New potent antihyperglycemic agents in db/db mice: Synthesis and structure-activity relationship studies of (4-substituted benzyl)(trifluoromethyl)pyrazoles and -pyrazolones

Article abstract of DOI:10.1021/jm960444z

The synthesis, structure-activity relationship (SAR) studies, and antidiabetic characterization of 1,2-dihydro-4-[[4- (methylthio)phenyl]methyl]-5-(trifluoromethyl)-3H-pyrazol-3-one (as the hydroxy tautomer; WAY-123783, 4) are described. Substitution of 4-methylthio, methylsulfinyl, or ethyl to a benzyl group at C₄, in combination with trifluoromethyl at C₅ of pyrazol-3-one, generated potent antihyperglycemic agents in obese, diabetic db/db mice (16-30% reduction in plasma glucose at 2 mg/kg). The antihyperglycemic effect was associated with a robust glucosuria (>8 g/dL) observed in nondiabetic mice. Chemical trapping of four of the seven possible tautomeric forms of the heterocycle by mono- and dialkylation at the acidic hydrogens provided several additional potent analogs (39-43% reduction at 5 mg/kg) of the lead 4 as well as a dialkylated pair of regioisomers that showed separation of the associated glucosuric effect produced by all of the active analogs in normal mice. Further pharmacological characterization of the lead WAY-123783 (ED₅₀ = 9.85 mg/kg, po in db/db mice), in oral and subcutaneous glucose tolerance tests, indicated that unlike the renal and intestinal glucose absorption inhibitor phlorizin, pyrazolone 4 does not effectively block intestinal glucose absorption. SAR and additional pharmacological data reported herein suggest that WAY-123783 represents a new class of potent antihyperglycemic agents which correct hyperglycemia by selective inhibition of renal tubular glucose reabsorption.

Full text of DOI:10.1021/jm960444z

3920
J . Med. Chem. 1996, 39, 3920-3928
New P oten t An tih yp er glycem ic Agen ts in d b/d b Mice: Syn th esis a n d
Str u ctu r e-Activity Rela tion sh ip Stu d ies of (4-Su bstitu ted
ben zyl)(tr iflu or om eth yl)p yr a zoles a n d -p yr a zolon es
Kenneth L. Kees,*,† J ohn J . Fitzgerald, J r.,† Kurt E. Steiner,§ J ames F. Mattes,‡ Brenda Mihan,§ Theresa Tosi,§
Diane Mondoro,§ and Michael L. McCaleb∇
Departments of Medicinal Chemistry and Analytical Chemistry, Global Chemical Sciences, and Cardiovascular and Metabolic
Diseases Pharmacology, Wyeth-Ayerst Research, Princeton, New J ersey 08543-8000
Received J une 20, 1996X
The synthesis, structure-activity relationship (SAR) studies, and antidiabetic characterization
of 1,2-dihydro-4-[[4-(methylthio)phenyl]methyl]-5-(trifluoromethyl)-3H-pyrazol-3-one (as the
hydroxy tautomer; WAY-123783, 4) are described. Substitution of 4-methylthio, methylsulfinyl,
or ethyl to a benzyl group at C4, in combination with trifluoromethyl at C5 of pyrazol-3-one,
generated potent antihyperglycemic agents in obese, diabetic db/db mice (16-30% reduction
in plasma glucose at 2 mg/kg). The antihyperglycemic effect was associated with a robust
glucosuria (>8 g/dL) observed in nondiabetic mice. Chemical trapping of four of the seven
possible tautomeric forms of the heterocycle by mono- and dialkylation at the acidic hydrogens
provided several additional potent analogs (39-43% reduction at 5 mg/kg) of the lead 4 as
well as a dialkylated pair of regioisomers that showed separation of the associated glucosuric
effect produced by all of the active analogs in normal mice. Further pharmacological
characterization of the lead WAY-123783 (ED50 ) 9.85 mg/kg, po in db/db mice), in oral and
subcutaneous glucose tolerance tests, indicated that unlike the renal and intestinal glucose
absorption inhibitor phlorizin, pyrazolone 4 does not effectively block intestinal glucose
absorption. SAR and additional pharmacological data reported herein suggest that WAY-
123783 represents a new class of potent antihyperglycemic agents which correct hyperglycemia
by selective inhibition of renal tubular glucose reabsorption.
Ch a r t 1. Genesis of Potent Antihyperglycemic
Pyrazolones
Recently, we described the discovery of 5-alkyl-4-
(arylmethyl)pyrazol-3-ones (hydroxy tautomers) as po-
tential new oral antidiabetic agents, based on their
ability to lower plasma glucose when administered
orally to obese, diabetic db/db mice.1 A wide variety of
2-naphthyl and benzo heterocycles were found to possess
oral activity at C4 of the heterocycle, while the alkyl
substituent at C5 was limited to lower alkyl groups and
amino. However, the best activity found belonged to the
simple naphthalene analog 1 (Chart 1). During the
course of structure-activity relationship (SAR) inves-
tigations on the aryl component of these structures, it
was also found that phenyl analog 2 showed statistically
significant activity, provided trifluoromethyl was at C5.1
Because the potency of the bicyclic arenes appeared to
reach a plateau (20-40% decrease in plasma glucose)
at a dose level of ∼20 mg/kg/per day × 4, we sought to
increase potency2 by exploring the effects of additional
substituents on the phenyl ring.
Initially, a 4-methylthio group was added to the
phenyl ring since sulfur requires about the same volume
element as the annulated (2nd) ring comprising 2-naph-
thyl and the hetero rings of the benzo heterocycles.3 In
vivo in the db/db mouse this approximation was born
out as 4-[[4-(methylthio)phenyl]methyl]-5-methylpyra-
zolone (3) (Chart 1) showed comparable activity to 1
without a trifluoromethyl group at C5. But pyrazolone
3 did not normalize the plasma glucose at high doses
a
Figures are percent mean ( SE change in plasma glucose of
db/db mice (n ) 6-10) at the dose (mg/kg/day × 4) indicated.
b
Mean of two experiments. *p e 0.05 for all data.
(Chart 1), nor did 3 maintain glucose lowering for 18-
24 h after the last dose as desired. These features were
attained when the trifluoromethyl group was placed at
C5, generating a new lead structure, pyrazolone 4 (Table
1).
In this paper, we report the structure-activity rela-
tionship studies in db/db mice that identified a potent
series of glucose-lowering analogs of 4, including the
separation of an associated glucosuric effect produced
by these compounds in normoglycemic mice. In addi-
tion, results from ancillary pharmacologic profiling of
* Address for correspondence: Wyeth-Ayerst Research, 145 King
of Prussia Rd., Radnor, PA 19087.
† Department of Medicinal Chemistry.
‡ Department of Analytical Chemistry.
§ Wyeth-Ayerst Research.
∇ Current address: Bayer Research Center, West Haven, CT 06516.
X Abstract published in Advance ACS Abstracts, September 1, 1996.
S0022-2623(96)00444-X CCC: $12.00 © 1996 American Chemical Society

New Potent Antihyperglycemic Agents in Mice
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 3921
Sch em e 1. Synthetic Routes to 3H-Pyrazol-3-ones and
“Trapped” Tautomers I-IVa
to give a mixture of pyrazolones 7 and 25, which were
separated by reverse phase chromatography (RP-C18
silica gel, gradient 50:50 H2O:MeOH f 90:10 elution).
The starting material for isopropyl sulfide derivative 9
was prepared from 4-bromobenzyl alcohol by sequential
treatment with n-BuLi (2 equiv, TMEDA, -78 °C) and
diisopropyl disulfide.
Oxidation of alkyl phenyl sulfide 4 with 30% hydrogen
peroxide at room temperature gave the sulfoxide 5,
whereas oxidation of 4 with excess oxone gave the
sulfone 6. Exposure of 4-(4-acetylbenzyl)pyrazole 205
to hydroxylamine (hydrochloride, pyridine, room tem-
perature) gave oximes 21 (1:1 syn:anti). Reduction of
20 with NaBH4 (EtOH, room temperature) gave carbinol
22. 4-[4-(Ethylamino)benzyl]pyrazolone 28 was pre-
pared as in Scheme 1 (a, c) starting from 4-(chloro-
methyl)phenylacetamide followed by reduction with
diborane (THF, 0 °C).
