Synthesis, characterization, and biological evaluation of oxadiazole derivatives bearing 5-phenyl-tetrazole as osteoclast differentiation inhibitors

Article abstract of DOI:10.1002/bkcs.10436

Novel oxadiazoles bearing 5-phenyl-tetrazole (5a-k) were designed and efficiently synthesized by treating 2-(5-phenyl-2H-tetrazole-2-yl)acetohydrazide (4) with aromatic carboxylic acids in POCl₃, and their in vitro anti-osteoclastogenic activities were evaluated. In the cell-based osteoclast differentiation model, all compounds (5a-k) inhibited the formation of osteoclasts. In addition, the potential target molecules of compound 5 analogs were predicted with their chemical substructures via a web-based interface, and some of them were found to be related to osteoclast differentiation. Consequently, the scaffold containing oxadiazole-tetrazole in a single molecule and their analogs are of potential use in the design of novel anti-osteoclastogenic therapeutics.

Full text of DOI:10.1002/bkcs.10436

Article
DOI: 10.1002/bkcs.10436
BULLETIN OF THE
S.-H. Moon et al.
KOREAN CHEMICAL SOCIETY
Synthesis, Characterization, and Biological Evaluation
of Oxadiazole Derivatives Bearing 5-Phenyl-tetrazole as
Osteoclast Differentiation Inhibitors
Seong-Hee Moon,† Muhammad Latif, Muhammad Qasim, Sik-Won Choi, Joo Yun Lee,
,k
‡,k
§
†
¶
¶
§,
†,
*
*
Byung Jin Byun, Aamer Saeed, and Seong Hwan Kim
†Laboratory of Translational Therapeutics, Korea Research Institute of Chemical Technology, Daejeon
305-600, Republic of Korea. *E-mail: hwan@krict.re.kr
‡
Center for Catalytic Hydrocarbon Functionalization, Institute for Basic Science (IBS) and Department of
Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea
§
Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan.
*
E-mail: aamersaeed@yahoo.com
Drug Discovery Platform Technology Group, Korea Research Institute of Chemical Technology, Daejeon
05-600, Republic of Korea
Received April 24, 2015, Accepted May 15, 2015, Published online August 12, 2015
¶
3
Novel oxadiazoles bearing 5-phenyl-tetrazole (5a–k) were designed and efficiently synthesized by treating 2-
5-phenyl-2H-tetrazole-2-yl)acetohydrazide (4) with aromatic carboxylic acids in POCl , and their in vitro
(
3
anti-osteoclastogenic activities were evaluated. In the cell-based osteoclast differentiation model, all com-
pounds (5a–k) inhibited the formation of osteoclasts. In addition, the potential target molecules of compound
5
analogs were predicted with their chemical substructures via a web-based interface, and some of them were
foundtoberelatedtoosteoclastdifferentiation. Consequently, thescaffoldcontainingoxadiazole–tetrazoleina
single molecule and their analogs are of potential use in the design of novel anti-osteoclastogenic therapeutics.
Keywords: Bone, Osteoclastogenesis, Oxadiazole, Tetrazole
Introduction
biologically active molecules has resulted in the never-ending
search to generate libraries for biological screening. The oxa-
7
Continuous bone remodeling by osteoclast-mediated resorp-
tion and osteoblast-mediated mineralization (or bone forma-
tion) maintains the bone's density and strength. However, an
imbalance of bone remodeling due to the overactivation of
osteoclasts and/or their weakness results in bone loss, which
consequently leads to pathological bone-related disorders,
such as osteoporosis, rheumatoid arthritis, periodontal dis-
diazole ring skeleton is an attractive structural motif due to its
biological and pharmacological activity and is generally con-
sidered among the privileged heterocyclics due to its large
impact in drug discovery programs against numerous
8–12
diseases.
