Efficient one-pot synthesis of 5-perfluoroalkylpyrazoles by cyclization of hydrazone dianions

Article abstract of DOI:10.1039/c5ob01151e

A highly selective and efficient method for the synthesis of 5-trifluoromethylated and 5-perfluoroalkylated pyrazoles has been developed which relies on the cyclization of hydrazine dianions with ethyl perfluorocarboxylates. The pyrazoles prepared were ev

Full text of DOI:10.1039/c5ob01151e

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DOI: 10.1039/C5OB01151E
Efficient Oneꢀpot Synthesis of 5ꢀPerfluoroalkylpyrazoles by
Cyclization of Hydrazone Dianions
Thang Ngoc Ngo,a Syeda Abida Ejaz,b Tran QuangHung,a,e Tuan Thanh Dang,a,*
Jamshed Iqbal,b Joanna Lecka,d Jean Sévigny,d Peter Langera,c*
aInstitut für Chemie, Universität Rostock, AlbertꢀEinsteinꢀStr. 3a, 18059 Rostock, Germany. Eꢀmail: peter.langer@uniꢀ
rostock.de; thanhtuandang@hotmail.com, Fax: +49 381 4986412; Tel: +49 381 4986410
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad, Pakistan
cLeibniz Institut für Katalyse an der Universität Rostock e. V. (LIKAT),AlbertꢀEinsteinꢀStr. 29a, 18059 Rostock, Germany.
dDepartment of MicrobiologyꢀInfectiology and Immunology, Faculty of Medicine, Centre de Recherche du CHU de Québec –
Université Laval, Québec, QC, G1V 4G2, Canada
eInstitute of chemistry, vietnam academy of science and technology, 18 Hoang Quoc Viet, Vietnam
Abstract: A highly selective and efficient method for the synthesis of 5ꢀtrifluoromethylated and
5ꢀperfluoroalkylated pyrazoles has been developed which relies on the cyclization of hydrazine
dianions with ethyl perfluorocarboxylates. The pyrazoles prepared were evaluated as potential
inhibitors of alkaline phosphatases, namely human tissue nonꢀspecific alkaline phosphatase (hꢀ
TNAP) and tissue specific intestinal alkaline phosphatase (IAP). Most pyrazole derivatives
inhibited hꢀIAP more markedly than hꢀTNAP and had minor effects on nucleotide
1

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pyrophosphatase/phosphodiesterases. Therefore, the compounds appear as potential selective
inhibitors of hꢀIAP.
Introduction
Pyrazole derivatives represent important building blocks in pharmaceutical and agrochemical
industry.1 The pyrazole ring is present in many leading drugs, such as Zometapine2 and Viagra3
and in agrochemicals, such as Tolfenpyrad4 and Fenpyroximate.5 Recently, the development of
efficient methods for the introduction of the trifluoromethyl group into heterocycles allowed the
preparation of new fluorinated heterocycles which exhibit significant changes in their chemical,
physical and biological properties.6,7 In fact, most of the recently developed drugs and
agrochemicals contain a fluorine atom or a fluorinated side chain.6,7 Therefore, new methods for
the synthesis of organofluorine molecules play an important role for the development of
bioactive molecules. Trifluoromethylated and perfluoroalkylated pyrazoles represent
pharmacologically relevant core structures8 which are present in many important drugs and
agrochemicals, such as Celecoxib (antiarthritic), Mavacoxib (antiarthritic), Razaxaban
(anticoagulant), Fluazolate (herbicide), Penthiopyrad (fungicide).9,10
2

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Figure 1. Drugs and agrochemicals containing trifluoromethylated pyrazoles.
The development of new methods for the synthesis of pyrazole derivatives has become
extremely important in recent years.1 Conventional methods for the synthesis of pyrazoles are
based on the cyclocondensation of hydrazines with 1,3ꢀdielectrophiles, such as 1,3ꢀdicarbonyl or
α,βꢀunsaturated carbonyl compounds.1,11 A convenient method is based on the 1,3ꢀdipolar
cycloaddition of nitrile imines with alkynes.12 The disadvantage of these methods is the
formation of a mixture of regioisomers of 3ꢀ and 5ꢀsubstituted pyrazoles which need to be
separated.1,11 Another interesting approach is the cyclization of hydrazone dianions with esters.13
Hauser and coꢀworkers were the first to report the synthesis of 5ꢀsubstituted pyrazoles by
cyclization of hydrazone 1,4ꢀdianions with esters.14 The same strategy was applied by our group
to prepare pyrazoleꢀ5ꢀcarboxylates and pyrazoleꢀ1,5ꢀdicarboxylates.15
Trifluoromethylated pyrazoles have been prepared by cyclocondensation of trifluoromethylꢀ
1,3ꢀdiketones with phenylhydrazine, however, mixtures of regioisomers were formed.1,16 5ꢀ
Trifluoromethylpyrazoles could be regioselectively synthesized by cyclization of
phenylhydrazones with Nꢀarylꢀtrifluoroacetimidoyl iodides. However, this method is limited by
the fact that the starting materials have to be prepared in two steps which proceed in
unsatisfactory yields.17 In order to find an atomꢀeconomic approach for the synthesis of
trifluoromethylated pyrazoles, several research groups have focused on the development of new
methods which address the issue of regioselectivity. Frizzo et al. reported a useful method for the
synthesis of 5ꢀ(trifluoromethyl)pyrazoles based on the condensation of 4ꢀalkoxyꢀ1,1,1ꢀtrifluoroꢀ
3ꢀalkenꢀ2ꢀones with phenylhydrazine using the ionic liquid [BMIM][BF4] as the solvent.18 In
general, this method gave very good yields of 5ꢀ(trifluoromethyl)pyrazoles, but for some
derivatives still regioisomeric mixtures were obtained. Recently, some useful methods have been
developed to overcome this problem, e.g., the use of fluorinated alcohols (TFE and HFIP)19 in
the cyclocondensation of trifluoromethylꢀ1,3ꢀdiketones with phenylhydrazine or the employment
of 4ꢀtrifluoromethylꢀsydnones as starting materials in the cycloaddition reaction with alkynes.20
In early 2014, Mykhailiuk and coworkers reported an interesting approach for the synthesis of 3ꢀ
trifluoromethylpyrazoles in very good yields based on the [3+2] cycloaddition of CF3CHN2 with
alkynes.21 Very recently, a new method for the synthesis of 3ꢀtrifluoromethylpyrazoles was
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described via trifluoromethylation/cyclization of α,βꢀalkynic hydrazone with a hypervalent
iodine reagent.22
Herein, we report a short, convenient and efficient method for the synthesis 5ꢀtrifluoromethylꢀ
and 5ꢀperfluoroalkylpyrazoles by oneꢀpot cyclization of hydrazone 1,4ꢀdianions with fluorinated
esters. In addition, we report the activity of the pyrazoles prepared as inhibitors of two alkaline
phosphatases: human tissueꢀnonspecific alkaline phosphatase (hꢀTNAP) and human intestinal
alkaline phosphatase (hꢀIAP). The effects of these molecules were also tested on two other
human ectonucleotidases, ectoꢀnucleotide pyrophosphatase/phosphodiesteraseꢀ1 (hꢀNPP1) and hꢀ
NPP3.
Alkaline Phosphatases (APs; EC 3.1.3.1) are homodimeric enzymes that require three metal
ions for its catalytic activity, i.e. Ca and two Zn cations. These enzymes catalyze the hydrolysis
of the phosphate ester bonds and the transphosphorylation reaction.23 Different mammalian
isozymes are present in the body and they are encoded by four genes. They are classified under
two categories; tissue specific alkaline phosphatases and tissue nonꢀspecific alkaline phosphatase
(TNAP).24 The tissue specific APs are expressed at restricted areas, such as placenta, germ cells
and intestine, whereas the tissue nonꢀspecific isozymes are predominant in bone, liver and
kidney tissues and are expressed at lower level at other sites. These four isozymes differ from
each other in relation to their response to specific inhibitors and their genes of origin. The main
physiological role of TNAP is the hydrolysis of extracellular inorganic pyrophosphate (PPi) to
inorganic phosphate.25
The inorganic pyrophosphate act as a potent inhibitor of calcification. Therefore, TNAP plays
an important role in the maintenance of the PPi level in the body and is helpful in the normal
process of bone mineralization.26 The potent inhibitors of TNAP act as a useful therapeutic agent
in different disease conditions, such as arterial calcification, ankylosis and osteoarthritis.27
Because of a high homology between tissueꢀspecific IAP and TNAP, very few selective
inhibitors of IAP have been reported.28 The tissue specific intestinal alkaline phosphatase is
enriched in surfactantꢀlike particles (SLP) and is located at the brush boarder of intestine. It plays
an important role in the intestinal absorption of lipids and nutrients through its association with
SLP. After a fatty meal the IAP level increases significantly. Therefore, IAP plays a major role
in the maintenance of intestinal homeostasis.29
4

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EꢀNPPsꢀtype ectophosphodiesterases are transmembrane bounded and cell surface proteins.
NPP2 can also be found extracellularly as a shedded or secreted enzyme. NPPs play an important
role in the hydrolysis of pyrophosphate or phosphodiester bonds in a variety of compounds
including nucleotides, (lyso) phospholipids and choline phosphate esters and prevent the
activation of P2X and P2Y receptors. While NPP1 and NPP3 are mainly involved in the control
of purinergic signaling, the other members of the NPP family rather hydrolyze phospholipids.
Although NPP1ꢀ3 have 50% structural relationship with each other, they show broad substrate
specificity for each enzyme.30
At a physiological level, NPPs mainly affect a number of processes like bone mineralization,
cell proliferation, motility and digestion. The isozymes of NPPs are involved in the
pathophysiology of different disease conditions, such as calcification, cancer and insulin
resistance. Therefore, efforts are being made to discover selective inhibitors of NPPs. NPP1
plays an important role in bone mineralization as it acts as the main enzyme responsible for the
production of PPi for chondrocytes and osteoblast. It also affects the insulin signaling process by
inhibiting the tyrosine kinase activity. NPP3 expression is considered as a tumor marker and is
associated with metastasis of cancer cells and carcinogenesis.31
Results and discussion
Synthesis of perfluoroalkylated pyrazoles:
Hydrazones 2 were prepared by condensation of ketones 1 with hydrazine derivatives. This
reaction proceeded under solvent free (‘green’) conditions at room temperature and is catalyzed
by acetic acid and provided nearly quantitative yields of hydrazones. Then, 2 were converted to
o
their dianions by treatment with 2 equivalents of nꢀBuLi in THF at ꢀ78 C. Subsequently, ethyl
perfluorocarboxylates 3 were added to the reaction mixture. After warming to ambient
temperature, either trifluoroacetic acid (TFA) or pꢀtoluenesulfonic acid (PTSA) was added to the
reaction mixture to give perfluoroalkylated pyrazoles 4 or 5, respectively (based on 1) (Scheme
1). The formation of the product proceeds by attack of the carbon of dianion A onto 3 to give
intermediate B, cyclization by attack of the nitrogen atom onto the carbonyl group to give
5

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intermediate C, and subsequent acidꢀmediated dehydration. Treatment of intermediate C with
TFA under reflux in dioxane allowed 5ꢀperfluoromethylated pyrazoles 4 in good to excellent
isolated yields. On the other hand, treatment ofintermediate C with PTSA (reflux, toluene) gave
the Nꢀdeprotected 3ꢀperfluoromethylpyrazoles 5. This could be explained by the acidity of TFA
and PTSA. PTSA is a stronger acid than TFA. The carbamate protecting group was removed
when PTSA was used but remained intact when TFA was used. It is noteworthy that the
synthesis of compound 4f could be scaled up to gram quantities. Starting with 10 mmol of 1f,
product 4f was isolated in 88% yield (2.67 gram).