Alkylation of hydroxypyrazoles of type II (Scheme 1)
with 1 equiv of alkyl halides at room temperature in
acetonitrile usually gave separable mixtures of N1- and
3-O-alkylated products (IIa ,b, Scheme 1) along with
starting material. TLC monitoring of the reaction with
methyl and ethyl iodides indicated that N1-alkylation
occurred first, but after 5-48 h O-alkyl is the major
product along with N1,3-O-dialkylated and N1-alkylated
(5-hydroxy) products in minor amounts. Using excess
alkylating agent and/or refluxing acetonitrile conditions,
a separable mixture of N1,3-O- (IIc, Scheme 1) and N1,5-
O- (IV, Scheme 1) dialkylated products was obtained,
the former predominating.6 However, the N1,5-O-di-
alkylated materials could be obtained in higher yield
as the sole product of alkylation of 5-hydroxypyrazoles
of type IV (R ) H, Scheme 1, vide infra). In this way
the mono- and dialkylation products 32-39 (Table 4)
and 42 (Table 5) were prepared.
a
(a) Ethyl 4,4,4-trifluoroacetoacetate NaH, DME, reflux; (b)
MeNHNHR‚2HCl, Et(i-Pr)2N, 3 Å molecular sieves, toluene reflux;
(c) anhydrous hydrazine, toluene or DME, (powdered 3 Å molec-
ular sieves, reflux; (d) MeI, NaH, DME, room temperature; (e) MeI,
K2CO3, CH3CN, room temperature; (f) BuLi (2 equiv), THF, -78
°C, then MeI (1 equiv) f room temperature; (g) MeI, K2CO3,
CH3CN, reflux.
4 (WAY-123783) in several rodent models of non-insulin-
dependent diabetes and in normal and insulin-depend-
ent diabetic rats are reported which indicate that 4 and
its analogs represent a new class of antihyperglycemic
agents that correct hyperglycemia by selective inhibition
of renal tubular glucose reabsorption.
In the case of iodoethane alkylation of 4-[4-(meth-
ylthio)benzyl]-substituted hydroxypyrazole 4 under re-
flux conditions, the two main dialkylated products 38
and 42 were contaminated with inseparable S-ethyl
impurities (see footnotes, Tables 4 and 5) via alkyl
exchange.7 In order to obtain greater quantities of
minor alkylation products 31, 40, and 41, alternative
synthetic routes were employed.
Ch em istr y
The majority of the compounds, 3H-pyrazol-3-ones
(1-4, 7-20, 23-27, Tables 1-3), were prepared by a
two-step sequence starting from 4-substituted benzyl
bromides as shown in Scheme 1 (Sequences a, c).
Reaction of ethyl 4,4,4-trifluoroacetoacetate with NaH
in anhydrous DME at ice temperature followed by
addition of the benzyl halide and reflux provided the
corresponding â-keto esters, which were treated with
hydrazine to give the trifluoromethyl-substituted hy-
droxy pyrazoles II (Scheme 1). Aprotic conditions were
employed throughout to avoid hemiketal formation at
the trifluoromethyl ketone center.4
The benzyl halides which were not commercially
available were routinely prepared by reduction of car-
boxylic acids/derivatives to carbinols followed by bro-
mination with HBr, BBr3, or CBr4/Ph3P. Alternatively,
the corresponding 4-substituted tolyl compounds were
brominated with NBS in refluxing carbon tetrachloride
(e.g., 7, 8, 20, 25). Quenching of 4-tolylmagnesium
chloride (4-chlorotoluene, Mg, aluminum triisopro-
poxide, THF) with trifluoromethyl disulfide gave 4-[(tri-
fluoromethyl)thio]toluene and starting chloride (∼3:2,
respectively (NMR)). This mixture was brominated
(NBS, CCl4, reflux) and carried on as described above
Treatment of pyrazolone 4 with 2 equiv of n-BuLi (1
equiv of TMEDA, THF, -78 °C) and quenching of the
dianion with 1 equiv of MeI gave N1-methyl-5-hydroxy-
pyrazole 31. Reaction of ethyl 3-[4-(methylthio)phenyl]-
R-(trifluoromethyl)propionate with methylhydrazine in
refluxing toluene (b, Scheme 1) gave N1-methylpyrazol-
5-one 40, as the aromatic enol tautomer (type IV,
Schemes 1 and 2). None of the less polar (TLC, 70-30
hexane-EtOAc) isomer 31 was detected.8 Assignments
of the tautomeric forms of these structures were made
on the basis of the solution cell (CHCl3) infrared spectra,
which indicated the presence of an OH stretching
absorption and the complete absence of a CdO stretch-
ing band, and NOE experiments.9 Confirmation of the
regioisomeric assignments of 31 and the more polar
isomer 40 were made on the basis of 13C NMR. The
chemical shift of the N-methyl group in 31 (δ 37.4,
CDCl3) is downfield from that of isomer 40 (δ 33.8,
CDCl3) and split into a quartet (J ) 2.3 Hz) by long
range coupling to the trifluoromethyl group, whereas
the methyl group in 40 is a singlet.7 Likewise, assign-

3922 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Kees et al.
Ta ble 1. SAR of 4-(4-Sulfur-substitutedbenzyl)-5-
(trifluoromethyl)pyrazol-3-ones
Sch em e 2. Potential Tautomeric Forms of
4-(Arylmethyl)-5-(trifluoromethyl)pyrazol-3-ones
yielda
(%)
dose
db/dbc
no.
R
n
mp (°C)b
(mg/kg) change (%)
4
Me
0
20
147.5-148.5
100
20
5
2
0.5
5
2
5
20
5
20
5
-68 ( 2**
-57 ( 1**d
-43 ( 4**e
-29 ( 2*f
-13 ( 5
5
Me
1
29
200.5-201.5
-33 ( 2**
-25 ( 2*
2 ( 5
6
7
Me
CF3
2
0
56
6
223-225
125-127
-15 ( 6e
5 ( 6
8
Et
0
7
133-134
-38 ( 2**
-25 ( 3*
-23 ( 3
-32 ( 5**
-3 ( 9
2
20
20
9
10
i-Pr
n-Bu
0
0
9
18
169-170
95-97.5
a
Yields are for analytically pure material obtained in the last
b
step and are not optimized. Analyses (C,H,N) were within (0.4%
of theoretical values unless otherwise indicated. c Groups of db/
db mice (n ) 4-6) were administered either drug or vehicle (Tween
80/saline) once daily po × 4 days. Values (mean ( SE) are the
percent change in plasma glucose concentration of drug-treated
mice relative to vehicle controls at the given dose (mg/kg); NA )
not active, generally less than -15% change; *p < 0.05 compared
ments were made to the dialkylated isomeric pairs 36
(least polar isomer),41 and 38,42 (more polar isomer).
N-Benzylpyrazolone 43 was prepared as in 40 using
benzylhydrazine. Like 40, compound 43 exists in solu-
tion as the enol tautomer (type IV, R ) H, Scheme 1),
and alkylation with methyl iodide occurred exclusively
at oxygen to give 44. Reaction of ethyl 2-[4-(methyl-
thio)benzyl]-3-oxo-4,4,4-trifluorobutyrate with N1,2-di-
methyl hydrazine gave hydrated pyrazolone 45 (Table
5). NMR, IR, MS, and CHN data indicate this com-
pound was isolated covalently bound to water, appar-
ently via the Michael addition of water.10
d
to vehicle control, **p < 0.01 compared to vehicle control. Mean
of three experiments. e Mean of two experiments. f Mean of six
experiments.
Ta ble 2. SAR of 4-(Carbon-substitutedbenzyl)-5-
(trifluoromethyl)pyrazol-3-ones (Tautomers)
yielda
(%)
dose
(mg/kg) change (%)
db/dbc
no.
R
mp (°C)b
Alkylation of ethyl 2-[4-(methylthio)benzyl]-3-oxo-
4,4,4-trifluorobutyrate (NaH, MeI, room temperature)
followed by hydrazine treatment gave pyrazolone 46 (d,
c, Scheme 1; IR (CHCl3) ν 1740 cm-1). Alkylation of 46
(e, Scheme 1) gave 47.
