1 H), 4.72 (d, J = 13.2 Hz, 1 H), 4.64 (d, J = 13.2
Hz, 1 H), 1.30 (d, J = 6.8 Hz, 3H). LCMS: tR = 1.095 min. MS m/z:
364, 366 [M + H]+.
Step E. To a 0 °C solution of 6-bromo-4-methyl-7-trifluoromethyl-
2,10-dihydro-9-oxa-1,2,4a-triazaphenanthren-3-one (0.364 g, 1.0
mmol) in THF (50 mL) was added sodium hydride (60% in mineral
oil, 0.080 g, 2 mmol), and the reaction mixture was stirred for 0.5 h.
(2-(Chloromethoxy)ethyl)trimethylsilane (0.250 g, 1.5 mmol) was
added, and the mixture was stirred at ambient temperature for 2 h. The
reaction was quenched by the addition of water (100 mL), and the
aqueous mixture was extracted with EtOAc (2 × 30 mL). The
combined organic layers were washed with brine (30 mL), dried over
Na2SO4, filtered, and evaporated in vacuo. The residue was purified by
column chromatography on silica gel (eluting with 3−20% EtOAc in
petroleum ether) to give 6-bromo-4-methyl-7-trifluoromethyl-2-(2-
trimethylsilanylethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triazaphe-
1
nanthren-3-one (compound 25, 0.330 g, 67%) as a white solid. H
NMR (CDCl3, 400 MHz): δ 7.32 (s, 1H), 7.16 (s, 1H), 5.17−5.09 (m,
2H), 4.75 (d, J = 12.6 Hz, 1H), 4.69 (q, J = 6.8 Hz, 1H), 4.57 (d, J =
12.6 Hz, 1H), 3.65 (t, J = 8.4 Hz, 2H), 1.51 (d, J = 6.8 Hz, 3H), 0.99
(t, J = 8.4 Hz, 2H), 0.01 (s, 9H). LCMS: tR = 1.462 min. MS m/z: 394,
396 [M − 100 + H]+.
Step F. A mixture of 6-bromo-4-methyl-7-trifluoromethyl-2-(2-
trimethylsilanylethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triazaphe-
nanthren-3-one (2.0 g, 4.05 mmol), tert-butyl 3-aminoazetidine-1-
K
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phase, ethanol (0.05% DEA) in supercritical CO2 from 5% to 40%;
flow rate, 2.35 mL/min; wavelength, 220 nm), tR = 10.53 min, 0.260 g,
21%). 1H NMR (CDCl3, 400 MHz): δ 8.18 (s, 1H), 7.18 (s, 1H), 5.91
(s, 1H), 4.68 (q, J = 6.8 Hz, 1H), 4.61 (d, J = 12.8 Hz, 1H), 4.52 (m,
1H), 4.51 (d, J = 12.8 Hz, 1H), 4.36 (m, 2H), 4.22 (m, 1H), 3.80 (m,
2H), 1.55 (d, J = 6.8 Hz, 3H), 1.49 (m, 9H).
temperature, and the solvent was removed in vacuo. The residue was
purified by preparative HPLC (column, YMC-Actus Triart C18 150
mm × 30 mm, 5 μm; mobile phase, MeCN in water (0.075% TFA),
from 60% to 77% within 11 min; flow rate, 25 mL/min; wavelength,
220 nm) and further separated by chiral SFC (column, Chiralpak AD
250 mm × 30 mm i.d., 5 μm; mobile phase, supercritical CO2/MeOH
(0.2% NH3·H2O) = 75/25; flow rate, 80 mL/min; wavelength, 220
nm) to give 3-methyl-3-((R)-4-methyl-3-oxo-7-trifluoromethyl-
2,3,4,10-tetrahydro-9-oxa-1,2,4a-triazaphenanthren-6-ylamino)-
azetidine-1-carboxylic acid tert-butyl ester (enantiomer 1, SFC
(column, Chiralpak AD-3, 3 μm, 150 mm × 4.6 mm i.d.; mobile
phase, MeOH (0.05% DEA) in supercritical CO2 from 5% to 40%;
flow rate, 2.5 mL/min; wavelength, 220 nm), tR = 6.686 min; 0.600 g,
19%) as a white solid. LCMS: tR = 1.192 min. MS m/z: 492 [M +
Na]+.
