Synthesis and structure–activity relationship studies of MI-2 analogues as MALT1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.04.059

Recent studies revealed that MALT1 is a promising therapeutic target for the treatment of ABC-DLBCL. Among several reported MALT1 inhibitors, MI-2 as an irreversible inhibitor represents a new class of ABC-DLBCL therapeutics. Due to its inherent potential cross-reactivity, further structure–activity relationship (SAR) study is imperative. In this work, five focused compound libraries based on the chemical structure of MI-2 are designed and synthesized. The systematic SARs revealed that the side chain of 2-methoxyethoxy has little impact on the activity and can be replaced by other functionalized groups, providing new MI-2 analogues with retained or enhanced potency. Compounds 81–83 with terminal hydroxyl group as side chain displayed enhanced activities against MALT1. Replacement of triazole core with pyrazole is also tolerant, while structural modifications on other sites are detrimental. These findings will facilitate further development of small-molecule MALT1 inhibitors.

Full text of DOI:10.1016/j.bmc.2018.04.059

Accepted Manuscript
Synthesis and Structure–Activity Relationship Studies of MI-2 Analogues as
MALT1 Inhibitors
Guolin Wu, Haixia Wang, Wenhui Zhou, Bihua Zeng, Wenhui Mo, Kejie Zhu,
Rong Liu, Jia Zhou, Ceshi Chen, Haijun Chen
PII:
S0968-0896(18)30517-0
https://doi.org/10.1016/j.bmc.2018.04.059
BMC 14342
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
12 March 2018
25 April 2018
30 April 2018
Please cite this article as: Wu, G., Wang, H., Zhou, W., Zeng, B., Mo, W., Zhu, K., Liu, R., Zhou, J., Chen, C.,
Chen, H., Synthesis and Structure–Activity Relationship Studies of MI-2 Analogues as MALT1 Inhibitors,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.04.059
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Synthesis and Structure–Activity Relationship Studies of MI-2
Analogues as MALT1 Inhibitors
a,1
Guolin Wu , Haixia Wang , Wenhui Zhou , Bihua Zeng , Wenhui Mo , Kejie
b,c,1
b,d,1
a
a
a
Zhu , Rong Liu , Jia Zhou , Ceshi Chen *, Haijun Chen *
b
e
b,
a,
a
College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China
b
Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese
Academy of Sciences & Yunnan Province, Kunming Institute of Zoology Kunming,
Yunnan 650223, China
c
University of Science and Technology of China, Hefei, Anhui 230027, China
d
Hubei Key Laboratory of Embryonic Stem Cell Research, Biomedical Research
Institute, Hubei University of Medicine, Shiyan, Hubei 442000, China
e
Chemical Biology Program, Department of Pharmacology and Toxicology,
University of Texas Medical Branch, Galveston, Texas 77555, USA
1
These authors contribute equally to this work.
Corresponding author
For H.C.: Fax, 86-591-22866227; e-mail, chenhaij@gmail.com.
For C.C.: Fax, 86-871-65181945; e-mail, chenc@mail.kiz.ac.cn.

Highlights
ꢀ
ꢀ
ꢀ
ꢀ
Various MI-2 analogues have been designed and synthesized.
Systematic SARs of the MI-2 were investigated.
The side chain of 2-methoxyethoxy is flexible and tolerable for modifications.
81-83 with terminal hydroxyl group show enhanced activities against MALT1.

Table of Contents (TOC) Graphic

ABSTRACT
Recent studies revealed that MALT1 is a promising therapeutic target for the
treatment of ABC-DLBCL. Among several reported MALT1 inhibitors, MI-2 as an
irreversible inhibitor represents a new class of ABC-DLBCL therapeutics. Due to its
inherent potential cross-reactivity, further structure−activity relationship (SAR) study
is imperative. In this work, five focused compound libraries based on the chemical
structure of MI-2 are designed and synthesized. The systematic SARs revealed that
the side chain of 2-methoxyethoxy has little impact on the activity and can be
replaced by other functionalized groups, providing new MI-2 analogues with retained
or enhanced potency. Compounds 81-83 with terminal hydroxyl group as side chain
displayed enhanced activities against MALT1. Replacement of triazole core with
pyrazole is also tolerant, while structural modifications on other sites are detrimental.
These findings will facilitate further development of small-molecule MALT1
inhibitors.
Keywords: MALT1; MI-2 analogues; Structure–activity relationships; Cancer
Therapeutics

1. INTRODUCTION
Lymphomas are one class of the most common cancers, while non-Hodgkin
1
lymphoma (NHL) accounts for 90% of all lymphomas. Diffuse large B-cell
lymphoma (DLBCL) is the most common subtype of NHL, representing about a third
2
of cases. Among all cases of DLBCL, the activated B cell-like (ABC) DLBCL is
,3
4
-7
the most dangerous one, with the highest rate of complications and mortality.
Patients with ABC-DLBCL often experience a rapid disease progression and most of
8
,9
them survive less than five years due to the lethal combination of resistance.
Accumulating evidence demonstrates that mucosa-associated lymphoid tissue
protein-1 (MALT1) plays a key role in maintaining proliferation of ABC-DLBCL
1
0-12
cells via activating NF-кB signaling.
Upon antigen receptor stimulation, MALT1
paracaspase forms part of CARMA1-BCL10-MALT1 (CBM) complex, which serves
as a signaling scaffold that recruits and activates the IкB kinase b (IKKb) and
1
3-16
stimulates NF-кB.
MALT1 protease activity potentiates NF-кB signaling by
cleaving and inactivating negative regulators such as IKK, A20 (TNFAIP3) and
1
7-20
CYLD.
All these findings suggest that the activity of MALT1 is required for
2
1
survival of ABC-DLBCL. Thus, inhibition of MALT1 proteolytic activity is
considered as a promising and powerful therapeutic approach for the treatment of
2
1,22
ABC-DLBCL.