Typically, the privileged scaffolds of 1,3,4-oxa-
diazoles are very good bioisosteres (surrogates) of amides and
esters, which contribute substantially to increased pharmaco-
logical activity. Because of the promising potential of 1,3,4-
oxadiazoles to engage in H-bonding interactions with
the receptors and promising metabolic profiles, numerous
1–4
ease, and cancer bone metastasis. Especially, bone-related
disorders accompanied by subsequent fractures impact clini-
cal and public health because fractures are one of the most
commoncausesofdisabilityandareassociated withenormous
1
3
biological activities such as antitumor,
antimicrobial,
5
14
15
16
healthcare expenditure.
antimitotic,
anticonvulsant, antiinflammatory,
antifungal,
antihypertensive,
and muscle relaxant
17
18–20
21
Since mature tartrate-resistant acid phosphatase (TRAP)-
+
22
positive multinucleated osteoclast cells (TRAP -MNCs)
have been reported. Interestingly, among the four isomers of
oxadiazoles, 1,3,4- oxadiazole core is a key building block in
several therapeutic agents, such as furamisole (as nitrofuran
antibacterial agent), nesapidil (antiarrhythmic agent), tiodazo-
sin (antihypertensive agent), raltegravir (antiretroviral agent
used for the treatment of HIV infection), translarna (used for
treating patients with nonsense mutation Duchenne muscular
derived from bone-marrow-derived macrophages (BMMs)
by receptor activator of nuclear factor-κB ligand (RANKL)
mediate bone resorption and their lifespan is shorter than that
of osteoblasts, the therapeutic strategy to inhibit osteoclast dif-
ferentiation and/or resorption is important to prevent and treat
6
bone-related disorders.
23
The synthesis of the heterocyclic core is of immense value
in medicinal chemistry, and the intense drive for novel
dystrophy), and zibotentan (anticancer agent).
Tetrazole with highest nitrogen content is another privi-
leged stable scaffold. Despite the fact that tetrazole does not
occur naturally, several novel drugs such as TAK-456
k These authors contributed equally to this work.
Correction added on 01 October 2015, after first online publication: ISSN (Print) has been corrected.
Bull. Korean Chem. Soc. 2015, Vol. 36, 2247–2253
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Article
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ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
2
4,25
+
(
antifungal agent),
losartan, irbesartan (angiotensin II
induced the differentiation of BMMs into 181 TRAP -MNCs,
2
6
27
receptor antagonists),
antimycobacterial compounds,
anti-inflammatory compounds,
but 5a–k at 10 μM significantly inhibited the formation of
TRAP -MNCs. Compound 5k exhibited the strongest anti-
2
8
+
and antihypertensive
29
agents contain tetrazole ring as a structural fragment. How-
ever, the use of 2,5-disubstituted tetrazoles as biological agents
has not been reported well, but, because of its resistance to bio-
logical degradation, it is possible to use this structural motif as
isosteric substituents of various functional groups in the devel-
opment of biologically active substances. To this end, 2,5-
osteoclastogenic activity.
The dose-dependent anti-osteoclastogenic activity of 5k
was further evaluated. When BMMs differentiated into
TRAP -MNCs by RANKL, 5k was shown to inhibit the for-
+
+
mation of TRAP -MNCs in a dose-dependent manner
(Figure 1(a)). No cytotoxicity in BMMs by 5k was observed
at theconcentrations usedin this study, suggesting that its anti-
osteoclastogenic activity could not be due to the cytotoxicity
(Figure 1(b)). Anti-osteoclastogenic activity of 5k was also
30
disubstituted tetrazoles as glutamate receptor modulators
and antiviral viral agents have been reported a decade ago.
31,32
Keeping in view the individual significance of both oxadia-
zole and tetrazole pharmacophores, here we report the synthe-
sis of tetrazole–oxadiazole conjugates (5a–k) incorporating
the tetrazole and oxadiazole units connected through one car-
bon bridge as anti-osteoclastogenic agents. The newly synthe-
sized oxadiazoles decorated with 5-phenyltetrazoles hybrids
were investigated for unmapped region of these privileged
pharmacophores as osteoclast differentiation inhibitors.
Table 1. Inhibitory effect of 5a–k on the formation of osteoclasts
.
Results and Discussion
+
No. of TRAP -MNCs (at 10 μM) (vehicle
Cpds
R
control; 181.67 ꢀ 7.64)
Chemistry. Our study commenced with the preparation of the
intermediate 5-phenyl-2H-tetrazole (2) from readily available
benzonitrile (1), as shown in Scheme 1. The substrate, 5-
phenyl-2H-tetrazole (2), was further alkylated with methyl
chloroacetate to provide methyl 2-(5-phenyl-2H-tetrazol-2-
yl)acetate (3) in 73% yield, followed by its conversion into
the corresponding 2-(5-phenyl-2H-tetrazol-2-yl)acetohydra-
zide (4) in excellent yield. The heterocyclization to asymmet-
rical 2,5-diaryl(alkyl)-1,3,4-oxadiazoles (5a–k) was
accomplished through the condensation of acyl hydrazide
5a
135.00 ꢀ 2.65
5
b
c
102.33 ꢀ 24.42
100.33 ꢀ 1.15
5
(
4) with suitably substituted aromatic carboxylic acids in pres-
ence of phosphorous oxychloride.