11 Me
12 Et
34
44
150-151.5
128-130
20
5
2
0.5
20
5
-36 ( 3*
-39 ( 4**
-28 ( 12**
-11 ( 3
-44 ( 3**
-17 ( 3
-7 ( 5
13 n-Pr
14 i-Pr
15 n-Bu
16 t-Bu
17 n-hexyl
18 CF3
19 (CF3)2CF
20 acetyl
21 CH3CdNOH
22 CH3CHOH
23 (CH3)3CCO
54
13
9
27
25
56
25
6
114-116d
139-141
105.5-108
194.5-196
102-103f
123-124.5
170-172
218.5-219.5
194-196
154-155
204-206
5
20
20
5
20
5
5
5
100
-7 ( 4e
Resu lts a n d Discu ssion
5 ( 4
-15 ( 6**
1 ( 5
-19 ( 7**
-28 ( 5**
-18 ( 6
-23 ( 4
SAR. Oxidation of the sulfide 4 to sulfoxide 5 (Table
1) surprisingly did not affect low-dose efficacy, whereas
oxidation to the sulfone 6 destroyed activity. Addition-
ally, none of the regioisomeric sulfides, sulfoxides, and
sulfones showed significant glucose-lowering activity in
the db/db mouse.11 Substitution of trifluoromethyl for
methyl on the sulfide moiety of 4 (7) significantly
decreased activity, but increasing the alkyl chain length
to ethyl (8) was tolerated. Addition of a branching
methyl (isopropyl, 9) or further chain lengthening to
n-butyl (analog 10) was deleterious. Provided that the
sulfides, not sulfoxides, are the active species in vivo,12
these results suggested that appropriate hydrocarbon
units might function in the role of the alkyl sulfide group
to yield potent analogs of 4 and avoid potential meta-
bolic issues such as in vivo transformation of 4 to chiral
5.
74
64
88
a-c See Table 1 footnotes. Analysis for 0.25H2O. e Mean of two
d
experiments. f Analysis for 0.9H2O.
(12), stands out as clearly the most potent analog,
comparable to 4, while shorter (11) and longer (13, 15,
17) or branched (14, 16) chains markedly decreased
activity, but perfluorocarbon substitution offered no
dramatic effects (compare 11 vs 18, 14 vs 19). With the
optimum 2-carbon chain length, polar groups which are
capable of being either hydrogen bond donors or accep-
tors were evaluated (20-23, Table 2). Acetyl analog 20
and its oxime derivative 21 were efficacious at 5 mg/
kg, while carbinol 22 and tertiary aryl ketone 23 showed
no significant activity at 5 and 100 mg/kg, respectively.
We thus prepared a number of hydrocarbon analogs
of 4. The results (11-23, Table 2) are consistant with
those in the sulfide series. One hydrocarbon unit, ethyl

New Potent Antihyperglycemic Agents in Mice
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 3923
Ta ble 3. SAR of 4-(Heteroatom-substitutedbenzyl)-5-
Ta ble 4. SAR of N1- and 3-O-Alkylated
(trifluoromethyl)pyrazol-3-ones (Tautomers)
(Trifluoromethyl)pyrazoles and -pyrazol-3-ones (Tautomers)
yielda
(%)
dose
(mg/kg)
db/dbc
change (%)
yielda
(%)
dose
(mg/kg) change (%)
db/dbc
no. R1
R2
R3
mp (°C)b
no.
X
mp (°C)b
24
F
8
159-160
20
5
5
20
5
2
20
120
5
5
5
-20 ( 5
31 Me
H
MeS
18
12
141.5-145.5
20
5
20
2
-29 ( 6**
-7 ( 2d
-3 ( 2
25
26
Cl
Br
28
42
151-152
135-136.5
-6 ( 7
32
H
Me
MeS
115-117
-57 ( 3**
-16 ( 6*
-36 ( 4**
-40 ( 9**
-34 ( 3**
-49 ( 3**
-41 ( 5**
-20 ( 5
-47 ( 3**
-24 ( 7**
-5 ( 9
33
34
35
H
H
H
Me
i-Pr MeS
n-Bu Et
Et
27
36
46
36
108-109e
92-93.5
oil
5
20
20
20
5
2
5
27
28
I
45
40
132-134
169-171e
-48 ( 2**d
-61 ( 4 **
-11 ( 3*d
-22 ( 7**
-4 ( 5
EtN
36 Me
Me
MeS
31-34
29
30
MeO
CF3O
45
46
181-183
90-91
37 Me
38 Et
39 n-Bu n-Bu Et
Me
Et
Et
MeS
40
38
17
oil
-38 ( 0**
5 ( 8
-3 ( 4
oilf
oilg
5
20
a-c See Table 1 footnotes. Mean of two experiments. e Analysis
for HCl(salt)‚0.52-propanol‚0.75H2O. Compound sinters at 104 °C,
liquifies over the range indicated.
d
a-c See Table 1 footnotes. Mean of two experiments. e Analysis
d
for 1H2O. f Inseparable mixture containing ∼25% SEt (NMR).
g
With two distinct potent compounds in hand (4, 12),
we sought to further diversify the nature of the 4-benzyl
substituent by examining the effects of various halogen
and heteroalkyls and -aryls. A summary of some of our
findings are presented in Table 3. In the halogen series
(24-27, Table 3), only the larger halogen atoms bromine
(26) and iodine (27) retained efficacy, but at best these
analogs appear to belong to a group of second-tier
compounds in potency compared to 4 and 12. Likewise
active analogs containing other heteroatoms were lim-
ited to short alkyl chains, but none of these analogs
could match 4 and 12 in both efficacy and potency. For
example, ethylamino analog 28 normalizes glucose at
a high dose but loses potency at 5 mg/kg (Table 3).
Similarly, methoxy analog 29 retains low-dose efficacy
but appears to belong to the second-tier group of less
potent compounds than 4 and 12. Trifluoromethoxy 30
abolished low-dose activity, a result comparable to the
sulfide pair 4,7 but in contrast to the alkyl pair 11,18
(compare also 24 vs 21). It appears from the SAR results
in Tables 1-3 that steric effects are the more dominant
factor controlling activity rather than electronic factors.
Continuing our search for additional potent com-
pounds, working back toward the heterocycle in lead 4,
we found that the methylene linker between the 4-sub-
stituted phenyl and pyrazolone rings was critical for
activity,11 and so we returned our attention to the
heterocyclic ring. As addressed previously,1 these 4-ben-
zyl-5-(trifluoromethyl)pyrazol-3-ones exist exclusively as
the aromatic hydroxy tautomer in solution. Since, in
principle, seven tautomeric forms are possible (Scheme
2), we sought to trap these tautomeric forms by alky-
lation at the acidic hydrogens. Using a variety of
synthetic methods (Scheme 1), several sets of mono- and
dialkylated materials were obtained (see the Chemistry
section), corresponding to four of the seven tautomers
(see I-IV, Schemes 1 and 2).
Analysis for 0.5H2O.
Ta ble 5. SAR of N1,5-O, N1,2-, and N1,C4-Alkylated
(trifluoromethyl)pyrazoles and -pyrazol-5-ones
yielda
(%)
dose
mp (°C)b (mg/kg) change (%)
db/dbc
no.
R1
R2
40 Me
H
38
138-139
-40 ( 4**
-11 ( 3
-39 ( 3**
-23 ( 3*
10 ( 4
5
5
5
41 Me
42 Et
43 C6H5CH2
Me
Et
H
70
10
16
15
oil
oild
169-170
oil
20
44 C6H5CH2 Me
NT
45 Me
Me
12
glasse
20
-6 ( 5
46
47 Me
H
Me
Me
30
31
117-118
oil
20
20
-9 ( 2
6 ( 5
d
a-c See Table 1 footnotes. Inseparable mixture (∼94:6) of MeS
and EtS (NMR). e Analysis for 2H2O. C: calcd, 50.30; found, 50.80.
IR, NMR, and MS suggest 1 mol of covalently bound water
(Micheal addition product).10
and O-n-butyl analogs 34 and 35 (Table 4) retained
significant, but decreased, activity compared to 4 at 20
mg/kg.
From the results in the series of dimethylated pyra-
zolones 36, 37 (Table 4), 41, 45, and 47 (Table 5), it is
apparent either nitrogen can be methylated without loss
in potency, provided the oxygen is also methylated
(compare 36, 37 vs 41, 4, 12). In contrast, when oxygen
is not alkylated, activity is abolished (N1,2-dimethyl 45,
N1,C4-dimethyl 47, Table 5). Homologs of the potent
dimethylated compounds gave more dramatic effects.
