Synthesis and biological evaluation of biarylamide derivatives as inhibitors of phagocytosis in macrophages

Full text of DOI:10.1002/bkcs.11036

Note
DOI: 10.1002/bkcs.11036
BULLETIN OF THE
H. J. Kang et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Biological Evaluation of Biarylamide Derivatives as
Inhibitors of Phagocytosis in Macrophages
Hae Ju Kang, Jiho Song, Kee Hyeob Hyun, Seung Hwarn Jeong, Jung Wuk Lee,
*
*
Kwang Woo Hwang, and Kyung Hoon Min
College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea.
E-mail: khwang@cau.ac.kr; khmin@cau.ac.kr
*
Received November 1, 2016, Accepted November 8, 2016, Published online December 28, 2016
Keywords: Phagocytosis, Macrophage, Small molecule, Inhibitor, Biarylamide
Macrophages are a kind of immune cells that play impor-
tant roles in the innate immune system. Their phagocytic
ability contributes not only to the removal of pathogens
and dead cells, but also to tissue remodeling for tissue
KOH gave acid 3. Conversion of acid 3 to acid chloride
and subsequent amide formation with diethylaminopropyla-
mine provided the desired biarylamides 4. Dibutoxy deriva-
tive 8 was synthesized according to the same procedure as
that used to obtain compounds 4a–e.
1
homeostasis. Phagocytosis is a highly complicated process
that involves a number of biomolecules including cell sur-
face receptors and cytoskeletal proteins are involved. Great
Flow cytometry-based phagocytosis assay was used to
evaluate the effects of all synthesized MPS03 derivatives in
RAW264.7 cells, a mouse microphage cell line, as our pre-
2
efforts have been made to discover novel factors involved3
in phagocytosis to elucidate its molecular mechanism.
Immune thrombocytopenia (ITP) is an autoimmune disor-
der, characterized by an increased tendency to bleed and is
caused by the low number of platelet. Patients with ITP
produce antibodies against their own platelets thereby
7,8
viously reported method. All derivatives had increased
activity compared to the parent compound MPS03 and
showed no detectable cytotoxicity at 20 μM (Figure 2). Pen-
tyl derivative 4a exhibited most potent activity among
synthesized compounds. Cyclohexylmethyl compound 4c
was equipotent to compound 4a. However, hexyl derivative
4d and heptyl compound 4e showed less activity than pentyl
compound 4a, indicating that inhibitory activity for phago-
cytosis decrease as the chain length of region A increases.
We have previously reported that butyl derivative was8
slightly less potent compared to 5 μM of cytochalasin D
4
destroying most of them. It has been suggested that plate-
let destruction mainly results from phagocytosis, and that
inhibition of phagocytosis may prevent the loss of plate-
5
lets. Now therefore, it is suggested that small molecule
inhibitors for phagocytosis could be developed as first-in-
class drugs to combat the increased platelet destruction that
6
7
occurs in ITP.
and methyl derivative almost no inhibitory activity. There-
With regard to discover small molecules that regulate
phagocytosis, we have recently reported biarylamide com-
pounds, including MPS03, inhibit macrophage phagocyto-
fore, the optimal alkyl group of region A deemed most
effective is the pentyl group. Dibutoxy derivative 8 showed
activity similar to 5 μM of cytochalasin D. Additional intro-
duction of alkoxy group at meta-position did not have any
significant effect on activity. Although further optimization,
including modification for region B, remains to be carried
out, the finding of this study provide useful information to
establish SAR of this scaffold as a basis to develop more
potent compounds. We believe that this series of compounds
deserve further development as inhibitors of phagocytosis.
7
,8
sis of zymosan (Figure 1). In addition, we demonstrated
that the biarylamides reduce the expression level of Rac1,
Rac2, and Cdc42, which are the key regulators of phagocy-
7
tosis. Furthermore, biarylamides suppressed the production
of pro-inflammatory cytokines by downregulation of
8
dectin-1 in macrophages.
We attempted to find additional potent small molecules
against phagocytosis to further explore structure–activity
relationship (SAR) of the various biaryl amide compounds.
