New azolidinediones as inhibitors of protein tyrosine phosphatase lb with antihyperglycemic properties

J . Med. Chem. 2000, 43, 995-1010

995

New Azolid in ed ion es a s In h ibitor s of P r otein Tyr osin e P h osp h a ta se 1B w ith

An tih yp er glycem ic P r op er ties

Michael S. Malamas,* J anet Sredy,^†Iwan Gunawan, Brenda Mihan, Diane R. Sawicki,^†Laura Seestaller,

Donald Sullivan, and Brenda R. Flam

Wyeth-Ayerst Research, Inc., CN 8000, Princeton, New J ersey 08543-8000

Received September 17, 1999

Insulin resistance in the liver and peripheral tissues together with a pancreatic cell defect are

the common causes of type 2 diabetes. It is now appreciated that insulin resistance can result

from a defect in the insulin receptor signaling system, at a site post binding of insulin to its

receptor. Protein tyrosine phosphatases (PTPases) have been shown to be negative regulators

of the insulin receptor. Inhibiton of PTPases may be an effective method in the treatment of

type 2 diabetes. A series of azolidinediones has been prepared as protein tyrosine phosphatase

1B (PTP1B) inhibitors. Several compounds were potent inhibitors against the recombinant

rat and human PTP1B enzymes with submicromolar IC₅₀values. Elongated spacers between

the azolidinedione moiety and the central aromatic portion of the molecule as well as

hydrophobic groups at the vicinity of this aromatic region were very important to the inhibitory

activity. Oxadiazolidinediones 87 and 88 and the corresponding acetic acid analogues 119 and

120 were the best h-PTP1B inhibitors with IC₅₀values in the range of 0.12-0.3 µM. Several

compounds normalized plasma glucose and insulin levels in the ob/ ob and db/ db diabetic mouse

models.

Ch a r t 1

In tr od u ction

Insulin resistance in the liver and peripheral tissues

together with a pancreatic cell defect are the common

causes of type 2 diabetes.¹The prevalence of insulin

resistance in prediabetic or glucose-intolerant subjects

has long been recognized.²The failure of insulin to

adequately suppress hepatic glucose output postpran-

dially combined with the reduced glucose disposal by

the peripheral tissues lead to abnormal glycemic control

after feeding. The increased and sustained plasma

glucose levels gradually progress into a number of

debilitating diabetic complications as retinopathy, neu-

ropathy, nephropathy, atherosclerosis, and coronary

artery disease.³Therefore, it is essential to control blood

glucose at the early stages of the disease.

Treatment of type 2 diabetes usually consists of diet,

exercise, and hypoglycemic agents. Sulfonylureas are

the most widely used antidiabetic agents. These agents

act by increasing insulin secretion but often are inef-

fective after 5 years of treatment.⁴Since the pioneering

discovery of ciglitazone (1) (Chart 1) by Takeda scien-

tists,⁵which reduced insulin resistance and normalized

plasma glucose levels in genetically diabetic and/or

obese animal models, a plethora of new thiazolidinedi-

ones have been developed.⁶Troglitazone (2), a thiazo-

lidinedione-type pharmacological agent, has been effec-

tively used as an insulin-enhancing agent in a large

number of insulin-resistant patients.⁷However, trogli-

tazone has produced severe liver toxic effects in a small

number of patients. Recently, SmithKline Beecham’s

thiazolidinedione BRL-49653 (rosiglitazone, 3) has been

approved for marketing in the United States, and

patients on this new agent will also be monitored for

potential liver toxicity.

It is now appreciated that insulin resistance is the

result of a defect in the insulin receptor signaling

system, at a site post binding of insulin to its receptor.

Accumulated scientific evidence demonstrating insulin

resistance in the major tissues which respond to insulin

(muscle, liver, adipose) strongly suggests that the defect

in insulin signaling resides at an early step in the signal

transduction cascade, specifically at the level of the

insulin receptor kinase activity, which appears to be

diminished.⁸

Protein tyrosine phosphatases (PTPases) play an

important role in the regulation of signal transduction

pathways and the phosphorylation of proteins. The

interaction of insulin with its receptor leads to the

phosphorylation of certain tyrosine molecules (1146,

1150, and 1151) within the receptor protein, thus

activating the receptor kinase. PTPases dephosphory-

late the activated insulin receptor, attenuating the

* To whom correspondence should be addressed. Tel: 732-274-4428.

Fax: 732-274-4505. E-mail: malamam@war.wyeth.com.

^†Present address: Institute for Diabetes Discovery, 23 Business

Park Dr., Branford, CN 06405.

Published on Web 02/09/2000

996 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5

Malamas et al.

Sch em e 1

tyrosine kinase activity. PTPases can also modulate

postreceptor signaling by catalyzing the dephosphory-

lation of cellular substrates of the insulin receptor

kinase. The enzymes that appear most likely to closely

associate with the insulin receptor and therefore most

likely to regulate the insulin receptor kinase activity

include PTP1B, LAR, PTPR, and SH-PTP2.⁹

elongated spacers were introduced between the azoli-

dinedione moiety and the central aromatic region of the

compounds. In the present study, we are reporting a

detailed systematic structure-activity relationship (SAR)

study of azolidinediones as PTPase inhibitors and the

identification of potent PTP1B inhibitors. Several azoli-

dinediones normalized plasma glucose and insulin levels

in genetically diabetic and/or obese animal models of

type 2 diabetes.

McGuire et al.¹⁰demonstrated that nondiabetic glu-

cose-intolerant subjects possessed significantly elevated

levels of PTPase activity in muscle tissue vs normal

subjects and that insulin infusion failed to suppress

PTPase activity as it did in insulin-sensitive subjects.

Meyerovitch et al.¹¹observed significantly increased

PTPase activity in the livers of two rodent models of

type 1 diabetes, the genetically diabetic BB rat and the

STZ-induced diabetic rat. Sredy et al.¹²observed similar

increased PTPase activity in the livers of obese, diabetic

ob/ ob mice, a genetic rodent model of type 2 diabetes.

Vanadium-containing inhibitors of PTPase have been

shown to increase insulin receptor tyrosine phosphory-

lation, exert insulin-like effects in vitro and in vivo, and

decrease hyperglycemia in insulin-deficient animals.¹³

PTP1B, an intracellular nonreceptor PTPase, has

shown to play a major role in the dephosphorylation of

the insulin receptor in many cellular and biochemical

studies.¹⁴A recent study with PTP1B knockout mice¹⁵

has also demonstrated that loss of PTP1B activity

resulted in an enhancement of the insulin sensitivity

and resistance to weight gain. Potent and orally active

PTP1B inhibitors could be potential pharmacological

agents for the treatment of type 2 diabetes and obesity.

We have evaluated a number of azolidinediones

previously synthesized in our laboratories and found

that several azolidinediones exhibited inhibitory activity

against a preparation of rat hepatic membrane, a source

of PTPase enzyme(s). The active compounds belonged

to an unexplored subclass of azolidinediones, where

Ch em istr y

The azolidinediones described in this paper were

prepared according to the synthetic Schemes 1-5. The

ethoxy-linked oxadiazolidinediones (Table 1; n ) 2) were

prepared according to Scheme 1. Refluxing benzamide

4 with 4-bromopropionyl acetate produced ester 5.¹⁶

Reduction of ester 5 with lithium aluminum hydride

afforded alcohol 6. Coupling of compound 6 with either

hydroxybenzaldehyde or hydroxyacetophenone 7, using

the Mitsunobu protocol,¹⁷produced phenoxy azoles 8.

Azoles 8, where R³) H, were treated with Grignard

reagents (alkyl or aromatic) followed by J ones oxidation

to afford azoles 9. Elongation of azoles 8 and 9 (R³) H,

alkyl, aryl) was achieved by the Horner-Wadsworth-

Emmons modification¹⁸of the Witting reaction using

various commercially available phosponates 10 to gen-

erate 11. Reduction of esters 11 with diisobutylalumi-

num hydride produced alcohols 12, which were con-

verted to hydroxylamines 14 by a two-step process.

First, application of the Mitsunobu protocol using tert-

butyl N-(tert-butoxycarbonyloxy)carbamate produced di-

carboxylated hydroxylamines 13, which upon treatment

with trifluoroacetic acid afforded hydroxylamines 14.

The hydroxylamines 14 were converted to the oxadi-

azolidinediones 15 with N-(chlorocarbonyl)isocyanate at

low temperatures (0-5 °C).

New Azolidinediones as PTP1B Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 997

Sch em e 2

Sch em e 3

The methoxy-linked oxadiazolidinediones (Tables 1

and 2; n ) 1) were prepared according to Schemes 1

and 2. Treatment of benzaldehyde 16 (Scheme 2) with

2,3-butanedione monoxime or 1-phenyl-1,2-propanedi-

one 2-oxime produced oxazole N-oxides 17. Deoxygen-

ation of 17 with phosphorus oxychloride afforded ox-

998 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5

Malamas et al.

Sch em e 4

azoles 18.¹⁹The desmethyl oxazoles 20 were prepared

from the appropriately substituted amides 19 upon

condensation with 1,3-dichloroacetone and subsequent

dehydration with thionyl chloride. The oxadiazoles 23

were prepared from amidoxime 22 upon condensation

with benzoyl chlorides 21.²⁰Phenols 24 were alkylated

with either chloromethyl oxazoles 18 and 20 or oxadi-

azoles 23 in the presence of potassium carbonate to

afford 25. Oxa(dia)zoles 25 were converted to the final

products 26 in a similar manner as described in Scheme

1.

ylphosphonium bromide and potassium tert-butoxide to

produce acetylene 35. Compound 35 was treated with

lithium bis(trimethylsilyl)amide and acetaldehyde to

produce alcohol 36. Conversion of 36 to the final product

37 was accomplished according to Scheme 1. The iodo

analogue 40 was prepared from acetylene 35 upon

treatment with lithium bis(trimethylsilyl)amide and

formaldehyde to afford alcohol 38, which was converted

to vinyl iodide 39 with lithium aluminum hydride,

sodium methoxide, and iodine.²¹Conversion of 39 to the

final product 40 was accomplished according to Scheme

1.

The methoxy-linked thia-, oxa-, thiadia-, and imid-

azolidinediones (Table 1) were prepared according to

Scheme 3. Alcohol 27a was treated with phosphorus

pentachloride and calcium carbonate to produce allylic

chloride 28a , which was further coupled with either the

thiazolidinedione or oxazolidinedione dianions to pro-

duce the thia- and oxazolidinediones 29 and 30. 2,4-

Thiazolidinedione was first treated with n-buyllithium

to generate the dilithio-2,4-thiazolidinedione and then

treated with chloride 28a to afford 29. The dilithio-2,4-

oxazolidinedione was generated with tert-butyllithium.

Lithium chloride was very essential for the coupling of

the dilithio-2,4-oxazolidinedione with chloride 28a. Treat-

ment of the allylic chloride 28a with sodium azide

produced the corresponding allylic azide, which was

converted to amine 31 with lithium aluminum hydride.

Amine 31 was first reacted with trimethylsilyl isocy-

anate to afford urea 32, which upon further reaction

with chlorocarbonylsulfenyl chloride produced thiadi-

azolidinedione 33.

