Discovery of tricyclic dipyrrolopyridine derivatives as novel JAK inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.07.043

Janus kinases (JAKs) play a crucial role in cytokine mediated signal transduction. JAK inhibitors have emerged as effective immunomodulative agents for the prevention of transplant rejection. We previously reported that the tricyclic imidazo-pyrrolopyridinone 2 is a potent JAK inhibitor; however, it had poor oral absorption due to low membrane permeability. Here, we report the structural modification of compound 2 into the tricyclic dipyrrolopyridine 18a focusing on reduction of polar surface area (PSA), which exhibits potent in vitro activity, improved membrane permeability and good oral bioavailability. Compound 18a showed efficacy in rat heterotopic cardiac transplants model.

Full text of DOI:10.1016/j.bmc.2017.07.043

Accepted Manuscript
Discovery of tricyclic dipyrrolopyridine derivatives as novel JAK inhibitors
Hiroaki Yamagishi, Takayuki Inoue, Yutaka Nakajima, Jun Maeda, Hiroaki
Tominaga, Hiroyuki Usuda, Takeshi Hondo, Ayako Moritomo, Fumihiro
Nakamori, Misato Ito, Koji Nakamura, Hiroki Morio, Yasuyuki Higashi,
Masamichi Inami, Shohei Shirakami
PII:
S0968-0896(17)31360-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.043
BMC 13880
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 July 2017
25 July 2017
26 July 2017
Please cite this article as: Yamagishi, H., Inoue, T., Nakajima, Y., Maeda, J., Tominaga, H., Usuda, H., Hondo, T.,
Moritomo, A., Nakamori, F., Ito, M., Nakamura, K., Morio, H., Higashi, Y., Inami, M., Shirakami, S., Discovery
of tricyclic dipyrrolopyridine derivatives as novel JAK inhibitors, Bioorganic & Medicinal Chemistry (2017), doi:
http://dx.doi.org/10.1016/j.bmc.2017.07.043
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Discovery of tricyclic dipyrrolopyridine derivatives as novel JAK inhibitors
*
Hiroaki Yamagishi, Takayuki Inoue, Yutaka Nakajima, Jun Maeda, Hiroaki Tominaga,
Hiroyuki Usuda, Takeshi Hondo, Ayako Moritomo, Fumihiro Nakamori, Misato Ito,
Koji Nakamura, Hiroki Morio, Yasuyuki Higashi, Masamichi Inami, and Shohei
*
Shirakami
Drug Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka, Tsukuba, Ibaraki
3
05-8585, Japan
*
Corresponding authors:
H. Yamagishi
Tel: +81 29 829 6194; Fax: +81 29 852 5387
E-mail: hiroaki.yamagishi@astellas.com
S. Shirakami
E-mail: shohei.shirakami@astellas.com
1

Abstract
Janus kinases (JAKs) play a crucial role in cytokine mediated signal transduction. JAK
inhibitors have emerged as effective immunomodulative agents for the prevention of
transplant
rejection.
We
previously
reported
that
the
tricyclic
imidazo-pyrrolopyridinone 2 is a potent JAK inhibitor; however, it had poor oral
absorption due to low membrane permeability. Here, we report the structural
modification of compound 2 into the tricyclic dipyrrolopyridine 18a focusing on
reduction of polar surface area (PSA), which exhibits potent in vitro activity, improved
membrane permeability and good oral bioavailability. Compound 18a showed efficacy
in rat heterotopic cardiac transplants model.
2

Keywords
Janus kinase inhibitor, IL-2, organ transplant rejection, autoimmune diseases,
immunomodulator
3

