Discovery of a Janus Kinase Inhibitor Bearing a Highly Three-Dimensional Spiro Scaffold: JTE-052 (Delgocitinib) as a New Dermatological Agent to Treat Inflammatory Skin Disorders

Article abstract of DOI:10.1021/acs.jmedchem.0c00450

Dermatologic disorders such as atopic dermatitis arise from genetic and environmental causes and are complex and multifactorial in nature. Among possible risk factors, aberrant immunological reactions are one of the leading etiologies. Immunosuppressive agents including topical steroids are common treatments for these disorders. Despite their reliability in clinical settings, topical steroids display side effects, typified by skin thinning. Accordingly, there is a need for alternate effective and well-tolerated therapies. As part of our efforts to investigate new immunomodulators, we have developed a series of JAK inhibitors, which incorporate novel three-dimensional spiro motifs and unexpectedly possess both excellent physicochemical properties and antidermatitis efficacy in the animal models. One of these compounds, JTE-052 (ent-60), also known as delgocitinib, has been shown to be effective and well-tolerated in human clinical trials and has recently been approved in Japan for the treatment of atopic dermatitis as the first drug among Janus kinase inhibitors.

Full text of DOI:10.1021/acs.jmedchem.0c00450

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Article
Discovery of a Janus Kinase Inhibitor Bearing a Highly Three-
Dimensional Spiro Scaffold: JTE-052 (Delgocitinib) as a New
Dermatological Agent to Treat Inflammatory Skin Disorders
Satoru Noji, Yoshinori Hara, Tomoya Miura, Hiroshi Yamanaka, Katsuya Maeda,
Akimi Hori, Hiroshi Yamamoto, Shingo Obika, Masafumi Inoue, Yasunori Hase,
Takuya Orita, Satoki Doi, Tsuyoshi Adachi, Atsuo Tanimoto, Chika Oki, Yukari Kimoto,
Yoshihiro Ogawa, Tamotsu Negoro, Hiromasa Hashimoto, and Makoto Shiozaki
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00450 • Publication Date (Web): 08 Jun 2020
Downloaded from pubs.acs.org on June 9, 2020
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Discovery of a Janus Kinase Inhibitor Bearing a
Highly Three-Dimensional Spiro Scaffold: JTE-
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52 (Delgocitinib) as a New Dermatological Agent
to Treat Inflammatory Skin Disorders
,
†
†
†
†
Satoru Noji,* Yoshinori Hara, Tomoya Miura, Hiroshi Yamanaka, Katsuya
†
†
†
†
†
Maeda, Akimi Hori, Hiroshi Yamamoto, Shingo Obika, Masafumi Inoue, Yasunori
†
†
†
†
§
§
Hase, Takuya Orita, Satoki Doi, Tsuyoshi Adachi, Atsuo Tanimoto, Chika Oki,
§
§
¶
†
Yukari Kimoto, Yoshihiro Ogawa, Tamotsu Negoro, Hiromasa Hashimoto, and
Makoto Shiozaki*,†
†
§
Chemical Research Laboratories, Biological Pharmacological Research
¶
Laboratories, Drug Metabolism & Pharmacokinetics Research Laboratories, Central
Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1, Murasaki-cho,
Takatsuki Osaka, 569-1125, Japan.
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KEYWORDS. Janus kinase, SAR, drug-likeness, ligand efficiency, fraction of sp , JAK
inhibitor, spiro scaffold, JTE-052.
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ABSTRACT
Dermatologic disorders such as atopic dermatitis arise from genetic and
environmental causes, and are complex and multi-factorial in nature. Among possible
risk factors, aberrant immunological reactions are one of the leading etiologies.
Immunosuppressive agents including topical steroids are common treatments for
these disorders. Despite their reliability in clinical settings, topical steroids display side
effects, typified by skin thinning. Accordingly, there is a need for alternate effective
and well-tolerated therapies. As part of our efforts to investigate new
immunomodulators, we have developed a series of JAK inhibitors, which incorporate
novel three-dimensional spiro motifs and unexpectedly possess both excellent
physicochemical properties and anti-dermatitis efficacy in the animal models. One of
these compounds, JTE-052 (ent-60), also known as delgocitinib, has been shown to
be effective and well-tolerated in human clinical trials, and has recently been approved
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in Japan for the treatment of atopic dermatitis as the first drug among Janus kinase
inhibitors.