O
R
R
R
NH
NH EtO
RF
N
N
O
HN
H2N
N
N
3
RF
RF
or
Ar
Ar
Ar
Ar
i
ii
5
4
1
2
TFA
Reflux (6h)
(R= Ph, COOEt)
PTSA
Reflux (8h)
(R= COOEt)
2.2 n-BuLi
R
R
R
NLi
N
Li
N
3a
N
O
N
N
RF
OLi
Li
Ar
RF
Ar
Ar
B
C
A
Scheme 1. Synthesis of pyrazoles 4 and 5. Conditions:i, neat, acetic acid, 20 oC; ii, 1) 2.2 equiv. nꢀBuLi,
THF, ꢀ78 oC to 20 oC. 2) 1.5 equiv. 3a, ꢀ78 oC to 20 oC. 3) TFA, reflux 2h (or PTSA, reflux, 8h).
Table 1. Sythesis of pyrazoles 4 and 5
No
4a
4b
Ar
R
RF Yield (%)a
C6H5
C6H5 CF3
89
79
4ꢀ(MeO)C6H4 C6H5 CF3
6

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3ꢀ(MeO)C6H4 C6H5 CF3
2ꢀ(MeO)C6H4 C6H5 CF3
81
61
74
87
90
82
78
64
95
91
93
85
83
76
57
65
51
56
57
4c
4d
4e
4f
4ꢀPhC6H4
4ꢀMeC6H4
2ꢀFC6H4
C6H5 CF3
C6H5 CF3
C6H5 CF3
C6H5 CF3
C6H5 CF3
4g
4h
4i
4ꢀFC6H4
2ꢀNaphthyl
4ꢀ(F3C)C6H4 C6H5 CF3
C6H5 C6H5 C2F5
4ꢀ(MeO)C6H4 C6H5 C2F5
4j
4k
4l
2ꢀNaphthyl
4ꢀPhC6H4
C6H5
C6H5 C2F5
C6H5 C2F5
C6H5 C3F7
4m
4n
4o
4p
4q
4r
5a
5b
5c
4ꢀ(MeO)C6H4 C6H5 C3F7
4ꢀ(MeO)C6H4 CO2Et CF3
2ꢀFC6H4
CO2Et CF3
4ꢀ(MeO)C6H4
4ꢀ(MeO)C6H4
4ꢀ(MeO)C6H4
H
H
H
CF3
C2F5
C3F7
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2ꢀNaphthyl
2ꢀNaphthyl
2ꢀNaphthyl
4ꢀ(F3C)C6H4
4ꢀ(F3C)C6H4
4ꢀ(F3C)C6H4
H
H
H
H
H
H
CF3
C2F5
C3F7
CF3
78
86
64
75
74
67
5d
5e
5f
5g
5h
5i
C2F5
C3F7
aIsolated yields
The cyclization of the dianion of the hydrazone of cyclododecanone 2s with ethyl 2,2,2ꢀ
trifluoroacetate afforded the annulated trifluoromethylated pyrazole 4s in 80% yield (Scheme 2).
The cyclizations of the hydrazones of cyclohexanone 2t, cyclohexꢀ2ꢀenꢀ1ꢀone 2u and tetralone
2v afforded the corresponding products 4tꢀv.
o
o
Scheme 2. Synthesis of 4sꢀv.Conditions:i, neat, acetic acid, 20 C; ii, 1) 2.2 equiv. nꢀBuLi, THF, ꢀ78 C
to 20 oC. 2) 1.5 equiv. 3a, ꢀ78 oC to 20 oC. 3) TFA, reflux 2h.
8

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Recently, it has been reported that trifluoromethylated indazole D32 represents a highly
selective ligand for the estrogen receptor β. Trifluoromethylated indazole E33 is a useful agent
for the treatment of obesity and diabetes (Figure 2). Therefore, we were interested in the
preparation of trifluoromethylated indazoles starting from ringꢀfused pyrazoles.
Figure 2. Bioactive trifluoromethylated indazoles D and E
The dehydrogenation of pyrazoles 4s and 4t with DDQ afforded the desired indazoles 6a and
6b in high yields, respectively. These experiments show that our methodology can be
successfully applied also for the synthesis of trifluorinated indazoles.
Scheme 3. Synthesis of 6a and 6b.Conditions:i, 2.0 equiv. DDQ, toluene, reflux, 3 h.
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The cyclization of the dianions of oximes 7a, b with ethyl trifluoroacetate and subsequent
treatment with TFA (reflux, 8h) afforded 5ꢀtrifluoromethylated isoxazoles 8a and 8b in 57% and
62% isolated yields, respectively.
Scheme 4. Synthesis of isoxazoles 8a, b. Conditions:i, 1) 2.2 equiv. nꢀBuLi, THF, ꢀ78 oC to 20 oC. 2) 1.5
equiv. CF3COOEt, ꢀ78 oC to 20 oC. 3) TFA, reflux 2h.
Alkaline Phosphatase and Nucleotide Pyrophosphatase Activity and SAR:
All the fluorinated pyrazole derivatives were tested for human recombinant APs and NPPs
and they were found that these compounds, in comparison to NPPs, to be selective inhibitors of
APs. Against hꢀNPP1 and hꢀNPP3, these compounds exhibited low response of inhibition. The
data obtained showed that all the values were below 50%. Compound 4i was found to be the
potent inhibitor of hꢀTNAP having IC50 value of IC50 ±SEM = 0.45±0.01. It can be suggested
that the activity of this compound might be due to the presence of phenyl and a trifluoromethyl
group at the parent pyrazole ring. When the activity of this compound was compared with the
other derivatives containing naphthalene ring attached to parent pyrazole it was clearly observed
that compound having phenyl and side chain with less carbon atom, has more impact on activity
against hꢀTNAP. When the number of carbon and fluorine increased the activity of the
compound was decreased as it was reflected in activity values of 4m, 5d, 5e and 5f. Levamisole
was used as a standard inhibitor against hꢀTNAP. Other compounds inhibited hꢀTNAP with IC50
values in the range of IC50 ±SEM = 0.449±0.001 to 50.3±3.28 µM. Compound 4n exhibited the
10

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most potent inhibition of hꢀIAP with an IC50value of IC50 ±SEM= 0.65±0.04 µM [sic] which is
over 120 folds more efficient than the known standard inhibitor Lꢀphenylalanine. The
comprehensive study of the compound structure was justified by comparing with Lꢀ
phenylalanine (known reference standard) which contain only one phenyl ring. This confirmed
that the presence of biphenyl group on the pyrazole ring might be responsible for its high activity
against hꢀIAP. On the other hand, when this compound was compared with 4e containing
biphenyl ring with the different carbon side chain it was observed that with a decreased in the
side chain of carbon atom the activity of compound was decreased against hꢀIAP. Other pyrazole
derivatives displayed hꢀIAP inhibition activity in the range IC50 ±SEM = 0.647±0.04 to
7.36±0.25 µM. The above mentioned data showed that most pyrazole derivatives were better
inhibitors of hꢀIAP than of hꢀTNAP.
Table 2. Alkaline phosphatase AP (hꢀTNAP & hꢀIAP) and NPP (hꢀNPP1 & hꢀNPP3) inhibition
in presence of the synthesized compounds
hꢀTNAP
IC50
hꢀIAP
hꢀNPP1
hꢀNPP3
a
IC50 a(µM)±SEM (% inhibition)b (% inhibition)b
No
(µM)±SEM
9.52±1.53
11.1±1.06
10.46±.65
26.6±2.56
3.23±0.48
1.48±0.72
2.59±0.38
10.1±1.72
0.45±0.01
3.11±0.84
2.11±0.28
5.01±0.79
1.35±0.06
48.6±3.22
50.3±3.28
25.9±1.38
11.6±1.28
13.1±0.63
1.49±0.38
1.22±0.22
1.63±0.41
2.31±0.13
1.41±0.05
3.52±0.98
5.78±0.74
1.62±0.23
4.46±0.78
2.37±0.79
5.12±0.84
3.71±0.37
2.19±0.05
0.65±0.04
1.35±0.14
7.11±0.98
7.36±0.25
10.5±1.02
18.9%
23.4%
21.2%
1.65%
12.4%
7.98%
13.8%
4.89%
23.2%
6.98%
34.5%
2.89%
38.4%
6.98%
28.2%
11.5%
3.08%
14.8%
12.5%
27.6%
22.6%
4.87%
13.5%
19.8%
6.87%
2.76%
2.87%
1.09%
24.8%
4.67%
14.6%
9.87%
12.7%
17.8%
6.08%
18.9%
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
5a
5b
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5c
5d
5e
5f
5g
5h
5i
6b
6a
4.34±0.03
2.91±0.35
1.95±0.26
4.47±0.97
2.23±0.32
2.39±0.36
2.57±0.77
1.96±0.33
6.03±0.75
5.84±0.37
39.1%
22.4%
6.87%
15.9%
19.3%
25.6%
28.2%
27.6%
12.8%
34.6%
18.7%
7.98%
10.8%
14.7%
29.8%
31.2%
12.6%
16.7%
8.09±1.28
13.9±1.08
12.9±1.38
1.62±0.11
47.1±3.11
2.04±0.17
21.2±1.78
17.2±0.89
19.21±0.001
ꢀꢀꢀꢀꢀꢀꢀ
Levamisole
LꢀPhenylalanine
80.21±0.001
Values are expressed as mean ± SEM of n = 3. a The IC50 is the concentration at which 50% of
b
the enzyme activity is inhibited. The % inhibition of the enzyme activity caused by 0.1 mM of
the tested compound.
Conclusion
In conclusion, we have demonstrated that a series of 5ꢀtrifluoromethylated and 5ꢀ
perfluoroalkylated pyrazoles, including deprotected derivatives, could be efficiently and
selectively synthesized by oneꢀpot cyclization of hydrazone dianions with ethyl
perfluorocarboxylates. In addition, two trifluoromethylated indazoles were prepared from the
corresponding bicyclic hydrazones. The cyclization of oxime dianions afforded trifluoromethylꢀ
substituted isoxazoles. All the compounds were selective inhibitors of APs with little effect on hꢀ
NPP1 and hꢀNPP3. In addition, our data showed that most of the compounds presented here
inhibited hꢀIAP more efficiently than hꢀTNAP. Therefore these compounds appear as selective
inhibitors of hꢀIAP. The results reported herein are of considerable interest for further
applications in medicinal chemistry.