Biology. Inhibitory effect of 5a–k on the formation of osteo-
clasts. The effect of 5a–k on osteoclast differentiation was
5d
5e
85.67 ꢀ 6.03
84.67 ꢀ 22.03
+
evaluated by counting TRAP -MNCs after BMMs were trea-
ted with each compound in the presence of RANKL and cul-
tured for 4 days. TRAP is a well-known biomarker of
5
f
75.00 ꢀ 8.72
70.33 ꢀ 12.66
3
3
osteoclast differentiation, and the fusion of monocyte
BMMs to form giant MNCs is required for mature osteoclasts
5g
3
4
to functionally resorb bone. As shown in Table 1, RANKL
5
h
i
65.33 ꢀ 4.51
64.00 ꢀ 7.00
5
5
5
j
52.33 ꢀ 7.51
28.00 ꢀ 16.09
k
Scheme 1. Synthesis of 5a-k.
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Antiosteoclastic Activity of Oxadiazole Derivatives
KOREAN CHEMICAL SOCIETY
Figure 1. Anti-osteoclastogenic activity of 5k. (A) After BMMs were cultured for 4 days in the presence of RANKL (10 ng/mL) and M-CSF (30
ng/mL) withthe vehicle (0.1% DMSO)or 5k, multinucleated osteoclasts were visualized byTRAPstaining. Also, theeffect of 5k onthe viability
+
of BMMs (B), the formation of TRAP -MNCs (C), and mRNA expression levels of gene related to osteoclast differentiation (D) were evaluated
#
##
as described in “Materials and Methods”. , P < 0.001 (vs. the negative control); *, P < 0.05; **, P < 0.01; ***, P <0.001 (vs. the RANKL-
treated group).
+
confirmed by counting the number of TRAP -MNCs
(
identified as the potential binding partners of compound 5 ana-
logs, and five proteins, namely histone-lysine N-methyltrans-
ferase, H3 lysine-9 specific 3 (G9a), glucagon-like peptide
1 receptor (GLP-1R), prelamin-A/C, DNA polymerase β,
and voltage-gated T-type calcium channel α-1H subunit, were
identified as potential functional molecules to show the bio-
logical activityofcompound 5analogs. Amongthem, theinhi-
bition of P2X7 or G9a has been shown to block osteoclast
differentiation. P2X7 expression in osteoclasts has been
Figure 1(c)) and measuring the mRNA expression levels of
the gene related to osteoclast differentiation such as c-Fos,
nuclear factor of activated T cells c1 (NFATc1), TRAP, the
dendritic cell-specific transmembrane protein (DC-STAMP),
and the d2 isoform of vacuolar (H+) ATPase V0 domain
(ATP6v0d2).
Two main transcription factors, c-Fos and NFATc1, are
induced by RANKL in the early-to-middle and middle-to-late
3
5
40
stages of osteoclast differentiation, respectively. The tempo-
ral induction of these transcription factors plays a role in the
transcriptional regulation of specific genes related to osteo-
clast differentiation, migration, fusion, and/or resorption.
Especially, DC-STAMP and ATP6v0d2 contain multiple
reported, and RAW264.7 cells that lacked P2X7 function
have been shown to fail to fuse to form multinucleated osteo-
41
clasts in response to RANKL. The structural similarity
between 5k and the P2X7 antagonist CHEMBL1096099 was
calculated to be about 61%. BIX01294, a specific inhibitor of
3
6
42
NFATc1 binding sites in their promoter regions, andthepro-
teins encoded by both of these genes are reported to play roles
G9a, suppressed osteoclast differentiation. In a further study,
the involvement of P2X7 or G9a in the anti-osteoclastogenic
action of compound 5 analogs would be investigated in detail.
37,38
in osteoclast fusion.