Chloride 28a was converted to the imidazolidinedione

34b by a three-step process. First, chloride 28a was

treated with glycine methyl ester in the presence of

triethylamine, followed by urea formation (34a ) with

potassium isocyanate, and finally cyclization with so-

dium hydride afforded 34b.

The acetylenic-linked oxadiazolidinediones 77 and 78

(Table 2) were prepared according to Scheme 4. Benz-

eldehyde 34 was treated with (bromomethyl)triphen-

The cyclopropane analogues 79 and 80 (Table 2) were

prepared from alcohols 27a (Scheme 3). Treatment of

27a with diethylzinc and chloroiodomethane produced

the corresponding cyclopropane alcohols 27b, which

were further converted to the final products according

to Scheme 1.

The saturated analogues 69 and 76 (Table 2) were

prepared from the corresponding olefins by catalytic

hydrogenation.

Benzofuran 101 (Table 3) was prepared according to

Scheme 5. Treatment of benzaldehyde 41 with 2,3,4-

pentanetrione 3-oxime produced oxazole N-oxide 42.

Deoxygenation of 42 with phosphorus oxychloride af-

forded oxazole 43. Treatment of 43 with bromine

furnished 44, which upon condensation with 3-bromo-

2-hydroxybenzaldehyde in the presence of sodium meth-

oxide afforded benzofuran 45. Reduction of the ketone

first with sodium borohydride to the corresponding

alcohol and second with triethylsilane/trifluoroacetic

acid produced benzofuran 46. Conversion of the aro-

matic bromide of 46 to a formyl group was achieved in

a two-step process: first, generation of nitrile 47a with

copper cyanide in N,N-dimethylformamide from 46, and

second reduction of 47a with Al-Ni in formic acid

afforded 47b. Aldehyde 47b was converted to the final

product 48c according to Scheme 1.

The N-acetic acid analogues 118-122 (Table 4) were

prepared by alkylation of the corresponding oxadiazoli-

New Azolidinediones as PTP1B Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 999

Sch em e 5

dinediones with tert-butyl bromoacetate/K₂CO₃and

subsequent acidic hydrolysis with trifluoroacetic acid.

Tetrazole 123 (Table 4) was prepared from the allylic

chloride 28a (Scheme 3) upon treatment with sodium

cyanide to furnish the allylic nitrile 28b and subsequent

conversion of 28b to tetrazole 28c with sodium azide

and ammonium chloride.

ob mice has been observed, which may cause or con-

tribute to the decline in receptor and postreceptor

tyrosine phosphorylation.¹²The db/ db model is also

glucose intolerant with fasting hyperglycemia and oc-

casional hyperinsulinemia. The in vivo activity was

assessed at a daily dose of 10-100 mg/kg (po) for 4 days

and measured as a specified decrease in plasma glucose

and insulin levels of the drug-treated group relative to

a vehicle-treated control group. A 50-60% (db/ db) and

30-40% (ob/ ob) decrease in plasma glucose level gener-

ally normalizes glucose to levels that are similar to the

nondiabetic controls. Ciglitazone was the reference

standard in all of the assays.

In Vitr o Stu d ies. In the early stage of our discovery

program, we evaluated a number of azolidinediones

previously synthesized in our laboratories and found

that several among them exhibited inhibitory activity

against a preparation of rat hepatic membrane, a source

of PTPase enzyme(s), with pNPP as the substrate.

Ciglitazone-like compounds, where a methylene group

linked the thiazolidinedione ring with the central phenyl

ring of the molecule, were either weakly active at 250

µM or inactive (data not shown). The propene-elongated

analogues (Table 1) exhibited promising inhibitory

activity against rat liver PTPase enzyme(s) at 50 µM

concentrations. These early findings, combined with the

good in vivo activity of these molecules in the db/ db

and ob/ ob diabetic mouse models, prompted us to

further explore the SAR of such compounds as PTPase

inhibitors. In the initial phase of our studies we used a

rat hepatic membrane preparation as the source of the

enzyme(s), with pNPP as the substrate. As our program

progressed, we used either recombinant rat or human

PTP1B enzyme as the enzyme source and phospho-

tyrosyl dodecapeptide TRDI(P)YETD(P)Y(P)YRK as the

substrate. This phosphopeptide, corresponding to the

major site of autophosphorylation of the insulin recep-

tor, has been shown to be an attractive substrate in

Resu lts a n d Discu ssion

The test compounds were evaluated for their in vitro

inhibitory activity against recombinant human²²and

rat²³PTP1B, as well as a rat hepatic membrane

preparation as the source of PTPase enzyme (s), with

phosphotyrosyl dodecapeptide TRDI(P)YETD(P)Y(P)-

YRK (corresponds to the 1142-1153 insulin receptor

kinase regulatory domain, phosphorylated on the 1146,

1150, and 1151 tyrosine residues; IR-triphosphopeptide),

and p-nitrophenyl phosphate (pNPP) as sources of the

substrate.³⁰Enzyme reaction progression was monitored

via the release of inorganic phosphate as detected by

the malachite green-ammonium molybdate method for

the phosphopeptide and the amount of p-nitrophenolate

ion released from pNPP.^24,27

The in vitro activity was expressed either as the

concentration of the test compound which inhibited

enzyme activity by 50% (IC₅₀) or as the average inhibi-

tion of the test compounds at 50, 10, and 2.5 µM

concentrations. Samples were prepared in quadrupli-

cates.

The test compounds were evaluated in vivo for their

ability to decrease plasma glucose levels in the geneti-

cally obese C57 B1/6J ob/ ob and diabetic C57BL/KsJ

db/ db mouse models. The ob/ ob animal model is

severely insulin resistant, hyperinsulinemic, and glu-

cose intolerant. Insulin resistance in this model has

been associated with a reduction in insulin-induced

protein tyrosine phosphorylation in tissues such as liver.

Also, elevated PTPase activity in the liver of obese ob/

1000 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5

Malamas et al.

New Azolidinediones as PTP1B Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1001

varying degrees for several PTPases, including PTP1B,

LAR, CD45, and TCPTPase.²⁶

phenyl moieties with either electron-withdrawing or

electron-donating groups (90-96) produced r-PTP1B

inhibitors with IC₅₀values in the range of 0.8-1.4 µM.

The only exception was the 4-methoxyphenyl analogue

96, which was modestly active at 2.5 µM. Elongation of

the propene linker with pentadienes (97 and 98) pro-

duced similar in vitro activity to 59.

Results with the propene-linked oxazole azolidinedi-

ones are shown in Table 1. Oxadiazolidinedione 49

exhibited modest inhibitory PTPase activity at 50 µM

and was similar to oxazolidinedione 50, while the

thiazolidinedione 51 was inactive. The ethoxy-linked (n

) 2) oxadia- and oxazolidinediones 52 and 53 demon-

strated similar in vitro activity to the methoxy-linked

(n ) 1) analogues 49 and 50, respectively. Substitutions

on the 2-phenyl group of the oxazole tail produced more

active in vitro compounds. The trifluoromethoxy-sub-

stituted thiazolidinedione 56 inhibited PTPase activity

by 46% at 50 µM, while the unsubstituted analogue 51

was inactive. Compound 56 was one of the our initial

analogues that inhibited r-PTP1B at a micromolar

concentration (IC₅₀) 1.5 µM). The analogous oxazoli-

dinedione 55 was weaker than 56. The trifluoromethyl

analogue 59 of 49 was more potent with an IC₅₀value

of 1.9 µM against r-PTP1B. The recombinant r-PTP1B

enzyme/IR-triphosphate assay proved to be more sensi-

tive to our compounds, as depicted by the greater degree

of inhibition for examples 56 and 59. The thiadiazo-

lidinedione 60 and the p-oxadiazolidinedione 61 were

2-fold less active than 59. The ethoxy-linked para-

substituted analogue 62 was similar to 59 in vitro. The

ortho-substituted oxadiazolidinedione 63 was much

weaker than the meta-substituted analogue 59. Sub-

stitutions at position-5 of the oxazole ring appeared to

be more critical to the enzyme inhibition. The 5-phenyl

analogue 65 was 6-fold better than the 5-methyl ana-

logue 59 with an IC₅₀value of 0.3 µM, while the

unsubstituted analogue 66 was only weakly active at

10 µM.

Our initial assessment indicated that meta-substi-

tuted oxadiazolidinediones have demonstrated a better

in vitro profile, and we further focused our SAR studies

on this series of compounds. Modifications of the linkers

between the oxadiazolidinedione ring and the central

aromatic region are shown in Table 2. The E- and

Z-isomers 49 (Table 1) and 67 were similar in vitro.

Various monomethyl and dimethyl analogues 70-76

were similar in vitro to 59. The substituted trifluoro-

methylacetylene 78 was better than 77 in vitro. The

cyclopropane analogue 80 was similar to 59 in vitro.

Replacement of the methyl group, attached to the

propene linker of 59, with longer alkyl chains resulted

in a marked increase in the inhibitory activity. The E-

and Z-octyl analogues 87 and 88 were submicromolar

inhibitors of r-PTP1B, with IC₅₀values of 0.4 and 0.2

µM, respectively. Shorter alkyl groups (ethyl, propyl,

butyl) were less active in vitro. Compounds 84, 86, and

88 also demonstrated comparable potency against re-

combinant h-PTP1B with IC₅₀values in the range of

0.3-1.9 µM. The 5-phenyl oxazole analogue 117 (Table

4) of 86 exhibited a 4-fold increase in inhibitory potency

(IC₅₀) 0.37 µM) against h-PTP1B. The bulky tert-butyl

analogue 89 had an IC₅₀value of 0.6 µM against

r-PTP1B. The long and/or bulky lipophilic substituents

at the vicinity of the central aromatic region, which

produced marked increases in inhibitory potency, may

play a key role in inhibitor/protein hydrophobic interac-

tions. Further replacement of the methyl group with

iodine, cyclohexyl, phenyl, benzyl, and substituted

Modifications of the central aromatic region produced

weaker in vitro inhibitors (Table 3). Benzofuran 100,

where the propene linker of 59 was connected to the

central phenyl ring via benzofuran formation, was found

to be inactive in vitro. However, the benzofuran ana-

logue 101, where the propene linker is attached to the

phenyl portion of the molecule, demonstrated good

activity against r-PTP1B. These findings suggest that

space constraints between the oxadiazolidinedione ring

and the central aromatic region may be critical to the

inhibitory activity of these compounds. The indan

analogue 102 was also active in vitro.

Further modifications included the replacement of the

oxazole tail of 59 with aromatic or heteroaromatic

moieties (Table 4). Analogues 106-109, where phenoxy

or benzyloxy groups with electron-withdrawing groups

(chlorine, trifluoromethyl) were introduced in the mol-

ecule, were active against r-PTP1B with IC₅₀values in

the range of 0.8-4.1 µM. Unsubstituted (103) or sub-

stituted phenoxy (104-105) analogues with electron-

donating groups (methyl, methoxy) were inactive against

r-PTP1B. The naphthyl analogue 112 was weakly active

at 2.5 µM. Benzofuran analogues 113 and 114 had IC₅₀

values of 2.7 and 1.3 µM, respectively. The pyridine 115

and oxadiazole 116 were inactive at 2.5 µM.