Graphical abstract
4

1
. Introduction
Immunosuppressive drugs are commonly used in the control of organ transplant
rejection. Calcineurin inhibitors, such as tacrolimus and cyclosporin, block the
phosphatase activity of calcineurin and the translocation of nuclear factor of activated
1
T-cells (NF-AT) into the nucleus. Mycophenolate mofetil (MMF) inhibits inosine
monophosphate dehydrogenase (IMPDH), which plays an important role in the
2
proliferation of immune cells. Although these drugs are effective for organ transplant
rejection, the ubiquitous expression of their targets leads to a range of adverse effects,
including nephrotoxicity and neurotoxicity (tacrolimus and cyclosporin) and
3
gastrointestinal toxicity (MMF). Pharmacological targets with limited expression in the
immune system might therefore have potential as immunomodulative agents with
reduced adverse effects.
Due to their unique role in the immune system, Janus kinases (JAKs) have emerged as
attractive targets to counter transplant rejection and treat autoimmune diseases. JAKs
are cytoplasmic protein tyrosine kinases that consist of four subtypes (JAK1, JAK2,
JAK3 and TYK2) and play key roles in various cytokine-mediated signal transduction
4
pathways. The binding of cytokines to their corresponding receptors activates JAKs,
which then phosphorylate signal transducers and activators of transcription (STAT) to
5
promote cytokine-responsive gene expressions. JAK3 and JAK1 are involved in the
signaling pathways of common γ-chain (γc) cytokines such as interleukin (IL)-2, -4, -7,
-9, -15 and -21, which play important roles in T-cell differentiation, proliferation and
survival. In contrast, JAK2 mediates signaling via hematopoietic cytokines such as
erythropoietin (EPO).
6
Several JAK inhibitors have been reported to date. In preclinical studies, the pan-JAK
5

inhibitor tofacitinib (Figure 1, 1, CP-690,550, in-house data: IC = 0.8 nM, 3.1 nM, 3.7
5
0
nM for JAK3, JAK2, JAK1, respectively) demonstrated efficacy in a nonhuman primate
7
transplant rejection model. Compound 1 has also demonstrated efficacy in the
treatment of various autoimmune diseases and was recently approved for the treatment
8
of rheumatoid arthritis (RA). In transplant rejection, therapies that inhibit JAK3,
thereby blocking IL-2 signal transduction, may be effective, since the localized
expression of JAK3 to hematopoietic cells, such as T-cells and B-cells, suggests a low
risk of adverse effects. We have screened compounds for the inhibition of not only
JAK3, but also JAK1, since IL-2 is also associated with JAK1, and JAK2, because it
may be associated with adverse hematopoietic effects such as anemia.
We previously characterized the tricyclic imidazo-pyrrolopyridinone 2, which
exhibited potent inhibitory activity against JAK3 and JAK1 with favorable metabolic
9
stability and reduced CYP3A4 time-dependent inhibition (TDI) (Figure 2). Our
docking study with human JAK3 revealed that the pyrrolopyridine scaffold of 2 might
form two hydrogen bonds with the hinge residues Glu903 and Leu905 (Figure 3). In
addition, the cyano group of 2 appeared to extend toward the glycine-rich loop.
Although we were concerned about a potential risk of anemia resulting from JAK2
inhibition, clinical trials of 1 revealed that anemia was not the most common adverse
effect and most cases of anemia were mild to moderate, in spite of its JAK2 inhibitory
1
0
activity. Since the selectivity of 2 for JAK3 and JAK1 over JAK2 is similar to that of
1
, it is likely that 2 may also have a low risk of inducing anemia.
Despite its favorable JAK inhibition, metabolic stability and TDI, compound 2 did
not exhibit sufficient oral absorption, probably because of its low permeability. Since
membrane permeability often correlates with lipophilicity and polar surface area (PSA),
6

medicinal chemistry efforts were focused toward 1) increasing lipophilicity to a
favorable level and 2) reducing PSA. Herein, we report the design, synthesis, and
structure-activity relationship (SAR) of novel tricyclic JAK inhibitors.
7

Figure 1. Structure of tofacitinib (1).
Figure 2. Structure of compound 2.
8

hinge region
glycine-rich loop
Figure 3. Predicted binding mode of compound 2 to human JAK3.
9