Figure 1. Chemical structures of recently reported MALT1 inhibitors.
The peptide Z-VRPR-FMK is the first reported MALT1 inhibitor that is proven
1
9,23
to be effective for interfering with survival of ABC-DLBCL cells in vitro.
However, Z-VRPR-FMK is not an ideal lead compound due to its poor pharmacologic
properties such as high molecular weight, relatively poor cell permeability, and
2
3,24
potential metabolic instability.
tors showed apparent anticancer activity in vitro and in vivo.
phenothiazines such as mepazine and thioridazine were identified as selective MALT1
Recently, several small-molecule MALT1 inhibi-
25,26
For example, some
2
4,27
inhibitors
via
mediating
reversible
MALT1
cleavage
inhibition.
β-Lapachone-based scaffold was identified as an irreversible, covalent MALT1
28
inhibitor. MI-2 is another irreversible MALT1 inhibitor featuring direct binding to
2
9
MALT1 and suppression of its protease function. Although traditional drug
development has shied away from irreversible inhibitors owing to the safety concerns,
the resurgence of covalent drugs with outstanding phamacodynamic properties of
MI-2 continues to drive medicinal chemists to discover more effective leads through
3
comprehensive SARs. For example, Overkleeft and coworkers explored the effect of
0
different substituents on C5-phenyl ring of MI-2 and found that para-substituent is of
3
1
influence on inhibitory potency, whereas meta-substituent is preferably avoided.
Furthermore, recent findings show that MI-2 is effective against ibrutinib-resistant

3
2-34
chronic lymphocytic leukemia (CLL) and inflammation diseases.
Those studies
greatly expand the potential application of MI-2. In order to determine more detailed
structural features which contribute to potency, we generated a focused set of 70
analogues to systematically explore the SARs of MI-2. The structural modification
directions of MI-2 were focused on five chemical sites (Figure 2): chloromethyl
amide moiety (site A), N-phenyl group (site B), C5-aryl moiety (site C), the side chain
of 2-methoxyethoxy (site D), the key core of 1H-1,2,4-triazole (site E). Herein, we
report the synthesis, pharmacological evaluation, and SARs of MI-2 analogues as
MALT1 inhibitors.
Figure 2. Five structural modification sites based on the chemical structure of MI-2.
2. RESULTS AND DISCUSSION
2.1. Chemistry
The first library was designed to explore the Site A and Site B. As shown in
Scheme 1, 3,4-dichlorobenzoic acid was reacted with oxalyl dichloride in CH
°C to give the corresponding benzoyl chloride in quantitative yield. Compound 1
was then reacted with potassium thiocyanate (KSCN) in acetone to afford benzoyl
2 2
Cl at
0

isothiocyanate (2). The intermediate 2 was treated with 2-methoxyethan-1-ol at 60 °C
to provide O-(2-methoxyethyl) benzoylcarbamothioate (3). Compound 3 was then
treated with substituted phenylhydrazine in ethanol to give the cyclized products 4-7.
The nitro containing 6 was reduced to phenylhydroxylamine 8 in Zn/NH
then converted to aniline 9 in Zn/AcOH/MeOH system. MI-2 (11) and its analogues
12-16) were obtained by employing a conventional amide coupling protocol. As
MI-2 contained the para-substituted moiety, MI-2 analogues with other positions
meta or ortho) were designed. However, only compound 17 with meta-position was
4
Cl system,
(
(
obtained, the synthesis of MI-2 analogues with ortho-position was unsuccessful due to
its steric hindrance.
a
Scheme 1. Design and synthesis of two focused compound libraries A and B.
a
2 2 2
Reagents and conditions: (a) (COCl) , CH Cl , cat. DMF, r.t. 12 h, 100%; (b) KSCN,

o
acetone, 60 C, 1 h, 83%; (c) 2-methoxyethan-1-ol, acetone, 60 C, 3 h, 45%; (d)
o
o
substituted phenylhydrazine, EtOH, 90 C, 6-12 h, 42-74%; (e) Zn, NH
’
r.t., 1 h, 77%; (f) Zn, AcOH, MeOH, r.t., 12 h, 79-98%; (g) R COOH, CH
4
Cl, MeOH,
Cl , EDCI,
2
2
r.t., 0.5-12 h, 42-99%; (h) ClCH COOH, CH Cl , EDCI, r.t., 5 h, 58%.
2
2
2
To explore dichlorophenyl ring C, a series of aroyl acids were reacted in the
same reaction conditions as described in Scheme 1, including chloroformylation,
followed by condensation with KSCN and 2-methoxyethan-1-ol, subsequently ring
closure with 4-nitrophenylhydrazine. The nitro-containing derivatives were reduced to
afford the corresponding anilines, followed by final coupling with 2-chloroacetic acid
to produce compounds 46-52 (Scheme 2).
a
Scheme 2. Design and synthesis of the focused compound library C.
a
2 2 2
Reagents and conditions: (a) (COCl) , CH Cl , cat. DMF, r.t., 2-12 h, 100%; (b)
o
KSCN, acetone, 60 C, 1-3 h; then 2-methoxyethan-1-ol, 3-8 h, 43%; (c)
o
-nitrophenylhydrazine, EtOH, 90 C, 6-12 h, 38-72%; (d) Zn, AcOH, MeOH, r.t., 12
4
h, 49-98%; (e) ClCH COOH, CH Cl , EDCI, r.t., 2-6 h, 60-96%.
2
2
2