Experimental Section
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General information: All chemicals used are commercially available and were used without
further purification. Column chromatography was performed using Merck Silicagel 60 (0.043ꢀ
0.06 mm). NMR data were recorded on a Bruker AC 250, Bruker ARX 300, Bruker ARX 500
spectrometers. Gas chromatographyꢀmass spectrometric analysis was carried out on an
AgilentHPꢀ5890 instrument with an Agilent HPꢀ5973 Mass Selective Detector (EI) and HPꢀ5
capillary column using helium carrier gas. ESI HRꢀMS measurements were performed on an
Agilent 1969A TOF massꢀspectrometer. A Finnigan MAT95 XP was used for High Resolution
MS (HRMS). Only the measurements with an average deviation from the theoretical mass of
±2mDa were accounted as correct.Infrared Spectra were recorded on a Nicolet 550 FT – IR
spectrometer with ATR sampling technique. Signal characterization: w = weak, m = medium, s =
strong, vs = very strong.
General procedure for the synthesis of pyrazoles:
Synthesis of hydrazones:
Ketone (0.5 mmol, 1 eq) and phenylhydrazine (0.525 mmol, 1.05 eq) were dissolved in 2 mL
of DCM. The solution was stirred for 1 min at room temperature and then the solvent was
removed under reduced pressure to obtain a “wellꢀmixed” mixture of starting materials. Then 3
drops of acetic acid was added to the reaction mixture while stirring at room temperature (20 oC).
The mixture was stirred for 5 min to ensure the reaction was complete (in most cases, the product
was solid and the reaction mixture became solid as the reaction became complete). The reaction
carried out nearly qualitative. Then the crude product was dissolved in 10 mL of DCM
(dissolving reaction mixture in DCM helped removing all volatiles easier). After that, all
volatiles (DCM, water, and acetic acid) were removed under reduced pressure and hydrazone
was transferred to the next step without further purification.
Synthesis of pyrazoles:
To a solution of hyrazone (0.5 mmol) in THF was added nꢀbutyllithium (0.44 mL, 2.2 eq, 2.5
o
M solution in hexane) slowly at ꢀ78 C under Ar. Then the mixture was allowed to rise to 20 oC
o
and stirred for 15 min. The reaction was cooled to ꢀ78 C again and ethyl perfluorocarboxylate
o
(1.5 eq, solution in 1 mL THF) was added. The temperature of the reaction was risen to 20 C
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again and stirred for 30 min, subsequently, 1 mL of TFA was added. The mixture was stirred
under reflux for 2 h. After cooling to room temperature, a saturated aqueous solution of NaHCO3
was added until no bubble of CO2 was observed. Then THF was removed and the remained
aqueous mixture was extracted with ethyl acetate (10 mLx3). Combined organic layers were
dried with MgSO4 and the solvent was removed under reduced pressure. The residue was
chromatographed (silica gel, nꢀheptane/DCM) to obtain pure product.
Synthesis of 1,3ꢀdiphenylꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (4a)19:
Compound 4a was synthesized following the general procedure using acetophenone for the
first step and ethyl trifluoroacetate for the next step. The product was isolated as a white solid
o
(128 mg, 89%). M.p.: 53 – 54 C. IR (ATR, cmꢀ1): ν = 549 (s), 615 (m), 685 (vs), 760 (s), 773
(s), 812 (s), 956 (m), 9872 (s), 1028 (s), 1072 (s), 1118 (vs), 1138 (m), 1211 (s), 1232 (s), 1288
(s), 1363 (m), 1444 (s), 1502 (m), 1556 (m), 1593 (m), 3054 (w), 3070 (w), 3139 (w). 1H NMR
(300 MHz, CDCl3) δ 7.92 – 7.83 (m, 2H), 7.65 – 7.32 (m, 8H), 7.12 (s, 1H). 19F NMR (282
13
MHz, CDCl3) δ ꢀ57.59 (s). C NMR (75 MHz, CDCl3) δ 151.8, 139.3, 134.0 (q, J = 39.2 Hz),
131.9, 129.4, 129.26 (2C), 128.9 (2C), 128.8, 126.0 (2C), 125.9, 125.9, 119.9 (q, J = 269.2 Hz),
106.2 (q, J = 2.4 Hz). GCꢀMS (EI, 70 eV): m/z (%) = 288 (100), 267 (37), 219 (9), 77 (20).
HRMS (EI): calcd. for C16H11F3N2 ([M]+): 288.08688, found: 288.08678.
Synthesis of 3ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀphenylꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (4b):
Compound 4b was synthesized following the general procedure using 4ꢀmethoxyacetophenone
for the first step and ethyl trifluoroacetate for the next step. The product was isolated as a yellow
o
solid (127 mg, 79%). M.p.: 77ꢀ78 C. IR (ATR, cmꢀ1): ν = 548 (m), 617 (m), 685 (s), 767 (vs),
810 (vs), 833 (m), 956 (m), 987 (s), 1031 (s), 1080 (s), 1088 (vs), 1115 (vs), 1151 (s), 1209 (s),
1248 (s), 1290 (s), 1359 (w), 1435 (s), 1450 (s), 1502 (s), 1558 (m), 1595 (m), 1614 (m), 2845
(w), 2943 (w), 2970 (w), 3022 (w), 3068 (w), 3130 (w). 1H NMR (300 MHz, CDCl3) δ 7.86 –
7.73 (m, 2H), 7.62 – 7.44 (m, 5H), 7.04 (s, 1H), 7.02 – 6.90 (m, 2H), 3.85 (s, 3H). 19F NMR (282
MHz, CDCl3) δ ꢀ57.59 (s). 13C NMR (75 MHz, CDCl3) δ 160.2, 151.6, 139.4, 133.9 (q, J = 39.0
Hz), 129.3, 129.2 (2C), 127.3 (2C), 125.9, 125.9, 124.5, 119.9 (q, J = 269.1 Hz), 114.3 (2C),
105.8 (q, J = 2.4 Hz), 55.47. GCꢀMS (EI, 70 eV): m/z (%) = 318 (100), 303 (26), 275 (12), 77
(12).HRMS (EI): calcd. for C17H13F3N2O ([M]+): 318.09745, found: 318.09788.
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Synthesis of 3ꢀ(3ꢀmethoxyphenyl)ꢀ1ꢀphenylꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (4c):
Compound 4c was synthesized following the general procedure using 3ꢀmethoxyacetophenone
for the first step and ethyl trifluoroacetate for the next step. The product was isolated as pale
yellow oil (129 mg, 81%). IR (ATR, cmꢀ1): ν = 544 (m), 627 (m), 688 (vs), 766 (s), 816 (m), 847
(m), 916 (w), 987 (s), 1041 (s), 1089 (s), 1124 (vs), 1143 (s), 1197 (s), 1224 (s), 1257 (m), 1284
(m), 1354 (m), 1433 (s), 1464 (m), 1500 (s), 1556 (m), 1597 (m), 2835 (w), 2939 (w), 3003 (w),
1
3063 (w), 3138 (w). H NMR (300 MHz, CDCl3) δ 7.63 – 7.47 (m, 5H), 7.47 – 7.40 (m, 2H),
7.35 (t, J = 8.1 Hz, 1H), 7.10 (s, 1H), 6.93 (ddd, J = 8.1, 2.5, 1.2 Hz, 1H), 3.87 (s, 3H). 19F NMR
13
(282 MHz, CDCl3) δ ꢀ57.60 (s). C NMR (75 MHz, CDCl3) δ 160.2 (s), 151.6 (s), 139.3 (s),
134.0 (q, J = 39.2 Hz), 133.2, 130.0, 129.5, 129.3 (2C), 125.9, 125.9, 119.9 (q, J = 269.2 Hz),
118.5, 114.8, 111.1, 106.4 (q, J = 2.4 Hz), 55.5. GCꢀMS (EI, 70 eV): m/z (%) = 318 (100), 297
(8), 267 (8), 205 (3), 77 (20). HRMS (EI): calcd. for C17H13F3N2O ([M]+): 318.09745, found:
318.09718.
Synthesis of 3ꢀ(2ꢀmethoxyphenyl)ꢀ1ꢀphenylꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (4d):
Compound 4d was synthesized following the general procedure using 2ꢀmethoxyacetophenone
for the first step and ethyl trifluoroacetate for the next step. The product was isolated as pale
yellow solid (109 mg, 61%). M.p.: 70 ꢀ 71oC. IR (ATR, cmꢀ1): ν = 542 (m), 692 (s), 750 (s), 777
(s), 814 (s), 989 (s), 1028 (s), 1068 (s), 1084 (s), 1113 (vs), 1157 (s), 1201 (s), 1230 (s), 1246 (s),
1290 (s), 1354 (m), 1421 (m), 1437 (s), 1456 (m), 1473 (s), 1504 (m), 1556 (m), 1585 (m), 1595
1
(m), 2841 (w), 2943 (w), 3057 (w), 3180 (w). H NMR (300 MHz, CDCl3) δ 8.04 (dd, J = 7.6,
19
1.7 Hz, 1H), 7.64 – 7.42 (m, 5H), 7.41 – 7.30 (m, 2H), 7.08 – 6.91 (m, 2H), 3.96 (s, 3H). F
13
NMR (282 MHz, CDCl3) δ ꢀ57.38 (s). C NMR (75 MHz, CDCl3) δ 156.9, 148.6, 139.4, 132.9
(q, J = 39.0 Hz), 129.8, 129.1, 129.1 (2C), 128.8, 125.8, 125.8, 120.9, 120.6, 120.0 (q, J = 269.0
Hz), 111.3, 110.3 (q, J = 2.5 Hz), 55.5. GCꢀMS (EI, 70 eV): m/z (%) = 318 (100), 289 (64), 267
(27), 249 (28), 221 (14), 77 (60). HRMS (EI): calcd. for C17H13F3N2O ([M]+): 318.09745, found:
318.09699.
Synthesis of 3ꢀ([1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ1ꢀphenylꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (4e):
Compound 4e was synthesized following the general procedure using 4ꢀphenylacetophenone
for the first step and ethyl trifluoroacetate for the next step. The product was isolated as pale
o
yellow solid (134 mg, 74%). M.p.: 94 – 95 C. IR (ATR, cmꢀ1): ν = 546 (m), 685 (vs), 727 (s),
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758 (vs), 820 (s), 845 (m), 987 (s), 1086 (s), 1117 (vs), 1149 (s), 1159 (s), 1201 (m), 1226 (s),
1290 (s), 1358 (m), 1410 (m), 1443 (s), 1502 (s), 1566 (w), 1595 (m), 2850 (w), 2920 (w), 3032
1
(w), 3053 (w), 3144 (w). H NMR (300 MHz, CDCl3) δ 7.99 – 7.90 (m, 2H), 7.72 – 7.63 (m,
4H), 7.63 – 7.33 (m, 8H), 7.16 (d, J = 0.3 Hz 1H). 19F NMR (282 MHz, CDCl3) δ ꢀ57.57 (s). 13C
NMR (75 MHz, CDCl3) δ 151.4, 141.5, 140.7, 139.4, 134.1 (q, J = 39.2 Hz), 130.8, 129.5, 129.3
(2C), 128.9 (2C), 127.6, 127.6 (2C), 127.2 (2C), 126.4 (2C), 125.9, 125.9, 119.9 (q, J = 269.1
Hz), 106.3 (q, J = 2.4 Hz). GCꢀMS (EI, 70 eV): m/z (%) = 364 (100), 343 (11), 152 (10), 77(10).
HRMS (EI): calcd. for C22H15F3N2 ([M]+): 364.11818, found: 364.11765.