N1,5-O-diethyl analog 42 (Table 5) shows significant
glucose-lowering activity at 5 mg/kg dose, but the
response is diminished relative to that of the corre-
sponding dimethyl 41, and the isomeric N1,3-O-diethyl
38 showed no significant glucose-lowering activity at 5
In the series of monomethylated pyrazolones 31-33
(Table 4), 40, and 46 (Table 5), O-methyl analog 32 (and
most likely O-methyl 33) retains potency and efficacy
comparable to 4 and 12. In contrast both N1-methyl-
3-hydroxypyrazole 31 and the N1-methyl-5-hydroxy
isomer 40 are inactive at 5 mg/kg. The monomethyl
analog at C4 (46) did not show significant activity at 20
mg/kg dose, nor did N1-benzyl 43 (Table 5). O-Isopropyl

3924 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Kees et al.
mg/kg. As expected, N1,3-O-di-n-butyl analog 39 was
inactive at 20 mg/kg.
In summary, investigations into the SAR of mono- and
dialkylated pyrazolone tautomers resulted in the iden-
tification of four additional potent antihyperglycemic
agents: O-methyl derivative 32, N1,3-O-dimethyl ana-
logs 36 and 37, and N1,5-O-dimethyl 41, yielding a
series (including 4 and 12) of six potent glucose-lowering
pyrazoles and pyrazolones for further pharmacological
characterization.
P h a r m a cology P r ofile of WAY-123783 (4). As can
be seen from dose-response data in Table 1, WAY-
123783 produced a 68% reduction in plasma glucose in
db/db mice at 100 mg/kg. It essentially normalized
glucose levels at 20 mg/kg (57% reduction) and retained
significant reduction at 2 mg/kg (∼30% decrease).
However, no significant glucose reduction occurred at
0.5 mg/kg. The dose-response data indicate that WAY-
123783 has an oral ED50 of 9.85 mg/kg. At a dose of 20
mg/kg, WAY-123783 normalized plasma glucose in db/
db mice 4 h after the initial dose and maintained
significant reduction for 24 h.13 In another experiment
to measure acute effects, streptozotocin-induced diabetic
rats (a model of insulin deficiency) were fasted for 3 h
prior to administration of WAY-123783 (25 mg/kg, po).
WAY-123783 showed no significant reduction in plasma
glucose during a period of 0.5-3 h after drug adminis-
tration (data not shown). In no cases was frank hy-
poglycemia observed in these diabetic rodents.
In order to assess the effects of WAY-123783-induced
plasma glucose reduction on plasma insulin levels, we
administered the drug to obese, diabetic ob/ob mice.
Unlike db/db mice, which have very high plasma glucose
levels but normal or slightly elevated circulating plasma
insulin levels at the age used in these studies,1 ob/ob
mice have only modest hyperglycemia but very high
insulin levels. Both models represent severe insulin
resistance and obesity and neither respond to the
clinically efficacious insulin-releasing sulfonylurea
agents.14
F igu r e 1. Effect of drugs on plasma glucose and insulin in
ob/ob mice. a2% Tween 80/saline. bCiglitazone (in vehicle I)
administered po once a day for 4 days at 100 mg/kg. cPhlorizin
(in vehicle I) administered ip once a day for 4 days at 200 mg/
e
kg. d1% aqueous NaHCO3 solution. WAY-123783 (in vehicle
II) administered po once a day for 4 days at 20 mg/kg. *p <
0.01.
by us and others19 produced significant glucosuria in
normal mice but were not active at high (e.g., 100 mg/
kg) doses in the db/db mice. Consistent with these
results, the SAR reported here produced several ex-
amples where the glucose-lowering effect in db/db mice
could be titrated away (e.g., 10, 17, 19, 39, 43) while a
robust glucosuria in the normal mouse was maintained.
One significant example of this trend concerned the
pyrazole isomers 38 and 42. Whereas compound 42
significantly lowered plasma glucose levels in db/db mice
at 5 mg/kg, 38 showed no glucose-lowering effect at this
dose, yet each produced approximately equivalent glu-
cosuria in normal mice.20 Our data suggest that if an
association exists between glucosuria and plasma glu-
cose lowering produced by these azoles, glucose lowering
must occur secondarily to a marked increase in glucose
excretion into the urine.
Because glucosuria in normal mice indicates altered
renal function, possibly altered tubular reabsorption of
glucose, it was of interest to compare the effects of WAY-
123783 with phlorizin on glucose tolerance tests (GTTs)
in normal and obese, insulin-resistant rats. Phlorizin
has long been recognized as an inhibitor of the active
sodium-glucose cotransporter (SGLT) present in both
kidney and gut.21 Inhibition of SGLT in the kidney
WAY-123783, at a dose of 20 mg/kg/day × 4, admin-
istered orally as a solution in 1% aqueous NaHCO3,
significantly decreased (-30%) plasma glucose in the
ob/ob mouse (Figure 1A), comparable to ciglitazone15
and phlorizin,16 two known antihyperglycemic agents
which do not release insulin. Similar to ciglitazone, the
reduction in plasma glucose induced by WAY-123783
was accompanied by even greater reductions in plasma
insulin concentrations (g-50%, Figure 1B), which were
not statistically significant due to variability in the
control group.
During the course of continued pharmacological char-
acterization of WAY-123783, we observed a robust
glucosuric effect produced by the compound in normal
mice.17 Subsequent to this finding, virtually all of the
compounds reported in this and previous work1 were
screened in the normal (Swiss CD) mouse for glucosuria
at 100 mg/kg/day × 4. The results of this study can be
summarized as follows. All compounds that showed
significant plasma glucose reduction in the db/db mouse
were positive in the glucosuria screen.18 There did not
appear to be a correlation between the magnitude of
glucosuria induced in normal mice and potency or
efficacy of the compounds as glucose-lowering agents in
db/db mice. In addition, many acidic azoles prepared

New Potent Antihyperglycemic Agents in Mice
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 3925
ylthio lead pyrazolone 4 (WAY-123783), including O-
methyl analog 32, N1,3-O-dimethyl analogs 36 and 37,
and N1,5-O-dimethylpyrazole 41.
Pharmacological characterization of the lead WAY-
123783 revealed a robust glucosuric effect produced by
the compound when administered to normal mice.
Subsequent evaluation of all of the analogs indicated
that plasma glucose lowering in diabetic animals is
likely a (secondary) consequence of glucosuria induction,
but there did not appear to be a correlation between the
magnitude of the glucosuria (qualitatively determined
as 0.1-12 g/dL) produced in normal mice and the
potency or efficacy of glucose lowering in db/db mice.
Oral glucose tolerance tests in normal, 18 h fasted rats
with a single, high dose of WAY-123783 (100 mg/kg)
administered 1 h prior to glucose challenge indicated
that, unlike phlorizin, the pyrazolone does not appear
to block intestinal glucose absorption. Our data suggest
that WAY-123783 represents a new class of low-dose
effective antihyperglycemic agents which correct hyper-
glycemia by selective inhibition of renal glucose reab-
sorption. The recent identification of SGLT2, a low-
affinitity, high-Km Na+-glucose contransporter respon-
sible for ∼90% of filtered glucose reabsorption in the
early proximal tubule, provides a possible molecular
mechanism for the observed effects of the compound on
basal circulating glucose levels in diabetic rodents and
the changes in plasma glucose following administration
of an oral glucose load.
F igu r e 2. Drug effects on plasma glucose during an OGTT
in normal rats. Rats were administered either vehicle (0.5%
methylcellulose) or drug in the morning. One hour later,
D-glucose was administered by oral gavage. Blood was collected
from the tail tip at 0 (pre), 30, 60, 90, and 120 min after
carbohydrate administration, and plasma glucose levels were
determined by an Hibachi 911 analyzer. **p < 0.01 (Dunnett’s
one-tailed t-test).
causes marked glucosuria due to inhibition of renal
tubular reabsorption of filtered glucose (from urine),
whereas inhibition of SGLT in the intestine blocks
absorption of dietary glucose. The effects of phlorizin
on intestinal glucose absorption can be measured by
monitoring the appearance of glucose in the plasma
following the administration of a glucose load.
Oral administration of glucose (2 g/kg) to 18 h fasted
Sprague-Dawley (normal) rats caused a characteristic
steep rise in plasma glucose concentration (Figure 2)
that was significantly blunted by administration of
phlorizin (400 mg/kg, po22 1 h prior to glucose challenge).