A brief SAR for region C of MPS03 has been previously
Experimental
7
reported. In this study, we describe inhibitory effect of
General Procedure for Synthesis of 4a–e and 8
small molecules, derived from MPS03, on phagocytosis,
with a focus on modification within region A. The synthetic
procedures for MPS03 derivatives are outlined in
Schemes 1. and 2. Various alkyl groups were introduced to
region A by O-alkylation of bromophenol. The alkoxy bro-
mobenzene 1 was converted to nitrile 2 through Suzuki
coupling reaction. Hydrolysis of the nitrile group with
Synthesis of 1a–e and 5. A mixture of bromophenol (1.0
equiv.), alkylbromide (2.0 equiv.), and K CO (excess) in
2
3
ꢀ
DMF was stirred for 1–3 h at 90 C. The reaction mixture
was diluted with acetone, filtered, and the filtrate was con-
centrated in vacuo. The crude mixture was purified by flash
column chromatography on silica gel to afford title com-
pounds 1a–e and 5.
Bull. Korean Chem. Soc. 2017, Vol. 38, 120–122
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filtrate was concentrated and purified by flash column chroma-
tography on silica gel to afford title compounds 2a–e and 6.
A
B
C
O
Synthesis of 3a–e and 7. Biphenyl-3-carbonitrile com-
O
pound was dissolved in EtOH, and mixed with KOH in
ꢀ
N
N
water. The mixture was stirred for 20–60 min at 150 C
H
under microwave irradiation. The reaction mixture was con-
centrated to remove EtOH, acidified by HCl, and extracted
by DCM. Combined organic layer was washed by brine,
dried over Na SO anhydrous, filtered, and concentrated in
MPS03
2
4
Figure 1. Structure of MPS03.
vacuo to afford biphenyl-3-carboxylic acid 3a–e and 7. The
crude product was used next reaction without further
purification.
Synthesis of 4a–e and 8. To a solution of appropriate
biphenyl-3-carboxylic acid in DCM, was added oxalyl
chloride (1.0–2.0 equiv.) and DMF (0.02 equiv.) in DCM
successively. The mixture was stirred for 1–2 h at room
temperature. From the reaction mixture, volatiles were eva-
porated to dryness under reduced pressure. The crude prod-
uct was used next reaction without further purification.
To a solution of appropriate biphenyl-3-carbonyl chlo-
ride (1.0 equiv.) n DCM, N,N-diethyl-1,3-diaminopropane
(1.5 equiv.), and trimethylamine (3.0 equiv.) was added.
The mixture was stirred for 12 h at room temperature. the
reaction mixture was diluted by DCM, washed by water
and brine, dried over Na SO anhydrous, filtered, and con-
Scheme 1. Reagents and conditions: (a) alkyl bromide, K
DMF, 90 C, 1–3 h, 1a (78%), 1b (83%), 1c (69%), 1d (79%), 1e
2
CO
3
,
ꢀ
3 4 2 3 2
(93%); (b) 3-cyanophenylboronic acid, Pd(PPh ) , K CO , H O,
2
4
THF, reflux, 12–24 h, 2a (43%), 2b (35%), 2c (43%), 2d (47%),
centrated under reduced pressure. The crude mixture was
purified by flash column chromatography on silica gel to
ꢀ
2
e (20%); (c) KOH, H
2
O, EtOH, microwave 150 C, 20–60 min,
3
a (75%), 3b (54%), 3c (50%), 3d (87%), 3e (87%); (d) (i) oxalyl
Cl , 1–2 h; (ii) N,N-diethyl-1,3-diaminopro-
N, THF, 12 h, 4a (93%), 4b (99%), 4c (64%), 4d (92%),
e (15%) for two steps.
afford title compounds 4a–e and 8.
chloride, DMF, CH
pane, Et
4
2
2
1
Compound 4a: H NMR (300 MHz, CDCl ): δ 8.68 (br,
3
3
1
H), 8.05 (s, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.65 (d,
J = 7.8 Hz, 1H), 7.55 (d, J = 8.7 Hz, 2H), 7.44 (t,
J = 7.8 Hz, 1H), 6.96 (d, J = 8.7 Hz, 2H), 3.99 (t,
J = 6.6 Hz, 2H), 3.57–3.63 (m, 2H) 2.67–2.77 (m, 6H),
1.76–1.90 (m, 4H), 1.33–1.49 (m, 4H), 1.1 (t, J = 7.2 Hz,
Synthesis of 2a–e and 6. A mixture of 3-cyanophenyl boro-
nic acid (1.2–1.5 equiv.), appropriate aryl bromide (1.0
equiv.), and 2 N-K CO (2.0–5.0 equiv.) in THF was
2
3
6H), 0.94 (t, J = 6.9 Hz, 3H).