The synthesis of MI-2 analogues with exchange of ring B and ring C was
described in scheme 3 according to the similar reaction conditions. Nitro-substituted
benzoic acids were reacted with sulfurous dichloride to provide the corresponding
benzoic chlorides. Benzoic chlorides were subjected to two different routes to afford
compounds 57 and 64.
a
Scheme 3. The exchange of ring B and ring C.
a
o
o
Reagents and conditions: (a) SOCl
-methoxyethan-1-ol, 5 h, 19%; (c) 3,4-dichlorophenylhydrazine, EtOH, 90 C, 12-18
h, 70-73%; (d) Zn, AcOH, MeOH, r.t., 12 h, 29%; (e) ClCH COOH, CH Cl , EDCI,
r.t., 16 h, 13-62%. (f) KSCN, acetone, 60 C, 1 h; then MeOH, 3 h, 61%; (g) 33%
2
, 80 C, 2 h; (b) KSCN, acetone, 60 C, 1 h; then
o
2
2
2
2
o
o
o
HBr/AcOH, 100 C, 12 h, 80%; (h) 2-methoxyethan-1-ol, PPh
3
, DIAD, THF, 0 C to
r.t., 2 h, 50%.

Synthesis of the focused compound library D was followed the expedient route
shown in Scheme 4A. The key intermediate 69 was obtained in five steps from
benzoyl isothiocyanate 2. This route included methyl substitution, cyclocondensation,
demethylation, reduction, coupling reaction and last-stage diversification. MI-2
3
analogues 70-109 were conveniently prepared by Mitsunobu reaction. Click
5
chemistry is frequently employed in drug discovery and greatly helps advance
3
6,37
research programs in the pharmaceutical industry.
-(1-aryl-1H-1,2,3-triazol-4-yl)alkoxy-1,2,4-triazoles have been designed and
synthesized via click reaction (Scheme 4B). The aryl azides were synthesized from
the commercially available corresponding arylamine treated with NaNO and NaN
In this work, a series of
3
2
3
.
All azides were directly used in the next step without further purification. Click
reaction was performed with an excess of azides to give compounds 110-114.
a
Scheme 4. Design and synthesis of the focused compound library D.

a
(
A) Mitsunobu reaction; (B) click reaction. Reagents and conditions: (a) MeOH,
o
acetone, 60 C, 3 h, 83%; (b) 4-nitrophenylhydrazine, MeOH, 70 C, 18 h, 89%; (c)
o
o
3% HBr/AcOH, 100 C, 6 h, 99%; (d) Zn, AcOH, r.t., MeOH, 12 h; (e)
3
o
ClCH
2
COOH, EDCI, r.t., CH
.5-24 h, 27-96%; (g) Ar-N
3-60%.
2
Cl
2
3
, 5 h; (f) ROH, PPh , DIAD, THF, 0 C to r.t.,
0
2
3
, CuSO
4
·5H O, sodium ascorbate, r.t., DMSO, 1 h,
2
3
-(2-Methoxyethyl)amino-1H-1,2,4-triazole 118 and 3-pentyl-1H-1,2,4-triazole
1
22 were synthesized according to the similar procedures (Scheme 1). Isothiocyanate
was condensed with 2-methoxyethan-1-amine in acetone to afford intermediate 115
2
(
Scheme 5). In addition, compound 119 was synthesized from the
3
,4-dichlorobenzamide according to
a
reported procedure with minor
and 119 were cyclized with
3
8,39
modifications.
Compounds
115
4
-nitrophenylhydrazine in different solvent (DMF for 115, pyridine for 119), followed
by two general final steps to yield compounds 118 and 122.

Scheme 5. Design and synthesis of 3-(2-methoxyethyl)amino-1H-1,2,4-triazole (118)
a
and 3-pentyl -1H-1,2,4-triazole (122).
a
o
Reagents and conditions: (a) 2-methoxyethan-1-amine, acetone, 60 C, 4.5 h, 46%; (b)
o
-nitrophenylhydrazine, DMF, 135 C, 13 h, 37%; (c) Zn, AcOH, MeOH, r.t., 12 h,
4
7
3%; (d) ClCH
2
COOH, CH
, 100 C, 2 h, 55%; (f) 4-nitrophenylhydrazine hydrochloride, pyridine, 120 C,
h, 11%.
2 2
Cl , EDCI, r.t., 2 h, 64-68%. (e) hexanoic anhydride,
o
o
H
2 4
SO
2
Scheme 6 depicts the structural modification of site E (the core) in which
H-1,2,4-triazole moiety is replaced with 1H-pyrazole core in the light of the
1
4
high-impact of nitrogen atom. The starting material 123 was prepared from the
0
4
1
phenylethanone according to a reported procedure. The subsequent compound
library was established by two different facile routes allowing for comprehensive
explorations of the SARs. Firstly, phenyl β-ketoester 123 reacted with the
4
-nitrophenylhydrazine to give intermediate 124. 1H-Pyrazol-5-ol 124 was then
treated with 2-methoxyethan-1-ol under Mitsunobu reaction condition to afford 125.
After reduction of nitro group and subsequently condensation, compound 127 was
obtained in moderate yield. Another route was started from the cyclization of phenyl
β-ketoester 123 and hydrazine hydrate to give 128, which was coupled with
4
-bromo-4-nitrobenzene to afford 129. Compound 129 was subjected to Mitsunobu
2
1
reaction with 2-methoxyethan-1-ol to afford 130, the structure of which was further

