Synthesis and characterization of 3-arylquinazolinone and 3-arylquinazolinethione derivatives as selective estrogen receptor beta modulators

2440

J. Med. Chem. 2006, 49, 2440-2455

Synthesis and Characterization of 3-Arylquinazolinone and 3-Arylquinazolinethione Derivatives

as Selective Estrogen Receptor Beta Modulators

Timur Gu¨ngo¨r,*^,†Ying Chen,^†Rajasree Golla,^‡Zhengping Ma,^‡James R. Corte,^†John P. Northrop,^†Bin Bin,^†

John K. Dickson,^†Terry Stouch,^§Rong Zhou,^‡Susan E. Johnson,^‡Ramakrishna Seethala,^‡and Jean H. M. Feyen^‡

Department of Chemistry, Department of Metabolic Diseases and CardioVascular Drug DiscoVery, and Department of Modeling,

Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543-4000

ReceiVed September 20, 2005

On the basis of the stucture of genistein, a new series of 3-arylquinazolines was prepared and tested for

their estrogen receptor (ER) R and â affinities. 5,7-Dihydroxy-3-(4-hydroxyphenyl)-4(3H)-quinazolinone

(1aa) acts as an agonist on both ER subtypes. It has 62-fold higher binding affinity [IC₅₀(ERâ) ) 179 nM]

and 38-fold higher functional potency in a transcription assay [EC₅₀(ERâ) ) 76 nM] with ERâ than with

ERR, thus improving upon the selectivity of genistein. All of the analogues showed preferential binding

affinity for ERâ. Many are also more potent in activating transcription by ERâ than by ERR. Transformation

of the CdO functionality at position 4 into a CdS group provided 5,7-dihydroxy-3-(4-hydroxyphenyl)-

4(3H)-quinazolinethione (1ba), which acts as an agonist on both ER subtypes but has 56-fold higher binding

affinity for ERâ over ERR [IC₅₀(ERâ) ) 47 nM] and 215-fold higher potency in the transcription assay

[EC₅₀(ERâ) ) 13 nM]. These ERâ-selective compounds may represent valuable tools in understanding the

differences in structure and biological function of ERâ and ERR.

Introduction

Estrogens are a family of naturally occurring steroid hormones

that exert most of their biological effects via interaction with

the estrogen receptor (ER), a member of the nuclear hormone

receptor superfamily.¹For the past decade, the physiological

effects of estrogens were attributed to a single receptor of the

ligand-activated transcription factor family, now known as ERR.

The discovery of a second estrogen receptor, ERâ, in 1996^2,3

resulted in a large research effort to elucidate differences in

function and to evaluate the pharmacological potential of

selective ligands.^4-6

ERâ and ERR share about a 60% identity in the ligand-

binding domain (LBD) and only 20% homology in their amino-

Figure 1.

terminal transactivation domain.^2,3,7However, the crystal struc-

tures of ERâ⁸and ERR⁹LBDs in the presence of a ligand show

little variation in the direct vicinity of the ligand, explaining

the high affinity of 17â-estradiol to both ERâ and ERR and

published.^16a-dHere we report our findings for a series of

3-arylquinazolines (Figure 1, compounds 1a and 1b).¹⁷

emphasizing why attempts to find selective ligands have met

with limited success. Several steroidal and nonsteroidal estrogen

ligands^10-12were studied and differences both in potency and

response in cell based transcriptional assays were observed,

suggesting that it may be possible to develop subtype-selective

estrogen receptor modulators. Although functional selectivity

of greater than 100-fold for ERR has been observed with certain

ligands,^13a,bpotent and selective ligands for ERâ have remained

elusive until recently and the isoflavanoid genistein (Figure 1)

remained for a long time the only agonist with modest ERâ

selectivity reported in the literature.^5,14

Chemistry

Compounds of general formula 1a and 1b were prepared by

different methods depending on the nature and the positions of

the substituents. Compounds 1aa-al (R₁) R₂) H) described

in Table 1, were prepared via the general synthetic pathway

depicted in Scheme 1.

The commercially available 3,5-dimethoxyaniline 2 was

transformed to 4,6-dimethoxyisatin 3 and then to the corre-

sponding 4,6-dimethoxyanthranilic acid 4 using literature pro-

cedures.¹⁸The reaction of triphosgene gave the key intermediate

5,7-dimethoxy isatoic anhydride 5. The desired anilines 6a-i

were then reacted with compound 5 at elevated temperature to

yield benzamides 7a-i. Reaction of 7a-i with triethyl ortho-

formate afforded 3-aryl-5,7-dimethoxyquinazolines 8aa-ak.

Cleavage of the methoxy groups with either an excess of BBr₃,

NaSEt, or LiCl gave the target compounds 1aa-al.

A few years ago, arylbenzothiophenes^15aand 1,3,5-triazine-

based compounds^15bwere described as having some selectivity

for ERâ. More recently, several papers describing compounds

with >100-fold selectivity for ERâ relative to ERR were

* To whom correspondence should be addressed. Telephone: (609) 818

4946. E-mail: timur.gungor@bms.com.

Compounds where R₂is not H (1am-ar; Table 1) were

obtained by the sequence described in Scheme 2. Compound

1aa was iodinated to yield 1am and 1an. Iodination of 8aa,

followed by coupling with MeB(OH)₂in the presence of Pd-

^†Department of Chemistry.

^‡Department of Metabolic Diseases and Cardiovascular Drug Discovery.

^§Department of Modeling.

