Novel benzofuran and benzothiophene biphenyls as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties

J . Med. Chem. 2000, 43, 1293-1310

1293

Novel Ben zofu r a n a n d Ben zoth iop h en e Bip h en yls 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,^†Christopher Moxham, Alan Katz, Weixin Xu, Robert McDevitt,

Folake O. Adebayo, Diane R. Sawicki,^†Laura Seestaller, Donald Sullivan, and J oseph R. Taylor

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

Received November 9, 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. Inhibition of PTPases may be an effective method in the

treatment of Type 2 diabetes. We have identified two novel series of benzofuran/benzothiophene

biphenyl oxo-acetic acids and sulfonyl-salicylic acids as potent inhibitors of PTP1B with good

oral antihyperglycemic activity. To assist in the design of these inhibitors, crystallographic

studies have attempted to identify enzyme inhibitor interactions. Resolution of crystal complexes

has suggested that the inhibitors bind to the enzyme active site and are held in place through

hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. In

the oxo-acetic acid series, hydrophobic substitutents at position-2 of the benzofuran/ben-

zothiophene biphenyl framework interacted with Phe182 of the catalytic site and were very

critical to the intrinsic activity of the molecule. The hydrophobic region of the catalytic-site

pocket was exploited and taken advantage by hydrophobic substituents at either the R-carbon

or the ortho aromatic positions of the oxo-acetic acid moiety. Similar ortho aromatic substitutions

on the salicylic acid-type inhibitors had no effect, primarily due to the different orientation of

these inhibitors in the catalytic site. The most active inhibitors of both series inhibited

recombinant human PTP1B with phosphotyrosyl dodecapeptide TRDI(P)YETD(P)Y(P)YRK as

the source of the substrate with IC₅₀values in the range of 20-50 nM. Compound 68 was one

of the most active compounds in vivo, normalizing plasma glucose levels at the 25 mg/kg dose

(po) and the 1 mg/kg dose (ip). Compound 68 was also selective against several other PTPases.

In tr od u ction

first of such a therapeutic agent used for the treatment

of Type 2 diabetes, has produced severe liver toxic

effects in a small number of patients. Most recently,

rosiglitazone, a second member of the glitazone class,

has been approved for marketing in the United States,

and patients on this new agent will also be monitored

for potential liver toxicity. The glitazone-type therapeu-

tic agents are not effective in all Type 2 diabetes

patients; therefore, there still remains a great need for

more effective and safe agents.

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 pre-diabetic 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 progresses into a number of

debilitating diabetic complications: retinopathy, neur-

opathy, 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.⁴Recently, a new class

of pharmacological agents, the glitazones, have been

effectively used as insulin enhancing agents in a large

number of insulin resistant patients.⁵Troglitazone, the

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.⁶

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 dephosphoryl-

* Wyeth-Ayerst Res., CN 8000, Princeton, NJ 08543-8000. Tel: (732)

274-4428. Fax: (732) 274-4505. E-mail address: malamam@war.

wyeth.com.

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

Park Drive, Branford, CT 06405.

Published on Web 02/25/2000

1294 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7

Malamas et al.

Sch em e 1^a

a

Reagents: (a) K₂CO₃, acetone; (b) H₃PO₄, xylenes; (c) 4-OCH₃-C₆H₄B(OH)₂, Pd(PPh₃)₄, Na₂CO₃, toluene; (d) n-BuLi, TMEDA, Et₂O;

(e) Br₂, KOAc, AcOH; (f) Pd(OAc)₂, K₂CO₃, 4′-OCH₃-biphenyl-4-boronic acid; (g) BBr₃, CH₂Cl₂; (h) R²-CHO or R²-CON(CH₃)OCH₃, n-BuLi,

THF; (i) NH₂NH₂, KOH; (j) NaBH₄, TFA; (k) BrCH₂CO₂CH₃, NaH (R⁵) H); HOCH(CHR⁵)CO₂CH₃, PPh₃, EtO₂CNdNCO₂Et [R⁵) alkyl,

aryl, alkyl(aryl)]; (l) NaOH, MeOH, THF; (m) ClSO₂C₆H₃R₇R₈, NaOH, THF.

ate the activated insulin receptor, attenuating the

tyrosine kinase activity. PTPases can also modulate

post-receptor signaling by catalyzing the dephosphoryl-

ation 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-PTP2.⁷

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.

In the present study, we are reporting a detailed

systematic structure-activity relationship (SAR) study

of novel benzofuran/benzothiophene biphenyls as highly

potent and orally active PTP1B inhibitors.

McGuire et al.⁸demonstrated that nondiabetic glucose

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.⁹observed 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.¹⁰observed

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 phosphoryl-

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

decrease hyperglycemia in insulin-deficient animals.¹¹

PTP1B, an intracellular nonreceptor PTPase, has

been shown to play a major role in the dephosphoryl-

ation of the insulin receptor in many cellular and

biochemical studies.¹²A recent study with PTP1B

knockout mice¹³has also demonstrated that loss of

PTP1B activity resulted in an enhancement of the

Ch em istr y

The benzofuran and benzothiophene biphenyls de-

scribed in this paper were prepared according to the

synthetic Schemes 1-3. In Scheme 1, alkylation of

phenols 1 with 4-bromophenacyl bromide 2 in the

presence of potassium carbonate produced ethanones 3,

which upon treatment with polyphosphoric acid at high

temperatures (150 °C) afforded benzofurans 4.¹⁴Cou-

pling of benzofurans 4 with commercially available

boronic acids, using the Suzuki protocol¹⁵Ph(PPh₃)₄/

Na₂CO₃, produced benzofuran biphenyls 5a (X ) O). The

analogous benzothiophene biphenyls 5b (X ) S) were

prepared by two synthetic routes. In route a, thioani-

soles 6 were first dilithiated, in the methylthio group

and in the ortho position of the benzene ring, with 2

equiv of n-butyllithium¹⁶and, second, treated with

equimolecular amounts of 4′-methoxy[1,1′-biphenyl]-4-

carbonyl chloride 7 to produce benzothiophene biphenyls

5b (X ) S). In route b, benzothiophenes 8 were first

brominated with bromine/potassium acetate to give 9

and, second, treated with 4′-methoxy[1,1′-biphenyl]-4-

Benzofuran Biophenyls as PTPase Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1295

Sch em e 2^a

a

Reagents: (a) Br₂, KOAc, AcOH; (b) aryl-boronic acid, (dppf)PdCl₂, K₂CO₃, DME.

Sch em e 3^a

a

Reagents: (a) DMF, POCl₃; (b) n-BuLi, 4′-Br-biphenyl-4-ol, THF; (c) Et₃SiH, TFA, CH₂Cl₂; (d) PCC, CH₂Cl₂; (e) BrCH₂CO₂Me, NaH,

DMF; (f) NaOH, MeOH, THF; (g) BrCH₂CN, NaH, DMF; (h) NaN₃, NH₄Cl, DMF; (i) n-BuLi, 6-Br-2-naphthol, THF; (j) Br₂, KOAc, AcOH.

ylboronic acid, Ph(PPh₃)₄, and Na₂CO₃to furnish 5b (X

) S). The phenolic compounds 10 were prepared from

5a and 5b upon demethylation with boron tribromide.

Lithiation of 5a , 5b, or 10 first with n-BuLi, followed

by subsequent addition of alkyl or aromatic aldehydes

or the analogous Weinreb amides¹⁷afforded biphenyls

11 and 12. Reduction of 11 (R³) H) with sodium

borohydride/trifluoroacetic acid¹⁸and that of 12 with

hydrazine/KOH¹⁹produced 13. When R³) CH₃in 11

and 12, boron tribromide deprotection of the methoxy

group was applied after the reduction steps, to furnish

13. Biphenyls 13 were either alkylated with methyl

bromoacetate/NaH or coupled with the required R-hy-

droxy carboxylates using the Mitsunobu protocol²⁰to

produce esters 16. Esters 16 were saponified with

sodium hydroxide to produce acids 17a . The 2-bro-

mobenzofuran biphenyl 15 was prepared from benzo-

furan 5a upon bromination with bromine (Br₂, KOAc,

AcOH) and subsequent demethylation with boron tri-

bromide. The biphenyl-4-yloxysulfonyl benzoic acids 17b

were prepared from biphenyls 13 and the corresponding

chlorosulfonyl benzoic acids in the presence of aqueous

sodium hydroxide.

In Scheme 2, biphenyls 13 were treated with bromine

and potassium acetate at low temperatures (5 °C) under

high dilution concentration (0.05 M) to give predomi-

nantly the monobromo-biphenyl 18a . Bromination of 13

with bromine at room temperature gave the dibromo-

biphenyl 18b. Intermediates 18a and 18b were coupled

with commercially available boronic acids, using the

Suzuki protocol [(dppf)PdCl₂, K₂CO₃], to produce ter-

phenyls 19. Intermediates 19 were converted to the

acids 20 according to Scheme 1. In Scheme 3, benzofu-

21

ran 21 was formylated with DMF/POCl₃to produce

22. Treatment of 4′-bromo[1,1′-biphenyl]-4-ol or 6-bromo-

2-naphthol with n-buyllthium followed by the addition

of 22 furnished benzofurans 23 and 27, respectively.

Reduction of 23 and 27 with triethylsilane/trifluroacetic

acid²²afforded 24 and 28, while oxidation with pyri-

dinium chlorochromate afforded ketones 25 and 29,

respectively. Bromination of 28 and 29 with bromine/

potassium acetate furnished naphthalenes 30a (X )

CH₂, CO). Intermediates 24, 25, and 30a were converted

to the acids 26a and 31a according to Scheme 1 and to

the tetrazoles 26b and 31b by alkylation with bromoac-

1296 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7

Malamas et al.

Sch em e 4^a

1150, and 1151 tyrosine residues; IR-tri-phosphopep-

tide) as the source of the substrate.²⁷Enzyme reaction

progression was monitored via the release of inorganic

phosphate as detected by the malachite green-am-

monium molybdate method.²⁸

The in vitro activity was expressed either as the

concentration of the test compound which inhibited

enzyme activity by 50% (IC₅₀) or the average inhibition

of the test compounds at 2.5 and 1 µM concentrations.

Samples were prepared in quadruplicates.

The test compounds were evaluated in vivo for their

ability to decrease plasma glucose and insulin levels in

the genetically obese C57 B1/6J ob/ob diabetic mouse

model. The ob/ob animal model is severely insulin

resistant, hyperinsulinemic, and glucose 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/ob mice has been

observed, which may cause or contribute to the decline

in receptor and post-receptor tyrosine phosphorylation.¹⁰

The in vivo activity was assessed at daily doses 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. Lower doses of 1 mg/kg were evaluated intrap-

eritoneally. A 30-40% (ob/ob) decrease in the plasma

glucose level generally normalizes the glucose level to

the nondiabetic control level. Ciglitazone was the refer-

ence standard in all of the assays.

a

Reagents: (a) I₂, Ce(NH₄)₂(NO₃)₆, CH₃CN; (b) 1-(2-propynyl-

)benzene, Pd(OAc)₂, CuI, n-BuNH₂; (c) Br₂, CCl₄; (d) 4′-OCH₃-

biphenyl-4-B(OH)₂, Pd(PPh₃)₄, Na₂CO₃, toluene; (e) BBr₃, CH₂Cl₂;

(f) (R)-(+)-3-phenyllactic acid methyl ester, EtO₂CNdNCO₂Et,

PPh₃, benzene; (g) NaOH, MeOH, THF.

etonitrile/NaH and subsequent conversion of the nitriles

to tetrazoles with sodium azide and ammonium chloride.

The furo[2,3-b]pyridine 36b was prepared according

to Scheme 4. 1,1′-Thiocarbonyldi-2(1H)-pyridone 33 was

treated with iodine and ammonium cerium nitrate to

afford iodo-pyridone 34.²³Palladium catalyzed coupling

of 34 with 1-(2-propynyl)benzene followed by cyclization

in the presence of cuprous iodide and n-butylamine

furnished furo[2,3-b]pyridine 35a .²⁴Intermediate 35a

was first brominated (Br₂/CCl₄) to 35b and then con-

verted to the final product 36b according to Scheme 1.

The imidazole[4,5-b]pyridine 45 and benzimidazole 46

were prepared according to Scheme 5. Treatment of

pyridine 37 with benzylamine afforded 38, which was

reduced with stannous chloride to amine 39. Cyclization

of 39 with ethyl chloroformate produced imidazo[4,5-b]-

pyridin-2-one 40, which was converted to 2-chloro-

imidazo[4,5-b]pyridine 41 upon treatment with phos-

phorus oxychloride. The analogous 2-chloro-benzimidazole

43 was prepared from benzimidazole 42 upon alkylation

with benzyl bromide. Both intermediates 41 and 43

underwent Suzuki coupling reaction with 4′-methoxy-

[1,1′-biphenyl]-4-ylboronic acid in the presence of Pd-

(PPh₃)₄and sodium carbonate to produce 44 (X ) N,

C). Intermediates 44 (X ) N, C) were converted to the

final products 45 and 46 according to Scheme 1.