4
confirmed by X-ray crystallographic analysis. As shown in Scheme 6B, 133 was
3
4
prepared according to the method described in the literature, and then reacted with
4
2-methoxyethan-1-ol to afford compound 134. Finally, the nitro-containing products
130 and 134 were reduced to corresponding anilines, which were coupled with
2-chloroacetic acid to give compounds 132 and 136, respectively.
Scheme 6. Design and synthesis of the focused compound library E with
a
N,3,5-trisubstituted pyrazole scaffold.
a
Reagents and conditions: (a) Me
2
CO
3
, NaH, THF, r.t., 2.5 h, 85%; (b)
o
-nitrophenylhydrazine, EtOH, 90 C, 3.5 h, 86%; (c) 2-methoxyethan-1-ol, PPh ,
4
3
DIAD, THF, r.t., 1 h, 47-80%; (d) Zn, AcOH, MeOH, r.t., 12 h, 83-98%; (e)
.
o
O, EtOH, 90 C, 0.5 h,
ClCH
2
COOH, EDCI, CH
2
Cl
2
, r.t., 1 h, 69-80%; (f) N
2
H
4
H
2
o
, DMSO, 90 C, 2.5 h, 59%;
85%; (g) 1-bromo-4-nitrobenzene, L-proline, CuI, Cs
2
CO
3
o
h) AcOH, toluene, 0 C for 1 h, then stirred at 100 C for 4 h, 65%.
o
(
2.2. Antiproliferative activity against ABC-DLBCL cell line
We first evaluated the antiproliferative activity of all MI-2 analogues on the
proliferation of Toledo (ABC-DLBCL cell line, T-cell lymphoma) by employing a

CCK-8 (cell counting kit-8) assay. The experimental results were presented as IC50
values (µM) and summarized in Table 1-4.
2.2.1. Effect of modification on site A and B
The previous studies indicated that MI-2 covalently binds to MALT1 with its
2
9
chloromethyl amide group and acts as an irreversible inhibitor. To determine the
importance of each portion of the compound for potency, the focused compound
library A was firstly synthesized and evaluated (Table 1). Compound 4 without
chloromethyl amide group was inactive. Similarly, replacement of chloromethyl
amide with other related functional group (5, 6, 8, 12-16) led to a significant loss of
antiproliferative activity. Notably, compound 17 with meta-position of chloromethyl
amide displayed 2-fold higher potency than MI-2. The results further suggest that
chloromethyl amide moiety on C1-phenyl is crucial for potency.
Table 1. Antiproliferative activity of the focused compound libraries A, B and C.
IC50
IC50
(µM)
0.40
Compd R
Compd R
(
µM)
>160
>160
>5.0
24.5
1
R = H
1
R = 4-COOH
1
R = 4-NO
1
R = 4-NHOH
1
4
5
6
8
17
46
47
48
2
R = 3-NHCOCH Cl
2
R = 2,4-dichlorophenyl 2.71
2
R = 3,5-dichlorophenyl 1.47
2
2
R = 2-chlorophenyl
3.26
2.76
1.72
1
1
1
R = 4-NHCOCH
2
R = 3-chlorophenyl
2
Cl
0.80
>5
49
50
(
MI-2)
1
R = 4-NHCOCH
2
R = 4-chlorophenyl
1
2
3

1
R =
2
R =
1
1
1
1
3
4
5
6
>5
51
52
57
64
3.13
2.97
0.59
0.98
4
-NHCOCH
2
OH
3,4,5-trimethoxyphenyl
1
R =
2
R = 2-thiophenyl
>3.2
54.5
>160
4
2 2
-NHCO(CH ) Cl
1
R =
3
R =
4
-NHCOCH(CH
3
)Cl
Cl)
4-(2-chloroacetamido)
3
R =
1
R =
4
-NHCO-(2-C
6
H
4
3-(2-chloroacetamido)
2.2.2. Effect of modification on site C
Previous studies on site C suggest that para-substituted phenyl group show much
3
1
better inhibitory activities than the corresponding meta-substituted analogues. Thus,
our modifications of this region were aimed to explore the SARs using more typical
analogues. We obtained a series of MI-2 analogues bearing general moieties at various
positions (46-52, Table 1). Among this focused compound library, the potency
decreases in the order of 47 > 50 > 46 ≈ 49 > 52 > 51 > 48. Both chloro-containing
compounds and trimethoxy substituted analogue displayed moderate potency.
Compound 52 with thiophene ring suffered a significant loss in potency. Our work
also showed that compounds with para-chlorophenyl substituent exhibited better
inhibitory activities than ortho- and/or meta-substituents. To further investigate the
position impact of site B and C, we synthesized two MI-2 analogues with N-1
dichlorophenyl substituent on 1H-1,2,4-triazole (57, 64). Notably, compound 57 with
4
-(2-chloroacetamido)phenyl substituted on C-5 exhibited better inhibitory activity
than MI-2, while compound 64 with 3-(2-chloroacetamido)phenyl moiety was
equipotent to MI-2.
2.2.3. Effect of modification on site D