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INTRODUCTION
Inflammatory skin disorders are some of the most common forms of
dermatological disease. Inflammatory skin disorders are characterized by epidermal
1
hyperplasia along with abnormal keratinocyte differentiation. These disorders
encompass a wide range of pathological conditions such as eczema, rosacea, atopic
dermatitis (AD), contact dermatitis, pruritus, psoriasis (PS), which afflict roughly 6.1
million patients and impose medical costs of $3 billion annually in the United States
alone.2
Many of these inflammatory skin disorders are triggered by aberrant immune
responses within cells on the surface of the skin, in which cytokine and lymphokine
expression is excessively up-regulated.3,4 Recently, it has become clear that
keratinocytes play a critical role in producing those immune mediators, causing them
to spread throughout the entire epidermis.5,6 A primary function of epidermal
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keratinocytes is providing a first-line defense against microorganisms and/or other
environmental threats; keratinocytes are alerted and activated once diverse stimuli
such as pathogens, ultraviolet (UV) light and allergens attack the skin barrier. Despite
the diverse and complex etiology of these disorders, topical steroids, which are
typically known as immunosuppressive agents, have been used broadly to treat
them.7–9 While the efficacy of topical steroids has been confirmed in the clinic, their
side effects, such as skin thinning,10,11 suggest a need for alternative effective and
well-tolerated therapies.
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Following the appearance of work by Darnell et al,12 Janus kinase (JAK)
inhibition has come to be regarded as a potential pathway for the treatment of certain
inflammatory and autoimmune diseases.13–15 The JAK family of proteins (JAK1, JAK2,
JAK3, and Tyk2) possess two kinase domains, a genuine kinase domain and a
pseudo-kinase domain having no catalytic activity; hence these proteins are named
after the two-faced Roman god Janus. Among the JAK family, critical mutations in
JAK3 have been found to lead to severe combined immune deficiency (SCID).16
Several research groups have investigated JAK inhibition, including Pfizer.17–20
21
In 2006, Pfizer published the results of a Phase II clinical study on the compound
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CP-690,550, also known as tofacitinib (1) (which was originally reported to be a JAK3
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inhibitor, but later was reported to be a pan-JAK inhibitor ). In this study, 264 patients
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with active rheumatoid arthritis (RA) were randomized and subsequently received
placebo or three different doses of CP-690,550. CP-690,550 achieved both primary
and secondary endpoints in this study, with minor adverse events such as headache
and nausea.21
Me
Me
N
N
N
N
N
O
N
H
1
Figure 1. Structure of CP-690,550 (tofacitinib).
Structurally, tofacitinib may be regarded as comprising two motifs: a
heteroaromatic ring, which adopts a relatively “flat” conformation, and an
azacycloalkane group, which, by comparison, displays a relatively greater degree of
“three dimensionality”. Prior to our work, little had been published about the structure-
activity relationships of JAK inhibitors such as tofacitinib. Thus, in our efforts to develop
a new series JAK3 inhibitor compounds, we investigated numerous modifications to
both these motifs in an attempt to elucidate the SAR of JAK inhibitors.
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RESULTS AND DISCUSSIONS
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Exploration of the hinge-binding motif
It is currently known that several compounds having ATP-competitive kinase
inhibitory activity are typically composed of two units: an adenine mimetic (a so-called
“hinge binder”) and an accompanying “scaffold” or “head” region. Tofacitinib falls in
this category: its pyrrolopyrimidine ring functions as a hinge binder, effecting
hydrogen-bonding with the Glu903 and Leu905 residues of the JAK3 protein.22
As part of our work, we synthesized and tested multiple compounds with
alternative hinge-binding motifs. For these efforts, compound 2 (shown in Table 1)
served as our reference compound, and several analogs thereof were synthesized in
an attempt to improve upon compound 2’s JAK3 inhibitory properties. Additionally, to
assess the tendency of these compounds to bind off-target, we also measured the
inhibitory effects of these compounds against another kinase: lymphocyte-specific
protein tyrosine kinase (LCK).17 Some of these compounds are shown in Table 1
below.
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Surprisingly, most of these analogs proved to be largely ineffective against
JAK3. From this, we surmise that the binding domain of JAK3 demonstrates a
preference for pyrrolopyrimidine. We also note that neither the compounds containing
monodentate aromatics (3 and 4) nor aliphatic heterocycles (11–13) show appreciable
JAK3 inhibition in vitro, and that the inclusion of certain steric or electrostatic features
is rarely tolerated (Me, c-Pr, and 2-Py for 7, 8, and 9, respectively).