In Vivo Stu d ies. The oxadiazolidinediones 49 and

52 (Table 1) were active in vivo at a dose of 100 mg/kg,

decreasing plasma glucose levels in the db/ db mouse

by 28% and 38%, respectively. Compound 49 also

normalized plasma glucose levels in the ob/ ob mouse

at 100 mg/kg. The substituted trifluoromethyl analogue

59 which was better in vitro decreased plasma glucose

levels in the ob/ ob mouse by 20% at a dose of 10 mg/kg

and normalized glucose levels in the db/ db mouse at

the 100 mg/kg dose. Compound 65, while being one of

our best compounds against h-PTP1B (IC₅₀) 0.3 µM),

was only weakly active in vivo. One reason of this poor

correlation may due to the higher lipophilic properties

of 65 (phenyl analogue) versus 59 (methyl analogue).

Substitutions on the olefinic linker of the compounds

(Table 2) played a more critical role on their in vivo

efficacy. The monosubstituted analogues appeared to be

more potent in vivo, normalizing plasma glucose levels

at 100 mg/kg, while the disubstituted analogues were

either weakly active or inactive. Lower alkyl groups

(methyl, ethyl, propyl, butyl) were superior to the more

hydrophilic groups (octyl, tert-butyl). The decline in the

in vivo potency of the analogues with the longer lipo-

philic tails (octyl analogues; 87, 88), despite their

superior in vitro properties, may be due to the higher

degree of lipophilicity of these compounds (CLog P )

7.4). The alkyl analogues with chain lengths of 1-4

carbons (59, 80, 84, 86) had CLog P values in the range

of 3.8-5.3. In an attempt to improve the oral efficacy

of our compounds, we prepared the acetic acid analogues

118-122 (Table 4) as less hydrophobic compounds. This

modification produced compounds with lower lipophilic

1002 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5

Ta ble 2. Chemical and Biological Data of Oxazole Azolidinediones^g

Malamas et al.

New Azolidinediones as PTP1B Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1003

Ta ble 2 (Continued)

a

pNPP used as the substrate and rat hepatic membrane as the source of PTPase enzyme(s). ¹IR-triphosphopeptide used as the substrate

b

and rat hepatic membrane as the source of PTPase enzyme(s). IR-triphosphopeptide used as the substrate and recombinant r-PTP1B

as the enzyme. ^cIR-triphosphopeptide used as the substrate and recombinant h-PTP1B as the enzyme. Compounds without melting

d

points were viscous oils. ^eNS: not significant, generally less than -15% change. ^fValues (mean ( SE) are percent change relative to

g

vehicle-treated group with use of 4-6 mice/group; *p < 0.05 and **p < 0.01 when compared to vehicle-treated mice. All compounds

were prepared according to the synthetic Schemes 1 and 2.

Ta ble 3. Chemical and Biological Data of Oxazole Azolidinediones^d

a

pNPP used as the substrate and rat hepatic membrane as the source of PTPase enzyme(s). ¹IR-triphosphopeptide used as the substrate

b

and rat hepatic membrane as the source of PTPase enzyme(s). IR-triphosphopeptide used as the substrate and recombinant r-PTP1B

as the enzyme. ^cValues (mean ( SE) are percent change relative to vehicle-treated group with use of 4-6 mice/group; *p < 0.05 when

d

compared to vehicle-treated mice. All compounds were prepared according to the synthetic Schemes 1, 2, and 5.

properties (CLog P values in the range of 1.6-3.7).

However, even though the acetic acid analogues 119 and

120 were found to be very potent inhibitors against

h-PTP1B, with IC₅₀values of 0.16 and 0.12 µM, they

were inactive in vivo (ob/ ob). The other acetic acid

analogues maintained their in vitro inhibitory activity,

with the exception of 122 which was lower. The tetrazole

analogue 123 was found to be inactive against h-PTP1B.

a possible dual mechanism of our compounds, future

work will be necessary to elucidate this hypothesis.

In summary, we have prepared a new series of

azolidinediones as PTPase inhibitors. Several com-

pounds were potent inhibitors against recombinant rat

and human PTP1B enzymes with submicromolar IC₅₀

values. Most of the compounds were also active against

a less sensitive rat hepatic membrane preparation at

higher concentrations (50 µM). Elongated spacers be-

tween the azolidinedione moiety and the central aro-

matic portion of the molecule appeared to be very

important to inhibitory activity. Large hydrophobic

groups at the vicinity of this aromatic central region

produced the most potent inhibitors. The octyl ana-

logues 87 and 88 and the corresponding acetic acid

analogues 119 and 120 were the best h-PTP1B inhibi-

tors with IC₅₀values in the range of 0.12-0.3 µM. The

tail portion of these molecules was also important to the

in vitro activity. In the case of the oxazole tail analogues,

There have been a number of reports that the thi-

azolidinediones are high-affinity peroxisome prolifera-

tor-activated receptor γ (PPARγ) agonists, and there is

a direct correlation between the in vitro potency at the

PPARγ receptor and the in vivo antihyperglycemic

potency in ob/ ob mice.³¹Since our compounds possess

some structural similarities with the thiazolidinedione-

type antihyperglycemic agents, an additional component

(PPARγ effect) to the PTP1B inhibition may contribute

to the in vivo activity of the compounds. While our

present studies are outside the scope of addressing such

1004 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5

Ta ble 4. Chemical and Biological Data of Azolidinediones^e

Malamas et al.

a

b

¹IR-triphosphopeptide used as the substrate and rat hepatic membrane as the source of PTPase enzyme(s). IR-triphosphopeptide

used as the substrate and recombinant r-PTP1B as the enzyme. ^cIR-triphosphopeptide used as the substrat and recombinant h-PTP1B

as the enzyme. Compounds without melting points were viscous oils. ^eAll compounds were prepared according to the synthetic Schemes

d

1 and 2.

substitutions at position-5 of the oxazole moiety with

hydrophobic substituents produced very good inhibitors.

The 5-phenyl analogues 65 and 117 had IC₅₀values

against h-PTP1B of 0.3 and 0.37 µM, respectively. The

oxadiazolidinedione analogues exhibited the most ap-

propriate in vitro and in vivo profile. Several compounds

New Azolidinediones as PTP1B Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1005

Hz, 1H, Ar-H), 7.56-7.59 (m, 2H, Ar-H), 7.85 (d, J ) 8.3 Hz,

1H, Ar-H), 8.1 (d, J ) 8.09 Hz, 2H, Ar-H); IR (KBr, cm^-1) 1690

(CO); MS m/e 375 (M⁺). Anal. (C₂₀H₁₆F₃NO₃) C, H, N.

normalized plasma glucose and insulin levels in the ob/

ob and db/ db diabetic mouse models. The in vitro

enhanced activity of several compounds did not trans-

late to higher in vivo potency. Although we did not have

the opportunity to examine the pharmacokinetic prop-

erties of these compounds, our data would suggest that

the increased lipophilic properties of these compounds

(higher ClogP values) might have negatively attributed

to their poor in vivo profile.

Eth yl (E)-3-[3-({5-Meth yl-2-[4-(tr iflu or om eth yl)ph en yl]-

1,3-oxa zol-4-yl}m et h oxy)p h en yl]-2-bu t en oa t e (11, R ¹

)

CF ₃, R³) CH₃, R⁴) H, n ) 0, m -m eth oxy lin k er ). Triethyl

phosphonoacetate (4.2 mL, 21.3 mmol) was added dropwise

into a mixture of sodium hydride (80% in mineral oil, 0.57 g,

19 mmol) and toluene (100 mL). The reaction mixture was

stirred for 1 h, and then 1-[3-({5-methyl-2-[4-(trifluoromethyl)-

phenyl]-1,3-oxazol-4-yl}methoxy)phenyl]-1-ethanone (5.7 g,

15.2 mmol) in THF (20 mL) was added dropwise. The mixture

was stirred for 48 h, poured into water, acidified with HCl (1

N) and extracted with ethyl acetate. The organic extracts were

dried over MgSO₄. Evaporation of the volatiles and purification

by flash chromatography on silica gel (hexane/ethyl acetate

Exp er im en ta l Section

Ch em istr y. Melting points were determined in open capil-

lary tubes on a Thomas-Hoover apparatus and are reported

uncorrected. ¹H NMR spectra were determined in the cited

solvent on a Bruker AM 400 (400 MHz) or a Varian XL-300

(300 MHz) instrument, with tetramethylsilane as an internal

standard. Chemical shifts are given in ppm and coupling

constants are in hertz. Splitting patterns are designated as

follows: s, singlet; br s, broad singlet; d, doublet; t, triplet; q,

quartet; m, multiplet. The infrared spectra were recorded on

a Perkin-Elmer 781 spectrophotometer as KBr pellets or as

solutions in chloroform. Mass spectra were recorded on either

a Finnigan model 8230 or a Hewlett-Packard model 5995A

spectrometer. Elemental analyses (C, H, N) were performed

on a Perkin-Elmer 240 analyzer and all compounds are within

(0.4% of theory unless otherwise indicated. All products,

unless otherwise noted, were purified by “flash chromatogra-

phy” with use of 220-400 mesh silica gel. Thin-layer chroma-

tography was done on silica gel 60 F-254 (0.25 mm thickness)

plates. Visualization was accomplished with UV light and/or

10% phosphomolybdic acid in ethanol. Unless otherwise noted,

all materials were obtained commercially and used without

further purification. All reactions were carried out under an

atmosphere of dried nitrogen.

1

5/1) gave a white solid (5.5 g, 81% yield): mp 88-89 °C; H

NMR (DMSO-d₆, 400 MHz) δ 1.22 (t, J ) 7.05 Hz, 3H, CH₃),

4.12 (q, J ) 7.05 Hz, 2H, CH₂), 5.08 (s, 2H, CH₂), 6.17 (s, 1H,

CH), 7.05-7.07 (m, 1H, Ar-H), 7.15 (d, J ) 7.9 Hz, 1H, Ar-H),

7.23 (m, 1H, Ar-H), 7.32 (t, J ) 8.09 Hz, 1H, Ar-H), 7.84 (d, J

) 8.5 Hz, 2H, Ar-H), 8.1 (d, J ) 8.3 Hz, 1H, Ar-H); IR (KBr,

cm^-1) 1705 (CO); MS m/e 445 (M⁺). Anal. (C₂₄H₂₂F₃NO₄) C, H,

N.