The oxazole biphenyls (Table 4) were prepared ac-

cording to synthetic Scheme 6. 1-(4-Bromophenyl)-1-

propanone was first converted to oxime 48 (NH₂OH/

NaOAc), followed by treatment with 4-trifluromethyl-

benzoyl chloride and pyridine to produce oxazole 49.²⁵

Suzuki coupling of 49 with benzeneboronic acid fur-

nished biphenyl 50a , which was further converted to

the final products 51a and 51b according to Schemes 1

and 3.

In the early stage of our discovery program, we have

identified that the benzofuran nucleus substituted at

position-3 with phenolic moieties exhibited weak inhibi-

tory activity against recombinant rat PTP1B (unpub-

lished results). Modeling studies, using the X-ray crystal

structure of h-PTP1B,²⁹have suggested that placing the

phenolic group of these inhibitors deeper into the

catalytic binding pocket (catalytic sequence residues

V₂₁₃HCSAGIGR₂₂₁SG) of the enzyme would result in

tighter inhibitor-protein interactions: hydrogen bond-

ing interactions of the phenolic hydroxyl group with

Arg221. Introduction of the biphenyl-4-ol group at

position-3 of the benzofuran ring produced 52 (Table 1),

a sub-micromolar inhibitor against h-PTP1B. Masking

of the hydroxyl group of 52 resulted in a decrease of

the in vitro activity (52 vs 53). Substitutions at posi-

tion-2 of the benzofuran moiety were also crucial to the

inhibitory activity. The unsubstituted analogue 54 was

inactive at 2.5 µM, while the ethyl analogue 55 was

much weaker than 52. The benzyl and benzoyl ana-

logues 56 and 57 were similar to 52. The analogous

benzothiophene analogues 58 and 59, however, were

quite different. The butyl analogue 58 exhibited inhibi-

tory activity similar to 52, while the benzyl analogue

59 was much weaker (59 vs 56). Introduction of hy-

droxyl group(s) on the benzyl group (analogues 60, and

61) resulted in marked increases of the inhibitory

activity (60, 61, vs 59). Replacement of the phenolic

group 52 with an oxo-acetic acid functionality (62)

resulted in a 3-fold loss of activity. We expected that

the acetic acid residue would be closer and bind much

tighter to Arg221, via a charge-charge interaction, than

the phenolic group of 52. Modeling studies, however,

indicated that the oxo-acetic acid-type inhibitors did not

Resu lts a n d Discu ssion

The test compounds were evaluated for their in vitro

inhibitory activity against recombinant human²⁶PTP1B

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

(P)YRK (corresponds to the 1142-1153 insulin receptor

kinase regulatory domain, phosphorylated on the 1146,

Benzofuran Biophenyls as PTPase Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1297

Sch em e 5^a

a

Reagents: (a) C₆H₅CH₂NH₂, toluene; (b) SnCl₂‚2H₂O, EtOAc; (c) ClCO₂Et, CHCl₃; (d) POCl₃, PCl₅; (e) C₆H₅CH₂Br, NaH, DMF; (f)

4′-OCH₃-biphenyl-4-B(OH)₂, Pd(PPh₃)₄, Na₂CO₃, toluene; (g) BBr₃, CH₂Cl₂.

Sch em e 6^a

a

Reagents: (a) NH₂OH, NaOAc, EtOH; (b) 4-CF₃-C₆H₄COCl, pyridine; (c) 4-OCH₃C₆H₄B(OH)₂, Pd(PPh₃)₄, Na₂CO₃, toluene; (d) BBr₃,

CH₂Cl₂.

take advantage of a hydrophobic pocket in the vicinity

of Arg221. To test this finding, a phenyllactic moiety

replaced the oxo-acetic acid group in 62. The benzofu-

ran-phenyllactic acid analogue 63 was 5-fold more

potent than the acetic acid analogue 62, with an IC₅₀

value of 0.44 µM. Furthermore, we examined the

enantioselectivity of the phenyllactic acid-type inhibitors

against enzyme inhibition. The R- and S-enantiomers

67 and 68 were prepared and evaluated against

h-PTP1B. Both enantiomers exhibited identical intrinsic

activity that was indistinguishable from that of the

racemate. Masking of the carboxylate group of 68 as the

methyl ester (70) was found to be detrimental to the

inhibitory activity. Compound 70 was only marginally

active at 2.5 µM. Replacement of the phenyllactic group

with various aromatic or heteroaromatic groups 71-

74 produced compounds similar to 68. The lactic acid

analogue 75 was 4-fold weaker than 67, indicative of

the loss of the additional hydrophobic interactions

between the inhibitor and enzyme at the vicinity of the

catalytic site by eliminating the phenyl group (benzyl

changes to methyl) in 75. The hydroxymethyl analogue

77 was 3-fold better than 67. Additional hydrogen

bonding interactions of the hydroxymethyl group may

have contributed to the increased intrinsic activity of

77. Interestingly, the benzoic acid analogue 78 (a

combination of the carboxylic acid and phenyl groups

of 66) produced similar results with 66. Substitutions

on the benzofuran portion of the molecule had various

effects on the inhibitory activity. The fluoro analogue

90 exhibited a 3-fold increase in inhibitory activity (90

vs 68), while the methyl analogue 91 was practically

unchanged. The furo-pyridine analogue 92 was 2-fold

weaker than 68, while the benzimidazole analogues 93

and 94 were very weak h-PTP1B inhibitors. The regio-

isomeric analogue 95 of 67, where the biphenyl is

attached at position-2 of the benzofuran nucleus, dem-

onstrated similar inhibitory activity to that of 67.

The benzothiophene analogous compounds were found

to be more potent against PTP1B. Both the butyl and

benzyl analogues 79 and 80 inhibited h-PTP1B with

IC₅₀values of 0.17 and 0.09 µM, respectively. Modifica-

tions on the benzyl group, attached at position-2 of the

benzothiophene ring, produced inhibitors 82-87 with

small differences in the their intrinsic activity than the

parent compound 80. Replacement of the benzyl group

with either a thiazole 88 or a pyridine 89 moiety

resulted in marked loss of the intrinsic activity. Fur-

thermore, truncation of the phenyl portion of the ben-

zothiophene nucleus produced much weaker inhibitors.

1298 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7

Malamas et al.

Ta ble 1. Chemical and Biological Data of Benzofuran and Benzothiophene Biphenyls^b

Benzofuran Biophenyls as PTPase Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1299

Ta ble 1 (Continued)

a

b

IR-tri-phosphopeptide used as the substrate, and recombinant h-PTP1B as the enzyme. All compounds were prepared according to

the synthetic Schemes 1, 4, and 5.

F igu r e 2. Schematic representation showing compound 80

(with an additional magenta-colored phenyl ring) in the

composite volume of the catalytic site. Replacement of the

other ortho hydrogen with a phenyl ring requires removal of

the benzyl side chain from the acetic acid head group.

F igu r e 1. Schematic representation showing compound 80

and its interactions with key residues within the binding

catalytic cavity. Key hydrogen bonding interactions between

the ligand and the bound waters (WAT1 and WAT2) are shown

as yellow dotted lines.

volume of the ligands was created using the MVOLUME

procedure. This volume represents the total volume

occupied by all of the ligands determined in house.

Figure 2 shows compound 80 (with an additional

magenta-colored phenyl ring) in this composite volume.

The magenta phenyl ring, added to the position ortho

to the phenyllactic acid head piece, clearly occupies a

hydrophobic region, not reached by our initial inhibitors.

Substitution in this position was therefore predicted to

contribute to the enhancement of ligand binding affinity.

Both thiophene analogues 96 and 97 were markedly less

potent than benzothiophene 80.

To further improve the intrinsic activity of our

compounds, we felt compelled to better understand the

inhibitor-protein interactions. Compound 80 was coc-

rystallized with h-PTP1B, and the crystal structure of

the binary complex was refined at 1.85 Å resolution. The

bound compound 80 extends deep into the active-site

pocket (Figure 1), making several hydrogen bonding and

hydrophobic interactions with key residues of the cata-

lytic site. The carboxylic acid portion of the ligand does

not interact directly with Arg221 as originally expected,

but participates in hydrogen bonds with two water

molecules which bridge the ligand with Arg221. Residue

Arg221 is responsible for substrate phosphate binding.

The phenyl ring of the biphenyl, closest to the carboxylic

acid, is sandwiched between residues Tyr46 and Phe182.

The A face of the second phenyl of the biphenyl is

Exploring the findings of the X-ray studies, hydro-

phobic groups (Table 2) were introduced at the ortho

position of the phenyllactic acid pharmacophore (ter-

phenyl-type inhibitors). The monobromo analogue 103

was found to be 2-fold better than 80, and the dibromo

analogue 104 was 4-fold better as well with IC₅₀values

of 0.058 and 0.025 µM, respectively. Similar increases

of the intrinsic activity were also observed for the

phenyl-substituted analogues 105 and 106. Modeling

studies (based on the ligand-composite volume overlap,

Figure 2) also suggested that an unsubstituted acetic

acid moiety instead of the phenyllactic acid might be

required for high inhibitory activity, since the terphenyl-

type inhibitors (ortho-ortho-disubstituted analogues)

already take advantage of the hydrophobic pocket at the

vicinity of the catalytic site. In agreement with our

modeling studies, terphenyl-oxo-acetic acid analogues

112-122 were potent h-PTP1B inhibitors with IC₅₀

values in the range of 0.025 to 0.1 µM. The ortho-

+

stacked against Lys120, interacting with its NH₃

group. There are several strong van der Waals contacts

between the 2-benzyl-benzothiophene tail-portion of the

molecule and Lys116. Additionally, the benzyl group

interacts with the π face of Phe182.

The complex of compound 80 and PTP1B was further

overlapped with several other in-house PTP1B com-

plexes, and the ligands were extracted into separate

work areas using the Sybyl software.³⁰A composite

1300 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7

Malamas et al.

Ta ble 2. Chemical and Biological Data of 2-Benzyl Benzofuran and Benzothiophene Biphenyls^b

a

b

IR-tri-phosphopeptide used as the substrate, and recombinant h-PTP1B as the enzyme. All compounds were prepared according to

the synthetic Schemes 1 and 2.

Benzofuran Biophenyls as PTPase Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1301

Ta ble 3. Chemical and Biological Data of 2-Butyl Benzofuran Biphenyls^b

a

b

IR-tri-phosphopeptide used as the substrate, and recombinant h-PTP1B as the enzyme. All compounds were prepared according to

the synthetic Scheme 3.

ortho-disubstituted acetic acids (118-122) were better

than the analogous monosubstituted analogues, since

they occupied a larger hydrophobic region at the vicinity

of the catalytic site. The second ortho-substituent oc-

cupies the hydrophobic region previously occupied by

the benzyl portion of the phenyllactic head piece (Figure

2). This site is defined on one side by the loop containing

residues 215-221 (including Arg221) and on the other

by Phe182 and Asp181). These hydrophobic portions of

the active site are somewhat larger than the phenyl ring

(modeling calculations), further explaining the greater

inhibitory activity of the substituted ortho-phenyl ana-

logues than the unsubstituted analogues (i.e., 115 vs

113). Alkoxy groups may also take advantage of favor-

able hydrogen bond contacts with Arg221. The butyric

acid analogue 123 was found to be 2-fold better than

the acetic acid analogue 111. The oxo-butyric acid chain

appears to extend deeper into the binding pocket, closer

to Arg221, resulting in tighter interactions at the active

site.

Similarly, the benzofuran nucleus produced low na-

nomolar inhibitors (124-135, 141 and 142) against

h-PTP1B with IC₅₀values in the range of 0.02-0.1 µM.

The exception was the nitro and cyclopentyl analogues

127 and 134 which were weaker. Several N-carboxylic

acid analogues (136-140), substituted at the meta-

position of the terminal phenyl group, were also good

h-PTP1B inhibitors, with the glycine analogue 136 being

the most active (IC₅₀) 0.082 µM).

Introduction of a carbon spacer between the benzo-

furan and the biphenyl moiety had only a small effect

on the inhibitory activity. The methylene-spaced oxo-

acetic acid analogue 147 (Table 3) was about 2-fold

better than 62, while the hydroxymethyl-spaced ana-

logue 148 was 4-fold better than 62. The hydroxylmethyl

spacer was also better for the phenolic compound 145

(IC₅₀) 0.2 µM), which was 3-fold better than 52. The

higher inhibitory activity of the hydroxylmethyl-spaced

analogues might be the result of additional hydrogen

bonding interactions between inhibitor and enzyme.

Surprisingly, the ortho-substituted dibromo analogue

146 was weaker than 145. This finding was in contrast

to the earlier results with the ortho-disubstituted

analogues (Table 2), where a marked increase of the

inhibitory activity had been observed. Bioisosteric re-

placement of the carboxylic acid with a tetrazole moiety

produced slightly better h-PTP1B inhibitors (147 vs

149).