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Table1. SAR exploration of the hinge binder starting from compound 2.a
Me
N
R
IC50 (µM)
IC50 (µM)
ID
R
ID
R
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LCKe
JAK3d
LCKe
2
3
N
N
0.079
>10
>10
3.0
>10
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N
N
>10
1.8
3.3
N
H
N
N
N
N
N
H
N
>10
>10
>10
>10
10
11c
>10
>10
>10
>10
N
H
4b
5
>10
>10
>10
O
N
N
H
O
1
2b,
H2N
N
N
c
NH
N
H
N
O
O
13c
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>10
N
N
Me
H
O
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>10
>10
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>10
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H
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a
See Experimental Section for assay protocols. The IC values are the average of at
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c
d
least two determinations. HCl salt form. Racemate. Reference compound 1 gave
e
mean (± SD) IC50 = 0.0024 (± 0.00069) µM, n = 48. Reference compound 1 gave
mean (± SD) IC = 0.34 (± 0.064) µM, n = 36.
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Modification of substituents on the hinge binder of compound 10
Based upon our experiments in modifying the hinge binding motif, compound
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0, bearing a phenylamino-pyrimidine substituent in the hinge binding region, was
selected for further investigation and modification due to the fact that its JAK3
inhibitory activity and its selectivity for JAK3 over LCK were comparable to those of
compound 2. As further modifications, we investigated a series of hydrophilic
substitutions at the para position of the aniline ring (Table 2, R2).
Several of these compounds achieved JAK3 inhibition at sub-micromolar
concentrations, and also inhibited T cell proliferation at similar concentrations (14, 16,
and 17). Compound 15, however, showed no efficacy against T cell proliferation
despite its JAK3 inhibitory activity. We believe that this lack of activity against T cell
may be attributable to compound 15’s poor physical properties (e.g. its low solubility),
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a belief that is supported by compound 15’s inability to inhibit the growth of normal
human lung fibroblasts (NHLF)—an assay that we used to measure the off-target
toxicity of our compounds against cells other than T cells.
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Additional modifications were undertaken at the R position of the pyrimidine
ring of compound 10 to explore their potential effects on JAK3 potency and specificity.
For these experiments, compound 16 served as our reference compound. Larger
1
substitutions at R generally resulted in decreasing JAK3 potency, with the methyl
group showing better JAK3 potency than the ethyl or trifluoromethyl group (18–20).
However, we found the superior LCK selectivity of the trifluoromethyl-bearing
compound 20 to be noteworthy.
Table 2. Substitution effects on the hinge binder.a
Me
N
R1
R2
N
N
N
H
Hinge Binder
IC50 (µM)
T cell (IL-2)d
ID
R1
R2
JAK3b
LCKc
NHLFe
H
H
10
1.8
>10
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H
OH
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8
0.41
0.073
0.11
4.2
0.55
0.46
2.0
0.78
>10
0.48
0.37
0.22
0.92
1.4
4.6
>10
O
H
H
OH
O
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1.1
NH2
Me
O
H
N
0.21
<1.0(65%)
2.0
Me
O
Me
0.044
0.79
0.34
0.40
>10
NH2
NH2
O
O
19
20
3.0
Me
F
F
0.077
2.2
NH2
F
a
See Experimental Section for assay protocols. The IC values are the average of at
50
b
least two determinations. Reference compound 1 gave mean (± SD) IC = 0.0024 (±
50
c
0.00069) µM, n = 48. Reference compound 1 gave mean (± SD) IC = 0.34 (± 0.064)
50
d
µM, n = 36. Reference compound 1 gave mean (± SD) IC = 0.016 (± 0.087) µM, n
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e
=
34. Reference compound 1 gave IC = > 10 µM, n = 29.
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In an attempt to improve the properties of compound 20 modifications to the
“
head” region were explored. Specifically, several aliphatic heterocycles were
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introduced at position R . Some of these are shown in Table 3. As shown in Table 3,
there was virtually no difference in JAK3 inhibition between compounds 21 and 22.
The effect of substituents on the aliphatic ring were also examined. A 2-methylated
analogue (25) showed better JAK3 potency than a 3-methylated isomer (23).