In contrast, WAY-123783, at 100 mg/kg, did not block
the sharp rise in plasma glucose following an oral
glucose challenge (Figure 2).23 These results indicate
that WAY-123783 differs from phlorizin by its apparent
incapacity to effectively block intestinal glucose absorp-
tion.24
The SAR data reported here support the notion that,
like phlorizin, WAY-123783 and, presumably, its ana-
logs are antihyperglycemic agents which act by blocking
SGLT in the kidney. The data are thus consistent with
WAY-123783 acting in a renal specific manner to block
tubular glucose reabsorption. The recent discovery of
SGLT2, an isoform of SGLT1 (intestinal Na+-glucose
cotransporter), as the principal mediator of renal glucose
reabsorption,21 provides an explanation for the observed
differential effects of phlorizin and WAY-123783 on
intestinal glucose absorption.
In summary, substitution of 4-methylthio, methyl-
sulfinyl, or ethyl to a benzyl group at C4, in combination
with trifluoromethyl at C5, of pyrazol-3-one (hydroxy
tautomer) generated potent antihyperglycemic agents
in obese, diabetic db/db mice (16-30% reduction in
plasma glucose levels at 2 mg/kg dose). Chemical
“trapping” of four of the seven possible tautomeric forms
of the heterocycle by mono- and dialkylation at the
acidic hydrogens provided several additional potent
analogs (39-43% reduction at 5 mg/kg) of the meth-
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover ap-
paratus and are uncorrected. Spectra were recorded for all
compounds and were consistent with assigned structures.
NMR spectra were recorded on a Varian XL-200, Varian VXR-
300, or Bruker AM-400 instrument. IR spectra were recorded
on a Perkin-Elmer diffraction grating or Perkin-Elmer 784
spectrophotometer. Mass spectra were recorded on a LKB-
9000S, Kratos MS 50, or Finnigan 8230 mass spectrometer.
Elemental analyses were obtained with a Perkin-Elmer 2400
elemental analyzer, and all compounds were within 0.4% of
theoretical values unless otherwise noted. All reactions were
carried out under inert atmosphere (N2 or Ar). HPLC puri-
fications were carried out on a Waters Prep 500 or Prep 500A
instrument. “Standard aqueous workup” involves separation
of an organic phase, washing it with saturated aqueous NaCl
solution, drying over anhydrous MgSO4, filtration, and con-
centration in vacuo on a rotary evaporator.
1,2-Dih yd r o-4-[[4-(m et h ylt h io)p h en yl]m et h yl]-5-(t r i-
flu or om eth yl)-3H-p yr a zol-3-on e (Ta u tom er , 4). A solution
of 4-(methylthio)benzyl alcohol (20 g, 0.13 mol) and carbon
tetrabromide (47.4 g, 0.14 mol) in dichloromethane (450 mL)
was cooled to 0 °C under N2 atmosphere. Triphenylphosphine
(37.4 g, 0.14 mol) was added portionwise over 0.5 h and the
resulting mixture allowed to warm gradually to ambient
temperature. The reaction mixture was poured onto saturated
aqueous NaCl solution. Following standard aqueous workup,
the residue was filtered through a silica gel column with the
aid of dichloromethane. Volatile materials were removed on
the rotary evaporator, and the residue was triturated with hot
heptane. The heptane solution was allowed to stand at room
temperature overnight, filtered, and concentrated. This pro-
cess was repeated twice with petroleum ether to give a
quantitative yield of [4-(methylthio)phenyl]methyl bromide as
a yellow mobile oil.
Sodium hydride (2.76 g, 69 mmol, 60% oil dispersion) in 1,2-
dimethoxyethane (50 mL) was cooled to 0 °C. Ethyl 4,4,4-
trifluoroacetoacetate (12.7 g, 69 mmol) was added dropwise
at a rate so as to control H2 evolution. When the addition was
complete the homogeneous solution was warmed to reflux
temperature and a solution of [4-(methylthio)phenyl]methyl

3926 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Kees et al.
bromide (15 g, 69 mmol) in 1,2-dimethoxyethane (100 mL) was
added. The reaction mixture was refluxed 15 h and cooled to
room temperature, and volatile materials were removed in
vacuo on the rotary evaporator. The residue was partitioned
between 1 N aqueous HCl solution and ethyl acetate followed
by standard aqueous workup. The residue was Kugelrohr
distilled at reduced pressure (∼0.5 mm Hg). Excess starting
â-keto ester was collected below 100 °C oven temperature, and
the desired ethyl 2-[4-(methylthio)benzyl]-3-oxo-4,4,4-trifluo-
robutyrate (12.2 g, 38 mmol, 55%) was collected at 115-140
°C oven temperature.
4-(Isopropylthio)benzyl bromide was prepared from the
above alcohol (15.0 g, 82.3 mmol), carbon tetrabromide (32.8
g, 98.8 mmol), and triphenylphosphine (25.9 g, 98.8 mmol) as
described for compound 4, which provided 17.1 g (69.8 mmol,
85%) of the product as an amber semisolid: MS (EI) m/z 245
(M+).
A solution of the bromide (15.0 g, 61.1 mmol) in DME was
added to a warm solution of â-keto ester anion (from 9.7 mL,
66.2 mmol, of ethyl 4,4,4-trifluoroacetoacetate and 2.60 g, 66.2
mmol, of NaH, 60% oil dispersion) in DME, as described for
compound 4, and gave 8.5 g (24.4 mmol, 40%) of the [4-(iso-
propylthio)benzyl]acetoacetate, as an amber oil: IR (film) υ
(cm-1) 1770, 1745; 1H NMR (200 MHz, CDCl3) δ 3.25 (d, 2H),
overlapping with outer lines of heptet at 3.3; MS (EI) m/z 348
(M+).
A mixture of ethyl 2-[4-(methylthio)benzyl]-3-oxo-4,4,4-
trifluorobutyrate (6.0 g, 19 mmol), anhydrous hydrazine (0.92
mL, 29 mmol), and toluene was refluxed for 15 h. The reaction
mixture was cooled to ambient temperature and concentrated
to a tan solid. The residue was triturated with hot toluene
(steam bath). The solution was decanted, and the product
crystallized on standing. The title compound, as white crys-
tals, weighed 1.08 g (3.8 mmol) after 15 h in an Abderhalden
apparatus (ethanol, reflux): IR (KBr) υ (cm-1) 3230, 1600,
1540, 1515, 1490; 1H NMR (400 MHz, DMSO-d6) δ 2.41 (s, 3H),
3.68 (s, 2H), 7.03 (d, 2H, J ) 8.2 Hz), 7.15 (d, 2H, J ) 8.2 Hz),
10.79 (s, br, 1H), 12.84 (s, br, 1H); MS (EI) m/z 288 (M+).
1,2-Dih yd r o-4-[[4-(m et h ylsu lfin yl)p h en yl]m et h yl]-5-
(tr iflu or om eth yl)-3H-p yr a zol-3-on e (Ta u tom er , 5). Com-
pound 4 (2.5 g, 8.7 mmol) in acetone (40 mL) was treated with
aqueous hydrogen peroxide solution (1 mL, 30% solution, 9
mmol) at room temperature for 24 h. The precipitate was
collected on a Buchner funnel, washed with H2O and diethyl
ether, and dried under vacuum to give 1 g (3 mmol) of white
solid: IR (KBr) υ (cm-1) 2860 (br), 1585, 1540, 1510, 1395,
1290, 1280, 1230, 1165, 1125, 1110, 1075, 1010, 995, 975, 950,
A mixture of the above acetoacetate (8.5 g, 24.4 mmol),
anhydrous hydrazine (1 mL, 31.3 mmol), and powdered 3A
sieves (4.5 g) was heated in toluene (475 mL) at reflux
overnight. The reaction mixture was filtered hot and concen-
trated in vacuo on a rotary evaporator and the residue
partitioned between dichloromethane and 5 N HCl solution
followed by standard aqueous workup. The residue was
treated with hot benzene (steam bath) and filtered while hot,
and the product deposited as white crystals upon cooling to
room temperature, to give 0.695 g (2.2 mmol) of 9: 1H NMR
(400 MHz, DMSO-d6) δ 1.18 (d, 6H, J ) 6.6 Hz), 3.37 (m, 2H,
overlapping with H2O peak), 3.7 (s, br, 2H), 7.05 (d, 2H, J )
8 Hz), 7.26 (d, 2H, J ) 8 Hz); MS (EI) m/z 316 (M+).
1,2-Dih yd r o-1-m eth yl-4-[4-(m eth ylth io)p h en yl]-5-(tr i-
flu or om eth yl)-3H-p yr a zol-3-on e (Ta u tom er , 31). A mix-
ture of compound 4 (2.50 g, 8.68 mmol), tetramethylethylene-
diamine (1.44 mL, 9.5 mmol, freshly distilled from calcium
hydride), and tetrahydrofuran (30 mL) was cooled to -78 °C
under N2 atmosphere. A solution of n-butyllithium (9.10 mL,
18.2 mmol, 2 M in pentane) was added dropwise to the
vigorously stirred reaction mixture. The mixture was held at
-78 °C for 0.3 h, warmed to ice temperature for 0.25 h, and
cooled to -78 °C, and methyl iodide (0.8 mL) was added
dropwise. The reaction mixture was allowed to warm to
ambient temperature, stirred for 15 h, and cooled in ice, and
the reaction was quenched with 1 N aqueous HCl solution.