A comprehensive series of analogues with O-substituent sidechain focused on
the site D have been prepared via Mitsunobu reaction (Scheme 4). We initially
focused on the introduction of alkyl moieties such as aliphatic chains (70-77) or cyclic
alkyl fragments (78-80), and the results revealed that almost all compounds displayed
comparable potency. Compounds 81-83 with hydroxyl moiety at terminal showed a
slightly improved potency. Notably, compounds 84-87 with ether chain are
approximately two-fold more potent. Additionally, introduction of sulfide (88),
fluorine atom (89) and heterocyclic (90-91) also led to nearly two-fold increased
activity. In particular, compound 93 with ethyl acetate moiety exhibits improved
activity, while acetylation of 1H-1,2,4-triazol-3-ol ring (92) dramatically reduced
potency. Following this trend, unsaturated hydrocarbon substituted analogues (94-107)
also showed improved activity with IC50 values ranging from 0.30 µM to 0.72 µM.
Moreover, compound 106, which was linked with the functional group tryptamine,
displayed the similar potency. To further confirm the impact of the replacement of the
drug-like fragment on site D, a series of analogues possessing 1,2,3-triazole moiety
4
5,46
through click reactions were synthesized (Table 3).
The results showed that
almost all compounds (110-114) displayed a slightly improved potency. The effect of
oxygen element linkage between 1,2,4-triazole and D site chain was also
4
7,48
examined.
The replacement of oxygen with nitrogen (118) or methylene group
(
122) did not affect the potency, further indicating that this region can be combined
with other functional groups to provide practical probes for further biological
mechanism investigation.

Table 2. Antiproliferative activity of the focused compound library D (part 1).
IC50
IC50
Compd R
Compd R
89
(
µM)
(µM)
11
0
.80
0.37
0.46
0.47
>2
(
MI-2)
69
70
71
72
73
74
75
76
77
H
>5
90
91
92
93
94
95
96
97
98
Me
Et
0.51
0.81
0.34
0.61
0.69
0.77
1.56
1.69
0.50
0.40
0.30
0.38
0.41
0.39

78
79
80
81
82
83
84
0.47
0.49
0.69
0.43
0.53
0.75
0.43
99
0.32
0.43
0.72
0.37
0.34
0.39
0.44
Ph
100
101
102
103
104
105
85
0.43
106
0.38
86
87
88
0.40
0.46
0.42
107
118
122
0.45
0.75
0.96
/
/
Table 3. Antiproliferative activity of the focused compound library D (part 2).

Compd
1 (MI-2)
10
11
n
/
Ar
/
IC50 (µM)
0.80
Compd
112
n
4
1
1
Ar
IC50 (µM)
0.60
1
Ph
1
1
2
Ph
Ph
0.40
113
2-ClC
6
H
4
0.31
1
0.38
114
2-MeOC
6
H
4
0.40
2.2.4. Effect of modification on site E
Further investigation on the role of 1H-1,2,4-triazole core were carried out to
explore the key core for potency. Analogues 127, 132 and 136 with 1H-pyrazole core
were prepared from phenyl β-ketoester (123) or 4-nitrophenylhydrazine as outlined in
Scheme 6. As shown in Table 4, compounds 127 and 132 exhibited a nearly
equipotent antiproliferative effect and compound 136 without dichlorophenyl
substituent at C5 position displayed a slightly lower potency. Our work indicated that
the triazole ring is not indispensable.
Table 4. Antiproliferative activity of the focused compound library E.
Compd
11 (MI-2)
127
132
136
IC50 (µM)
0.80
1.11
0.86
7.61

2.3. MI-2 analogues inhibit MALT1 activity determined by western blot assay
Previous findings supported that cleavage of RelB could reflect the activity of
4
9
MALT1 via Western blot assay. To confirm that these new MI-2 analogues might
inhibit the functions of MALT1 in ABC-DLBCL cells, several notable compounds
were selected for further evaluation. As shown in Figure 3 and Figure S1 (see
Supporting Information), compounds such as 57, 81-83, 86, 87, 111, especially
compounds 81-83 with terminal hydroxyl moiety as side chain exhibited better
inhibitory activity against MALT1.
Figure 3. Western blot analysis of RELB cleavage product after Jurkat T cells were
treated with MI-2 analogues (2 µM) (See Figure S1 in the Supporting Information for
more details) .
2
.4. SARs
The SARs of MI-2 analogous as MALT1 inhibitors are summarized in Figure 4.
Firstly, both sites A and B are essential fragments to maintain the inhibitory potency,

while changing site C might slightly decrease the activity. In addition, the side chain
of 2-methoxyethoxy is more flexible and can be replaced by many functionalized
groups, suggesting that MI-2 can assembly combine with other drugs via different
linker to produce new compounds with more potent pharmacological effects.
Furthermore, MI-2 analogues with terminal hydroxyl moiety on site D exhibit better
inhibitory activity against MALT1. While, replacement of triazole core with pyrazole
moiety is also tolerated, more extensive chemical modifications on this core are
needed.
Figure 4. SARs of MI-2 analogous as MALT1 inhibitors.
3. CONCLUSIONS
In summary, our systematic SAR studies identified multiple structural features
that affect the potency of MI-2 analogues. We found that analogues with
chloromethethyl amide substituent on C1-phenyl are important to maintain efficacy of
MI-2, and replacement of the C5-aryl ring may slightly decrease the activity.
Additionally, modifications of the 2-methoxyethoxy chain and triazole core often lead
to comparable or enhanced potency against MALT1. This work will facilitate further