Unfortunately, the selectivity of compound 25 to JAK3 over LCK was significantly
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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5
6
reduced. More disappointingly, a strong cytotoxic effect was observed for compound
25 in NHLF assay, with that compound inhibiting the proliferation of NHLF cells at a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
concentration of 1 M. Compound 23, as well as compound 24 (bearing a 3-hydroxyl
substituent) likewise inhibited NHLF growth without any improvement in JAK3
inhibitory activity. In view of these results, we decided to discontinue our further
exploration of compounds which, like compound 10, incorporated a phenylamino-
pyrimidine moiety in the hinge binding region.
Table 3. Head motif exploration of 20.a
Head Motif
R3
F
O
F
F
N
NH2
N
N
H
IC50 (µM)
ID
R3
N
JAK3c
LCKd
T cell (IL-2)e
NHLFf
21
0.11
>10
2.9
1.3
2
2
3
0.12
>10
>10
5.9
2.1
0.45
1.3
1.5
1.4
1.8
N
Me
2
b
0.061
N
N
OH
24
0.053
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25
b
<1.0
Me
0.018
0.22
0.27
N
(
96%)
a
See Experimental Section for assay protocols. The IC values are the average of at
50
b
c
least two determinations. Racemate. Reference compound 1 gave mean (± SD) IC
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d
= 0.0024 (± 0.00069) µM, n = 48. Reference compound 1 gave mean (± SD) IC =
50
e
0
.34 (± 0.064) µM, n = 36. Reference compound 1 gave mean (± SD) IC = 0.016 (±
50
f
0
.0087) µM, n = 34. Reference compound 1 gave IC = > 10µM, n = 29.
50
Exploration of the head motif
In parallel with our investigations of the hinge binding region, we also
investigated modifications to the head region, using compound 2 as a reference
compound (Table 4). Rather than limit our investigation to relatively “flat” aromatic
moieties, a decision was made also to investigate moieties having relatively greater
degrees of “three dimensionality”. With this consideration in mind, we synthesized and
tested numerous compounds, including the three cyclic amines (30–32) and the other
“
head” motifs (26–29) summarized in Table 4. These
compounds
showed
acceptable levels of JAK3 potency and selectivity over LCK. Additionally, we note that
the ligand efficiencies (LEs) of compounds 30 and 31 exceed that of compound 2 (0.64,
0
.62 vs 0.57). Ligand efficiency (LE) is a theoretical concept that was introduced by
Kuntz in 199923 and has been characterized as a one potential measure of a
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound’s “drug-likeness”.24–26 The LE values of 30 and 31 are superior to those of
26–29. In view of the promising JAK3, LCK, and LE values for compounds 30 and 31,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
we decided to investigate those compounds further.
Table 4. SAR of the head
R
Head Motif
N
motif
comprised
of
N
N
H
pyrroropyrimidine.a
IC50 (µM)
IC50 (µM)
ID
R
ID
R
JAK3d
LCKe
LEf
JAK3d
LCKe
LEf
2
Me
0.079 >10
0.57
0.48
0.43
0.45
29c
30
31
32
0.33
>10
0.55
0.64
0.62
0.58
N
O
26
1.1
>10
>10
>10
0.26
0.16
0.42
>10
>10
>10
N
2
7b,
Me
3.7
c
N
O
N
28
O
2.7
a
See Experimental Section for assay protocols. The IC values are the average of at
50
b
c
d
least two determinations. Racemate. HCl salt form. Reference compound 1 gave
e
mean (± SD) IC50 = 0.0024 (± 0.00069) µM, n = 48. Reference compound 1 gave
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f
mean (± SD) IC = 0.34 (± 0.064) µM, n = 36. LE = −1.37 log IC (JAK3) / number
50
50
of heavy atoms.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As shown in Table 5, methyl scanning27 of compounds 30 and 31 was
conducted. 2- or 3-methylation of the cyclic amines led to a slight improvement of JAK3
potency (33, 34, 36, and 37), whereas the 4-methylpieridine derivative 35 showed a
significant drop in potency. The inclusion of a cyanoacetamide group at the 2- or the
3
-position also was examined, with the nitrogen atom therein masked by an additional
methyl group (38 and 39). Interestingly, compound 38, bearing a five-membered ring,
boosted T cell inhibitory activity whereas compound 39, bearing a six membered ring,
did not show significant T cell inhibitory activity.