Replacement of the benzofuran nucleus with a phenyl-

substituted oxazole moiety produced several sub-micro-

molar inhibitors (Table 4). The dibromo-phenyllactic

analogue 159 was the best compound of this series with

an IC₅₀value of 0.13 µM. Attachment of the oxazole

group at regioisomeric positions 4′ or 3′ on the biphenyl

nucleus produced similar results (152 vs 156).

Modeling studies have also suggested that a naph-

thalene nucleus, substituted at positions 2 and 6, could

replace the biphenyl group of our inhibitors. Several

compounds were prepared (Table 5) using similar SAR

approaches developed in the previous series of h-PTP1B

inhibitors. The halogen (bromine, iodine)-substituted

phenyllactic acid analogues 167 and 169 were the best

compound of this series with IC₅₀values of 0.37 and 0.32

µM, respectively. The analogous oxazole analogue 172

was less active.

The good h-PTP1B inhibitory activity of the benzoic

acid analogue 78 (Table 1; IC₅₀) 0.36 µM) served as

the starting point for the design of PTP1B inhibitors

with similar structural framework and a distinct phar-

macophore group. Introduction of a sulfone spacer

between the biphenyl central aromatic region and the

benzoic acid resulted in a 5-fold improvement of the

intrinsic activity (174 vs 77; Table 6). The meta-

substituted analogous compound 175 was only incre-

mentally weaker (IC₅₀) 0.1 µM) than 174. Addition of

a hydroxyl group on the benzoic acid nucleus further

improved the inhibitory activity of the compounds. Both

salicylic acid analogues 176 and 177 were 2- and 4-fold

better than the corresponding benzoic acids 174 and

175, respectively. Applying similar SAR approaches that

were developed for the acetic acid-type inhibitors, we

prepared several salicylic acid-type inhibitors (178-185)

with excellent inhibitory activity against h-PTP1B in

the low nanomolar range (0.02-0.04 µM). However, the

1302 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7

Malamas et al.

Ta ble 4. Chemical and Biological Data of Substituted Oxazole Biphenyls^b

a

b

IR-tri-phosphopeptide used as the substrate, and recombinant h-PTP1B as the enzyme. All compounds were prepared according to

the synthetic Scheme 6.

Ta ble 5. Chemical and Biological Data of 2-Butyl Benzofuran Naphthalenes^b

a

b

IR-tri-phosphopeptide used as the substrate, and recombinant h-PTP1B as the enzyme. All compounds were prepared according to

the synthetic Schemes 3 and 6.

findings with the salicylic acid-type inhibitors were in

variance to the acetic acid-type inhibitors. While, ortho-

substitutions in the oxo-acetic acid series had signifi-

cantly contributed to the enhancement of the inhibitor’s

intrinsic activity, similar substitutions in the salicylic

acid series of compounds (181-183) had essentially no

effect. A different orientation of this class of inhibitors

in the active site may play a role in inhibitor-protein

interactions and explain these discrepancies. Compound

181, a member of the salicylic acid-type inhibitors, was

Benzofuran Biophenyls as PTPase Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1303

Ta ble 6. Chemical and Biological Data of Sulfono Biphenyls^b

a

b

IR-tri-phosphopeptide used as the substrate, and recombinant h-PTP1B as the enzyme. All compounds were prepared according to

the synthetic Schemes 1 and 2.

cocrystallized with h-PTP1B, and the crystal structure

of the binary complex was refined at 2.0 Å resolution.

This high-resolution structure revealed the binding

orientation of the inhibitor in the active site (Figure 3).

In contrast to the compound 80 X-ray structure findings,

the carboxylic acid of compound 181 makes direct

hydrogen bond interactions with the protein at residues

Lys120 and Tyr46 and interacts only with one of the

two water molecules that interact with Arg221. The

aromatic portion of the salicylic acid moiety is sand-

wiched between residues Tyr46 and Phe182. The lipo-

philic 2-benzyl-benzothiophene tail-piece of the molecule

forms nonspecific van der Waals interactions with the

protein. For compound 181, even though its pharma-

cophore portion of the molecule (salicylic acid) occupies

similar space in the active site as the acetic acid-type

inhibitors, the inhibitor orientation is almost opposite

to the acetic acid-type inhibitors. Ortho-aromatic sub-

stitutents to the sulfonyl-salicylic acid moiety are not

accessing the same hydrophobic region as had been

previously observed with the similarly substituted acetic

acid analogues (Figure 4).

The different orientations of the acetic acid-type (80)

and the salicylic acid (181) ligands in the X-ray crystal

structure with PTP1B are shown in Figure 5. Compound

80 was merged with the X-ray crystal structure of

compound 181, and only protein atoms 10 Å from either

of the ligands were displayed. A MOLCAD surface³¹was

computed for this portion of the protein and was color

coded by the lipophilicity of the underlying residues

(brown being very lipophilic, blue and green hydro-

philic). The acidic pharmacophore portions of the mol-

ecules orient to the catalytic site of the protein, while

the lipophilic tail-portions of the molecules occupy

opposite regions of the protein.

1304 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7

Malamas et al.

F igu r e 5. Schematic representation showing compound 80

(red) and 181 (blue) merged with PTP1B (catalytic region) in

a MOLCAD surface in which brown represents very lipophilic

and blue/green represents hydrophilic environments. Only

protein atoms 10 Å from either of the ligands are displayed.

F igu r e 3. Schematic representation showing compound 181

and its interactions with key residues within the binding

catalytic cavity.

Ta ble 7. In Vivo Data of Selected Compounds

F igu r e 4. Schematic representation showing compound 181

(with an additional magenta-colored phenyl ring) in the

composite volume of the catalytic site. This view highlights

the difference in ligand orientation of 181 versus those found

for the previously studied ligands.

The compounds were also evaluated in vivo for their

ability to decrease plasma glucose and insulin levels in

the genetically obese C57 B1/6J ob/ob diabetic mouse

model. Initially, the compounds were evaluated at 100

mg/kg dose (po) for 4 days, where the majority among

them normalized plasma glucose levels (Table 7). Sub-

sequently, as our compounds had demonstrated anti-

hyperglycemic properties and a proof-of-concept had

been established, we used lower, 10 and 25 mg/kg, oral

doses for in vivo screening. Several compounds signifi-

cantly decreased plasma glucose levels at the 25 mg/kg

dose, while all the compounds tested at 10 mg/kg dose

were either marginally active or inactive (data not

shown). Compound 68 was one of the most active in vivo

compounds, lowering glucose levels by 27% at the oral

dose of 25 mg/kg and normalizing glucose levels at the

intraperitoneal dose of 1 mg/kg (Table 7). The lower oral

potency of 68 may due to the low absorption of the

compound. Compound 68 exhibited mean plasma con-

a

Values are percent change relative to vehicle-treated group

with use of four to six mice per group; *p < 0.05 when compared

to vehicle-treated mice. ^bNot tested. ^cNS not significant, generally

d

less than -15% change. Mean of 38 experiments, p < 0.05.

centration (AUC_0-∞) 32 µg/mL) after a single 10 mg/

kg oral dose in mice, in comparison to a 1 mg/kg

intraperitoneal dose, with an AUC_0-∞value of 54 µg/

mL. The half-life (t_1/2) of 68 was found to be 6 to 8 h in

mice.

The protein tyrosine phosphatase domains of receptor

and nonreceptor PTPases are highly conserved with

∼35% mean sequence identity between known phos-

phatases.³²Therefore, it is essential that PTP1B inhibi-

tors intended for chronic therapy demonstrate a high

level of selectivity. The selectivity of compound 68 was

Benzofuran Biophenyls as PTPase Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1305

Ta ble 8. h-PTP1B Specificity of Compound 68

commercially and used without further purification. All reac-

tions were carried out under an atmosphere of dried nitrogen.

Gen er a l P r oced u r e for th e Syn th esis of th e Ben zofu -

r a n a n d Ben zoth iop h en e Bip h en yls. All 4,4′-substituted

biphenyl compounds were synthesized according to the fol-

lowing representative procedures.

a

PTPases implicated in the development of Type 2 diabetes.⁷

3-(4-Br om op h en yl)-1-ben zofu r a n (4, R¹) H). Potassium

carbonate (24.8 g, 179.8 mmol) was added into a mixture of

4-bromophenacyl bromide (50.0 g, 179.8 mmol), phenol (16.9

g, 179.8 mmol), and dry acetone (200 mL). The reaction

mixture was refluxed for 12 h, cooled to room temperature,

poured into water, and extracted with ethyl ether. The organic

extracts were dried over MgSO₄. Evaporation gave ω-phenoxy-

4-bromoacetophenone as a yellow solid (49.6 g). A mixture of

ω-phenoxy-4-bromoacetophenone (49.0 g, 167.8 mmol), poly-

phosphoric acid (100 g), and xylenes (300 mL) was refluxed

for 12 h. The reaction mixture was cooled to room temperature,

poured into water, and extracted with ethyl ether. The organic

extracts were dried over MgSO₄. Evaporation and purification

by flash chromatography on silica gel (hexanes/EtAOc 40:1)

gave 3-(4-bromophenyl)-1-benzofuran as a yellow solid (36.2

g, 74% yield): mp 70-71 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ

7.35-7.4 (m, 2H, Ar-H), 7.62-7.7 (m, 5H, Ar-H), 7.85-7.9 (m,

1H, Ar-H), 8.4 (s, 1H, CH); MS m/e 272 (M⁺). Anal. (C₁₄H₉-

BrO) C, H.

3-(4′-Meth oxy-bip h en yl-4-yl)-ben zofu r a n (5a , R¹) H,

X ) O). 4-Methoxy-benzeneboronic acid (14.17 g, 70.5 mmol)

in ethyl alcohol (10 mL) was added into a mixture of 3-(4-

bromophenyl)-1-benzofuran (17.5 g, 64.1 mmol), sodium car-

bonate (2 N, 64.1 mL), tetrakis(triphenylphosphine)palladium-

(0) (2.23 g, 1.92 mmol), and toluene (200 mL). The reaction

mixture was refluxed for 12 h, cooled to room temperature,

and treated with hydrogen peroxide (30%, 5 mL) for 1 h. The

mixture was poured into water and extracted with ethyl

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

ration and crystallization from acetone/ethyl ether gave 3-(4′-

methoxy-biphenyl-4-yl)-benzofuran as a white solid (14.9 g,

77% yield): mp 137-138 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ

3.79 (S, 3H, OCH₃), 7.05 (d, J ) 8.78 Hz, 2H, Ar-H), 7.36-

7.42 (m, 2H, Ar-H), 7.62-7.64 (m, 3H, Ar-H), 7.74 (d, J ) 8.56

Hz, 2H, Ar-H), 7.8 (d, J ) 8.56 Hz, 2H), Ar-H), 7.94-7.98 (m,

1H, Ar-H), 8.4 (s, 1H, CH); MS m/e 300 (M⁺). Anal. (C₂₁H₁₆O₂)

C, H.

4′-Ben zofu r a n -3-yl-bip h en yl-4-ol (10, R¹) H, X ) O).

Boron tribromide (1.0 M, 6.67 mL, 6.67 mmol) was added

dropwise into a cold (-78 °C) mixture of 3-(4′-methoxy-

biphenyl-4-yl)-benzofuran (2.0 g, 6.67 mmol) and dichlo-

romethane (25 mL). The reaction mixture was gradually

allowed to come to room temperature and stirred for 10 h. The

mixture was cooled to 0 °C, and methyl alcohol (5 mL) was

added dropwise. After being stirred for 10 min, the mixture

was poured into water and extracted with ethyl ether. The

organic extracts were dried over MgSO₄. Evaporation and

crystallization from ethyl ether/hexanes gave 4′-benzofuran-

3-yl-biphenyl-4-ol as a yellow solid (1.69 g, 87% yield): mp

174-175 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 6.87 (d, J ) 8.78

Hz, 2H, Ar-H), 7.34-7.41 (m, 2H, Ar-H), 7.56 (d, J ) 8.78,

2H, Ar-H), 7.66 (m, 1H, Ar-H), 7.71 (d, J ) 8.57 Hz, 2H, Ar-

H), 7.8 (d, J ) 8.57 Hz, 2H), Ar-H), 7.94-7.96 (m, 1H, Ar-H),

8.39 (s, 1H, CH), 9.58 (S, 1H, OH); IR (KBr, cm^-1) 3350 (OH);

MS m/e 286 (M⁺). Anal. (C₂₀H₁₄O₂) C, H.

evaluated in vitro against several PTPases (Table 8)

using the triphosphorylated insulin receptor peptide as

the substrate.⁷Compound 68 demonstrated 10 to 100-

fold selectivity against the tested PTPases. Compound

68 was least selective (10-fold) over LAR, a PTPase also

implicated as a negative regulator of the insulin receptor

and development of Type 2 diabetes.

