Identification of an imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model

Article abstract of DOI:10.1016/j.bmcl.2017.08.022

Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients.

Full text of DOI:10.1016/j.bmcl.2017.08.022

Accepted Manuscript
Identification of an imidazopyridine scaffold to generate potent and selective
TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model
Jun Liang, Anne Van Abbema, Mercedesz Balazs, Kathy Barrett, Leo
Berezhkovsky, Wade S. Blair, Christine Chang, Donnie Delarosa, Jason
DeVoss, Jim Driscoll, Charles Eigenbrot, Simon Goodacre, Nico Ghilardi,
Calum MacLeod, Adam Johnson, Pawan Bir Kohli, Yingjie Lai, Zhonghua Lin,
Priscilla Mantik, Kapil Menghrajani, Hieu Nguyen, Ivan Peng, Amy Sambrone,
Steven Shia, Jan Smith, Sue Sohn, Vickie Tsui, Mark Ultsch, Karen Williams,
Lawren C. Wu, Wenqian Yang, Birong Zhang, Steven Magnuson
PII:
S0960-894X(17)30823-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.022
BMCL 25216
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 July 2017
10 August 2017
11 August 2017
Please cite this article as: Liang, J., Van Abbema, A., Balazs, M., Barrett, K., Berezhkovsky, L., Blair, W.S., Chang,
C., Delarosa, D., DeVoss, J., Driscoll, J., Eigenbrot, C., Goodacre, S., Ghilardi, N., MacLeod, C., Johnson, A., Bir
Kohli, P., Lai, Y., Lin, Z., Mantik, P., Menghrajani, K., Nguyen, H., Peng, I., Sambrone, A., Shia, S., Smith, J.,
Sohn, S., Tsui, V., Ultsch, M., Williams, K., Wu, L.C., Yang, W., Zhang, B., Magnuson, S., Identification of an
imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo
psoriasis model, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.08.022
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Identification of an imidazopyridine scaffold to
generate potent and selective TYK2 inhibitors that
demonstrate activity in an in vivo psoriasis model
Jun Liang,a Anne Van Abbema,b Mercedesz, Balazs,c Kathy Barrett,b Leo Berezhkovsky,d Wade S. Blair,b Christine
Chang,b Donnie Delarosa,b Jason DeVoss,c Jim Driscoll,d Charles Eigenbrot,e Simon Goodacre,i Nico Ghilardi,f
Calum MacLeod,i Adam Johnson,b Pawan Bir Kohli,b Yingjie Lai,a Zhonghua Lin,c Priscilla Mantik,g Kapil
Menghrajani,d Hieu Nguyen,c Ivan Peng,c Amy Sambrone,g Steven Shia,e Jan Smith,b Sue Sohn,f Vickie Tsui,a Mark
Ultsch,d Karen Williams,i Lawren C. Wu,f Wenqian Yang,h Birong Zhang,a Steven Magnusona*
a Department of Discovery Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
b Department of Biochemical and Cellular Pharmacology, Genentech, Inc., 1 DNA Way, South San Francisco,
California 94080, USA.
c Department of Translational Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080,
USA.
d Department of Drug Metabolism and Pharmacokinetics, Genentech, Inc., 1 DNA Way, South San Francisco,
California 94080, USA.
e Department of Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
f Department of Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
g Department of Small Molecule Pharmaceutical Sciences, Genentech, Inc., 1 DNA Way, South San Francisco,
California 94080, USA.
hChemPartner Co. Ltd. 998 Halei Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, P. R. China.
iArgenta Discovery Services, CRL, 8-9 Spire Green Centre, Harlow, Essex, CM19 5TR, United Kingdom.