In view of these unexpected results, we continued our investigations into the
effects that structural changes to cyclic amino head groups might have on T cell
inhibitory activity. Our research contrasts with the work of Pfizer, who in 2010 reported
that their JAK inhibitor compounds bearing cyclic amino head motifs were less potent
than compounds having an N-methyl-cycloalkyl head motif.18
Compound 40 is an analogue of 38, with an extra carbon present between the
ring and the cyanoacetimide substituent. However, compound 40 exhibited no ability
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
to inhibit T cell proliferation. These differences in T cell inhibitory activity between 38–
40 were surprising in view of the general level of structural similarity among these
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds and their comparable JAK3 inhibitory activity.
In an effort to examine the potential effects of conformational freedom vs.
conformational constraints within the head region, a series of fused bicyclic
compounds was synthesized. Of these, only compound 42 achieved T cell inhibitory
activity approaching that of compound 38 while retaining a comparable degree of JAK3
inhibition (IC = 0.20 M). The JAK3 potencies of the fused bicyclic compounds 41
50
and 43 were significantly reduced, with a concomitant loss of T cell potency.
As part of our exploration of conformationally constrained groups, we also
synthesized a series of compounds possessing spiro rings. Some of these compounds
are shown in Table 5. As demonstrated by compounds 44 and 45, JAK3 inhibitory
activity improved as the size of the terminal ring became larger, though these
improvements did not necessarily translate to improved T cell inhibitory activities.
Additionally, reduced in vitro potencies were observed for compound 46, in which the
cyanoacetyl moiety is located at the gamma position toward the nitrogen atom of the
inner pyrrolidine ring; these reduced potencies for compound 46 suggested that the
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relative position of the cyanoacetyl moiety within the head region could have important
effects on activity. Collectively, these results suggested to us that there might be some
appropriate combination of ring sizes within a spiro group that might achieve
acceptable levels of JAK3 inhibition. In view of those results, we conducted further
experiments on compounds that included a piperidine ring as part of the spiro group,
while varying the size of the terminal ring. Some of these spiro compounds are shown
in Table 5. Among compounds 47–49, compound 47 exhibited the best inhibitory
activity against both JAK3 and T cell proliferation.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. SAR of the head motif focused on cyclic amines.a
R
Head Motif
N
N
N
H
IC50 (µM)
Fsp3f
ID
R
JAK3b
LCKc
T cell (IL-2)d
NHLFe
Me
33
0.094
>10
3.0
>10
0.46
N
Me
34
N
0.082
>1.0
>10
5.5
3.5
>10
>10
0.46
0.50
Me
N
35
>10
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Me
36
0.13
0.14
0.40
0.30
0.66
1.2
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.1
2.4
2.4
>10
>10
>10
>10
>10
>10
>10
>10
>10
3.9
0.50
0.50
0.43
0.50
0.47
0.47
0.50
0.47
0.50
0.53
0.50
0.53
0.56
N
37
38
39
N
Me
R'
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Me
N
0.15
1.7
N
Me
N
R'
N
Me
N
R'
40
>10
>10
0.22
>10
0.15
0.25
1.8
N
R'
N
41
42
43
N
N
R'
0.20
2.7
N
R'
N
N
N
44
R'
R'
0.088
0.048
0.64
0.036
0.041
N
N
45
46
47
N
R'
N
>10
6.3
>10
>10
3.9
N
N
R'
0.048
0.086
N
N
N
48
0.63
R'
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9
49
N
0.18
5.0
2.2
3.4
0.58
N
R'
aAll compounds are racemates. R’ = cyanoacetyl. See Experimental Section for assay
b
protocols. The IC values are the average of at least two determinations. Reference
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
c
compound 1 gave mean (± SD) IC50 = 0.0024 (± 0.00069) µM, n = 48. Reference
d
compound 1 gave mean (± SD) IC50 = 0.34 (± 0.064) µM, n = 36. Reference
e
compound 1 gave mean (± SD) IC50 = 0.016 (± 0.0087) µM, n = 34. Reference
f
3
3
compound 1 gave IC50 = > 10 µM, n = 29. Fsp = number of sp hybridized
carbons/total carbon count.28
In an attempt to improve upon the activity of compound 47, we adopted a two-
pronged approach, on the one hand synthesizing compounds incorporating other spiro
rings, and on the other hand investigating other acyl substituents. Our efforts to
synthesize other spiro rings created significant synthetic challenges for our chemists.
