A phenotypic drug discovery study on thienodiazepine derivatives as inhibitors of T cell proliferation induced by CD28 co-stimulation leads to the discovery of a first bromodomain inhibitor

Article abstract of DOI:10.1016/j.bmcl.2016.01.084

A phenotypic screening of thienodiazepines derived from a hit compound found through a binding assay targeting co-stimulatory molecules on T cells and antigen presenting cells successfully led to the discovery of a thienotriazolodiazepine compound (7f) po

Full text of DOI:10.1016/j.bmcl.2016.01.084

Bioorganic & Medicinal Chemistry Letters 26 (2016) 1365–1370
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A phenotypic drug discovery study on thienodiazepine derivatives as
inhibitors of T cell proliferation induced by CD28 co-stimulation
leads to the discovery of a first bromodomain inhibitor
Junichi Endo, Hidemasa Hikawa, Maiko Hamada, Seigo Ishibuchi, Naoto Fujie, Naoki Sugiyama,
Minoru Tanaka, Haruhito Kobayashi, Kunio Sugahara, Koichi Oshita, Kazunori Iwata, Shinsuke Ooike,
⇑
Meguru Murata, Hiroshi Sumichika, Kenji Chiba, Kunitomo Adachi
Research Unit B, Research Division, Mitsubishi Tanabe Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A phenotypic screening of thienodiazepines derived from a hit compound found through a binding assay
targeting co-stimulatory molecules on T cells and antigen presenting cells successfully led to the
discovery of a thienotriazolodiazepine compound (7f) possessing potent immunosuppressive activity.
A chemical biology approach has succeeded in revealing that 7f is a first inhibitor of epigenetic
bromodomain-containing proteins. 7f is expected to become an anti-cancer agent as well as an
immunosuppressive agent.
Received 30 September 2015
Revised 9 December 2015
Accepted 30 January 2016
Available online 2 February 2016
Keywords:
CD28
Ó 2016 Elsevier Ltd. All rights reserved.
Phenotypic drug discovery
Chemical biology
BET family
Bromodomain
Immunosuppressant
Anti-cancer
BET family proteins (bromodomain-containing protein 2, 3, 4
and T) bind to acetyl-lysine residues in the tail of histones H3
and H4.1 Bromodomain-containing protein 4 (BRD4) has been
reported to relate to regulation of epigenetic expression of genes,
cell proliferation, and cell division.2–5 BET inhibitors have been
expected to provide new therapies for cancer and a selective BET
inhibitor JQ1 has been reported to show antitumour efficacy in sev-
eral cancers including the nuclear protein of the testis (NUT) mid-
line carcinoma and multiple myeloma in animals.6,7 Since the early
2000s we have been searching for novel immunosuppressants
showing immunological tolerance focusing on a co-stimulatory
molecule on T cells based on a phenotypic drug discovery strategy,
At the beginning of 2000s, we were interested in a co-stimula-
tory molecule, cluster of differentiation 28 (CD28) because the
molecule had been elucidated to be involved in immunological tol-
erance and expected to be a key to development of low-molecular
weight immunosuppressants for the treatment of various
immunological disorders including rheumatoid arthritis.9 A high
throughput screening focused on binding between B7 proteins on
antigen presenting cells and CD28 or CD152 (CTLA-4) on T cells
resulted in the discovery of thienodiazepine compound 1 as one
of the hit compounds. We immediately executed both in vitro (a
cell-based assay using mixed lymphocyte reaction10) and in vivo
(a mouse collagen-induced arthritis (CIA) model11) evaluation of
1. Interestingly, the compound showed considerably strong thera-
peutic effects in the CIA model, suggesting that 1 would possess an
unidentified mechanism of action in addition to the inhibition of
the binding between B7 and CD28 because the inhibitory activity
in vitro was weak and we could not explain its in vivo effects. This
encouraged us to optimize the compound using a phenotypic assay
but not the binding assay. We constructed a cell-based assay sys-
tem for evaluating the inhibition of T cell proliferation induced
by CD28 co-stimulation and started a drug discovery study using
this assay system.12 It is well known that thienodiazepines have
obtaining
a potent immunosuppressive thienodiazepine com-
pound.8 A chemical biology approach has successfully elucidated
that the compound is a first small molecule inhibiting the pro-
tein–protein interaction between histones and BRDs. We herein
describe the discovery of novel thienodiazepines and their mode
of action (MOA).
⇑
Corresponding author. Tel.: +81 045 963 7235; fax: +81 045 963 7257.
E-mail address: Adachi.Kunitomo@mc.mt-pharma.co.jp (K. Adachi).
http://dx.doi.org/10.1016/j.bmcl.2016.01.084
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