In summary we have identified two novel series of

benzofuran/benzothiophene biphenyl oxo-acetic acids

and sulfonyl-salicylic acids as potent inhibitors of

PTP1B with good oral antihyperglycemic activity. To

assist in the design of these inhibitors, crystallographic

studies have attempted to identify enzyme inhibitor

interactions. Resolution of crystal complexes has sug-

gested that the inhibitors bind to the enzyme active site

and are held in place through hydrogen bonding and

van der Waals interactions formed within two hydro-

phobic pockets. In the oxo-acetic acid series, hydrophobic

substitutents at position-2 of the benzofuran/ben-

zothiophene biphenyl framework interacted with Phe182

of the catalytic site and were very critical for the

intrinsic activity of the molecule. The hydrophobic

region of the active-site pocket was exploited and taken

advantage of by hydrophobic substituents at either the

R-carbon or the ortho aromatic positions of the oxo-acetic

acid moiety. Similar ortho aromatic substitutions on the

salicylic acid-type inhibitors had no effect, primarily due

to the different orientation of these inhibitors in the

catalytic site. The most active inhibitors of both series

inhibited recombinant human PTP1B with phosphoty-

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

source of the substrate with IC₅₀values in the range of

20-50 nM. Compound 68 was one of the most active

compounds in vivo, normalizing plasma glucose levels

at an oral dose of 25 mg/kg (po) and 1 mg/kg (ip).

Compound 68 was also selective against several other

PTPases.

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. Optical rotations

were determined in the cited solvent on a Perkin-Elmer model

241 MC polarimeter. All products, unless otherwise noted,

were purified by “flash chromatography” with use of 220-400

mesh silica gel. Thin-layer chromatography 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

[3-(4′-Hyd r oxy-bip h en yl-4-yl)-ben zofu r a n -2-yl]-p h en yl-

m eth a n on e (12, R¹, R³) H, R²) P h , X ) O). n-Butyllithium

(2.5 N, 8.4 mL, 20.98 mmol) was added dropwise into a cold

(-78 °C) mixture of 4′-benzofuran-3-yl-biphenyl-4-ol (3.0 g,

10.49 mmol) and tetrahydrofuran (50 mL). The mixture was

gradually allowed to warm to -40 °C and stirred for 3 h.

N-Methoxy N-methyl benzamide (1.6 mL, 10.49 mmol) was

added dropwise into the mixture. The new reaction mixture

was allowed to warm to 0 °C and stirred for 30 min. The

reaction was quenched with aqueous ammonium chloride,

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

ethyl acetate. The organic extracts were dried over MgSO₄.

1306 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7

Malamas et al.

Evaporation and crystallization from ethyl ether/hexanes gave

[3-(4′-hydroxy-biphenyl-4-yl)-benzofuran-2-yl]-phenyl-metha-

none as a yellow solid (2.9 g, 71% yield): mp 231-232 °C; ¹H

NMR (DMSO-d₆, 400 MHz) δ 6.87 (d, J ) 8.57 Hz, 2H, Ar-H),

7.44-7.46 (m, 3H, Ar-H), 7.51-7.64 (m, 8H, Ar-H), 7.78-7.87

(m, 4H, Ar-H), 9.6 (s, 1H, OH); IR (KBr, cm^-1) 3350 (OH), 1650

(CO); MS m/e 390 (M⁺). Anal. (C₂₇H₁₈O₃) C, H.

4′-(2-Ben zyl-ben zofu r a n -3-yl)-bip h en yl-4-ol (13, R¹) H,

R²) P h , X ) O). Hydrazine monohydrate (1.38 g, 27.68 mmol)

was added into a mixture of [3-(4′-hydroxy-biphenyl-4-yl)-

benzofuran-2-yl]-phenyl-methanone (2.7 g, 6.92 mmol) and

diethylene glycol (20 mL). The reaction mixture was stirred

at 180 °C for 1 h. The mixture cooled to room temperature,

and potassium hydroxide (1.16 g, 20.76 mmol) was gradually

added. The mixture was stirred at 130 °C for 10 h, cooled to

room temperature, poured into water, and extracted with ethyl

ether. The organic extracts were dried over MgSO₄. Evapora-

tion and crystallization from ethyl ether/hexanes/acetone gave

4′-(2-benzyl-benzofuran-3-yl)-biphenyl-4-ol as a white solid

(2.45 g, 94% yield): mp 151-153 °C; ¹H NMR (DMSO-d₆, 400

MHz) δ 4.26 (s, 2H, CH₂), 6.87 (d, J ) 8.78 Hz, 2H, Ar-H),

7.2-7.36 (m, 7H, Ar-H), 7.52-7.62 (m, 6H, Ar-H), 7.75 (d, J

) 8.57 Hz, 2H, Ar-H)9.58 (s, 1H, OH); IR (KBr, cm^-1) 3360

(OH); MS m/e 376 (M⁺). Anal. (C₂₇H₂₀O₂) C, H.

(2R)-2-{4′-[2-(H yd r oxy-p h en yl-m et h yl)-b en zofu r a n -3-

yl)-bip h en yl-4-yloxy]-3-p h en yl-p r op ion ic Acid (74). So-

dium borohydride (0.15 g, 4.06 mmol) was added portionwise

into a cold (0 °C) mixture of (2R)-2-[4′-(2-benzoyl-benzofuran-

3-yl)-biphenyl-4-yloxy]-3-phenyl-propionic acid methyl ester

(1.5, 2.7 mmol), methyl alcohol (20 mL), and tetrahydrofuran

(5 mL). The reaction mixture was allowed to come to room

temperature and stirred for 30 min. The mixture was then

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

ethyl acetate. The organic extracts were dried over MgSO₄.

Evaporation gave a yellow oil (1.4 g), which was taken in

methyl alcohol (20 mL) and tetrahydrofuran (20 mL) and

treated with sodium hydroxide (2.5 N, 5.0 mL) for 30 min. The

mixture was poured into water, acidified with HCL (2 N), and

extracted with ethyl ether. The organic extracts were dried

over MgSO₄. Evaporation and purification by flash chroma-

tography on silica gel (hexanes/EtAOc 5:1) gave an off-white

1

solid (1.16 g, 79% yield): mp 95-97 °C; H NMR (DMSO-d₆,

400 MHz) δ 3.2-3.3 (m, 2H, CH₂), 5.05 (s, 2H, CH₂), 5.4 (m,

1H, CH), 5.9 (s, 1H, OH), 6.96 (m, 2H, Ar-H), 7.2-7.38 (m,

12H, Ar-H), 7.57-7.7 (m, 6H, Ar-H), 7.76 (d, J ) 8.35, 2H,

Ar-H); IR (KBr, cm^-1) 3400-2700 (CO₂H), 1740 (CO); MS m/e

539 (M - H)⁺; Anal. (C₃₆H₂₈O₅) C, H.

3-(4′-Meth oxy-bip h en yl-4-yl)-ben zo[b]th iop h en e (5b,

R¹) H, X ) S). (Rou te a ). n-Butyllithium (99.9 mL, 249.8

mmol) was added dropwise into a cold (-20 °C) mixture of

thioanisole (13.98 mL, 119.3 mmol), tetramethylenediamine

(18.8 mL, 124.9 mmol), and ethyl ether (300 mL). The mixture

was allowed to come to room temperature and stirred for 12

h. Then, the mixture was cooled to -78 °C, and 4′-methoxy-

[1,1′-biphenyl]-4-carbonyl chloride (28.0 g, 113.6 mmol) in ethyl

ether (50 mL) was added over a 30 min period. The new

mixture was stirred at -78 °C for 2 h and then was allowed

to come to room temperature and stirred for an additional 20

h. The reaction was quenched with aqueous ammonium

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

ether. The organic extracts were dried over MgSO₄. Evapora-

tion gave a brown oil, which was dissolved in benzene (200

mL). Camphorsulfonic acid (2 g) was added. The mixture was

refluxed for 12 h, poured into water, and extracted with ethyl

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

ration and crystallization of the residue from ethyl ether/

acetone gave 3-(4′-methoxy-biphenyl-4-yl)-benzo[b]thiophene

(2S)-2-[4′-(2-Ben zyl-ben zofu r an -3-yl)-biph en yl-4-yloxy]-

3-p h en yl-p r op ion ic Acid Meth yl Ester (16, R ¹) H, R²

)

P h , R⁵) CH₂P h , X ) O). Diethylazodicarboxylate (1.67 mL,

10.64 mmol) in benzene (20 mL) was added dropwise into a

cold (0 °C) mixture of 4′-(2-benzyl-bnzofuran-3-yl)-biphenyl-

4-ol (2.0 g, 5.32 mmol), (R)-(+)-3-phenyllactic acid methyl ester

(1.91 g, 10.64 mmol), triphenylphosphine (2.8 g, 10.64 mmol),

and benzene (50 mL). The reaction mixture was stirred at room

temperature for 30 min, poured into water, and extracted with

ethyl ether. The organic extracts were dried over MgSO₄.

Evaporation and purification by flash chromatography on silica

gel (hexanes/EtAOc 8:1) gave (2S)-2-[4′-(2-benzyl-benzofuran-

3-yl)-biphenyl-4-yloxy]-3-phenyl-propionic acid methyl ester as

a yellow oil (2.56 g, 89% yield): ¹H NMR (DMSO-d₆, 400 MHz)

δ 3.2 (m, 2H, CH₂), 3.65 (s, 3H, CO₂CH₃), 4.26 (s, 2H, CH₂),

5.17 (m, 1H, CH), 6.97 (d, J ) 8.78, 2H, Ar-H), 7.2-7.37 (m,

12H, Ar-H), 7.57-7.71 (m, 6H, Ar-H), 7.76 (d, J ) 8.57, 2H,

Ar-H); IR (KBr, cm^-1) 1750 (CO); MS m/e 538 (M⁺). Anal.

(C₃₇H₃₀O₄) C, H.

1

as an off-white solid (7.8 g, 23% yield): mp 134-136 °C; H

(2S)-2-[4′-(2-Ben zyl-ben zofu r an -3-yl)-biph en yl-4-yloxy]-

3-p h en yl-p r op ion ic Acid (68). Sodium hydroxide (2.5 N, 10

mL) was added into a mixture of (2S)-2-[4′-(2-benzyl-benzo-

furan-3-yl)-biphenyl-4-yloxy]-3-phenyl-propionic acid methyl

ester (2.5 g, 4.65 mmol), methyl alcohol (40 mL), and tetrahy-

drofuran (40 mL). The reaction mixture was stirred for 1 h,

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

ethyl ether. The organic extracts were dried over MgSO₄.

Evaporation and crystallization from ethyl ether/hexanes gave

(2S)-2-[4′-(2-benzyl-benzofuran-3-yl)-biphenyl-4-yloxy]-3-phen-

yl-propionic acid as a white solid (2.32 g, 95% yield): mp 167-

169 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 3.2-3.3 (m, 2H, CH₂),

3.65 (s, 3H, CO₂CH₃), 4.26 (s, 2H, CH₂), 5.01 (m, 1H, CH), 6.96

(d, J ) 8.57, 2H, Ar-H), 7.2-7.38 (m, 12H, Ar-H), 7.57-7.7

NMR (DMSO-d₆, 400 MHz) δ 3.8 (s, 3H, OCH₃), 7.06 (d, J )

8.78 Hz, 2H, Ar-H), 7.4-7.42 (m, 2H, Ar-H), 7.64-7.67 (m,

4H, Ar-H), 7.77 (d, J ) 8.35 Hz, 2H, Ar-H), 7.85 (s, 1H, CH),

7.94 (m, 1H, Ar-H), 8.09 (m, 1H, Ar-H); MS m/e 316 (M⁺). Anal.

(C₂₁H₁₆OS) C, H.

(Rou te b). Palladium(II) acetate (2.9 mg, 0.01 mmol) was

added into a mixture of 3-bromo-benzo[b]-thiophene (1.4 g, 6.58

mmol), 4′-methoxy-biphenyl-4-boronic acid (1.5 g, 6.58 mmol),

potassium carbonate (2.27 g, 16.45 mmol), acetone (20 mL),

and H₂O (20 mL). The reaction mixture was stirred at 65 °C

for 2 h, poured into water, and extracted with ethyl acetate.

The organic extracts were dried over MgSO₄. Evaporation and

crystallization from ethyl ether/hexanes gave 3-(4′-methoxy-

biphenyl-4-yl)-benzo[b]thiophene as an off-white solid (1.76 g,

85% yield).

4′-(2-Ben zyl-ben zo[b]th iop h en -3-yl)-3-br om o-bip h en yl-

4-ol; 4′-(2-Ben zyl-ben zo[b]th iop h en -3-yl)-3,5-d ibr om o-bi-

p h en yl-4-ol [18a R²) H, 18b (R²) Br ), R¹) H, X ) O].