2
. Chemistry
9
Compounds 4a-e were synthesized as shown in Scheme 1. Condensation of 3 with
carboxylic acids gave the desired 4a-b. Treatment of 3 with carbamoyl chlorides
resulted in 4c-d. S Ar displacement of chloride in 6-chloronicotinonitrile with 3 gave
N
4
e.
Compound 14 was synthesized as summarized in Scheme 2. Protection of
pyrrolopyridine 5 with 2-(trimethylsilyl)ethoxymethyl (SEM) group provided 6, which
was
followed
by
SNAr
displacement
of
chloride
in
6
with
9
(
±)-cis-1-benzyl-4-methylpiperidin-3-amine (7) gave 8. Reduction of the cyano group
by DIBAL gave aldehyde 9, which was converted to oxime 10. Treatment with isobutyl
chloroformate resulted in pyrazolopyrrolopyridine 11, followed by deprotection of the
Bn group in the presence of 1-chloroethyl chloroformate and the SEM group under
acidic condition to afford 13. Condensation with cyanoacetic acid gave the desired
compound 14.
Compound 18 and 18a-b were synthesized as described in Scheme 3. Wittig reaction
of aldehyde 9 with (methoxymethyl)triphenylphosphonium chloride provided enol ether,
which was converted to dipyrrolopyridine 15 under acidic condition. Deprotection of
the SEM group and the Bn group led to 17. Condensation with cyanoacetic acid gave
the desired compound 18. The racemate 16 was separated by chiral column
chromatography to give 16a and 16b, which were converted to 18a and 18b,
respectively.
Compound 25 was prepared as shown in Scheme 4. Treatment of 17 with Boc O gave
2
1
9, followed by bromination to afford 20. Deprotection of the Boc group under basic
1
0

condition and protection of the SEM group provided 22. Treatment with Zn(CN) and
2
Pd(PPh ) resulted in 23, which was followed by deprotection of the Boc group and the
3
4
SEM group to give 24. Condensation with cyanoacetic acid provided the desired
compound 25.
Compound 32 was synthesized by using Corey-Fuchs alkynylation as shown in
Scheme 5. Aldehyde 9 was converted into dibromoolefine 26, which was treated with
DBU to give bromoalkyne 27. Treatment of 27 with n-BuLi and following trapping of
the intermediate acetylenic anion with MeI provided the methyl-substituted acetylene 28.
Treatment of 28 with t-BuOK gave dipyrrolopyridine 29. Deprotection of the SEM
group and the Bn group provided 31. Condensation with cyanoacetic acid gave the
desired compound 32.
Compound 37 and 37a were synthesized as outlined in Scheme 6. Hydrogenation of 15
in the presence of Boc O gave 33, which was treated with NBS to provide 34.
2
Treatment with Zn(CN) and Pd(PPh ) resulted in 35, followed by deprotection of the
2
3 4
SEM group and the Boc group under acidic condition to afford 36. Condensation with
cyanoacetic acid gave the desired compound 37. The racemate 36 was purified by chiral
column chromatography to give 36a and 36b, respectively. Condensation of 36a with
cyanoacetic acid gave compound 37a.
Compound 41 was synthesized as summarized in Scheme 7. Treatment of 15 with
Eschenmoser's salt provided 38, followed by deprotection of the SEM group to give 39.
Hydrogenation with Pd(OH) provided 40, which was converted into the desired
2
compound 41.
1
1

Scheme 1. Synthesis of 4a-e. Reagents and conditions: (a) carboxylic acid, EDC·HCl,
HOBt, DIPEA, DMF; (b) 1-piperidinecarbonyl chloride or 4-morpholinecarbonyl
chloride, DIPEA, CH Cl ; (c) 6-chloronicotinonitrile, Et N, n-BuOH, microwave,
2
2
3
1
30 °C for 0.5 h, 76%.
1
2

Scheme 2. Synthesis of 14. Reagents and conditions: (a) SEMCl, NaH, DMF-THF,
°C to rt for 3 h, 88%; (b) DIPEA, NMP, 180 °C for 2 h, 91%; (c) DIBAL, THF, 0 °C
0
for 1 h, 82%; (d) NH OH·HCl, pyridine, EtOH, rt for 3 h, 96%; (e) isobutyl
2
chloroformate, Et N, CH Cl , 0 °C to rt for 1 h; evaporation; DMF, 150 °C for 1.5 h,
3
2
2
5
6%; (f) 1-chloroethyl chloroformate, DCE, 65 °C for 1.5 h; evaporation; MeOH, reflux
for 3 h, 84%; (g) TFA, CH Cl , rt for 3 h; evaporation; ethylenediamine, MeOH, 55 °C
2
2
for 0.5 h, 68%; (h) cyanoacetic acid, EDC·HCl, HOBt, DIPEA, DMF, rt, overnight,
2%.
3
1
3