Step C. To a solution of 3-methyl-3-((R)-4-methyl-3-oxo-7-
trifluoromethyl-2,3,4,10-tetrahydro-9-oxa-1,2,4a-triazaphenanthren-6-
ylamino)azetidine-1-carboxylic acid tert-butyl ester (enantiomer 1,
0.270 g, 0.55 mmol) in DCM (10 mL) was added TFA (2 mL), and
the reaction mixture was stirred at ambient temperature for 2 h. The
solvent was removed in vacuo to give (R)-4-methyl-6-(3-methyl-
azetidin-3-ylamino)-7-trifluoromethyl-2,10-dihydro-9-oxa-1,2,4a-triaza-
phenanthren-3-one trifluoroacetic acid (compound 41, SFC (column,
Chiralpak AD-3, 5 μm, 150 mm × 4.6 mm i.d.; mobile phase,
isopropanol (0.05% DEA) in supercritical CO2 from 5% to 40%; flow
rate, 2.4 mL/min; wavelength, 220 nm), tR = 9.62 min; 0.275 g, 99%)
as a pale solid. 1H NMR (CD3OD, 400 MHz): δ 7.17 (s, 1H), 5.85 (s,
1H), 5.04 (m, 1H), 4.55 (q, J = 13.2 Hz, 2H), 4.24 (m, 2H), 4.14 (m,
2H), 1.71 (s, 3H), 1.44 (d, J = 6.8 Hz, 3H). LCMS (column, Ultimate
XB-C18, 3 μm, 30 mm × 2.1 mm; mobile phase, acetonitrile (0.02%
TFA) in water (0.04% TFA), from 0% to 60% within 4 min; flow rate,
1.2 mL/min; wavelength, 220 nm), tR = 2.005 min. MS m/z: 370 [M +
H]+.
Biology. Kinase Inhibition Assays. PKCθ TR-FRET fragment
screening is performed as follows. The concentrations of individual
components in PKCθ are 5 nM enzyme (Life Technologies catalog no.
P2996, HIS-tagged); 100 nM Oregon Green-labeled fluorescent probe
based on staurosporine (Kd of probe of 29 nM); 2 nM terbium-anti-
HIS antibody (Life Technolgoies catalog no. PV5895). The reaction
buffer consists of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 0.0075%
Triton X-100/100 μM sodium orthovanadate, 1 mM dTT.
Compounds are tested starting at 300 μM. The final concentration
of DMSO in the reaction does not exceed 2%. Reactions are carried
out in a 20 μL volume in PerkinElmer Proxiplate-Plus 384-well white
assay plates (catalog no. 6008289) by combining kinase/probe/
antibody/test compound, mixing, and finally allowing the reaction to
come to equilibrium (incubation for 2.5 h). Fluorescence is measured
on a PerkinElmer Envision plate reader (emission + excitation
parameters: Oregon Green/terbium-340 + 520/495 nm). All results
calculations are performed on the ratio of the two emission values
(probe/antibody). Successful competition by the test compound vs
the fluorescent probe for the ATP binding site of each kinase is
quantified by calculating the IC50 value based on a four-parameter
logistic curve fit.
PKC kinase activity was assayed by time-resolved fluorescence in
vitro kinase assay. Unless otherwise indicated, all reagents used for
enzyme assays were obtained from Sigma. Specifically, 5 μL of N- or
C-terminal His6-tagged, recombinant, human PKC, expressed by
baculovirus in Sf21 cells (Millipore) was mixed with 5 μL of inhibitor
(various concentrations, 5% final DMSO), 5 μL of Bio-cdc peptide
(biotin-Ahx-AKVSRSGLYRSPSMPENLNRPR, 2 μM final concen-
tration, New England Peptide), and 5 μL of ATP (100 μM final
concentration) in 1× reaction buffer, 20 μL final volume (PKC 1× RB:
20 mM MOPS, pH 7.2, 10 mM MgCl2, 5 mM EGTA, 1.1 mM DTT,
0.01% Triton-X-100, 7.5 mM β-phosphoglycerol, 1.1 mM Na3VO4,
0.05 mg/mL phosphatidylserine, 0.005 mg/mL diacylglycerol, 0.1 mM
CaCl2, 5% Millipore PKC lipid activator (catalog no. 20133)). PKCα
or PKCθ 1× RB was as described above except CaCl2 concentration
was 1.1 mM. Reactions were initiated by addition of enzyme in a black
1/2 area 96- or 384-well flat-bottomed polystyrene plate (Corning).