(E)-3-[3-({5-Met h yl-2-[4-(t r iflu or om et h yl)p h en yl]-1,3-

oxa zol-4-yl}m eth oxy)p h en yl]-2-bu ten -1-ol (12, R¹) CF ₃,

R³) CH₃, R⁴) H, n ) 0, m -m eth oxy lin k er ). Diisobutyl-

aluminum hydride (1 M, 28.1 mL, 28.1 mmol) was added

dropwise into a cold (-78 °C) solution of ethyl (E)-3-[3-({5-

methyl-2-[4-(trifluoromethyl)phenyl]-1,3-oxazol-4-yl}methoxy)-

phenyl]-2-butenoate (5.0 g, 11.23 mmol) in ethyl ether (100

mL). The mixture was stirred for 30 min, quenched with

MeOH, poured into HCl (1 N), and extracted with ethyl

acetate. The organic extracts were dried over MgSO₄. Evapo-

ration of the volatiles and purification by flash chromatography

on silica gel (hexane/ethyl acetate 2/1) gave a clear oil (4.3 g,

95% yield): ¹H NMR (DMSO-d₆, 400 MHz) δ 1.97 (s, 3H, CH₃),

2.48 (s, 3H, CH₃), 4.14 (d, J ) 6.2 Hz, 1H, OH), 5.05 (s, 2H,

CH₂), 5.93 (m, 1H, CH), 6.91-6.94 (m, 1H, Ar-H), 7.0-7.02

(m, 1H, Ar-H), 7.08-7.09 (m, 1H, Ar-H), 7.25 (t, J ) 8.09 Hz,

1H, Ar-H), 7.87 (d, J ) 8.09, 1H, Ar-H), 8.13 (d, J ) 8.3 Hz,

2H, Ar-H); IR (KBr, cm^-1) 3400 (OH); MS m/e 404 (M⁺). Anal.

(C₂₂H₂₀F₃NO₃) C, H, N.

Gen er a l P r oced u r e for th e Syn th esis of th e Oxa zole

Azolid in ed ion es. The oxadiazolidinediones described in this

paper (Tables 1-5) were synthesized from alcohols 6 (Scheme

1), chloromethyl oxazoles 18 and 20 (Scheme 2), and oxadi-

azoles 23 (Scheme 2) by the following representative proce-

dures. Alcohols 6 were obtained from commercially available

benzamides 4 according to literature methods.¹⁶

4-(Ch lor om eth yl)-5-m eth yl-2-[4-(tr iflu or om eth yl)ph en -

yl]-1,3-oxa zole (18, R¹) CF ₃, R²) CH₃). Hydrogen chloride

(gas, 16.0 g) was passed through a cold (0 °C) solution of

4-trifluorobenzaldehyde (50.0 g, 287 mmol) and 1,3-butadione

monoxime (26.4 g, 261 mmol) in ethyl acetate (100 mL). The

reaction mixture was then warmed to 5 °C and stirred for 3

h. Cold (0 °C) ethyl ether was added and the precipitated solid

filtered and dried to give a white solid (54.8 g, 65% yield). The

solid was dissolved in chloroform (280 mL), cooled to 5 °C, and

phosphorus oxychloride (19.7 mL, 211 mmol) in chloroform

(125 mL) was added dropwise over a 15 min period. The

reaction mixture was refluxed for 2.5 h, poured into cold water,

and the organic layer was washed with NaOH (1 N). The

organic extracts were dried over MgSO₄. Evaporation of the

volatiles and crystallization of the residue from ethyl ether/

hexane gave a yellow solid (30.0 g, 30% yield): mp 84-85 °C;

¹H NMR (DMSO-d₆, 400 MHz) δ 2.46 (s, 3H, CH₃), 4.77 (s,

2H, CH₂), 7.88 (d, J ) 8.09 Hz, 2H, Ar-H), 8.1 (d, J ) 8.09 Hz,

2H, Ar-H); MS m/e 275 (M⁺). Anal. (C₁₂H₉F₃ClNO) C, H, N.

1-[3-({5-Meth yl-2-[4-(tr iflu or om eth yl)ph en yl]-1,3-oxazol-

4-yl}m eth oxy)p h en yl]-1-eth a n on e (25, R¹) CF ₃, R², R³

) CH₃, W ) C). Potassium carbonate was added into a

mixture of 4-(chloromethyl)-5-methyl-2-[4-(trifluoromethyl)-

phenyl]-1,3-oxazole (8.0 g, 29 mmol), 3-hydroxyacetophenone

(3.95 g, 29 mmol) and DMF (100 mL). The reaction mixture

was stirred at 65 °C for 10 h and then poured into water and

extracted with ethyl acetate. The organic extracts were dried

over MgSO₄. Evaporation of the volatiles and purification by

flash chromatography on silica gel (hexane/ethyl acetate 4/1)

ter t-Bu tyl (ter t-Bu toxyca r bon yl)oxy{(E)-3-[3-({5-m eth -

yl-2-[4-(tr iflu or om eth yl)ph en yl]-1,3-oxazol-4-yl}m eth oxy)-

p h en yl]-2-bu ten yl}ca r ba m a te (13, R¹) CF ₃, R³) CH₃,

R⁴) H, n ) 0, m -m eth oxy lin k er ). Diethyl azodicarboxylate

(1.45 mL, 9.23 mmol) in THF (10 mL) was added dropwise

into a cold (-20 °C) solution of (E)-3-[3-({5-methyl-2-[4-

(trifluoromethyl)phenyl]-1,3-oxazol-4-yl}methoxy)phenyl]-2-

buten-1-ol (3.1 g, 7.69 mmol), triphenylphosphine (2.42 g, 9.23

mmol), tert-butyl N-(tert-butoxycarbonyloxy)carbamate (2.15

g, 9.23 mmol), and THF (50 mL). After the addition, the

mixture was allowed to come to 0 °C, stirred for 1 h, poured

into water and extracted with ethyl acetate. The organic

extracts were dried over MgSO₄. Evaporation of the volatiles

and purification by flash chromatography on silica gel (hexane/

ethyl acetate 6/1) gave a light yellow oil (4.6 g, 97% yield): ¹H

NMR (DMSO-d₆, 400 MHz) δ 1.41 (s, 9H, tert-butyl), 1.42 (s,

9H, tert-butyl), 2.01 (s, 3H, CH₃), 2.49 (s, 3H, CH₃), 4.38 (brs,

2H, CH₂), 5.05 (s, 2H, CH₂), 5.82 (m, 1H, CH), 6.92-7.1 (m,

2H, Ar-H), 7.08-7.1(m, 1H, Ar-H), 7.27 (t, J ) 8.09 Hz, 2H,

Ar-H), 8.12 (d, J ) 8.09 Hz, 2H, Ar-H); IR (KBr, cm^-1) 1780

(CO), 1710 (CO); MS m/e 618 (M⁺). Anal. (C₃₂H₃₇F₃N₂O₇) C,

H, N.

H yd r oxy{(E)-3-[3-({5-m et h yl-2-[4-(t r iflu or om et h yl)-

phenyl]-1,3-oxazol-4-yl}methoxy)phenyl]-2-butenyl}carbamic

Acid (14, R¹) CF ₃, R³) CH₃, R⁴) H, n ) 0, m -m eth oxy

lin k er ). Trifluoracetic acid (10 mL) was added into a solution

of tert-butyl (tert-butoxycarbonyl)oxy{(E)-3-[3-({5-methyl-2-[4-

(trifluoromethyl)phenyl]-1,3-oxazol-4-yl}methoxy)phenyl]-2-

butenyl}carbamate (4.5 g, 7.28 mmol) and dichloromethane

(40 mL). The reaction mixture was stirred for 8 h, and the

volatiles were removed in vacuo. The residue was taken in

1

gave a white solid (8.9 g, 82% yield): mp 90-91 °C; H NMR

(DMSO-d₆, 400 MHz) δ 2.49 (s, 3H, CH₃), 2.57 (s, 3H, CH₃),

5.1 (s, 2H, CH₂), 7.29-7.32 (m, 1H, Ar-H), 7.45 (t, J ) 8.09

1006 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5

Malamas et al.

ethyl acetate and washed with NaOH (1 N). The organic

extracts were dried over MgSO₄. Evaporation of the volatiles

and purification by flash chromatography on silica gel (methyl

alcohol/ethyl acetate 1/10) gave a clear oil (2.7 g, 89% yield):

¹H NMR (DMSO-d₆, 400 MHz) δ 1.98 (s, 3H, CH₃), 2.47 (s,

3H, CH₃), 3.5 (d, J ) 6.4 Hz, 2H, CH₂), 5.02 (s, 2H, CH₂), 5.8

(brs, 1H, NH), 5.95 (m, 1H, CH), 6.93-7.1 (m, 2H, Ar-H), 7.18

(m, 1H, Ar-H), 7.5-7.6 (m, 2H, Ar-H, OH), 7.88 (d, J ) 8.3

(E)-4-[3-(3-Ch lor o-1-m eth ylp r op en yl)p h en oxym eth yl]-

5-m eth yl-2-p h en yloxa zole (28a , R¹) CF ₃, R³) CH₃). 3-[3-

(5-Methyl-2-phenyloxazol-4-ylmethoxy)phenyl]but-2-en-1-ol (10.0

g, 29.9 mmol) in ethyl ether (50 mL) was added into a cold (0

°C) suspension of phosphorus pentachloride (9.31 g, 44.7

mmol), calcium carbonate (4.47 g, 44.7 mmol), and ethyl ether

(300 mL). The reaction mixture was stirred for 30 min and

poured into water. The organic layer was separated and

washed with water and brine. The organic extracts were dried

over MgSO₄. Evaporation of the volatiles and purification by

flash chromatography on silica gel (hexane/ethyl acetate 5/1)

gave a clear oil (9.1 g, 86% yield): ¹H NMR (DMSO-d₆, 400

MHz) δ 2.1 (s, 3H, CH₃), 2.45 (s, 3H, CH₃), 4.42 (d, J ) 8.09

Hz, 2H, CH₂), 5.04 (, S, 2H, CH₂), 6.05 (t, J ) 8.09 Hz, 1H,

CH), 6.97 (dd, J ) 8.3, 2.49 Hz, 1H, Ar-H), 7.04 (d, J ) 7.7

Hz, 1H, Ar-H), 7.13 (t, J ) 2.49 Hz, 1H, Ar-H), 7.28 (t, J ) 7.9

Hz, 1H, Ar-H), 7.5 (m, 3H, Ar-H), 7.94 (m, 2H, Ar-H); MS m/e

353 (M⁺). Anal. (C₂₁H₂₀ClNO₂) C, H, N.

(E)-5-{3-[3-(5-Meth yl-2-ph en yloxazol-4-ylm eth yl)ph en yl]-

b u t -2-en yl}oxa zolid in e-2,4-d ion e (30). tert-Butyllithium

(17.5 mL, 29.7 mmol) was added dropwise in to a rapidly

stirred cold (-78 °C) solution of lithium chloride (3.6 g, 84.8

mmol) and oxazolidine-2,4-dione (1.43 g, 14.1 mmol) in THF

(90 mL). The mixture was stirred at -78 °C for 30 min and

then gradually warmed to 0 °C. After recooling to -78 °C, (E)-

4-[3-(3-chloro-1-methylpropenyl)phenoxymethyl]-5-methyl-2-

phenyloxazole (5.0 g, 14.1 mmol) in THF (5 mL) was added

all at once. After stirring for 10 min at -78 °C, the mixture

was gradually warmed to room temperature and stirred for 5

h. Then, the reaction mixture was quenched with aqueous

NH₄Cl, poured into water, acidified with HCl, and extracted

with ethyl acetate. The organic extracts were dried over

MgSO₄. Evaporation of the volatiles and purification by flash

chromatography on silica gel (hexane/ethyl acetate 3/1), gave

a white solid (3.5 g, 59% yield): mp 138-139 °C; ¹H NMR

(DMSO-d₆, 400 MHz) δ 2.0 (s, 3H, CH₃), 2.45 (, 3H, CH₃), 2.76-

2.8 (m, 2H, CH₂), 5.02 (s, 2H, CH₂), 5.12 (t, J ) 5.2 Hz, 1H,

CH), 5.76 (m, 1H, CH), 6.85-6.95 (m, 2H, Ar-H), 7.02 (t, J )

2.07 Hz, 1H, Ar-H), 7.26 (t, J ) 7.9 Hz, 1H, Ar-H), 7.56 (m,

3H, Ar-H), 7.94 (m, 2H, Ar-H), 11.87 (brs, 1H, NH); IR (KBr,

cm^-1) 1750 (CO); MS m/e 419 (M + H)⁺. Anal. (C₂₄H₂₂N₂O₅)

C, H, N.