Abstract:
Herein we report identification of an imidazopyridine class of potent and selective TYK2
inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting
the pyridine and aryl rings of compound 1. Further optimization led to generation of compound
30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity
against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30
demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an
imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was
accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a
therapeutic approach for psoriasis patients.
Keywords:
TYK2
kinase
psoriasis
imidazopyridine
IL-23

Tyrosine Kinase 2 (TYK2) is a member of the Janus kinase (JAK) family. It associates
with the cytoplasmic tails of numerous cytokine receptors and induces intracellular signaling,
through activation of signal transducers and activators of transcription (STAT) proteins, to
regulate gene expression and transcription.1,2 IL-12 and IL-23 are two TYK2 dependent
cytokines of particular interest, because antibodies against IL-12 and IL-23 have proven
therapeutic utility in several diseases. In the case of the IL-12 pathway, phosphorylation of
STAT4 leads to expression of IFNγ3,4 while in the IL-23 pathway, phosphorylation of STAT3
leads to increased IL-17 levels (Figure 1).5,6 The IL-12 and IL-23 pathways have been
implicated in diseases of the immune system including psoriasis and inflammatory bowel
diseases (IBD).7-12 These chronic inflammatory disorders of epithelial cells triggered by an
overly activated immune system, including Th1and Th17 immune responses, require new and
safe therapies.
The other members of the Janus Kinase family (JAK1, JAK2, and JAK3) are highly
homologous to TYK2.13 As we set forth to find tool compounds suitable for in vivo studies, we
were particularly aware of the need for selectivity against JAK2, as this related kinase is required
for the signaling of several hematopoietic growth factors, including the erythropoietin (EPO)
receptor that is responsible for the production of red blood cells and the thrombopoeitin (TPO)
receptor that is required for platelet formation.14 Complete inhibition of the kinase activity of
JAK2 will lead to thrombocytopenia. We set forth to identify TYK2 tool compounds with a
range of selectivity against JAK2 and set a minimum of 10-fold selectivity against JAK2 based
on testing in relevant cell based assays.15
Previously, we have described the lead identification of cyclopropyl analog 116 and its
subsequent optimization to fluorocyclopropane 2.17 During the course of those studies we

followed IL-12 pathway activity, by monitoring IL-12 mediated STAT4 phosphorylation in a
cell assay and IFNγ production in a pharmacokinetics/pharmacodynamics (PK/PD) animal
model. Around the time of completing these efforts, we modified our assay cascade to evaluate
activity in the IL-23 pathway, measuring IL-23-mediated STAT3 phosphorylation in cells15 and
IL-17 production in a PK/PD animal model. As measured in this IL-23-mediated cell assay,
compound 2 had an EC50 of 1.2 µM, around 5-fold less potent than it was in our IL-12-dependent
cell assay (Figure 1). This erosion in cell-based potency coincided with a decrease in the cellular
selectivity relative to the JAK2-based cellular readout (measuring EPO-mediated STAT5
phosphorylation). There are several possible reasons why a compound might be less potent in
the IL-23-dependent assay as compared with the IL-12-driven assay such as the use of different
cell types (Th17 memory vs NK92) or different requirements of TYK2 and JAK2 inhibition to
fully block the IL-23 or IL-12 pathways.15 Nevertheless, improving potency against TYK2 (and
thereby improving activity in the IL-23-mediated cell assay) became a priority for the project
team.
Figure 1: Comparison of the effects of compound 2 on IL-12 mediated STAT4 phosphorylation
and IL-23-mediated STAT3 phosphorylation.

To improve TYK2 potency, we evaluated constraint of the amide bond between the aryl
and pyridyl rings through construction of another ring.18 When we considered the bound
confirmation of amides 1 and 2 in TYK2 (and those of other amide crystal structures from the
same series), constraining the amide to carbon 5 of the pyridine ring, which sit in-plane with
each other, appeared to be a promising idea (Figure 2, cyclization direction “a”). However, we
were mindful of the fact that the alternate amide rotamer could also be constrained to carbon 3 of
the pyridine ring (cyclization direction “b”).
Figure 2: Possible cyclization directions of different amide rotamers.
We proceeded by making of a number of constrained analogs of amide 1 to determine
which cyclization strategy might present a path forward to improved potency (Table 1).19
Comparison of isoquinolones 3 and 4 illustrated two very important trends. First, cyclization
direction “b” (isoquinoline 4) afforded better potency than cyclization direction “a” (isoquinoline
3). More importantly, isoquinoline 4 has much better JAK2 selectivity (8-fold) than isoquinoline
3 (0.4-fold). These differences are even more pronounced comparing the 5,6-ring system for
imidazopyridines 5 and 6. Imidazopyridine 6 is 2000-fold more potent against TYK2 and 90-
fold more JAK2 selective than imidazopyridine 5. The loss of potency for 5-member ring 5 vs 6-
member ring 3 can be attributed to a compacting of the smaller ring system requiring an