In silico Target Prediction. In order to predict the biological
targets of compound 5 analogs, the chemicals with the similar
substructure as compound 5 analogs were identified in the
ChEMBL database. As shown in Table 2, two proteins, car-
boxypeptidase B and P2X purinoceptor 7 (P2X7), were
Experimental
39
Chemistry. Allchemicals werereagentgrade andusedaspur-
chased. Reactions were monitored by TLC. Flash column
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Antiosteoclastic Activity of Oxadiazole Derivatives
KOREAN CHEMICAL SOCIETY
Table 2. Target prediction for 5k.
CHEMBLID
Structure
Target protein (UnitProtKB)
Carboxypeptidase B (P15086)
1
. Assay plate form: Binding assay
CHEMBL552336
CHEMBL1096099
P2X purinoceptor 7 (Q99572)
2
. Assay plate form: Functional assay
Histone-lysine N-methyltransferase, H3 lysine-9 specific
CHEMBL1318385
CHEMBL1310954
3
(Q96KQ7)
Glucagon-like peptide 1 receptor (P43220)
Prelamin-A/C (P02545)
DNA polymerase beta (P06746)
Glucagon-like peptide 1 receptor (P43220)
Voltage-gated T-type calcium channel alpha-1H subunit
CHEMBL1408371
(
O95180)
chromatographywascarried out onsilica gel (230–400 mesh).
1
13
H NMR and C NMR spectra were recorded in δ units
relative to deuterated solvent as an internal reference using a
00 MHz NMR instrument. Liquid chromatography-tandem
5
mass spectrometry analysis was performed on an electrospray
ionization (ESI) mass spectrometer with photodiode-array
detector (PDA) detection (Scheme 2).
Scheme 2. Synthesis of probe 2.
Scheme 3. Synthesis of probe 3.
Synthesis of 5-Phenyl-2H-tetrazole (2). A mixture of
sodium azide (17.05 g, 262 mmol), benzonitrile (25.75 g,
2
50 mmol), and zinc (II) chloride (17 g, 120 mmol) was sus-
+
pended in H O (250 mL). The reaction mixture was refluxed
8.02–8.06 (m, 2H). LC-MS (EI) for C
H N (M+H) : m/z =
7 6 4
2
for 15 h. After consumption of benzonitrile, the mixture was
cooled to room temperature and filtered. The solid residue
147.06, Exact mass:146.06 (Scheme 3).
Preparation of Methyl 2-(5-phenyl-2H-tetrazole-2-yl)ace-
tate (3). A mixture of 5-phenyl-tetrazole 2 (18.26 g, 120
mmol), triethyl amine (50.59 g, 500 mmol), and methyl chlor-
oacetate (27.13 g, 250 mmol) was refluxed in 150 mL of
was treated with 3 N HCl (250 mL) to give the desired product
ꢁ
2
as a white crystalline solid (21.0 g, 78% yield). Mp 216 C:
1
H NMR (300 MHz, DMSO-d ) δ 7.59–7.62 (m, 3H),
6
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Antiosteoclastic Activity of Oxadiazole Derivatives
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acetonitrile was refluxed for 2 h. The completion of reaction
was checked by TLC. On completion of the reaction, the sol-
vent was removed under reduced pressure and the solid prod-
uct obtained was recrystallized from n-hexane to furnish the
pure methyl 2-(5-phenyl-2H-tetrazole-1-yl)acetate 3 (25 g,
2H), 7.44–7.75 (m, 7H), 8.09 (m, 2H); LC-MS (EI) for
+
C H FN O (M+H) : m/z = 323.30, Exact mass: 322.0978.
16
11
6
2-(2-Bromophenyl)-5-((5-phenyl-2H-tetrazol-2-yl)
methyl)-1,3,4-oxadiazole (5b): Light brown solid (Yield:
ꢁ
−1
90%), mp. 257–260 C; FT-IR (neat) ν (cm ): 1159 (C–O–
ꢁ
white crystalline solid) in 73% yield. Mp 98–100 C; FT-IR
C), 1285 (N N–N), 1540 (Ar–C C), 695 (−C–Br), 1611
(C N), 3057 (Ar–CH); H NMR (300 MHz, DMSO-d ) δ
5.63 (s, 2H), 7.32–8.01 (m, 7H), 8.03 (m, 2H); LC-MS
−
1
1
(
(
neat) ν (cm ): 1281 (N N–N), 1547 (Ar–C C), 1606
6
C N), 1756 (C O), 2854 (CH ), 3067 (Ar–CH); 1H
3
+
NMR (300 MHz, DMSO-d ) δ 3.75 (s, 2H), 5.93 (s, 2H),
(EI) for C H BrN O (M+H) : m/z = 383.12, Exact mass:
6
16 11
6
7
.56-7.59 (m, 3H), δ 8.06-8.10 (m, 2H). LC-MS (EI) for
382.0177.