Synthesis of 1ꢀphenylꢀ3ꢀ(pꢀtolyl)ꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (4f)34:
Compound 4f was synthesized following the general procedure using 4ꢀmethylacetophenone
for the first step and ethyl trifluoroacetate for the next step. M.p: 71 – 72 oC. IR (ATR, cmꢀ1): ν =
544 (m), 623 (m), 687 (vs), 768 (s), 798 (vs), 827 (m), 989 (s), 1086 (vs), 1122 (vs), 1155 (s),
1203 (m), 1232 (s), 1290 (m), 1440 (s), 1504 (m), 1556 (m), 1595 (w), 2357 (w), 2860 (w), 2922
1
(w), 3022 (w), 3061 (w). H NMR (250 MHz, CDCl3) δ 7.82 – 7.65 (m, 2H), 7.63 – 7.44 (m,
19
5H), 7.33 – 7.18 (m, 2H), 7.08 (s, 1H), 2.40 (s, 3H). F NMR (282 MHz, CDCl3) δ ꢀ57.58 (s).
13C NMR (63 MHz, CDCl3) δ 151.8, 139.4, 138.7, 133.9 (q, J = 39.2 Hz), 129.6 (2C), 129.4,
129.2 (2C), 129.1, 125.9 (4C), 119.9 (q, J = 269.2 Hz), 106.1 (q, J = 2.4 Hz), 21.4. GCꢀMS (EI,
70 eV): m/z (%) = 302 (100), 281 (18), 267 (8), 233 (7), 77 (14).HRMS (ESI): calcd. for
C17H15F3N2 ([M+H]+): 303.11036, found: 303.11038.
Synthesis of 3ꢀ(2ꢀfluorophenyl)ꢀ1ꢀphenylꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (4g):
Compound 4g was synthesized following the general procedure using 2ꢀfluoroacetophenone
for the first step and ethyl trifluoroacetate for the next step. The product was isolated as pale
o
yellow solid (138 mg, 90%). M.p. 73 – 74 C. IR (ATR, cmꢀ1): ν = 553 (m), 625 (m), 661 (m),
686 (s), 750 (vs), 771 (s), 820 (s), 833 (s), 947 (m), 960 (m), 989 (s), 1028 (m), 1070 (s), 1115
(vs), 1165 (s), 1201 (s), 1238 (s), 1261 (m), 1290 (s), 1358 (m), 1423 (m), 1444 (s), 1471 (m),
1500 (m), 1554 (m), 1581 (m), 1587 (m), 1913 (w), 3056 (w), 3076 (w), 3165 (w).1H NMR (300
MHz, CDCl3) δ 8.19 – 8.07 (m, 1H), 7.68 – 7.50 (m, 5H), 7.50 – 7.35 (m, 1H), 7.35 – 7.15 (m,
3H). 19F NMR (282 MHz, CDCl3) δ ꢀ57.59 (s), ꢀ116.00 (s). 13C NMR (126 MHz, CDCl3) δ 160.5
(d, J = 250.0 Hz), 146.5, 139.3, 134.3 – 133.3 (m), 130.2 (d, J = 8.5 Hz), 129.5, 129.3 (2C),
128.6 (d, J = 3.3 Hz), 125.9, 125.9, 124.6 (d, J = 3.5 Hz), 119.9 (q, J = 269.2 Hz), 119.9 (d, J =
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11.8 Hz), 116.3 (d, J = 22.0 Hz), 109.6 (m, 1C). GCꢀMS (EI, 70 eV): m/z (%) = 306 (100), 285
(38), 267 (9), 237 (7), 77 (15).
Synthesis of 3ꢀ(4ꢀfluorophenyl)ꢀ1ꢀphenylꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (4h)34:
Compound 4h was synthesized following the general procedure using 4ꢀfluoroacetophenone
for the first step and ethyl trifluoroacetate for the next step. The product was isolated as a yellow
o
solid (125 mg, 82%). M.p.: 64 – 65 C. IR (ATR, cmꢀ1): ν = 544 (m), 619 (m), 692 (s), 750 (m),
769 (vs), 808 (vs), 837 (s), 956 (m), 987 (s), 1028 (m), 1066 (s), 1076 (s), 1086 (vs), 1111 (vs),
1155 (s), 1207 (s), 1228 (s), 1290 (s), 1440 (s), 1502 (s), 1525 (m), 1558 (m), 1595 (m), 1606
1
(m), 1888 (w), 3058 (w), 3141 (w). H NMR (300 MHz, CDCl3) δ 7.91 – 7.75 (m, 2H), 7.63 –
7.44 (m, 5H), 7.20 – 7.07 (m, 2H), 7.06 (s, 1H). 19F NMR (282 MHz, CDCl3) δ ꢀ57.65 (s), ꢀ
13
112.97 (s). C NMR (75 MHz, CDCl3) δ 163.2 (d, J = 247.9 Hz), 150.9, 139.3, 134.2 (q, J =
39.2 Hz), 129.5, 129.3 (2C), 128.1 (d, J = 3.2 Hz), 127.8 (d, J = 8.2 Hz, 2C), 125.8, 125.8, 119.8
(q, J = 269.2 Hz), 115.9 (d, J = 21.8 Hz, 2C), 106.0 (q, J = 2.4 Hz). GCꢀMS (EI, 70 eV): m/z (%)
= 306 (100), 285 (40), 237 (9), 77 (17). HRMS (EI): calcd. for C16H10F4N2 ([M]+): 306.07746,
found: 306.07713.
Synthesis of 3ꢀ(naphthalenꢀ2ꢀyl)ꢀ1ꢀphenylꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (4i):
Compound 4i was synthesized following the general procedure using 2ꢀacetonaphthone for the
first step and ethyl trifluoroacetate for the next step. The product was isolated as a pale yellow
o
solid (102 mg, 78%). M.p.: 71 – 71 C. IR (ATR, cmꢀ1): ν = 542 (m), 687 (s), 742 (s), 767 (s),
798 (s), 804 (s), 819 (m), 856 (s), 885 (m), 945 (m), 989 (s), 1028 (m), 1057 (s), 1074 (s), 1086
(s), 1113 (vs), 1153 (s), 1230 (s), 1246 (s), 1290 (s), 1431 (m), 1485 (m), 1504 (s), 1556 (w),
1595 (m), 3028 (w), 3055 (w). 1H NMR (300 MHz, Acetone) δ 8.50 (s, 1H), 8.13 (d, J = 8.3 Hz,
1H), 8.04 – 7.88 (m, 3H), 7.72 – 7.47 (m, 8H). 19F NMR (282 MHz, Acetone) δ 119.44 (s). 13C
NMR (75 MHz, Acetone) δ 152.5, 140.4, 134.6, 134.6, 134.5 (q, J = 39.0 Hz), 130.6, 130.3,
130.3 (2C), 129.5, 129.3, 128.8, 127.6, 127.4, 126.9, 126.9, 125.7, 124.6, 121.1 (q, J = 268.4
Hz), 107.6 (q, J = 2.5 Hz). GCꢀMS (EI, 70 eV): m/z (%) = 338 (100), 317 (14), 127 (17), 77 (20).
HRMS (EI): calcd. for C20H13F3N2 ([M]+): 338.10253, found: 338.10259.
Synthesis of 1ꢀphenylꢀ5ꢀ(trifluoromethyl)ꢀ3ꢀ(4ꢀ(trifluoromethyl)phenyl)ꢀ1Hꢀpyrazole (4j):
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Compound 4j was synthesized following the general procedure using 4ꢀ
trifluoromethylacetophenone for the first step and ethyl trifluoroacetate for the next step. The
product was isolated as a yellow solid (113 mg, 64%). M.p.: 50 – 51 oC. IR (ATR, cmꢀ1): ν = 552
(m), 592 (s), 625 (m), 687 (vs), 766 (s), 804 (s), 845 (s), 991 (s), 1062 (vs), 1068 (vs), 1089 (s),
1093 (s), 1109 (vs), 1132 (vs), 1232 (s), 1288 (s), 1323 (s), 1419 (w), 1446 (m), 1504 (m), 1531
(w), 1558 (w), 1595 (m), 1622 (m), 3063 (w), 3145 (w). 1H NMR (300 MHz, CDCl3) δ 7.98 (d, J
19
= 8.1 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.62 – 7.46 (m, 2H), 7.16 (s, 1H). F NMR (282 MHz,
13
CDCl3) δ ꢀ57.71 (s), ꢀ62.63 (s). C NMR (75 MHz, CDCl3) δ 150.3, 139.1, 135.3 (d, J = 1.3
Hz), 134.5 (q, J = 39.6 Hz), 130.7 (q, J = 32.5 Hz), 129.7, 129.4 (2C), 126.17 (2C), 125.9 (q, J =
3.8 Hz, 2C), 125.9, 125.8, 124.3 (q, J = 272.3 Hz), 119.7 (q, J = 269.3 Hz), 106.5 (q, J = 2.4
Hz).GCꢀMS (EI, 70 eV): m/z (%) = 356 (100), 335 (51), 287 (10), 267 (10), 77 (27).HRMS (EI):
calcd. for C17H10F6N2 ([M]+): 356.07427, found: 356.07482.
Synthesis of 5ꢀ(perfluoroethyl)ꢀ1,3ꢀdiphenylꢀ1Hꢀpyrazole (4k):
Compound 4k was synthesized following the general procedure using acetophenone for the
first step and ethyl pentafluoropropionate for the next step. The product was isolated as a pale
yellow solid (95%). M.p.: 140 – 142 oC. IR (ATR, cmꢀ1): ν = 544 (w), 580 (w), 602 (m), 619 (m),
630 (m), 688 (vs), 711 (m), 750 (s), 765 (vs), 777 (s), 814 (m), 914 (m), 937 (s), 958 (s), 1020
(m), 1041 (s), 1076 (m), 1092 (s), 1134 (s), 1190 (s), 1203 (s), 1223 (s), 1331 (m), 1444 (m),
1
1498 (m), 1595 (w), 3076 (w), 3151 (w). H NMR (300 MHz, CDCl3) δ 7.90 – 7.82 (m, 2H),
7.55 – 7.48 (m, 5H), 7.48 – 7.32 (m, 3H), 7.10 (d, J = 0.9 Hz, 1H). 19F NMR (282 MHz, CDCl3)
13
δ ꢀ83.38 (t, J = 2.9 Hz), ꢀ106.28 (q, J = 2.9 Hz). C NMR (63 MHz, CDCl3) δ 152.1, 139.9,
132.0 (t, J = 27.6 Hz), 131.7, 129.7, 129.1 (2C), 128.9 (2C), 128.9, 126.9 (2C), 126.0 (2C), 122.8
– 108.2 (CF2CF3), 107.4 – 107.0 (m, 1C). GCꢀMS (EI, 70 eV): m/z (%) = 338 (100), 319 (6), 269
(14), 219 (8), 77 (24). HRMS (EI): calcd. for C17H11F5N2 ([M]+): 388.08369, found: 388.08322.