+
-
Scheme 3. Synthesis of 18 and 18a-b. Reagents and conditions: (a) MeOCH P Ph Cl ,
2
3
NaHMDS, THF, 0 °C to rt for 13 h; (b) AcCl, MeOH, rt to 80 °C for 0.5 h; H O, 80 °C
2
for 1 h, 83% (2 steps); (c) TFA, CH Cl , rt, overnight; evaporation; ethylenediamine, 1
2
2
M NaOH aq., MeOH, CH Cl , rt for 2 h, quant.; (d) cat. Pd(OH) , HCO NH , EtOH,
2
2
2
2
4
reflux; (e) cyanoacetic acid, EDC·HCl, HOBt, DIPEA, DMF, rt; (f) separation by chiral
column chromatography.
1
4

Scheme 4. Synthesis of 25. Reagents and conditions: (a) Boc O, DMAP, CH Cl , rt for
2
2
2
0
8
.5 h, 92%; (b) NBS, CH Cl , 0 °C for 10 min, 90%; (c) K CO , MeOH, 50 °C for 0.5 h,
2 2 2 3
8%; (d) SEMCl, NaH, DMF, 0 °C to rt for 0.5 h, 70%; (e) Zn(CN) , cat. Pd(PPh ) ,
2
3 4
DMF, 100 °C for 2 h, 75%; (f) TFA, CH Cl , rt for 1.5 h; evaporation; ethylenediamine,
2
2
MeOH, 50 °C for 1 h, 85%; (g) cyanoacetic acid, HATU, DIPEA, DMF-CH Cl , 50 °C
2
2
for 1 h, 55%.
1
5

Scheme 5. Synthesis of 32. Reagents and conditions: (a) CBr , PPh , CH Cl , 0 °C to rt
4
3
2
2
for 1 h, 89% (b) DBU, DMSO, 0 °C to rt for 1 h, 85%; (c) n-BuLi, THF, –50 °C for 0.5
h; MeI, –50 °C to rt for 0.5 h, 52%; (d) t-BuOK, THF, rt to 50 °C for 4 h, 38%; (e) TFA,
CH Cl , rt for 2 h; evaporation; ethylenediamine, 1 M NaOH aq., MeOH, CH Cl ,
2
2
2
2
6
0 °C for 1 h, 90%; (f) cat. Pd(OH) , HCO NH , EtOH, reflux for 12 h, 89%; (g)
2 2 4
cyanoacetic acid, EDC·HCl, HOBt, Et N, DMF, 50 °C for 2 h, 88%.
3
1
6

Scheme 6. Synthesis of 37 and 37a. Reagents and conditions: (a) H (1 atm), cat,
2
Pd(OH) , Boc O, MeOH, rt for 2.5 h, quant.; (b) NBS, CH Cl , 0 °C for 10 min, 35%;
2
2
2
2
(
c) Zn(CN) , cat. Pd(PPh ) , DMF, microwave, 100 °C for 1 h, 65%; (d) TFA, CH Cl ,
2 3 4 2 2
rt for 2 h; evaporation; ethylenediamine, 1 M NaOH aq., MeOH, CH Cl , rt for 1 h,
2
2
quant.; (e) cyanoacetic acid, EDC·HCl, HOBt, DIPEA, DMF, rt; (f) separation by chiral
column chromatography.
1
7

Scheme 7. Synthesis of 41. Reagents and conditions: (a) Eschenmoser's salt, DMF,
0 °C for 1.5 h, 96%; (b) TFA, CH Cl , rt for 2 h; evaporation; ethylenediamine, 1 M
6
2
2
NaOH aq., MeOH, CH Cl , rt, overnight, quant.; (c) cat. Pd(OH) , HCO NH , EtOH,
2
2
2
2
4
reflux for 2 h, 71%; (d) cyanoacetic acid, EDC·HCl, HOBt, Et N, DMF, rt, 49%.
3
1
8