+
C H N O (M+H) : m/z = 219.09, Exact mass:218.0804.
2-(2-Chlorophenyl)-5-((5-phenyl-2H-tetrazol-2-yl)methyl)-
1,3,4-oxadiazole (5c): Yellowish solid (Yield: 82%),
1
0 10 4 2
Preparation of 2-(5-Phenyl-2H-tetrazole-2-yl)acetohydra-
zide (4). To a solution of methyl 2-(5-phenyl-2H-tetrazole-2-
yl)acetate 3 (27.25 g, 125 mmol) in 150 mL of anhydrous
methanol was added hydrazine hydrate (12.50 g, 250 mmol).
The reaction mixture was stirred at room temperature and the
progress monitored by TLC. On completion, the product pre-
cipitated out, which was filtered and washed with methanol to
ꢁ
−1
Mp. 232–235 C; FT-IR (neat) ν (cm ): 1060 (C–O–C),
1280 (N N–N), 1553 (Ar–C C), 780 (−C–Cl), 1610
(C N), 3060 (Ar–CH); H NMR (300 MHz, DMSO-d ) δ
5.71 (s, 2H), 7.42–7.31 (m, 6H), 8.10 (m, 3H); LC-MS
1
6
+
(EI) for C H C N O (M+H) : m/z = 339.07, Exact mass:
1
6 11 l 6
338.0682.
ꢁ
leave product 4 as white crystalline powder. Mp 212 C: FT-
2-(2-Naphthyl)-5-((5-phenyl-2H-tetrazol-2-yl)methyl)-
1,3,4-oxadiazole (5d): White crystalline solid (Yield: 95%),
−
1
IR (neat) ν (cm ): 1280 (N N–N), 1549 (Ar–C C), 1608
C N), 1659 (C O), 3057 (Ar–CH), 3132 (N–H), 3307
NH2); H NMR (300 MHz, DMSO-d ) δ 4.46 (s, 2H), 5.45
s, 2H), 7.55–7.61 (m, 3H), 8.05–8.08 (m, 2H), 9.65 (s,
ꢁ
−1
(
(
(
Mp. 242–245 C; FT-IR (neat) ν (cm ): 1055 (C–O–C),
1
1280 (N N–N), 1549 (Ar–C C), 1608 (C N), 3057 (Ar–
6
1
CH); H NMR (300 MHz, DMSO-d ) δ 5.61 (s, 2H),
6
+
1
H). LC-MS (EI) for C H N O (M+H) : m/z = 219.09, Exact
7.82–8.18 (m, 9H), 8.26 (m, 2H), 8.91 (s, 1H); LC-MS
9
10 6
+
mass: 218.0916 (Scheme 4).
(EI) for C H N O (M+H) : m/z = 355.36, Exact mass:
2
0 14 6
General Procedure for the Synthesis of 2-Phenyl-5-((5-
phenyl-2H-tetrazol-2-yl)methyl)-1,3,4-oxadiazoles (5a–
k). An equimolar mixture of 2-(5-phenyl-2H-tetrazole-2-yl)
acetohydrazide 4 and the suitable carboxylic acid in phospho-
rous oxychloride was refluxed for 7–8 h. The progress of reac-
tion was continuously monitored by TLC. On completion, the
reaction mixture was poured onto ice slowly. The reaction
mixture was neutralized with a 5% solution of potassium car-
bonate and was allowed to stand in a freezer for 1 h. The prod-
uct that precipitated out was filtered and washed with cold
water several times to afford the desired series of compounds
354.1229.
2-(4-Nitrophenyl)-5-((5-phenyl-2H-tetrazol-2-yl)methyl)-
1,3,4-oxadiazole (5e): Light green solid (Yield: 95%),
Mp. 245–247 C; FT-IR (neat) ν (cm ): 1020 (C–O–C),
1282 (N N–N), 1549 (Ar–C C), 1564 (−NO2), 1616
(C N), 3057 (Ar–CH); H NMR (300 MHz, DMSO-d ) δ
5.75 (s, 2H), 7.57 (m, 3H), 8.01-8.21 (m, 4H), 8.36 (d, J =
5.5 Hz, 2H); LC-MS (EI) for C H BrN O (M+H) : m/z
= 350.10, Exact mass: 349.0923.