Synthesis of 3ꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀ(perfluoroethyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole (4l):
Compound 4l was synthesized following the general procedure using 4ꢀmethoxyacetophenone
for the first step and ethyl pentafluoropropionate for the next step. The product was isolated as a
o
pale yellow solid (168 mg, 91%). M.p.: 82 – 83 C. IR (ATR, cmꢀ1): ν = 532 (s), 576 (m), 602
(m), 617 (m), 634 (m), 694 (s), 748 (s), 771 (vs), 804 (s), 835 (s), 935 (s), 958 (s), 1026 (s), 1036
(s), 1092 (vs), 1134 (s), 1176 (s), 1190 (vs), 1215 (vs), 1250 (s), 1292 (m), 1331 (m), 1443 (m),
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1502 (m), 1552 (w), 1596 (m), 1614 (m), 2839 (w), 2943 (w), 3010 (w), 3139 (w). 1H NMR (300
MHz, CDCl3) δ 7.91 – 7.68 (m, 2H), 7.57 – 7.44 (m, 5H), 7.02 (s, 1H), 7.00 – 6.92 (m, 2H), 3.85
(s, 3H). 19F NMR (282 MHz, CDCl3) δ ꢀ83.39 (t, J = 2.7 Hz), ꢀ106.26 (q, J = 2.6 Hz). 13C NMR
(63 MHz, CDCl3) δ 160.2, 151.9, 139.9, 131.9 (t, J = 27.6 Hz), 129.6, 129.0 (2C), 127.3 (2C),
126.9 (2C), 124.5, 114.3 (2C), 121.8 ꢀ 109.6 (CF2CF3), 106.9 – 106.5 (m, 1C), 55.5. GCꢀMS (EI,
70 eV): m/z (%) = 368 (100), 353 (20), 77 (14). HRMS (EI): calcd. for C18H13F5N2O ([M]+):
368.09426, found: 368.09390.
Synthesis of 3ꢀ(naphthalenꢀ2ꢀyl)ꢀ5ꢀ(perfluoroethyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole (4m):
Compound 4m was synthesized following the general procedure using 2ꢀacetonaphthone for
the first step and ethyl pentafluoropropionate for the next step. The product was isolated as a pale
brown solid (181 mg, 93%). M.p.: 104 – 106 oC. IR (ATR, cmꢀ1): ν = 544 (w), 575 (m), 617 (m),
628 (m), 642 (m), 685 (s), 748 (vs), 771 (s), 804 (vs), 860 (s), 887 (m), 941 (vs), 980 (m), 1020
(s), 1043 (s), 1095 (s), 1134 (s), 1190 (vs), 1329 (m), 1431 (m), 1487 (m), 1504 (m), 1594.92
(m), 3043 (w), 3058 (w). 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 8.01 (dd, J = 8.6, 1.7 Hz,
19
1H), 7.95 – 7.79 (m, 3H), 7.64 – 7.41 (m, 7H), 7.23 (d, J = 0.7 Hz, 1H). F NMR (282 MHz,
13
CDCl3) δ ꢀ83.31 (t, J = 2.9 Hz), ꢀ106.28 (q, J = 2.9 Hz). C NMR (75 MHz, CDCl3) δ 152.1,
139.9, 133.6 (2C), 132.2 (t, J = 27.9 Hz), 129.8, 129.1 (3C), 128.7, 128.4, 127.9, 126.9 (2C),
126.6, 126.5, 125.0, 123.9, 120.6 – 106.3 (CF2CF3), 107.6 – 107.3 (m, 1C). GCꢀMS (EI, 70 eV):
m/z (%) = 388 (100), 269 (7), 194 (6), 127 (16), 77 (18). HRMS (EI): calcd. for C21H13F5N2
([M]+): 388.09934, found: 388.09878.
Synthesis of 3ꢀ([1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ5ꢀ(perfluoroethyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole (4n):
Compound 4n was synthesized following the general procedure using 4ꢀphenylacetophenone
for the first step and ethyl pentafluoropropionate for the next step. The product was isolated as
o
pale yellow solid (178 mg, 85%). M.p.: 140 ꢀ142 C. IR (ATR, cmꢀ1): ν = 694 (vs), 727 (s), 746
(s), 765 (vs), 819 (s), 846 (m), 937 (s), 958 (s), 1020 (m), 1041 (s), 1072 (m), 1093 (s), 1134 (s),
1186 (vs), 1219 (vs), 1278 (w), 1331 (m), 1409 (w), 1441 (m), 1502 (m), 1597 (m), 3061 (w),
1
3132 (w). H NMR (300 MHz, CDCl3) δ 7.86 (d, J = 8.0 Hz, 2H), 7.73 – 7.51 (m, 4H), 7.51 –
7.21 (m, 8H), 7.04 (s, 1H). 19F NMR (282 MHz, CDCl3) δ ꢀ83.33 (t, J = 2.5 Hz), ꢀ106.24 (q, J =
2.5 Hz). 13C NMR (63 MHz, CDCl3) δ 151.7, 141.6, 140.7, 139.9, 132.1 (t, J = 27.4 Hz), 130.7,
129.7, 129.1 (2C), 129.0 (2C), 127.7, 127.6 (2C), 127.2 (2C), 126.9 (2C), 126.4 (2C), 125.4 –
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110.9 (CF2CF3), 107.4 – 107.1 (m, 1C). GCꢀMS (EI, 70 eV): m/z (%) = 414 (100), 395 (3), 345
(5), 295 (4), 207 (3), 152 (8), 116 (1), 77 (8). HRMS (EI): calcd. for C23H15F5N2 ([M]+):
414.11499, found: 414.11460.
Synthesis of 5ꢀ(perfluoropropyl)ꢀ1,3ꢀdiphenylꢀ1Hꢀpyrazole (4o):
Compound 4o was synthesized following the general procedure using acetophenone for the
first step and ethyl heptafluorobutyrate for the next step. The product was isolated as a white
solid (161 mg, 83%). M.p.: 90 – 91 oC. IR (ATR, cmꢀ1): ν = 532 (m), 590 (m), 648 (s), 692 (vs),
746 (s), 765 (vs), 777 (s), 814 (s), 874 (vs), 957 (m), 1003 (m), 1026 (m), 1078 (m), 1109 (vs),
1
1138 (s), 1182 (s), 1223 (vs), 1344 (s), 1443 (m), 1500 (s), 1595 (w), 3053 (w), 3157 (w). H
NMR (300 MHz, CDCl3) δ 7.91 – 7.82 (m, 2H), 7.50 (s, 5H), 7.48 – 7.32 (m, 3H), 7.12 (s, 1H).
19F NMR (282 MHz, CDCl3) δ ꢀ80.04 (t, J = 10.1 Hz), ꢀ103.84 – ꢀ104.15 (m), ꢀ124.86 – ꢀ125.05
(m). 13C NMR (63 MHz, CDCl3) δ 152.1, 139.9, 132.3 (t, J = 28.7 Hz), 131.7, 130.0, 129.3 (2C),
129.2 (2C), 129.1, 127.4 (2C), 126.3 (2C), 125.8 – 92.5 (CF2CF2CF3), 108.0 – 107.6 (m, 1C).
GCꢀMS (EI, 70 eV): m/z (%) = 388 (100), 269 (39), 77 (18). HRMS (EI): calcd. for C18H11F7N2
([M]+): 388.08050, found: 388.07991.
Synthesis of 3ꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀ(perfluoropropyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazole (4p):
Compound 4p was synthesized following the general procedure using 4ꢀmethoxyacetophenone
for the first step and ethyl heptafluorobutyrate for the next step. The product was isolated as a
o
pale solid (160 mg, 76%). M.p.: 97 – 98 C. IR (ATR, cmꢀ1): ν = 644 (m), 692 (s), 744 (s), 773
(s), 800 (vs), 872 (vs), 904 (m), 958 (m), 1006 (s), 1030 (s), 1070 (m), 1078 (m), 1109 (vs), 1138
(s), 1178 (s), 1184 (s), 1223 (vs), 1251 (s), 1344 (m), 1435 (s), 1446 (s), 1502 (s), 1523 (m),
1552 (w), 1596 (w), 1614 (m), 2835 (w), 2939 (w), 2964 (w), 3001 (w), 3066 (w). 1H NMR (300
MHz, CDCl3) δ 7.83 – 7.79 (m, 1H), 7.79 – 7.76 (m, 1H), 7.49 (s, 3H), 7.03 (s, 1H), 7.01 – 6.96
19
(m, 1H), 6.96 – 6.92 (m, 1H), 3.85 (s, 2H). F NMR (282 MHz, CDCl3) δ ꢀ80.05 (t, J = 10.1
13
Hz), ꢀ103.83 – ꢀ104.18 (m), ꢀ124.90 – ꢀ125.02 (m). C NMR (63 MHz, CDCl3) δ 160.2, 151.9,
139.9, 132.9 – 131.6 (m, 1C), 129.6, 128.9 (2C), 127.3 (2C), 127.1 (2C), 124.5, 114.3 (2C),
107.2 – 106.9 (m, 1C), 55.5 (signals of CF2CF2CF3 could not be detected). GCꢀMS (EI, 70 eV):
m/z (%) = 418 (100), 375 (4), 299 (12), 77 (9). HRMS (EI): calcd. for C19H13F7N2O ([M]+):
418.09106, found: 418.09036.
Ethyl 3ꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazoleꢀ1ꢀcarboxylate (4q):
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Compound 4q was synthesized following the general procedure using 4ꢀ
methoxyacetophenone, ethyl carbazatefor the first step and ethyl trifluoroacetate for the next
o
step. The product was isolated as a white (90mg, 57%). M.p.: 73 – 74 C. IR (ATR, cmꢀ1): ν =
3519.0 (w), 3117.7 (w), 2966.8 (w), 2842.0 (w), 1766.3 (s), 1613.6 (m), 1587.0 (m), 1464.0 (m),
1441.8 (m), 1293.7 (s), 1143.8 (s), 1006.6 (m), 946.1 (m), 834.4 (s), 746.3 (m), 680.4 (m), 554.2
(m). 1H NMR (300 MHz, CDCl3) δ 7.87 – 7.71 (m, 2H), 7.10 (s, 1H), 7.01 – 6.89 (m, 2H), 4.57
(q, J = 7.1 Hz, 2H), 3.85 (s, 3H), 1.49 (t, J = 7.1 Hz, 3H). 19F NMR (282 MHz, CDCl3) δ ꢀ60.03.
13C NMR (75 MHz, CDCl3) δ 161.1, 153.5, 148.3, 135.8 (q, J = 41.3 Hz), 128.0 (2C), 123.1,
119.3 (q, J = 269.1 Hz), 114.4 (2C), 110.8 (q, J = 3.2 Hz), 65.5, 55.5, 14.1. GCꢀMS (EI, 70 eV):
m/z (%) = 314 (47), 270 (12), 255 (17), 242 (100), 227 (58), 213 (17), 199 (36), 170 (12), 151
(22), 120 (5), 101 (5), 75 (6), 63 (5). HRMS (+EI): calcd. for C14H13O3F3N2 ([M]+): 314.08728,
found: 314.08732.
Ethyl 3ꢀ(2ꢀfluorophenyl)ꢀ5ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazoleꢀ1ꢀcarboxylate (4r):
Compound 4r was synthesized following the general procedure using 2ꢀfluoroacetophenone,
ethyl carbazatefor the first step and ethyl trifluoroacetate for the next step. The product was
o
isolated as a white (98mg, 57%). M.p.: 84 – 85 C. IR (ATR, cmꢀ1): ν = 3505.3 (w), 3171.4 (w),
3076.4 (w), 2996.6 (w), 2918.6 (w), 1761.2 (s), 1620.7 (w), 1591.2 (w), 1446.9 (s), 1304.1 (m),
1229.2 (m), 1141.8 (s), 1026.1 (m), 1032.2 (m), 949.0 (m), 840.6 (m), 747.9 (m), 676.2 (m),
1
552.1 (w). H NMR (300 MHz, CDCl3) δ 8.10 (td, J = 7.7, 1.8 Hz, 1H), 7.47 – 7.34 (m, 1H),
7.30 (d, J = 3.5 Hz, 1H), 7.27 – 7.10 (m, 2H), 4.59 (q, J = 7.1 Hz, 2H), 1.50 (t, J = 7.1 Hz, 3H).
