Discovery, structure-activity relationship studies, and anti-nociceptive effects of 1-phenyl-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one as novel opioid receptor agonists

Article abstract of DOI:10.1016/j.bmc.2014.07.012

The μ-opioid receptor (MOR) is the major opioid receptor targeted by most analgesics in clinical use. However, the use of all known MOR agonists is associated with severe adverse effects. We reported that the 1-phenyl-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-ones are novel opioid receptor agonists. Subsequent structural modification resulted in the potent MOR/KOR (κ-opioid receptor) agonists 19, 20, and 21. Testing the analgesic effect of these in WT B6 mice (tail-flick test) gave ED₅₀ values of 8.4, 10.9, and 26.6 mg/kg, respectively. The 1-phenyl-3,6,6-trimethyl-1,5,6,7- tetrahydro-4H-indazol-4-one core could be addressed in 1 or 2 synthetic steps with moderate to high percent of yield. In the adenylyl cyclase assay, compound 19 displayed a MOR/KOR agonist profile, with IC₅₀ values of 0.73 and 0.41 μM, respectively. Current results suggest that compound 19 is a promising lead to go further development and in vitro/in vivo adverse effects studies.

Full text of DOI:10.1016/j.bmc.2014.07.012

Bioorganic & Medicinal Chemistry 22 (2014) 4694–4703
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery, structure–activity relationship studies, and
anti-nociceptive effects of 1-phenyl-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one as novel opioid receptor agonists
Ming-Fu Cheng a, Li-Chin Ou a, Shu-Chun Chen a, , Wan-Ting Chang a, , Ping-Yee Law b, Horace H. Loh b,
Yu-Sheng Chao a, Chuan Shih a, Shiu-Hwa Yeh a, , Shau-Hua Ueng a,
⇑
⇑
a Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Miaoli County 35053, Taiwan, ROC
b Department of Pharmacology, University of Minnesota, Medical School, Minneapolis, MN 55455, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The
l-opioid receptor (MOR) is the major opioid receptor targeted by most analgesics in clinical use.
Received 13 May 2014
Revised 7 July 2014
Accepted 8 July 2014
Available online 16 July 2014
However, the use of all known MOR agonists is associated with severe adverse effects. We reported that
the 1-phenyl-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-ones are novel opioid receptor agonists.
Subsequent structural modification resulted in the potent MOR/KOR (j-opioid receptor) agonists 19,
20, and 21. Testing the analgesic effect of these in WT B6 mice (tail-flick test) gave ED50 values of 8.4,
10.9, and 26.6 mg/kg, respectively. The 1-phenyl-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one
core could be addressed in 1 or 2 synthetic steps with moderate to high percent of yield. In the adenylyl
cyclase assay, compound 19 displayed a MOR/KOR agonist profile, with IC50 values of 0.73 and 0.41 lM,
respectively. Current results suggest that compound 19 is a promising lead to go further development
Keywords:
l/j opioid receptor agonist
Structure–activity relationship
Anti-nociceptive effects
Tail-flick test
and in vitro/in vivo adverse effects studies.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
such as dysphoria, water diuresis, salivation and emesis, which
may limit the utility of KOR agonists for clinical pain manage-
Opioids, including morphine, primarily act on opioid receptors,
of which there are three well-defined subtypes: MOR, DOR (d-opi-
oid receptor) and KOR. These opioid receptors are Gi-coupled
receptors, possessing the same general structure of an extracellular
N-terminal region, seven transmembrane domains, and an intra-
cellular C-terminal tail structure. Neuronal cells in the central ner-
vous system (CNS) broadly express these receptors, which regulate
numerous complex, partially overlapping physiological and neuro-
biological functions.1–5 Currently, most opioids in clinical use for
analgesia are either non-selective or selective MOR agonists,6 and
it has been proven that the analgesic effect of opioids is mediated
principally by activation of MOR. However, use of MOR also results
in tolerance, dependence, and addiction.7–10 DOR are implicated in
morphine tolerance, dependence and rewarding effects, and DOR
agonists have been regarded as very weak analgesics.11–13 Activa-
tion of KOR in non-human primates causes severe adverse effects
ment.14 However, recent studies of mixed MOR/DOR agonists for
analgesia with reduced tolerance was reported.15 In addition, the
relevance of that mixed MOR/KOR activity is useful in reducing
cocaine self-administration, and displays fewer side effects has
been established recently.16–20
Most of the non-peptide MOR/DOR and MOR/KOR agonists are
morphine-like compounds or morphine analogs. High-throughput
screening (HTS) has the potential to discover new scaffolds of
non-peptide opioid receptor agonists, which may result in novel
analgesics that work without severe adverse effects. 1-(2-Chloro-
phenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one (1)
was identified to activate
micromolar range (Fig. 1) by HTS. The EC50 of compound 1 in the
FLIPRÒ calcium assay at MOR is 2.28
M. Compound 1 is based on
l-opioid receptor with activity in the
l
a tetrahydro-4H-indazol-4-one core structure, and can be synthe-
sized by the condensation of hydrazines and 2-acylcyclohexane-
1,3-diones under acidic conditions. The 1-phenyl-3,6,6-trimethyl-
1,5,6,7-tetrahydro-4H-indazol-4-one core is not a morphine-like
structure, may has the potential to distinguish from the morphine-
like MOR/DOR or MOR/KOR agonists, and the synthesis of it is easy
to address. Although some of the 1-phenyl-3,6,6-trimethyl-1,5,6,
7-tetrahydro-4H-indazol-4-one analogs in this study are
⇑
Corresponding authors. Tel.: +886 37 246166x35759; fax: +886 37 586456
(S.-H.Y.); tel.: +886 37 246166x35791; fax: +886 37 586456 (S.-H.U.).
E-mail addresses: bau9763@bp.nhri.org.tw (S.-H. Yeh), shueng@nhri.org.tw
(S.-H. Ueng).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.07.012
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

M.-F. Cheng et al. / Bioorg. Med. Chem. 22 (2014) 4694–4703
4695
from the corresponding hydrazinobenzoic acids through esterifica-
tion. According to literature procedures, several specific phen-
ylhydrazines (2,4-dibromo 23a, 2-bromo-4-chloro 23b, 2-bromo-
4-methyl 23c) were obtained from diazotization of the correspond-
ing anilines with sodium nitrite, followed by reduction of the result-
ing diazonium salts with tin(II) chloride.24 Phenylhydrazines 23a–
23c were utilized to prepare compound 19–21, respectively.
Scheme 2 depicts the synthesis of 3-trifluoromethyl- and 3-
cyclopropyl-tetrahydroindazol-4-ones 26 and 27 from the corre-
sponding 2-acylcyclohexane-1,3-diones (25a and 25b) under acidic
and basic conditions, respectively. Compounds 25a and 25b were
prepared by C-acylation of 5,5-dimethyl-1,3-cyclohexanedione
with trifluoroacetic acid and cyclopropanecarbonyl chloride,
respectively.25,26 In addition, compound 28 was synthesized from
hydrazine and 2-acetyl-5,5-dimethyl-1,3-cyclohexanedione under
neutral conditions.27 Tetrahydroindazol-4-ol 29 was synthesized
by reduction of 19 (Scheme 3). The purities of all final compounds
were measured by reverse phase HPLC, and determined to be more
than 95%.