development of new small-molecule MALT1 inhibitors.
4
4
4
. EXPERIMENTAL SECTION
.1. Chemistry
.1.1. General Experimental
All reagents were purchased from commercial sources and used without further
purification. Thin layer chromatography (TLC) on precoated aluminum plates (silica
gel 60 F254, Merck, Darmstadt) was used to monitor reaction progress. Visualization
of the developed chromatograms was detected by UV (254 nm and 365 nm).
Preparative column chromatography was performed using silica gel (300–400 mesh,
flash). Preparative thin layer chromatography (PTLC) separations were carried out on
0.20 mm Yantai Jiangyou silica gel plates (HSGF254). Proton Nuclear Magnetic
1 13
Resonance NMR ( H NMR) spectra and carbon nuclear magnetic resonance ( C
1
13
NMR) spectra were recorded on a Bruker‐400 ( H, 400 MHz; C, 101 MHz)
spectrometer. Chemical shifts for protons are reported in parts per million and
referenced to the NMR solvent peak (CDCl
.31). Chemical shifts for carbons are reported in parts per million and referenced to
the carbon resonances of the NMR solvent (CDCl : δ77.16; DMSO‐d : δ 39.52;
CD OD: δ 49.00). Signals are listed in ppm, and multiplicity identified as s = singlet,
3
: δ 7.26; DMSO‐d
6
: δ 2.50; CD
3
OD: δ
3
3
6
3
d = doublet, t = triplet, q = quartet, m = multiplet. Chemical shifts were expressed in
ppm, and J values were given in Hz. High resolution mass spectra (HRMS) were
obtained from Thermo Fisher Scientific Exactive Plus mass spectrometer. Melting

point was determined using the X‐4A melting point apparatus (Shanghai Yidian Co.,
Ltd.) and uncorrected.
General procedure A (synthesis of aroyl chloride from aroyl acid). To a solution of
2 2
aroyl acid (1.0 eq) in CH Cl was added oxalyl dichloride (1.1 eq) and DMF (2-5
o
drops) at 0 C. The mixture was allowed to stir at room temperature for 2-12 h. Then
the mixture was concentrated to give compounds 1, 18-24. The products were used
directly in the next reaction.
General procedure B (Synthesis of O-alkyl aroylcarbamothioate from aroyl chloride).
To a solution of aroyl chloride (1.0 eq) in acetone was added KSCN (1.0 eq). The
o
mixture was stirred at 60 C for 1-3 h until the starting material was completely
consumed. The reaction mixture was then cooled to room temperature and ROH (2.5
o
eq) in acetone was added to the reaction mixture. After stirring at 60 C for another
3
-8 h, the reaction mixture was cooled to room temperature and removed the solvent
in vacuo. The crude product was dissolved with EtOAc and extracted with H O. The
organic layer was dried over anhydrous Na SO , and then concentrated to give crude
2
2
4
product under reduced pressure. The residue was purified by silica gel
chromatography to give compounds 25-31, 54 and 59.
General procedure C (Synthesis of 1,5-diaryl-1H-1,2,4-triazole). To a solution of
O-alkyl aroylcarbamothioate (1.0 eq) in EtOH was added substituted phenylhydrazine
o
1.0 eq). The mixture was stirred at 90 C for 8-24 h, and then the solvent was
(
removed in vacuo. The crude product was dissolved with EtOAc and washed with
portions of H O. The organic layer was dried over anhydrous Na SO , and then
2
2
4

concentrated to give crude product under reduced pressure. The residue was purified
by silica gel chromatography to give the desired product.
General procedure D (Reduction of nitroarenes). To a solution of nitro-containing
derivatives (1.0 eq) in MeOH was added AcOH and Zn (5.0-10.0 eq). The mixture
was stirred at r.t. for 12-24 h. The mixture was diluted with EtOAc and washed with
sat. NaHCO
3
, H
2
O and brine, respectively. The organic layer was dried over
, and then concentrated to give crude product under reduced
anhydrous Na
2
SO
4
pressure. The residue was purified by silica gel chromatography to give the desired
product.
General procedure E (Coupling reaction of aniline with carboxylic acid). To a
solution of arylamine (1.0 eq) in CH
2
2
Cl was added the appropriate carboxylic acid
(
1.5 eq) and EDCI (2.0 eq). The mixture was stirred at r.t. for 5-12 h. The mixture was
diluted with EtOAc, washed with H
2
3
O, sat. NaHCO , brine, respectively. The organic
layer was dried over anhydrous Na
2
SO , and then concentrated to give crude product
4
under reduced pressure. The residue was purified by silica gel chromatography to give
the desired product.
General procedure F (Mitsunobu reaction). To a solution of nucleophile (1.0 eq),
PPh
dropwise at 0 °C. Then the mixture was allowed to warm to room temperature and
stirred for 0.5-24 h. The mixture was diluted with EtOAc, washed with H O. The
organic layer was dried over anhydrous Na SO , filtered, and concentrated under
reduced pressure. The residue was purified by silica gel chromatography to give the
3
(1.5-3.0 eq) in THF was added ROH (2.0-5.0 eq) and DIAD (1.5-3.0 eq)
2
2
4