19F NMR (282 MHz, CDCl3) δ ꢀ60.00, ꢀ115.67. 13C NMR (63 MHz, CDCl3) δ 160.8 (d, J =
251.0 Hz), 148.8, 148.1, 135.5 (q, J = 42.0 Hz), 131.5 (d, J = 8.6 Hz), 129.2 (d, J = 2.8 Hz),
124.7 (d, J = 3.5 Hz), 119.3 (d, J = 269.2 Hz), 118.5 (d, J = 11.5 Hz), 116.4 (d, J = 21.9 Hz),
114.5 – 113.4 (m), 65.7, 14.1.
Synthesis of 2ꢀphenylꢀ3ꢀ(trifluoromethyl)ꢀ4,5,6,7,8,9,10,11,12,13ꢀdecahydroꢀ2Hꢀ
cyclododeca[c]pyrazole (4s):
Compound 4s was synthesized following the general procedure using cyclododecanone for the
first step and ethyl trifluoroacetate for the next step. The product was isolated as a white solid
o
(140 mg, 80%). M.p.: 56 – 57 C. IR (ATR, cmꢀ1): ν = 546 (m), 692 (s), 771 (s), 993 (s), 1086
(s), 1111 (vs), 1168 (s), 1242 (m), 1309 (m), 1329 (m), 1350 (m), 1452 (m), 1504 (s), 1556 (w),
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1595 (m), 2856 (m), 2904 (m), 2933 (m). 1H NMR (250 MHz, CDCl3) δ 7.44 (s, 5H), 2.73 – 2.47
19
(m, 4H), 1.90 – 1.62 (m, 4H), 1.59 – 1.30 (m, 12H). F NMR (282 MHz, CDCl3) δ ꢀ55.61 (s).
13C NMR (63 MHz, CDCl3) δ 153.2, 140.1, 129.0 (2C), 129.0 (q, J = 37.2 Hz), 128.9, 126.2,
126.1, 123.2 – 123.0 (m), 120.9(q, J = 269.4 Hz), 28.9, 28.3, 26.0, 25.8, 25.4, 25.2, 23.0 (2C),
22.9, 20.8. GCꢀMS (EI, 70 eV): m/z (%) = 350 (100), 331 (9), 307 (50), 293 (41), 281 (71), 267
(36), 253 (43), 240 (79), 77 (40). HRMS (EI): calcd. for C20H25F3N2 ([M]+): 350.19643, found:
350.19626.
Synthesis of 2ꢀphenylꢀ3ꢀ(trifluoromethyl)ꢀ4,5,6,7ꢀtetrahydroꢀ2Hꢀindazole (4t)17:
Compound 4t was synthesized following the general procedure using cyclohexanone for the
first step and ethyl trifluoroacetate for the next step. The product was isolated as a yellow oil
(109 mg, 82%). IR (ATR, cmꢀ1): ν = 546 (m), 681 (m), 692 (s), 744 (m), 766 (s), 914 (m), 964
(m), 993 (s), 1028 (m), 1074 (s), 1097 (vs), 1116 (vs), 1149 (s), 1170 (s), 1213 (m), 1296 (m),
1352 (m), 1377 (m), 1464 (m), 1504 (s), 1574 (w), 1598 (m), 2854 (w), 2939 (m). 1H NMR (300
19
MHz, CDCl3) δ 7.49 – 7.37 (m, 5H), 3.07 – 2.53 (m, 4H), 2.13 – 1.61 (m, 4H). F NMR (282
MHz, CDCl3) δ ꢀ55.92 (s). 13C NMR (75 MHz, CDCl3) δ 150.2, 139.8, 129.0 (2C), 128.9, 128.1
(q, J = 38.2 Hz), 125.9, 125.9, 120.9 (q, J = 269.4 Hz), 119.8 (q, J = 1.7 Hz), 23.4, 22.8, 22.8,
20.9 (d, J= 1.3 Hz). GCꢀMS (EI, 70 eV): m/z (%) = 266 (100), 247 (6), 238 (48), 217 (4), 197
(52), 77(39). HRMS (EI): calcd. For C14H13F3N2 ([M]+): 266.10253, found: 266.10212.
Synthesis of 2ꢀphenylꢀ3ꢀ(trifluoromethyl)ꢀ4,5ꢀdihydroꢀ2Hꢀindazole (4u):
Compound 4u was synthesized following the general procedure using 2ꢀcyclohexenꢀ1ꢀone for
the first step and ethyl trifluoroacetate for the next step. The product was isolated as a brown oil
(87 mg, 66%). IR (ATR, cmꢀ1): ν = 536 (m), 596 (m), 661 (m), 690 (s), 765 (s), 993 (s), 1095
(vs), 1117 (vs), 1163 (s), 1207 (m), 1304 (m), 1325 (m), 1377 (m), 1462 (m), 1502 (s), 1577 (w),
1
1597 (m), 1705 (w), 2837 (w), 2898 (w), 2937 (w), 3060 (w). H NMR (250 MHz, CDCl3) δ
7.53 – 7.32 (m, 5H), 6.60 (dt, J = 9.9, 2.0 Hz, 1H), 6.14 (dt, J = 9.9, 4.3 Hz, 1H), 3.08 – 2.78 (m,
2H), 2.55 – 2.36 (m, 2H). 19F NMR (282 MHz, CDCl3) δ ꢀ56.16 (s). 13C NMR (63 MHz, CDCl3)
δ 148.6, 139.7, 131.5, 129.1 (2C), 128.9, 127.7 (q, J = 38.4 Hz), 125.9, 125.8, 120.8 (q, J = 269.6
Hz), 119.7, 118.7 (q, J = 1.8 Hz), 23.4, 18.5 (d, J = 1.3 Hz). GCꢀMS (EI, 70 eV): m/z (%) = 314
(100), 245 (33), 218 (10), 77 (27), 51 (12). HRMS (EI): calcd. for C18H13F3N2 ([M]+):
314.10253, found: 314.10234.
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Synthesis of 2ꢀphenylꢀ3ꢀ(trifluoromethyl)ꢀ4,5ꢀdihydroꢀ2Hꢀbenzo[g]indazole (4v):
Compound 4v was synthesized following the general procedure using αꢀtetralones for the first
step and ethyl trifluoroacetate for the next step. The product was isolated as a brown solid (130
mg, 82%). M.p.: 78 – 79 oC. IR (ATR, cmꢀ1): ν = 549 (m), 648 (m), 696 (s), 731 (s), 781 (s), 897
(m), 945 (m), 987 (s), 1026 (m), 1047 (s), 1103 (vs), 1157 (s), 1174 (s), 1226 (s), 1267 (m), 1307
(m), 1331 (m), 1352 (m), 1377 (m), 1446 (s), 1500 (s), 1595 (m), 2848 (w), 2901 (w), 2964 (w).
1H NMR (300 MHz, CDCl3) δ 8.07 – 7.75 (m, 1H), 7.67 – 7.35 (m, 5H), 7.36 – 7.26 (m, 3H),
19
13
3.16 – 2.77 (m, 4H). F NMR (282 MHz, CDCl3) δ ꢀ56.04 (s). C NMR (75 MHz, CDCl3) δ
148.9, 139.8, 136.5, 129.2, 129.2(2C), 128.6, 128.5, 128.5, 128.3 (q, J = 38.3 Hz), 127.2, 126.2,
126.2, 122.8, 120.7 (q, J = 269.6 Hz), 120.1 (q, J = 1.8 Hz), 28.9, 19.4 (d, J = 1.3 Hz). GCꢀMS
(EI, 70 eV): m/z (%) = 314 (100), 245 (33), 218 (10), 142 (13), 115 (10), 77 (27). HRMS (EI):
calcd. For C18H13F3N2 ([M]+): 314.10253, found: 314.10234.
5ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (5a)35:
Compound 5a was synthesized following the general procedure using 4ꢀmethoxyacetophenone,
ethyl carbazate for the first step and ethyl trifluoroacetate for the next step. After adding ethyl
trifluoroacetate, the reaction was allowed to rise to 20 oC and stirred for 30 min. Then the solvent
was removed under reduced pressure. After that, the residue was dissolved in toluene and 2
mmol (4 eq) of PTSA was added. The reaction mixture was stirred under reflux for 8 h. After
cooling, a saturated aqueous solution of NaHCO3 was added until no bubble of CO2 was
observed. Then THF was removed and the remained water was extracted with ethyl acetate (10
mLx3). Combined organic layers were dried with MgSO4 and the solvent was removed under
reduced pressure. The residue was purified by chromatography (silica gel, nꢀheptane/DCM). The
o
product was isolated as a white solid (62 mg, 51%). M.p.: 147 – 148 C. IR (ATR, cmꢀ1): ν =
3225.7 (w), 2974.4 (w), 2845.7 (w), 1614.5 (m), 1574.2 (m), 1516.7 (m), 1490.1 (m), 1458.8 (s),
1440.5 (m), 1274.9 (s), 1243.7 (s), 1109.9 (s), 1056.2 (s), 980.9 (m), 836.3 (s), 795.3 (s), 742.4
1
(m). H NMR (300 MHz, CDCl3) δ 7.95 – 7.31 (m, 2H), 7.11 – 6.77 (m, 2H), 6.66 – 6.62 (m,
1H), 3.85 (s, 3H).19F NMR (282 MHz, CDCl3) δ ꢀ62.17 (s). 13C NMR (75 MHz, CDCl3) δ 160.7,
145.2, 143.7 (q, J = 38.1 Hz), 127.3 (2C), 121.3 (q, J = 268.6 Hz), 120.7, 114.8 (2C), 100.5,
55.5. GCꢀMS (EI, 70 eV): m/z (%) = 242 (100), 227 (41), 223 (10), 199 (41), 169 (3), 151 (24).
HRMS (+EI): calcd. for C11H9O1F3N2 ([M]+): 242.06615, found: 242.0660.
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5ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀ(perfluoroethyl)ꢀ1Hꢀpyrazole (5b):
Compound 5b was synthesized following the procedure for compound 5a using 4ꢀ
methoxyacetophenone for the first step and ethyl pentafluoropropionate for the next step. The
product was isolated as pale yellow solid (82 mg, 56%). M.p.: 137 – 138 oC. IR (ATR, cmꢀ1): ν =
3185.5 (w), 3142.1 (w), 3033.4 (w), 1619.0 (m), 1513.2 (s), 1330.6 (m), 1258.5 (s), 1185.6 (s),
1029.1 (s), 933.0 (s), 830.5 (m), 747.8 (m), 614.4 (m). 1H NMR (300 MHz, MeOD) δ 7.90 – 7.49
(m, 2H), 7.16 – 6.94 (m, 2H), 6.86 (s, 1H), 3.86 (s, 3H). 19F NMR (282 MHz, MeOD) δ ꢀ86.12, ꢀ
114.10. 13C NMR (63 MHz, MeOD) δ 161.9, 128.3 (2C), 115.6 (2C), 102.2, 55.8 (signals of
CF2CF3 could not be detected). GCꢀMS (EI, 70 eV): m/z (%) = 292 (199), 277 (30), 249 (39),
223 (12), 151 (20), 111 (6). HRMS (EI): calcd. for C12H9O1N2F5 ([M]+): 292.06296, found:
292.06263.
5ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀ(perfluoropropyl)ꢀ1Hꢀpyrazole (5c):
Compound 5c was synthesized following the procedure for compound 5a using 4ꢀ
methoxyacetophenone for the first step and ethyl heptafluorobutyrate for the next step. The
product was isolated as pale yellow solid (98 mg, 57%). M.p.: 127 – 128 oC. IR (ATR, cmꢀ1): ν =
3144.1 (w), 3031.2 (w), 2947.1 (w), 1890.6 (w), 1617.6 (m), 1512.5 (s), 1427.8 (m), 1348.6 (m),
1311.7 (m), 1255.9 (m), 1229.3 (s), 1178.2 (s), 1106.6 (s), 1029.6 (m), 1000.1 (m), 874.2 (s),
1
829.8 (m), 746.3 (s), 652.1 (m), 620.4 (m). H NMR (300 MHz, MeOD) δ 7.78 – 7.47 (m, 2H),
19
7.22 – 6.91 (m, 2H), 6.85 (s, 1H), 3.85 (s, 3H). F NMR (282 MHz, MeOD) δ ꢀ81.15 – ꢀ82.64
(m), ꢀ111.79 (d, J = 9.5 Hz), ꢀ127.00 – ꢀ130.06 (m). 13C NMR (63 MHz, MeOD) δ 161.9, 146.4,
143.0, 128.3, 122.2, 115.6, 102.4, 55.8 (signals of CF2CF2CF3 could not be detected). GCꢀMS
(EI, 70 eV): m/z (%) = 342 (100), 327 (18), 299 (25), 223 (29), 208 (4), 180 (5), 151 (15), 111
(6). HRMS (EI): calcd. for C13H9O1N2F7 ([M]+): 342.05976, found: 342.05959.
5ꢀ(naphthalenꢀ2ꢀyl)ꢀ3ꢀ(trifluoromethyl)ꢀ1Hꢀpyrazole (5d)35:
Compound 5d was synthesized following the procedure for compound 5a using 2ꢀ
acetonaphthone for the first step and ethyl trifluoroacetate for the next step. The product was
o
isolated as white solid (102 mg, 78%). M.p.: 180 – 181 C. IR (ATR, cmꢀ1): ν = 3227.5 (w),
3043.6 (w), 2923.5 (w), 1631.9 (w), 1608.5 (w), 1582.5 (w), 1567.3 (w), 1500.0 (m), 1474.0 (w),
1255.6 (m), 1150.1 (s), 1120.6 (s), 1103.6 (s), 992.0 (m), 860.5 (m), 803.6 (s), 741.1 (s), 713.1
(m), 679.4 (m), 601.7 (m). 1H NMR (300 MHz, MeOD) δ 8.26 (s, 1H), 8.11 – 7.72 (m, 4H), 7.56
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(dd, J = 6.3, 3.0 Hz, 2H), 7.10 (s, 1H). 19F NMR (282 MHz, MeOD) δ ꢀ63.58. 13C NMR (75
MHz, Acetone) δ 145.5, 144.3 (q, J = 37.5 Hz), 134.4, 134.4, 129.9, 129.1, 128.7, 127.8, 127.8,
126.7,125.6, 124.3, 122.9 (q, J = 267.5 Hz), 102.1. GCꢀMS (EI, 70 eV): m/z (%) = 262 (100),
243 (7), 214 (10), 183 (6), 165 (22), 152 (1), 139 (4), 127 (7), 69 (3). HRMS (+ESI): calcd. for
C14H10F3N2 ([M+H]+): 263.07906, found: 263.07925.
5ꢀ(naphthalenꢀ2ꢀyl)ꢀ3ꢀ(perfluoroethyl)ꢀ1Hꢀpyrazole (5e):
Compound 5e was synthesized following the procedure for compound 5a using 2ꢀ
acetonaphthone for the first step and ethyl pentafluoropropionate for the next step. The product
o
was isolated as pale yellow solid (134 mg, 86%). M.p.: 124 – 126 C. IR (ATR, cmꢀ1): ν =
3147.0 (w), 3114.4 (w), 2933.1 (w), 1955.6 (w), 1906.3 (w), 1785.8 (w), 1679.0 (w), 1583.7 (w),
1566.5 (w), 1513.6 (w), 1431.1 (w), 1332.5 (s), 1214.1 (s), 1183.3 (s), 1131.7 (s), 1069.4 (m),
1028.9 (s), 940.5 (s), 801.5 (m), 745.4 (s), 619.4 (m). 1H NMR (300 MHz, MeOD) δ 8.21 (d, J =
4.6 Hz, 1H), 8.08 – 7.70 (m, 4H), 7.62 – 7.42 (m, 2H), 7.07 (s, 1H). 19F NMR (282 MHz,
MeOD) δ ꢀ86.02, ꢀ114.00. 13C NMR (63 MHz, MeOD) δ 146.4, 134.8, 130.1, 129.3, 128.8,
127.9, 126.9, 125.8, 124.4, 103.3 (some signals could not be detected). GCꢀMS (EI, 70 eV): m/z
(%) = 312 (100), 293 (5), 243 (18), 214 (13), 194 (4), 165 (17), 121 (11), 82 (3), 69 (2). HRMS
(+ESI): calcd. for C15H10F5N2 ([M+H]+): 313.07587, found: 313.07617.
3ꢀ(3,3,3,3,3,3,3ꢀheptafluoroꢀ3l8ꢀpropꢀ1ꢀynꢀ1ꢀyl)ꢀ5ꢀ(naphthalenꢀ2ꢀyl)ꢀ1Hꢀpyrazole (5f):
Compound 5f was synthesized following the procedure for compound 5a using 2ꢀ
acetonaphthone for the first step and ethyl heptafluorobutyrate for the next step. The product was
o
isolated as pale yellow solid (116 mg, 64%). M.p.: 181 – 182 C. IR (ATR, cmꢀ1): ν = 3183.6
(w), 3067.3 (w), 2877.8 (w), 1565.2 (w), 1512.8 (w), 1415.8 (w), 1349.1 (m), 1224.1 (m),
1
1176.5 (s), 1103.7 (m), 1000.7 (m), 874.8 (m), 791.3 (m), 744.3 (m), 651.2 (m). H NMR (300
MHz, MeOD) δ 8.26 (s, 1H), 8.12 – 7.72 (m, 4H), 7.68 – 7.44 (m, 2H), 7.11 (s, 1H).19F NMR
13
(282 MHz, MeOD) δ ꢀ81.78 (t, J = 9.5 Hz), ꢀ111.71 (d, J = 8.6 Hz), ꢀ128.28 (s). C NMR (75
MHz, Acetone) δ 145.7, 134.4, 134.4, 129.9, 129.2, 128.7, 127.8, 127.8, 125.7, 124.3, 103.6
(some signals could not be detected). GCꢀMS (EI, 70 eV): m/z (%) = 362 (100), 343 (9), 243
(34), 214 (17), 194 (4), 165 (14), 122 (13), 83 (4), 69 (4). HRMS (+ESI): calcd. for C16H10F7N2
([M+H]+): 363.007267, found: 363.07305.
3ꢀ(trifluoromethyl)ꢀ5ꢀ(4ꢀ(trifluoromethyl)phenyl)ꢀ1Hꢀpyrazole (5g)35:
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Compound 5g was synthesized following the procedure for compound 5a using 4ꢀ
trifluoromethylacetophenone for the first step and ethyl trifluoroacetate for the next step. The
o
product was isolated as white solid (105 mg, 75%). M.p.: 135 – 136 C. IR (ATR, cmꢀ1): ν =
3239.9 (w), 3161.7 (w), 3066.3 (w), 2987.4 (w), 1914.8 (w), 1790.5 (w), 1738.4 (w), 1671.9 (w),
1622.3 (w), 1587.6 (w), 1494.7 (w), 1325.4 (m), 1253.0 (m), 1172.6 (m), 1109.0 (s), 1068.0 (m),
982.9 (m), 916.7 (m), 813.6 (m), 747.3 (m), 661.5 (m), 592.3 (m). 1H NMR (300 MHz, MeOD) δ
7.95 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.12 (s, 1H). 19F NMR (282 MHz, MeOD) δ ꢀ
13
63.66, ꢀ64.34. C NMR (75 MHz, CDCl3) δ 144.2, 143.5 (q, J = 38.0 Hz), 131.5 (q, J = 33.0
Hz), 131.2, 126.4 (2C, q, J = 3.8 Hz), 125.8 (2C), 123.7 (q, J = 272.3 Hz), 120.7 (q, J = 269.0
Hz), 102.1. GCꢀMS (EI, 70 eV): m/z (%) = 280 (100), 261 (23), 231 (7), 211 (20), 201 (5), 182
(15), 164 (4), 145 (8), 133 (49, 87 (2), 69 (6). HRMS (+ESI): calcd. for C11H7F6N2 ([M+H]+):
281.05579, found: 281.05099.
5ꢀ(perfluoroethyl)ꢀ3ꢀ(4ꢀ(trifluoromethyl)phenyl)ꢀ1Hꢀpyrazole (5h):
Compound 5h was synthesized following the procedure for compound 5a using 4ꢀ
trifluoromethylacetophenone for the first step and ethyl pentafluoropropionatefor the next step.
The product was isolated as white solid (122 mg, 74%). M.p.: 142 – 143 oC. IR (ATR, cmꢀ1): ν =
3191.2 (w), 3032.7 (w), 2887.0 (w), 1623.7 (w), 1590.6 (w), 1465.6 (w), 1429.3 (w), 1325.2 (s),
1225.8 (s), 1194.1 (s), 1125.4 (s), 1063.8 (m), 1030.5 (m), 973,8 (m), 939.2 (m), 841.6 (m),
807.8 (m), 750.1 (m), 693.5 (m), 622.0 (w), 591.5 (w). 1H NMR (300 MHz, MeOD) δ 7.96 (d, J
19
= 8.2 Hz, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.14 (s, 1H). F NMR (282 MHz, MeOD) δ ꢀ64.34, ꢀ
86.12, ꢀ114.07. 13C NMR (75 MHz, CDCl3) δ 144.6, 131.7 (q, J = 32.9 Hz), 126.4 (2C, q, J = 3.8
Hz), 123.8 (q, J = 272.2 Hz), 103.9 (some signals could not be detected). GCꢀMS (EI, 70 eV):
m/z (%) = 330 (81), 311 (21), 261 (100), 232 (4), 213 (9), 182 (6), 164 (18), 145 (5), 121 (5), 105
(5), 69 (6). HRMS (+ESI): calcd. for C12H7F8N2 ([M+H]+): 331.04760, found: 331.04806.