Cl
1
N
2
N
3
FLIPR calcium assay EC = 2.28 µM
50
7
4
6
O
5
1
Figure 1. Initial hit from HTS.
commercially available, the application of these analogs is unappar-
ent.21–23 In this study, 1-phenyl-3,6,6-trimethyl-1,5,6,7-tetrahydro-
4H-indazol-4-ones are reported as novel opioid receptor agonists,
and their structure–activity relationships and pharmacological
studies discussed.
2. Chemistry
The regioselective condensation of phenylhydrazines with 2-
acylcyclohexane-1,3-diones in the presence of a catalytic amount
of acid at room temperature or under reflux temperature of metha-
nol or ethanol gave tetrahydroindazol-4-ones 2–22, Scheme 1. A
variety of commercially available phenylhydrazines were utilized
to this reaction, and a series of tetrahydroindazol-4-one derivatives
2–21 bearing phenyl substituents at N1-, and methyl groups at
C3- and C6-positions were prepared (Scheme 1). Phenylhydrazines
for preparing compound 10 and compound 11 were synthesized
3. Biological evaluation
3.1. Structure–activity relationship (SAR) studies of tetrahydro-
4H-indazol-4-ones
A variety of tetrahydro-4H-indazol-4-one derivatives were pre-
pared and their structure–activity relationships explored using a
R2
R3
R1
R1
H
R4
acid, rt ~ 78 o
EtOH or MeOH
C
O
O
O
R2
R3
N
NH2
N
N
R6
R5
R7
R6
R7
R8
R5
R4
O
R8
1−22
R6, R7, R8 = Me (1−21)
11 R1, R2 = H, R3 = COOMe, R4, R5 = H
12 R1 = Me, R2 = H, R3 = Me, R4, R5 = H
13 R1, R2 = Me, R3 ~ R5 = H
22 R1 = Cl, R2 = H, R3 = Cl, R4, R5 = H,
6 = Me, R7, R8 = H
1 R1 = Cl, R2 ~ R5 = H
2 R1 ~ R5 = H
3 R1 = H, R2 = Cl, R3 ~ R5 = H
4 R1, R2 = H, R3 = Cl, R4, R5 = H
5 R1 = Br, R2 ~ R5 = H
6 R1, R2 = H, R3 = Br, R4, R5 = H
7 R1 = Me, R2 ~ R5 = H
8 R1 = Et, R2 ~ R5 = H
9 R1, R2 = H, R3 = OMe, R4, R5 = H
10 R1 = COOEt, R2 ~ R5 = H
R
14 R1 = Me, R2, R3 = H, R4 = Me, R5 = H
15 R1 = Me, R2 = H, R3 = Cl, R4, R5 = H
16 R1 = F, R2 = H, R3 = F, R4, R5 = H
17 R1 = Cl, R2, R3 = H, R4 = Cl, R5 = H
18 R1 = Cl, R2 = H, R3 = Cl, R4, R5 = H
19 R1 = Br, R2 = H, R3 = Br, R4, R5 = H
20 R1 = Br, R2 = H, R3 = Cl, R4, R5 = H
21 R1 = Br, R2 = H, R3 = Me, R4, R5 = H
Scheme 1. Synthesis of tetrahydroindazol-4-one derivatives.
Cl
O
Cl
H
Cl
N
NH2
O
O
O
N
N
O
Cl
O
R2
R2
R1
R2
a for 25a
b for 25b
c for 26
d for 27
O
24a R1 = OH, R2 = CF3
24b R1 = Cl, R2 = cyclo-C3H5
25a R2 = CF3
25b R2 = cyclo-C3H5
26 R2 = CF3
27 R2 = cyclo-C3H5
Scheme 2. Modification of 2-acylcyclohexane-1,3-diones and synthesis of tetrahydroindazol-4-ones. Reagents and conditions: (a) CDI, imidazole, CHCl3, rt; (b) DMAP, DIPEA,
CH2Cl2, reflux; (c) H2SO4, EtOH, reflux; (d) NaOH, EtOH, rt.

4696
M.-F. Cheng et al. / Bioorg. Med. Chem. 22 (2014) 4694–4703
O
O
O
(3: EC50 >100 lM) or para- (4: EC50 = 13.6 lM) position of the ben-
zene ring resulted in a significant drop in potency. Replacing the
ortho-chlorine atom of 1 with bromine to gave analog 5, the
N
HN
hydrazine (aq)
THF, reflux
potency of which was 2-fold higher than 1 (5: EC50 = 1.12
1: EC50 = 2.28 M). In comparison, substituting the chlorine for
methyl (to give 7) and ethyl (to give 8) devastated the potency
EC50 = 4.72 and 19 M, respectively. Introduction of methoxy and
ester groups was found to have no benefit; the EC50 values for com-
pounds 9–11 was in double digit M range (9: EC50 = 18.4 M, 10:
EC50 = 30.2 M, 11: EC50 = 10 M). To expand the scope of SAR
lM vs
O
l
28
l
Br
Br
Br
Br
l
l
l
l
N
NaBH4
N
N
N
study, ortho- and para-di-substituted phenyl groups were intro-
duced in tetrahydro-4H-indazol-4-one and a dramatic enhance-
CH2Cl2 / MeOH
ment of activation of the
example, compound 12 (EC50 = 0.37
than compounds 13 (R1, R2 = Me, EC50 = 8.9
R4 = Me, EC50 = 10
M). Replacing the methyl groups with halogens
to give compounds 15 and 18 resulted in an improvement in EC50
values (0.37 and 0.15 M, respectively). Introduction of a bromine
l
-opioid receptor was observed. For
M) exhibited higher potency
M) and 14 (R1,
O
OH
l
l
19
29
l
Scheme 3. Synthesis of 28 and 29.
l
atom at the ortho-position of an ortho-, para-di-substituted ben-
zene ring gave compounds 19, 20, and 21 (19, 20, 21: EC50 = 0.041 -
FLIPRÒ calcium assay in CHO-K1 cells, stably expressing hMOR and
15. Activation of MOR elicits an intracellular calcium release
l
M), which were 27-fold more potent than compound 5, bearing
only one bromo substituent at the ortho-position of benzene ring.
These results suggest that the -opioid receptor favors ortho-,
Ga
leading to an increase in the relative fluorescence units (RFU).28
As shown in Table 1, the substituted groups at C3- and C6-positions
of tetrahydro-4H-indazol-4-one were methyl groups and various
substituents of phenyl group at N1-position were introduced to
investigate the relationships between substituents R1–R5 and bio-
logical activity. Compound 2, without substituents on the benzene
ring, eliminated agonistic activity. This result reveals the impor-
tance of substituents on the benzene ring. Then, several analogs
bearing mono-substituents at either R1, R2 or R3, were synthesized
(3–11). Changing the position of the chlorine atom to the meta-
l
para-di-substituted benzene on the N1-position of tetrahydro-4H-
indazol-4-one. The activation ability of ortho-, para-di-halogen
benzene on the N1-position of tetrahydro-4H-indazol-4-one was
increased in the order; F, Cl, Br (16: EC50 = 10.3
EC50 = 0.15 M, 19: EC50 = 0.041 M).
To investigate additional SARs, several more derivatives were
synthesized, Table 2. Analog 22 (EC50 = 13.6 M), in which the
lM, 18:
l
l
l
methyl groups at the R2 and R3 positions were replaced with
hydrogen atoms, displayed 91-fold less potency than compound
18, revealing the di-methyl group at the C6-position of tetrahy-
dro-4H-indazol-4-one to be necessary for agonistic activity.
