Synthesis, biological evaluation, and metabolic stability of chlorogenic acid derivatives possessing thiazole as potent inhibitors of α-MSH-stimulated melanogenesis

Article abstract of DOI:10.1016/j.bmcl.2017.09.044

A series of catechol and dioxolane analogs containing thiazole CGA derivatives have been synthesized and evaluated for their inhibitory activity against α-MSH. The inhibitory activity was improved by replacing an α,β-unsaturated carbonyl of previously reported caffeamides with thiazole motif. Surprisingly, compound 7d, one of the derivatives of dioxolane analogs, displayed the most potent inhibitory activity with an IC₅₀ of 0.90 μM. Further studies on metabolic stability and bioactivation potential were also accomplished.

Full text of DOI:10.1016/j.bmcl.2017.09.044

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation, and metabolic stability of chlorogenic
acid derivatives possessing thiazole as potent inhibitors of
stimulated melanogenesis
a-MSH-
Hyeju Jo a,e, Yuanyuan Zhou b,e, Mayavan Viji a,e, Minho Choi a, Jae Young Lim a, Jaeuk Sim a,
Jeongtae Rhee a, Youngsoo Kim a, Seung-Yong Seo c, Wun-Jae Kim d, Jin Tae Hong a, Heesoon Lee a,
a,
Kiho Lee b, , Jae-Kyung Jung
⇑
⇑
a College of Pharmacy and Medicinal Research Center (MRC), Chungbuk National University, Cheongju 28160, Republic of Korea
b College of Pharmacy, Korea University, Sejong 30019, Republic of Korea
c College of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 21936, Republic of Korea
d College of Medicines, Chungbuk National University, Cheongju 28644, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of catechol and dioxolane analogs containing thiazole CGA derivatives have been synthesized and
Received 14 August 2017
Revised 14 September 2017
Accepted 20 September 2017
Available online xxxx
evaluated for their inhibitory activity against
an ,b-unsaturated carbonyl of previously reported caffeamides with thiazole motif. Surprisingly, com-
pound 7d, one of the derivatives of dioxolane analogs, displayed the most potent inhibitory activity with
a-MSH. The inhibitory activity was improved by replacing
a
an IC50 of 0.90
accomplished.
lM. Further studies on metabolic stability and bioactivation potential were also
Keywords:
Chlorogenic acid (CGA)
Melanogenesis
Ó 2017 Elsevier Ltd. All rights reserved.
a-MSH
Caffeamide
Thiazole
Chlorogenic acid (CGA) is a natural polyphenolic compound
found in various agricultures such as coffee, beans, and particularly
Lonicera japonica.1 According to previous studies, CGA exhibits var-
ious biological activities such as anti-bacterial, anti-inflammatory,
anti-oxidant, and anti-carcinogenic activities.2–5 In addition, CGA
mation.9–11 Thus, seeking a novel structure replacing the undesired
,b-unsaturated carbonyl is of great significance. In this regard, to
improve drug-like properties, we have replaced the ,b-unsatu-
rated carbonyl subunit of caffeamide with thiazole scaffold
(Fig. 1).12–14 Thiazole has structural similarities with the
,b-unsat-
a
a
a
and caffeic acid derivatives were found to inhibit
a
-MSH-induced
urated carbonyl and has one of the most important structural
motifs of heteroaromatic compounds in medicinal chemistry, due
to its wide range of biological activities. Its derivatives showed
anti-bacterial, antifungal, anti-HIV, anti-inflammatory and antitu-
mor activities.15–19
Moreover, Kim et al. demonstrated that a derivative of 2-
aminothiazole inhibits melanogenesis in B16 mouse melanoma
cells.20 We also proposed to replace the catechol with the diox-
olane for pharmacophore identification. In addition, we selected
trifluoromethyl- and chlorobenzylamine which have significant
melanogenesis.6,7
Recently, our group reported CGA derivatives such as caffeate
and caffeamide to show the strong inhibitory activity against
MSH-induced melanogenesis (up to 0.2
M).8 However, these com-
pounds might have some problems in terms of chemical stability
a
-
l
and drug-like properties, mainly due to the
bonyl moiety.
a,b-unsaturated car-
Furthermore, the a,b-unsaturated carbonyl group in drug candi-
date could act as the strong electrophile for Michael addition with
a variety of nucleophiles in living system (e.g. cysteine, nucleic acid
etc.), resulting in the formation of non-selective covalent bond for-
inhibitory activities against a-MSH-induced melanogenesis in the
previous caffeamide series.8 Herein, we report our design and bio-
logical evaluation of novel derivatives possessing the thiazole moi-
ety. Besides, its derivatives were characterized for their
metabolism properties.
⇑
Corresponding authors.
E-mail addresses: kiholee@korea.ac.kr (K. Lee), orgjkjung@chungbuk.ac.kr (J.-K.
Jung).
e
These authors contributed equally to this work
https://doi.org/10.1016/j.bmcl.2017.09.044
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jo H., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.09.044