2-(tert-Butyl)-5-((5-phenyl-2H-tetrazol-2-yl)methyl)-
1,3,4-oxadiazole (5f ): Light greenish solid (Yield: 97%),
Mp. 175–177 C; FT-IR (neat) ν (cm ): 1026 (C–O–C),
1280 (N N–N), 1608 (C N), 3125, 3160 (N–H); H NMR
(300 MHz, DMSO-d ) δ 1.20 (s, 9H), 5.60 (s, 2H),
7.55–7.61 (m, 3H), 8.07 (m, 2H); LC-MS (EI) for
ꢁ
−1
1
6
+
16
11
7 3
ꢁ
−1
(
5a–k) (Scheme 5).
-(3-Fluorophenyl)-5-((5-phenyl-2H-tetrazol-2-yl)
methyl)-1,3,4-oxadiazole (5a): White crystalline solid
1
2
6
ꢁ
−1
(
(
Yield: 91%), Mp. 233–236 C; FT-IR (neat) ν (cm ): 1058
+
C–O–C), 1280 (N N–N), 1549 (Ar–C C), 1608 (C N),
C H N O (M+H) : m/z = 285.14, Exact mass: 284.1385.
14 16 6
1
3
057 Ar-CH); H NMR (300 MHz, DMSO-d ) δ 5.55 (s,
2-(Furan-2-yl)-5-((5-phenyl-2H-tetrazol-2-yl)methyl)-
,3,4-oxadiazole (5g): White solid (Yield: 88%).
6
1
ꢁ
−1
Mp. 198–200 C; FT-IR (neat) ν (cm ): 1142 (C–O–C),
285 (N N–N), 1551 (Ar–C C), 1605 (C N), 3072 (Ar–
1
1
CH); H NMR (300 MHz, DMSO-d ) δ 5.64 (s, 2H), 6.64
6
(d, J = 3Hz, 1H), 7.2 (d, J = 3Hz, 1H), 7.62–7.90 (m, 4H),
+
8
2
.04 (m, 2H); LC-MS (EI) for C H N O (M+H) : m/z =
14 10 6 2
95.11, Exact mass: 294.0865.
-((5-Phenyl-2H-tetrazol-2-yl)methyl)-5-(m-tolyl)-1,3,4-
oxadiazole (5h): White crystalline solid (Yield: 95%),
Scheme 4. Synthesis of probe 4.
2
ꢁ
−1
Mp. 243–245 C; FT-IR (neat) ν (cm ): 1056 (C–O–C),
280 (N N–N), 1549 (Ar–C C), 1609 (C N), 3057 (Ar–
1
1
CH); H NMR (300 MHz, DMSO-d ) δ 3.06 (s, 3H), 5.62
6
(s, 2H), 7.20–7.36 (m, 7H), 8.01 (m, 2H); LC-MS (EI) for
+
Scheme 5. Synthesis of 5a–k.
C H N O (M+H) : m/z = 319.11, Exact mass: 318.1229.
17 14 6
Bull. Korean Chem. Soc. 2015, Vol. 36, 2247–2253
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Antiosteoclastic Activity of Oxadiazole Derivatives
KOREAN CHEMICAL SOCIETY
2
-(4-Chloro-2-nitrophenyl)-5-((5-phenyl-2H-tetrazol-
Technologies, Grand Island, NY, USA) with 10 ng/mL of
mouse recombinant M-CSF (R&D Systems, Minneapolis,
MN, USA) for 1 day. Then, after nonadherent BMCs were
re-plated on a Petri dish and cultured for 3 days in the presence
of M-CSF (30 ng/mL), the adherent BMMs were used for oste-
2
(
(
-yl)methyl)-1,3,4-oxadiazole (5i): Light yellowish solid
Yield: 80%). Mp. 265–267 C; FT-IR (neat) ν (cm ): 1035
C–O–C), 1280 (N N–N), 1549 (Ar–C C), 782 (−C–Cl),
560 (−NO2), 1610 (C N), 3057 (Ar–CH); H NMR (300
MHz, DMSO-d ) δ 5.60 (s, 2H), 7.53-8.17 (m, 5H), 8.24
m, 2H), 8.43 (s, 1H); LC-MS (EI) for C H C N O (M
ꢁ
−1
1
1
4
oclast differentiation. For osteoclastogenesis, BMMs (1 × 10
6
5
(
+
cells/well in a 96-well plate or 3 × 10 cells/well in a 6-well
1
6 10 l 7 3
+
H) : m/z = 384.75, Exact mass: 383.0533.