Introduction of trifluoromethyl and cyclopropyl groups at the
C3-position of tetrahydro-4H-indazol-4-one gave compounds 26
Table 1
Structure–activity relationships of tetrahydroindazol-4-ones 1–21
2
R
3
R
(EC50 = 1.54
active than compound 18. Removing the benzene ring to give com-
pound 28 (EC50 >100 M) eliminated all agonistic activity, empha-
lM) and 27 (EC50 = 0.37 lM), both of which were less
1
R
4
R
l
N
N
5
sizing the importance of the benzene ring at the N1-position. When
the carbonyl group at the C4 position was reduced to a hydroxyl
group to give compound 29, the agonistic activity was 2-fold less
R
O
than compound 19 (EC50 = 0.1 lM). In summary, the ortho, para-
di-halogen/dimethyl-substituted (except fluorine atom) benzene
ring at the N1-position of the tetrahydro-4H-indazol-4-one, and
1−21
a
Compound
R1
R2
R3
R4
R5
EC50
2.28
(lM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Cl
H
H
H
Br
H
Me
Et
H
CO2Et
H
Me
Me
Me
Me
F
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
Br
H
H
OMe
H
CO2Me
Me
H
H
Cl
F
H
Cl
Br
Cl
Me
—
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
Cl
H
H
H
H
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
—
Table 2
>100
>100
13.6
1.12
9.91
4.72
19
Structure–activity relationships of tetrahydroindazol-4-ones 22, 26–29
Cl
Br
Cl
Br
N
N
N
N
N
HN
R1
18.4
30.2
10
0.37
8.9
R2
R3
O
O
OH
Me
H
H
22, 26, 27
28
29
10
0.37
10.3
2.89
0.15
0.041
0.041
0.041
0.01
a
Compound
R1
R2
R3
EC50 (lM)
H
22
26
27
28
29
Morphine
Me
CF3
Cyclo-C3H5
—
—
—
H
H
13.6
1.54
0.37
>100
0.1
17
Cl
H
Me
Me
—
—
—
Me
Me
—
—
—
18
Cl
H
19
20
21
Morphine
Br
Br
Br
—
H
H
H
—
0.01
a
a
EC50 values are the mean of at least two independent experiments.
EC50 values are the means of at least two independent experiments.

M.-F. Cheng et al. / Bioorg. Med. Chem. 22 (2014) 4694–4703
4697
M)
Table 3
[3H]naloxone displacement assay and cAMP inhibition assay for functional selectivity
a
b
b
b
Compound
Ki (l
M)
hMOR IC50
(
lM)
hDOR IC50
(lM)
hKOR IC50 (l
12
15
18
19
20
21
27
29
Naloxone
DAMGO
DPDPE
U50488
3.93 0.14
>100
NDc
NDc
7.89
0.73
3.3
NDc
NDc
10
NDc
NDc
2.65
0.41
0.57
0.37
NDc
1.1
NDc
0.47 0.028
0.61 0.074
0.74 0.036
1.3 0.88
>100
1.69
3.38
>100
NDc
>100
NDc
NDc
0.0009
NDc
6
NDc
3.38
NDc
0.005
NDc
NDc
0.002 0.0005
NDc
NDc
NDc
0.0003
NDc
NDc
NDc
a
b
c
Ki values are the average SEM from three independent experiments.
IC50 values are the mean of at least two independent experiments.
ND = not determined.
the dimethyl groups at the C6-position, contribute greatly to the
human opioid receptor agonism.
l
105
100
95
(A)
3.2. Binding affinities determination and measurement of
adenylyl cyclase-mediated effects
19
19 in naloxone
0.3% DMSO
0.3% DMSO in naloxone
Binding affinities of the new compounds for MOR were deter-
mined by displacement of [3H]naloxone binding in HEK cells
expressing recombinant rat MOR. Compounds 19, 20, 21 had sig-
nificant binding for the MOR with Ki value in submicromolar range
(19, Ki = 0.47 lM; 20, Ki = 0.61 lM; 21, Ki = 0.74 lM) (Table 3) in
90
radioligand displacement studies. The poor correlation between
Ki of [3H]naloxone displacement assay and EC50 of FLIPRÒ calcium
assay revealed that the cellular response to a pharmacological ago-
nist was related to the efficacy of agonist, which is the intrinsic
character for individual agonist, and the sensitivity of the
assay.29,30 The typical signaling pathway of opioid receptors is
through Gi/Go proteins for inhibiting adenylyl cyclase.31 To investi-
gate the selectivity of new compounds in activating opioid recep-
tors, the ability of new compounds in affecting forskolin-
stimulated adenylyl cyclase activity in HEK293 cells stably
85
0
25
50
75
100
125
150
175
Time (sec)
105
(B)
20
20 in naloxone
0.3% DMSO
0.3% DMSO in naloxone
100
95
expressing the
shown in Table 3, compound 19 was both MOR and KOR agonist
(MOR IC50 = 0.73 M; KOR IC50 = 0.41 M), whereas compound
20 and 21 were selective KOR agonist (20, MOR IC50 = 3.3 M,
KOR IC50 = 0.57 M; 21, MOR IC50 = 6 M, KOR IC50 = 0.37 M).
l, d, and j opioid receptors was measured. As
l
l
90
l
l
l
l
Thus compounds 19, 20 and 21 were novel agonists of both MOR
and KOR.
85
0
25
50
75
100
125
150
175
Time (sec)
105
100
95
3.3. Compounds 19, 20 and 21 activate G protein-coupled
inwardly-rectifying potassium channel in the AtT-20 pituitary
cells
(C)
21
21 in naloxone
0.3% DMSO
0.3% DMSO in naloxone
Upon activation of the opioid receptor and coupling to trimeric
G proteins, the bc subunit of the G protein is released from Gabc
complex to activate the G protein-coupled inwardly-rectifying
potassium (GIRK) channels.32 Spinal GIRK1/GIRK2 heterotetramer-
ic channels modulate nociception and contribute to morphine
analgesia.33 In this study, AtT-20 cells endogenously expressing
GIRK1/GIRK2,34 were transiently transfected with myc-MOR
expression plasmid, to detect GIRK-mediated membrane potential
hyperpolarization after morphine treatment. By using the FLIPRÒ
membrane potential assay, 19, 20 and 21 treatment was found to
alter the membrane potential in myc-tagged MOR expressing
90
85
0
25
50
75
100
125
150
175
Time (sec)
Figure 2. Effects of compound-mediated membrane potential hyperpolarization in
pituitary AtT-20 cells. Compounds 19, 20 and 21 induced a membrane potential
hyperpolarization in MOR expressing AtT-20 cells. The pituitary AtT-20 cells were
transiently transfected with vehicle or myc-MOR, the effects of compounds on membrane
AtT-20 cells (Fig. 2). The EC50 value of 19 was 5.49
of 20 and 21 was 0.1 M and 0.2 M, respectively. Finally, nalox-
one was found to reverse compound-stimulated membrane poten-
lM, and that
l
l
potential were detected in the presence or absence of naloxone (1 lM) treatment.

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M.-F. Cheng et al. / Bioorg. Med. Chem. 22 (2014) 4694–4703
tial hyperpolarization in myc-tagged MOR expressing AtT-20 cells,
indicating that the effects of compounds were dependent on opioid
receptor activation.