Step B. A solution of 3-(4(R)-methyl-3-oxo-7-trifluoromethyl-
2,3,4,10-tetrahydro-9-oxa-1,2,4a-triazaphenanthren-6-ylamino)azeti-
dine-1-carboxylic acid tert-butyl ester (enantiomer 2, 0.260 g, 0.57
mmol) in DCM (10 mL) and TFA (2 mL) was stirred at ambient
temperature for 1 h. The solvent was removed in vacuo to give 6-
(azetidin-3-ylamino)-4(R)-methyl-7-trifluoromethyl-2,10-dihydro-9-
oxa-1,2,4a-triazaphenanthren-3-one trifluoroacetic acid as a pale yellow
solid (compound 39, SFC (column, Chiralcel OJ-H 250 mm × 4.6
mm i.d., 5 μm; mobile phase, ethanol in supercritical CO2 from 5% to
40%; flow rate, 2.35 mL/min; wavelength, 220 nm), tR = 7.98 min,
0.260 g, 97%). 1H NMR (DMSO-d6, 400 MHz): δ 10.90 (s, 1H), 8.91
(br s, 1H), 8.70 (br s, 1H), 7.11 (s, 1H), 6.26 (s, 1H), 5.83 (br s, 1H),
5.05 (q, J = 6.8 Hz, 1H), 4.55 (m, 3H), 4.32 (m, 2H), 4.01 (m, 2H),
1.30 (d, J = 6.8 Hz, 3H). LCMS (column, Ultimate XB-C18, 3 μm, 30
mm × 2.1 mm; mobile phase, acetonitrile (0.02% TFA) in water
(0.04% TFA), from 0% to 60% within 4 min; flow rate, 1.2 mL/min;
wavelength, 220 nm), tR = 2.068 min. MS m/z: 356 [M + H]+.
Step C. A solution of 3-(4(S)-methyl-3-oxo-7-trifluoromethyl-
2,3,4,10-tetrahydro-9-oxa-1,2,4a-triazaphenanthren-6-ylamino)-
azetidine-1-carboxylic acid tert-butyl ester (enantiomer 1, 0.270 g, 0.59
mmol) in DCM (10 mL) and TFA (2 mL) was stirred at ambient
temperature for 1 h. The solvent was removed in vacuo to give 6-
(azetidin-3-ylamino)-4(S)-methyl-7-trifluoromethyl-2,10-dihydro-9-
oxa-1,2,4a-triazaphenanthren-3-one trifluoroacetic acid as a pale yellow
solid (compound 40, SFC (column, Chiralcel OJ-H 250 mm × 4.6
mm i.d., 5 μm; mobile phase, ethanol in supercritical CO2 from 5% to
40%; flow rate, 2.35 mL/min; wavelength, 220 nm), tR = 6.44 min,
0.275 g, 99%). 1H NMR (DMSO-d6, 400 MHz): δ 11.05 (s, 1 H), 8.93
(br s, 1H), 8.72 (br s, 1H), 7.10 (s, 1H), 6.26 (s, 1H), 5.83 (br s, 1H),
5.04 (q, J = 6.8 Hz, 1H), 4.55 (m, 3H), 4.32 (m, 2H), 4.01 (m, 2H),
1.30 (d, J = 6.8 Hz, 3H). LCMS (column, Ultimate XB-C18, 3 μm, 30
mm × 2.1 mm; mobile phase, acetonitrile (0.02% TFA) in water
(0.04% TFA), from 0% to 60% within 4 min; flow rate, 1.2 mL/min;
wavelength, 220 nm), tR = 2.070 min. MS m/z: 356 [M + H]+.