Following standard aqueous workup, the residue (a thick black
oil) was dissolved in a minimum amount of warm ethyl acetate
and left to stand at room temperature for 13 days. Upon
partial evaporation of solvent during the period, the title
compound, as pale violet crystals, grew out of the sludge. The
crystals were collected, washed with diethyl ether, and air-
1
935, 895, 805, 770; H NMR (400 MHz, DMSO-d6) δ 2.68 (s,
3H), 3.8 (s, 2H), 7.28 (d, 2H, J ) 8 Hz), 7.56 (d, 2H, J ) 8 Hz),
10.88 (s, br, 1H), 12.89 (s, br, 1H); MS (EI) m/z 304 (M+).
1,2-Dih yd r o-4-[[4-(m et h ylsu lfon yl)p h en yl]m et h yl]-5-
(tr iflu or om eth yl)-3H-p yr a zol-3-on e (Ta u tom er , 6). To a
solution of 4 (2.50 g, 8.68 mmol) in acetic acid (20 mL), ethanol
(20 mL), water (20 mL), and concentrated sulfuric acid (10 mL)
was added potassium peroxymonosulfate (oxone; 5.29 g, 8.6
mmol) in one portion. The mixture was stirred at room
temperature for 15 h, another 2.6 g (4.3 mmol) of oxone was
added, and the reaction mixture was stirred for an additional
24 h at room temperature. The reaction mixture was filtered
and the filtrate was subjected to standard aqueous workup.
The product, homogeneous by TLC (60-40 hexane-ethyl
acetate + 1% acetic acid), was dissolved in a minimum amount
of ethyl acetate (hot, steam bath), diluted with hexane until
turbid, and then cooled in ice. The title compound, a white
solid, was collected by vacuum filtration and air-dried: IR
(KBr) υ (cm-1) 3280, 3025, 2930, 2670, 1600, 1560, 1525, 1485,
1430, 1410, 1395, 1330, 1290, 1255, 1185, 1150, 1115, 1090,
dried to give 0.472 g, 1.56 mmol, of 31: IR (CHCl3) υ (cm-1
)
3050, 2980, 1580, 1530, 1480, 1425, 1270, 1165, 1120, 1080,
1020, 1000, 955; 1H NMR (400 MHz, CDCl3) δ 2.45 (s, 3H),
3.78 (s, 5H, overlapping N-Me and CH2; note: with 15 drops
of C6D6 added, NMe shifts to 3.53, CH2 @ 3.76), 7.16 (ABq,
4H, J ) 8.8 Hz); 13C NMR (100 MHz, CDCl3) δ 16.2, 26.8, 37.5
(q J ) 2.3 Hz), 106.2 (q, J ) 1.5 Hz), 120.2 (q, J ) 270.4 Hz),
127.1, 128.6, 129.4 (q, J ) 37.4 Hz), 135.8, 137, 159.4; MS (EI)
m/z 302 (M+).
1
1010, 1000, 985, 980, 910, 820, 790, 765, 690, 650; H NMR
(400 MHz, DMSO-d6) δ 3.16 (s, 3H), 3.84 (s, 2H), 7.34 (d, 2H,
J ) 8.4 Hz), 7.81 (d, 2H, J ) 8.4 Hz), 10.87 (br, 1H); MS (EI)
m/z 320 (M+).
4-[(4-E t h ylp h e n yl)m e t h yl]-3-m e t h oxy-5-(t r iflu or o-
m eth yl)-1H-p yr a zole (33). A mixture of compound 12 (2 g),
anhydrous potassium carbonate (1.44 g, pulverized), and
acetonitrile (25 mL) was refluxed for 1 h and cooled to room
temperature, and methyl iodide was added to the stirred
mixture in portions (4 × 0.44 mL) over 2 days. The reaction
mixture was diluted with enough water to dissolve all salts
and the mixture partitioned between ethyl acetate and satu-
rated brine solution. The extracts were washed with saturated
brine, dried over MgSO4, and concentrated and the residue
chromatographed on silica gel (35 wt equiv). Elution with 10%
ethyl acetate/hexane provided the title compound as a white
solid: IR (KBr) υ (cm-1) 3240, (br OH/H2O), 3020, 2980, 2920,
1510, 1470, 1450, 1420, 1405, 1315, 1240, 1225, 1165, 1120,
1025, 965, 935, 900, 835, 820, 775, 755, 720, 700, 660, 630; 1H
NMR (400 MHz, CDCl3) δ 1.21 (t, 3H, J ) 7.6 Hz), 2.6 (q, 2H,
J ) 7.6 Hz), 3.78 (s, 2H), 3.93 (s, 3H), 7.11 (q, 4H, J ) 8.2 Hz),
8.8-9.6 (1H); MS (EI) m/z 284 (M+).
1,2-Dih yd r o-4-[[4-(isop r op ylth io)p h en yl]m eth yl]-5-(tr i-
flu or om eth yl)-3H-p yr a zol-3-on e (Ta u tom er , 9). To a -78
°C solution of 4-bromobenzyl alcohol (25.0 g, 0.134 mol) in
tetrahydrofuran (350 mL) and tetramethylethylenediamine
(42.5 mL, 0.281 mol) was added a solution of n-butyllithium
in hexane (202 mL, 1.6 M, 0.323 mol). The reaction mixture
was held at -78 °C for 1.5 h, warmed to 0 °C for 0.5 h, and
then recooled to -78 °C and a solution of diisopropyl disulfide
(20 g) in tetrahydrofuran (300 mL) added dropwise. The
mixture was allowed to warm to room temperature gradually
and stirred for 15 h. The reaction mixture was cooled in ice,
and the reaction was quenched with aqueous 1 N HCl solution
followed by standard aqueous workup. The crude amber oil
was partially purified by distillation on a Kugelrohr apparatus
(undesired material collected at oven temperature 23-65 °C,
∼0.5 mmHg). The pot residue contained 20.5 g (0.113 mol,
84%) of 4-(isopropylthio)benzyl alcohol which was used without
further purification: IR (film) υ (cm-1) 3350; 1H NMR (200
MHz, CDCl3) δ 1.28 (d, 6H), 2.6 (t, 1H), 3.35 (heptet, 2H), 4.68
(d, 2H); MS (EI) m/z 182 (M+).