1
degassed for 5 min. To the mixture, Pd(PPh ) (0.02 equiv)
Compound 4b: H NMR (300 MHz, CDCl ): δ 8.73
3
4
3
was added and the mixture was refluxed for 12–24 h. The
reaction mixture was diluted with EtOAc, washed by water
and brine, dried over Na SO anhydrous, and filtered. The
(br, 1H), 8.05 (s, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.65
(d, J = 7.8 Hz, 1H), 7.55 (d, J = 8.7 Hz, 2H), 7.44
(t, J = 7.8 Hz, 1H), 6.96 (d, J = 8.7 Hz, 2H), 4.02
2
4
ꢀ
Scheme 2. Reagents and conditions: (a) 1-bromobutane, K
CO , H O, THF, reflux, 12 h, 45 %; (c) KOH, H
–2 h; (ii) N,N-diethyl-1,3-diaminopropane, Et
2
CO
3
, DMF, 90 C, 2 h, 83 %; (b) 3-cyanophenylboronic acid, Pd(PPh
3
)
4
,
,
ꢀ
K
1
2
3
2
2
O, EtOH, microwave 150 C, 20 min, 96 %; (d) (i) oxalyl chloride, DMF, CH
N, THF, 12 h, 32 % for two steps.
2
Cl
2
3
Bull. Korean Chem. Soc. 2017, Vol. 38, 120–122
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Note
KOREAN CHEMICAL SOCIETY
1
.69–1.91 (m, 8H), 1.19–1.33 (m, 5H), 1.09 (t,
J = 7.2 Hz, 6H).
1
Compound 4d: H NMR (300 MHz, CDCl ): δ 8.69 (br,
3
1
H), 8.06 (s, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.66 (d,
J = 7.8 Hz, 1H), 7.56(d, J = 8.7 Hz, 2H), 7.45 (t,
J = 7.8 Hz, 1H), 6.96 (d, J = 8.7 Hz, 2H), 3.99 (t,
J = 6.6 Hz, 2H), 3.58–3.64 (m, 2H), 2.67–2.77 (m, 6H),
1
6
.76–1.92 (m, 4H), 1.30–1.50 (m, 6H), 1.11 (t, J = 7.2 Hz,
H), 0.91 (t, J = 7.2 Hz, 3H).
1
Compound 4e: H NMR (300 MHz, CDCl ): δ 8.59 (s,
3
1
H), 8.09 (s, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.67 (d,
J = 7.5 Hz, 1H), 7.56 (d, J = 8.7 Hz, 2H), 7.46 (t,
J = 7.5 Hz, 1H), 6.97 (d, J = 8.7 Hz, 2H), 3.99 (t,
J = 6.3 Hz, 2H), 3.60–3.65 (m, 2H), 2.78–2.85 (m, 4H),
1
.94–2.00 (m, 2H), 1.76–1.85 (m, 2H), 1.25–1.47 (m, 8H),
1
.17 (t, J = 7.2 Hz, 6H), 0.90 (t, J = 6.3 Hz, 3H).
1
Compound 8: H NMR (300 MHz, CDCl ): δ 8.62 (s,
3
1
H), 8.13 (s, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.67 (d,
J = 8.1 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.16–7.19 (m,
2
3
H), 6.95 (d, J = 8.7 Hz, 1H), 4.02–4.11 (m, 4H),
.60–3.66 (m, 2H), 2.82–2.89 (m, 6H), 2.00 (quint,
J = 6.0 Hz, 1H), 1.78–1.87 (m, 4H), 1.49–1.59 (m, 4H),
.20 (t, J = 7.2 Hz, 6H), 0.96–1.01 (m, 6H).
1
Acknowledgment. This work was supported by the
National Research Foundation of Korea (NRF) grant
funded by the Korea government (MSIP) (NRF-
2
015R1A5A1008958)
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Figure 2. Inhibitory effect of derivatives on phagocytosis of
zymosan by RAW264.7 cells. (a) Phagocytosis of zymosan was
analyzed using flow cytometry. All compounds were evaluated at
1
2
2
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1
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2
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8
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Bull. Korean Chem. Soc. 2017, Vol. 38, 120–122
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