(R)-4-Methyl-6-(3-methylazetidin-3-ylamino)-7-trifluoro-
methyl-2,10-dihydro-9-oxa-1,2,4a-triazaphenanthren-3-one
Trifluoroacetic Acid or Hydrochloride Acid (41). Step A. A
mixture of 6-bromo-4-methyl-7-trifluoromethyl-2-(2-trimethylsilanyl-
ethoxymethyl)-2,10-dihydro-9-oxa-1,2,4a-triazaphenanthren-3-one
(compound 25, 2.0 g, 4.05 mmol), tert-butyl 3-amino-3-methylazeti-
dine-1-carboxylate (0.904 g, 4.85 mmol), diacetoxypalladium (0.091 g,
0.405 mmol), Cs2CO3 (2.64 g, 8.10 mmol), and BINAP (0.252 g,
0.405 mmol) in toluene (10 mL) was heated at reflux for 10 h. The
reaction mixture was cooled to ambient temperature and filtered. The
filtrate was concentrated in vacuo and the residue was purified by
column chromatography on silica gel (eluting with 10% EtOAc in
petroleum ether) to give 3-methyl-3-[4-methyl-3-oxo-7-trifluorometh-
yl-2-(2-trimethylsilanylethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-
1,2,4a-triazaphenanthren-6-ylamino]azetidine-1-carboxylic acid tert-
1
butyl ester (2.2 g, 91%) as a pale yellow solid. H NMR (CDCl3,
400 MHz): δ 7.16 (s, 1H), 5.96 (br s, 1H), 5.13 (q, J = 9.2 Hz, 2H),
4.61 (m, 1H), 4.56 (q, J = 13.2 Hz, 2H), 4.41 (br s, 1H), 4.04 (m, 2H),
3.95 (d, J = 12.4 Hz, 2H), 3.68 (m, 2H), 1.66 (s, 3H), 1.47 (d, J = 6.4
Hz, 3H), 1.46 (s, 9H), 0.99 (m, 2H), 0.02 (s, 9H). LCMS (column,
Ultimate XB-C18, 3 μm, 30 mm × 2.1 mm; mobile phase, MeCN
(0.02% TFA) in water (0.04% TFA), from 30% to 90% within 2 min;
flow rate, 1.2 mL/min; wavelength, 220 nm), tR = 1.276 min. MS m/z:
622 [M + Na]+.
Step B. To a solution of 3-methyl-3-[4-methyl-3-oxo-7-trifluor-
omethyl-2-(2-trimethylsilanylethoxymethyl)-2,3,4,10-tetrahydro-9-oxa-
1,2,4a-triazaphenanthren-6-ylamino]azetidine-1-carboxylic acid tert-
butyl ester (4 g, 6.67 mmol) in THF (10 mL) was added a solution
of TBAF (1 M in THF, 80 mL, 80 mmol). The reaction mixture was
heated at 80 °C for 15 h. The reaction mixture was cooled to ambient
L
dx.doi.org/10.1021/jm500669m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Following a 60 min incubation at room temperature, the reaction was
quenched with 5 μL of 0.5 M EDTA, pH 7.5. Detection buffer is
added to each well (50 mM HEPES, pH 7.0, 0.01% Tween (BioRad),
0.1% BSA, 0.9 ng/well batch 2 Phospho-(Ser) 14-3-3 binding motif
4E2 monoclonal antibody-K (Cell Signaling no. 9606, labeled by
PerkinElmer W1024), and 0.055 μg/well CR130-100 (Perkin Elmer)).
Plates are incubated for 10 min at room temperature and read on the
RubyStar HTRF microplate analyzer, measuring fluorescence counts at
665 and 620 nm (20 counts/s). Background counts from a minus
enzyme control are subtracted from all data. Data (after background
correction) are converted to percent activity by dividing by the signal
obtained from PKC without drug sample. IC50 values are determined
by fitting the percent activity vs inhibitor concentration data set to
percent activity = 1/1 + [I]/IC50 by nonlinear least means squares
curve fitting.
Cellular Assays: T-Blast CD3/CD28 Induced IL-2 Cell Assay.
Human T-blasts are generated using phytohemaglutinin (PHA) per
previously published protocols.35 To set up the assay, T-blasts were
thawed and cells were washed using standard growth medium (RPMI
1640 medium with 2 mM L-glutamine, 10 mM HEPES, 100 U/mL
Pen/Strep, and 10% FBS). Cells were counted, resuspended at 2 ×
106/mL in growth medium, and seeded at 2 × 105/100 μL cell
suspension per well in flat-bottomed 96-well plate. Compound
dilutions were prepared from 100% DMSO stocks. A total of eight
serial dilutions are made (1/3 in 100% DMSO). Anti-CD3 + anti-
CD28 mix was added to the cells/compounds suspension at 5 μg/mL
final concentration. Plates were transferred to 37 °C/CO2 incubator
for 24 h. Supernatant was collected for IL-2 determination, while MTT
toxicity analyses were performed on remaining cells/medium.
Concanavalin A (Con A) Induced Cytokine Production in Balb/c
Mice. Eight-week-old female Balb/c mice (Charles River Laboratories,
Wilmington, MA) were challenged intravenously with 10 mg/kg
concanavalin A (Amersham Biosciences). Two hours later, the mice
were bled via cardiac puncture and their plasma was analyzed for IL-2
(ELISA kit, R&D Systems). Inhibitor and cyclosporin A (Novartis)
were dosed orally 2 h prior to challenge. The vehicle used for the
inhibitor was 0.5% hydroxypropylmethylcellulose/0.02% Tween 80 in
water.