(E )-5-{3-[3-(5-Me t h yl-2-p h e n yloxa zol-4-ylm e t h oxy)-

p h en yl]bu t-2-en yl}th ia zolid in e-2,4-d ion e (29). n-Butyl-

lithium (16.6 mL, 41.6 mmol) was added dropwise in to a cold

(-78 °C) solution of thiazolidine-2,4-dione (2.31 g, 19.8 mmol)

and THF (80 mL). The mixture was stirred at -78 °C for 15

min and then gradually warmed to 0 °C and stirred for 30

min, to complete the dianion formation. After recooling to -78

°C, 4-[3-(3-chloro-1-methylpropenyl)phenoxymethyl]-5-methyl-

2-phenyloxazole (7.0 g, 19.8 mmol) in THF (15 mL) was added

all at once. The mixture was stirred for 30 min at -78 °C and

then was gradually warmed to room temperature and stirred

for 2 h. The reaction mixture was quenched with aqueous NH₄-

Cl, poured into water, acidified with HCl, and extracted with

ethyl acetate. The organic extracts were dried over MgSO₄.

Evaporation of the volatiles and purification by flash chroma-

tography on acid washed (5% H₃PO₄/MeOH) silica gel (hexane/

ethyl acetate 3/1), gave a white solid (2.9 g, 33% yield): mp

48-49 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 2.01 (s, 3H, CH₃),

2.45 (s, 3H, CH₃), 2.82 (m, 2H, CH₂), 4.7 (t, J ) 5.6 Hz, 1H,

CH), 5.01 (s, 2H, CH₂), 5.74 (m, 1H, CH), 6.98 (m, 2H, Ar-H),

7.02 (t, J ) 1.86 Hz, 1H, Ar-H), 7.28 (t, J ) 8.09 Hz, 1H, Ar-

H), 7.51 (m, 3H, Ar-H), 7.94 (m, 2H, Ar-H), 12.06 (brs, 1H,

NH); IR (KBr, cm^-1) 1700 (CO); MS m/e 435 (M + H)⁺. Anal.

(C₂₄H₂₂N₂O₄S‚0.25H₂O) C, H, N.

Hz, 2H, Ar-H), 8.12 (d, J ) 8.09 Hz, 1H, Ar-H); IR (KBr, cm^-1

)

3280 (NH), 2590 (OH); MS m/e 419 (M + H)⁺. Anal. (C₂₂H₂₁F₃-

N₂O₃) C, H, N.

2-{(E)-3-[3-({5-Met h yl-2-[4-(t r iflu or om et h yl)p h en yl]-

1,3-oxa zol-4-yl}m eth oxy)p h en yl]-2-bu ten yl}-1,2,4-oxa d i-

a zolid in e-3,5-d ion e (59). N-(Chlorocarbonyl)isocyanate (0.5

mL, 6.22 mmol) was added dropwise into a cold (-5 °C)

solution of hydroxy{(E)-3-[3-({5-methyl-2-[4-(trifluoromethyl)-

phenyl]-1,3-oxazol-4-yl}methoxy)phenyl]-2-butenyl}carbamic

acid (2.6 g, 6.22 mmol) and THF (25 mL). The reaction mixture

was stirred for 30 min, poured into HCl (1 N), and extracted

with ethyl acetate. The organic extracts were dried over

MgSO₄. Evaporation of the volatiles and purification by flash

chromatography on acid washed (5% H₃PO₄/MeOH) silica gel

(hexane/ethyl acetate 3/1) gave a white solid (1.85 g, 62%

yield): mp 136-138 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 2.06

(s, 3H, CH₃), 2.48 (s, 3H, CH₃), 4.43 (d, J ) 7.05 Hz, 2H, CH₂),

5.06 (s, 2H, CH₂), 5.9 (t, J ) 7.05 Hz, 1H, CH), 6.9 (dd, J )

8.09, 1.87 Hz,0.1H, Ar-H), 7.03 (d, J ) 8.3 Hz, 1H, Ar-H), 7.12

(t, J ) 1.87 Hz, 1H, Ar-H), 7.28 (t, J ) 7.9 Hz, 1H, Ar-H), 7.88

(d, J ) 8.3 Hz, 2H, Ar-H), 8.12 (d, J ) 8.09 Hz, 2H, Ar-H),

12.4 (brs, 1H, NH); IR (KBr, cm^-1) 3020 (NH), 1750 (CO); MS

m/e 488 (M + H)⁺. Anal. (C₂₄H₂₀F₃N₃O₅) C, H, N.

3-([5-Meth yl-2-(4-(tr iflu or om eth yl)p h en yl)oxa zol-4-yl]-

m eth oxy)ben za ld eh yd e [25(1), R¹) CF ₃, R²) CH₃, R³

)

H, W ) C, m eta -su bstitu ted ]. Potassium carbonate (3.77 g,

27.3 mmol) was added into a mixture of 4-chloromethyl-5-

methyl-2-(4-trifluoromethylphenyl)oxazole (5.25 g, 19.1 mmol),

3-hydroxylbenzaldehyde (2.33 g, 19.1 mmol), and DMF (50

mL). The mixture was stirred at 80 °C for 3 h, poured into

water, acidified with HCl (2 N) and extracted with ethyl

acetate. The organic extracts were dried over MgSO₄. Evapo-

ration of the volatiles and crystallization from ethyl ether/

hexane gave a yellow solid (4.47 g, 65% yield): mp 104-105

°C; ¹H NMR (DMSO-d₆, 400 MHz) δ 2.49 (s, 3H, CH₃), 5.13 (s,

2H, CH₂), 7.37-7.4 (m, 1H, Ar-H), 7.53-7.6 (m, 2H, Ar-H),

7.87 (d, J ) 8.5 Hz, 2H, Ar-H), 8.13 (d, J ) 8.09 Hz, 2H, Ar-

H), 9.98 (s, 1H, CHO); IR (KBr, cm^-1) 1695 (CO); MS m/e 361

(M⁺). Anal. (C₁₉H₁₄F₃NO₃) C, H, N.

1-{3-([5-Meth yl-2-(4-(tr iflu or om eth yl)p h en yl)oxa zol-4-

yl]m eth oxy)p h en yl}p r op a n -1-on e [25(2), R¹) CF ₃, R²

)

CH₃, R³) CH₂CH₃, W ) C, m eta -su bstitu ted ]. Ethylmag-

nesium bromide (11.1 mL, 33.24 mmol) was added dropwise

in to a cold (0 °C) solution of 3-[5-methyl-2-(4-trifluorometh-

ylphenyl)oxazol-4-ylmethoxy)benzaldehyde (12.0 g, 33.24 mmol)

and THF (50 mL). After stirring for 30 min, the reaction

mixture was quenched with aqueous NH₄Cl, poured into water,

acidified with HCl (2 N) and extracted with EtOAc. The

organic extracts were dried over MgSO₄. Evaporation gave a

yellowish oil (13.0 g), which was dissolved in acetone (200 mL).

The mixture was cooled to 5 °C, and freshly prepared J ones

reagent (40 mL) was added dropwise. After the addition, the

mixture was stirred for 30 min, poured into water and

extracted with EtOAc. The organic extracts were dried over

MgSO₄. Evaporation and crystallization from ethyl ether/

hexane (after cooling to 0 °C) gave a white solid (9.6 g, 74%

1

yield): mp 73-74 °C; H NMR (DMSO-d₆, 400 MHz) δ_1.07

(t, J ) 7.25 Hz, 3H, CH3), 2.49 (s, 3H, CH3), 3.02 (q, J ) 7.26

Hz, 2H, CH2), 5.11 (s, 2H, CH2), 7.3 (m, 1H, Ar-H), 7.45 (t,

J ) 7.9 Hz, 1H, Ar-H), 7.58-7.6 (m, 2H, Ar-H), 7.87 (d, J )

8.09 Hz, 2H, Ar-H), 8.12 (d, J ) 8.3 Hz, 2H, Ar-H); IR (KBr,

cm^-1) 1695 (CO); MS m/e 389 (M⁺). Anal. (C₂₁H₁₈F₃NO₃) C, H,

N.

The thiadiazolidinedione 60 was prepared according to the

following procedure.

(E)-3-{3-[5-Meth yl-2-(4-tr iflu or om eth ylp h en yl)oxa zol-

ylm eth oxy]p h en yl}bu t-2-en ylu r ea (32, R¹) CF ₃, R³

)

CH₃, m eta -su bstitu ted ). (E)-3-{3-[5-Methyl-2-(4-trifluoro-

methylphenyl)oxazol-4-ylmethoxy]phenyl}but-2-en-1-ol (6.0 g,

14.9 mmol) in ethyl ether (50 mL) was added to a cold (0 ×b0°C)

suspension of phosphorus pentachloride (4.4 g, 20.8 mmol),

The thia- and oxazolidinediones 29 and 30 (Scheme 3) were

prepared according to the following procedures.

New Azolidinediones as PTP1B Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1007

calcium carbonate (1.5 g, 14.9 mmol), and ethyl ether (50 mL).

The reaction mixture was stirred for 30 min and then poured

into water. The organic layer was separated, washed with

water and brine. The organic extracts were dried over MgSO₄.

Evaporation of the volatiles and purification by flash chroma-

tography on silica gel (hexane/ethyl acetate 20/1) gave a clear

oil (2.1 g, 35% yield). The oil (2.1 g, 4.97 mmol), sodium azide

(0.9 g, 14.2 mmol) and DMF (40 mL) was stirred at 80 ×b0°C

for 18 h. The reaction mixture was then poured into water

(100 mL), acidified to pH 3 with HCl (2 N), and extracted with

ethyl acetate. The organic extracts were dried over MgSO₄.

Evaporation gave a brown oil (1.8 g), which was dissolved in

THF (30 mL) and cooled to 0 ×b0°C. Lithium aluminum

hydride (2.2 mL, 1.0 M, 2.2 mmol) was added dropwise over a

20 min period. The reaction mixture was quenched with ethyl

acetate (10 mL) and NaOH (30 mL, 2.5 N) and extracted with

ethyl ether. The organic extracts were dried over MgSO₄.

Evaporation and purification by flash chromatography on silica

gel (ethyl acetate/methyl alcohol 1:1) gave a yellow oil (0.8 g,

42% yield). The amine (0.8 g) was dissolved in dioxane (10 mL)

and treated with trimethylsilyl isocyanate (0.67 mL, 4 mmol).

