Design and synthesis of tricyclic cores for kinase inhibition

Article abstract of DOI:10.1016/j.bmcl.2012.11.108

Interest in therapeutic kinase inhibitors continues to grow beyond success in oncology. To date, ATP-mimetic kinase inhibitors have focused primarily on monocyclic and bicyclic heterocyclic cores. We sought to expand on the repertoire of potential cores for kinase inhibition by exploring tricyclic variants of classical bicyclic hinge binding motifs such as pyrrolopyridine and pyrrolopyrazine. Herein we describe the syntheses of eight alternative tricyclic cores as well as in vitro screening results for representative kinases of potential therapeutic interest.

Full text of DOI:10.1016/j.bmcl.2012.11.108

Bioorganic & Medicinal Chemistry Letters 23 (2013) 693–698
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of tricyclic cores for kinase inhibition
Stacy Van Epps a, , Bryan Fiamengo a, , Jeremy Edmunds a, Anna Ericsson d, Kristine Frank b,
Michael Friedman a, Dawn George a, Jonathan George a, Eric Goedken a, Brian Kotecki c, Gloria Martinez a,
Philip Merta b, Michael Morytko a, Shashank Shekhar c, Barbara Skinner e, Kent Stewart b, Jeffrey Voss a,
⇑
⇑
Grier Wallace a, Lu Wang a, Lu Wang a, Neil Wishart a,
⇑
a Abbott Bioresearch Center, 381 Plantation St, Worcester, MA 01605, USA
b Abbott Laboratories, 100 Abbott Park Rd, Abbott Park, IL 60064, USA
c Abbott Laboratories, 1401 Sheridan Rd, North Chicago, IL 60064, USA
d Forma Therapeutics, 500 Arsenal St, Watertown, MA 02472, USA
e Abbott Laboratories, 381 Plantation St. Worcester MA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Interest in therapeutic kinase inhibitors continues to grow beyond success in oncology. To date,
ATP-mimetic kinase inhibitors have focused primarily on monocyclic and bicyclic heterocyclic cores.
We sought to expand on the repertoire of potential cores for kinase inhibition by exploring tricyclic
variants of classical bicyclic hinge binding motifs such as pyrrolopyridine and pyrrolopyrazine. Herein
we describe the syntheses of eight alternative tricyclic cores as well as in vitro screening results for
representative kinases of potential therapeutic interest.
Received 11 October 2012
Revised 21 November 2012
Accepted 26 November 2012
Available online 5 December 2012
Keywords:
Kinase inhibitors
Ó 2012 Elsevier Ltd. All rights reserved.
Tricyclic pyrrolopyridines
Tricyclic pyrrolopyrazines
In recent years drug discovery organizations have taken an
increased interest in kinases inhibitors to treat multiple diseases
beyond cancer, such as rheumatoid arthritis (RA), psoriasis, inflam-
matory bowel disease, and other autoimmune diseases.1,2 Recent
clinical results from Pfizer’s Jak inhibitor tofacitinib3 and AstraZen-
eca and Rigel’s kinase inhibitor fostamatinib4 (Fig. 1) indicate that
kinase inhibitors can demonstrate clinical efficacy as well as drug
tolerability in chronic inflammatory indications.
Additionally, by fusing a third ring to such kinase hinge binding
cores, we are able to incorporate conformationally preferred sub-
stituents that are able to interact in a complementary manner to
the protein target.
The majority of the kinase inhibitors identified to date bind to
the ATP binding site in the active conformation, and are typically
referred to as type 1 inhibitors. As a result of extensive activities
in this area of research it is becoming increasingly more challeng-
ing to identify novel type 1 chemotypes as starting points for small
molecule kinase drug discovery programs. Limited by the specific-
ities of the ATP pharmacophore, particularly the ability to interact
with the hinge region of the kinase, as well as the need to maintain
drug-like properties within the molecule, a variety of monocyclic
and bicyclic heterocyclic hinge binders have been developed.5
Given the expansion of prior art around such chemotypes, we
investigated the ability to modify commonly adopted kinase hinge
binders, such as 1H-pyrrolo[2,3-d]pyridine and 5H-pyrrolo[2,3-
b]pyrazine, to identify several novel tricyclic heterocycles (Fig. 2).