3-Eth oxy-1-eth yl-4-[[4-(m eth ylth io)p h en yl]m eth yl]-5-
(tr iflu or om eth yl)p yr a zole (38) a n d 5-Eth oxy-1-eth yl-4-

New Potent Antihyperglycemic Agents in Mice
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 3927
[[4-(m eth ylth io)p h en yl]m eth yl]-3-(tr iflu or om eth yl)p yr a -
zole (42). A mixture of compound 4 (2.0 g, 6.9 mmol),
anhydrous potassium carbonate (pulverized, 2.44 g, 17 mmol),
and acetonitrile (25 mL) was refluxed for 1 h. Ethyl iodide
(1.4 mL, 17 mmol) was added, and the mixture was refluxed
overnight. The reaction mixture was cooled to room temper-
ature and diluted with enough water to dissolve all salts
followed by standard aqueous workup and chromatography on
silica gel (35 wt equiv, elution with 10% ethyl acetate/hexane)
to provide the title compound 38 (least polar isomer, 861 mg,
2.5 mmol) as a white solid. Further elution provided the more
polar isomer 42 (237 mg, 0.69 mmol), as a yellow oil. (Note:
both isomers were contaminated with inseparable SEt analogs
as indicated in the tables.) 38: IR (film) υ (cm-1) 2980, 2930,
1580, 1510, 1490, 1475, 1405, 1390, 1360, 1305, 1210, 1175,
1115, 1095, 1045, 1015, 800, 765, 740; 1H NMR (400 MHz,
CDCl3) δ 1.28* (t, 3H, J ) 7.4 Hz), 1.36 (m (overlapping
triplets), 6H), 1.55 (s, 3H), 2.89* (q, 2H, J ) 7.4 Hz), 3.74 (s,
br, 2H), 4.08 (q, J ) 7.1 Hz), 4,23 (q, J ) 7 Hz, 2H, 2H), 7.15
(m, 4H, overlapping with 2H*), 7.24* (d, 2H); 13C NMR (100
MHz, CDCl3) δ 14.4*, 14.8, 15.5, 16.2, 26.8*, 28, 46 (partially
resolved quartet), 64.7, 106.1, 120.6 (q, J ) 270 Hz), 126.9,
127.9 (q, J ) 37 Hz), 128.7, 129.5*, 133.6*, 135.5, 137.5, 138.3*,
160.1 (*absorptions assigned to SEt impurity); MS (EI) m/z
344 (M+).
the mixture was stirred at reflux for 15 h. The mixture was
filtered, triturated with petroleum ether (hot, steam bath),
concentrated in vacuo on the rotary evaporator, and dried
under vacuum, to give 9.10 g (27.2 mmol, 87%) of ethyl 2-[4-
(methylthio)benzyl]-3-oxo-4,4,4-trifluorobutyrate, as a mobile
yellow oil: IR (film) υ (cm-1) 1735; 1H NMR (400 MHz, CDCl3)
δ 1.39 (s, 3H), 2.46 (s, 3H), 3.31 (dd, J ab ) 14 Hz); MS (EI) m/z
334 (M+).
The title compound was prepared from the above â-keto
ester (8.60 g, 25.7 mmol), anhydrous hydrazine (1.63 mL, 51.4
mmol), and 3 Å molecular sieves (powdered, 4.0 g) in toluene
(350 mL) as for compound 4. The reaction mixture was filtered
hot and concentrated on the rotary evaporator and the residue
crystallized from toluene to give (after drying on an abderh-
alden apparatus, refluxing acetone, 20 h) 2.30 g (7.62 mmol)
of 46, as yellow crystals: IR (CHCl3) υ (cm-1) 3430, 3220
(broad), 3020, 2990, 2820, 1740, 1600, 1490, 1450, 1425, 1405,
1
1395, 1385, 1185, 1140, 1060, 1035, 1010, 835, 800, 720; H
NMR (400 MHz, CDCl3) δ 1.5 (s, 3H), 2.44 (s, 3H), 3.10 (ABq,
2H, J ) 13.8 Hz), 7.04 (d, 2H, J ) 8.4 Hz), 7.09 (d, 2H, J )
8.4 Hz), 8.6 (s, br, 1H).
1,4-Dim eth yl-4-[[4-(m eth ylth io)p h en yl]m eth yl]-5-(tr i-
flu or om eth yl)-2H-p yr a zol-5-on e (47). A mixture of 46 (3.0
g, 9.9 mmol), anhydrous potassium carbonate (pulverized, 12
g, 87 mmol), and acetonitrile (200 mL) was refluxed for 1 h.
Methyl iodide (15.4 mL, 248 mmol) was added; the mixture
was refluxed for 15 h followed by addition of methyl iodide
(15.4 mL) and continued reflux for 24 h. The reaction mixture
was cooled to ambient temperature, filtered, and concentrated.
The crude product was passed through a short column of silica
gel with the aid of hexane to give the title compound (960 mg,
3 mmol) as an amber syrup: IR (film) υ (cm-1) 2975, 2920,
1725, 1600, 1585, 1490, 1450, 1435, 1420, 1400, 1380, 1345,
1310, 1230, 1170, 1135, 1120, 1100, 1035, 1020, 1010, 960, 930,
920, 830, 800, 755, 735, 710, 690; 1H NMR (400 MHz, CDCl3)
δ 1.47 (s, 3H), 2.43 (s, 3H), 3.08 (s, 2H), 3.12 (s, 3H), 7.0 (d,
2H, J ) 8.3 Hz), 7.09 (d, 2H, J ) 8.4 Hz); MS (CI) m/z 317 (M
+ H)+.
P ostp r a n d ia l ob/ob Mou se Assa y. On the morning of day
1 (baseline), 56 ob/ob mice (C57Bl/6J , J ackson Laboratories,
3-4 months of age, body weight of 43-61 g) were randomly
assigned into seven groups (n ) 8) of equivalent mean body
weight. Drug or vehicle (0.2 mL) was administered once daily
for 3 days to the ad libitum fed mice. On the morning of day
4, food was removed from all cages, drug or vehicle was
administered, and mice were fasted for the remainder of the
experiment. Four hours later, mice were anesthetized with
fluothane (halothane) and then quickly decapitated for blood
collection into fluoride-containing tubes. Tubes were mixed
and maintained on ice until centrifuged. Plasma was sepa-
rated, and the levels of glucose in the plasma were determined
by an Abbott VP analyzer. The remainder of the plasma was
frozen until insulin could be determined by RIA, using the
double-antibody technique.
42: IR (film) υ (cm-1) 2980, 2930, 1575, 1510, 1495. 1485,
1470, 1440, 1275, 1260, 1165, 1120, 1090, 1055, 1025, 980, 925,
1
890, 790. 720; H NMR (400 MHz, CDCl3) δ 1.27* (t, 3H, J )
7 Hz), 1.28 (t, 3H, J ) 7.1 Hz), 1.42 (t, 3H, J ) 7.1 Hz), 2.46
(s, 3H), 2.9* (q, 2H, J ) 7 Hz), 3.82 (s, br, 2H), 3.93 (q, 2H, J
) 7.1 Hz), 4.05 (q, 2H, J ) 7.3 Hz), 7.08 (d, 2H, J ) 8.4 Hz),
7.18 (d, 2H, J ) 8.4 Hz); 13C NMR (100 MHz, CDCl3) δ 14.4*,
14.9, 15.4, 16, 27.4, 27.9*, 42.8, 70.8, 102.8, 121.6 (q, J ) 269
Hz), 126.9, 128.4, 129.4*, 134*, 135.9, 136.8, 137.7*, 138.8 (q,
J ) 36 Hz), 151.3 (*absorptions assigned to SEt impurity); MS
(EI) m/z 344 (M+).
1-Meth yl-4-[[4-(m eth ylth io)p h en yl]m eth yl]-3-(tr iflu o-
r om eth yl)-2H-p yr a zol-5-on e (Ta u tom er , 40). The title
compound was prepared as in compound 4 synthesis except
that methylhydrazine was used instead of hydrazine. Recrys-
tallization from toluene-hexane mixture provided the title
compound as off-white crystals: IR (CHCl3) υ (cm-1) 3100 (br),
1570, 1550, 1480, 1420, 1395, 1290, 1270, 1240, 1150, 1115,
1
1055; H NMR (400 MHz, CDCl3) δ 2.45 (s, 3H), 3.49 (s, 3H),
3.78 (s, 2H), 7.07 (d, J ) 8.4 Hz), 7.17 (d, J ) 8.4 Hz); 13C
NMR (100 MHz, CDCl3) δ 15.9, 27.3, 33.8, 98.9, 121.4 (q, J )
269.7 Hz), 127.1, 128.7, 135.3, 136.8, 138 (q, J ) 36.6 Hz), 150;
MS (EI) m/z 302 (M+).