3
. Results and Discussion
3
.1 in vitro structure-activity relationships
We evaluated the inhibitory activity of newly synthesized compounds against human
JAK1, JAK2 and JAK3 enzymes. We also used an IL-2-induced rat T-cell proliferation
assay to assess the cellular potency of selected compounds that reflects JAK1 and JAK3
inhibitory activity. In addition, we evaluated the compounds’ metabolic stability in rat
liver microsomes, CYP3A4 TDI and membrane permeability.
PK studies in rat revealed that compound 2 had low plasma concentration (Table 1,
AUC0-24 h = 11.8 ng·h/mL at 1.0 mg/kg p.o.) and low bioavailability (F = 11.2%).
Further, compound 2 displayed low membrane permeability in parallel artificial
-6
membrane permeability assay (PAMPA) (Table 2, PAMPA permeability = <0.7 × 10
cm/s), providing evidence that the poor oral exposure of 2 may be due to its low
membrane permeability.
a
Table 1. Pharmacokinetic parameters of compound 2
i.v. (1 mg/kg)
Vd t1/2
p.o. (1 mg/kg)
t1/2
CL
AUC0-24 h
(ng·h/mL)
Cmax
(ng/mL)
Tmax
(h)
AUC0-24 h
F
(%)
(
mL/min/kg) (L/kg)
(h)
(h)
(ng·h/mL)
113 3.62
1.13
148
2.98
0.25
11.8
11.8
11.2
a
Pharmacokinetics were performed in SD rats (female).
1
9

Our first attempt to identify more desirable compounds was to improve the membrane
permeability of compound 2 by increasing lipophilicity. However, increased
lipophilicity can lead to high metabolic clearance and increase the risk of CYP3A4 TDI.
In a previous study, metabolic stability of compounds decreased dramatically when
9
CLogP exceeded 3.0. Therefore, we designed compounds with CLogP value below 3.0.
In addition, since organ transplant rejection is generally treated with a combination of
calcineurin inhibitors and MMF, and JAK inhibitors could be added to this combination
of treatments, it is essential to reduce the risk of drug-drug interactions (DDI) to prevent
organ transplant rejection. Therefore, we evaluated compounds according to the
-6
following criteria: (i) PAMPA permeability >5.0 × 10 cm/s, and (ii) CYP3A4 TDI %
remaining >85%.
To increase lipophilicity, we modified the amide linkage of compound 2. Since our
docking study suggested that the amide linkage was located at some distance from the
9
hinge binding region of JAK, modifications here were expected to be acceptable.
Introduction of a dimethyl group to the α-position of the amide led to a decrease in
JAK3 and JAK1 inhibitory activity (Table 2, 4a). In contrast, 4a displayed improved
membrane permeability due to increased lipophilicity (PAMPA permeability = 6.3 ×
-6
1
1
0 cm/s, CLogP = 1.7). However, 4a exhibited TDI (CYP3A4 TDI % remaining =
8%). While conversion of the cyanomethyl group to a piperidine ring (4c) resulted in a
slight decrease in JAK3 and JAK1 inhibitory activity and improved membrane
permeability, 4c also exhibited CYP3A4 TDI (TDI % remaining = 11%). To reduce the
lipophilicity, we synthesized the morpholine analogue 4d. However, 4d exhibited
decreased JAK3 and JAK1 inhibitory activity and did not satisfy our criteria (PAMPA
-6
permeability = 1.3 × 10 cm/s, TDI % remaining = 61%). Although the tetrazole
2
0

derivative 4b retained JAK3 and JAK1 inhibitory activity, it had low permeability
-6
11
(
PAMPA permeability = <0.8 × 10 cm/s). Introduction of cyanopyridine also
resulted in the retention of JAK3 inhibitory activity (4e, JAK3 IC = 1.6 nM) and an
5
0
-6
improvement in membrane permeability (PAMPA permeability = 8.8 × 10 cm/s).
However, 4e did not satisfy our criteria for TDI (TDI % remaining = 29%) due to
increased lipophilicity (CLogP = 2.8). Therefore, although modification of the amide
linkage increased lipophilicity to improve membrane permeability, it was difficult to
balance JAK potency and risk of TDI.
2
1