undesired shift in the positioning of the dichlorophyenyl ring. Conversely, the 20-fold
improvement in TYK2 potency for the smaller ring system (imidazopyridine 6 vs isoquinoline 4)
can be easily understood as the 5-member ring system relieves a steric clash with the amide
carbonyl of isoquinoline 4, and installs a favorable intramolecular hydrogen bond between the
imidazole NH and the amide carbonyl. We obtained a crystal structure of this prototype
constrained imidazopyridine 6 in TYK2 and compared it to the TYK2 co-crystal structure of
amide 1 (Figure 3a). The aforementioned intramolecular hydrogen bond is apparent from the co-
crystal structure. Furthermore, we noted that the constrained analog sits deeper in the binding
pocket, packing more closely with Ile960 than amide 1. This improved van der Waals
interaction may also contribute to the potency of imidazopyridine 6. The hinge backbone does
shift slightly to accommodate the ligand 6, but productive hydrogen bond distances with the
hinge are maintained. We also compared the TYK2 co-crystal structure of analog 6 to its JAK2
co-crystal structure (Figure 3b). Here, too, we see that analog 6 sits deeper in the TYK2 binding
pocket, creating more favorable interactions with Ile980 than it does with the corresponding
residue, Val911, of JAK2. The significant improvement in biochemical selectivity over JAK2
for this constrained core is a definite advantage over the unconstrained amide core.

Figure 3: a) overlay of compound 1 (beige, PDB code 4GIH) and compound 6 (green, PDB code
5WAL) in TYK2; b) overlay of compound 6 in TYK2 (beige) and JAK2 (blue, PDB code
5WEV).
With the pronounced preference for cyclization direction “b” for 5,6-bicyclo systems, we
continued our exploration of other heterocycles (Table 1). Analogs that are not able to form a
productive intramolecular hydrogen bond with the amide carbonyl (7-11) all have decreased
TYK2 potency relative to imidazopyridine 6. Conversely, analogs capable of productive
hydrogen bonds, either through sulfur (12 and 13) or through CH (14) were all very potent
against TYK2 with varying levels of biochemical selectivity against JAK2.20 Indeed,
thiazolopyridine 13, and pyrazolopyridine 14 both represented new cores as starting points for
further optimization.21,22 For the purpose of this report, we will focus on optimization of the
imidazopyrdine 6.
Table 1. Analogs with different constrained heterocycles in place of the pyridine amide
aTYK2 Ki
aJAK2 Ki (µM)
Constrained
Heterocycle
Cyclization
Direction
Ex
3
(Fold Selectivity)b
(µM)
0.016
(0.4 fold)
a
b
a
0.042
0.15
(8.3 fold)
4
0.017
3.5
N
3.2
(0.9 fold)
5

0.15
(88 fold)
6
7
8
9
b
b
b
b
0.0017
0.013
0.13
(10 fold)
2.1
(111 fold)
0.019
0.90
(103 fold)
0.0088
0.59
(29 fold)
10
11
12
13
14
b
b
b
b
b
0.020
0.031
0.37
(12 fold)
0.0066
(7.5 fold)
0.0008
0.0005
0.0013
0.026
(52 fold)
0.19
(142 fold)
a Biochemical assays. Arithmetic mean of at least 3 separate determinations (n ≥ 3). On average, the coefficients of
b
variation were less than 0.3 times the mean for these assays. Fold selectivity for TYK2 inhibition vs. JAK2
inhibition as measured by JAK2 Ki ÷ TYK2 Ki.
With the imidazopyridine core selected for further profiling, we explored alternatives to
the cyclopropyl amide to further enhance TYK2 potency. Initial SAR was largely consistent
with SAR observed in the open amide series,16 such that pyrimidine 15 was identified with
submicromolar biochemical potency and good JAK2 selectivity (Table 2). We next explored a
number of different substitutions on the pyrimidine focusing primarily on the C6-position, which
we felt, based on crystal structures, would allow us to explore a range of substitutions to tune
both selectivity and physicochemical properties. Functional groups of varying sizes were
tolerated (analogs 16-26), many with sub-nanomolar TYK2 biochemical potency, but with
variable JAK2 selectivity. Small substituents (16-21) afforded good JAK2 selectivity, while