desired product.
General procedure G (Click reaction). To a solution of terminal alkynes (1.0 eq),
azides (6.0 eq) and sodium ascorbate (0.5 eq) in DMSO/H
CuSO ·5H O (0.5 eq). The mixture was stirred at r.t. for 0.5-3 h. The mixture was
diluted with EtOAc, washed with H O. The organic layer was dried over anhydrous
Na SO , filtered, and concentrated under reduced pressure. The residue was purified
by silica gel chromatography to give the desired product.
2
O (5/1, v/v) was added
4
2
2
2
4
4.1.2. 3,4-Dichlorobenzoyl isothiocyanate (2). To a solution of 1 (12.9 g, 60 mmol) in
o
acetone (50 mL) was added KSCN (5.8 g, 60 mmol). The mixture was stirred at 60 C
for 2 h. Then the solvent was evaporated under vacuum. The resulting residue was
2
dissolved EtOAc (50 mL), washed with H O (2 × 20 mL) and brine (20 mL),
respectively. The organic layer was dried over anhydrous Na
2
4
SO and then
concentrated under reduced pressure. The crude product was purified by silica gel
chromatography (PE/EtOAc = 4:1) to give the desired product (11.9 g, 83%) as a
o
white solid. Melting point: 42.4-43.3 C. TLC: R
1
f
= 0.78 (PE). H NMR (400 MHz,
CDCl ) δ 8.13 (d, J = 2.1 Hz, 1H), 7.88 (dd, J = 8.4, 2.1 Hz, 1H), 7.58 (d, J = 8.4 Hz,
3
1
3
1
1
4
2
H). C NMR (101 MHz, CDCl
3
) δ 160.6, 150.2, 140.1, 133.8, 132.2, 131.2, 130.7,
+
NOS [M + H] m/z 233.0855, found 233.0860.
29.3. HRMS (ESI): calcd for C
8
H
3
Cl
2
.1.3. O-(2-Methoxyethyl) (3,4-dichlorobenzoyl)carbamothioate (3). To a solution of
(11.6 g, 50 mmol) in acetone (30 mL) was added 2-methoxyethan-1-ol (9.5 g, 125
o
mmol). The mixture was stirred at 60 C for 5 h. The reaction was diluted with EtOAc
(
2
50 mL) and washed with portions of H O (3 × 20 mL). The organic layer was dried

2 4
over anhydrous Na SO , and then concentrated to give crude product under reduced
pressure. The residue was purified by silica gel chromatography (PE/EtOAc = 8:1) to
give the desired product (6.9 g, 45%) as a pale yellow oil. Physical state: pale yellow
1
= 0.67 (PE/EtOAc = 2:1). H NMR (400 MHz, CDCl
oil. TLC: R
f
3
) δ 9.15 (s, 1H),
7
2
1
.93 (d, J = 2.0 Hz, 1H), 7.69 – 7.63 (m, 1H), 7.57 (d, J = 8.4 Hz, 1H), 4.76 – 4.66 (m,
1
3
H), 3.79 – 3.70 (m, 2H), 3.42 (s, 3H).
3
C NMR (101 MHz, CDCl ) δ 188.0, 161.2,
37.9, 133.8, 132.9, 131.1, 130.0, 126.8, 100.1, 71.8, 69.7, 59.3. HRMS (ESI): calcd
+
S [M + H] m/z 307.9909, found 307.9912.
for C11
H
11Cl
2
NO
3
4.1.4. 5-(3,4-Dichlorophenyl)-3-(2-methoxyethoxy)-1-phenyl-1H-1,2,4-triazole (4).
Following the general procedure C, the reaction was conducted from the starting
material (81 mg, 0.26 mmol) and phenylhydrazine (34 mg, 0.32 mmol) to give the
f
desired product (71 mg, 74%). Physical state: yellow oil. TLC: R = 0.38 (PE/EtOAc
1
4:1). H NMR (400 MHz, CDCl
=
3
) δ 7.64 (s, 1H), 7.49 – 7.37 (m, 3H), 7.37 – 7.27
(
m, 3H), 7.18 (d, J = 8.8 Hz, 1H), 4.56 – 4.40 (m, 2H), 3.85 – 3.69 (m, 2H), 3.43 (s,
1
3
3
1
3
H). C NMR (101 MHz, CDCl ) δ 167.8, 150.8, 137.7, 134.5, 133.0, 130.7, 130.5,
29.6, 129.3, 127.7, 127.5, 125.5, 70.7, 69.0, 59.2. HRMS (ESI): calcd for
+
[M + H] m/z 364.0614, found 364.0623.
C
17
H15Cl
2
N
3
O
2
4.1.5. 4-(5-(3,4-Dichlorophenyl)-3-(2-methoxyethoxy)-1H-1,2,4-triazol-1-yl)benzoic
acid (5). Following the general procedure C, the reaction was conducted from the
starting material (51 mg, 0.16 mmol) and 4-hydrazinylbenzoic acid (30 mg, 0.20
mmol) to give the desired product (28 mg, 42%). Physical state: pale yellow solid;
o
1
f 2 2
Melting point: 180.6-181.2 C. TLC: R = 0.34 (CH Cl /MeOH = 10:1). H NMR

