Malerich JP, Lam JS, Hart B, Fine RM, Klebansky B, Tanga MJ, D’Andrea, A. Diamino-1,2,4-triazole derivatives are selective inhibitors of TYK2 and JAK1 over JAK2 and JAK3. Bioorg Med Chem Lett. 2010; 20(24):7454-7457. DOI: 10.1016/j.bmcl.2010.10.026.

Abstract

Tyrosine kinase 2 (TYK2) is required for signaling of interleukin-23 (IL-23), which plays a key role in rheumatoid arthritis. Presented is the design and synthesis of 1,2,4-triazoles, and the evaluation of their inhibitory activity against the Janus associated kinases TYK2 and JAKs 1–3.

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Diamino-1,2,4-triazole derivatives are selective inhibitors of TYK2 and JAK1
over JAK2 and JAK3
Jeremiah P. Malericha,?, Jennifer S. Lama,?, Barry Hartb, Richard M. Finec, Boris Klebanskyc,
Mary J. Tangaa, Annalisa D’Andreaa,⇑
aSRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, United States
bInnovation Pathways, Palo Alto, CA 94301, United States
cBioPredict, Inc., 660 Kinderkamack Road, Oradell, NJ 07649, United States
a r t i c l ei n f o
Article history:
Received 24 August 2010
Revised 30 September 2010
Accepted 5 October 2010
Available online 13 October 2010
Keywords:
Tyrosine kinase 2
TYK2
Janus associated kinase
JAK1
JAK2
JAK3
a b s t r a c t
Tyrosine kinase 2 (TYK2) is required for signaling of interleukin-23 (IL-23), which plays a key role in rheu-
matoid arthritis. Presented is the design and synthesis of 1,2,4-triazoles, and the evaluation of their inhib-
itory activity against the Janus associated kinases TYK2 and JAKs 1–3.
? 2010 Elsevier Ltd. All rights reserved.
Blocking cytokine activity is an established strategy for the
treatment of rheumatoid arthritis (RA) and other inflammatory
diseases.1–3Biologic therapies such as etanercept (Enbrel) inhibit-
ing tumor necrosis factor (TNF) have emerged as routine treat-
ments for RA over the last decade. In addition to high costs and
the requirement for clinical administration, these agents suffer
from a high degree of non-responders. The small-molecule metho-
trexate (MTX), an antifolate agent, has been used for the treatment
of RA for over two decades. MTX remains one of the front-line
treatments for early RA, although many patients discontinue use
due to lack of efficacy, toxicity or compliance issues. The current
understanding of cytokine networks in vivo and the lack of re-
sponse to current biologic therapies by certain patients indicate
that new cytokines should be considered as therapeutic targets.
Interleukin 23 (IL-23) has been shown to be a critical factor in
the pathogenesis of animal models of RA.4,5IL-23 signaling re-
quires activation of Janus associated kinase 2 (JAK2) and tyrosine
kinase 2 (TYK2). Mutant mice with a naturally defective TYK2 gene
do not develop arthritis.6Although either JAK2 or TYK2 could be
targeted for blocking IL-23 activity, JAK2 is used more broadly in
cytokine signaling. We therefore selected TYK2 as the target for
our research, with an immediate goal of understanding the rela-
tionships between inhibitor structure and selectivity across the Ja-
nus associated kinases, JAKs 1–3 and TYK2. Here we present our
initial studies toward selective TYK2 inhibitors.
Despite the recognition of TYK2 as a potential target in various
inflammatory diseases, compounds that selectively inhibit TYK2
over JAKs 1–3 are nearly absent from the literature. The binding
properties of a collection of drugs and drug candidates with a panel
of kinases were recently reported.7Of these, a single compound,
JNJ-7706621 (1),8,9is selective for TYK2 and JAK1 over JAK2 and
JAK3. Previously, TG101348 (2),10,11INCB018424 (3),12–15and CP-
690550 (4)16were designed as selective JAK inhibitors (Table 1).