Figure 1. Recent non-oncology kinase inhibitors in the clinic.
⇑
Corresponding authors.
E-mail address: stacy.vanepps@abbott.com (S. Van Epps).
Figure 2. Location of third ring.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.108

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S. Van Epps et al. / Bioorg. Med. Chem. Lett. 23 (2013) 693–698
Herein we describe the synthesis of eight different tricyclic
saturated ring (Scheme 4). Starting from commercially available
pyrrolopyridine 25, intermediate 26 was synthesized using
ortho-lithiation followed by trapping with ethyl chlorofomate.11
A protecting group exchange and traditional functional group
modifications allowed access to key aldehyde 29. Wittig product
30 was formed using ((1,3-dioxolan-2-yl)methyl)triphenyl-phos-
phonium bromide and was reduced by hydrogenation to give 31.
Acidic dioxolane deprotection and subsequent intramolecular
imine formation followed by reduction yielded protected tricycle
32. The 2,3,4,7-tetrahydro-1H-pyrrolo[2,3-h][1,6]naphthyridine
analog 33 was obtained after removal of tosyl protection under
basic conditions.
cores starting from commercially available heterocycles. Detailed
experimental procedures for the synthesis of such cores have pre-
viously been described.6 Our interests focused on the synthesis of
unique tricyclic pyrrolopyridines and pyrrolopyrazines. All cores
were initially synthesized with an unadorned cyclohexyl substitu-
ent. This was done in order to simplify the initial chemistry efforts
and to allow for ready comparisons across the series. Starting with
tricyclic variants of pyrrolopyridines the synthetic route to 3,6-
dihydropyrazolo[4,3-d]pyrrole[2,3-b]pyridines 5 and 6 as well as
6H-isoxazolo[4,5-d]pyrrole[2,3-b]pyridine
8
is presented in
Scheme 1. Treatment of commercially available aldehyde 1 with
cyclohexylmagnesium chloride and subsequent oxidation of the
alcohol afforded ketone 2. Hydrazone formation using either
hydrazine or methyl-hydrazine followed by cyclization gave final
compounds 5 and 6, respectively.7 Oxime formation from 2 using
hydroxylamine provided intermediate 7 which was cyclized in
the presence of potassium tert-butoxide to afford tricycle 8.
The synthesis of 1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyri-
dine cores ( 15, 23, 24) are exemplified in Schemes 2 and 3.
Nitration of 4-Chloro-3-iodopyridin-2-amine 9 with potassium ni-
trate resulted in 10 (Scheme 2). Sonogashira cross coupling of 10
with TMS-acetylene followed by desilylation and modified rho-
dium-catalyzed Larock cyclization led to common intermediate
12.8 SNAr displacement of chloride 12 with cyclohexylamine
gave 13, which underwent tin chloride-promoted reduction to dia-
mine 14. Subsequent ring closure utilizing triethyl orthoformate
generated the desired product 15 in low yield. Further investiga-
tion of additional analogs in this series led to protection of the pyr-
role nitrogen as a means to optimize reaction yields (Scheme 3).
Thus azaindole 12 was tosyl-protected prior to nitro-assisted
chloro displacement with cyclohexylamine providing 17 (Scheme
3). In similar fashion, tin chloride-promoted reduction of the nitro
group led to diamine 18 which readily underwent acylation to fur-
nish 19 and 20. Cyclization of 19 with POCl3 and subsequent
deprotection furnished tricycle 23. Intramolecular dehydrative ring
closure of 20 using propylphosphonic anhydride led to 22 in an im-
proved 68% yield. Hydrolysis of the tosyl protected pyrrole pro-
vided final compound 24. These types of tricyclic kinase
inhibitors have also very recently been reported via nitration of
4-chloro-1-phenylsulfonyl-1H-pyrrolo[2,3-b]pyridine.9,10
In addition to our efforts on azaindole-containing tricycles,
additional tricycles derived from pyrrolopyrazine were explored.