Bromine (1.47 mL, 28.7 mmol) in acetic acid (50 mL) was

added dropwise over a 30 min period into a cold (5 °C) mixture

of 4′-(2-benzyl-benzo[b]thiophen-3-yl)-biphenyl-4-ol (7.5 g, 19.1

mmol), potassium acetate (18.6 g, 190.1 mmol), and acetic acid

(300 mL). After the addition, the mixture was stirred for 10

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

organic extracts were washed with aqueous sodium bisulfite

and dried over MgSO₄. Evaporation and purification by flash

chromatography on silica gel (hexanes/EtAOc/CH₂Cl₂3:1:1)

gave 4′-(2-benzyl-benzo[b]thiophen-3-yl)-3-bromo-biphenyl-4-

ol as a light yellow solid (4.7 g): mp 54-56 °C; ¹H NMR

(DMSO-d₆, 400 MHz) δ 4.22 (s, 2H, CH₂), 7.09 (s, 1H, 8.35

(1H, Ar-H), 7.2-7.24 (m, 3H, Ar-H), 7.3-7.4 (m, 4H, Ar-H),

(m, 6H, Ar-H), 7.76 (d, J ) 8.35, 2H, Ar-H); IR (KBr, cm^-1

)

3500-2700 (CO₂H), 1745 (CO); MS m/e 524 (M⁺); [R]²⁵

-13.4 (1.0, THF). Anal. (C₃₆H₂₈O₄) C, H.

)

D

(2S)-2-[4′-(2-Ben zyl-ben zofu r an -3-yl)-biph en yl-4-yloxy]-

3-ph en yl-pr opion ic Acid Tr om eth am in e Salt (68,Tr om eth -

a m in e Sa lt). A mixture of (2S)-2-[4′-(2-benzyl-benzofuran-3-

yl)-biphenyl-4-yloxy]-3-phenyl-propionic acid (1.0 g, 1.91 mmol),

tromethamine (0.23 g, 1.91 mmol), tetrahydrofuran (10 mL),

and water (1.0 mL) was stirred at 60 °C for 1 h. The volatiles

were removed in vacuo, and the residue was washed with

water and dried to give a white solid (1.1 g, 89% yield): mp

1

147-148 °C; H NMR (DMSO-d₆, 400 MHz) δ 3-3.1 (m, 1H,

CH₂), 3.2-3.23 (m, 1H, CH₂), 4.26 (s, 2H, CH₂), 4.48 (m, 1H,

CH), 6.88 (d, J ) 8.78, 2H, Ar-H), 7.17-7.39 (m, 12H, Ar-H),

7.57-7.7 (m, 6H, Ar-H), 7.76 (d, J ) 8.35, 2H, Ar-H); MS m/e

523 (M - H)⁺. Anal. (C₃₆H₂₇O₄‚tromethamine‚1.5H₂O) C, H,

N.

Benzofuran Biophenyls as PTPase Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1307

7.5-7.56 (m, 3H, Ar-H), 7.6 (dd, J ) 8.57, 2.42, 1H, Ar-H), 7.8

(d, J ) 8.57 Hz, 2H, Ar-H), 7.88 (d, J ) 2.42 Hz, 1H, Ar-H),

7.93 (m, 1H, Ar-H), 10.45 (s, 1H, OH); IR (KBr, cm^-1) 3440

(OH); MS m/e 477 (M⁺). Anal. (C₂₇H₁₉BrOS) C, H. 4′-(2-Benzyl-

benzo[b]thiophen-3-yl)-3,5-dibromo-biphenyl-4-ol was also ob-

basic), diluted with EtOAc (350 mL), stirred overnight, and

filtered. The biphasic filtrate was separated, and the aqueous

phase was extracted with ethyl acetate. The combined organic

extracts were dried over MgSO₄. Evaporation and purification

by flash chromatography on silica gel (EtOAc/petroleum ether,

1/1) gave benzyl-pyridine-2,3-diamine as a light red solid: mp

1

tained as a light yellow solid (1.4 g): mp 59-61 °C; H NMR

1

(DMSO-d₆, 400 MHz) δ 4.21 (s, 2H, CH₂), 7.18-7.22 (m, 3H,

Ar-H), 7.3-7.4 (m, 4H, Ar-H), 7.45 (m, 1H, Ar-H), 7.52 (d, J )

8.35 Hz, 2H, Ar-H), 7.84 (d, J ) 8.35 (, 2H, Ar-H), 7.91-7.93

(m, 1H, Ar-H), 7.94 (S, 2H, Ar-H), 10.12 (s, 1H, OH); IR (KBr,

cm^-1) 3470 (OH); MS m/e 548(M⁺). Anal. (C₂₇H₁₈Br₂OS) C, H.

88 °C; H NMR (DMSO-d₆, 400 MHz) δ 4.55 (d, J ) 5.93 Hz,

2H, CH₂), 4.73 (brs, 2H, NH₂), 6.02 (t, J ) 5.93 Hz, 1H, NH),

6.35 (m, 1H, Ar-H), 6.67 (m, 1H, Ar-H), 7.2 (m, 1H, Ar-h),

7.23-7.33 (m, 5H, Ar-H); IR (KBr, cm^-1) 3400 (NH); MS m/e

200 (M+H)⁺. Anal. (C₁₂H₁₃N₃) C, H, N.

4,4′′-Dim eth oxy-5′-{4-[2-(p h en ylm eth yl)ben zo[b]th ien -

3-Ben zyl-1,3-d ih yd r o-im a d a zo[4,5-b]p yr id in -2-on e (40).

Ethyl chloroformate (3.34 g, 30.7 mmol) was added to a

solution of benzyl-pyridine-2,3-diamine (2.78 g, 14.0 mmol) in

chloroform (70 mL), and the mixture was refluxed for 1.5 h.

The reaction mixture was washed with aqueous NaHCO₃and

water, and evaporated to dryness. The residue was purified

by flash chromatography on silica gel (EtOAc/chloroform, 1/1)

to give a brown oil, which was dissolved in absolute ethanol

(15 mL) and added into a solution of sodium ethoxide (10

mmol) in absolute ethanol (15 mL). The mixture was refluxed

for 3 h and concentrated in vacuo, and the residue was diluted

with water, neutralized with HCl (2 N), and extracted with

ethyl acetate. The extracts were washed with water and dried

over MgSO₄. Evaporation and purification by flash chroma-

tography (EtOAc/petroleum ether, 1/3) gave 3-benzyl-1,3-

dihydro-imadazo[4,5-b]pyridin-2-one as an orange solid: mp

172 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 5.02 (s, 2H, CH2),

7.02 (m, 1H, Ar-H), 7.25 (m, 1H, Ar-H), 7.33 (m, 5H, Ar-H),

7.94 (m, 1H, Ar-H), 11.2 (brs, 1H, NH); IR (KBr, cm^-1) 1710

(CO); MS m/e 225 (M + H)⁺. Anal. (C₁₃H₁₁N₃O) C, H, N.

3-Ben zyl-2-(4′-m eth oxy-bip h en yl-4-yl)-3H-im a d a zo[4,5-

b]p yr id in e (44, X ) N). Phosphorus pentachloride (0.92 g,

4.44 mmol) was added into a refluxing suspension of 3-benzyl-

1,3-dihydro-imadazo[4,5-b]pyridin-2-one (1.0 g, 4.44 mmol) in

phosporus oxychloride (15 mL). The mixture was refluxed for

12 h. The solvent was then removed in vacuo, and the residue

was treated with water and basified with sodium hydroxide

(5 N) under external cooling. The solution was extracted with

ethyl acetate, washed with brine, and dried over MgSO₄.

Evaporation and purification by flash chromatography (EtOAc/

petroleum ether 1/3) gave a yellow solid (423 mg). The

haloimidazopyridine (0.423 g, 1.74 mmol) and tetrakis(tri-

phenylphosphine)palladium(0) (100 mg, 0.09 mmol) were

dissolved in the 1,2-dimethoxyethane (5 mL) and stirred at

room temperature for 10 min. 4′-methoxy-biphenyl-4-boronic

acid (0.61 g, 1.91 mmol) was then added, followed by aqueous

sodium carbonate (2 M, 3.5 mL). The mixture was refluxed

for 12 h, diluted with water, and extracted with dichlo-

romethane. The organic extracts were dried over MgSO₄.

Evaporation and purification by flash chromatography (EtOAc/

dichloromethane 1/10) gave 3-benzyl-2-(4′-methoxy-biphenyl-

4-yl)-3H-imadazo[4,5-b]pyridine as a white solid: mp 159 °C;

¹H NMR (DMSO-d₆, 400 MHz) δ 3.81 (s, 3H, OCH₃), 5.69 (s,

2H, CH₂), 7.02 (m, 4H, Ar-H), 7.21-7.26 (m, 3H, Ar-H), 7.46

(m, 1H, Ar-H), 7.7-7.8 (m, 7H, Ar-H), 8.5 (d, J ) 6.15 Hz, 1H,

Ar-H); MS m/e 391 (M + H)⁺. Anal. (C₂₆H₂₁N₃O) C, H, N.

3-yl]p h en yl}-[1,1′;3′,1′′-ter p h en yl]-2′-ol (19, R¹) H, R³

)

OCH₃, R⁴) 4-OCH₃-P h , X ) O). Palladium(II) acetate (81

mg, 0.036 mmol) was added into a mixture of 4′-(2-benzyl-

benzo[b]thiophen-3-yl)-3,5-dibromo-biphenyl-4-ol (1.0 g, 1.82

mmol), 4-methoxy-benzeneboronic acid, barium hydroxide

(0.93 g, 5.46 mmol), 1,2-dimethoxyethane (10 mL), and water

(10 mL). The mixture was stirred at 75 °C for 10 h, poured

into water, and extracted with ethyl ether. The organic

extracts were dried over MgSO₄. Evaporation and purification

by flash chromatography on silica gel (hexanes/EtAOc 4:1)

gave 4,4′′-dimethoxy-5′-{4-[2-(phenylmethyl)benzo[b]thien-3-

yl]phenyl}[1,1′;3′,1′′-terphenyl]-2′-ol as a yellow solid (0.45 g,

1

41% yield): mp 80-82 °C; H NMR (DMSO-d₆, 400 MHz) δ

3.82 (s, 6H, OCH₃, OCH₃), 4.23 (s, 2H, CH₂), 7.04 (d, J ) 8.78

Hz, 4H, Ar-H), 7.2-7.24 (m, 3H, Ar-H), 7.3-7.4 (m, 4H, Ar-

H), 7.5-7.56 (m, 5H, Ar-H), 7.61 (d, J ) 8.78 Hz, 2H, Ar-H),

7.9-7.97 (m, 3H, Ar-H), 8.32 (s, 1H, OH); IR (KBr, cm^-1) 3420

(OH); MS m/e 604 (M⁺). Anal. (C₄₁H₃₂O₃S) C, H.

[(4,4′′-Dim eth oxy-5′-{2-(p h en ylm eth yl)ben zo[b]th ien -

3-yl]p h en yl}-[1,1′;3′,1′′-ter p h en yl]-2′-yl)oxy]-a cetic Acid

(121). Methyl bromoacetate (0.16 mL, 1.66 mmol) was added

dropwise into a mixture of 4,4′′-dimethoxy-5′-{4-[2-(phenyl-

methyl)benzo[b]thien-3-yl]phenyl}[1,1′;3′,1′′-terphenyl]-2′-ol (1.0

g, 1.66 mmol), potassium carbonate (0.23 g, 1.66 mmol), and

N,N-dimethylformamide (10 mL). The reaction mixture was

stirred at 75 °C for 2 h, poured into water, and extracted with

ethyl acetate. The organic extracts were dried over MgSO₄.

Evaporation and purification by flash chromatography on silica

gel (hexanes/EtAOc 4:1) gave a yellow oil (1.05 g). The crude

product was taken in methyl alcohol (10 mL) and tetrahydro-

furan (10 mL) and treated with NaOH (2.5 N 3.0 mL) for 30

min. Then, the reaction mixture was poured into water,

acidified with HCl (2 N), and extracted with ethyl ether. The

organic extracts were dried over MgSO₄. Evaporation and

purification by flash chromatography on silica gel (hexanes/

EtAOc 3:1) gave [(4,4′′-mimethoxy-5′-{2-(phenylmethyl)benzo-

[b]thien-3-yl]phenyl}-[1,1′;3′,1′′-terphenyl]-2′-yl)oxy]-acetic acid

1

as a white solid (0.79 g, 72% yield): mp 99-101 °C; H NMR

(DMSO-d₆, 400 MHz) δ 3.82 (s, 8H, OCH₃, OCH₃, CH₂), 4.23

(s, 2H, CH₂), 7.04 (d, J ) 8.78 Hz, 4H, Ar-H), 7.2-7.24 (m,

3H, Ar-H), 7.3-7.4 (m, 4H, Ar-H), 7.5-7.56 (m, 3H, Ar-H),

7.62-7.68 (m, 6H, Ar-H), 7.9-7.97 (m, 3H, Ar-H), 12.6 (brs,

1H, CO₂H); IR (KBr, cm^-1) 3400-2700 (CO₂H), 1740 (CO); MS

m/e 662 (M⁺). Anal. (C₄₃H₃₄O₅S) C, H.