When we dock compounds 1–4 into the crystal structure of
TYK2,17the binding interactions of these compounds with TYK2
define a common region extending from the hinge of the kinase do-
main to the phosphate binding region (Fig. 1). The hinge of the ki-
nase domain consists of the stretch of peptide that connects the
two lobes of the domain, providing an array of hydrogen bond do-
nors and acceptors. These ligands also interact with the phosphate
binding region of ATP, adjacent to the conserved DFG motif. This
DFG sequence forms the start of the kinase activation loop and is
within reach of the flexible G-rich loop that forms a flap over the
top of ATP and substrates to shield water from the site of phos-
phate transfer. JNJ-7706621 and TG101348 project tail groups to-
ward the other end of ATP binding site, where it opens toward
solvent.
0960-894X/$ - see front matter ? 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.026
⇑Corresponding author. Tel.: +1 (650) 859 3323; fax: +1 (650) 859 3444.
E-mail address: annalisa.dandrea@sri.com (A. D’Andrea).
?These authors contributed equally to the work.
Bioorganic & Medicinal Chemistry Letters 20 (2010) 7454–7457
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl

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The near-perfect conservation of amino acids within the active
site of the JAK family is well known. This conservation has the
implication that the development of selective TYK2 inhibitors is
not easily accomplished, but the examples of TG101348 and
CP690550 prove that selectivity can be achieved within this kinase
family. In our program, two observations were incorporated into
the development of the compounds with the aim of achieving
TYK2 selectivity. First, there is considerable sequence variability
in the G-rich loop of JAK kinases and differences in its conforma-
tion might offer an opportunity for achieving differential response
to TYK2. However, the inherent flexibility of this region makes it
difficult to predict what chemical structures might offer selective
inhibition across the JAKs. Therefore we turned our attention to a
second observation. Residue Arg901 in Tyk2 is different in
Jak2(Gln) and JAK3(Ser). Compounds that can form productive
electrostatic interactions with this Arg could result in isoform
selectivity. Much of the work on the compounds reported herein
was executed prior to the release of the crystal structures of
TYK2. In a homology model built from the JAK2 crystal structure,
we considered Arg901 to be flexible, and thus available to be tar-
geted by rationally designed inhibitors. This was called into ques-
tion by TYK2 crystal structures that show Arg901 forming a
hydrogen bond to Tyr980 from the hinge.17Of course, the crystal
structure is a static representation and may not accurately describe
the dynamic nature of the kinase.
Given the modest selectivity of JNJ-7706621, we used this com-
pound as a starting point to develop SAR. The compound was
Table 1
Kinase inhibitors with activity against JAKsa
CompoundTYK2JAK1JAK2JAK3
N
N
N
H2N
SO2NH2
N
H
OF
F
1: JNJ-7706621
Kd(Ref. 7) 32 nM21 nM220 nM 180 nM
N
N
N
H
O
N
N
2: TG101348
N
H
S
H
N
OO
tBu
IC50(Ref. 10)150 nM 100 nM3 nM1 lM
N
N
NH
N
N
3: INCB018424
O
N
IC50(Ref. 15) 19 nM3 nM3 nM430 nM
N
Me
N
N
N
NH
Me
4: CP690550
Kd(Ref. 7)620 nM>10 lM5 nM2.2 nM
aValues listed are either dissociation constants (Kd) or enzyme inhibitory activities (IC50) reported in the literature.
Figure 1. JNJ-7706621 docked into crystal structure of TYK2.
N
N
N
H2N
S
N
H
OF
F
NH2
O O
tail
core - designed to
interact with hinge
head
JNJ-7706621
N
N
N
H2N
N
H
O
R2
R1
tail
core
head
Figure 2. Deconstruction of JNJ-7706621.