3.4. Compounds 19, 20, 21 elicited anti-nociceptive effects in B6
mice
To further evaluate the activity of compounds 19–21, mice were
injected with 19, 20, and 21 and their tail-flick latencies at the indi-
cated time points (0, 0.5 and 1 h) measured. The extent of thermal
withdrawal was evaluated by measuring the AUC of the tail-flick test
to study the effect of the compounds on pain-related behavior in B6
mice. All three compounds, especially 19, were found to exert strong
analgesia compared to vehicle control (Fig. 3). The ED50 of three
compounds were listed in Table 4. It was also found that this effect
could be attenuated using the opioid antagonist naloxone (Fig. 3).
Table 4
ED50 of 19, 20, and 21
19
20
21
ED50 (mg/kg)
8.4
10.9
26.6
(A)
20 mg/kg compound 19 (i.v.)
8
4. Conclusions
6
4
2
The tetrahydro-4H-indazol-4-one containing opioid receptor
agonist 1 was identified by high-throughput screening. Structural
modification of this scaffold resulted in the novel opioid receptor
agonist 19, a >50 fold level potency improvement compared with
compound 1, in the FLIPRÒ assay at MOR. Radioligand displacement
study of 19 exhibited moderate binding affinity. The effect of 19 on
cAMP accumulation assay in HEK293 cells stably expressing the
l, d,
0
0
10
20
30
Time (min)
20 mg/kg compound19 (n=6)
40
50
60
70
and opioid receptors revealed that 19 possesses a mixed MOR/KOR
j
agonistic activity. Recent studies emerged the relevance of MOR/
KOR agonists or partial agonists in treating cocaine abuse or in pain
relief with fewer adverse effects.16–20 The membrane potential
hyperpolarization assay in pituitary AtT-20 cell indicated that 19
altered the cell membrane potential, suggesting that 19 effectively
activated MOR. Compound 19 elicited strong anti-nociceptive effect
in WT B6 mice (ED50 = 8.4 mg/kg). Synthesis of 19 could be easily
addressed in 2 synthetic steps with 61% of yield. These results dem-
onstrate that compound 19 is a promising lead for further develop-
ment and in vitro/in vivo adverse effects studies.
10 mg/kg naloxone + 20 mg/kg compound 19 (n=6)
(B)
18.4 mg/kg compound 20 (i.v.)
8
6
4
2
5. Experimental
5.1. Chemistry methods
0
0
20
40
60
80
Time (min)
18.4 mg/kg compound 20 (i.v.) (n=6)
10 mg/kg naloxone (s.c.) + 18.4 mg/kg compound 20 (i.v.) (n=6)
All the reagents and solvents were reagent grade and were used
without further purification unless otherwise specified. All materi-
als used were commercially available and used as supplied. Merck
silica gel 60 F254 sheets were used for analytical thin-layer chroma-
tography (TLC). Column chromatography was performed on silica
gel (230–400 mesh) or used an Isco CombiFlash Companion system
(Teledyne Isco) with prepacked silica gel cartridges. 1H NMR spec-
tra were recorded on a Varian Mercury-300 or Varian Mercury-400
spectrometers. Chemical shifts (d) are given in parts per million
(ppm) relative to the solvent peak. High-resolution electrospray
ionization mass spectra were measured with a VARIAN 901-MS
(FT-ICR Mass) mass spectrometer. Purity of all tested compounds
was over 95% and determined on a Hitachi 2000 series HPLC sys-
tem equipped with a C-18 column (Agilent ZORBAX Eclipse XDB-
(C)
25 mg/kg compound 21 (i.v.)
8
6
4
2
0
C18 5
l
m, 4.6 mm  150 mm) at 254 nm, eluting with mobile
0
10
20
30
Time (min)
25 mg/kg compound 21 (i.v.) (n=6)
10 mg/kg naloxone (s.c.) + 25 mg/kg compound 21 (i.v.) (n=6)
40
50
60
70
phase A (acetonitrile) and mobile phase B (10 mM NH4OAc aque-
ous solution containing 0.1% formic acid) from 0 min (A/B, 10%)
to 45 min (A/B, 90%) at 25 °C.
Figure 3. Compounds 19, 20 and 21 show analgesic effects through opioid receptor
activation in B6 mice. Analgesic effect of 19 (A), 20 (B) and 21 (C) in the WT B6 mice.
After detection of basal latencies, each group of mice were injected with 19 (20 mg/
kg, iv), 20 (18.4 mg/kg, iv) and 21 (25 mg/kg, iv), respectively, to detect tail-flick
latencies at the indicated time points (10 min, 20 min, 30 min, 40 min, 50 min, and
60 min). Mice were injected with naloxone (10 mg/kg, sc) 5 min before compound
injection. Quantitative results were calculated from tail-flick test (tail-flick latency-
basal latency). Neither the vehicle nor naloxone alone was found to alter tail-flick
latency (Supplementary Fig. S1). ⁄⁄⁄p <0.001 versus sham-control group. (Statistical
analysis was carried out using one-way ANOVA with appropriate post hoc tests.)
5.2. Representative procedure for the synthesis of
tetrahydroindazol-4-ones
To a mixture of 1-(2-bromo-4-chlorophenyl)hydrazine hydro-
chloride 23b (103.2 mg, 0.40 mmol) and 2-acetyl-5,5-dimethyl-
1,3-cyclohexanedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL)
was treated with a few drops of concd H2SO4. The mixture was
stirred at room temperature for 24 h. After the reaction was

M.-F. Cheng et al. / Bioorg. Med. Chem. 22 (2014) 4694–4703
4699
finished, the mixture was diluted with EtOAc (8.0 mL) and washed
with water (4.0 mL), satd NaHCO3(aq) (4.0 mL) and brine (4.0 mL).
The organic layer was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by Isco CombiFlash
Companion column chromatography (silica gel, 0–70% EtOAc/hex-
ane) to give 1-(2-bromo-4-chlorophenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one 20 (65.0 mg, 44%) as a powder. 1H
NMR (CD3OD, 400 MHz) d 7.93 (d, J = 2.0 Hz, 1H), 7.61 (dd, J = 2.0,
8.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 2.54 (s, 2H), 2.46 (s, 3H), 2.40
(s, 2H), 1.10 (s, 6H); HRMS calcd for C16H17BrClN2O (M+H)
367.0213, found 367.0211.
anedione (72.9 mg, 0.40 mmol), by a procedure similar to that for
20, gave 1-(2-bromophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-
4H-indazol-4-one 5 (92.4 mg, 69%) as a powder. 1H NMR (CD3OD,
400 MHz) d 7.84 (dd, J = 2.4, 7.6 Hz, 1H), 7.60–7.50 (m, 3H), 2.53
(s, 2H), 2.47 (s, 3H), 2.40 (s, 2H), 1.10 (s, 6H); HRMS calcd for
C16H18BrN2O (M+H) 333.0602, found 333.0606.
5.2.6. 1-(4-Bromophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-
4H-indazol-4-one (6)
The reaction of 1-(4-bromophenyl)hydrazine hydrochloride
(89.4 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol), by a procedure similar to that for
20, gave 1-(4-bromophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-
4H-indazol-4-one 6 (34.6 mg, 26%) as a powder. 1H NMR (CD3OD,
300 MHz) d 7.71 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 2.86
(s, 2H), 2.47 (s, 3H), 2.41 (s, 2H), 1.08 (s, 6H); HRMS calcd for
5.2.1. 1-(2-Chlorophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-
4H-indazol-4-one (1)
The reaction of 1-(2-chlorophenyl)hydrazine hydrochloride
(71.6 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) at room tem-
perature overnight, by a procedure similar to that for 20 using
concd HCl (0.40 mL) instead of H2SO4 and the crude product was
purified by flash chromatography on silica gel eluting with
EtOAc/hexane (4:1), gave 1-(2-chlorophenyl)-3,6,6-trimethyl-
1,5,6,7-tetrahydro-4H-indazol-4-one 1 (45.2 mg, 39%) as a powder.