Ben zyl-(3-n itr o-p yr id in -2-yl)-a m in e (38). To a stirred

solution of 2-chloro-3-nitropyridine (10 g, 63.1 mmol) in toluene

(100 mL) was added in one portion benzylamine (13.5 g, 126

mmol), and the mixture refluxed overnight. The reaction was

cooled to room temperature and filtered. The solvent was

evaporated, and the residue was purified by flash chromatog-

raphy (EtOAc/petroleum ether 1/10) to give benzyl-(3-nitro-

pyridin-2-yl)-amine as a yellow solid: mp 78 °C: ¹H NMR

(DMSO-d₆, 400 MHz) δ 4.8 (d, J ) 5.93 Hz, 2H, CH₂), 6.78

(m, 1H, Ar-H), 7.2-7.23 (m, 1H, Ar-H), 7.3-7.4 (m, 4H, Ar-

H), 8.44 (m, 2H, Ar-H), 8.95 (t, J ) 6.93 Hz, 1H, NH); IR (KBr,

cm^-1) 3400 (NH); MS m/e 229 (M⁺). Anal. (C₁₂H₁₁N₃O₂) C, H,

N.

2-Ben zylfu r o[2,3-b]p yr id in e (35a ). Iodine (7.6 g, 69.3

mmol) in acetonitrile (300 mL) was added dropwise into a

suspension of 1,1-thiocarbonyldi-2(1H)-pyridone (16.6 g, 69.3

mmol), ammonium cerium(IV) nitrate (28.5 g, 51.97 mmol) and

acetonitrile (700 mL). The reaction mixture was stirred at 50

°C for 24 h and concentrated to give a brown solid. The residue

was partitioned in ethyl acetate and cold (0 °C) aqueous

sodium bisulfite. The aqueous phase was recovered and

extracted twice with ethyl acetate. The combined organic

extracts were washed with water and brine and dried over

MgSO₄. Evaporation and purification by flash chromatography

(dichloromethane/isopropyl alcohol 20/1) gave 3-iodo-2-pyri-

done as a yellow solid (2.1 g). A mixture of 3-iodo-2-pyridone

(1.2 g, 5.43 mmol), palladium acetate (12 mg), triphenylphos-

phine (24 mg), copper(I) iodide (24 mg), n-butylamine (1.08

mL, 10.86 mmol), 3-phenyl-1-propyne (0.83 mL, 6.5 mmol), and

tetrahydrofuran (9 mL) was stirred at 55 °C for 48 h. The

Ben zyl-p yr id in e-2,3-d ia m in e (39). A solution of benzyl-

(3-nitro-pyridin-2-yl)-amine (5 g, 21.8 mmol) and tin(II) chlo-

ride dihydrate (24.6 g, 109.1 mmol) in EtOAc (100 mL) was

refluxed for 2 h. The reaction mixture was cooled to room

temperature, quenched with saturated aqueous NaHCO₃(until

1308 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7

Malamas et al.

reaction was quenched with water and extracted with ethyl

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

ration and purification by flash chromatography (hexanes/

ethyl acetate 4/1) gave 2-benzylfuro[2,3-b]pyridine as a brown

oil (0.74 g, 65% yield): ¹H NMR (DMSO-d₆, 400 MHz) δ 4.13

(s, 2H, CH2), 6.33 (s, 1H, CH), 7.15 (m, 1H, Ar-H), 7.26 (m,

1H, Ar-H), 7.28 (m, 4H, Ar-H), 7.78 (dd, J ) 7.5, 1.54 Hz, 1H,

Ar-H), 8.22 (dd, J ) 6.6, 1.76 Hz, 1H, Ar-H); MS m/e 209 (M⁺).

Anal. (C₁₄H₁₁NO) C, H, N.

(7.3 g, 43% yield): mp 82-84 °C; ¹H NMR (DMSO-d₆, 400

MHz) δ 2.63 (s, 3H, CH₃), 7.58 (m, 4H, Ar-H), 7.9 (d, J ) 8.35

Hz, 2H, Ar-H), 8.18 (d, J ) 8.35 Hz, 2H, Ar-H); MS m/e 381

(M⁺). Anal. (C₁₇H₁₁BrF₃NO) C, H, N.

4-(4′-Met h oxy-b ip h en yl-4-yl)-5-m et h yl-2-(4-t r iflu or o-

m eth yl-p h en yl)-oxa zole (50a , R¹) 4-CF ₃-P h ). 4-Methoxy-

benzeneboronic acid (1.44 g, 7.19 mmol) in ethyl alcohol (5 mL)

was added into a mixture of 4-(4-bromo-phenyl)-5-methyl-2-

(4-trifluoromethyl-phenyl)-oxazole (2.5 g, 6.54 mmol), sodium

carbonate (2 N, 6.5 mL), tetrakis(triphenylphosphine)palladium-

(0) (0.23 g, 0.196 mmol), and toluene (200 mL). The reaction

mixture was refluxed for 12 h, cooled to room temperature,

and treated with hydrogen peroxide (30%, 5 mL) for 1 h. The

mixture was poured into water and extracted with ethyl

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

ration and crystallization from hexanes/ethyl ether gave 4-(4′-

methoxy-biphenyl-4-yl)-5-methyl-2-(4-trifluoromethyl-phenyl)-

oxazole as a white solid (2.2 g, 82% yield): mp 167-168 °C;

¹H NMR (DMSO-d₆, 400 MHz) δ 2.68 (s, 3H, CH₃), 3.8 (s, 3H,

OCH₃), 7.05 (d, J ) 8.78 Hz, 2H, Ar-H), 7.65 (d, J ) 8.78 Hz,

2H, Ar-H), 7.68 (d, J ) 8.57 Hz, 2H, Ar-H), 7.73 (d, J ) 7.57

Hz, 2H, Ar-H), 7.9 (d, J ) 8.35 Hz, 2H, Ar-H), 8.2 (d, J ) 8.35

Hz, 2H, Ar-H); MS m/e 409 (M⁺). Anal. (C₂₄H₁₈F₃NO₂) C, H,

N.

2-Ben zyl-3-(4′-m eth oxy[1,1′-bip h en yl]-4-yl)fu r o[2,3-b]-

p yr id in e (36a ). Bromine (1.6 mL, 31.7 mmol) was added

dropwise into mixture of 2-benzylfuro[2,3-b]pyridine (1.57 g,

31.7 mmol), sodium acetate (5.2 g, 63.4 mmol), and acetic acid

over a 50 min period. The mixture was stirred for 6 h, poured

into water, and extracted with dichloromethane. The organic

extracts were washed with NaOH (0.01 N), water, and brine

and dried over MgSO₄. Evaporation and purification by flash

chromatography on silica gel (hexanes/ethyl acetate 9/1) gave

3-bromo-2-benzylfuro[2,3-b]pyridine as a yellow solid (0.8 g).

The product was dissolved in acetone (7 mL) and added into a

mixture of 4′-methoxy-biphenyl-4-boronic acid (0.69 g, 3.06

mmol), potassium carbonate (0.96 g, 6.95 mmol), palladium

acetate (2.5 mg), and water 7 (mL). The reaction was stirred

at 65 °C for 24 h, poured into water, and extracted with

dichloromethane. The organic extracts were dried over MgSO₄.

Evaporation and purification by flash chromatography (hex-

anes/ethyl acetate 9/1) gave 2-benzyl-3-(4′-methoxy[1,1′-biphen-

yl]-4-yl)furo[2,3-b]pyridine as an off-white solid (0.26 g, 24%

yield): ¹H NMR (DMSO-d₆, 400 MHz) δ 3.87 (s, 3H, OCH₃),

4.27 (s, 2H, CH₂), 7.03 (d, J ) 8.78 Hz, 2H, Ar-H), 7.24 (m,

2H, Ar-H), 7.33 (m, 4H, Ar-H), 7.55 (8.56 Hz, 1H, Ar-H), 7.61

(d, J ) 7.56 Hz, 2H, Ar-H), 7.69 (d, J ) 8.78 Hz, 2H, Ar-H),

7.78 (dd, J ) 7.5, 1.54 Hz, 1H, Ar-H), 8.22 (dd, J ) 6.6, 1.76

Hz, 1H, Ar-H); MS m/e 391 (M⁺). Anal. (C₂₇H₂₁NO₂) C, H, N.

4′-(2-Ben zyl-ben zofu r an -3-yl)-3-n itr o-biph en yl-4-ol. Iron-

(III) nitrate nonahydrate (8.04 g, 19.9 mmol) was added to a

solution of 4′-(2-benzyl-benzofuran-3-yl)-biphenyl-4-ol (6.8 g,

18.1 mmol) in absolute ethanol (80 mL), and the mixture was

stirred at 45 °C for 1.5 h. The reaction mixture was cooled to

room temperature, poured into HCl (0.1 N), and extracted with

ethyl acetate. The extracts were washed with brine and dried

over MgSO₄. Evaporation and purification by flash chroma-

tography on silica gel (EtOAc/petroleum ether 1/10) gave 4′-

(2-benzyl-benzofuran-3-yl)-3-nitro-biphenyl-4-ol as a yellow

solid: mp 75 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 4.26 (s, 2H,

CH₂), 7.2-7.36 (m, 8H, Ar-H), 7.6-7.64 (m, 4H, Ar-H); 7.8 (d,

J ) 8.35 Hz, 2H, Ar-H), 7.9 (dd, J ) 8.78, 2.41 Hz, 2H, Ar-H),

8.2 (d, J ) 2.63 Hz, 1H, Ar-H)11.16 (brs, 1H, OH); IR (KBr,

cm^-1) 3350 (OH); MS m/e 420 (M - H)⁺. Anal. (C₂₇H₁₉NO₄‚

0.5H₂O) C, H, N.

6-[(2-Bu tyl-ben zofu r a n -3-yl)-h yd r oxy-m eth yl-n a p h th a -

len -2-ol (27, R¹) bu tyl). n-Butyllithium (17.9 mL) was added

dropwise into a cold (-78 °C) mixture of 6-bromo-2-naphthol

(5.0 g, 22.42 mmol), and tetrahydrofuran (100 mL). The

reaction mixture was stirred for 2 h, and then 2-butyl-

benzofuran-3-carboxaldehyde (4.53 g, 22.42 mmol) in tetrahy-

drofuran (5 mL) was added dropwise. The mixture was stirred

for 30 min, quenched with aqueous ammonium chloride,

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

ethyl ether. The organic extracts were dried over MgSO₄.

Evaporation and purification by flash chromatography on silica

gel (hexanes/EtOAcc 3:1) gave 6-[(2-butyl-benzofuran-3-yl)-

hydroxy-methyl-naphthalen-2-ol as a yellow solid (6.8 g): mp

1

38-40 °C; H NMR (DMSO-d₆, 400 MHz) δ 0.88 (t, J ) 7.25

Hz, 3H, CH₃), 1.38-1.4 (m, 2H, CH₂), 1.6-1.64 (m, 2H, CH₂),

1.9 (t, J ) 7.47 Hz, 2H, CH₂), 7.03-7.05 (m, 3N, Ar-H), 7.12

(m, 1H, Ar-H), 7.36 (dd, J ) 8.57, 1.76 Hz, 1H, Ar-H), 7.41

(8.13 Hz, 2H, Ar-H), 7.59 (d, J ) 8.57 Hz, 1H, Ar-H), 7.75 (d,

J ) 7.69 (1H, Ar-H), 7.9 (s, 1H, Ar-H), 9.64 (s, 1H, OH); IR

(KBr, cm^-1) 3370 (OH); MS m/e 346 (M⁺). Anal. (C₂₃H₂₂O₃) C,

H.

6-[(2-Bu tyl-ben zofu r an -3-ylm eth yl)-n aph th alen -2-ol (28,

R¹) bu tyl). Triethylsilane (7.4 mL, 46.38 mmol) was added

into a cold (0 °C) mixture of 6-[(2-butyl-benzofuran-3-yl)-

hydroxy-methyl-naphthalen-2-ol (8.0 g, 23.19 mmol) and dichlo-

romethane (100 mL). After 10 min, trifluoroacetic acid (10 mL)

was added into the reaction mixture, and the reaction was

stirred for 30 min, poured into water, and extracted with ethyl

ether. The organic extracts were dried over MgSO₄. Evapora-

tion gave 6-[(2-butyl-benzofuran-3-ylmethyl)-naphthalen-2-ol

as a brown oil (6.2 g): ¹H NMR (DMSO-d₆, 400 MHz) δ 0.85

(t, J ) 7.25 Hz, 3H, CH₃), 1.15-1.5 (m, 2H, CH₂), 1.6-1.63

(m, 2H, CH₂), 2.81 (t, J ) 7.47 Hz, 2H, CH₂), 4.07 (s, 2H, CH₂),

7.0-7.05 (m, 3H, Ar-H), 7.17 (t, J ) 7.25 Hz, 1H, Ar-H), 7.34

(d, J ) 7.7 Hz, 1H, Ar-H), 7.45 (d, J ) 8.13 Hz, 1H, Ar-H),

7.57 (d, J ) 8.56 Hz, 1H, Ar-H), 7.62 (m, 2H, Ar-H), 9.6 (s,

1H, OH); IR (KBr, cm^-1) 3400 (OH); MS m/e 330 (M⁺). Anal.