increasing size of the substituent, particularly those with more polar atoms, led to erosion of
JAK2 biochemical selectivity. With this series of analogs we were also able to generate
meaningful cellular SAR and selectivity data using our IL-23-mediated cell-based assay and the
corresponding JAK2 (EPO-mediated) cell assay. Relative to amide 2 (IL-23-mediated cell IC50
= 1.2 µM), we observed that the enhanced biochemical potency of the constrained compounds
such as amine 18 gave a corresponding improvement in cell potency (0.044 µM). In general
there was a reasonable correlation between TYK2 biochemical potency and IL-23-mediated cell
activity15 with occasional outliers such as methyl ether 20, in which poor solubility may be
limiting its cellular potency readout. When assessing cellular JAK2 selectivity, it was clear that
for most analogs there was erosion in the fold-selectivity observed relative to biochemical
selectivity. The reduced cellular selectivity could potentially be attributed to a number of factors
including the use of different cell types in the assays, translation of kinase-domain constructs
used in the biochemical assay to the full length enzymes in the cell assays, and poor compound
solubility. Nevertheless, we were happy to see that analogs such as 16, 18, 19 and 21 displayed
good IL-23-mediated cell potency and reasonable cellular selectivity (> 10-fold). We also
explored SAR at the 2-position of the pyrimidine, albeit not as extensively. While small
substitutions were tolerated, they usually led to a loss of TYK2 biochemical potency (cf 15 and
27; 21 and 28; 16 and 29). This loss of TYK2 potency can possibly be attributed to disruption of
the intramolecular hydrogen bond between the nitrogen on the pyrimidine and the NH of the
imidazole as a result of the 2-substitution.
Table 2. Heterocycle replacement of cyclopropyl amide

aJAK2 Ki
(µM)
IL-23-
pSTAT3c
EC50 (µM)
EPO-pSTAT5d
EC50 (µM)
aTYK2
Constrained
Heterocycle
Ex
15
16
17
18
Ki (µM)
(Fold Sel.)e
(Fold Sel.)b
0.095
(158 fold)
0.0006
0.0004
0.0003
0.0005
0.021
(52 fold)
1.9
(11 fold)
0.17
0.26
0.014
(47 fold)
1.3
(5.0 fold)
0.026
(52 fold)
0.64
(16 fold)
0.044
0.093
(133 fold)
7.7
(220 fold)
19
20
0.0007
0.0007
0.035
3.6
0.11
(157 fold)
6.5
(1.8 fold)
0.034
(56 fold)
2.1
(15 fold)
21
22
0.0006
0.0005
0.14
0.52
0.016
(32 fold)
1.0
(1.9 fold)
0.0065
(11 fold)
0.46
(2.3 fold)
23
24
0.0006
0.0010
0.20
0.36
0.0054
(5.4 fold)
1.1
(3.1 fold)
0.014
(12 fold)
0.82
(14 fold)
25
0.0012
0.059
N
N
0.010
(8.3 fold)
1.1
(7.8 fold)
26
27
28
0.0012
0.0051
0.0032
0.14
9.4
N
N
OH
0.44
(86 fold)
10
(1.1 fold)
0.043
(13 fold)
n.d.
n.d.
n.d.
N
0.49
(49 fold)
29
0.0099
n.d.
N
N