Published on Web 03/25/2006

ERâ Modulators Based on Genistein

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2441

Table 1. Structure and Physical-Chemical Characteristic of

Compounds 1aa-au (R₁) H)^a

Table 2. Structure and Physical-Chemical Characteristic of

Compounds 1av-aag (R₁* H)

compd

no.

preparation

method

compd

no.

preparation

method

formula^a

R₂

R₃

R

R′

R′′

formula^c

R₁

CH₃

C₂H₅

nPr

nBu

iPr

iBu

SH

Cl

OH

R

R′

1av

1aw

1ax

1ay

1az

1aaa

1aab

1aac

1aad

1aae

1aaf

1aag

OH

H

CH₃

H

Scheme 3

Scheme 4

Scheme 3

C₁₅H₁₂N₂O₄

C₁₆H₁₄N₂O₄

C₁₇H₁₆N₂O₄

C₁₈H₁₈N₂O₄

C₁₇H₁₆N₂O₄

C₁₈H₁₈N₂O₄

C₁₅H₁₂N₂O₄S

C₁₄H₁₀N₂O₄S

C₁₄H₉ClN₂O₄

C₁₄H₁₀N₂O₅

C₁₅H₁₂N₂O₃

C₂₀H₁₄N₂O₄

1aa

1ab

1ac

1ad

1ae

1af

1ag

1ah

1ai

H

OH

H

CH₃

H

Scheme 1 C₁₄H₁₀N₂O₄

Scheme 1 C₁₅H₁₂N₂O₄

Scheme 1 C₁₄H₉ClN₂O₄

Scheme 1 C₁₄H₉FN₂O₄

Scheme 1 C₁₅H₁₂N₂O₄

Scheme 1 C₁₄H₉FN₂O₄

Scheme 1 C₁₄H₉ClN₂O₄

Scheme 1 C₁₅H₁₂N₂O₄

H

2-CH₃OH

3-Cl

3-F

H

2-F

2-Cl

OH

OCH₃

OH

3-CH₃OH

H

1aj

H

OH

H

CH₃CH₃Scheme 1 C₁₆H₁₄N₂O₄

1ak^a

H

Scheme 1 C₁₃H₉N₃O₄

Scheme 2 C₁₆H₁₄N₂O₄

Scheme 2 C₁₄H₈I₂N₂O₄

Scheme 2 C₁₅H₁₂N₂O₄

Scheme 2 C₁₄H₉IN₂O₄

Scheme 2 C₁₇H₁₆N₂O₄

CH₃

4-OHPh

1al^a

H

1am 8-I

^aThe purities of the final compounds were assessed by HPLC and were

g97%, except for 1ay and 1aae (95%), 1aac (90%), and 1az (96%). The

formulas were assessed by HRMS and were within (2 ppm. The NMR

spectra are described either in the Experimental Section or in the Supporting

Information.

1an

1ao

1ap

1aq

1ar

6,8-di-I

8-CH₃

6-I

6-nPr

6-allyl

H

CH₃CH₃Scheme 2 C₁₉H₁₈N₂O₄

1as^b

1at^d

1au^e

H

H^d

e

Scheme 1 C₁₄H₁₀N₂O₃

Scheme 1 C₁₄H₁₀N₂O₄

Scheme 1 C₁₄H₁₀N₂O₃

Compounds 1as-1au were similarly prepared in order to

complete the SAR (see footnotes of Table 1).

H

OH

^aFor all compounds A ) B ) CH, except for 1ak, where A ) N, B )

CH, and 1al, where A ) CH, B ) N. ^bCompound obtained by the same

sequence as described in Scheme 1 starting with the commercial p-Me

aniline. ^cThe purities of the final compounds were assessed by HPLC and

were g97%, except for 1aq (93%), 1as (96%), 1ay (95%), and 1az (96%).

The formulas were assessed by HRMS and were within (2 ppm. The NMR

spectra are described either in the Experimental Section or in the Supporting

Information. ^dThe OR′′ substituent is in the para position, except for 1at,

where the substitution is on the meta position. ^eCompound 1au does not

contain any OR′′ group.

Compounds 1av-aaa (Table 2, R₁* H) were prepared by

the alternative general method depicted in Scheme 3. The 4,6-

dimethoxyanthranilic acid 4 was transformed to the key

intermediates 5,7-dimethoxy-3,1-benzoxazin-4-ones 9b-f by

reaction with the desired alkanoylanhydride (R₁* H) or

alkanoyl chloride. (Compound 9a was prepared by the reaction

of 4 with triethylorthoformate). Further reaction with 4-meth-

oxyaniline 6a gave the corresponding derivatives 10aa-af. The

cleavage of the methoxy groups by BBr₃or NaSEt yielded the

desired derivatives 1av-aaa (Table 2).

(dppf)Cl₂, afforded 8am. Cleavage of the methoxy groups

according to the procedure described above gave 1ao. The

5-methoxy group of compound 8aa (A ) B)CH, R₃) H) was

selectively cleaved by LiCl. The resulting phenol (1aj) was

alkylated with allyl bromide to yield 8ao. Compound 1ar was

obtained by Claisen rearrangement, and 1aq was obtained by

hydrogenation of 1ar followed by cleavage of the methoxy

groups.

Compounds 1aab-aae of Table 2 were prepared according

to Scheme 4.

Compounds 4 and 11 were reacted respectively with 1-iso-

cyanato-4-methoxybenzene 12a (X ) O) and 1-isothiocyanato-

4-methoxybenzene 12b (X ) S) to yield compounds 10ak and

10ag. The corresponding derivatives 1aae and 1aac were

obtained after ether cleavage as described earlier. Alkylation

Scheme 1^a

^a(i) oxalyl chloride, 165 °C 30 min; (ii) 33% NaOH/30% H₂O₂; (iii) triphosgene, 0 °C; (iv) N,N-(dimethylamino)pyridine, anhydrous dimethylacetamide,

110 °C, 24 h; (v) anhydrous triethylorthoformate, reflux 4 h; (vi) BBr₃, CH₂Cl₂, or EtSNa, DMF, reflux, or LiCl, DMF, 135 °C.