(C₂₃H₂₂O₂) C, H.

1-(4-Br om o-p h en yl)-p r op a n on e Oxim e (48). Sodium

acetate (80.0 g, 0.97 mol) was added into a mixture of 1-(4-

bromo-phenyl)-propanone (52.0 g, 0.24 mol), hydroxylamine

hydrochloride (50.8 g, 0.73 mol), ethyl alcohol (500 mL), and

water (100 mL). The reaction mixture was stirred at 60 °C for

1 h, poured into water, and extracted with ethyl ether. The

organic extracts were dried over MgSO₄. Evaporation and

crystallization from ethyl ether/ hexanes gave 1-(4-bromo-

phenyl)-propanone oxime as a white solid (49.6 g, 89% yield):

¹H NMR (DMSO-d₆, 400 MHz) δ 1.0 (t, J ) 7.69 Hz, 3H, CH₃),

2.67 (q, J ) 7.69 Hz, 2H, CH₂), 7.5 (m, 4H, Ar-H), 11.2 (s, H,

OH), IR (KBr, cm^-1) 3200 (OH); MS m/e 227 (M⁺). Anal. (C₉H₁₀

-

BrNO) C, H, N.

1-Br om o-6-(2-bu tyl-ben zofu r a n -3-ylm eth yl)-n a p h th a -

len -2-ol (30a , R¹) Bu tyl, X ) CH₂). Bromine (0.96 mL, 18.78

mmol) in acetic acid (10 mL) was added dropwise over a 30

min period into a cold (5 °C) mixture of 6-[(2-butyl-benzofuran-

3-ylmethyl)-naphthalen-2-ol (6.2 g, 18.78 mmol) and acetic acid

(50 mL). After the addition, the mixture was poured into water

and extracted with ethyl ether. The organic extracts were

washed with aqueous sodium bisulfite and dried over MgSO₄.

Evaporation gave 1-bromo-6-(2-butyl-benzofuran-3-ylmethyl)-

naphthalen-2-ol as a brown oil (5.6 g): mp 54-56 °C); ¹H NMR

(DMSO-d₆, 400 MHz) δ 0.85 (t, J ) 7.25 Hz, 3H, CH₃), 1.2-

1.35 (m, 2H, CH₂), 1.6-1.65 (m, 2H, CH₂), 2.83 (t, J ) 7.47

4-(4-Br om o-ph en yl)-5-m eth yl-2-(4-tr iflu or om eth yl-ph en -

yl)-oxa zole (49). Pyridine (3.55 mL, 43.86 mmol) was added

into a mixture of 1-(4-bromo-phenyl)-propanone oxime (10.0

g, 43.86 mmol) and toluene (20 mL). The reaction mixture was

stirred for 30 min, and 4-trifluoromethyl-phenyl acetyl chloride

(16.27 mL, 109.6 mmol) was added dropwise. The mixture was

stirred at 100 °C for 24 h, poured into water, and extracted

with ethyl acetate. The organic extracts were dried over

MgSO₄. Evaporation and purification by flash chromatography

on silica gel (hexanes/EtoAc 40:1) gave 4-(4-bromo-phenyl)-5-

methyl-2-(4-trifluoromethyl-phenyl)-oxazole as a white solid

Benzofuran Biophenyls as PTPase Inhibitors

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1309

Hz, 2H, CH₂), 4.1 (s, 2H, CH₂), 7.04 (t, J ) 7.69 Hz, 1H, Ar-

H), 7.13 (t, J ) 7.25 Hz, 1H, Ar-H), 7.23 (d, J ) 8.78 Hz, 1H,

Ar-H), 7.3 (d, J ) 7.67 Hz, 1H, Ar-H), 7.42 (m, 2H, Ar-H), 7.67

(m, 2H, Ar-H), 7.89 (d, 8.18 Hz, 1H, Ar-H), 10.42 (s, 1H, OH);

maintained at pH 11. The reaction was stirred for 30 min,

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

ethyl ether. The organic extracts were dried over MgSO₄.

Evaporation and purification by flash chromatography on

acidic silica gel (hexanes/EtAOc 2:1) gave 4-[4′-(2-benzyl-

benzofuran-3-yl)-biphenyl-4-yloxysulfonyl]-2-hydroxy-benzo-

ic acid as a white solid, (0.67 g, 43% yield): mp 96-98 °C; ¹H

NMR (DMSO-d₆, 400 MHz) δ 4.26 (s, 2H, CH₂), 7.18-7.39 (m,

11H, Ar-H), 7.6-7.64 (m, 4H, Ar-H), 7.77-7.82 (m, 4H, Ar-

H), 7.99 (d, J ) 8.56 Hz, 1H, Ar-H); IR (KBr, cm^-1) 3420 (OH),

3600-2700 (CO₂H); MS m/e 575 (M - H)⁺. Anal. (C₃₄H₂₄O₇S‚

0.5‚H₂O) C, H.

IR (KBr, cm^-1) 3480 (OH); MS m/e 408 (M⁺). Anal. (C₂₇H₁₁

-

BrO2) C, H.

5-[1-Br om o-6-(2-b u t yl-b en zofu r a n -3-ylm et h yl)-n a p h -

th a len -2-yloxy]-1H-tetr a zole (170). Sodium hydride (0.16

g, 3.96 mmol) was added into a mixture of 1-bromo-6-(2-butyl-

benzofuran-3-ylmethyl)-naphthalen-2-ol (1.35 g, 3.3 mmol) and

N,N-dimethylformamide (10 mL). The mixture was stirred for

1 h, and bromoacetonitrile (0.27 mL, 3.96 mmol) was added

dropwise. The mixture was stirred for 1 h, poured into water,

acidified with HCl (2 N), and extracted with ethyl ether.

Evaporation gave a yellow oil (1.15 g). The residue was taken

in N,N-dimethylformamide (20 mL) and treated with sodium

azide (1.29 g, 19.8 mmol), and ammonium chloride (1.05 g, 19.8

mmol). The mixture was stirred at 120 °C for 10 h, poured

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

ether. The organic extracts were dried over MgSO₄. Evapora-

tion and purification by flash chromatography on acidic silica

gel (hexanes/EtOAc 2:1) gave 5-[1-bromo-6-(2-butyl-benzofu-

ran-3-ylmethyl)-naphthalen-2-yloxy]-1H-tetrazole as a white

solid (0.98 g): mp 148-150 °C; ¹H NMR (DMSO-d₆, 400 MHz)

δ 0.85 (t, J ) 7.25 Hz, 3H, CH₃), 1.2-1.35 (m, 2H, CH₂), 1.6-

1.65 (m, 2H, CH₂), 2.83 (t, J ) 7.47 Hz, 2H, CH₂), 4.16 (s, 2H,

CH₂), 5.66 (s, 2H, CH₂), 7.06 (t, J ) 7.69 Hz, 1H, Ar-H), 7.13

(t, J ) 7.25 Hz, 1H, Ar-H), 7.32 (d, J ) 7.67 Hz, 1H, Ar-H),

7.44 (d, J ) 8.78 Hz, 1H, Ar-H), 7.51 (d, J ) 8.78 Hz, 1H, Ar-

H), 7.57 (d, J ) 8.78 Hz, 2H, Ar-H), 7.8 (s, 1H, Ar-H), 7.92 (d,

J ) 8.78 Hz, 1H Ar-H), 7.98 (d, J ) 8.78 Hz, 1H, Ar-H); MS

m/e 490 (M⁺). Anal. (C₂₅H₂₃BrN₄O₂) C, H, N.

(2-Bu tyl-ben zofu r a n -3-yl)-(4′-h yd r oxy-bip h en yl-4-yl)-

m eth a n on e (25, R¹) Bu tyl). Pyridinium chlorochromate

(1.74 g, 8.06 mmol), was added into a mixture of 4′-[(2-butyl-

benzofuran-3-yl)-hydroxy-methyl]-biphenyl-4-ol (2.0 g, 5.37

mmol) and dichloromethane (20 mL). The reaction mixture was

stirred for 1 h, diluted with ethyl ether, and filtered through

a florisil pad. The organic extracts were dried over MgSO4.

Evaporation and purification by flash chromatography on silica

gel (hexanes/EtOAc 3:1) gave (2-butyl-benzofuran-3-yl)-(4′-

hydroxy-biphenyl-4-yl)-methanone as a yellow solid (1.1 g): ¹H

NMR (DMSO-d₆, 400 MHz) δ 0.73 (t, J ) 7.25 Hz, 3H, CH₃),

1.14-1.18 (m, 2H, CH₂), 1.62-1.69 (m, 2H, CH₂), 2.78 (t, J )

7.47 Hz, 2H, CH₂), 7.21 (t, J ) 7.87 Hz, 1H, Ar-H), 7.35-7.4

(m, 3H, Ar-H), 7.66 (d, J ) 8.13 Hz, 1H, Ar-H), 7.95-8.02 (m,

2H, Ar-H), 8.14 (d, J ) 8.78 1H, Ar-H), 8.34 (s, 1H, Ar-H),

11.05 (s, 1H, OH); IR (KBr, cm^-1) 3200 (OH), 1600 (CO); MS

m/e 370 (M⁺). Anal. (C₂₃H₂₂O₃) C, H.

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 Recom bin a n t h -P TP 1B. Human recombi-

nant PTP1B (321 aa form) was prepared as described.²⁶

Mea su r em en t of P TP a se Activity. The malachite green-

ammonium molybdate method, as described by Lanzetta et

al.²⁸and adapted for the plate reader, was used for the

nanomolar detection of liberated phosphate by recombinant

h-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

h-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 was preincubated for 10 min at

37 °C with or without test compound and 81.83 mM HEPES

reaction buffer, pH 7.4. Peptide substrate, at a final concentra-

tion of 50 µM, was equilibrated to 37 °C in a LABLINE Multi-

Blok heater. The preincubated recombinant enzyme prepara-

tion with or without drug was added to initiate the dephos-

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

reaction was terminated by the addition 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 tet-

rahydrate in 4 N HCl, and 0.5% Tween 20. Sample blanks were

prepared by the addition of MG/AM/Tw to substrate and 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 nanomoles of

phosphate released/min/mg protein. Inhibition of recombinant

h-PTP1B by test compounds was calculated as percent of

phosphatase control. A four-parameter nonlinear logistic

regression of PTPase activities using SAS release 6.08, PROC

NLIN, was used for determining IC₅₀values of the test

compounds. The reported IC₅₀values show significant fit to

the regression curve (p < 0.05).

6-(2-Bu tyl-ben zofu r a n -3-ylm eth yl)-1-iod o-n a p h th a len -

2-ol (30b, R¹) Bu tyl, X ) CH₂). Iodine (1.16 g, 4.55 mmol)

was added portionwise into a cold (0 °C) mixture of 6-[(2-butyl-

benzofuran-3-ylmethyl)-naphthalen-2-ol (1.5 g, 4.55 mmol),

sodium hydroxide (0.35 g, 9.1 mmol), and methyl alcohol (20

mL). The reaction mixture was stirred 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 and purification by flash chromatography on silica gel

(hexanes/EtOAc 2:1) gave 6-(2-butyl-benzofuran-3-ylmethyl)-

1-iodo-naphthalen-2-ol as a brown oil (1.85 g): ¹H NMR

(DMSO-d₆, 400 MHz) δ 0.86 (t, J ) 7.25 Hz, 3H, CH₃), 1.3-

1.35 (m, 2H, CH₂), 1.6-1.67 (m, 2H, CH₂), 2.85 (t, J ) 7.47

Hz, 2H, CH₂), 4.1 (s, 2H, CH₂), 7.05 (t, J ) 7.69 Hz, 1H, Ar-

H), 7.14-7.18 (m, 2H, Ar-H), 7.31 (d, J ) 7.67 Hz, 1H, Ar-H),

7.39 (d, J ) 8.56 Hz, 1H, Ar-H), 7.42 (d, J ) 8.13 Hz, 1H, Ar-

H), 7.63 (s, 1H, Ar-H), 7.67 (d, J ) 8.57 Hz, 1H, Ar-H), 7.84

ob/ob Dia betic Mou se Mod el. Experimental details for

the ob/ob diabetic mouse model were previously described.³³

X-r a y Cr ysta llogr a p h ic Stu d ies: P r otein P r od u ction ,

Cr ysta lliza tion , a n d Da ta Collection . Human recombinant

PTP1B catalytic domain (residues 1-299) was prepared as

described.³⁴Complex solution was made by mixing excess of

inhibitor dissolved in DMSO with protein (protein:inhibitor

) 1:1.5 ∼ 3.0), incubated at room temperature for at least 3

h, then filtered with 0.1 µm filters. Crystals of h-PTP1B-

inhibitor complex were obtained by vapor diffusion at 4 °C

using 0.1 M HEPES, (pH 7.5), 50 mM MgCl₂, 18% PEG 4000,

3 mM DTT, and 10∼15 mg/mL protein. Typically, rodlike

crystals appeared overnight and continued to grow to a

maximum size of 0.4 mm × 0.2 mm × 0.7 mm within 10 days.