Table 2. Modification of amide linkage
JAK3 JAK2 JAK1
in vitro
CLint
CYP3A4
TDI
PAMPA
b
c
d
e
Compound
R
-
IC50a
IC50a
IC50a
(nM)
permeability CLog P
-6
(
nM) (nM)
(mL/min/kg) % remaining (× 10 cm/s)
1
2
0.8
1.1
7.1
2.1
5.5
1.4
1.6
3.1
2.6
15
3.7
1.5
58
63
100
90
18
11
61
88
29
5.0
<0.7
6.3
0.92
1.0
No
Depletion
f
4
a
44
1.7
4
c
7.0
12
13
249
11
3.0
No
Depletion
4
4
d
b
19
f
f
1.3
1.5
No
Depletion
3.5
4.5
3.2
9.0
<0.8
8.8
0.86
2.8
4
e
195
a
b
Experiments were performed in duplicate. In vitro metabolism with rat liver
c
microsomes in the presence of the NADPH generating system. Calculated as the
percentage of difference in the rate of CYP3A4-mediated conversion of midazolam to
1
’-hydroxymidazolam between 0 min and 30 min pre-incubation in the presence of a
2
2

d
e
test compound. PAMPA was performed at pH = 6.5 condition. CLogP values
f
calculated by ACD/Percepta. Stable against rat liver microsomal metabolism.
2
3

Second, we attempted to improve membrane permeability by reducing the PSA of
molecules. Since PSA is generally correlated with the number of hydrogen bond donors
1
2
(HBDs) and hydrogen bond acceptors (HBAs),
we modified the tricyclic
imidazopyrrolopyridinone scaffold. Further, given that pyrrolopyridine interacts with
the hinge region of JAK, we attempted to modify the imidazolone unit. Since the
imidazolone unit has one HBD and three HBAs, we speculated that modification of this
unit would significantly reduce PSA. Removal of one HBD by methylation of the NH of
9
imidazolone resulted in a slight decrease in JAK3 inhibitory activity (Table 3, 42 ,
JAK3 IC = 5.0 nM). However, as expected, membrane permeability was improved
5
0
-6
compared to 2 (PAMPA permeability = 2.5 × 10 cm/s), suggesting that decreasing
PSA by reducing HBD counts was effective for improving permeability. Encouraged by
2
this data, we designed the pyrrazole ring analogue 14 (TPSA = 91 Å ), which had only
one HBA on this moiety. Although 14 also had slightly reduced JAK3 inhibitory
activity (JAK3 IC = 4.1 nM), its membrane permeability was improved compared to 2
5
0
-6
(
PAMPA permeability = 5.9 × 10 cm/s). To further remove HBA, we designed the
2
pyrrole ring analogue 18 (TPSA = 78 Å ), which exhibited both increased JAK3 and
JAK1 inhibitory activity and improved membrane permeability compared to 2 (JAK3,
-6
JAK1 IC = 0.44 nM, 1.1 nM, respectively, PAMPA permeability = 13 × 10 cm/s).
5
0
2
4

Table 3. Modification of imidazopyrrolopyridinone scaffold
JAK3
IC50a
JAK2
IC50a
(nM)
JAK1
PAMPA
c,d
b
c
HBDc
HBAc TPSA
Compound
R1
IC50a permeability CLog P
2
-6
counts counts
(Å )
(
nM)
(nM)
1.5
(×10 cm/s)
2
1
5
4
.1
.0
.1
2.6
7.3
11
<0.7
1.0
1.1
2
1
1
1
1
8
8
7
6
7
105
(
(
racemate)
42
29
7.5
1.1
3.7
2.5
5.9
13
96
91
78
89
racemate)
14
1.4
(
(
racemate)
18
0
.44
1.0
3.1
2.0
racemate)
1
0.80
5.0
0.92
a
b
Experiments were performed in duplicate. PAMPA was performed at pH = 6.5
c
condition. CLogP values, HBD counts, HBA counts, and TPSA were calculated by
d
ACD/Percepta. TPSA means topological polar surface area.
2
5