2442 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8

Gu¨ngo¨r et al.

Scheme 2^a

^a(i) I₂/H₅IO₆, EtOH, rt; (ii) I₂/H₅IO₆, EtOH, 50 °C; (iii) MeB(OH)₂, [PdCl₂(dppf)]CH₂Cl₂, K₃PO₄, THF, reflux; (iv) LiCl; DMA, 100-120 °C; (v) NaH,

allyl bromide, DMF, 0° to rt; (vi) diphenyl ether, 150 °C; (vii) CH₂Cl₂, BBr₃, rt, or EtSNa, DMF, reflux; (viii) H₂, 5% Pd/C, MeOH.

Scheme 3^a

Scheme 4^a

^a(i) Triethylorthoformate, 140 °C, 4 h; or alkanoyl chloride or anhydride,

TEA, CH₂Cl₂, 40 °C; (ii) xylene/reflux 4 h, or AcOH, 60 °C, 24 h; (iii)

CH₂Cl₂, BBr₃, rt, or EtSNa, DMF, reflux.

of the 2-mercapto derivative 10ag by methyl iodide followed

by cleavage of the methoxy groups, gave compound 1aab.

Compound 1aad was obtained from 10ak by POCl₃reaction

and ether cleavage. Compounds 1aaf and 1aag were prepared

similarly in order to complete the SAR.

^a(i) Trimethylsilyldiazomethane, THF, MeOH, rt; (ii) toluene, reflux,

15 h; (iii) 1 N NaOH, MeOH; CH₃I; rt, 3 days; (iv) POCl₃; (v) CH₂Cl₂,

BBr₃or EtSNa, DMF, reflux.

Compound 1aah was obtained by the alkylation of 1aj

followed by the cleavage of the 7- and 4′-methoxy groups using

EtSNa. 1aak was obtained in three steps from 1aa by benzyl-

ation of the 7- and 4′-hydroxy groups and methylation of the

5-hydroxy, followed by hydrogenolysis of the benzyl groups.

Compounds of formula 1ba-bj (X ) S) represented in Table

4 were prepared according to the classical thionylation procedure

using P₂S₅or Lawesson’s reagent (Scheme 7).

Cleavage of the methoxy groups under the BBr₃or NaSEt

reaction gave the corresponding derivatives 1ba-bj. To com-

plete the SAR, commercially available biochanin A was also

thionylated and the 4′-methoxy group cleaved in order to obtain

the “thiogenistein” analogue 13.

Compounds of formula 1aah to 1aas (with the exception of

1aah and 1aak) presented in Table 3 were prepared according

to Scheme 5. The 5-methoxy group of compound 8aa was

selectively cleaved as previously described (see Scheme 2). The

free 5-OH group was triflated with the N-phenyltrifluoromethane-

sulfonimide according to the literature.^{19a, b}

Palladium-based coupling chemistry then yielded compounds

8aq-ay. Ether cleavage reaction, according to one of the

previously described methods, gave compounds 1aai-aas.

Compounds 1aah and 1aak were obtained according to the

reaction sequences described in Scheme 6.

ERâ Modulators Based on Genistein

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2443

Scheme 6^a

Table 3. Structure and Physical-Chemical Characteristic of

Compounds 1aah-aas (R₁) H)

preparation

method

compd

R

formula^a

1aah^b

1aai

1aaj

1aak

1aal

1aam

1aan

1aao

OCH₂CH₃

NH₂

CH₃

OCH₃

CN

OTf

Scheme 6

Scheme 5

Scheme 6

Scheme 5

C₁₆H₁₄N₂O₄

C₁₄H₁₁N₃O₃

C₁₅H₁₂N₂O₃

C₁₅H₁₂N₂O₄

C₁₅H₉N₃O₃

C₁₅H₉F₃N₂O₆S

C₁₆H₁₄N₂O₃

C₁₆H₁₂N₂O₃

^a(i) Anhydrous DMA, NaH, 0 °C to rt; (ii) 0 °C, benzyl bromide 1 h

then CH₃I, 1 h at 0 °C, and 1 h at rt; (iii) Pd/C, H₂, EtOAc/EtOH (1:1), 4

h; (iv) DMF, K₂CO₃, C₂H₅I, 120 °C, 3 h; (v) DMF, EtSNa.

CH₂CH₃

1aap

1aaq

1aar

1aas

CO₂CH₃

Ph

CH₂OH

Scheme 5

C₁₆H₁₂N₂O₅

C₂₀H₁₄N₂O₃

C₁₅H₁₂N₂O₄

C₁₇H₁₄N₂O₃

Table 4. Structure and Physical-Chemical Characteristic of

Compounds 1ba-bj (X ) S) Prepared According to Scheme 7^a

^aThe purities of the final compounds were assessed by HPLC and were

g97%, except for 1aao (95%). The formulas were assessed by HRMS and

were within (2 ppm. The NMR spectra are described either in the

Experimental Section or in the Supporting Information. ^bCompound 1aah

(see Experimental Section) was obtained by alkylation of 1aj followed by

cleavage of the methoy groups by EtSNa.

compd

no.