The crystals belonged to space group P3(1)21, a ) b ) 88 Å, c

) 104 Å, R ) â ) 90°, γ ) 120°. The crystals were cryoprotected

by transferring into the crystallization solution plus 30%

glycerol. Cryoprotected crystals were flash cooled in a gaseous

nitrogen stream at 100 K prior to data collection.

(d, J ) 8.78 Hz, 1H, Ar-H), 10.54 (s, 1H, OH); IR (KBr, cm^-1

3450 (OH); MS m/e 476 (M⁺). Anal. (C₂₃H₂₁IO₂) C, H.

)

4-[4′-(2-Ben zyl-b en zofu r a n -3-yl)-b ip h en yl-4-yloxysu l-

fon yl]-2-h yd r oxy-ben zoic Acid (175). Sodium hydroxide

(50%) was added dropwise into cold (0 °C) mixture of 4′-(2-

benzyl-benzofuran-3-yl)-biphenyl-4-ol (1.0 g, 2.66 mmol), 4-chlo-

rosulfonyl-2-hydroxy-benzoic acid (1.25 g, 5.32 mmol), and

tetrahydrofuran (50 mL) in a rate that the reaction was

1310 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7

Malamas et al.

(11) (a) Evans, J . L.; J allal, B. Protein Tyrosine Phosphatases: Their

Role in Insulin Action and Potential Drug Targets. Exp. Opin.

Invest. Drugs 1999, 8, 139-160. (b) Brichard, S. M.; Bailey, C.

J .; Henquin, J . C. Vanadate Improves Glucose Homeostasis in

Insulin-Resistant ob/ob Mice. Fundam. Clin. Pharmacol. 1990,

4, Suppl. 1, 54S. (c) Cro, G.; Serrano J . J .; Mongold, J . J .; Tep,

A.; Vian, L.; Lazaro, R. Effects of Vanadyl Derivatives on Animal

Models of Diabetes. Fundam. Clin. Pharmacol. 1990, 4, Suppl.

1, 20S.

Diffraction data of the h-PTP1B-inhibitor complex crystals

were collected by in-house conventional X-ray generator using

Raxis-II image detectors. All data were collected at 100 K and

processed with DENZO/SCALPACK in Data Collection and

Procession.³⁵R-merge for h-PTP1B-compound 182 is 4%

(resolution 40-2.0 Å, coverage ) 94.6%) and for h-PTP1B-

compound 80 is 4.6% (resolution 40-1.85 Å, coverage )

92.2%).

(12) Byon, J . C. H.; Kusari, A. B.; Kusari, J . Protein-Tyrosine

Phosphatase-1B Acts as a Negative Regulator of Insulin Signal

Transduction. Mol. Cell Biochem. 1998, 182, 101-108.

(13) Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins,

S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-Hagen, J .;

Chan, C. C.; Ramachandran, C.; Gresser, M. J .; Tremblay, M.

L.; Kennedy, B. P. Increased Insulin Sensitivity and Obesity

Resistance in Mice Lacking the Protein Tyrosine Phosphatase-

1B. Science 1999, 283, 1544-1548.

(14) Durani, N.; J ain, R.; Saeed, A.; Dikshit, D. K.; Durani, S.; Kapil,

R. S. Structure-Activity Relationship of Antiestrogens: A Study

Using Triarylbutenone, Benzofuran, and Triarylfuran Analogues

as Models for Triarylethylenes and Triarylpropenones. J . Med.

Chem. 1989, 32, 1700-1707.

Refin em en t. These structures were solved by molecular

replacement with the h-PTP1B catalytic domain structure

model (PDB code). The area around residues 179-185 was

rebuilt based on the difference density, since there was a large

conformational change upon the inhibitor binding. Cycles of

model rebuilding and refinement were carried out with a

X-PLOR (Version 3.1, Brunger, A. T., 1992), a system for X-ray

crystallography and NMR (New Haven: Yale University Press)

and QUANTA. Inhibitor was built into the difference density

after the first cycle of refinement. Water molecules were

assigned according to F_o-F_cmaps. The final model for h-PTP1B-

compound 182 contained residues 1-299 and 77 waters. It

exhibited good stereochemistry, with an averaged bond length

and bond angle deviation from ideal geometry of 0.017 Å and

1.868°, respectively. One hundred percent of all the residues

were in the most favorable orientation and the allowed regions

of a Ramachandran plot. The overall R-factor is 22.3%, and

R-free is 26.2% with 8% test reflections using diffraction data

between 20 and 2.0 Å. The final model for h-PTP1B-compound

80 contained residues from 1 to 298 and 165 water molecules.

The rms deviation for bond length and angle are 0.006 Å and

1.35°, respectively. One hundred percent of all residues are

in the most favorable orientation and the allowed regions of a

Ramachandran plot. The overall R-factor is 22.2% using

diffraction data between 40 and 1.85 Å.

(15) Miyaura, N.; Suzuki, A. Palladium-Catalyzed Cross-Coupling

Reactions of Organoboron Compounds. Chem. Rev. 1995, 95,

2457-2483.

(16) Cabiddu, S.; Cancellu, C.; Floris, C.; Gelli, G.; Melis, S. Meta-

lation Reactions; Part XI. A Novel, One-Step Synthesis of Benzo-

[b]thiophene derivatives. Synthesis 1998, 888-890.

(17) Keck, G. E.; McHardy, S. F.; Murry, J . A. Some Unusal Reactions

of Weinreb Amides. Tetrahedron Lett. 1993, 34, 6215-6218.

(18) Mutaitis, C. F.; Patragnoni, R.; Goodkin, G.; Neighbour, B.;

Obaza-Nutaitis, J . Reduction of Heterocyclic Alcohols With

Sodium Borohydride-Trifluoroacetic Acid. Preparetaion of bis-

Heterocyclic Methanes. Org. Prep. Proc. Int. 1991, 23, 403-411.

(19) Todd, D. The Wolff-Kishner Reduction. Org. React. 1948, 4, 378.

(20) Hughes, D. L. The Mitsunobu Reaction. Organic Reactions; J ohn

Wiley & Sons Publishers: New York, 1992; pp 335-656.

(21) Goldenberg, C.; Binon, F.; Gillyns, E. Recherches Dans La Serie

Des Benzofurannes. XVIII. Synthese De Benzofuryl-3, -4, -5, -6,

-7 Aminoethanols. Chim. Three. 1966, 221-227.

(22) Kursanov, D. N.; Parnes, Z. N.; Loim, N. M. Applications of ionic

Hydrogenation to Organic Synthesis. Synthesis 1974, 633-655.

(23) Asakura, J .; Robins, M. J . Cerium(IV)-Mediated Halogenation

at C-5 of Uracil Derivatives. J . Org. Chem. 1990, 55, 4928-4933.

(24) Houpis, I. N.; Choi, W. B.; Reider, P. J .; Molina, A.; Churchill,

H.; Lynch, J .; Volante, R. p. Synthesis of Functionalized Furo-

[2,3-b]pyridines via the Pd-Catalyzed Coupling of Acetylenes to

Iodopyridones. Preparation of a Key Intermediate to a New HIV

Protease Inhibitor L-754,394. Tetrahedron Lett. 1994, 25, 9355-

9358.

(25) Bhatt, M. V.; Reddy, G. S. A New Synthesis of Oxazoles.

Tetrahedron Lett. 1980, 21, 2359-2360.

(26) Barford, D. P.; Keller, J . C.; Flint, A. J .; Tonks, N. K. Purification

and Crystallization of the Catalytic Domain of Human Protein

Tyrosine Phosphatase 1B Expressed in Escherichia coli. J . Mol.

Biol. 1994, 239, 726-730.

(27) Ramachandran, C.; Aebersold, R.; Tonks, N. K.; Pot, D. A.

Sequential Dephosphorylation of a Multiply Phosphorylated

Insulin Receptor Peptide by Protein Tyrosine Phosphatases.

Biochemistry 1992, 31, 4232-4238.

(28) Lanzetta, P. A.; Alvarez, L. J .; Reinach, P. S.; Candia, O. A. An

Improved Assay for Nanomolar Amounts of Inorganic Phosphate.

Anal. Biochem. 1979, 100, 95-97.

(29) Barford, D. J .; Flint, A. J .; Tonks, N. K. Crystal Structure of

Human Protein Tyrosine Phosphatase 1B. Science 1994, 263,

1397-1404.

(30) SYBYL 6.5, Tripos Inc., 1699 South Hanley Rd., St. Louis,

Missouri, 63144, USA.

(31) Heiden, W.; Goetze, T.; Brickmann, J . J . Comput. Chem. 1993,

14, 246.

(32) Barford, D.; J ia, Z.; Tonks, N. K. Protein Tyrosine Phosphatases

Take Off. Nature Struct. Biol. 1995, 2, 1043-1053.

(33) Wrobel, J .; Li, Z.; Dietrich, A.; McCaleb, M.; Mihan, B.; Sredy,

J .; Sullivan, D. Novel 5-(3-Aryl-2-propynyl)-5-(arylsulfonyl)-

thiazolidine-2,4-diones as Antihyperglycemic Agents. J . Med.

Chem. 1998, 41, 1084-1091.

(34) Hoppe, E.; Berne, P. F.; Stock, D.; Rasmussen, J . S.; Moller, N.

P. H.; Ullrich, A.; Huber, R. Expression, Purification and

Crystallization of Human Phosphotyrosine Phosphatase 1B. Eur.

J . Biochem. 1994, 223, 1069-1077.

Ack n ow led gm en t. We gratefully acknowledge Dr.

A. Grinfeld and Mr. Z. Li for their synthetic contribution

in the preparation of three compounds.

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.

(4) (a) Goldman, J . M. Oral Hypoglycemic Agents: An Update of

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.

(5) (a) Matsumoto, K.; Miyake, S.; Yano, M.; Tominaga, Y. Increase

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

1749. Additional information in Scrips 1997, 2282, 21, and Scrips

1997, 2292, 20. (b) Troglitazone is being marketed in the United

States as Rezulin and in J apan as Noscal.

(6) Haring, H. U. The Insulin Receptor: Signaling Mechanism and

Contribution to the Pathogenesis of Insulin Resistance Diabe-

talogia 1991, 34, 848.

(7) (a) Goldstein, B. J . Protein-Tyrosine Phosphatases and the

Regulation of Insulin Action. J . Cell. Biochem. 1992, 31, 33-

42. (b) Goldstein, B. J . Regulation of Insulin Receptor Signaling

by Protein-Tyrosine Dephosphorylation. Receptor 1993, 3, 1-15.

(c) Ahmad, F.; Goldstein, B. J . Purification, Identification and

Subcellular Distribution of Three Predominant Protein-Tyrosine

Phosphatase Enzymes in Skeletal Muscle Tissue. Biochim.

Biophys. Acta 1995, 1248, 57-69.

(8) McGuire M. C.; Fields, R. M.; Nyomba, B. L.; Raz, I.; Bogardus,

C.; Tonks, N. K.; Sommercorn, J . Abnormal Regulation of Protein

Tyrosine Phosphatase Activities in Skeletal Muscle of Insulin-

Resistant Humans. Diabetes 1991, 40, 939.

(9) Myerovitch, J .; Baker, J . M.; Kahn, C. R. Hepatic Phosphaty-

rosine Phosphatase Activity and Its Alterations in Diabetic Rats.

J . Clin. Invest. 1989, 84, 976.

(10) Sredy, J .; Sawicki, D. R.; Flam, B. R.; Sullivan, D. Insulin

Resistance Is Associated With Abnormal Dephosphorylation of

a Synthetic Phosphopeptide Corresponding to the Major Auto-

phosphorylation Sites of the Insulin Receptor. Metabolism 1995,

44, 1074.

(35) Otwinowski, Z., Sawyer, L., Isaacs, N., Bailey, S. W., Eds.

Daresbury, U.K. Science and Engineering Council, pp 56-62,

1993.

J M990560C

Article Doi

Novel benzofuran and benzothiophene biphenyls as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties

DOI: 10.1021/jm990560c

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm990560c

Full text of DOI:10.1021/jm990560c

Products guided by the article

R&D Labs maybe for 897808-35-4

Relevant to this article

Hot Product