Synthesis and structure–activity relationship studies of LLY-507 analogues as SMYD2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127598

SET and MYND domain-containing protein 2 (SMYD2), a lysine methyltransferase, is reported to catalyze the methylation of lysine residues on histone and non-histone proteins. As a potential target for cancer therapy, there are several SMYD2 inhibitors are reported, LLY-507 as a cell-active inhibitor exhibits submicromolar potency against SMYD2 in several cancer cell lines. To know which structural fragment of LLY-507 is suitable for chemical modification, three sites are chosen for structure–activity relationship studies (SARs). Among our focused library, compounds 43 and 44 with amide link on site C showed reasonably improved potency indicating that modification on this fragment is more flexible and introduction of electrophilic warheads in this position might provide lysine-targeting covalent inhibitors for SMYD2.

Full text of DOI:10.1016/j.bmcl.2020.127598

Journal Pre-proofs
Synthesis and structure–activity relationship studies of LLY-507 analogues as
SMYD2 inhibitors
Bin Zhang, Liping Liao, Fan Wu, Fengcai Zhang, Zhongya Sun, Haijun Chen,
Cheng Luo
PII:
S0960-894X(20)30709-5
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127598
BMCL 127598
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Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 September 2020
24 September 2020
28 September 2020
Please cite this article as: Zhang, B., Liao, L., Wu, F., Zhang, F., Sun, Z., Chen, H., Luo, C., Synthesis and
structure–activity relationship studies of LLY-507 analogues as SMYD2 inhibitors, Bioorganic & Medicinal
Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127598
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Synthesis and structure–activity relationship studies of LLY-507 analogues as
SMYD2 inhibitors
Bin Zhanga,#, Liping Liaob,c,#, Fan Wua,#, Fengcai Zhangb,c, Zhongya Sunb,c, Haijun
Chena,*, Cheng Luob,c,
*
aKey Laboratory of Molecule Synthesis and Function Discovery (Fujian Province
University), College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China
bState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China
cUniversity of Chinese Academy of Sciences, 19 Yuquan Road, Beijing, 100049, China
*E-mail addresses:
chenhaij@gmail.com (H. Chen)
cluo@simm.ac.cn (C. Luo)
#These authors contribute equally to this work.

Abstract
SET and MYND domain-containing protein
2
(SMYD2),
a
lysine
methyltransferase, is reported to catalyze the methylation of lysine residues on
histone and non-histone proteins. As a potential target for cancer therapy, there are
several SMYD2 inhibitors are reported, LLY-507 as a cell-active inhibitor exhibits
submicromolar potency against SMYD2 in several cancer cell lines. To know which
structural fragment of LLY-507 is suitable for chemical modification, three sites are
chosen for structure–activity relationship studies (SARs). Among our focused library,
compounds 43 and 44 with amide link on site C showed reasonably improved
potency indicating that modification on this fragment is more flexible and
introduction of electrophilic warheads in this position might provide lysine-targeting
covalent inhibitors for SMYD2.
Keywords: SMYD2 inhibitors; Thermal shift assay; Structure-activity relationships.

Protein lysine methyltransferases play a critical role in epigenetic gene
regulation.1,2 The SMYD family, containing SET and MYND domains, is one special
and important class of protein lysine methyltransferases.3,4 Accumulating evidence
has shown that SMYD2, one of the SMYD members, is overexpressed in various types
of human cancers.5-7 SMYD2 has been proposed as a potential oncogene that affects
the proliferation, apoptosis, and metastasis of cancer cells via histone or non-histone
methylation.8-11 Therefore, there are intensive ongoing efforts to identify SMYD2
inhibitors as valuable chemical probes to elucidate the role of SMYD2 in cancer and
other diseases.12,13
O
HO
N
NC
R
NH
N
N
O
N
HN
N
HN
O
F
F
OH
Cl
NH
O
Cl
Cl
O
Cl
AZ505, R = H; A-893, R = OH
BAY-598
O
N
N
N
N
N
N
N
Cl
H
O
N
N
HN
N
N
N
LLY-507
EPZ033294
Fig. 1. Chemical structures of recently reported SMYD2 inhibitors.
The currently reported small-molecule inhibitors of SMYD2 are shown in Fig. 1.14
AZ505 is the first reported inhibitor of SMYD2 with an inhibitory potency in the
submicromolar range.15,16 The crystal structures of SMYD2 in complex with these
inhibitors show that except EPZ033294, they all bind in a similar fashion, occupying
the peptide-binding groove of SMYD2.15-17 Among them, LLY-507 is the first
cell-active, selective small-molecule inhibitor of SMYD2 with an IC50 of 70 nM and
inhibits the proliferation of various cancer cell lines.18,19 Considering the large
number of lysine residues in SMYD2 especially the area around the ligand binding
sites, it is suitable to design covalent inhibitors by introduction of electrophilic
warheads.18,19 Previous findings show that replacement of the propyl pyrrolidine