Glucose 6-Phosphate Isomerase (G6PI) Induced Arthritis in DBA/J
Mice. Eight-week-old male DBA/J mice (Jackson Labs, Bar Harbor,
ME) were immunized intradermally (i.d.) at the base of the tail with
100 μL of 1:1 (v/v) emulsion containing 300 μg of G6PI (see
Supporting Information) and 200 μg of heat-inactivated Mycobacte-
rium tuberculosis H37Ra (complete Freund’s adjuvant, Difco, Laurence,
KS). The mice were dosed orally according to the study design with
vehicle (0.5% hydroxypropylmethylcellulose/0.02% Tween 80 in
water) or inhibitor twice a day throughout the course of the study
(days 0−17). Rat anti-mouse CD4 (GK1.5.4 ra IgG2b/k, AbbVie,
Worcester, MA) was dosed twice per week at a concentration of 12
mg/kg intraperitoneal (ip) in 200 μL of PBS. Disease activity was
assessed by measuring rear paw thickness using calipers on days 7, 10,
13, 15, and 17. Data represented as change in paw thickness compared
to paw thickness measured on day 7. Statistical significance was
determined by two-way ANOVA.
P.H., Q.L., S.M., L.R., and J.J.E. are employees at AbbVie. The
design, study conduct, and financial support for the research
were provided by AbbVie. Y.Z. and J.W. are employees at WuXi
AppTec (Shanghai) Co., Ltd. M.B. and D.P. are employees at
Inventiva. AbbVie, WuXi, and Inventiva participated in the
interpretation of data, review, and approval of the publication.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the following from AbbVie: Li Li, Yuanyuan
Sun, and Zheng Zhou for in vitro testing; Kent Stewart for
helpful design and modeling discussions; Chaohong Sun, Steve
Swann (currently at Takeda), and Maricel Torrent for fragment
screening; Uri Ladror for FAK protein purification; Kerren
Swinger (currently at Epizyme) for FAK crystallization; Rodger
Henry for small molecule X-ray structure of compound 35; Phil
Merta for kinome and fragment screening data; Donna
McCarthy for Con A data; Shaughn Bryant for GPI data;
Silvino Sousa, Bernhard Sielaff, Medha Tomlinson, and
Ramkrishan Sadhukhan for GPI cloning, expression, and
purification; Brett Herzberg and Jason Segreti for rat and dog
cardiovascular data; Lisa Schaffter for optical rotation data. The
́ ̂
authors thank the following from Inventiva: Jerome Amaudrut
for modeling discussions. The X-ray crystal structure of PKCθ
with compound 35 was determined by Proteros Biostructures
GmbH (Martinsried, Germany). The FAK-compound 1 data
set was collected at IMCA. Use of the IMCA-CAT beamline
17-ID (or 17-BM) at the Advanced Photon Source was
supported by the companies of the Industrial Macromolecular
Crystallography Association through a contract with Haupt-
man-Woodward Medical Research Institute. Use of the
Advanced Photon Source was supported by the U.S. Depart-
ment of Energy, Office of Science, Office of Basic Energy
Sciences, under Contract DE-AC02-06CH11357.
ABBREVIATIONS USED
■
AP-1, activator protein 1; b.i.d., bis in diem (twice a day);
CD28, cluster of differentiation 28; CFA, complete Freund’s
adjuvant; CIA, collagen-induced arthritis; Con A, concanavalin
A; CsA, cyclosporin A; EAE, experimental autoimmune
encephalomyelitis; FAK, focal adhesion kinase; GPI, glucose
phosphate isomerase; HTRF, homogeneous time-resolved
fluorescence; IL-2, interleukin 2; MDCK-MDR, Madin−
Darby canine kidney cell with the MDR gene; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFAT,
nuclear factor of activated T cell; NFκB, nuclear factor κ light-
chain enhancer of activated B cell; PHA, phytohaemaglutinin;
PKC, protein kinase C; RA, rheumatoid arthritis; Treg,
regulatory T cell; TR-FRET, time-resolved fluorescence
resonance energy transfer
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional experimental details and a csv file containing
molecular formula strings. This material is available free of
charge via the Internet at http://pubs.acs.org.
REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*Phone: 1-508-688-8001. E-mail: dawn.george@abbvie.com.
■
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