R₃

R

R′

R′′

formula^b

1ba

1bb

1bc

1bd

1be

1bf^a

1bg

1bh

1bi

H

OH

H

C₁₄H₁₀N₂O₃S

C₁₄H₉FN₂O₃S

C₁₅H₁₂N₂O₃S

C₁₅H₁₂N₂O₂S

C₁₆H₁₄N₂O₂S

C₁₃H₉N₃O₃S

Scheme 5^a

3-F

3-CH₃OH

H

CH₃

CH₂CH₃

OH

OCOCH₃

OSO₂CF₃CH₃

OH CH₃

COCH₃COCH₃C₂₀H₁₆N₂O₆S

CH₃

C₁₇H₁₃F₃N₂O₅S₂

C₁₆H₁₄N₂O₃S

13^c

^aA is CH for all compounds except for 1bf, where A ) N. ^bThe purities

of the final compounds were assessed by HPLC and were g97%, except

for 1bf, 1bh (93%), and 1bi (95%). The formulas were assessed by HRMS

and were within (2 ppm. The NMR spectra are described either in the

Experimental Section or in the Supporting Information. ^c“Thiogenistein”.

Scheme 7^a

^a(i) LiCl, DMF, 100-120 °C; (ii) BBr₃, CH₂Cl₂; (iii) NaH, DMF,

N-phenyltrifluoromethanesulfonimide; (iv) Pd(OAc)₂BINAP, Cs₂CO₃; (vi)

CH₂Cl₂, BBr₃or EtSNa, DMF, reflux.

Biological Evaluation

To determine the affinity of compounds and selectivity for

ERR versus ERâ, an in vitro ligand binding assay with [³H]-

estrone as radioligand and biotinylated ligand binding domains

of ERâ and ERR were used in a scintillation proximity assay

(SPA). For the functional transcription activation assay, HeLa

cells were stably transfected with a construct expressing full

length human ERâ or ERR receptor cotransfected with a

luciferase reporter gene construct driven by an ERE-TK minimal

promoter. The potency of the compounds in the transactivation

assay was measured by their ability to activate the estrogen

response element (ERE) in a luciferase reporter gene assay.

^a(i) P₂S₅or Lawesson’s reagent, xylene, reflux; (ii) BBr₃, CH₂Cl₂; EtSNa,

DMF, reflux or Py/HCl, heat; (iii) Ac₂O, DMAP, Py, rt, 5 h.

(MolSoft, Inc., San Diego, CA) for automated docking, and

LOOK (Molecular Applications Group) for homology modeling.

An ERâ homology model was built from the then-known R

isozyme complexed with 17-â-estradiol.⁹

Molecular Modeling

Molecular modeling was performed using InsightII (Accelrys,

Inc., San Diego, CA) for graphical docking, Discover with the

CFF98 force field for conformational analysis (Accelrys), ICM

We developed our binding model for the quinazolines by first

overlaying the phenol ring of genistein with the A ring of

estradiol, found in the ERR crystal structure, and then transfer-

2444 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8

Gu¨ngo¨r et al.

effect. A similar steric effect was observed when substituents

were introduced at positions 6 and 8 (1am-1ar). Both electron-

withdrawing (1ag, 1ah) and electron-donating groups (1ac) at

the 2′-position were well-tolerated. Replacing either the 2′-

carbon or 3′-carbon with a nitrogen (1ak, 1al) resulted in a loss

of activity.

As mentioned in the molecular modeling section, the C2 and

C5 positions of the quinazolinone are oriented in the regions

were the ERR and ERâ differ in the binding pocket. Modifica-

tions of substituents at positions C2 and C5 of the quinazoline

may offer the opportunity to improve ERâ/ERR selectivity. We

initially focused on the C2-position of the quinazolinone.

Introduction of a number of alkyl groups, as well as heteroatoms,

at C2 (1av-1aag) led to a significant loss in activity. It was

hypothesized that these substituents at C2 were altering the

preferred dihedral angle of the distal phenyl moiety and thus

affecting the binding affinity. Nevertheless, it is interesting to

note that the binding affinity improved as the length of the alkyl

chain increased in size (compare 1ax with 1ay). However, this

trend was not observed in the transactivation assay.

Figure 2. 1ba docked to the ERâ pocket (ligand and residues colored

by atom type). Only the key residues of the ERâ binding site are shown

for simplicity (starting from the upper left corner and in a counter-

clockwise manner: Glu305, Arg346, Ile373, His475, and Met336).

ring the information to our ERâ homology model. Subsequent

analysis of the 3-arylquinazoline series proceeded through an

overlay onto genistein, and the result is shown in Figure 2. It

should be noted that our original genistein docked orientation

was subsequently confirmed by crystallographic analysis⁸

Our binding model (Figure 2) indicated that the quinazolines

are of an ideal length for forming tight hydrogen bonds between

the 4′-hydroxyl and Glu305 and Arg346 at one end of the pocket

and between the 7-hydroxyl and His475 at the other end.

Conformational analysis (Discover/CFF98) of genistein and the

quinazoline series suggest that the ring systems are at lowest

energy in an off-planar conformation, with planarity being

limited likely by steric repulsion between the ortho substituents.

Our model suggests that positions C2 and C5 of the quinazoline

are oriented in regions where ERR and ERâ differ in the ligand

binding domain. Specifically, Leu384 and Met421 in ERR are

Met336 and Ile373 in ERâ, respectively. Modifications of

substituents at positions C2 and C5 of the quinazoline may offer

the opportunity to improve ERâ/ERR selectivity.

We turned out attention to the C5-position of the quinazoli-

none. The 5-hydroxyl presumably participates in an intramo-

lecular hydrogen-bonding interaction with the 4-keto group.