(
400 MHz, CDCl
3
) δ 8.82 (s, 1H), 8.19 – 8.04 (m, 2H), 7.67 (s, 1H), 7.39 (t, J = 7.6
Hz, 3H), 7.19 (d, J = 8.1 Hz, 1H), 4.56 – 4.48 (m, 2H), 3.84 – 3.76 (m, 2H), 3.45 (s,
1
3
3
1
3
H). C NMR (101 MHz, CDCl ) δ 168.0, 151.3, 141.4, 135.1, 133.4, 131.5, 130.9,
2 3 4
30.8, 127.9, 127.3, 124.8, 70.7, 69.3, 59.2. HRMS (ESI): calcd for C18H15Cl N O
+
M + H] m/z 408.0512, found 408.0513.
[
4
.1.6.
-(3,4-Dichlorophenyl)-3-(2-methoxyethoxy)-1-(4-nitrophenyl)-1H-1,2,4-triazole (6).
5
Following the general procedure C, the reaction was conducted from the starting
material (3.0 g, 10 mmol) and 4-nitrophenylhydrazine (1.5 g, 10 mmol) to give the
f
desired product (2.3 g, 56%). Physical state: yellow oil. TLC: R = 0.53 (PE/EtOAc =
1
:1). H NMR (400 MHz, CDCl
2
7
–
1
7
3
) δ 8.26 (d, J = 9.0 Hz, 2H), 7.66 (d, J = 1.9 Hz, 1H),
.52 (d, J = 9.0 Hz, 2H), 7.43 (d, J = 8.4 Hz, 1H), 7.20 (dd, J = 8.4, 1.9 Hz, 1H), 4.59
1
3
4.41 (m, 2H), 3.87 – 3.70 (m, 2H), 3.44 (s, 3H). C NMR (101 MHz, CDCl ) δ
3
68.3, 151.7, 147.1, 142.3, 135.5, 133.6, 131.0, 130.9, 127.9, 127.1, 125.2, 125.0,
+
[M + H] m/z 409.0465,
0.6, 69.3, 59.2. HRMS (ESI): calcd for C17
H14Cl
2
N
4
O
4
found 409.0462.
4
.1.7.
-(3,4-Dichlorophenyl)-3-(2-methoxyethoxy)-1-(3-nitrophenyl)-1H-1,2,4-triazole (7) .
5
Following the general procedure C, the reaction was conducted from the starting
material (200 mg, 0.65 mmol) and 3-nitrophenylhydrazine (119 mg, 0.78 mmol) to
f
give the desired product (179 mg, 67%). Physical state: yellow oil. TLC: R = 0.46
1
PE/EtOAc = 2:1). H NMR (400 MHz, CDCl
(
3
) δ 8.31 (s, 1H), 8.29 – 8.23 (m, 1H),

7.69 – 7.65 (m, 1H), 7.60 (d, J = 4.9 Hz, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.23 – 7.18 (m,
1
3
1
H), 4.55 – 4.47 (m, 2H), 3.81 – 3.75 (m, 2H), 3.48 – 3.40 (m, 3H). C NMR (101
) δ 168.2, 151.5, 148.8, 138.6, 135.4, 133.6, 131.0, 130.9, 130.5, 130.5,
2 4 4
27.7, 126.9, 123.5, 120.3, 70.6, 69.3, 59.2. HRMS (ESI): calcd for C17H14Cl N O
MHz, CDCl
3
1
+
M + H] m/z 409.0465, found 409.0457.
[
4.1.8.
N-(4-(5-(3,4-Dichlorophenyl)-3-(2-methoxyethoxy)-1H-1,2,4-triazol-1-yl)phenyl)hydr
oxylamine (8). To a solution of 6 (2.3 g, 5.6 mmol) in MeOH (80 mL) was added sat.
4
NH Cl (20 mL) and Zn (3.68 g, 56 mmol). The mixture was stirred at r.t. for 1 h. The
mixture was poured into the cold water to give a brown solid (1.7 g, 77%) without
o
further purification. Physical state: brown solid; Melting point: 147.2-147.8 C. TLC:
1
= 0.64 (PE/EtOAc = 1:1). H NMR (400 MHz, CDCl
R
f
3
) δ 7.69 (d, J = 2.0 Hz, 1H),
7.31 (d, J = 8.4 Hz, 1H), 7.20 – 7.15 (m, 1H), 7.13 (d, J = 8.7 Hz, 2H), 6.97 – 6.86 (m,
1
3
3
H), 4.54 – 4.45 (m, 2H), 3.82 – 3.73 (m, 2H), 3.44 (s, 3H). C NMR (101 MHz,
CDCl
3
) δ 167.5, 151.4, 150.9, 134.5, 133.1, 130.8, 130.5, 127.6, 127.4, 126.6, 114.3,
+
7
0.8, 69.1, 59.3. HRMS (ESI): calcd for C17
found 395.0665.
.1.9. 4-(5-(3,4-Dichlorophenyl)-3-(2-methoxyethoxy)-1H-1,2,4-triazol-1-yl)aniline
9). Following the general procedure D, the reduction was conducted from the starting
H16Cl
2
N
4
O
3
[M + H] m/z 395.0672,
4
(
material (137 mg, 0.33 mmol) to give the desired product (124 mg, 98%). Physical
o
state: yellow solid; Melting point: 116.5-117.2 C. TLC: R
f
= 0.22 (PE/EtOAc = 2:1).
1
H NMR (400 MHz, CDCl ) δ 7.69 (d, J = 1.9 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.22
3