The reaction mixture was stirred for 18 h, poured into a

mixture of water (40 mL) and saturated NH₄Cl (40 mL). The

aqueous layer was extracted with ethyl acetate, and the

organic extracts were dried over MgSO₄. Evaporation of the

volatiles and purification by flash chromatography on silica

gel (ethyl acetate/ethanol) gave a white solid (0.5 g, 55%

yield): ¹H NMR (DMSO-d₆, 300 MHz) δ 2.07 (s, 3H, CH₃), 2.48

(s, 3H, CH₃), 3.6 (m, 2H, CH₂), 5.05 (s, 2H, CH₂), 5.4 (s, 2H,

NH2), 5.78 (t, J ) 6.8 Hz, 1H, NH), 6.03 (t, J ) 6.8 Hz, 1H,

CH), 6.95-7.01 (m, 2H, Ar-H), 7.03 (s, 1H, Ar-H), 7.22 (t, J

) 8.5 Hz, 1H, Ar-H), 7.82 (d, J ) 8.12 Hz, 2H, Ar-H), 8.12 (d,

J ) 8.13 Hz, 2H, Ar-H); MS m/e 445 (M⁺). Anal. (C₂₃H₂₂F₃N₃O₃)

C, H, N.

(E)-2-(3-{3-[5-Meth yl-2-(4-tr iflu or om eth ylph en yl)oxazol-

4-ylm et h oxy]p h en yl}b u t -2-en yl)-[1,2,4]t h ia d ia zolid in e-

3,5-d ion e (60). Chlorocarbonylsulfenyl chloride (0.54 mL, 6.4

mmol) was added dropwise into a mixture of (E)-3-{3-[5-

methyl-2-(4-trifluoromethylphenyl)oxazol-ylmethoxy]phenyl}-

but-2-enylurea (2.2 g, 4.94 mmol) and toluene (40 mL). The

reaction mixture was heated to 60 °C for 4 h, poured into

water, acidified with HCl (2 N) and extracted with ethyl

acetate. The organic extracts were dried over MgSO₄. Evapo-

ration of the volatiles and purification by flash chromatography

on acid washed (5% H₃PO₄/MeOH) silica gel (hexane/ethyl

acetate 3/1) gave an off-white solid (0.63 g, 25% yield): mp

60-61 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 2.07 (s, 3H, CH₃),

2.48 (s, 3H, CH₃), 4.33 (d, J ) 6.8 Hz, 2H, CH₂), 5.05 (s, 2H,

CH₂), 5.85 (t, J ) 6.8 Hz, 1H, CH), 6.95-7.05 (m, 3H, Ar-H),

7.29 (t, J ) 8.56 Hz, 1H, Ar-H), 7.87 (d, J ) 8.03 Hz, 2H, Ar-

H), 8.12 (d, J ) 8.13 Hz, 2H, Ar-H), 12.14 (brs, 1H, NH); IR

(KBr, cm^-1) 1710 (CO); MS m/e 503 (M⁺). Anal. (C₂₄H₂₀F₃N₃O₄S)

C, H, N.

gel (hexane/ethyl acetate/ethyl alcohol 6/4/0.5) gave an off-

white solid (1.3 g, 51% yield): ¹H NMR (DMSO-d₆, 400 MHz)

δ 1.92 (s, 3H, CH₃), 2.48 (, 3H, CH₃), 3.44 (s, 3H, CH₃), 3.52

(d, J ) 7.4 Hz, 2H, CH₂), 3.8 (s, 2H, CH₂), 5.03 (s, 2H, CH₂),

5.4 (t, J ) 7.4 Hz, 1H, CH), 5.87 (s, 2H, NH₂), 6.95 (m, 2H,

Ar-H), 7.2 (t, J ) 8.4 Hz, 1H, Ar-H), 7.26 (m, 1H, Ar-H), 7.89

(d, J ) 8.5 Hz, 2H, Ar-H), 8.12 (d, J ) 8.12 Hz, 2H, Ar-H); IR

(KBr, cm^-1) 1700 (CO), 1750 (CO); MS m/e 517 (M⁺). Anal.

(C₂₆H₂₆F₃N₃O₅) C, H, N.

1-{(E)-3-[3-({5-Met h yl-2-[4-(t r iflu or om et h yl)p h en yl]-

1,3-oxa zol-4-yl}m eth oxy)p h en yl]-2-bu ten yl}-2,4-im id a zo-

lid in ed ion e (64). Sodium hydride (60% in mineral oil, 0.1 g,

2.76 mmol) was added into a mixture of methyl 2-((amino-

carbonyl){(E)-3-[3-({5-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-

oxazol-4-yl}methoxy)phenyl]-2-butenyl}amino)acetate 91.3 g,

2.51 mmol) and N,N-dimethylformamide (20 mL). The mixture

was stirred for 1 h, poured into water, acidified with HCl (2

N), and extracted with ethyl acetate. The organic extracts were

dried over MgSO₄. Evaporation of the volatiles and purification

by flash chromatography on silica gel (hexane/ethyl acetate/

ethyl alcohol 6/4/0.5) gave an off-white solid (0.89 g, 73%

yield): ¹H NMR (DMSO-d₆, 400 MHz) δ 1.93 (s, 3H, CH₃), 2.46

(s, 3H, CH₃), 3.6 (d, J ) 7.4 Hz, 2H, CH₂), 3.76 (s, 2H, CH₂),

5.03 (s, 2H, CH₂), 5.4 (t, J ) 7.4 Hz, 1H, CH), 6.95 (d, J ) 7.5

Hz, Ar-H), 7.07 (dd, J ) 7.25, 1.75 Hz, 1H, Ar-H), 7.4 (t, J )

8.3 Hz, 1H, Ar-H), 7.36 (m, 1H, Ar-H), 7.89 (d, J ) 8.12 Hz,

2H, Ar-H), 8.12 (d, J ) 8.12 Hz, 2H, Ar-H); IR (KBr, cm^-1

)

1710 (CO); MS m/e 485 (M⁺). Anal. (C₂₅H₂₂F₃N₃O₄) C, H, N.

The acetylenic oxadiazolidinediones 77 and 78 were pre-

pared from alcohol 36 according to the above-described meth-

odology.

4-(3-E t h yn ylp h en oxym et h yl)-5-m et h yl-2-(4-t r iflu or o-

m eth ylp h en yl)oxa zole (35, R¹) CF ₃, m eta -su bstitu ted ).

(Bromomethyl)triphenylphosphonium bromide (21.2 g, 48.6

mmol) was added portionwise to a -78 °C mixture of potas-

sium tert-butoxide (10.9 g, 97.2 mmol) in THF (200 mL). The

mixture was stirred for 2 h, then 3-[5-methyl-2-(4-trifluoro-

methylphenyl)oxazol-4-ylmethoxy]benzaldehyde (11.7 g, 32.4

mmol) in THF (50 mL) was added dropwise. The mixture was

stirred for 1 h at -78 °C and then at room temperature for 2

days. The reaction mixture was quenched with aqueous NH₄-

Cl, poured into water, acidified with HCl (2 N), and extracted

with ethyl acetate. The organic extracts were dried over

MgSO₄. Evaporation of the volatiles and purification by flash

chromatography on silica gel (hexane/ethyl acetate 8/1) gave

a white solid (7.5 g, 67% yield): mp 62-64 °C; ¹H NMR

(DMSO-d₆, 400 MHz) δ 2.47 (s, 3H, CH₃), 4.18 (s, 1H, CH),

5.05 (, 2H, CH₂), 7.07 (m, 2H, Ar-H), 7.16 (m, 1H, Ar-H), 7.3

(t, J ) 7.9 Hz, 1H, Ar-H), 7.88 (d, J ) 8.09 Hz, 2H, Ar-H),

8.12 (d, J ) 8.09 Hz, 2H, Ar-H); MS m/e 357 (M⁺). Anal.

(C₂₀H₁₄F₃NO₂‚0.25 H₂O) C, H, N.

4-{3-[5-Met h yl-2-(4-t r iflu or om et h ylp h en yl)oxa zol-4-

ylm eth oxy]p h en yl}bu t-3-yn -2-ol (36, R¹) CF ₃, m eta -

su bstitu ted ). Lithium bis(trimethylsilyl)amide (10.5 mL, 10.5

mmol) was added into a cold (0 °C) solution of 4-(3-ethynylphe-

noxymethyl)-5-methyl-2-(4-trifluoromethylphenyl)oxazole (3.0

g, 8.7 mmol) in THF (100 mL). After 1 h at 0 °C, acetaldehyde

(0.59 mL, 10.5 mmol) was added dropwise. The mixture was

stirred for 30 min, quenched with aqueous NH₄Cl, poured into

water, acidified with HCl (2 N), and extracted with ethyl

acetate. The organic extracts were dried over MgSO₄. Evapo-

ration and purification by flash chromatography on silica gel

(hexane/ethyl acetate 4/1) gave a yellow solid (2.1 g, 59%

The imidiazolidinedione 64 was prepared according to the

following procedure.

Met h yl 2-((Am in oca r b on yl){(E)-3-[3-({5-m et h yl-2-[4-

(tr iflu or om eth yl)ph en yl]-1,3-oxazol-4-yl}m eth oxy)ph en yl]-

2-bu ten yl}a m in o)a ceta te (34a , R¹) CF ₃, R³) CH₃, m eta -

su bstitu ted ). Triethylamine (4.4 mL, 31.3 mmol) was added

into a mixture of 4-({3-[(E)-3-chloro-1-methyl-1-propenyl]-

phenoxy}methyl)-5-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-

oxazole (2.1 g, 5.21 mmol), glycine methyl ester hydrochloride

(0.66 g, 5.21 mmol), and THF (10 mL). The reaction mixture

was refluxed 18 h and the volatiles were removed in vacuo.

The residue was then dissolved in ethyl acetate and washed

with NH₄Cl. The organic extracts were dried over MgSO₄.

Evaporation of the volatiles and purification by flash chroma-

tography on silica gel (hexane/ethyl acetate/methyl alcohol 70/

12/1) gave brown oil (2.0 g). The oil was then dissolved in acetic

acid (6 mL), and then water (10 mL) and potassium cyanate

(0.7 g) were added. The mixture was stirred at 40 °C for 20

min, poured into water and extracted with ethyl acetate. The

organic extracts were dried over MgSO₄. Evaporation of the

volatiles and purification by flash chromatography on silica

1

yield): mp 86-87 °C; H NMR (DMSO-d₆, 400 MHz) δ 1.38

(d, J ) 6.64 Hz, 3H, CH₃), 2.47 (s, 3H, CH₃), 4.6 (m, 1H, CH),

5.04 (s, 2H, CH₂), 5.45 (d, J ) 4.98 Hz, 1H, OH), 7.01-7.08

(m, 3H, Ar-H), 7.29 (t, J ) 7.9 Hz, 1H, Ar-H), 7.89 (d, J ) 8.3

Hz, 2H, Ar-H), 8.12 (d, J ) 8.09, 1H, Ar-H); IR (KBr, cm^-1

3300 (OH); MS m/e 401 (M⁺). Anal. (C₂₂H₁₈F₃NO₃) C, H, N.

)

The cyclopropane analogues 79 and 80 were prepared from

alcohol 25b according to the above-described methodology.