1366
J. Endo et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1365–1370
Me
Cl
Cl
Me
N
N
N
Me
Me
Me
N
N
N
N
Me
S
N
O
S
N
N
S
Me
Me
1
2 (clotiazepam)
3 (etizolam)
Cl
Cl
N
N
Me
Me
Me
Me
N
N
N
N
N
S
S
Me
N
Me
Me
4 (israpafant)
5
Figure 1. Thienodiazepines.
Table 1
Table 2
Effect of substitution at the 6-position on inhibition of T cell proliferation induced by
CD28 co-stimulation
SAR of biphenyl derivatives 7a–m
R5
Cl
Me
Me
Me
N
N
CO2Me
Me
N
N
S
R4
N
S
N
Me
N
Me
N
Compound
R5
Cl
IC50 (lM)
Compound
R4
H
IC50 (lM)
6h
7a
7b
7c
7d
7e
7f
7g
7h
7i
0.038
0.22
0.053
0.036
0.19
0.033
0.026
0.18
0.027
0.015
0.032
0.0098
0.16
5
6a
6b
6c
6d
6e
2.5
Ph (racemate)
4-MeOPh
3-MeOPh
2-MeOPh
4-CNPh
3-CNPh
2-CNPh
3-AcNHPh
3-CH3COPh
3-HOPh
Me (racemate)
Et (racemate)
CH2Ph (racemate)
CH2CH2Ph (racemate)
CH2(2-Py) (racemate)
CH2(3-Py) (racemate)
CH2(4-Py) (racemate)
CH2CO2Me (S)
0.39
0.34
0.5
2.68
0.33
0.4
0.24
0.038
>10
0.012
0.03
0.044
0.11
0.036
0.39
0.58
0.1
6f
6g
6h
6i
6j
6k
6l
6m (JQ1)
6n
6o
6p
6q
CH2CO2Me (R)
CH2CO2Et (S)
7j
7k
7l
3-MsPh
3-CF3OPh
3-CN-4-HOPh
CH2CO2(n-Pr) (S)
CH2CO2(i-Pr) (S)
CH2CO2(t-Bu) (S)
CH2CO2Bn (S)
CH2CH2OMe (S)
CH2CH2OH (S)
7m
0.0025
alcohol, and cyano groups. Introduction of an (S)-methoxycar-
bonylmethyl group (6h) resulted in a drastic increase in activity,
while the antipode (6i) completely lost the activity, suggesting that
(S)-configuration at the 6-position would be critical for showing
biological activity. Other ester derivatives (6j–n) with the (S)-con-
figuration also showed potent activity, among which ethyl ester 6j
is the most potent. Derivatives with other functionality such as
methoxymethyl, hydroxymethyl, and cyanomethyl showed mod-
erate activity (6o–q). Methyl ester 6h showed better pharmacoki-
netic properties than ethyl ester 6j and we chose 6h as the lead for
further optimization.13
CH2CH2CN (S)
a variety of biological activity. Anxiolytic drugs, clotiazepam
(RizeÒ, 2) and etizolam (DepasÒ, 3); and an antiasthmatic plate-
let-activating factor (PAF) antagonist, israpafant (PafnolTM, 4) have
a common thienodiazepine scaffold (Fig. 1). These are our products
and we had a large library of this class of compounds. Firstly, we
screened 1-related compounds selected from the library, identify-
ing compound 5 with an IC50 of 2.5 lM as the lead.
We focused on the introduction of a substituent at the 6-posi-
tion of the thienodiazepine ring (Table 1). Introduction of an alkyl
or aralkyl group (6a–c, 6e–g) resulted in a five to ten-fold increase
in activity, while phenethyl derivative 6d retained the activity. We
investigated introduction of polar substituents such as ester, ether,
We turned our attention to modification of the 4-position of the
pendant phenyl group of compound 6h. We synthesized com-
pounds substituted at the 4-position of the pendant phenyl group
of 6h by another phenyl group with various substituents (Table 2).
The introduction of a phenyl group resulted in a five-fold reduction