5-Meth oxy-1-m eth yl-4-[[4-(m eth ylth io)p h en yl]m eth yl]-
3-(tr iflu or om eth yl)p yr a zole (41). A mixture of compound
40 (3.0 g, 9.9 mmol), anhydrous potassium carbonate (pulver-
ized, 12 g, 87 mmol), and acetonitrile (275 mL) was refluxed
for 2 h and cooled to room temperature, and methyl iodide
(neat, 2.0 mL) was added. The reaction mixture was stirred
at room temperature for 2.5 days. The mixture was filtered
and concentrated in vacuo to provide analytically pure 41 (2.2
g, 6.9 mmol), as a mobile yellow oil: IR (KBr) υ (cm-1) 3080,
3020, 2980, 2945, 2920, 1600, 1575, 1515, 1485, 1435, 1410,
1380, 1340, 1290, 1270, 1160, 1115, 1070, 1040, 1020, 990, 965,
950, 925, 825, 800, 775, 720, 715, 685; 1H NMR (400 MHz,
CDCl3) δ 2.46 (s, 3H), 3.73 (s, 6H, overlapping N-Me and O-Me;
in benzene-d6, OMe @ 2.97, NMe @ 3.03), 3.84 (s, 2H), 7.09 (d,
2H, J ) 8.4 Hz), 7.18 (d, 2H, J ) 8.4 Hz); 13C NMR (100 MHz,
C6D6) δ 15.6, 27.5, 34.1, 60.9, 103, 122.8 (q, J ) 269 Hz),
126.9-128.8 obscured by solvent), 136.9, 137.2, 138.9 (q, J )
36 Hz), 152.7; MS (+FAB) m/z 316 (M + H)+.
1,4-Dim eth yl-4-[[4-(m eth ylth io)p h en yl]m eth yl]-5-(tr i-
flu or om eth yl)-2H-p yr a zol-5-on e (46). To a slurry of sodium
hydride (60% oil dispersion, 1.37 g, 34.3 mmol) in anhydrous
DME (300 mL) at -25 °C was added a solution of ethyl
R-(trifluoroacetyl)-3-[4-(methylthio)phenyl]propionate (10.0 g,
31.2 mmol; see compound 4 synthesis) in DME (20 mL) at a
rate so as to control H2 evolution. When gas evolution ceased,
the mixture was allowed to warm to ambient temperature and
a solution of methyl iodide (5.50 g, 39.0 mmol) in DME (30
mL) was added dropwise. After 1.5 h at room temperature,
Ack n ow led gm en t. We thank Dr. Mike Malamas for
helpful suggestions, Bruce Hoffmann and staff for
analytical services, Kathryn Santilli (NOE experi-
ments), Marilyn Winkler, and Phyllis Totaro (manu-
script preparation).
Refer en ces
(1) Kees, K. L.; Caggiano, T. J .; Steiner, K. E.; Fitzgerald, J . J .;
Kates, M. J .; Christos, T. E.; Kulishoff, J . M.; Moore, R. D.;
McCaleb, M. L. Studies on New Acidic Azoles as Glucose-
Lowering Agents in Obese, Diabetic db/db Mice. J . Med. Chem.
1995, 38, 617-628.
(2) ED25 values, the effective oral dose for 25% reduction in plasma
glucose, have dropped from 40 mg/kg (ciglitazone) to 0.05 mg/
kg for some newer analogs; see: (a) Sohda, T.; Mizuno, K.;
Mamose, Y.; Ikeda, H.; Fujita, T.; Meguro, K. Studies on
Antidiabetic Agents. 11. Novel Thiazolidinedione Derivatives as
Potent Hypoglycemic and Hypolipidemic Agents. J . Med. Chem.
1992, 35, 2617-2626. (b) Another series of potent thiazolidene-

3928 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Kees et al.
diones in ob/ob mice have been recently described; see: Hulin,
B.; Clark, D. A.; Goldstein, S. W.; McDermott, R. E.; Dambek,
P. J .; Kappeler, W. H.; Lamphere, C. H.; Lewis, D. M.; Rizzi, J .
P. Novel Thiazolidene-2,4-diones as Potent Euglycemic Agents.
J . Med. Chem. 1992, 35, 1853-1864.
(15) Hulin, B.; McCarthy, P. A.; Gibbs, E. M. The Glitazone Family
of Antihyperglycemic Agents. Curr. Pharm. Des. 1996, 2, 85-
102.
(16) Rossetti, L.; Smith, D.; Schulman, G. I.; Papachristou, D.;
DeFronzo, R. A. Correction of Hyperglycemia with Phlorizin
Normalizes Tissue Sensitivity to Insulin in Diabetic Rats. J .
Clin. Invest. 1987, 79, 1510-1515.
(17) All drugs were administered to normal (Swiss CD) mice at 100
mg/kg, po/day × 4 (n ) 3). On day 4, mice were placed into
Nalgene metabolic cages (each group of three in a single cage)
with ad libitum access to food and water. Urine was collected
for 20-24 h, and urine glucose levels were qualitatively deter-
mined using an Abbot VP autoanalyzer.
(18) Urine glucose above 0.1 g/dL was considered “positive” for
glucosuria.
(19) M. Malamas, I. Gunawan, M. McCaleb, unpublished results.
See: Malamas, M. Naphthalenylmethyl Thiophenones as Anti-
hyperglycemic Agents. U.S. Patent 5,444,086, 1995.
(20) Glucosuria: 38, 7.65 g/dL; 42, 7.3 g/dL.
(21) Kanai, Y.; Lee, W.-S.; You, G.; Brown, D.; Hediger, M. A. The
Human Kidney Low Affinity Na+-glucose Cotransporter SGLT2.
Delineation of the Major Renal Reabsorptive Mechanism for a
D-Glucose. J . Clin. Invest. 1994, 93, 397-404 and references
therein.
(22) Administration of phlorizin at 200 mg/kg, ip gave similar results.
(23) Nor did WAY-123783 block absorption of a subcutaneous infusion
of glucose (1 g/kg) given to obese, insulin-resistant Zucker (fa/
fa) rats, after 4 days of drug administration (25 mg/kg/day × 4);
on the morning of the fourth day, Zucker rats (n ) 4) were fasted
for 2 h followed by the fourth administration of WAY-123783.
After an additional 1 h fast, glucose (1 g/kg) was administered
by subcutaneous infusion. Blood samples were collected from the
tail tip at 0 (pre-glucose), 30, 60, 90, and 120 min after glucose.
The area under the glucose curve was compared to that of
vehicle-treated rats.
(3) van der Waals radii for sulfur and half-thickness of a phenyl
ring are 1.85 Å; see: Cotton, F. A.; Wilkinson, G. Advanced
Inorganic Chemistry, 3rd ed.; Interscience: New York, 1972; p
120.
(4) Hemiketal formation at the trifluoromethyl ketone carbonyl was
facile in protic media, and this material was very slow to cyclize
with hydrazine.
(5) Compound 20 was prepared by starting from commercially
available 4-acetyltoluene and protection as the ethylene ketal
followed by NBS, steps a and b (Scheme 1), and aqueous HC1.
(6) Sodium hydroxide in methanol or ethanol gave similar results.
(7) IR and 1H and 13C NMR data are given in the Experimental
Section.
(8) A minor impurity in 40 was detected in the 13C NMR spectrum,
but the chemical shift (δ 38.8 (s), CDC13) indicates it is not 31.
(9) Irradiation of the hydroxyl proton of 40 at δ 11.0 (DMSO-d6)
produced an NOE at the N-methyl (δ 3.61), benzylmethylene (δ
3.7), and the two adjacent ortho hydrogens (δ 7.0).
(10) IR (KBr) ν (cm-1) 3240 (br), 2920, 1665 (very strong), 1495, 1165;
1H NMR (400 MHz, CDCl3) δ 2.46 (s, 3H), 2.59 (s, 3H), 3.02 (s,
3H), 3.13 (s, 3H), 3.28 (s, 1H); note: singlet at δ 3.28 ex-
changeswith D2O; the spectrum does not change upon addition
of 3 drops of C6D6, but sample in C6D6 alone shows an ABX
pattern (δ 2.9- 3.1), 3-3 proton singlets at higher field, and a
1 proton singlet at δ 3.6; MS (DCI) m/z 335 (M+ + H).
(11) M. L. McCaleb, unpublished results.
(12) We have no metabolic data concerning the possible in vivo
interconversion of 4 and 5.
(13) Four hours after administration of 4 (20 mg/kg), mean plasma
glucose levels in db/db mice were decreased 59%; 24 h later,
plasma glucose was decreased 22% vs controls.
(14) In our hands, neither first-generation (tolbutamide) nor second-
generation (glyburide) sulfonylurea agents lowered plasma
glucose in fed ob/ob or db/db mice. In db/db mice, glyburide (10
mg/kg/day × 4) did not significantly increase plasma insulin
levels.
(24) In one experiment, WAY-123783 administered (100 mg/kg)
simultaneously with glucose (2 g/kg, po) to 18 h fasted normal
rats significantly inhibited glucose absorption 30 min postdose
(∼15% inhibition).
J M960444Z