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deconstructed into three portions (Fig. 2): the core, which binds the
hinge region; the head region, which occupies the phosphate bind-
ing region; and the tail, which is oriented toward Arg901 in our
computational model. A series of compounds around the 1,2,4-tri-
azole core of JNJ-7706621 were synthesized to probe the SAR of
groups flanking the core according to the general scheme shown
in Scheme 1, using methods developed by Webb.18,19Treatment
of aniline 5 with diphenyl cyanocarbonimidate afforded the aniline
adduct 6, which was heated to reflux with hydrazine to give the
diaminotriazole 7. Reaction of 7 with benzoyl chloride 8 gave a
mixture of regioisomers 9 (major) and 10 (minor).
Kinase activity was determined by applying Invitrogen’s Lan-
thaScreen™ TR-FRET assays using the catalytic domain of recombi-
nant human kinase. The inhibitory activity of compounds 1 and
11–26 is shown in Table 2. From these data, we are able to assess
the potential of this scaffold to produce selective TYK2 inhibitors.
H2N
NCN
OPhPhO
NN
H
OPh
NC
H2NNH2
N
N
N
H2N
N
H
O
N
N
N
H2N
N
H
H
Cl
O
iPrOH
(72-91%)
THF, refl.
(50-73%)
Py
(60-80%,
~6:1 mixture)
N
N
H2N
N
H
O
N
567
9 (major regioisomer)
10 (minor regioisomer)
+
8
R1
R1
R1
R1
R1
R2
R2
R2
Scheme 1. General synthesis of TYK2 inhibitors.
Table 2
Inhibitory activity of 1,2,4-triazoles against TYK2 and JAKs 1–3
CompoundR1
R2
IC50(nM)
TYK2JAK1 JAK2JAK3
1
11
12
13
14
15
4-SO2NH2
3-SO2NH2
3-CO2Et
3-CO2H
4-CO2Me
4-CO2H
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
2,6-F2
39
70
1720
690
760
42
39
19
990
610
500
45
1700
2500
>10,000
>10,000
>10,000
2110
950
1250
>10,000
>10,000
>10,000
1470
16
N
N
H2N
N
H
O
N
F
F
OEt
O
>10,000ntntnt
17
N
N
H
N
H2N
N
H
OEt
O
>10,000ntntnt
18
19
20
H
3-NMe2
4-NMe2
2,6-F2
2,6-F2
2,6-F2
360
70
70
230
39
80
P10,000
5000
2500
3250
2000
850
21
N
N
N
N
H2N
N
H
O
F
F
300160>10,0005000
22
23
24
25
3-CO2Et
3-CO2Et
3-CO2Et
3-CO2Et
H
2-F
4-CF3
4-NMe2
>10,000
6040
>10,000
>10,000
nt
P10,000
nt
nt
nt
>10,000
nt
nt
nt
>10,000
nt
nt
26
N
N
N
H2N
N
H
O
OEt
O
O
>10,000ntntnt
7456
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The activity and selectivity of JNJ-7706621 (1) was confirmed.
TYK2 inhibitory activity was modulated by the tail group, where
para-substituted compounds were more potent than their meta-
congeners. This was most dramatic in carboxylates 15 versus 13,
with smaller effects evident in sulfonamides 1 versus 11 and esters
14 versus 12. Stronger hydrogen bond acceptors also increased
TYK2 inhibition; sulfonamides 1 and 11, carboxylate 15 and dim-
ethylamines 19 and 20 were better inhibitors than esters 12 and
14 and unsubstituted tail analog 18. While this was consistent with
the hypothesis that interacting with Arg901 would improve TYK2
activity, selectivity was relatively insensitive to these changes, ex-
cept for a modest increase in JAK1 versus TYK2 selectivity between
para and meta pairs (e.g. 1 vs11, 12 vs 14, and 19 vs 20). With re-
spect to the head group, the 2,6-difluorobenzoyl head group was
strongly preferred for TYK2 activity. Removal of the head group
(17) or a 1,2-shift of the 2,6-difluorobenzoyl head group (16) gave
inactive compounds. The 2,6-difluoro substitution appeared to be
critical as the monofluoro compound 23 was significantly less ac-
tive than 12 and non-halogenated 22 was inactive. The effect
was not purely electronic, as trifluoromethyl analog 24 was inac-
tive as well. Other head group variants showed no inhibition.