A linear synthetic approach to 6H-pyrrolo[2,3-e][1,2,4]triazol-
o[4,3-a]pyrazine 41 is represented in Scheme 5. Treatment of
aminopyrazine 34 with NBS generated 35 which underwent Sono-
gashira cross coupling with TMS-acetylene. One pot cyclization
and protection formed intermediate 37. Buchwald–Hartwig
cross-coupling of 37 with di-tert-butyl hydrazodicarboxylate
yielded 38 and acidic cleavage produced hydrazine 39. Hydrazide
40 was generated using cyclohexylcarbonyl chloride and was
subsequently cyclized by treatment with thionyl chloride and
deprotected to afford triazole 41.
Intermediate 37 gave access to multiple tricycles:
3H-imidazo[1,2-a]pyrrole[2,3-e]pyrazine 44 (Scheme 6), 3H-dipyr-
rolo[1,2-a:2’,3’-e]pyrazine 50 (Scheme 7), and 6H-imidazo[1,5-
a]pyrrolo[2,3-e]pyrazine 55 (Scheme 8). As shown in Scheme 6,
Buchwald-Hartwig cross-coupling of 37 with tert-butyl carbamate
followed by alkylation and Boc deprotection afforded ketone 43.
Acidic cyclization employing 2,2,3,3,3-pentafluoropropanoic anhy-
dride and tosyl deprotection provided the desired tricycle 44.
Suzuki cross-coupling of key intermediate 37 with styrylboron-
ic acid followed by oxidative cleavage using osmium tetraoxide
and sodium periodate produced aldehyde 46 (Scheme 7). Horner-
Wadsworth-Emmons homologation with diethyl 2-cyclohexyl-2-
oxoethylphosphonate and subsequent hydrogenation led to ketone
48. Sulfur-mediated cyclization using Belleau’s reagent and tosyl
hydrolysis generated final compound 50.
Additional utility of intermediate 37 also allowed access to the
6H-imidazo[1,5-a]pyrrolo[2,3-e]pyrazine core (Scheme 8). Palla-
dium-catalyzed cyanation of 37 and subsequent hydrogenation in
the presence of acid provided the HCl salt of amine 52. Amidation
As an alternative to aromatic azaindole-containing tricycles, we
expanded our focus to a tricyclic system containing a partially
Scheme 1. Reagents and conditions: (a) cyclohexylmagnesium chloride, THF, À20 °C to rt, 16 h, 51%; (b) Dess–Martin periodinane, DCM, 0 °C, 3 h, 73%; (c) for 3: hydrazine,
AcOH, EtOH, reflux, 20 h, 48%; for 4: methylhydrazine, AcOH, EtOH, reflux, 48 h, 72%; (d) hydroxylamine hydrochloride, AcOH, 100–130 °C, 72 h, 52%; (e) for 5: Pd(OAc)2,
CyPFt-Bu, NaOt-Bu, NMP, microwave 170 °C, 40 min, 33%; for 6: Pd(OAc)2, CyPFt-Bu, NaOt-Bu, NMP, microwave 160 °C, 45 min, 65%; (f) KOt-Bu, DMSO, microwave 125 °C,
1.5 h, 46%.

S. Van Epps et al. / Bioorg. Med. Chem. Lett. 23 (2013) 693–698
695
Scheme 2. Reagents and conditions: (a) KNO3, H2SO4, À10 °C, 15 h, 60%; (b) TMS-acetylene, PdCl2(PPh3)2, CuI, TEA, THF, 50 °C, 12 h, 70%; (c) KF, DMF, rt, 4 h, 87%; (d)
chloro(1,5-cyclooctadiene)rhodium(I) dimer, tris(4-fluorophenyl)phosphine, DMF, 80 °C, 2 h, 81%; (e) cyclohexylamine, DMF, rt, 18 h, 82%; (f) SnCl2Á2H2O, EtOH, 55 °C, 2 h,
82%; (g) triethyl orthoformate, p-TsOH-H2O, 80 °C, 5 h, 3%.