3ꢀ(3,3,3,3,3,3,3ꢀheptafluoroꢀ3l8ꢀpropꢀ1ꢀynꢀ1ꢀyl)ꢀ5ꢀ(4ꢀ(trifluoromethyl)phenyl)ꢀ1Hꢀpyrazole
(5i):
Compound 5i was synthesized following the procedure for compound 5a using 4ꢀ
trifluoromethylacetophenone for the first step and ethyl heptafluorobutyrate for the next step.
The product was isolated as white solid (127 mg, 67%). M.p. 109 – 110 oC. IR (ATR, cmꢀ1): ν =
3171.2 (w), 3034.4 (w), 2950.7 (w), 2887.4 (w), 1623.5 (w), 1591.8 (w), 1467.1 (w), 1424.8 (w),
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1326.4 (s), 1276.0 (w), 1232.3 (s), 1171.5 (m), 1109.2 (s), 1063.0 (s), 1001.4 (m), 876.1 (m),
842.4 (m), 810.1 (m), 747.9 (m), 652.6 (m), 592.2 (m). 1H NMR (300 MHz, MeOD) δ 7.96 (d, J
= 8.2 Hz, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.14 (s, 1H). 19F NMR (282 MHz, MeOD) δ ꢀ64.35 (s), ꢀ
81.84 (t, J = 9.6 Hz), ꢀ111.76 (s), ꢀ128.29 – ꢀ128.38 (m). GCꢀMS (EI, 70 eV): m/z (%) = 380
(64), 361 (23), 261 (100), 213 (8), 182 (5), 164 (17), 69 (6). HRMS (+ESI): calcd. for
C13H7F10N2 ([M+H]+): 381.04441, found: 381.04428.
Synthesis of 2ꢀphenylꢀ3ꢀ(trifluoromethyl)ꢀ2Hꢀindazole (6a):
To a solution of 4s (100 mg) in toluene (7 mL), DDQ (2 equiv.) was added. The reaction
mixture was stirred under reflux for 3h. Then the reaction mixture was cooled to room
temperature and ethyl acetate (10 mL) and water (10 mL) were added. The organic layer was
separated and washed with water three times. After drying and removal of solvent, the residue
was purified by chromatography (silica gel, nꢀheptane/DCM). The product was isolated as a
yellow oil (71 mg, 71%). IR (ATR, cmꢀ1): ν = 534 (m), 567 (m), 627 (m), 640 (m), 692 (s), 743
(vs), 768 (s), 829 (m), 914 (m), 933 (m), 989 (s), 1001 (s), 1030 (m), 1074 (s), 1103 (vs), 1147
(s), 1174 (s), 1223 (s), 1298 (s), 1381 (w), 1429 (s), 1469 (m), 1500 (s), 1522 (m), 1551 (w),
1
1597 (m), 2361 (w), 2858 (w), 2929 (w), 3066 (w). H NMR (300 MHz, CDCl3) δ 7.90 – 7.76
19
(m, 2H), 7.64 – 7.49 (m, 5H), 7.49 – 7.37 (m, 1H), 7.37 – 7.27 (m, 1H). F NMR (282 MHz,
CDCl3) δ ꢀ54.47 (s). 13C NMR (75 MHz, CDCl3) δ 148.3, 139.7, 130.1, 129.2 (2C), 127.4, 126.3,
126.3, 125.2, 123.8 (q, J = 39.5 Hz), 121.7, 121.1 (q, J = 269.0 Hz), 119.5 (q, J = 1.7 Hz), 118.6.
GCꢀMS (EI, 70 eV): m/z (%) = 262 (100), 236 (7), 193 (34), 166 (11), 77 (15), 51 (12). HRMS
(EI): calcd. for C14H9F3N2 ([M]+): 262.07123, found: 262.07106.
Synthesis of 2ꢀphenylꢀ3ꢀ(trifluoromethyl)ꢀ2Hꢀbenzo[g]indazole (6b):
To a solution of 4t (100 mg) in toluene (7 mL), DDQ (2 equiv.) was added. The reaction
mixture was stirred under reflux for 3h. Then the reaction mixture was cooled to room
temperature and ethyl acetate (10 mL) and water (10 mL) were added. The organic layer was
separated and washed with water three times. After drying and removal of solvent, the residue
was purified by chromatography (silica gel, nꢀheptane/DCM). The product was isolated as a pale
o
yellow solid (90 mg, 90%). M.p.: 100 – 102 C. IR (ATR, cmꢀ1): ν = 549 (s), 681 (s), 746 (s),
767 (s), 804 (s), 885 (m), 982 (s), 1045 (s), 1099 (vs), 1176 (s), 1217 (s), 1238 (m), 1269 (m),
1
1309 (m), 1385 (w), 1441 (s), 1473 (m), 1504 (s), 1558 (w), 1597 (m), 3024 (w), 3049 (w). H
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NMR (300 MHz, CDCl3) δ 8.66 (dd, J = 5.7, 3.5 Hz, 1H), 7.93 – 7.80 (m, 1H), 7.76 – 7.52 (m,
9H). 19F NMR (282 MHz, CDCl3) δ ꢀ54.74 (s). 13C NMR (63 MHz, CDCl3) δ 146.1, 139.7,
132.3, 129.8, 129.1 (2C), 128.5, 127.7, 127.5, 127.3, 126.4, 126.3, 125.0, 124.7 (q, J = 39.8 Hz),
122.5, 120.9 (q, J = 269.2 Hz), 119.3, 116.9 (q, J = 1.9 Hz). GCꢀMS (EI, 70 eV): m/z (%) = 312
(100), 242 (30), 77 (10). HRMS (EI): calcd. for C18H11F3N2([M]+): 312.08688, found:
312.08667.
3ꢀ(4ꢀmethoxyphenyl)ꢀ5ꢀ(trifluoromethyl)isoxazole (8a)36:
Compound 8a was synthesized following the general procedure using 4ꢀmethoxyacetophenone,
hydroxylamine for the first step and ethyl trifluoroacetate for the next step. The product was
o
isolated as a white (69 mg, 57%). M.p. 76 – 78 C. IR (ATR, cmꢀ1): ν = 3114.5 (w), 2963.7 (w),
2844.2 (w), 1611.5 (m), 1532.2 (w), 1459.8 (m), 1432.7 (m), 1319.7 (m), 1242.4 (m), 1174.7
(m), 1114.0 (s), 1023.3 (m), 966.8 (m), 915.7 (m), 837.2 (m), 821.7 (m), 747.2 (m), 680.0 (w).1H
NMR (300 MHz, CDCl3) δ 7.82 – 7.68 (m, 2H), 7.04 – 6.96 (m, 2H), 6.94 (d, J = 0.9 Hz, 1H),
3.87 (s, 3H). 19F NMR (282 MHz, CDCl3) δ ꢀ64.24. 13C NMR (75 MHz, CDCl3) δ 162.3, 161.8,
159.1 (q, J = 42.4 Hz), 128.6 (2C), 119.9, 118.1 (q, J = 270.3 Hz), 114.7 (2C), 103.3 (d, J = 2.1
Hz), 55.5. GCꢀMS (EI, 70 eV): m/z (%) = 243 (82), 174 (82), 146 (100), 131 (14), 119 (7), 103
(9), 92 (11), 76 (18), 63 (15), 50 (9). HRMS (+EI): calcd. for C11H8O2F3N1 ([M]+): 243.05016,
found: 243.05028.
3ꢀ(naphthalenꢀ2ꢀyl)ꢀ5ꢀ(trifluoromethyl)isoxazole (8b):
Compound 8b was synthesized following the general procedure using 2ꢀacetonaphthone,
hydroxylamine for the first step and ethyl trifluoroacetate for the next step. The product was
o
isolated as a white (82 mg, 62%). M.p. 103 – 104 C. IR (ATR, cmꢀ1): ν = 3232.3 (w), 3126.3
(w), 2921.0 (w), 1631.3 (w), 1486.7 (m), 1438.6 (m), 1303.8 (m), 1143.6 (s), 1104.9 (s), 1057.6
(m), 964.1 (m), 901.5 (m), 827.7 (s), 745.5 (m), 633.1 (w).1H NMR (300 MHz, CDCl3) δ 8.26 (d,
J = 0.7 Hz, 1H), 8.00 – 7.84 (m, 4H), 7.64 – 7.48 (m, 2H), 7.14 (d, J = 0.9 Hz, 1H).19F NMR
(282 MHz, CDCl3) δ ꢀ64.15. 13C NMR (75 MHz, CDCl3) δ 162.8, 159.4 (q, J = 42.7 Hz), 134.5,
133.2, 129.3, 128.7, 128.1, 127.7, 127.3, 127.2, 124.8, 123.7, 118.1 (d, J = 270.5 Hz), 103.7 (d, J
= 2.1 Hz). GCꢀMS (EI, 70 eV): m/z (%) = 263 (100), 194 (72), 166 (41), 139 (21), 127 (68), 115
(12), 97 (8), 69 (10). HRMS (+ESI): calcd. for C14H9O1F3N1 ([M+H]+): 264.06307, found:
264.06321.
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Biological protocols
Cell Transfection with Human APs and NPPs
COSꢀ7 cells were transfected with plasmids expressing human APs (TNAP & IAP)37 or
human NPPs ( (NPPꢀ1)38 or (NPPꢀ3)39) in 10ꢀcm plates, by using Lipofectamine. The confluent
cells were incubated for 5 hr at 37°C in DMEM/Fꢀ12 in the absence of fetal bovine serum and
with 6 µg of plasmid DNA and 24 µL of Lipofectamine reagent. The same volume of DMEM/Fꢀ
12 containing 20% FBS was added to stop the transfection and cells were harvested 48–72 h
later.
Preparation of membrane fractions
The transfected cells were washed three times at 4°C, with Tris–saline buffer, collected by
scraping in the harvesting buffer (95 mM NaCl, 0.1 mM PMSF, and 45 mM Tris buffer, pH 7.5)
and washed twice by centrifugation at 300×g for 5 min at 4°C37. Subsequently, cells were
resuspended in the harvesting buffer containing 10 µg/mL aprotinin and sonicated. Nuclear and
cellular debris were discarded by 10 min centrifugation (300×g at 4°C). Glycerol was added to
the resulting supernatant at a final concentration of 7.5%.
Samples were kept at ꢀ80°C until used. Protein concentration was estimated using Bradford
microplate assay and bovine serum albumin was used as a standard.
Protocol of Alkaline Phosphatase Assay (hꢀTNAP & hꢀIAP)
A chemiluminescent substrate, CDPꢀstar, was used for the determination of activity of hꢀ
TNAP and hꢀIAP. The conditions for the assay were optimized with the slight modifications in
previously used spectrophotometric method.40 The assay buffer was composed of 2.5 mM
MgCl2, 0.05 mM ZnCl2 and 8 M DEA (pH 9.8). Initial screening was performed at a
concentration of 0.2 mM of the tested compounds. The total volume of 50 µL contained 10 µL of
tested compound (0.2 mM with final DMSO 1% (v/v)), 20 µL of hꢀTNAP (46 ng of protein from
COS cell lysate in assay buffer) or of hꢀIAP (57 ng protein in assay buffer). The mixture was
o
preꢀincubated for 5ꢀ7 minutes at 37 C and luminescence was measured as preꢀread using
microplate reader (BioTek FLx800, Instruments, Inc. USA). Then, 20 µL of CDPꢀstar (final
concentration of 110 µM) was added to initiate the reaction and the assay mixture was incubated
29