-(2-Chloro-6-nitrophenyl)-5-((5-phenyl-2H-tetrazol-
-yl)methyl)-1,3,4-oxadiazole (5j): Light yellowish solid
plate) were seeded in triplicate and cultured in the presence
of 10 ng/mL of mouse recombinant RANKL (R&D Systems)
and M-CSF (30 ng/mL) for 4 days to differentiate into mature
2
2
(
(
1
ꢁ
−1
+
Yield: 80%). Mp. 275–278 C; FT-IR (neat) ν (cm ): 1130
C–O–C), 1280 (N N–N), 1549 (Ar–C C), 782 (–C–Cl),
560 (−NO ), 1608 (C N), 3057 (Ar–CH); H NMR (300
MHz, DMSO-d ) δ 5.74 (s, 2H), 7.35 (m, 1H), 7.57 (m,
H), 8.05 (m, 2H), 8.25 (d, J = 3Hz, 1H), 8.36 (d, J = 3Hz,
H); LC-MS (EI) for C H C N O (M+H) : m/z = 384.15,
Exact Mass: 383.0533.
TRAP -MNCs.
TRAP Staining and Osteoclast Counting. Mature osteoclasts
were visualized by staining TRAP, a biomarker of osteoclast dif-
ferentiation. Briefly, multinucleated osteoclasts were fixed with
3.7% formalin for 10 min, permeabilized with 0.1% Triton X-
100for10 min, andstainedwithTRAPsolution(Sigma-Aldrich,
1
2
6
3
1
+
1
6 10 l 7 3
+
MO, USA). TRAP -MNCs (≥5 nuclei) were then counted.
4
2-(3,5-Dimethoxyphenyl)-5-((5-phenyl-2H-tetrazol-2-
Cell Viability Assay. BMMs (1 × 10 cells/well) were seeded
yl)methyl)-1,3,4-oxadiazole (5k): White solid (Yield: 76%).
in a 96-well plate and incubated for 1 day. Then, cells were
treated with M-CSF (30 ng/mL) and 5k. After incubation
for 3 days, cell viability was measured by the CCK-8 assay
kit according to the manufacturer's protocol. All experiments
were performed in triplicate.
ꢁ
−1
Mp. 263–266 C; FT-IR (neat) ν (cm ): 1026 (C–O–C), 1280
N N–N), 1552 (Ar–C C), 1220 (ArC–O–C), 1608 (C N),
(
1
3
6
8
3
055 (Ar–CH); H NMR (300 MHz, DMSO-d ) δ 3.82 (s,
6
H), 5.70 (s, 2H), 6.69 (s, 1H), 7.03 (s, 2H), 7.57 (m, 3H),
.07 (m, 2 H); LC-MS (EI) for C H N O (M+H) : m/z =
65.15, Exact mass: 364.1283.