(E)-2-{3-[5-Meth yl-2-(4-tr iflu or om eth ylp h en yl)oxa zol-

4-ylm et h oxy]p h en yl}cyclop r op ylm et h a n ol (27b , R¹

)

CF ₃, R³) H, m eta -su bstitu ted ). Chloroiodomethane (3.37

1008 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5

Malamas et al.

mL, 46.3 mmol) was added dropwise in to a cold (0 °C) solution

of diethyzinc (23.14 mL, 23.14 mmol) and dichloroethane (40

mL). After stirring for 10 min (E)-{3-[5-methyl-2-(4-trifluo-

romethylphenyl)oxazol-4-ylmethoxy]phenyl}prop-2-en-1-ol (4.5

g, 11.57 mmol) in dichloromethane (10 mL) was added drop-

wise. The reaction mixture was stirred for 1 h and quenched

with aqueous NH₄Cl. The new mixture was stirred for 15 min,

poured into water and extracted with ethyl ether. The organic

extracts were dried over MgSO₄. Evaporation of the volatiles

and purification by flash chromatography on silica gel (hexane/

ethyl acetate 3/2) gave a clear oil (2.8 g, 80% yield): ¹H NMR

(DMSO-d₆, 400 MHz) δ 0.85 (t, J ) 6.9 Hz, 2H, CH₂), 1.26 (m,

1H, CH), 1.74 (m, 1H, CH), 2.47 (s, 3H, CH₃), 3.37 (m, 1H,

CH₂), 3.41 (m, 1H, CH₂), 4.6 (t, J ) 5.6 Hz, 1H, OH), 4.99 (s,

2H, CH₂), 6.68 (d, J ) 7.9 Hz, 1H, Ar-H), 6.73 (m, 1H, Ar-H),

6.8 (m, 1H, Ar-H), 7.14 (t, J ) 7.9 Hz, 1H, Ar-H), 7.89 (d, J )

8.09 Hz, 2H, Ar-H), 8.14 (d, J ) 8.3 Hz, 2H, Ar-H); IR (KBr,

cm^-1) 3360 (OH); MS m/e 403 (M⁺). Anal. (C₂₂H₂₀F₃NO₃) C,

H, N.

30 min, the mixture was poured into water and extracted with

ethyl ether/ethyl acetate 1/1. The organic extracts were dried

over MgSO₄. Evaporation of the volatiles and purification by

flash chromatography on silica gel (hexane/ethyl acetate 2/1)

gave a yellow solid (3.4 g, 97% yield): mp 108-109 °C; ¹H NMR

(DMSO-d₆, 400 MHz) δ 2.39 (s, CH₃), 2.6 (3H, CH₃), 4.12 (s,

2H, CH₂), 6.8 (s, 1H, CH), 7.48 (m, 3H, Ar-H), 7.6 9d, J ) 8.7

Hz, 1H, Ar-H), 7.85-7.9 (m, 3H, Ar-H), 8.2 (d, J ) 1.87 Hz,

1H, Ar-H); IR (KBr, cm^-1) 1675 (CO); MS m/e 331 (M⁺). Anal.

(C₂₁H₁₇F₃NO₃) C, H, N.

Tetrazole 123 was prepared according to the following

procedure.

(E)-4-[3-({5-Meth yl-2-[4-(tr iflu or om eth oxy)p h en yl]-1,3-

oxa zol-4-yl}m eth oxy)p h en yl]-3-p en ten en itr ile (28b, R¹)

CF ₃, R³) CH₃, m eta -su bstitu ted ). Sodium cyanide (0.1 g,

2.02 mmol) in water (0.5 mL) was added into a mixture of

4-({3-[(E)-3-chloro-1-methyl-1-propenyl]phenoxy}methyl)-5-

methyl-2-[4-(trifluoromethoxy)phenyl]-1,3-oxazole (0.68 g, 1.55

mmol) and acetonitrile (10 mL). The reaction mixture was

refluxed for 3 h, poured into water, acidified with HCl (2 N)

and extracted with ethyl acetate. The organic extracts were

dried over MgSO₄. Evaporation of the volatiles and purification

by flash chromatography on silica gel (hexane/ethyl acetate

2/1) gave a yellow oil (0.65 g, 66% yield): ¹H NMR (DMSO-d₆,

400 MHz) δ 2.03 (s, 3H, CH₃), 2.46 (s, 3H, CH₃), 3.5 (d, J )

7.03 Hz, 2H, CH₂), 5.04 (s, 2H, CH₂), 5.8 (t, J ) 7.03 Hz, 1H,

CH), 6.95 (dd, J ) 8.13, 2.4 1H, Ar-H), 7.02 (d, J ) 7.68 Hz,

1H, Ar-H), 7.1 (t, J ) 1.98 Hz, 1H, Ar-H), 7.25 (t, J ) 7.9 Hz,

1H, Ar-H), 7.5 (d, J ) 7.9 Hz, 2H, Ar-H), 8.05 (d, J ) 8.78 Hz,

2H, Ar-H); MS m/e 428 (M⁺). Anal. (C₂₁H₁₇F₃NO₃) C, H, N.

4-[5-Meth yl-4-({3-[(E)-1-m eth yl-3-(2H-1,2,3,4-tetr a zol-5-

yl)-1-p r op en yl]p h en oxy}m eth yl)-1,3-oxa zol-2-yl]p h en yl

Tr iflu or om eth yl Eth er (123). Sodium azide (1.6 g, 24.7

mmol) was added into a mixture of (E)-4-[3-({5-methyl-2-[4-

(trifluoromethoxy)phenyl]-1,3-oxazol-4-yl}methoxy)phenyl]-3-

pentenenitrile (2.12 g, 4.95 mmol), NH₄Cl (1.33 g, 24.7 mmol),

and N,N-dimethylformamide (50 mL). The reaction mixture

was stirred at 130 °C for 5 h, cooled to room temperature,

poured into water, acidified with HCl (2 N), and extracted with

ethyl acetate. The organic extracts were dried over MgSO₄.

Evaporation of the volatiles and purification by flash chroma-

tography on acid washed (5% H₃PO₄/methyl alcohol) silica gel

(hexane/ethyl acetate 5/1) gave an off-white solid (0.75 g, 33%

yield): mp 100-101 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 2.07

(s, 3H, CH₃), 2.44 (s, 3H, CH₃), 3.85 (d, J ) 7.25 Hz, 2H, CH₂),

5.03 (s, 2H, CH₂), 6.07 (t, J ) 7.03 Hz, 1H, CH), 6.95 (dd, J )

7.91, 2.4 1H, Ar-H), 7.02 (d, J ) 7.91 Hz, 1H, Ar-H), 7.1 (t, J

) 1.98 Hz, 1H, Ar-H), 7.25 (t, J ) 7.91 Hz, 1H, Ar-H), 7.5 (d,

J ) 8.13 Hz, 2H, Ar-H), 8.05 (d, J ) 8.56 Hz, 2H, Ar-H), 16.05

(brs, 1H, NH); MS m/e 471 (M⁺). Anal. (C₂₁H₁₇F₃NO₃) C, H,

N.

The iodooxadiazolidinedione 90 was prepared from alcohol

39 according to the above-described methodology.

(Z)-3-Iod o-3-[3-({5-m e t h yl-2-[4-(t r iflu or om e t h oxy)-

p h en yl]-1,3-oxa zol-4-yl}m et h oxy)p h en yl]-2-p r op en -1-ol

(39, R¹) CF ₃, m eta -su bstitu ted ). Lithium bis(trimethylsi-

lyl)amide (1 M, 14 mL, 14 mmol) was added into a cold (0 °C)

solution of 4-{4-[(3-ethynylphenoxy)methyl]-5-methyl-1,3-ox-

azol-2-yl}phenyl trifluoromethyl ether (5.0 g, 14 mmol) in THF

(80 mL). After 1 h formaldehyde (gas) was passed through the

mixture for 20 min. The mixture was stirred for 1 h, quenched

with aqueous NH₄Cl, poured into water, acidified with HCl (2

N), and extracted with ethyl acetate. The organic extracts were

dried over MgSO₄. Evaporation and purification by flash

chromatography on silica gel (hexane/ethyl acetate 4/1) gave

a yellow oil (2.8 g). The oil was dissolved in tetrahydrofuran

(60 mL) and added slowly into a mixture of sodium methoxide

(1.02 g, 18.8 mmol), lithium aluminum hydride (1 M, 9.4 mL,

9.4 mmol) and tetrahydrofuran (50 mL). The reaction mixture

was then refluxed for 1 h, cooled to -78 °C, and then iodine

(5.5 g, 43.4 mmol) in THF (20 mL) was added dropwise. The

mixture was allowed to come to room temperature, poured into

NH₄Cl, and extracted with ethyl acetate. The organic extracts

were washed with sodium bisulfite, and dried over MgSO₄.

Evaporation and purification by flash chromatography on silica

gel (hexane/ethyl acetate 4/1) gave a yellow oil (1.55 g, 22%

yield): ¹H NMR (DMSO-d₆, 300 MHz) δ 2.47 (s, 3H, CH₃), 4.2

(m, 2H, CH₂), 5.05 (s, 2H, CH₂), 5.2 (m, 1H, OH), 6.4 (m, 1H,

CH), 7.01-7.08 (m, 2H, Ar-H), 7.2 (t, J ) 1.9 Hz, 1H, Ar-H),

7.4 (t, J ) 7.9 Hz, 1H, Ar-H), 7.89 (d, J ) 8.3 Hz, 2H, Ar-H),

8.12 (d, J ) 8.09, 1H, Ar-H); IR (KBr, cm^-1) 3300 (OH); MS

m/e 515(M⁺). Anal. (C₂₁H₁₇F₃INO₃) C, H, N.

Benzofuran 101 was prepared from methyl ketone 48b

according to the above-described methodology.

Biologica l Meth od s. 1. In Vitr o In h ibition of p-Nitr o-

p h en ylp h osp h a ta se (p NP P a se, or th op h osp h or ic m on o-

ester p h osp h oh yd r ola se) Activity in Mem br a n es Iso-

la ted fr om Ra t Liver . P r ep a r a tion of m em br a n es: Male

Sprague-Dawley rats (Charles River, Kingston, NY) weighing

100-150 g, maintained on standard rodent chow (Purina),

were sacrificed by asphyxiation with CO₂. The liver was

removed and washed in cold 0.85% (w/v) saline and weighed.

The tissue was homogenized on ice in 10 volumes of buffer A

and the membranes were isolated essentially as described by

Meyerovitch et al.²⁵with minor modifications. The liver

homogenate was filtered through silk to remove any remaining

tissue debris and then was centrifuged at 10000g for 20 min.

The pellet, membrane fraction, was resuspended and lightly

homogenized in: 20 mM TRIS-HCl (pH 7.4), 50 mM mercap-

toethanol, 250 mM sucrose, 2 mM EDTA, 10 mM EGTA, 2 mM

AEBSF, 0.1 mM TLCK, 0.1 mM TPCK, 0.5 mM benzamidine,

25 µg/mL leupeptin, 5 µg/mL pepstatin A, 5 µg/mL antipain,

5 µg/mL chymostatin, 10 µg/mL aprotinin to a final concentra-

tion of approximately 750 µg protein/mL. Protein concentration

was determined by the Bio-Rad protein assay using crystalline

bovine serum albumin as a standard (Bio-Rad Laboratories,

Richmond, CA).