moiety of LLY-507 with a variety of amines is unable to improve the potency.20 In
order to determine which site of LLY-507 is suitable for structural modification, we
plan to choose other three chemical sites of LLY-507 through simple reactions for
more information about SARs (Fig. 2): 1,1'-biphenyl region (site A), the key core of
piperazine (site B), the side chain of 1-ethyl-3-methyl-1H-indole-methane (site C).
Herein, we report the synthesis, pharmacological evaluation, and SARs of LLY-507
analogues as SMYD2 inhibitors.
N
A
H
N
N
O
N
B
N
C
N
Fig. 2. Three structural modification sites based on the chemical structure of LLY-507:
A. 1,1'-Biphenyl region; B. Other linkers; C. Side indole group.
The synthesis of LLY-507 analogues was outlined in Schemes 1-4. As shown in
Scheme 1, coupling of ethyl acetoacetate with 3-hydrazineylbenzoic acid generated
compound 1. Amidation of compound 1 afforded amide 2. Compound 2 was
alkylated with tert-butyl 3-iodoazetidine-1-carboxylate to give the intermediate 3,
followed by deprotection to furnish 4. This intermediate was then treated with
3,4-dichlorophenethyl methanesulfonate to give compound 5. Compound 7 was
obtained via click reaction.21

O
O
O
O
A.
N
N
OH
H
H
a
b
c
O
O
O
N
N
N
1
OH
2
OH
N
N
N
NR
ethyl 3-oxobutanoate
R = Boc
R = H
3
4
d
B.
O
e
N
O
H
O
O
N
H
OH
f
N
g
N
N
H
N
N
O
N
N
N
Cl
Cl
5
3-ethynylbenzoic acid
6
7
o
Scheme 1. Reagents and conditions: (a) 3-hydrazinylbenzoic acid, AcOH, 130 C; (b)
3-(pyrrolidin-1-yl)propan-1-amine, HBTU, DIPEA, DMF, 25 °C; (c) tert-butyl
3-iodoazetidine-1-carboxylate, Cs2CO3, DMF, 80 °C; (d) HCl in dioxane, MeOH, 25 °C;
(e) 3,4-dichlorophenethyl methanesulfonate, K2CO3, CH3CN, 70 oC; (f)
3-(pyrrolidin-1-yl)propan-1-amine, EDCI, HOBt, Et3N, CH2Cl2, 25 °C; (g) 12, CuSO4,
VcNa, THF/H2O, 25 °C.
As shown in Scheme 2, the intermediates 14-16 were obtained by a simple
three-step
reaction.
Treatment
of
5-bromonicotinic
acid
with
3-pyrrolidin-1-ylpropan-1-amine provided 20. Compound 21 was synthesized by
Suzuki coupling reaction. The final products were obtained by employing a
conventional amide coupling protocol.
8, 11, 14: X = CH
9, 12, 15: X = CCH3
10, 13, 16: X = N
X
a
b
c
H2N
Br
X
N3
N
N
H
N
X
X
N
11-13
N
8-10
O
14-16
e
O
O
O
O
N
N
N
H
N
N
H
f
d
N
OH
N
N
N
O
H
X
N
H
N
OH
Br
Br
17: X = CH
18: X = CCH3
19: X = N
5-bromonicotinic
acid
20
21
O
O
O
O
N
N
N
H
N
N
N
H
N
N
H
N
g
e
h
O
O
H
N
NH2
N
H
N
H
N
H
O
22
23
24