1H NMR (CD3OD, 400 MHz) d 7.68 (d, J = 7.6 Hz, 1H), 7.60–7.52 (m,
3H), 2.54 (s, 2H), 2.47 (s, 3H), 2.40 (s, 2H), 1.09 (s, 6H); HRMS calcd
for C16H18ClN2O (M+H) 289.1108, found 289.1117.
C16H18BrN2O (M+H) 333.0602, found 333.0606.
5.2.7. 3,6,6-Trimethyl-1-(2-methylphenyl)-1,5,6,7-tetrahydro-
4H-indazol-4-one (7)
The reaction of 1-(2-methylphenyl)hydrazine hydrochloride
(63.5 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol), by a procedure similar to that for
20, gave 3,6,6-trimethyl-1-(2-methylphenyl)-1,5,6,7-tetrahydro-
4H-indazol-4-one 7 (55.0 mg, 51%) as a powder. 1H NMR (CD3OD,
400 MHz) d 7.49–7.36 (m, 3H), 7.28 (d, J = 7.6 Hz, 1H), 2.50 (s,
2H), 2.47 (s, 3H), 2.40 (s, 2H), 2.09 (s, 3H), 1.08 (s, 6H); HRMS calcd
for C17H21N2O (M+H) 269.1654, found 269.1656.
5.2.2. 3,6,6-Trimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-
4-one (2)
The reaction of 1-phenylhydrazine (43.3 mg, 0.40 mmol) with
2-acetyl-5,5-dimethyl-1,3-cyclohexanedione (72.9 mg, 0.40 mmol)
in ethanol (1.2 mL) at room temperature overnight, by a procedure
similar to that for 20 using concd HCl (0.40 mL) instead of H2SO4,
gave 3,6,6-trimethyl-1-phenyl-1,5,6,7-tetrahydro-4H-indazol-4-
one 2 (39.2 mg, 39%) as a powder. 1H NMR (CD3OD, 300 MHz) d
7.59–7.45 (m, 5H), 2.84 (s, 2H), 2.47 (s, 3H), 2.41 (s, 2H), 1.08 (s,
6H); HRMS calcd for C16H19N2O (M+H) 255.1497, found 255.1501.
5.2.8. 1-(2-Ethylphenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-
indazol-4-one (8)
The reaction of 1-(2-ethylphenyl)hydrazine hydrochloride
(69.1 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol), by a procedure similar to that for
20, gave 1-(2-ethylphenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-
indazol-4-one 8 (52.1 mg, 46%) as a powder. 1H NMR (CD3OD,
400 MHz)
d 7.52–7.47 (m, 2H), 7.40–7.36 (m, 1H), 7.26 (d,
5.2.3. 1-(3-Chlorophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-
4H-indazol-4-one (3)
J = 7.6 Hz, 1H), 2.51 (s, 2H), 2.48–2.42 (m, 5H), 2.40 (s, 2H), 1.09–
1.06 (m, 9H); HRMS calcd for C18H23N2O (M+H) 283.1810, found
283.1810.
The reaction of 1-(3-chlorophenyl)hydrazine hydrochloride
(71.6 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) under reflux
for 5 h, by a procedure similar to that for 20 using concd HCl
(0.40 mL) instead of H2SO4, gave 1-(3-chlorophenyl)-3,6,6-tri-
methyl-1,5,6,7-tetrahydro-4H-indazol-4-one 3 (45.5 mg, 39%) as
a powder. 1H NMR (CD3OD, 400 MHz) d 7.63 (dd, J = 1.2, 3.6 Hz,
1H), 7.60–7.47 (m, 3H), 2.88 (s, 2H), 2.47 (s, 3H), 2.41 (s, 2H),
1.09 (s, 6H); HRMS calcd for C16H18ClN2O (M+H) 289.1108, found
289.1108.
5.2.9. 1-(4-Methoxyphenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-
4H-indazol-4-one (9)
The reaction of 1-(4-methoxyphenyl)hydrazine hydrochloride
(69.9 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) under reflux
for 5 h, by a procedure similar to that for 20 using concd HCl
(0.40 mL) instead of H2SO4, gave 1-(4-methoxyphenyl)-3,6,6-tri-
methyl-1,5,6,7-tetrahydro-4H-indazol-4-one 9 (45.8 mg, 40%) as
an oil. 1H NMR (CD3OD, 300 MHz) d 7.42 (d, J = 9.0 Hz, 2H), 7.08
(d, J = 9.0 Hz, 2H), 3.87 (s, 3H), 2.77 (s, 2H), 2.46 (s, 3H), 2.40 (s,
2H), 1.08 (s, 6H); HRMS calcd for C17H21N2O2 (M+H) 285.1603,
found 285.1604.
5.2.4. 1-(4-Chlorophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-
4H-indazol-4-one (4)
The reaction of 1-(4-chlorophenyl)hydrazine hydrochloride
(71.6 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) at room tem-
perature for 2.5 days, by a procedure similar to that for 20 using
concd HCl (0.40 mL) instead of H2SO4, gave 1-(4-chlorophenyl)-
5.2.10. Ethyl 2-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indazol-1-yl)benzoate (10)
The reaction of 2-hydrazinobenzoic acid hydrochloride
(56.6 mg, 0.30 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (54.7 mg, 0.30 mmol) in ethanol (0.9 mL) under reflux
for 6 h, by a procedure similar to that for 20 using concd HCl
(0.30 mL) instead of H2SO4, gave ethyl 2-(3,6,6-trimethyl-4-oxo-
4,5,6,7-tetrahydro-1H-indazol-1-yl)benzoate 10 (27.7 mg, 28%) as
a powder. 1H NMR (CDCl3, 300 MHz) d 8.01 (dd, J = 1.5, 7.5 Hz,
1H), 7.67–7.54 (m, 2H), 7.36 (dd, J = 1.5, 7.5 Hz, 1H), 4.15 (q,
J = 7.2 Hz, 2H), 2.52 (s, 3H), 2.50 (s, 2H), 2.38 (s, 2H), 1.13 (t,
3,6,6-trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one
4 (45.5 mg,
39%) as a powder. 1H NMR (CD3OD, 300 MHz) d 7.56 (m, 4H),
2.86 (s, 2H), 2.47 (s, 3H), 2.41 (s, 2H), 1.09 (s, 6H); HRMS calcd
for C16H18ClN2O (M+H) 289.1108, found 289.1109.
5.2.5. 1-(2-Bromophenyl)-3,6,6-trimethyl-1,5,6,7-tetrahydro-
4H-indazol-4-one (5)
The reaction of 1-(2-bromophenyl)hydrazine hydrochloride
(89.4 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-

4700
M.-F. Cheng et al. / Bioorg. Med. Chem. 22 (2014) 4694–4703
J = 7.2 Hz, 3H), 1.09 (s, 6H); HRMS calcd for C19H23N2O3 (M+H)
327.1709, found 327.1703.
a powder. 1H NMR (CD3OD, 400 MHz) d 7.48 (s, 1H), 7.39 (d,
J = 8.4 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 2.51 (s, 2H), 2.46 (s, 3H),
2.40 (s, 2H), 2.07 (s, 3H), 1.08 (s, 6H); HRMS calcd for C17H20ClN2O
(M+H) 303.1264, found 303.1267.