While not a priority for the current research, these compounds
likely represent novel CDK inhibitors given their structural similar-
ity to JNJ-7706621 (1).
Next, we sought to determine whether these triazoles would
also be capable of inhibiting TYK2 in a biologically relevant setting.
We stimulated human PHA blasts with IL-12, a cytokine which re-
quires TYK2 and JAK2 for signaling, in the absence or presence of
inhibitors, and then examined the levels of IFNc, a downstream
product of IL-12 signaling. We found that compounds 11, 19, and
20 inhibited IFNc production similar to JNJ-7706621 (1) whereas
compounds 13 and 15 partially inhibited IFNc (Fig. 3). Compounds
16 and 17 were included as negative controls since they showed no
activity in the kinase assay. They showed minimal IFNc inhibition.
In conclusion, we have identified four compounds, 11, 15, 19,
and 20, with activity and selectivity similar to the most selective
compound described in the literature JNJ-7706621 (1). Compounds
11, 15, and 20 maintain activity in a cellular context. Our future ef-
forts will be directed toward developing compounds with higher
selectivity for TYK2 over JAK1-3. Work will be guided by the new
crystal structures, and we will seek new opportunities to achieve
selectivity in this family of kinases.
Supplementary data
Supplementary data (synthetic procedures, NMR spectra of
compounds 11–26, and methods for enzymatic inhibition assays)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.10.026.
References and notes
1. Waldburger, J. M.; Firestein, G. S. Arthritis Res. Ther. 2009, 11, 206.
2. Feldmann, M. J. Clin. Invest. 2008, 118, 3533.
3. Brennan, F. M.; McInnes, I. B. J. Clin. Invest. 2008, 118, 3537.
4. Cua, D. J.; Sherlock, J.; Chen, Y.; Murphy, C. A.; Joyce, B.; Seymour, B.; Lucian, L.;
To, W.; Kwan, S.; Churakova, T.; Zurawski, S.; Wiekowski, M.; Lira, S. A.;
Gorman, D.; Kastelein, R. A.; Sedgwick, J. D. Nature 2003, 421, 744.
5. Cornelissen, F.; van Hamburg, J. P.; Lubberts, E. Curr. Opin. Investig. Drugs 2009,
10, 452.
6. Shaw, M. H.; Boyartchuk, V.; Wong, S.; Karaghiosoff, M.; Ragimbeau, J.;
Pellegrini, S.; Muller, M.; Dietrich, W. F.; Yap, G. S. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 11594.
7. 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. Nat. Biotechnol. 2008,
26, 127.
8. Lin, R. H.; Connolly, P. J.; Huang, S. L.; Wetter, S. K.; Lu, Y. H.; Murray, W. V.;
Emanuel, S. L.; Gruninger, R. H.; Fuentes-Pesquera, A. R.; Rugg, C. A.; Middleton,
S. A.; Jolliffe, L. K. J. Med. Chem. 2005, 48, 4208.
9. Lin, R. H.; Huang, S. L.; Wetter, S. K.; Connolly, P. J.; Emanuel, S. L.; Gruninger, R.
H.; Middleton, S. A. U.S. Patent 7317031 B2, 2009.
10. Werning, G.; Kharas, M. G.; Okabe, R.; Moore, S. A.; Leeman, D. S.; Cullen, D. E.;
Gozo, M.; McDowell, E. P.; Levine, R. L.; Doukas, J.; Mak, C. C.; Noronha, G.;
Martin, M.; Ko, Y. D.; Lee, B. H.; Soll, R. M.; Tefferi, A.; Hood, J. D.; Gilliland, D. G.