Scheme 2. Reagents and conditions: (a) 1,4-dibromobutane, NaH, dry THF, 25 °C; (b)
o
NaN3, DMSO, 80 C; (c) PPh3, THF/H2O, 25 °C; (d) 2-carboxyphenylboronic acid,
Pd(PPh3)4, Na2CO3, CH3CN/H2O, 90 oC; (e) HBTU, DIPEA, DMF, 25 °C; (f)
3-(pyrrolidin-1-yl)propan-1-amine, EDCI, HOBt, TEA, CH2Cl2, 25 °C; (g) octan-1-amine,
HBTU, DIPEA, THF/CHCl3, 25 °C; (h) cyclopropanecarboxylic acid, EDCI, THF/CHCl3, 25
°C.
As shown in Scheme 3, compound 20 was subject to Suzuki coupling reaction with
2-hydroxyphenylboronic acid to afford 25. Compound 28 was obtained by Mitsnobu
reaction,22 deprotection and coupling reaction with 3,4-dichlorophenylacetic acid.
Compounds 31-34 were obtained by the similar reaction route with different organic
acids.
O
O
O
O
O
N
N
N
N
N
H
N
N
N
H
N
H
e
b
Br
20
N
N
Cl
Cl
R1
a
O
R1 = Boc
R1 = H
26
27
d
28
O
N
N
H
N
O
O
N
N
H
N
N
N
H
N
e
OH
c
O
O
N
N
25
R2
R3
31, R3 = COCH2CH3 32, R3 = COCH2CN
33, R3 = COCH2-(3,4-dichlorophenyl)
34, R3 = COCH2CH2-(3,4-dichlorophenyl)
R2 = Boc
R2 = H
29
30
d
Scheme 3. Reagents and conditions: (a) 2-hydroxyphenylboronic acid, Pd(PPh3)4,
Na2CO3, dioxane/H2O, 80 °C; (b) tert-butyl 4-hydroxypiperidine-1-carboxylate, PPh3,
DIAD, THF, 25 °C; (c) tert-butyl 3-iodoazetidine-1-carboxylate, Cs2CO3, DMF, 70 °C;
(d) HCl in dioxane, MeOH, 25 °C; (e) RCOOH, EDCI, CHCl3/THF, 25 °C.
As shown in Scheme 4, compound 36 was obtained via Buchwald-Hartwig
amination and Miyaura-Boronization reaction. Subsequent reaction with 20 afforded
37. Removal of Boc group provided piperazine 38, which was suitable for
derivatization under simple conditions to afford compounds 39-47.

O
N
Br
N
N
H
N
Br
Br
O
O
a
c
b
B
NBoc
NBoc
N
NBoc
N
1,2-dibromo
benzene
35
36
37
d
O
O
O
N
N
N
N
N
H
N
N
N
N
H
f
e or e, d
H
O
Cl
N
N
N
N
R2
N
NH
NH
Cl
39
38
40-47
Cl
Cl
O
R2 =
O
O
O
O
Cl
Cl
40
42
46
41
45
43
O
NH
O
O
Cl
Cl
NH
NH2
47
NH
44
Scheme 4. Reagents and conditions: (a) 1-Boc-piperazine, Pd2(dba)3, BINAP, KOtBu,
o
o
toluene, 80 C; (b) B2Pin2, Pd(dppf)Cl2, KOAc, 1,4-dioxane, 80 C; (c) 25, Pd(PPh3)4,
Na2CO3, dioxane/H2O, 80 °C; (d) HCl in dioxane, MeOH, 25 °C; (e) R2COOH, EDCI,
CHCl3/THF, 25 °C; (f) 1,2-dichloro-4-isocyanatobenzene, Et3N, CH2Cl2, 25 °C.
To determine whether all the newly synthesized LLY-507 analogues bind to
SMYD2, we utilized a fluorescence-based thermal shift assay to characterize the
interaction between SMYD2 and these compounds.23 The experimental results were
presented as melting temperature (oC) and summarized in Table 1. Besides, all the
newly synthesized LLY-507 analogues were assessed for their inhibitory activities
against SMYD2 via in vitro methylation transfer assay. LLY-507 was employed as the
positive control.20 The IC50 values were illustrated in Table 1.
Table 1. SMYD2 inhibitory activity of the focused compound.
PTS (oC) a
Compound
Compd. protein
:
IC50 (nM)