1-[2-(5-Meth yl-2-p h en yloxa zol-4-ylm eth yl)ben zofu r a n -

5-yl]eth a n ol (48a , R¹) H). Methylmagnesium chloride (4.2

mL, 12.6 mmol) was added into a cold (0 °C) solution of 2-(5-

methyl-2-phenyloxazol-4-ylmethyl)benzofuran-5-carbalde-

hyde (prepared according to EP 0 428 312 A2, 4.0 g, 12.6 mmol)

and THF (20 mL). The reaction was stirred at 0 °C for 20 min

and then at room temperature for 30 min. The mixture was

poured into water, acidified with HCl (2 N) and extracted with

ethyl acetate. The organic extracts were dried over MgSO₄.

Evaporation of the volatiles and purification by flash chroma-

tography on silica gel (hexane/ethyl acetate 2/1) gave a yellow

1

solid (3.75 g, 88% yield): mp 103-105 °C; H NMR (DMSO-

d₆, 400 MHz) δ 1.33 (d, J ) 6.2 Hz, 3H, CH₃), 2.37 (s, 3H,

CH₃), 4.06 (s, 2H, CH₂), 4.8 (q, J ) 6.3 (1H, CH), 5.14 (brs,

1H, OH), 6.61 (s, 1H, CH), 7.2 (dd, J ) 8.51, 1.66 Hz, 1H, Ar-

H), 7.4-7.5 (m, 5H, Ar-H), 7.91 (m, 2H, Ar-H); IR (KBr, cm^-1

3380 (OH); MS m/e 333 (M⁺). Anal. (C₂₁H₁₉F₃NO₃) C, H, N.

)

1-[2-(5-Meth yl-2-p h en yloxa zol-4-ylm eth yl)ben zofu r a n -

5-yl]eth a n on e (48b, R¹) H). Freshly prepared J ones reagent

(6.5 mL, 10.5 mmol) was added dropwise into a cold (10 °C)

solution of 1-[2-(5-methyl-2-phenyloxazol-4-ylmethyl)benzofu-

ran-5-yl]ethanol (3.5 g, 10.51 mmol) and acetone (50 mL). After

New Azolidinediones as PTP1B Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 1009

Mea su r em en t of p NP P a se a ctivity: The assay was

conducted as described by Moss²⁶and Tonks et al.²⁷with minor

modifications. The incubation mixture contains in a final

volume of 0.24 mL: 50 mM HEPES (pH 7.4), 6.33 mM pNPP,

and 5/50/150 µM compound suspended in 1.25% DMSO.

Membranes were preincubated with compound in HEPES

buffer for 10 minutes at 37 °C. The reaction was started by

the additions of pNPP and after 30 min at 37 °C, the reaction

was terminated by adding 1 mL of NaOH (0.1 N). The assay

was performed in triplicate. The reaction mixture contained

approximately 30 µg protein, of the above components, plus

3.33 mM TRIS-HC1, 8.33 mM 2-mercaptoethanol, 41.67 mM

sucrose, 0.33 mM EDTA, 1.67 mM EGTA, 0.33 mM AEBSF,

0.02 mM TLCK, 0.02 mM TPCK, 0.08 mM benzamidine, 4.17

µg/mL leupeptin, 0.83 µg/mL pepstatin A, 0.83 µg/mL antipain,

0.83 µg/mL chymostatin, and 1.67 µg/mL aprotinin. The

samples were read at 410 nm in a spectrophotometer and were

regression of PTPase activities using SAS release 6.08, PROC

NLIN, was used for determining IC₅₀values of the test

compounds.

3. In h ibition of Tr ip h osp h or yla ted In su lin Recep tor

Dodecaph osph opeptide Deph osph or ylation by h -P TP 1B.

P r ep a r a tion of r ecom bin a n t h -P TP 1B: Human recombi-

nant PTP1B (321 aa form) was prepared as described.²²

Measurements and calculations of h-PTP1B activity were

determined similar to the r-PTP1B assay.

4. ob/ob a n d d b/d b Mou se Mod els. Experimental details

for the ob/ ob and db/ db diabetic mouse models were previ-

ously described.²⁸

Refer en ces

(1) DeFronzo, R. A.; Bonadonna, R. C.; Ferrannini, E. Pathogenesis

of NIDDM. Diabetes Care 1992, 15, 318-368.

(2) Reaven, G.; Bernstein, R.; Davis, B.; Olefsky, J . M. Nonketonic

Diabetes Mellitus: Insulin Deficiency or Insulin Resistance? Am.

J . Med. 1976, 60, 80.

(3) Reaven, G. Role of Insulin Resistance in Human Disease.

Diabetes 1988, 37, 1595-1607.

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Sulfonylureas. Drugs Today 1989, 25, 689-695. (b) Kolterman,

O. G.; Prince, M. J .; Olefsky, J . M. Insulin Resistance in Non-

Insulin Dependent Diabetes Mellitus. Impact of sulfonylureas

agents in vivo and in vitro. Am. J . Med. 1983, 74 (Suppl. 1A),

82-101. (c) Ferrannini, E. The Insulin resistance Syndrome.

Curr. Opin. Nephrol. Hypertens. 1992, 1, 291-298.

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5[4-(1-Methylcyclohexylmethoxy)benzyl]thiazolidine-2,4-dione and

its Derivatives. Chem Pharm. Bull. 1982, 30, 3563-3573, 3580-

3600.

evaluated based on

standard solution.

a calibration curve of p-nitrophenol

Ca lcu la tion s: The results were expressed as percent of

control, in that the amounts of p-nitrophenol formed in the

compound treated samples (nmol/min/mg protein) were com-

pared to the amount (nmol/min/mg protein) formed in the

untreated samples. The mean and standard error of the mean

were calculated (Lotus 1-2-3) and the IC₅₀values were

calculated by a regression analysis of the linear portion of the

inhibition curves (STAT80 release 3.00, EDOSE version 2.00).

pNPPase activity was also determined in every experiment

and expressed per mg protein.

2. In h ibition of Tr ip h osp h or yla ted In su lin Recep tor

Dod eca p h osp h op ep tid e Dep h osp h or yla tion by r -P TP 1B.

P r ep a r a tion of r ecom bin a n t r -P TP 1B: Rat recombinant

PTP1B, prepared as described by Goldstein et al.,²³was

suspended in 33 mM TRIS-HCl, 2 mM EDTA, 10% glycerol

and 10 mM â-mercaptoethanol.

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Horikishi, H. Characterization of New Oral Antidiabetic Agent

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Antidiabetic Agents: Synthesis and Biological Activities of

Pioglitazone and Related Compounds. Chem. Pharm. Bull. 1991,

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as Potent Hypoglycemic Agents. J . Med. Chem. 1991, 34, 1538-

1544. (d) Clark, D. A.; Goldstein S. W.; Volkmann, R. A.; Eggler,

J . F.; Holland, G. F.; Hulin, B.; Stevenson, R. W.; Kreutter, D.

K.; Gibbs, E. M.; Krupp, M. N.; Merrigan, P.; Kelbaugh, P. L.;

Andrews, E. G.; Tickner, D. L.; Suleske, R. T.; Lamphere, C. H.;

Rajeckas, F. J .; Kappeler, W. H.; McDermott, R. E.; Hutson, N.

J .; J ohnson, M. R. Substituted Dihydrobenzopyran and Dihy-

drobenzofuran Thiazolidine-2,4-diones as Hypoglycemic Agents.

J . Med. Chem. 1991, 34, 319-325. (e) 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

Thiazolidine-2,4-diones as potent Euglycemic agents. J . Med.

Chem. 1992, 35, 1853-1864. (f) Goldstein, S. W.; McDermott,

R. E.; Gibbs, E. M.; Stevenson, R. W. Hydroxyurea Derivatives

as Hypoglycemic Agents. J . Med. Chem. 1993, 36, 2238-2240.

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M.; Smith, S. A.; Thurlby, P. L. The synthesis of BRL-49653 -

A Novel and Potent Antihyperglycemic agent. Bioorg. Med.

Chem. Lett. 1994, 4, 1181-1184. (h) Cantello, B. C.; Cawthorne,

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Lister, C. A.; Smith, S. A.; Thurrlby, P. L. [[ω-(Heterocyclo-

amino)alkoxy]banzyl]-2,4-thiazolidinediones as Potent Antihy-

perglycemic Agents. J . Med. Chem. 1994, 37, 3977-3985. (i)

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of Lipoprotein (a) with Troglitazone. Lancet 1997, 350, 1748-

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Mea su r em en t of P TP a se a ctivity: The malachite green-

ammonium molybdate method, as described by Lanzetta et

al.²⁴and adapted for the platereader, was used for the

nanomolar detection of liberated phosphate by recombinant

r-PTP1B. The assay used, as substrate, a dodecaphosphopep-

tide custom synthesized by AnaSpec, Inc. (San J ose, CA). The

peptide, TRDIYETDYYRK, corresponding to the 1142-1153

catalytic domain of the insulin receptor, is phosphorylated on

tyrosine residues 1146, 1150, and 1151. The recombinant

r-PTP1B was diluted with buffer, pH 7.4, containing 33 mM

TRIS-HCl, 2 mM EDTA and 50 mM â-mercaptoethanol to

obtain an approximate activity of 1000-2000 nmol/min/mg

protein. The diluted enzyme (83.25 mL) was preincubated for

10 min at 37 °C with or without test compound (6.25 µL) and

305.5 mL of the 81.83 mM HEPES reaction buffer, pH 7.4.

Peptide substrate, 10.5 µL at a final concentration of 50 µM,

was equilibrated to 37 °C in a LABLINE Multi-Blok heater.

The preincubated recombinant enzyme preparation (39.5 mL)

with or without drug was added to initiate the dephosphory-

lation reaction, which proceeded at 37 °C for 30 min. The

reaction was terminated by the addition of 200 µL of the

malachite green-ammonium molybdate-Tween 20 stopping

reagent (MG/AM/Tw). The stopping reagent consisted of 3

parts 0.45% malachite green hydrochloride, 1 part 4.2%

ammonium molybdate tetrahydrate in 4 N HCl, and 0.5%

Tween 20. Sample blanks were prepared by the addition of

200 mL MG/AM/Tw to substrate and followed by 39.5 µL of

the preincubated recombinant enzyme with or without drug.

The color was allowed to develop at room temperature for 30

min and the sample absorbances were determined at 650 nm

using a plate reader (Molecular Devices). Samples and blanks

were prepared in quadruplicates.

Ca lcu la tion s: PTPase activities, based on a potassium

phosphate standard curve, were expressed as nmol of phos-

phate released/min/mg protein. Inhibition of recombinant

r-PTP1B by test compounds was calculated as percent of

phosphatase control. A four-parameter nonlinear logistic

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J M990476X

Article Doi

New azolidinediones as inhibitors of protein tyrosine phosphatase lb with antihyperglycemic properties

DOI: 10.1021/jm990476x

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Article abstract of DOI:10.1021/jm990476x

Full text of DOI:10.1021/jm990476x

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