Specifically, the literature at the time was devoid of reliable methods for making a
diazaspirocycle incorporating an azetidine ring. We therefore had to develop a reliable
method by ourselves, for which our extensive efforts are described later in this
publication. In the meantime, our investigations into other acyl substituents yielded
results such as those shown in Table 6. Compounds 50–53 showed reduced cell
potency compared to the cyanoacetyl substituent of compound 47.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 6. SAR of the acyl group on the head group.a
N
R
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
H
IC50 (µM)
ID
R
JAK3b
LCKc
T cell (IL-2)d
NHLFe
*
*
4
7
0
1
2
3
0.036
6.3
0.048
>10
N
O
O
Me
N.Tf
N.Tf
5
5
5
5
0.25
0.067
0.17
>10
5.8
*
*
*
OH
0.19
>10
O
O
O
N.T.f
N.T.f
OMe
>10
H
N
N
0.010
4.5
0.22
>10
aAll compounds are racemates. See Experimental Section for assay protocols. The
b
IC values are the average of at least two determinations. Reference compound 1
50
c
gave mean (± SD) IC50 = 0.0024 (± 0.00069) µM, n = 48. Reference compound 1
d
gave mean (± SD) IC = 0.34 (± 0.064) µM, n = 36. Reference compound 1 gave
50
e
mean (± SD) IC = 0.016 (± 0.0087) µM, n = 34. Reference compound 1 gave IC =
50
50
f
>
10 µM, n = 29. Not tested.
Investigation of diazaspiroalkane-based JAK inhibitors
Compound 54 was synthesized according to a newly-developed method. This
compound, incorporating an azetidine ring as part of its spiro group, demonstrated
good T cell inhibitory activity (Table 7). Encouraged by this result, we made further
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modifications to this compound. The results are included in Table 7. Among four
methylated analogs (55–58), compound 57, bearing a methyl adjacent to the
quaternary carbon, displayed improved JAK3 potency. The gem-dimethyl analogue 58
displayed comparatively less JAK3 potency, though its difluoro analogue 59 achieved
JAK3 and T cell results comparable to those of 57. Compound 60 and 61, pyrrolidine
analogs of 57 and 59, respectively, displayed activity against T cell proliferation
roughly comparable to that of compounds 57 and 59.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 7. SAR of azetidine-associated diazaspirocycle derivatives.a
n
R
m
R'
N
N
O
N
N
N
H
N
IC50 (µM)
T cell (IL-2)f
ID
Structure
Fsp3h
JAK3d
LCKe
NHLFg
N
5
4
R'
0.038
3.7
0.051
>10
0.50
N
N
N
N
5b
N.T.i
N.T.i
5
R'
R'
0.41
8.6
5.2
0.53
0.53
Me
Me
56b
0.058
0.10
>10
N
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Me
N
7b,
c
5
N
0
.0070
1.5
>10
2.6
0.032
2.0
>10
>10
>10
>10
>10
0.53
0.56
0.48
0.50
0.47
R'
Me
Me
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
58
0.13
R'
N
F
F
N
N
59
0.0064
0.021
0.011
0.048
0.039
0.055
R'
R'
N
Me
6
0b,
>10
4.7
c
N
F
F
N
61
R'
N
aAll compounds are racemates. R’ = cyanoacetyl. See Experimental Section for assay
b
protocols. The IC50 values are the average of at least two determinations. Single
diastereomer (the absolute stereochemistry was not defined.) cBoth of the
diastereomers were obtained. Data for the more potent compound in terms of JAK3
d
inhibitory activity is shown. Reference compound 1 gave mean (± SD) IC = 0.0024
50
e
(± 0.00069) µM, n = 48. Reference compound 1 gave mean (± SD) IC = 0.34 (±
5
0
f
0
.064) µM, n = 36. Reference compound 1 gave mean (± SD) IC = 0.016 (± 0.0087)
50
g
h
3
3
µM, n = 34. Reference compound 1 gave IC > 10 µM, n = 29. Fsp = number of sp
50
28 i
hybridized carbons/total carbon count. Not tested.
The spiro-based compounds listed in Table 8 were isolated in enantiomerically
pure form and characterized in pharmacokinetic assays. Although these compounds
displayed comparable IC50 values in in vitro JAK3 inhibitory and T cell proliferation
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assays, meaningful differences appeared among them in assessing their
pharmacokinetic profiles.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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9
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Generally, compounds incorporating a piperidine ring (ent-47–ent-59) were
discovered to be more readily metabolized, while the pyrrolidine analogues, ent-60
and ent-61, were metabolically more stable across the tested species.