J. Endo et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1365–1370
1367
CN
Cl
Cl
a, b
c
N
N
N
S
S
S
N
CO2H
CO2Me
CO2Me
N
N
N
N
N
N
N
N
8
6h
7f
Scheme 1. Reagents and conditions: (a) PivCl, Et3N; (b) NaOMe, MeOH (58%); (c) 3-CNPhB(OH)2, Pd(OAc)2, JOHNPHOS, KF, THF (95%).
in activity (7a); however, methoxyphenyl substituted analogs 7b
and 7c and cyanophenyl substituted analogs 7e and 7f showed
comparable activity to 6h, while 2-substituted phenyl analogs 7d
and 7g showed considerably reduced activity as compared to 6h.
We next synthesized compounds with a 3-substitued phenyl group
at the 4-position of the pendant phenyl group of 6h (7h–m).
Although
a compound with an electron-withdrawing group
(CF3O) showed a four-fold reduction in activity, the other com-
pounds showed potent activity. In particular, compounds 7k and
7m were 4- and 25-fold more potent than 6h, respectively. We
evaluated potent inhibitors thus obtained by using the CIA model
and toxicity tests, identifying compound 7f, which showed the
most potent therapeutic effects in the CIA model and the least tox-
icity, as a preclinical candidate to progress into further studies.
Compound 7f was synthesized as shown in Scheme 1. Chiral
acid 814 was esterified to afford 6h, which was coupled with
3-cyanophenyl boronic acid to give 7f (Scheme 1).
Compound 7f was evaluated in vivo and proved to be effective
in animal models of autoimmune diseases and transplantation. For
example, in a mouse collagen-induced arthritis model, 7f demon-
strated a potent therapeutic effect at an oral dose of 0.3 mg/kg or
higher (Fig. 2).
We started a research program to elucidate the MOA of 7f using
Jurkat cells. We synthesized several chemical probes including 9
and 10 and succeeded in identifying the target protein of 7f by
using a biotin conjugate 10 (Fig. 3).
Figure 2. Therapeutic effect of 7f on chronic-progressing CIA in DBA/1 J mice.15
O
HN
NH
H
O
H
N
H
H
CN
O
N
S
O
O
O
O
O
HN
H
NH
H
O
H
N
N
Me
Me
N
S
O
S
S
N
H
CO2Me
CN
O
N
S
N
Me
9
N
Me
Me
CO2Et
N
N
S
N
Me
10
Figure 3. Chemical probes for the pull down assay.

1368
J. Endo et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1365–1370
HO
CN
O
O
O
O
NC
HO
B
O
O
O
B B
Br
Br
O
N
6h
N
a
c
b
S
CO2Me
N
S
N
CO2Me
N
N
N
N
11
12
O
O
CN
HN
H
NH
H
HO
H
N
O
NH2
O
S
O
O
O
N
9
S
d
CO2Me
N
N
N
13
Scheme 2. Reagents and conditions: (a) PdCl2(PCy3)2, KOAc, dioxane (100%); (b) PdCl2(PPh3)2, Na2CO3, THF, H2O (75%); (c) K2CO3, DMF, then TFA, CH2Cl2 (92%), (d) EDC HCl,
HOBt, Et3N, DMF (29%).
HO
CN
O
O
B
NC
HO
O
O
O
B B
BocHN
Br
Br
O
6j
N
N
a
c
b
S
S
CO2Et
CO2Et
N
N
N
N
N
N
O
14
15
HN
H
NH
H
O
CN
S
H2N
S
H
N
S
O
O
O
O
N
O
N
SO3Na
10
S
CO2Et
N
d
N
N
16
Scheme 3. Reagents and conditions: (a) PdCl2(PCy3)2, KOAc, dioxane (100%); (b) PdCl2(PPh3)2, Na2CO3, THF, H2O (52%); (c) Cs2CO3, MEK, then TFA, CH2Cl2 (28%), (d) THF,
DMSO (17%).
Chemical probes 9 and 10 were synthesized as outlined in
Schemes 2 and 3, respectively.
cytosol nucleus
For elution of the specific binding molecules to the compound,
Mr
9
10
9 10
we used reducing reagent tris(2-carboxyethyl)phosphine (TCEP).
Compound 10 has a disulfide bond in the linker, while compound
9 does not. As a result, the target molecules were successfully
eluted from the nucleus fraction using compound 10 and avidin-
resin. We identified nuclear proteins, bromodomain-containing
proteins BRD2, BRD3, and BRD4 as the target molecules (Fig. 4).16
Next, we examined whether addition of 7f competitively inhi-
bits the pull down of the BRDs induced by 10/avidin-resin. As a
result, 7f inhibited the pull down dose-dependently but the anti-
pode of 7f (anti-7f) did not inhibit the pull down, confirming that
7f binds to the BRDs and is a first inhibitor against BRDs (Figs. 5).16
It has been also verified that 7f inhibits the binding between BRD4
and an acetylated histone peptide (AcH4) with an IC50 value of
33.9 nmol/L (Fig. 6) (see Supplementary material). Based on these
findings, 7f is speculated to show its therapeutic effect on the
(kD)
250
150
BRD4
BRD2
BRD3 and BRD4 (shortform)
100
75
50
37
25
20
15
chronic-progressing CIA by inhibiting
T cell activation and
proinflammatory cytokine production which are mediated by the
BRD-acetylated histone interaction.
Figure 4. Results of the pull down assay.16