20:1 10:1
5:1
5
7
NEb
NE
3.1
2.7
NE
NE
NE
NE
1.2
NEb
NE
2.2
2.5
0
0.23 0.09
0.37 0.09
-1.3
3.5
3.7
NE
NTc
NT
3.1
NT
NE
NE
NE
NE
NT
NTc
NT
NT
NT
-1.0
NE 1699
NE >5000
NT 75
17
18
19
22
23
24
28
31
32
33
34
39
40
41
42
43
44
45
46
47
LLY-50
7
3
53
NT 1502
NT >5000
NT >5000
NT >5000
NE 81
NE 1914
NE 2415
1.6 48
NE 57
NT 522
NT 1002
NT 891
-0.61 NT >2500
NT
2.8
NE
3.0
1.0
3.6 17
NT 50
NT 1469
NT 24
NT 57
3.4
1.4
3.0
2.9
3.5 71
c
a PTS is a binding experiment between protein and compound. b NE: no effect.
NT: not tested.
As shown in Table 1, compounds 5 and 7 without 1, 1'-biphenyl group were
inactive, suggesting the 1,1'-biphenyl group was critical for maintaining the SMYD2
inhibition. Hence, we synthesized LLY-507 analogues by replacement of 1,1'-biphenyl
group with 2-(pyridin-3-yl)benzoic acid. Notably, compound 18 bearing a
4-(3-methyl-1H-indol-1-yl)butan-1-amine tail showed potent SMYD2 inhibitory
activity with an IC50 value of 53 nM and a fluorescence-based thermal shift assay
indicated that compound 18 dose-dependently shifted the melting temperature (Tm)
of SMYD2 for 2.73 oC (Fig. 3). The inhibitory activity was also substantially
maintained when the 4-(3-methyl-1H-indol-1-yl)butan-1-amine was replaced by
4-(1H-indol-1-yl)butan-1-amine. However, introducing other long-chain groups such

as 4-(1H-benzo[d]imidazol-1-yl)butan-1-amine (19), octan-1-amine (22), putrescine
(23), or N-(4-aminobutyl)cyclopropanecarboxamide (24) would lead to a dramatical
loss of SMYD2 inhibitory potency of compounds, indicating that the tolerance of this
position was limited. Comparing of compounds 33, 28 with 43, it can be suggested
that the piperazine group was critical for maintaining the SMYD2 inhibition. Hence,
we examined how the replacement of the side indole group (site C) might affect SAR.
We prepared analogues with various alternatives to the piperazine and observed
that some of these modifications were tolerated. Notably, an attempt to introduce
the 2-(3,4-dichlorophenyl)acetic acid led to compound 43 showing 4-fold improved
potency with an IC50 value of 17 nM, whereas replacement with 3,4-dichlorobenzoic
acid
(41)
was
inactive.
Furthermore,
compound
39
with
N-(3,4-dichlorophenyl)acetamide suffered a significant loss in potency. However,
compound 46 with 3-(3,4-dichlorophenyl)propanoic acid substituted on the
piperazine exhibited better inhibitory activity than LLY-507. Additionally,
introduction of 2-phenylacetic acid (40) and ibuprofen (42) also led to significantly
decreased in potency. We attributed this potency decrease to their unfavorable
interaction with hydrophobic pocket in the binding site of the protein. The
replacement of 1-ethyl-3-methyl-1H-indole with 2-(1H-indol-3-yl)acetic acid (44) or
tryptophan (47) did not affect the potency, further indicating that this region would
be combined with electrophilic warheads to provide Lysine-Targeting covalent
inhibitors for SMYD2.24