a Biochemical assays. Arithmetic mean of at least 3 separate determinations (n ≥ 3). On average, the coefficients of
b
variation were less than 0.3 times the mean for these assays. Fold selectivity for TYK2 inhibition vs. JAK2
inhibition as measured by JAK2 Ki ÷ TYK2 Ki. c Cell-based assay of TYK2 inhibition. Arithmetic mean of at least
3 separate runs (n > 3). On average, the coefficients of variation were less than 0.5 times the mean for cell-based
d
assays. Cell-based assay of JAK2 inhibition. Arithmetic mean of at least 3 separate runs (n> 3). Fold selectivity:
e
EPO pSTAT5 EC50 ÷ IL-12 pSTAT4 EC50.
Previously, we had shown in the original open amide series that placement of a cyano
group at the 4-position of the dichlorophenyl ring, as shown in amide 2, was tolerated from a
potency and selectivity point of view and, more importantly, led to improved in vivo clearance in
rodents.17 We took some of our favored analogs from Table 2 and made the cyano versions of
them (Table 3). Of these analogs, only 6-methyl pyrimidine 30 had appropriate potency and
selectivity to advance to further selectivity and pharmacokinetic profiling.
Table 3. Imidazopyridines with nitrile on the phenyl ring
aJAK2 Ki
(µM)
IL-23-
pSTAT3c
EC50 (µM)
EPO-pSTAT5d
EC50 (µM)
compare to
analog from
Table 3
aTYK2
Ex
R
Ki (µM)
(Fold Sel.)e
(Fold Sel.)b
0.022
(31 fold)
1.4
(21 fold)
30
31
-CH3
-NH2
16
17
18
0.0007
0.0006
0.0006
0.066
0.45
0.023
(38 fold)
0.036
1.6
(3.5 fold)
0.62
(8.6 fold)
32 -NHCH3
0.072
(60 fold)
a Biochemical assays. Arithmetic mean of at least 3 separate determinations (n ≥ 3). On average, the coefficients of
b
variation were less than 0.3 times the mean for these assays. Fold selectivity for TYK2 inhibition vs. JAK2
inhibition as measured by JAK2 Ki ÷ TYK2 Ki. c Cell-based assay of TYK2 inhibition. Arithmetic mean of at least
3 separate runs (n > 3). On average, the coefficients of variation were less than 0.5 times the mean for cell-based

d
assays. Cell-based assay of JAK2 inhibition. Arithmetic mean of at least 3 separate runs (n> 3). Fold selectivity:
e
EPO pSTAT5 EC50 ÷ IL-12 pSTAT4 EC50.
Regarding the other JAK family kinases, compound 30 has a JAK1 Ki of 24 nM and
JAK3 Ki of 58 nM, indicating good TYK2 selectivity (35-fold for JAK1 and 83-fold for JAK3).
Compound 30 was further profiled, at a concentration of 70 nM (100x the TYK2 Ki), against a
panel of 58 kinases that were known to be relevant to JAK family kinase inhibitors, where it
inhibited only two kinases by >50%. Full inhibitor titration curves were measured for these
kinases with CDK2 Ki = 89 nM (126-fold selective for TYK2) and CDK9 Ki = 108 nM (154-fold
selective). Compound 30 was also tested, at a concentration of 10 µM, against a panel of 40
pharmacologically important and safety-related receptors and channels, where it only showed
appreciable activity against NK1 (67% inhibition). Physicochemical and DMPK properties of
compound 30 are shown in Figure 4. The compound is highly permeable, and has moderate
stability in liver microsomes. Stability in hepatocytes is good and this correlates well with in
vivo clearance. In mice, the compound is cleared quickly, but appreciable exposure can be
obtained at higher doses enabling in vivo PD and efficacy evaluation.

Figure 4: Physicochemical and DMPK properties of compound 30
We progressed compound 30 for testing in our IL-23-induced PK/PD model (Figure 5).23
In this model, pathway activation was achieved by intravenous dosing of IL-23 and IL-1B,
followed 3 h later by collection of plasma for measurement of IL-17F (measured by cytokine
ELISA) and drug concentration. The positive control, anti-p40 antibody, was dosed
intraperitoneally 60 minutes prior to cytokine stimulation, while compound 30 was dosed orally
30 minutes before cytokine injection. Statistically significant reduction of plasma IL-17F was
observed at the 100 and 70 mg/kg doses (Figure 5a), but at 30 mg/kg inter animal variability was
evident and the reduction of IL-17F was not statistically different from vehicle. The
corresponding plasma free drug concentrations (Table 4) reveal that strong inhibition of the
pathway (87% at 100 mg/kg and 72% at 70 mg/kg) occurred when free drug concentrations at
Cmax were 39-fold and 23-fold over the TYK2-dependent IL-23 cell assay IC50 (66 nM).
Notably, at each of these doses there was also at least 1-fold coverage of the JAK2-related cell
IC50 (1400 nM). At 30 mg/kg, where there was not statistically significant reduction of IL-17F,
plasma exposure indicated 13-fold coverage of the TYK2-related cell IC50 but only 0.6-fold
coverage of the JAK2 cell IC50 at Cmax. It is curious that the 30 mg/kg dose was not active,
given the strong coverage to TYK2. One possible explanation is that some level of JAK2
inhibition, which is also coupled to the IL-23 receptor, along with strong coverage of TYK2, is
required to strongly inhibit IL-17F production in this assay.24 It is also worth noting that both IL-
23 and IL-1B are used to stimulate the pathway and so potentially TYK inhibition alone may not
be sufficient to inhibit this model.
Table 4. PK data for IL-23-induced PK/PD experiment with compound 30.