Among the two pyrrolidine analogs, ent-60 exhibited both greater potency
against T cells and a lower clearance rate in monkeys than ent-61, thereby suggesting
that ent-60 might be more potent and display longer-lasting effects in an in vivo setting.
Additionally, it was decided that the synthesis of ent-61 imposed cost ineffectiveness
that could be avoided with ent-60. Accordingly, ent-60 was selected as a candidate for
clinical development, and was renamed JTE-052.
Table 8. Representative JAK inhibitors possessing a spirodiazacycloalkane.a
IC50 (µM)
T cell (IL-2)c
MS (%, 60min)
CL monkeyd
(L/hr/kg)
N.T.e
ID
JAK3b
human
monkey
ent-47
ent-54
ent-57
ent-59
0.011
0.041
0.017
0.011
0.019
75.7
93.4
73.4
88.4
29.4
0.019
0.0040
0.0060
85.2
25.1
64.6
0.76
N.T.e
N.T.e
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1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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3
4
4
4
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4
4
4
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5
5
5
5
5
5
5
6
ent-60
0.013
0.011
0.0089
0.033
97.4
99.4
99.6
95.4
0.29
0.35
(
JTE-052)
ent-61
a
See Experimental Section for assay protocols. The IC values are the average of at
50
0
1
2
3
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9
0
1
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b
least two determinations. Reference compound 1 gave mean (± SD) IC = 0.0024 (±
5
0
c
0.00069) µM, n = 48. Reference compound 1 gave mean (± SD) IC = 0.016 (±
5
0
d
e
0
.0087) µM, n = 34. Administered intravenously at 0.1 mg/kg. Not tested.
In order to clarify the mechanism of high potency of JTE-052 against JAK3, X-
ray co-crystal analysis was performed with compound 60. Only one of the enantiomers
gave a good crystal with a high resolution of 1.98 Å, suggesting that this enantiomer
29
(
ent-60) was an active isomer (Figure 2). According to the structure, the cyanoacetyl
group made orthogonal dipolar interactions with main chain atoms (Gly829–Lys830
and Gly834–Ser835), while the azetidine motif occupied the lipophilic pocket
neighborhood the JAK3 hinge region with the 7H-pyrrolo[2,3-d]pyrimidine ring making
hydrogen bond interactions with JAK3 hinge region of Glu903 (2.6 Å) and Leu905 (2.9
Å). It was thus presumed that the potent JAK3 activity of JTE-052 was attributed to
those interactions.
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0
Figure 2. X-ray crystallographic structure of JAK3 (human) in complex with JTE-052
focusing on binding site (PDB code 7C3N). Hydrogen bonds are depicted as dashed
lines (yellow). Surface are showed around JTE-052 as meshed lines.
As a part of our further characterizations, the selectivity of JTE-052 for JAK3
was investigated in comparison with the other members of the JAK family and a panel
of 50 other human kinases. As shown in Table 9, JTE-052 was found to exhibit
inhibitory activity across all JAK family members (JAK3, JAK1, JAK2 and Tyk2) and
inhibited JAK1 and JAK2 at nanomolar concentrations. As can be seen from the data in
Table 9, the inhibitory activity of JTE-052 across the JAK family differs from that of
tofacitinib; interestingly, JTE-052 exhibits relatively greater inhibitory activity against JAK1
and JAK2 than JAK3 compared to tofacitinib, and its selectivity for the JAK family over LCK
is improved in comparison with tofacitinib. JTE-052 also proved to be highly selective for
the JAK family members, with no significant inhibition of non-JAK kinases under 1 M
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0
except over ROCK-II. Surprisingly, the potency of JTE-052 in whole-cell assays is
not linearly correlated with its potency against JAK kinases, as was sometimes
0
1
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0
31
observed in other kinase inhibitor programs. For example, we expected that the IC
50
values of JTE-052 against IL-6/GM-CSF signaling would be correlated with JTE-052’s
activity against each JAK isozyme. However, the results in Table 9 below show that
JTE-052 suppressed JAK1-dependent signaling at an IC value tenfold below the IC
50
50
value required to suppress JAK2-dependent signaling (IL-31/GM-CSF). The exact
mechanism of such discrepancy between the enzymatic inhibition and the cell potency
is not fully understood; it seemed that potency against JAK1 enzyme predominantly
accounted for the results of cytokine signaling inhibitions. This suggestion is supported,
for example, by the reported K value for JAK1, which is about ten times that of JAK3,
m
making JAK1 easier to inhibit than JAK3 at the same ATP concentration.32,33 Consequently, it
is likely that much of the efficacy observed in the in vivo models is driven by JAK1.