Removal of the C5-hydroxyl group (1as) resulted in a 48-fold

loss in ERâ binding and a 12-fold loss in ERâ transactivation.

To evaluate the importance of this 5-hydroxyl, several analogues

were prepared (1aah-1aas). Homologation of the C5-hydroxyl

(1aar) led to a 7-fold improvement in ERâ binding (IC₅₀) 24

nM), but the selectivity dropped and the transactivation potency

remained weak. It is interesting to note that the hydroxymethyl

substitution (1aar) resulted in a dramatic improvement in

efficacy against ERR, possibly aided by hydrogen bonding

between the hydroxyl and Met421 sulfur. When the hydroxyl

was replaced with an NH₂(1aai), the binding and transactivation

potency as well as selectivity dropped considerably. Further loss

of activity was observed in the case of methoxy (1aak), nitrile

(1aal), and a bulky group such as phenyl (1aaq). Only small

alkyl groups such as methyl [1aaj, IC₅₀(ERâ) ) 317 nM, R/â

selectivity ) 30] and ethyl [1aan, IC₅₀(ERâ) ) 265 nM, R/â

selectivity ) 50] maintained good binding and transactivation

potency and selectivity. On the basis of modeling studies, these

small groups could make tight contact with the wall of the

pocket. Larger substituents have steric conflicts with residues

Ile376 and Leu380. Overall, the described structural modifica-

tions did not optimize the effect of the lead structure 1aa.

Result and Discussion

On the basis of the structure of genistein, a series of

3-arylquinazolinones was prepared. The structure-activity of

compounds 1aa to 1aas is shown in Table 5. Quinazolinone

1aa, the direct analogue of genistein, exhibited a 3-fold loss in

ERâ binding [IC₅₀(ERâ) ) 179 nM] while maintaining similar

activity in ERâ transactivation potency [EC₅₀(ERâ) ) 76 nM].

The ERâ versus ERR binding and transactivation selectivity was

improved significantly (62-fold in the binding and 215-fold in

the transactivation assay).

It is known that the hydrogen-bonding network between the

A-ring phenol and Glu305/Arg346 and the 17â-hydroxyl and

His 475 contributes significantly to the binding of 17â-estradiol.

As a result, we investigated the role that the 4′- and 7-hydroxyl

groups had on binding affinity in the quinazolinones. Any

changes to the hydrogen-bonding network either by removing

the 4′-hydroxy (1au), shifting the hydroxyl to the 3′-position

(1at), or protecting the hydroxyl as a methyl ether (1ab, 1aj)

led to significant loss in ERâ binding. Next we explored the

role of sterics and electronics in the hydrogen bonding of the

4′- and 7-hydroxyl groups. Introduction of a fluoro group (1ae)

at the 3′-position resulted in a 3-fold loss in ERâ binding

compared to 1aa, but ERâ versus ERR functional selectivity

was maintained. Larger groups (1ad, 1ai) at the 3′-position led

to a 10-fold loss in ERâ activity, presumably due to a steric

To further assess the role of the carbonyl function, we

prepared the thiocarbonyl analogue of 1aa. The resulting

compound 1ba (Table 6) had 4-fold improved binding potency

with similar selectivity compared to the parent compound 1aa.

Moreover, 1ba showed a 6-fold improvement in transactivation

potency and the best transactivation selectivity observed for the

quinazolinone series [transactivation EC₅₀(ERâ) ) 13 nM,

selectivity R/â ratio ) 215). It is postulated that this improve-

ment in binding affinity is due to the increased van der Waals

contact between the protein and the sulfur. The most potent

and selective compounds from the previous SAR studies were

thionylated, and their biological activities are represented in

Table 6, together with reference derivatives such as 17â-

estradiol, estrone, and genistein. In general, the thiocarbonyl

provided an increase in binding affinity that was observed on

both receptor subtypes; therefore, no significant improvements

of binding selectivity were observed. On the other hand, the

transactivation potency increased for every thionylated com-

pound, often with improved selectivity. To our knowledge this

ERâ Modulators Based on Genistein

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2445

Table 5. Structure and in Vitro Binding of Compounds 1aa-aas^a

³H-estrone IC₅₀(nM)^c

select

transactivation EC₅₀(nM)^d

select

compd

no.