Scheme 3. Reagents and conditions: (a) NaH, TsCl, DMF, 0 °C to rt, 2 h, 98%; (b) cyclohexylamine, DIEA, DMF, rt, 30 min; (c) SnCl2Á2H2O, EtOH, 75 °C, 5 h, 79% over 2 steps; (d)
for 19: acetic anhydride, TEA, 0 °C to rt, 5 h; for 20: trifluoroacetic anhydride, TEA, DCM, rt, 10 min, 75%; (e) for 21: POCl3, 80 °C, 16 h; for 22: propylphosphonic anhydride,
DMF, 100 °C, 16 h, 68%; (f) for 23: 1 M aq NaOH, dioxane, 90 °C, 4 h, 6% over 3 steps; for 24: 1 M aq NaOH, dioxane, 80 °C, 1 h, 29%.
Scheme 4. Reagents and conditions: (a) sec-BuLi, ethyl chloroformate, THF, À78 °C to rt, 40 min, 98%; (b) TBAF, THF, 0 °C, 1 h, 52%; (c) NaH, TsCl, DMF, 0 °C to rt, 2 h, 92%; (d)
cyclohexylamine, n-BuOH, 110 °C, 16 h, 73%; (e) DIBAL-H, toluene, -78 °C to rt, 2 h, 80%; (f) MnO2, CHCl3, rt, 16 h, 87%; (g) ((1,3-dioxolan-2-yl)methyl)triphenyl-phosphonium
bromide, KOt-Bu, THF, 0 °C to rt, 16 h, 67%; (h) 10% Pd/C, H2, EtOAc, rt, 1.5 h, 97%; (i) conc. HCl, EtOH, 40 °C, 2 h, then NaBH4, 0 °C, 2 h, 68%; (j) 5 M aq NaOH, dioxane, 100 °C,
48 h, 60%.

696
S. Van Epps et al. / Bioorg. Med. Chem. Lett. 23 (2013) 693–698
Scheme 5. Reagents and conditions: (a) NBS, Na2CO3, DMSO-H2O, <15 °C to rt, 6 h, 76%; (b) TMS-acetylene, PdCl2(PPh3)2, CuI, TEA, THF, À5 to 0 °C, 1.5 h, 88%; (c) NaH, TsCl,
DMF, 0 °C to rt, 1 h, 54%; (d) di-tert-butyl hydrazodicarboxylate, Pd(OAc)2, Xantphos, K2CO3, t-AmOH/dioxane, 95 °C, 2 h, 91%; (e) (i)H3PO4, THF/heptane, 70 °C, 2 h, (ii) K2CO3,
H2O, rt, 1 h, 94%; (f) cyclohexylcarbonyl chloride, DIEA, dioxane, 0 °C to rt, 1 h; (g) SOCl2, dioxane, 90 °C, 1 h; (h) 2 M aq Na2CO3, MeOH, 80 °C, 72 h, 40% over 3 steps.
Scheme 6. Reagents and conditions: (a) tert-butyl carbamate, Pd(OAc)2, Xantphos, K2CO3, t-AmOH-dioxane, 95 °C, 3 h, 74%; (b) 2-bromo-1-cyclohexylethone, NaH, DMF, 0 °C,
1 h, 83%; (c) TFA, DCM, rt, 2 h, quant.; (d) 2,2,3,3,3-pentafluoropropanoic anhydride, acetonitrile, 60 °C, 2 h, 94%; (e) 1 M aq NaOH, dioxane, 50 °C, 1 h, quant.
Scheme 7. Reagents and conditions: (a) styrylboronic acid, PdCl2(dppf)–CH2Cl2, Na2CO3, THF–H2O, 65 °C, 24 h, 92%; (b) OsO4, NaIO4, dioxane/H2O, rt, 15 h, 92%; (c) diethyl 2-
cyclohexyl-2-oxoethylphosphonate, NaH, THF, rt, 2 h, 74%; (d) 10% Pd/C, H2, EtOAc, 1 h, quant.; (e) Belleau’s reagent, THF, rt, 6 h, 42%; (f) 2 M aq NaOH, dioxane, 90 °C, 15 h,
55%.