5.2.11. Methyl 4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indazol-1-yl)benzoate (11)
The reaction of 4-hydrazinobenzoic acid hydrochloride
(60.9 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in MeOH (1.2 mL) under reflux
for 3 days, by a procedure similar to that for 20, gave methyl 4-
(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzo-
ate 11 (27.7 mg, 28%) as a powder. 1H NMR (CD3OD, 400 MHz)
d 8.19 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 3.94 (s, 3H), 2.94
(s, 2H), 2.48 (s, 3H), 2.43 (s, 2H), 1.09 (s, 6H); HRMS calcd for
5.2.16. 1-(2,4-Difluorophenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (16)
The reaction of 1-(2,4-difluorophenyl)hydrazine hydrochloride
(72.2 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) under reflux
for 5 h, by a procedure similar to that for 20 using concd HCl
(0.40 mL) instead of H2SO4, gave 1-(2,4-difluorophenyl)-3,6,6-tri-
methyl-1,5,6,7-tetrahydro-4H-indazol-4-one 16 (66.5 mg, 57%) as
a powder. 1H NMR (CD3OD, 300 MHz) d 7.64–7.56 (m, 1H), 7.34–
7.27 (m, 1H), 7.20 (dd, J = 7.8, 7.8 Hz, 1H), 2.62 (s, 2H), 2.46 (s,
3H), 2.40 (s, 2H), 1.08 (s, 6H); HRMS calcd for C16H17F2N2O
(M+H) 291.1309, found 479.1360.
C18H21N2O3 (M+H) 313.1552, found 313.1558.
5.2.12. 1-(2,4-Dimethylphenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (12)
The reaction of 1-(2,4-dimethylphenyl)hydrazine hydrochloride
(69.1 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) at room tem-
perature overnight, by a procedure similar to that for 20 using
concd HCl (0.40 mL) instead of H2SO4 and the crude product was
purified by flash chromatography on silica gel eluting with
EtOAc/hexane (4:1), gave 1-(2,4-dimethylphenyl)-3,6,6-trimethyl-
1,5,6,7-tetrahydro-4H-indazol-4-one 12 (48.5 mg, 42%) as a pow-
der. 1H NMR (CD3OD, 400 MHz) d 7.25 (s, 1H), 7.18 (d, J = 7.6 Hz,
1H), 7.13 (d, J = 7.6 Hz, 1H), 2.48 (s, 2H), 2.46 (s, 3H), 2.39 (s, 5H),
2.03 (s, 3H), 1.07 (s, 6H); HRMS calcd for C18H23N2O (M+H)
283.1810, found 283.1817.
5.2.17. 1-(2,5-Dichlorophenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (17)
The reaction of 1-(2,5-chlorophenyl)hydrazine hydrochloride
(85.4 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) at room tem-
perature overnight, by a procedure similar to that for 20 using
concd HCl (0.40 mL) instead of H2SO4 and the crude product was
purified by flash chromatography on silica gel eluting with
EtOAc/hexane (4:1), gave 1-(2,5-dichlorophenyl)-3,6,6-trimethyl-
1,5,6,7-tetrahydro-4H-indazol-4-one 17 (60.1 mg, 46%) as a pow-
der. 1H NMR (CD3OD, 300 MHz) d 7.69–7.60 (m, 3H), 2.57 (s, 2H),
2.46 (s, 3H), 2.40 (s, 2H), 1.09 (s, 6H); HRMS calcd for C16H17Cl2N2O
(M+H) 323.0718, found 323.0719.
5.2.13. 1-(2,3-Dimethylphenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (13)
The reaction of 1-(2,3-dimethylphenyl)hydrazine hydrochloride
(69.1 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) at room tem-
perature overnight, by a procedure similar to that for 20 using
concd HCl (0.40 mL) instead of H2SO4 and the crude product was
purified by flash chromatography on silica gel eluting with
EtOAc/hexane (4:1), gave 1-(2,3-dimethylphenyl)-3,6,6-trimethyl-
1,5,6,7-tetrahydro-4H-indazol-4-one 13 (50.8 mg, 45%) as a pow-
der. 1H NMR (CD3OD, 400 MHz) d 7.37 (d, J = 7.6 Hz, 1H), 7.26
(dd, J = 7.6, 7.6 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 2.46 (s, 5H), 2.39
(s, 2H), 2.37 (s, 3H), 1.94 (s, 3H), 1.07 (s, 6H); HRMS calcd for
5.2.18. 1-(2,4-Dichlorophenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (18)
The reaction of 1-(2,4-chlorophenyl)hydrazine hydrochloride
(85.4 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) under reflux
for 5 h, by a procedure similar to that for 20 using concd HCl
(0.40 mL) instead of H2SO4, gave 1-(2,4-dichlorophenyl)-3,6,6-tri-
methyl-1,5,6,7-tetrahydro-4H-indazol-4-one 18 (62.1 mg, 48%) as
a powder. 1H NMR (CDCl3, 400 MHz) d 7.58 (d, J = 2.0 Hz, 1H),
7.42–7.35 (m, 2H), 2.53 (s, 3H), 2.49 (s, 2H), 2.38 (s, 2H), 1.10 (s,
6H); HRMS calcd for
479.1360.
C16H17Cl2N2O (M+H) 323.0718, found
C18H23N2O (M+H) 283.1810, found 283.1814.
5.2.14. 1-(2,5-Dimethylphenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (14)
5.2.19. 1-(2,4-Dibromophenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (19)
The reaction of 1-(2,5-dimethylphenyl)hydrazine hydrochloride
(69.1 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclohex-
anedione (72.9 mg, 0.40 mmol), by a procedure similar to that for
20, gave 1-(2,5-dimethylphenyl)-3,6,6-trimethyl-1,5,6,7-tetrahy-
dro-4H-indazol-4-one 14 (31.1 mg, 27%) as a powder. 1H NMR
(CD3OD, 300 MHz) d 7.30–7.29 (m, 2H), 7.10 (s, 1H), 2.50 (s, 2H),
2.46 (s, 3H), 2.40 (s, 2H), 2.37 (s, 3H), 2.02 (s, 3H), 1.08 (s, 6H);
HRMS calcd for C18H23N2O (M+H) 283.1810, found 283.1811.
The reaction of 1-(2,4-dibromophenyl)hydrazine hydrochloride
23a (121.0 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-cyclo-
hexanedione (72.9 mg, 0.40 mmol), by a procedure similar to that
for 20, gave 1-(2,4-dibromophenyl)-3,6,6-trimethyl-1,5,6,7-tetra-
hydro-4H-indazol-4-one 19 (100.2 mg, 61%) as a powder. 1H
NMR (CD3OD, 400 MHz) d 8.07 (d, J = 2.0 Hz, 1H), 7.76 (dd, J = 2.0,
8.4 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 2.54 (s, 2H), 2.46 (s, 3H), 2.40
(s, 2H), 1.10 (s, 6H); HRMS calcd for
410.9708, found 410.9709.