J. Endo et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1365–1370
1369
7f
anti-7f
10 10
competitor
(µmol/L)
0
1
5
-
CN
(kD)
250
BRD4
BRD2
150
N
S
CO2Me
N
N
100
75
N
BRD3 and
BRD4 (short form)
7f
CN
50
37
N
S
CO2Me
N
N
25
N
1
2
3
4
5
6
anti-7f
pull down with
probe 10
probe 9
Figure 5. Inhibition of the pull down by 7f and anti-7f.
10.0
䢳䢲䢲
䢷䢲
䢲
Placebo (
10 mg/kg (
30 mg/kg (
)
%inhibiꢀon
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
)
)
43
65
80
10 mg/kg B.I.D. (
)
** P<0.01
by Dunneꢁ's test
v.s. placebo group
**
**
**
䢲
䢳
䢳䢲
䢳䢲䢲
䢳䢲䢲䢲 䢳䢲䢲䢲䢲
7f (nmol/mL)
Figure 6. Inhibition of BRD4-AcH4 binding by 7f.
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
Days after administration [days]
Figure 7. Anti-tumor activity of 7f.18
Table 3
Effects of compound 7f on cell survival17
inhibitory activity on the in vitro growth of wide variety of tumor
cell lines with submicromolar potency, especially strong activity
against leukemia, lymphoma and multiple myeloma cell lines. In
solid tumors, prostate cancer, lung cancer and colorectal cancer
cell lines showed high sensitivity against 7f (Table 3).
Compound 7f was evaluated for its in vivo anti-tumor efficacy
by using xenograft of human promyelocytic leukemia HL-60 cell
line implanted onto nude mice (Fig. 7).18 Significant inhibition of
relative tumor growth was achieved to 43%, 65% and 80% in the
mice receiving 10, 30 mg/kg and 10 mg/kg twice a day of 7f,
respectively (p-value <0.01), suggesting that 7f would be useful
as an anticancer agent.
In summary, a phenotypic assay targeting the inhibition of T cell
proliferation induced by CD28 co-stimulation led to the discovery
of a novel immunosuppressive thienodiazepine compound 7f,
which has proved to be a first BRD inhibitor by using a chemical
biology method and is expected to become not only an immuno-
suppressive agent but also an anticancer agent.
Cell line
Tissue type
GI50 (lM)
M-1
Mouse myeloid leukemia
Premyelocytic leukemia
CML (Chronic Myeloid Leukemia)
Burkitt’s lymphoma
Burkitt’s lymphoma
Myeloma B-cell type lymphoma
ALL (Acute Lymphoblastic Leukemia)
Mouse melanoma
Hepatocellular cancer
Ovarian cancer
Ovarian cancer
Prostate cancer
Prostate cancer
0.003
0.007
0.019
0.067
0.829
0.069
0.106
0.080
0.104
0.130
5.27
0.199
0.201
0.207
>10
0.207
>10
HL-60
MV4-11
Daudi
U-937
RPMI-8226
MOLT-4
B16
Kim-1
A-2780
SKOV-3
PC-3
LNCap
EBC-1
A549
LN-18
U-87MG
Hela
NSCLC (Non Small Cellular Lung Cancer)
NSCLC
Brain cancer
Brain cancer
Cervical cancer
0.274
0.484
1.50
Colo-205
LLC
Colorectal cancer
Lewis lung carcinoma
Acknowledgments
Since it turned out that compound 7f specifically binds to
BRD2/3/4 proteins and inhibits their binding to acetylated his-
tones, we started investigating inhibitory activity of 7f against
the in vitro proliferation of tumor cell lines.17 7f exhibited
We thank Mr. Hikaru Nonaka and Ms. Junko Wada for con-
structing a high through-put screening system (binding between
B7 and CD28/CTLA-4) and executing the assay.