Fig. 3. The thermal shift assay displaying the stabilization of SMYD2 by compounds
18, 33, 43 and 44.
Accumulating evidence supports that SMYD2 was overexpressed in gastric cancer
cell lines, and inhibition of SMYD2 expression suppresses gastric cancer cell
proliferation, survival, and tumor growth in vitro and in vivo.25 Therefore, we further
tested whether compounds 17-19, 22, 33, 34, 43, 44, and 46 could inhibit the cell
proliferation rate of SMYD2 over-expressed gastric cancer cell lines AGS and NCI-87
(Table 2). Compounds 17-19 and 22 exhibited no inhibitory activity on AGS cells at
low concentrations, which could arise from the reduced cell membrane permeability.
As shown in Table 2, both compounds 43, 44, and 46, the most potent SMYD2
inhibitors, exhibited significantly anti-proliferative activity against gastric cancer cell
lines AGS (Fig. 4) and NCI-87. Among them, compound 44 (IC50 = 2.3 μM and 3 μM)
displayed the best potency and efficacy in AGS and NCI-87 cell inhibition assays
respectively, which was two-fold higher than LLY-507.
Table 2. In vitro anti-proliferative activity of newly synthesized compounds against
human gastric cancer cell lines.
IC50 (μM)
AGS NCI-N87
IC50 (μM)
AGS NCI-N87
Compd.
Compd
17
18
19
22
33
50
20
50
20
34
43
44
46
17
NT
5.8 6.0
2.3 3.0
4.6 5.0
100 100
50 50
17.6 NTa
LLY-507 5.5 7.0
a NT: not tested.

Fig. 4. The cell viability of the selected compounds in AGS cells.
The SARs of LLY-507 analogous as SMYD2 inhibitors were summarized in Fig. 5.
Firstly, site A was critical for maintaining SMYD2 inhibition. When the piperazine (site
B) group was replaced with 3-methoxyazetidine or 4-methoxypiperidine decreased
activity was observed against SMYD2. Inhibitory activities also revealed that the
substitution of the amino group leads to a significant loss in potency. An amino
group (site C) was indispensable for exerting inhibitory activity. Another finding was
that a bulkier terminal site C group resulted in reduced activity (43 to 47) from 17
nM to 57 nM. Activity gradually decreased when the site C group was larger than the
ethyl group; in particular, 46 and 42, with the largest group, showed no inhibitory
activity toward SMYD2. Furthermore, comparing 41, 45 with 43, it could be
concluded that the piperazine group linked by an ester group (n = 2) was essential
for activity. LLY-507 analogues with the hydrophobic group on site C appeared to be
more favorable in general and further indicating that this region was more flexible
and could be replaced by many functionalized groups.

N
A
H
N
N
O
C
Flexible sidechain
with hydrophobic
group enhanced potency
N
B
n
( )
N
N
1,1'-biphenyl: essential
group for the activity
n = 2 is critical for the activity
O
O
N
>
N
>
N
N
Fig. 5. SARs of LLY-507 analogous as SMYD2 inhibitors.
In summary, a series of novel LLY-507 analogs were synthesized and evaluated for
their SMYD2 inhibitory activity. Among them, compounds 43 and 44 could inhibit the
proliferation of gastric cancer cell lines AGS and NCI-87, which was equivalent to or
better than LLY-507. Our results showed that the sites A and B are indispensable for
exerting inhibitory activity, while site C was more flexible. Taken together, the site C
could be combined with electrophilic warheads such as acrylamides and vinyl
sulfones via amide reaction to provide targeting covalent inhibitors for SMYD2. This
work would facilitate further development of new targeting covalent inhibitors for
SMYD2.
Note
The authors declare no competing financial interest
Acknowledgments
This research was supported by the Natural Science Foundation of Fujian Province
(2019J01202 to H.C.), the Natural Science Foundation of China (81625022, 91853205,
81821005 to C.L.), K. C. Wong Education Foundation to C.L.
A supplementary date
Supplementary data associated with this article can be found, in the online

version, at https://doi.org/xx.
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Graphical abstract
N
N
H
N
N
H
O
N
N
N
O
N
O
N
Cl
Cl
N
N
LLY-507
SMYD2: IC50 = 71 nM
Compound 43
SMYD2: IC50 = 17 nM
Highlights
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A series of LLY-507 analogues were designed and synthesized.
A fluorescence-based thermal shift assay was utilized.
Six analogues exhibited improved potency.
This work would facilitate further development of covalent inhibitors for SMYD2.