C16H17Br2N2O (M+H)
5.2.15. 1-(4-Chloro-2-methylphenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (15)
5.2.20. 1-(2-Bromo-4-methylphenyl)-3,6,6-trimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one (21)
The reaction of 1-(4-chloro-2-methylphenyl)hydrazine hydro-
chloride (77.2 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-1,3-
cyclohexanedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL) at
room temperature overnight, by a procedure similar to that for
20 using concd HCl (0.40 mL) instead of H2SO4 and the crude prod-
uct was purified by flash chromatography on silica gel eluting with
EtOAc/hexane (4:1), gave 1-(4-chloro-2-methylphenyl)-3,6,6-tri-
methyl-1,5,6,7-tetrahydro-4H-indazol-4-one 15 (53.3 mg, 44%) as
The reaction of 1-(2-bromo-4-methylphenyl)hydrazine hydro-
chloride 23c (95.0 mg, 0.40 mmol) with 2-acetyl-5,5-dimethyl-
1,3-cyclohexanedione (72.9 mg, 0.40 mmol) in ethanol (1.2 mL)
at room temperature overnight, by a procedure similar to that for
20, gave 1-(2-bromo-4-methylphenyl)-3,6,6-trimethyl-1,5,6,7-tet-
rahydro-4H-indazol-4-one 21 (55.1 mg, 40%) as a powder. 1H
NMR (CD3OD, 400 MHz) d 7.67 (s, 1H), 7.37 (dd, J = 1.6, 1.6 Hz,

M.-F. Cheng et al. / Bioorg. Med. Chem. 22 (2014) 4694–4703
4701
2H), 2.51 (s, 2H), 2.46 (s, 3H), 2.44 (s, 3H), 2.39 (s, 2H), 1.09 (s, 6H);
HRMS calcd for C17H20BrN2O (M+H) 347.0759, found 347.0756.
ecarbonyl chloride (0.65 mL, 7.1 mmol) in CH2Cl2 (1.8 mL) slowly.
The reaction was refluxed for 2 h. After the reaction was finished,
CH2Cl2 was removed and the residue was purified by Isco Combi-
Flash Companion column chromatography (silica gel, 0–30%
EtOAc/hexane) to give compound 25b (1.1 g, 77%) as a powder.
1H NMR (CDCl3, 400 MHz) d 3.60–3.56 (m, 1H), 2.52 (s, 2H), 2.40
(s, 2H), 1.32–1.28 (m, 2H), 1.16–1.09 (m, 8H).
5.2.21. 1-(2,4-Dichlorophenyl)-3-methyl-1,5,6,7-tetrahydro-4H-
indazol-4-one (22)
The reaction of 1-(2,4-dichlorophenyl)hydrazine hydrochloride
(85.4 mg, 0.40 mmol) with 2-acetyl -1,3-cyclohexanedione
(61.7 mg, 0.40 mmol) in ethanol (1.2 mL) under reflux for 5 h, by
a procedure similar to that for 20 using concd HCl (0.40 mL)
instead of H2SO4, gave 1-(2,4-dichlorophenyl)-3-methyl-1,5,6,7-
tetrahydro-4H-indazol-4-one 22 (49.9 mg, 42%) as a powder. 1H
NMR (CD3OD, 300 MHz) d 7.78 (d, J = 2.1 Hz, 1H), 7.59–7.52 (m,
2H), 2.88–2.69 (m, 2H), 2.56–2.49 (m, 2H), 2.46 (s, 2H), 2.37 (s,
1H), 2.19–2.12 (m, 2H); HRMS calcd for C14H13Cl2N2O (M+H)
295.0405, found 295.0403.
5.3.5. 1-(2,4-Dichlorophenyl)-6,6-dimethyl-3-(trifluoromethyl)-
1,5,6,7-tetrahydro-4H-indazol-4-one (26)
The reaction of 1-(2,4-dichlorophenyl)hydrazine hydrochloride
(85.4 mg, 0.40 mmol) with 5,5-dimethyl-2-(trifluoroacetyl)cyclo-
hexane-1,3-dione 25a (94.5 mg, 0.40 mmol) in ethanol (1.2 mL)
under reflux for 5 h, by a procedure similar to that for 20, gave
1-(2,4-dichlorophenyl)-6,6-dimethyl-3-(trifluoromethyl)-1,5,6,7-
tetrahydro-4H-indazol-4-one 26 (71.5 mg, 47%) as a powder. 1H
NMR (CD3OD, 400 MHz) d 7.83 (d, J = 1.2 Hz, 1H), 7.61 (dd, J = 1.2,
1.2 Hz, 2H), 2.63 (s, 2H), 2.48 (s, 2H), 1.11 (s, 6H); HRMS calcd
for C16H14Cl2F3N2O (M+H) 377.0435, found 377.0442.
5.3. Representative procedure for the preparation of
phenylhydrazine hydrochlorides from anilines
A mixture of 2-bromo-4-chloroaniline (825.9 mg, 4.0 mmol)
and concd HCl (1.6 mL) was cooled at 0 °C and treated with a solu-
tion of NaNO2 (0.3 g, 4.4 mmol) in H2O (1.5 mL) precooled to 0 °C.
After 45 min, a solution of SnCl2 (2.2 g, 11.5 mmol) in concd HCl
(2.8 mL) was added and a precipitation was formed. The mixture
was stirred at room temperature for 2 h. After filtration, the precip-
itation was collected and washed with water (7.0 mL) and ether
(30.0 mL) to give 1-(2-bromo-4-chlorophenyl)hydrazine hydro-
5.3.6. 3-Cyclopropyl-1-(2,4-dichlorophenyl)-6,6-dimethyl-
1,5,6,7-tetrahydro-4H-indazol-4-one (27)
The reaction of 1-(2,4-dichlorophenyl)hydrazine hydrochloride
(234.9 mg, 1.1 mmol) with 2-(cyclopropylcarbonyl)-5,5-dimethyl-
cyclohexane-1,3-dione 25b (199.9 mg, 0.96 mmol) in ethanol
(5.0 mL) at room temperature overnight, by a procedure similar
to that for 20 using NaOH (44.0 mg, 1.1 mmol) instead of H2SO4,
gave 3-cyclopropyl-1-(2,4-dichlorophenyl)-6,6-dimethyl-1,5,6,7-
tetrahydro-4H-indazol-4-one 27 (36.2 mg, 11%) as a powder. 1H
NMR (CDCl3, 400 MHz) d 7.55 (d, J = 2.0 Hz, 1H), 7.39 (dd, J = 2.0,
8.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 2.62–2.58 (m, 1H), 2.45 (s,
2H), 2.40 (s, 2H), 1.10 (s, 6H), 1.03–1.00 (m, 4H); HRMS calcd for
chloride 23b (446.7 mg, 43%) as
300 MHz) d 10.05 (br, 2H), 7.96 (br, 1H), 7.69 (d, J = 2.4 Hz, 1H),
7.46 (dd, J = 2.4, 8.7 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H).
a
solid. 1H NMR (DMSO,
5.3.1. 1-(2,4-Dibromophenyl)hydrazine hydrochloride (23a)
Preparation of 1-(2,4-dibromophenyl)hydrazine hydrochloride
23a (0.5 g, 41%) from 2,4-dibromoaniline (1.0 g, 4.0 mmol) by a
procedure similar to that for 23b. 1H NMR (DMSO, 400 MHz) d
10.11 (br, 2H), 8.01 (br, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.88 (dd,
J = 2.0, 8.8 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H).
C18H19Cl2N2O (M+H) 349.0874, found 349.0875.
5.3.7. 3,6,6-Trimethyl-1,5,6,7-tetrahydro-4H-indazol-4-one (28)
To a solution of 2-acetyl-5,5-dimethyl-1,3-cyclohexanedione
(72.9 mg, 0.40 mmol) in THF (2.0 mL) was added hydrazine
(0.80 mmol, 65% in H2O). The reaction was stirred under reflux.