+
1
8 16 6 3
Evaluation of mRNA Expression. In the presence of M-CSF
(30 ng/mL), BMMs were treated with RANKL (10 ng/mL) or
its combination with 5k (3 μM) for 3 days. Then, total RNA
was isolated using TRIzol reagent (Life Technologies, Grand
Island, NY, USA) and the first-strand cDNA was synthesized
using 1 μg of total RNA, 1 μM of oligo-dT18 primer, 10 units
of the RNase inhibitor, RNasin (Promega, Madison, WI,
USA), and Omniscript Reverse Transcriptase (Qiagen,
Valencia, CA, USA) according to the manufacturer's instruc-
tion. SYBR green-based quantitative PCR was performed
using the Stratagene Mx3000P real-time PCR system in
20 μL reaction mixture containing 10 μL of TOPreal qPCR
2× PreMIX (Enzynomics, Daejeon, Korea), 2 pmol of forward
primer, 2 pmol of reverse primer, and 2 μL of 1:10-diluted
first-strand cDNA 1 μg of cDNA. Amplification parameters
Biology
Osteoclast Differentiation. This study was carried out in strict
accordance with the recommendations in the Standard Proto-
col for Animal Study of Korea Research Institute of Chemical
Technology (KRICT; No. 2012-7D-02-01). The protocol
(ID No. 7D-M1) was approved by the Institutional Animal
Care and Use Committee of KRICT. All efforts were made
to minimize suffering for the animals. Five-week-old male
ICR mice (Damul Science Co., Daejeon, Korea) were main-
tained in a room illuminated daily from 07:00 to 19:00
(
(
a 12:12 h light/dark cycle) under controlled temperature
23 ꢀ 1 C) and ventilation (10–12 times per hour). Humidity
ꢁ
was maintained at 55 ꢀ 5%, and the mice had free access to a
standard animal diet and tap water. To isolate bone-marrow-
derived cells (BMCs) from mice, after cervical dislocation,
femur and tibia were flushed with α-MEM (Invitrogen Life
Technologies, Carlsbad, CA, USA) supplemented with anti-
biotics (100 Units/mL penicillin and 100 μg/mL streptomycin;
Invitrogen Life Technologies, Grand Island, NY, USA).
BMCs were cultured on a culture dish in α-MEM supplemen-
ted with 10% fetal bovine serum (FBS; Invitrogen Life
ꢁ
consisted of an initial denaturation at 95 C for 10 min and
ꢁ
40 cycles of three-step PCR (a denaturation at 94 C for 40
s, annealing at 53 C for 40 s, and extension at 72 C for 1
min). All reactions were run in triplicate, and the data were
method. Primers were designed
using an online primer design program (Table 3). GAPDH
was used as the control gene. Significance was determined
ꢁ
ꢁ
–
ΔΔCT
43
analyzed using the 2
44
–ΔΔCT
with GAPDH-normalized 2
values.
Table 3. Primer sequences used in this study.
0
0
0 0
Reverse Primer (5 –3 )
Target Gene
Forward Primer (5 –3 )
c-Fos
NFATc1
TRAP
DC-STAMP
ATP6v0d2
GAPDH
CCAGTCAAGAGCATCAGCAA
GGGTCAGTGTGACCGAAGAT
GATGACTTTGCCAGTCAGCA
CCAAGGAGTCGTCCATGATT
AGACCACGGACTATGGCAAC
AACTTTGGCATTGTGGAAGG
AAGTAGTGCAGCCCGGAGTA
GGAAGTCAGAAGTGGGTGGA
ACATAGCCCACACCGTTCTC
GGCTGCTTTGATCGTTTCTC
CGATGGGTGACACTTGGCTA
ACACATTGGGGGTAGGAACA
Bull. Korean Chem. Soc. 2015, Vol. 36, 2247–2253
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de 2252

BULLETIN OF THE
Article
Antiosteoclastic Activity of Oxadiazole Derivatives
KOREAN CHEMICAL SOCIETY
ComputationalMethods. Based onthe chemical substructure,
target molecules for compound 5 analogs were predicted via a
web-based interface at https://www.ebi.ac.uk/chembl.
Active compounds containing the 1,3,4-oxadiazole moieties
connected to the five-membered heterocyclic rings through
a carbon linker were chosen for the target prediction. The
structural similarity was calculated by ECFC4 method in
Pipeline Pilot, version 8.5 (Accelrys, San Diego, CA, USA).
18. D. H. Boschelli, D. T. Connor, D. A. Bornemeier, R. D. Dyer,
J. A. Kennedy, P. J. Kuipers, G. C. Okonkwo, D. J. Schrier,
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39
1
9. M. D. Mullican, M. W. Wilson, D. T. Connor, C. R. Kostlan,
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Conclusions
23. H. Khalilullah, M. J. Ahsan, M. Hedaitullah, S. Khan, B. Ahmed,
Mini Rev. Med. Chem. 2012, 12, 789.
In conclusion, we synthesized novel oxadiazoles bearing 5-
phenyl-tetrazole analogs, found their anti-osteoclastogenic
activities, andsuggestedP2X7orG9aastheirtarget molecules
in silico. In future, we propose to study the structural tuning of
oxadiazoles bearing 5-phenyl-tetrazole to fit one of the poten-
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which might improve their target specificity and anti-osteo-
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Bull. Korean Chem. Soc. 2015, Vol. 36, 2247–2253
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de 2253