2
H. Jo et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 1. Design of new CGA derivatives using molecular modifications.
The synthesis of a series of compounds 3 was shown in
Scheme 1. Various benzylamine derivatives 1 were treated with
c-HCl, followed by addition of potassium thiocyanate, to afford
thiourea 2.21 Finally, the cycloaddition of thiourea 2 with commer-
cially
available
2-chloro-1-(3,4-dihydroxyphenyl)ethanone
afforded the desired compounds 3a–3g.22
The synthesis of derivatives having dioxolane was depicted in
Scheme 2.
a-Bromination of acetophenone 4 with NBS and p-TsOH
in acetonitrile afforded
a
-bromoacetophenone 5, which was con-
Scheme 2. Synthesis of dioxolane derivatives possessing thiazole. Reagent and
conditions: (a) NBS, p-TsOH, CH3CN, rt, 24 h, 76%; (b) Thiourea, EtOH, 80 °C, 24 h,
41%; (c) NaH, benzyl bromide, THF, 10 min, 17–21%.
verted to 2-aminothiazol 6 via cycloaddition with thiourea.23–25
N-Benzylation of 6 with various benzyl bromides in the presence
of sodium hydride gave the desired compounds 7a–7g.22
Synthesized analogs were evaluated for their inhibitory activi-
ties against a-MSH-induced melanogenesis in B16 melanoma cells
as shown in Tables 1 and 2. The compound 3a bearing a simple
benzyl group and the compound 3e bearing an o-chlorobenzyl
group showed slightly higher activities than the corresponding caf-
Table 1
The inhibitory activity of catechol analogs.
feamide analogs 8a and 8e, respectively (2.1
lM of 3a vs 8.0 lM of
8a and 2.0 M of 3e vs 6.5
l
l
M of 8e).8 The compounds 3b and 3c
bearing a trifluoromethyl group exhibited significant inhibitory
effects. However, these results presented reduced activities, com-
pared to the corresponding caffeamides 8b and 8c (2.6
lM of 3b
vs 1.8 M of 8b and 1.8 M of 3c vs 1.4
l
l
l
M of 8c).8 Generally,
the inhibitory potency was not significantly enhanced, when intro-
ducing thiazole to improve drug-likeness (Table 1).
Next, the effects of dioxolane on the activity were examined. All
the tested compounds showed reduced activities compared to the
series 3 with the exception of compound 7d. The compound 7d
with p-CF3 group exhibited outstanding inhibitory activity
(0.90 lM). Moreover, the effect of electron withdrawing sub-
stituent on the inhibitory activity would not be position-depen-
dent (Table 2). For example, the compounds 7d with p-CF3
compound
R1
R2
R3
IC50 (l
M)a
3a
3b
3c
3d
3e
3f
H
CF3
H
H
Cl
H
H
H
CF3
H
H
H
H
H
CF3
H
2.1 0.15
2.6 1.1
1.8 0.11
32.7 5.8
2.0 0.058
>50
showed more potent effect (0.90
lM) than that of the compound
7g (37.9 M) with p-Cl. On the other hand, there is no much differ-
l
ence in their activity when these groups existed in ortho or meta
positions.
Metabolic stability of 7d was compared to that of its corre-
sponding caffeamide analog 8d in vitro in human liver S9 factions
Cl
H
H
Cl
3g
H
11.5 4.4
a
Data are taken as a mean from 3 independent experiments.
(1 mg/mL) fortified with 1 mM nicotinamide adenine dinucleotide
phosphate (NADPH), acid
uridine-50-diphosphoglucuronic
(UDPGA) and 30-phosphoadenosine-50-phosphosulfate (PAPS) in
50 mM Tris buffer (pH 7.5) (Fig. 2). Sample analyses were con-
ducted by an Agilent 6530 Q-TOF LC-MS/MS system. Compound
8d was metabolized rapidly with a t1/2 of ꢀ5 min while compound
7d metabolism was much slower showing a t1/2 of 69.3 min (Fig. 2).
Hepatic clearance (CLH) values predicted from these metabolic sta-
bility data using ‘well-stirred’ model of the liver were 19.1 and
Scheme 1. Synthesis of catechol derivatives possessing thiazole. Reagent and
conditions: (a) (i) c-HCl; (ii) KSCN, THF, 80 °C, 24 h, 11–19% (two steps); (b) 2-