Scheme 8. Reagents and conditions: (a) Zn, Zn(CN)2, PdCl2(dppf)-CH2Cl2, DMA, 95 °C, 16 h, 70%; (b) 5% Pd/C, H2 40 psi, quinoline, 37% HCl, THF/H2O, rt, 2 h, 88%; (c)
cyclohexanecarbonyl chloride, Hunig’s base, DCM, rt, 4 h, 80%; (d) Lawesson’s reagent, dioxane, 60 °C to rt, 16 h; (e) Hg(OAc)2, dioxane, rt, 30 min, 80% over 2 steps; (f) 2 M aq
NaOH, dioxane, 90 °C, 5 h, 90%.

S. Van Epps et al. / Bioorg. Med. Chem. Lett. 23 (2013) 693–698
697
Table 1
12,13
in vitro enzyme inhibition (IC50
)
Compound
Aurorab 1 (
l
M)
FLTa-3 (
l
M)
Jakc2 (
l
M)
Kdbr (
lM)
PKCab
(lM)
TYK2c
(lM)
5
6
8
0.13
0.34
0.57
0.74d
0.22
0.44
1.8
na
na
0.01
0.05
0.03
5.7b
6.0
11.4
na
0.09
na
na
na
0.09
0.16
0.20
1.3
2.6
2.8
1.7
1.3
1.7
na
18.0
na
na
0.05
0.30
0.06
na
na
na
0.53
1.2
na
4.8
na
d
15
23
24
33
41
44
50
55
3.7
2.6
d
b
d
0.64
d
b
d
4.3
na
0.72
na
2.6
1.0
0.60
0.91
3.2
>50
27.3
0.24
0.15
0.89
15.3
0.04
0.03
0.27
na
Na = data not available.
a
1000
l
M ATP.
M ATP.
M ATP.
binding assay.
b
c
100
l
1
l
d
Table 2
in vitro Jak enzyme and cellular activity14,15
Compound
Jak 1 enzymea
(l
M)
Jak 1 cellb
(l
M)
Jak 2 cellb
(l
M)
Jak2/Jak1 Cellular selectivity
5
6
8
15
23
24
41
44
50
55
0.003
0.09
0.02
0.03
0.02
0.04
0.03
0.004
0.02
0.01
0.17
1.2
4.2
0.89
1.6
9.8
6.6
>20
15.8
4.8
>20
2.8
25Â
1Â
0.43
0.21
0.18
0.97
0.35
0.15
na
4Â
47Â
37Â
21Â
45Â
32Â
na
0.37
8Â
Na = data not available
a
Enzyme inhibition (IC50) at 1 lM ATP.
Cellular efficacy (EC50).
b
with cyclohexylcarbonyl chloride generated 53 which readily
underwent thioamide formation using Lawesson’s reagent fol-
lowed by mercury catalyzed intramolecular cyclization to afford
54. Finally, base mediated tosyl hydrolysis gave the desired tricycle
55.
identifying selective Jak1 inhibitors with compounds 15, 23, 41,
and 44 showing the highest level of cellular selectivity in these
assays.
In conclusion, modifying the common pyrrolopyridine and pyr-
rolopyrazine hinge binders led to the discovery of novel tricyclic
inhibitors. The heterocyclic compounds described herein exhibit
a wide range of activity for various kinases including Jak kinases.
To improve potency and selectivity for Jak1 over Jak2, alteration
of the cyclohexyl capping group to a more optimized Jak kinase
inhibitor was investigated. Subsequent reports will detail the
efforts that led to potent and selective Jak1 inhibitors.
By design, the tricyclic heterocycles exemplified in (Schemes 1–
8) possess hydrogen bond donor-acceptor hinge binding motifs,
that have the potential to competitively bind to the ATP active site
of kinases. Thus, the tricyclic inhibitors were evaluated for activity
against a panel of kinases. The in vitro enzymatic assay data for a
selected set of kinases for the final compounds described above
are presented in Table 1. With the exception of compounds 15,
23, and 24 all in vitro potencies were generated in enzyme activity
assays. The inhibitory activity of 15, 23, and 24 were determined
using a trFRET binding competition assay. While these tricyclic
hinge binders possess a range of binding activities and potencies
against different kinases we were particularly interested by how
many of them potently inhibit the Jak family kinase, Jak2. Due to
our interest in exploring kinase inhibitors in non-oncology indica-
tions the analogs with activity against Jak2 were then further
profiled against Jak1.