Regardless, JTE-052’s effective suppression of IL-2 and IL-31 signaling pathways
further bolstered its potential as a clinical candidate.
Table 9. In vitro profiles of JTE-052.a
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Enzymes IC50 (µM)
ID
JAK3
JAK1
JAK2
TyK2
LCK
JTE-052
0.013
0.0028
0.0026
0.058
5.8
Tofacitini
b
0.0011
0.0029
0.0012
0.042
0.44
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cytokine signaling IC50 (µM) in human PBMCs
IL-2b
IL-6c
IL-31d
IL-23e
GM-CSFf
INF-g
ID
JAK1/JAK3 JAK1/JAK2 JAK1/JAK2 JAK2/Tyk2 JAK2/JAK2 JAK1/Tyk2
JTE-052
0.040
0.033
0.025
0.084
0.30
0.018
a
b
The IC values are the average of at least two determinations. The phosphorylation
5
0
+
+
c
levels of Stat5 in CD3 CD4 cells stimulated with IL-2 were measured. The
-
+
phosphorylation levels of Stat3 in CD3 CD4 cells stimulated with IL-6 were measured.
dThe phosphorylation levels of Stat3 in A549 cells stimulated with IL31 were measured.
e
+
+
The phosphorylation levels of Stat3 in CD3 CD4 cells stimulated with IL-23 were
f
-
+
measured. The phosphorylation levels of Stat5 in CD3 CD4 cells stimulated with GM-
g
+
+
CSF were measured. The phosphorylation levels of Stat1 in CD3 CD4 cells
stimulated with INF- were measured.
Pharmacokinetic properties of JTE-052 were characterized (Table 10). In these
tests, the solubility of JTE-052 proved acceptable in several media, and protein binding
was low in the tested species.
Table 10. Physicochemical properties and PK profiles of JTE-052.
LogD
Caco2
Solubility (µM)
Protein Binding (%)
(
pH
Pap
Sol
µM
monke
y
PBS Fassif Fessif
human
rat
dog
-8
1
0cm
7)
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5
5
5
5
5
6
0
.6
3.3 1.0
>95 >475 >475
40.7
37.2
27.1
26.6
JTE-052 was also metabolically stable in both liver microsomes and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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9
0
1
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hepatocytes, with stability values in human cells greater than those in rat and monkey
cells (Table 11). The in vivo PK profiles of JTE-052 were also satisfactory showing
over 50% of bioavailability across the species (Table 12).
Table 11. Metabolic stability of JTE-052.
Metabolized stability (60min, %)
human
rat
dog
monkey
Microsom
9
7.4
9.3
90.2
97.2
99.6
e
Hepatocyt
e
N.T.a
9
94.7
84.8
aNot tested.
Table 12. In vivo PK profiles of JTE-052.
iv PK parametersa
po PK parametersb
dose
t1/2
Cltot
Vdss MRT
dose
tmax
(h)
Cmax
F
speci (mg/kg
(h)
(L/h/kg) (L/kg)
(h)
(mg/kg
(ng/mL)
(%)
rat
1.0
0.3
1.7
2.3
2.1
2.1
1.2
1.0
10
1.5
0.9
2.5
1.9
78
dog
0.37
3.2
1.0
124
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a
b
iv administration at corresponding dose in 10% DMSO. po administraion at
corresponding dose in 0.5% MC.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To explore the in vivo pharmacological effects of JTE-052, a rat adjuvant-
induced arthritis (AIA) model was chosen based on the robustness and the
reproducibility of this animal model.34,35 As shown in Figure 3a, dose-dependent
suppression of paw swelling was observed; complete suppression of paw swelling was
observed at a dose of 30 mg/kg of JTE-052 upon oral administration. The anti-
dermatitis effects of JTE-052 were confirmed in a rat 2,4-dinitrochlorobenzene
(
DNCB)-induced dermatitis model36,37, in this model, complete suppression of ear
swelling was achieved at an oral dose of 10 mg/kg (Figure 3b). Additionally, topical
formulations of JTE-052 for AD treatment were examined in the rat DNCB model
(Figure 3c). Treatment using 1% JTE-052 in a petroleum-based ointment produced
significant suppression of ear swelling; three-times-a-day administration of the JTE-
052 ointment almost completely suppressed ear swelling in this model.
(a)
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