Cancer Cell 2008, 13, 311.
11. Hood, J. D.; Noronha, G. U.S. Patent Application 2009/0286789.
12. Fridman, J. S.; Scherle, P. A.; Collins, R.; Burn, T. C.; Li, Y.; Li, J.; Covington, M. B.;
Thomas, B.; Collier, P.; Favata, M. F.; Wen, X.; Shi, J.; McGee, R.; Haley, P. J.;
Shepard, S.; Rodgers, J. D.; Yeleswaram, S.; Hollis, G.; Newton, R. C.; Metcalf, B.;
Friedman, S. M.; Vaddi, K. J. Immunol. 2010, 184, 5298.
13. Rodgers, J. D.; Wang, H.; Combs, A. P.; Sparks, R. B. U.S. Patent 7335667, 2008.
14. Rodgers, J. D.; Shepard, S. U.S. Patent 7598257, 2009.
15. Fridman, J. S. Presented at Cambridge Healthtech Institute’s Fifth Annual Drug
Discovery Chemistry Meeting, San Diego, CA, April 2010; Development of a
JAK-inhibitor for Rheumatoid Arthritis.
16. Changelian, P. S.; Flanagan, M. E.; Ball, D. J.; Kent, C. R.; Magnuson, K. S.; Martin,
W. H.; Rizzuti, B. J.; Sawyer, P. S.; Perry, B. D.; Brissette, W. H.; McCurdy, S. P.;
Kudlacz, E. M.; Conklyn, M. J.; Elliott, E. A.; Koslov, E. R.; Fisher, M. B.; Strelevitz,
T. J.; Yoon, K.; Whipple, D. A.; Sun, J. M.; Munchhof, M. J.; Doty, J. L.; Casavant, J.
M.; Blumenkopf, T. A.; Hines, M.; Brown, M. F.; Lillie, B. M.; Subramanyam, C.;
Shang-Poa, C.; Milici, A. J.; Beckius, G. E.; Moyer, J. D.; Su, C. Y.; Woodworth, T.
G.; Gaweco, A. S.; Beals, C. R.; Littman, B. H.; Fisher, D. A.; Smith, J. F.; Zagouras,
P.; Magna, H. A.; Saltarelli, M. J.; Johnson, K. S.; Nelms, L. F.; Des Etages, S. G.;
Hayes, L. S.; Kawabata, T. T.; Finco-Kent, D.; Baker, D. L.; Larson, M.; Si, M. S.;
Paniagua, R.; Higgins, J.; Holm, B.; Reitz, B.; Zhou, Y. J.; Morris, R. E.; O’Shea, J. J.;
Borie, D. C. Science 2003, 302, 875.
17. Chrencik, J. E.; Patny, A.; Leung, I. K.; Korniski, B.; Emmons, T. L.; Hall, T.;
Weinberg, R. A.; Gormley, J. A.; Williams, J. M.; Day, J. E. J. Mol. Biol. 2010, 413–
433.
18. Webb, R. L.; Labaw, C. S. J. Heterocycl. Chem. 1982, 19, 1205.
19. Webb, R. L.; Eggleston, D. S.; Labaw, C. S.; Lewis, J. J.; Wert, K. J. Heterocycl.
Chem. 1987, 24, 275.
Figure 3. Inhibitory effect of compounds on IFNc cytokine production. Human PHA
blasts, from two different donors were derived from PBMCs cultured in PHA (0.1 lg/
ml) and IL-2 (10 U/ml) for 7 days. PHA blasts were pre-incubated with inhibitors
(10 lM) for 15 min and then stimulated with IL-12 (10 ng/ml). After 48 h, cell
supernatants were collected and IFNc production was measured by ELISA. Data are
mean ± SEM of triplicate wells.
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