After 4 h, THF was removed and the residue was washed with
water (2.0 mL) and CH2Cl2 (1.0 mL) to give 3,6,6-trimethyl-
1,5,6,7-tetrahydro-4H-indazol-4-one 28 (30.1 mg, 42%) as a pow-
der. 1H NMR (DMSO, 300 MHz) d 2.59 (s, 2H), 2.33 (s, 3H), 2.21
(s, 2H), 0.98 (s, 6H); HRMS calcd for C10H15N2O (M+H) 179.1184.
5.3.2. 1-(2-Bromo-4-methylphenyl)hydrazine hydrochloride
(23c)
Preparation of 1-(2-bromo-4-methylphenyl)hydrazine hydro-
chloride 23c (311.5 mg, 33%) from 2-bromo-4-methylaniline
(744.2 mg, 4.0 mmol) by a procedure similar to that for 23b. 1H
NMR (DMSO, 300 MHz) d 10.00 (br, 2H), 7.68 (br, 1H), 7.40 (s,
1H), 7.16 (d, J = 8.1 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 2.49 (s, 3H).
5.3.8. 1-(2,4-Dibromophenyl)-3,6,6-trimethyl-4,5,6,7-
tetrahydro-1H-indazol-4-ol (29)
5.3.3. 5,5-Dimethyl-2-(trifluoroacetyl)cyclohexane-1,3-dione
(25a)
A
solution of tetrahydroindazol-4-one 19 (30.1 mg,
0.073 mmol) in CH2Cl2 (1.0 mL) and MeOH (0.30 mL) was treated
with NaBH4 (14.0 mg, 0.37 mmol), and stirred at room temperature
overnight. The reaction mixture was quenched with satd NH4Cl(aq)
(1.0 mL) and extracted with CH2Cl2 (2.0 mL). The organic layer was
dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by Isco CombiFlash Companion column chro-
matography (silica gel, 0–70% EtOAc/hexane) to give 1-(2,4-dib-
romophenyl)-3,6,6-trimethyl-4,5,6,7-tetrahydro-1H-indazol-4-ol 29
(19.0 mg, 63%) as a powder. 1H NMR (CD3OD, 300 MHz) d 8.00
(d, J = 1.8 Hz, 1H), 7.69 (dd, J = 1.8, 8.7 Hz, 1H), 7.31 (d, J = 8.7 Hz,
1H), 4.81 (dd, J = 7.8, 7.8 Hz, 1H), 2.34 (s, 3H), 2.29 (d, J = 15.9 Hz,
1H), 2.05 (d, J = 15.9 Hz, 1H), 1.90 (dd, J = 7.8, 13.2 Hz, 1H), 1.55
(dd, J = 7.8, 13.2 Hz, 1H), 1.09 (s, 3H), 0.96 (s, 3H); HRMS calcd
for C16H19Br2N2O (M+H) 412.9864, found 412.9864.
To a solution of CDI (1.2 g, 7.1 mmol) in CHCl3 (35.0 mL) was
added a solution of trifluoroacetic acid (1.1 mL, 14.2 mmol) in
CHCl3 (53.0 mL) slowly. Then the mixture was treated with a solu-
tion of 5,5-dimethyl-1,3-cyclohexanedione (504.7 mg, 3.6 mmol)
and imidazole (245.1 mg, 3.6 mmol) in CHCl3 (35.0 mL), and stirred
at room temperature for 45 min. After the reaction was finished,
the mixture was washed with 1 N HCl(aq) (50.0 mL) and water
(50.0 mL). The organic layer was dried over MgSO4 and concen-
trated under reduced pressure. The residue was purified by Isco
CombiFlash Companion column chromatography (silica gel, 0–
100% EtOAc/hexane) to give compound 25a (1.1 g, 77%) as a
powder.
5.3.4. 2-(Cyclopropylcarbonyl)-5,5-dimethylcyclohexane-1,3-
dione (25b)
5.4. FLIPRÒ calcium assay
To a solution of 5,5-dimethyl-1,3-cyclohexanedione (995.3 mg,
7.1 mmol), DMAP (256.6 mg, 2.1 mmol) and DIPEA (1.2 mL,
7.1 mmol) in CH2Cl2 (7.0 mL) was added a solution of cyclopropan-
One day before the assay, CORNINGÒ black with clear flat
bottom 96-well assay plates were coated with a 0.1 mg/mL

4702
M.-F. Cheng et al. / Bioorg. Med. Chem. 22 (2014) 4694–4703
Poly-L-Lysine solution. Chinese hamster ovary (CHO)-K1 cells, sta-
5.8. Tail-flick test
bly expressing hMOR and Ga15 (GenScript), were suspended in
the F12 medium and plated at a density of ꢀ8 Â 104 cells/well
Nociception of mice was evaluated by using a tail-flick instru-
ment (Columbus Instruments). The intensity of the light was
adjusted so that the averaged basal tail-flick latencies were 3–4 s.
A cut-off time of 10 s was used as the maximum possible effect
(MPE) to avoid tissue damage. A mouse that did not flick its tail
within the cut-off time was considered as fully analgesic. The area
under the time-response curve (AUC)35 and the ED50 value was
employed to evaluate the analgesic effect of the drug(s). The tail-
flick latency was examined at 10, 20, 30, 40, 50, and 60 min after
drug administration to plot time-response (test latency-basal
latency) curve and the AUC value was calculated. The ED50 was
determined by the up-and-down method.36
in 200 lL medium. Cells were incubated in a humidified atmo-
sphere of 10% CO2 at 37 °C overnight so as to reach an 80–90%
confluent cell monolayer before assay. At the day of assay,
150
50
lL medium/well was removed from plate. To each well,
l
L FLIPRÒ calcium assay reagent dissolved in 1Â assay buffer
(HBSS: KCl 5 mM, KH2PO4 0.3 mM, NaCl 138 mM, NaHCO3
4 mM, Na2HPO4 0.3 mM, d-glucose 5.6 mM, with additional
20 mM HEPES and 13 mM CaCl2, pH 7.4), with 2.5 mM probenecid
was added and the plate is incubated at 37 °C for 1 h. Compounds
and other reagents were dissolved in the assay buffer. Using a
FlexStationIII (Molecular Devices), the [Ca2+
]
fluorescence
i
increases after robotic injections of compounds or other reagents
were monitored every 1.52 s interval with excitation wavelength
Acknowledgments
at 485 nm and with emission wavelength at 525 nm. The [Ca2+
]
i
fluorescence was measured up to 90 s after agonist injection.
The fluorescence intensity from 6 to 12 wells of cells were
We are grateful to National Health Research Institutes and
National Science Council of the Republic of China (100NP1004)
for financial support.
averaged and the relative amount of [Ca2+
] release was deter-
i
mined by integrating the AUC of the [Ca2+]i fluorescence averages.
Supplementary data
5.5. Competition radioligand binding assay
Supplementary data (the in vivo data of vehicle control and nal-
oxone) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmc.2014.07.012.
Briefly, HEK-MOR cells were washed twice and scraped off the
culture plate with ice-cold phosphate-buffered saline (PBS), and
pelleted by centrifugation at 1000g, 10 min, 4 °C. Cell pellets were
homogenized in assay buffer (50 mM Tris–HCl, pH 7.5), at 4 °C. Cel-
lular membrane pellet was collected by centrifuge at 24,000g for
30 min at 4 °C, and resuspended in assay buffer. For competition
binding assay, cell membranes were incubated for 60 min in
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