Acknowledgments
The authors would like to thank Eric Dominguez, Lori Dowding,
Silvia Kwak, Sara Murdock, Takazvida Nyaundi, Denise Perron,
Jose-Andres Salmeron-Garcia and Carmen Szynal for their contri-
butions in obtaining in vitro enzyme activity and cellular data.
Additionally, we would also like to thank Matthew Kurnick and
Lemma Kifle for acquiring the in vitro binding data.
All of the compounds tested exhibited potencies <100 nM in the
Jak1 enzymatic assay at an ATP concentration close to its Km (Table
2). To facilitate a comparison of all the different tricycles, the com-
pounds were progressed into multiple Jak cellular assays for eval-
uation of potency and selectivity. The majority of the compounds
exhibited good inhibitory activity in the Jak1 (IL-6/pSTAT3) cellular
assay. Furthermore, most of the compounds showed modest Jak2
(Epo/pSTAT5) cellular activity leading to a defined cellular selectiv-
ity window for Jak1 over Jak2. This demonstrates the possibility of
References and notes
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Invitrogen), Oregon Green-labeled fluorescent probes (2Â Kd of probe; range
of 6.25–200 nM), terbium anti-HIS or -GST antibody (2 nM; invitrogen). The
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X-100/100
lM sodium orthovanadate/1 mM dTT. Reactions are carried out in a
20 L volume in 384-well plates, by combining kinase/probe/antibody/test
l
compound, mixing, and equilibrating 2.5 h. Fluorescence is measured on a
PerkinElmer Envision plate reader (excitation 340 nm and emission at 520/
495 nm).
14. In vitro Jak1 kinase activity assay used Jakl kinase domain (aa 845–1142),
biotin-TYR2 peptide (Biotin-(Ahx)-AEEEYFFLFA-amide; 2 lM) and 1 lM ATP in
50 mM MOPSO pH 6.5, 10 mM MgC12, 2 mM MnC12 (2 mM), DTT (2.5 mM),
BSA (0.01% w/v), 0.1 mM Na3VO4 and ATP (0.001 mM). After 60 min, the
enzymatic reaction was quenched by addition of EDTA (to final concentration
100 mM) and developed by addition of PT66 K (europium labeled anti-
11. L’Heureux, A.; Thibault, C.; Ruel, R. Tetrahedron Lett. 2004, 45, 2317.
12. In vitro kinase activity assays were performed typically at ATP concentrations
phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, MA) and 3.12 lg/
mL phycolink streptavidin-allophycocyanin acceptor (cat #PJ25S, Prozyme, San
Leandro, CA). Following incubation for P60 min, 96-well plates were read via a
Rubystar detector (BMG) using a 337 nm laser for excitation and emission
wavelength of 665 nm.
near measured ATP Km values (1–1000 lM) in the presence of small
biotinylated peptide substrates. Following an enzyme reaction at room
temperature in 96- or 384-well plates, reactions were stopped by addition of
excess EDTA and the phosphorylated product was detected by time-resolved
fluorescence energy transfer (trFRET) using fluorescent (donor) antibodies that
15. Jak1 cellular assays (IL-6 dependent pSTAT3 in TF-1 cells) and Jak2 cellular
assays (Epo-dependent pSTAT5 assays in UT-7 cells) were conducted as
described in the supplemental methods in Goedken, E. R.; Devanarayan, V.;
Harris, C. M.; Dowding, L. A.; Jakway, J. P.; Voss, J. W.; Wishart, N.; Jordan, D. C.;
Talanian, R. V. J. Biomol. Screen. 2012, 17, 857.
recognize the phosphorylation site on the peptide and
a streptavidin-
allophycocyanin (acceptor) conjugate that binds the peptide’s biotinylated
portion.