Chloro-1-(3,4-dihydroxyphenyl)ethanone, EtOH, 80 °C, 24 h, 32–35%.
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H. Jo et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 2
The inhibitory activity of dioxolane analogs.
compound
R1
R2
R3
IC50 (l
M)a
7a
7b
7c
7d
7e
7f
H
CF3
H
H
Cl
H
H
H
CF3
H
H
H
H
H
CF3
H
11.1 1.3
9.6 0.11
15.3 1.0
0.90 0.10
12.4 2.0
13.2 0.90
37.9 1.8
Cl
H
H
Cl
7g
H
a
Data are taken as a mean from 3 independent experiments.
Fig. 3. Proposed metabolic pathway of 8d (A) and 7d (B) in human liver
microsomes and S9 fractions. Gluc: glucuronide; SULT: sulfotransferase; UGT:
uridine-50-diphospho-glucuronosyltransferase.
8d produced a metabolite of m/z 641.1532 detected in negative ion
mode ([MꢁH]ꢁ), with a mass increase of 305 Da from that of the
parent. This mass change is characteristic of a GSH conjugation
with the oxidized metabolite (i.e. o-quinine) of a catechol-contain-
ing compound as in 8d (Fig. 3A). Furthermore, its MS/MS spectrum
also showed product ions characteristic of a GSH conjugate (i.e. m/z
272, 254, 143 and 128).32 On the contrary, no metabolites with m/z
value 307 Da greater than that of 8d were detected when incu-
bated in the absence of NADPH, suggesting that 8d itself was not
reactive enough to form a GSH conjugate, but was bioactivated
via oxidation. No GSH conjugates were detected for 7d in the
absence and presence of NADPH, indicating that this compound
was neither reactive nor bioactivated in the given incubation
condition.
Fig. 2. Metabolic stability of 7d and 8d in human liver S9 fractions fortified with
NADPH, UDPGA and PAPS. Data are expressed as mean S.D. (n = 3).
12.2 mL/min/kg for 8d and 7d, respectively; these CLH values corre-
spond to hepatic extraction ratio (EH) of 0.96 and 0.61, indicative of
a high and a moderate clearance for 8d and 7d, respectively.26–29
Metabolite identification was conducted using the high-resolu-
tion mass spectral data obtained from the analytical results of the
aforementioned metabolic stability studies.30 As shown in
Fig. 3A, 8d was metabolized by both sulfation (M1) and glu-
curonidation (M2) most likely by the catechol moiety. Compound
7d was shown to be metabolized by monooxidation of the thiazole
and the dioxolane ring leading to a sulfoxide metabolite (M1) and a
demethylenated metabolite (M2), respectively; M2 was shown to
be further metabolized by sulfation (M3) and glucuronidation
(M4) most likely by the catechol moiety (Fig. 3B).
In summary, a series of novel catechol and dioxolane analogs
possessing thiazole were synthesized and their inhibitory activity
was evaluated against
a-MSH-induced melanogenesis in B16
Glutathione (GSH) trapping assay was conducted with 8d and
7d to investigate their chemical reactivity and bioactivation poten-
tial.31 Test compounds were incubated with human liver micro-
somes (1 mg/mL) fortified with 1 mM NADPH and glutathione
(GSH) in 0.1 M potassium phosphate buffer (pH 7.4); the reaction
was terminated after 30-min incubation and the samples were
analyzed by an Agilent 6530 Q-TOF LC-MS/MS system. As a result,
mouse melanoma cells. Compound 3a, 3b, 3c, 3e and 7d were iden-
tified to show significant inhibition. Especially, 7d showed signifi-
cantly higher inhibitory effect compared to the previous series.
Additionally, it was demonstrated that protection of catechol moi-
ety with dioxolane ring formation and replacement of a,b-unsatu-
rated carbonyl with thiazole ring can mitigate the issues of poor
metabolic stability and bioactivation. Thus, these analogs represent
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4
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