The reduction of PSA by decreasing HBD and HBA counts led to the identification of
dipyrrolopyridine 18 with potent JAK inhibitory activity and improved membrane
permeability. While increasing lipophilicity was required to improve permeability when
modifying the amide linkage of 2, 18 exhibited good membrane permeability without
significant increase in lipophilicity (CLogP = 2.0). Therefore, the dipyrrolopyridine
scaffold 18 demonstrated simultaneously favorable metabolic stability and low risk of
TDI as expected (Table 4, in vitro CL = 110 mL/min/kg, TDI % remaining = 90%).
int
We explored the SAR of the dipyrrolopyridine scaffold and evaluated the metabolic
stability and CYP3A4 TDI. The results are summarized in Table 4.
Given that even the introduction of a methyl group into the tricyclic scaffold (Table 3,
4
2, JAK3 IC = 5.0 nM) led to a slight decrease in potency, we concluded that only
50
substituents that were limited in size could be introduced into the dipyrrolopyridine
scaffold. Introduction of a cyano group at R led to decreased JAK3 inhibitory activity
2
(
Table 4, 25, JAK3 IC = 1.1 nM). Additionally, although introduction of a methyl
50
group at the R position led to increased potency, compound 32 exhibited a potential
3
risk of TDI (TDI % remaining = 44%). Next, we investigated the introduction of
substituents to the R position. Introduction of a cyano group led to the identification of
1
3
7, which exhibited potent JAK3 inhibitory activity (37, JAK3 IC = 0.38 nM) and
50
satisfied our TDI criteria (TDI % remaining = 90%). On the other hand, the methyl
substituted analogue 41 exhibited decreased potency and TDI risk (JAK3 IC = 2.5 nM,
50
TDI % remaining = 62%). Therefore, based on JAK inhibitory activity, metabolic
stability, TDI, and membrane permeability, compounds 18 and 37 were selected for
further profiling.
2
6

Since 18 and 37 were racemates, each enantiomer was prepared. The
3R,4R)-enantiomer 18a showed more potent JAK inhibitory activity than the
(
corresponding (3S,4S)-enantiomer 18b (Table 5, JAK3 IC50 = 0.14 nM, 200 nM,
respectively). Therefore, the (3R,4R)-enantiomer 37a was prepared (JAK3 IC = 0.11
5
0
nM). Compound 18a and 37a showed more potent inhibitory activity compared to 1.
2
7

Table 4. SAR of dipyrrolopyridine scaffold
JAK3
IC50a
JAK2
IC50a
(nM)
JAK1
IC50a
in vitro
CLint
CYP3A4
TDI
PAMPA
b
c
d
e
Compound
R1
R2
R3
permeability CLog P
-6
(
nM)
(nM) (mL/min/kg) % remaining (×10 cm/s)
1
8
H
H
H
CN
H
H
H
0.44
1.1
1.0
2.2
0.3
4.1
4.9
1.1
110
47
90
91
44
90
62
13
3.8
26
13
23
2.0
1.5
2.5
2.2
2.5
(
racemate)
25
f
NT
(
(
(
(
racemate)
32
H
Me
H
0.17
0.38
2.5
0.43
154
419
309
racemate)
37
f
CN
Me
H
NT
racemate)
41
f
H
H
NT
racemate)
a
b
Experiments were performed in duplicate. In vitro metabolism with rat liver
c
microsomes in the presence of the NADPH generating system. Calculated as the
percentage of difference in the rate of CYP3A4-mediated conversion of midazolam to
1
’-hydroxymidazolam between 0 min and 30 min pre-incubation in the presence of a
d
e
test compound. PAMPA was performed at pH = 6.5 condition. CLogP values
f
calculated by ACD/Percepta. NT = not tested.
2
8

Table 5. Profile of 18a, 18b and 37a
JAK3
IC50a
JAK2
IC50a
(nM)
JAK1
IC50a
(nM)
T-cell
IC50
(nM)
in vitro
CLint
CYP3A4
TDI
PAMPA
c
d
e
Compound
permeability
b
-6
(
nM)
(mL/min/kg) % remaining (×10 cm/s)
1
8a
0
.14
0.30
390
1.4
0.46
4.3
138
89
89
88
20
21
14
5.0
(
3R, 4R)
18b
f
f
2
00
NT
NT
(
3S, 4S)
37a
f
0
.11
3.2
3.7
NT
389
63
93
(
3R, 4R)
1
0.80
3.1
23
100
a
c
b
Experiments were performed in duplicate. IL-2-induced rat T-cell proliferation IC .
5
0
In vitro metabolism with rat liver microsomes in the presence of the NADPH
d
generating system. Calculated as the percentage of difference in the rate of
CYP3A4-mediated conversion of midazolam to 1’-hydroxymidazolam between 0 min
e
and 30 min pre-incubation in the presence of a test compound. PAMPA was performed
f
at pH = 6.5 condition. NT = not tested.
2
9