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J. Endo et al. / Bioorg. Med. Chem. Lett. 26 (2016) 1365–1370
limbs; score 3, severe edema throughout the limbs; score 4, severe edema
Supplementary data
throughout the limbs and ankylosis.
12. Inhibition of T cell proliferation induced by CD28 co-stimulation was evaluated
as follows. Splenic T cells from BALB/c mice were stimulated with anti-CD28
Ab (10 ng/mL) and phorbol 12-myristate 13-acetate (PMA, 250 ng/mL) in the
presence of compounds. T cell proliferation after 24 h culture was determined
by [methyl-3H]thymidine incorporation assay. IC50 values were calculated by
linear regression.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.01.
084.
13. The pharmacokinetic parameters for 6h and 6j in rats are as follows. 6h (3 mg/kg,
iv), AUC0–1: 1185 ngꢀh/mL; CL: 2568 mL/h/kg; Vss: 4584 mL/kg, 6h (10 mg/kg,
po), Cmax: 663 ng/mL; Tmax: 0.83 h; AUC0–1: 2674 ng h/mL; BA: 67.7%. 6j (3 mg/
kg, iv), AUC0–1: 1081 ngꢀh/mL; CL: 2799 mL/h/kg; Vss: 1324 mL/kg, 6j (10 mg/kg,
po), Cmax: 1293 ng/mL; Tmax: 0.5 h; AUC0–1: 1778 ngꢀh/mL; BA: 49.3%.
14. Ehara, S.; Kobayashi, H.; Komatsu, H.; Sueoka, H. WO1998/011111, 1998.
15. The therapeutic effect of 7f on chronic-progressing CIA was evaluated as
follows. DBA/1J mice were immunized with bovine type II collagen and
Freund’s complete adjuvant once. After 9 weeks of the immunization, 7f was
orally administered to the mice for 8 weeks. Arthritis score were determined
weekly during administration periods. Each symbol represents the
mean SEM of arthritic score of 12 animals. Statistical significance versus
arthritis control group was calculated by Steel method. (*: p <0.05, **: p <0.01.)
16. The pull down assay method (Fig. 4) and the competition experiment (Fig. 5)
are as follows. The compound 9 or 10 was mixed with NeutrAvidin-resin
(Thermo Scientific; Pierce Protein Biology Products, Rockford, IL) at rt for
30 min with vortex. These complexes were washed with PBS and then
incubated with PMA/ionomycin activated Jurkat cell lysate (cytosol and
nucleus fractions), which were extracted by the Nuclear extract kit (Sigma–
Aldrich, St Louis, MO), at 4 °C, for 2 h with rotation. After washing the
complexes with TNE buffer (10 mM Tris–HCl pH 7.5/1% NP-40/0.15 M NaCl/
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11. The effects of compounds on mouse collagen-induced arthritis were evaluated
as follows. DBA/1J mice were immunized with type II collagen twice between
21 days interval. Compounds were orally administered to DBA/1J mice for
3 weeks from the day of secondary immunization with type II collagen.
Arthritis score was judged at 21–42 days after primary immunization
according to the following criteria. Score 0, no change; score 1, edema at one
joint; score 2 edema at two or more joints, or mild edema throughout the
1 mM EDTA), the complexes were incubated with 20 lmol/L of reducing
reagent TCEP (Thermo Scientific; Pierce Protein Biology Products, Rockford, IL)
at rt for 15 min with vortex. Removing the NeutrAvisin-resin by centrifugation,
the supernatants were analyzed by SDS–PAGE. In the competition experiment,
compound 7f or anti-7f was added in the Jurkat cell lysate before the pull
down assay.
17. Tumor cells were exposed 72 h at 37 °C, 5% CO2 to 7f at various concentrations.
At the end of incubation, cell viability and proliferation were quantified by
using a tetrazolium salt (WST-8) based colorimetric assay. We created dose-
growth inhibition curve, and calculate GI50 values (geometric mean value) and
95% confidential interval by non-linear regression analysis.
18. 7f was orally administered for 14 days at a dose of 10, 30 mg/kg/day once a day
or 10 mg/kg twice a day. The tumor mass was measured to calculate the
relative tumor growth rate.