R₁

R₂

R₃

R

R′

R′′ ^b

ERâ

ERR

R/â

ERâ

ERR

R/â

1aa

1ab

1ac

1ad

1ae

1af

1ag

1ah

1ai

H

CH₃

C₂H₅

nPr

nBu

iPr

iBu

SH

Cl

H

2′-CH₃

3′-Cl

3′-F

H

2′-F

2′-Cl

3′-CH₃

H

OH

H

CH₃

H

CH₃

H

CH₃

H

CH₃

H

CH ₃

H

CH₃

H

CH₃

H

179

9525

422

7211

650

39%

256

263

7487

2%

3531

51696

4081

12298

1275

3970

7305

NT^e

8609

3842

18472

11371

5752

3224

249

11097

25956

4382

55015

44175

10%

7485

6247

34626

0%

97982

66860

40%

28%

15297

29%

62

3

10

8

76 ( 8

2918

20%

5305

5137

3112

36%

1143

1427

4576

35%

3143

26%

8317

19%

9344

27%

12%

NT

39%

10082

17%

33%

9317

43%

17%

10%

NT

38

47%

111

48

4

27

1181 ( 220

117 ( 26

8506

190

251 ( 119

1254

14%

358

9763

593

1966

8870

1638

23%

NT

961

68

29

24

5

6

4

1aj

1ak^a

1al^a

1am

1an

1ao

28

1

9

14

1

8-I

6,8-di-I

8-CH₃

6-I

6-nPr

6-allyl

H

12

1ap

1aq

1ar

10649

NT

1.5

1as^b

1at^b

1au^b

1av

53640

43814

75588

33248

19437

5365

178

70399

NT

46%

11111

29%

70277

25%

48473

49%

15512

9635

10%

6

11

4

3

2

0.7

1.5

OH

H

904

11

1721

1583

1261

25%

7%

24%

OH

H

1aw

1ax

1ay

7.4

1az

48086

1aaa

1aab

1aac

1aad

1aae

1aaf

1aag

1aah

1aai

1aaj

1aak

1aal

1aam

1aan

1aao

NT^d

NT

72848

6994

13426

9379

46%

440553

44265

8242

317

3843

1985

8776

1313

26%

39%

983

2

0.5

7

18%

9268

14%

15%

12%

32%

2392

36%

29%

42%

4521

4624

OH

CH₃

4-OHPh

H

8

H

0.1

21%

25%

OCH₂CH₃

NH₂

CH₃

OCH₃

CN

H

2

30

2064

136

8506

7579 ( 511

3804

62

18

39%

H

73677

11979

265

13%

39%

13240

38%

OSO₂CF₃

CH₂CH₃

H

50

73

6

H

17004

750

1aap

1aaq

1aar

1aas

H

CO₂CH₃

Ph

CH₂OH

H

9536

30661

24

81324

101544

121

9

3

5

9406

11685

4457

729

6%

0%

16%

4261

2302

43968

19

6

^aFor all compounds A ) B ) CH, except for 1ak, where A ) N, B ) CH, and 1al, where A ) CH, B ) N. ^bFor all compounds the OR′′ substituent

is in the para position, except for 1at, where the substitution is on the meta position, and compound 1au, which does not contain any OR′′ group. ^cFor those

compounds the binding did not reach 50% inhibition at 100 µM, the percent inhibition at 100 µM is given, unless otherwise indicated. ^dTransactivation

values in bold repesent partial agonism, as these compounds “plateaued” at higher concentration and did not reach 100% activity even at 10 µM. For those

compounds where the transactivation was low and did not reach a plateau, and the EC₅₀could not be determined, the percent transactivation at 10 µM is

given, unless otherwise indicated. The values are an average of two to four experiments with standard deviation, except for few single assays that are the

average of duplicate determinations. ^eNT ) not tested.

“thio effect” has not been reported in the literature and offers

new insight in the SAR of ERâ and ERR modulators.

yl)-4(3H)-quinazolinone (1aa), an exact analogue of genistein,

acting as an agonist on both ER subtypes, exhibited improved

ERâ selectivity in binding and transactivation compared to

genistein [respectively 62-fold higher binding affinity [IC₅₀-

(ERâ) ) 179 nM] and 38-fold higher potency in the transcrip-

tion assay [EC₅₀(ERâ) ) 76 nM]]. To further investigate the

SAR, we prepared a series of analogues in which the substituents

in several positions were modified. To varying degrees, all of

the analogues showed preferential binding affinity for ERâ.

Conclusion

Several 3-arylquinazolinone analogues of genistein were

prepared and their biological activity toward ERR and ERâ

receptors evaluated. As expected, all three OH functionalities

play an important role, and blocking one of them gave poorer

binding (1ab or 1af vs 1aa). 5,7-Dihydroxy-3-(4-hydroxyphen-

2446 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8

Gu¨ngo¨r et al.

Table 6. Structure and in Vitro Binding of Compounds 1ba-bi (R₁) H) and Reference Compounds^a

³H-estrone IC₅₀(nM)^c

transactivation EC₅₀(nM)^d

compd

no.

select

R/â

select

R/â

R₃

R

R′

R′′

ERâ

47

102

658

79

156

515

26%

22%

11 ( 1

31 ( 6

61 ( 10

1340

ERR

2636

3080

2854

1753

2260

27569

11%

18%

ERâ^b

ERR

2793

776

2406

1669

1462

4859

18%

21%

25%

0.59 ( 0.17

166 ( 13

956 ( 8

1125

1ba

1bb

1bc

1bd

1be

1bf^a

1bg

1bh

1bi

H

OH

H

56

30

4

22

15

54

13 ( 6

215

39

15

8

28

3′-F

3′-CH₃

20

160

222

H

CH₃

CH₂CH₃

OH

OCOCH₃

OSO₂CF₃

OH

53

173

11342

23%

COCH₃

CH₃

COCH₃

CH₃

9%

28%

(+)-3,17â-estradiol

estrone

genistein

13, “thiogenistein ”

10 ( 2

29 ( 7

1973 ( 444

55883

1

32

42

0.84 ( 0.22

26 ( 11

73 ( 29

14

0.7

6

13

80

^aFor all compounds A ) CH, except for 1bf, where A ) N. ^bEC₅₀values for compounds in ERâ transactivation given in bold represent partial agonism,

as these compounds “plateaued” at higher concentration and did not reach 100% activity even at 10 µM. The values are the average of two to four experiments

with standard deviation, except for a few single determinations. ^cFor those compounds where the binding did not reach 50% inhibition at 100 µM, the

percent inhibition at 100 µM is given, unless otherwise indicated. ^dFor those compounds where the transactivation was low and did not reach a plateau and

the EC₅₀could not be determined, the percent transactivation at 10 µM is given, unless otherwise indicated.

roacetic acid, respectively. Solutions were dried with magnesium

sulfate unless otherwise noted. Melting points were recorded on a

Mel-Temp II capillary apparatus (Laboratory Devices Inc.) and are

uncorrected.