Dose of 30
(mg/kg)
[Free drug] of 30 Fold coverage of
at T = 3 ha (µM)
Fold coverage of
c
JAK2 cell EC50
b
TYK2 cell EC50
100
70
2.6
1.5
39 fold
23 fold
13 fold
1.9 fold
1.1 fold
0.61 fold
30
0.86
b
a Collection for plasma analysis 3.5 h after dose of compound 30 – approximately at Cmax. Fold coverage
c
calculated by free drug concentration/IL-23-pSTAT3 EC50 of compound 30 (0.066 µM). Fold coverage calculated
by free drug concentration/EPO-pSTAT5 EC50 of compound 30 (1.4 µM).
Serum IL-17F
b)
a)
300
250
200
150
100
50
p < 0.05
*
30%
*
*
87% 72%
*
100%
0
Serum IL-17F
d)
c)
2000
1000
p < 0.05
*
*
74%
*
98%
*
99%
100
40
*
100%
Figure 5: In vivo PD and efficacy data for compound 30. a) Serum IL-17F levels in IL-23-
induced PD assay; b) ear thickness reduction measured on day 5 in imiquimod-induced psoriasis

model; c) serum IL-17F levels measured on day 5 in imiquimod model; d) platelet levels
measured on day 5 in imiquimod model
With these positive PK/PD results we proceeded to an efficacy study using an
imiquimod-mediated psoriasis model.25,26 In this model imiquimod, a topical agent that
stimulates multiple pathways such as IL-1, IL-23/IL-17, and TLR7, is administered to the ear of
a mouse for 5 days, while the contralateral ear is used as the unstimulated control. Compound 30
was dosed orally at 100, 70, and 30 mg/kg BID for those same 5 days as imiquimod treatment,
and at the start of the sixth day, ear thickness was measured. Plasma was also collected to assess
serum IL-17F levels and other blood parameters. Statistically significant reductions in ear
thickness, comparable to the anti-p40 positive control, were observed at 100 and 70 mg/kg,
whereas the 30 mg/kg dose gave only a minimal reduction (Figure 5b). At the 30 mg/kg BID
dose, free drug trough levels are well below the TYK2 cellular EC50 and it is not surprising that
this dose is not active in this assay. The efficacy results are consistent with the reductions in
serum IL-17F observed in this 5-day model (Figure 5c) where complete reduction was observed
at the highest two doses and more modest reduction was attained at the 30 mg/kg dose. While
most other serum measurements showed little change relative to the naïve controls, a 50%
reduction in platelet levels was observed at the 100 and 70 mg/kg doses (Figure 5d). It is
tempting to speculate that this mild thrombocytopenia could potentially be related to combined
JAK2 and TYK2 inhbition and the resultant effects on the TPO pathway.27 However, the
relationship between JAK2 and/or TYK2 inhibition and the effects on the IL-23 and TPO
pathways needs to be probed further with compounds of different TYK2/JAK2 selectivity
profiles, and potentially compounds from other chemical scaffolds.28 Our efforts in this
investigation will be the subject of future publications.

Acknowledgement
We thank Mengling Wong, Chris Hamman, Michael Hayes, Baiwei Lin, Deven Wang, Yutao
Jiang, and Yanzhou Liu for purification and analytical support.
Supporting Information
Details of the crystallographic methods and sythetic and analytical information for compounds 3-
32 are available.
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JAK3 biochemical data was spot checked against certain compounds since this series
typically showed good JAK3 selectivity. Biochemical assay conditions can be found at:
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Graphic Abstract – Magnuson TYK2 Imidazopyridines