Many were also more potent in activating transcription through

ERâ than through ERR. Substitution of the 5-hydroxyl by an

ethyl group (e.g. 1aan) improved the ERâ selectivity in the

transcription assay by 2-fold, despite a 10-fold decrease of

selectivity in the binding mode [IC₅₀(ERâ) )265 nM; EC₅₀

(ERâ) ) 62].

Improvements in the potency and selectivity of these quinazoli-

none analogues were achieved with the discovery of the “thio

effect.” Specifically, 5,7-dihydroxy-3-(4-hydroxyphenyl)-4(3H)-

quinazolinethione (1ba) was found to be the most potent and

selective derivative both in the binding and transactivation assays

[respectively 56-fold higher binding affinity [IC₅₀(ERâ) ) 47

nM] and 215-fold higher potency in the transcription assay

[EC₅₀(ERâ) ) 13 nM]]. This “thio effect,” never reported in

the literature for ER modulators, was also observed in the thio

analogue of genistein itself, compound 13. These ERâ-selective

compounds may represent valuable tools in understanding the

differences in structure and biological function of ERâ and ERR

receptors.

Proton NMR (¹H NMR) and carbon NMR (¹³C NMR) spectra

were recorded on JEOL EC-400 or EC-500 spectrometers with

tetramethylsilane as an internal standard. Chemical shifts are

reported in parts per million (ppm, δ units). Coupling constants

are reported in units of hertz (Hz). Splitting patterns are designated

as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad.

Low-resolution mass spectra (MS) were recorded on a JEOL JMS-

AX505HA, JEOL SX-102, or SCIEX-API spectrometer. High-

resolution mass spectra were recorded on an AMD-604 (AMD

Electra GmbH) high-resolution double-focusing mass spectrometer

(Analytical Instrument Group, Raleigh, NC). Mass spectra were

acquired in the positive ion mode under electrospray ionization

(ESI) or fast atom bombardment (FAB) methods. Combustion

analyses were performed by Atlantic Microlabs, Inc., Norcross, GA.

General Procedures for the Synthesis of Compounds 1aa-

al, According to Scheme 1. 4,6-Dimethoxyindole-2,3-dione (3).

3,5-Dimethoxyaniline hydrochloride (2) (12 g, 63 mmol) and oxalyl

chloride (20 mL, 230 mol) were reacted at 165 °C for 30 min. The

excess oxalyl chloride was removed by distillation to give a green-

yellow residue. The reaction mixture was cooled and methanol was

added. The resulting suspension was heated, filtered, washed with

methanol, and dried to yield 13 g of 4,6-dimethoxyindole-2,3-dione

Experimental Section

Chemistry. All commercial chemicals and solvents are reagent

grade and were used without further purification, unless otherwise

specified. The following solvents and reagents have been abbrevi-

ated: tetrahydrofuran (THF), ethyl ether (Et₂O), dimethyl sulfoxide

(DMSO), ethyl acetate (EtOAc), dichloromethane (DCM), trifluo-

roacetic acid (TFA), dimethylformamide (DMF), methanol (MeOH),

N,N-(dimethylamino)pyridine (DMAP), triethylamine (TEA), and

sodium hexamethyldisilazane (NaHMDS). All reactions except

those in aqueous media were carried out with the use of standard

techniques for the exclusion of moisture. Reactions were monitored

by thin-layer chromatography on 0.25 mm silica gel plates (60F-

254, E. Merck) and visualized with UV light, iodine vapors, 12-

molibdatophosphoric acid hydrate (CAS No. 51429-74-4) or

potassium permanganate solution (CAS No. 7722-64-7). Final

compounds were typically purified by flash chromatography on

silica gel (E. Merck 0.040-0.063 mm) or by preparative reverse-

phase high-pressure liquid chromatography. Analytical and prepara-

tive HPLC analyses were performed on YMC columns (S-5, 120A

ODS, 4.6 × 150 mm; S-10, 120A ODS, 30 × 500 mm) with

MeOH:water gradients containing 0.2%H₃PO₄and 0.1% trifluo-

1

3 (100%): LC/MS (ESI) (M + H)⁺) 208; mp 300-304 °C; H

NMR (400 MHz, DMSO-d₆) δ 10.9 (bs, 1H), 6.16 (d, J ) 1.76

Hz, 1H), 6.00 (d, J ) 1.76 Hz, 1H), 3.91 (s, 3H), 3.88 (s, 3H).

4,6-Dimethoxyanthranilic Acid (4) To a heated mixture of 4,6-

dimethoxyindole-2,3-dione (3) (13 g, 63 mmol, in an oversized

flask) in 33% NaOH solution was added carefully 20 mL of 30%

solution of H₂O₂. A vigorous exothermic reaction occurs. After all

the H₂O₂was added, the reaction mixture was maintained at 100

°C for an additional 10 min. The pH of the solution was brought

to 8 with concentrated HCl and acidified to pH 5-6 with acetic

acid. The solid was filtered, washed with water, and dried to yield

6.2 g of 4,6-dimethoxyanthranilic acid (4) as a pale brown solid

(50%): LC/MS (ESI) (M + H)⁺) 198; mp 120-125 °C; ¹H NMR

(400 MHz, DMSO-d₆) δ 5.94 (d, J ) 1.76 Hz, 1H), 5.79 (d, J )

1.76 Hz, 1H), 3.75 (s, 3H), 3.71 (s, 3H).

1,2-Dihydro-5,7-dimethoxy-3,1-benzoxazine-2,4-dione (4,6-

Dimethoxyisatoic Anhydride) (5) To a cooled (0 °C) brown

ERâ Modulators Based on Genistein

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2447

solution of 4,6-dimethoxyanthranilic acid (4) (76.0 g, 0.385 mol)

in THF (1.3 L) was added triphosgene (40.0 g, 0.135 mol) in

portions over a 20-min period. After 30 min, the reaction was

warmed to room temperature and stirred for 1.5 h. The reaction

mixture was poured into cold (0 °C) water. Additional water was

added to facilitate the stirring of the thick solid formed. After stirring

for 30 min, the reaction mixture was filtered to give a beige solid.

The solid was washed with water, air-dried, and then dried under

high vacuum to give 79.2 g (92%) of the isatoic anhydride 5: LC/

1H), 3.88 (s, 3H), 3.83 (s, 3H); HRMS (ESI, M + H⁺) calcd for

C₂₈H₃₂N₂O₃445.2481, found 445.2592.

2-Amino-4,6-dimethoxy-N-(4-methoxyphenyl)benzamide (7a).

A suspension of 5 (3.75 g, 16.8 mmol), p-anisidine (5.15 g, 42.0

mmol), and DMAP (0.205 g, 1.68 mmol) in 37.3 mL of N,N-

dimethylacetamide was warmed to 110 °C for 24 h. The dark red-

brown solution was cooled to room temperature, diluted with 70

mL of water, and extracted with ethyl acetate. The combined organic

layers were washed with brine, dried, and concentrated to yield a

dark purple solid. Purification by column chromatography on silica

gel (2.5% of MeOH in CH₂Cl₂) yielded 3 g (59%) of 7a: ¹H NMR

(400 MHz, CDCl₃) δ 9.53 (s, 1H), 7.46 (d, J ) 8.8 Hz, 2H), 6.89

(d, J ) 8.8 Hz, 2H), 6.36 (bs, 2H), 5.85 (d, J ) 2.2 Hz, 1H), 5.82

(d, J ) 2.2 Hz, 1H), 3.93 (s, 3H), 3.81 (s, 3H), 3.79 (s, 3H).

2-Amino-4,6-dimethoxy-N-(4-methoxy-2-methylphenyl)benz-

amide (7b). A mixture of isatoic anhydride 5 (150 mg, 0.673

mmol), 4-methoxy-2-methylaniline (184 mg, 1.34 mmol), and

DMAP (5 mg) in N,N-dimethylacetamide (1.5 mL) was heated at

110 °C overnight. The solvent was removed in vacuo and the

residue was dissolved in EtOAc and water. The layers were

separated, and the aqueous layer was extracted with EtOAc (2×).

The combined organic layers were washed with water and brine,

dried over Na₂SO₄, and concentrated. Purification via column

chromatography on silica gel (35% EtOAc in hexane) gave 155

mg (72%) of 7b as a pink solid: LC/MS (ESI) (M + H)⁺) 317.

2-Amino-N-(2-fluoro-4-methoxyphenyl)-4,6-dimethoxybenz-

amide (7d). To a mixture of 4-amino-3-fluoroanisole (200 mg, 1.42

mmol) and NaHMDS (1 M) in THF (4.39 mmol) were added 5

(320 mg, 1.42 mmol) and 2 mL of N,N-dimethylacetamide. The

resulting suspension was heated at 90 °C for 4 h. The reaction

mixture was cooled to room temperature and diluted with EtOAc

and 1 N HCl. The layers were separated, and the aqueous layer

was extracted with EtOAc (2×). The combined organic layers were

washed with water, saturated NaHCO₃, and brine; dried over Na₂-

SO₄; and concentrated. Purification via column chromatography

on silica gel (30% EtOAc in hexane) gave 220 mg (48%) of 7d as

1

MS (ESI) (M - H)^-) 221.9; mp 287-293 °C (dec); H NMR

(400 MHz, DMSO-d₆) δ 11.51 (bs, 1H), 6.36 (d, J ) 1.76 Hz,

1H), 6.20 (d, J ) 1.76 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H).

5,7-Dimethoxy-3-(4-methoxyphenyl)-4(3H)-quinazolinone (8aa)

A mixture of the isatoic anhydride 5 (50 g, 0.224 mol), p-anisidine

(68.9 g, 0.560 mol), DMAP (2.73 g, 0.022 mol), and 500 mL of

anhydrous dimethylacetamide was warmed to 110 °C. A clear

brown solution formed at 80-90 °C, and bubbling was observed

(CO₂loss). After 24 h, the reaction was cooled to room temperature,

diluted with water (1 L), and extracted with EtOAc (3 × 500 mL).

The combined organic layers were washed with brine (1 × 250

mL), dried over MgSO₄, filtered, and concentrated to give a brown

residue weighing 85.2 g. The brown residue was dissolved in

anhydrous triethylorthoformate (720 mL) and warmed to reflux for

4 h. Upon cooling to room temperature an off-white solid

precipitated. Filtration, washing with triethyl orthoformate (1 ×

1L), air-drying, and drying under high vacuum gave 55.1 g (79%)

of 5,7-dimethoxy-3-(4-methoxyphenyl)quinazoline-4-one (8aa), as

1

an off white solid: LC/MS (ESI) (M + H)⁺) 313.1; H NMR

(400 MHz, DMSO-d₆) δ 8.17 (s, 1H), 7.37 (d, 2H, J ) 8.8 Hz),

7.06 (d, 2H, J ) 8.8 Hz), 6.73 (d, 1H, J ) 2.2 Hz), 6.62 (d, 1H,

J ) 2.2 Hz), 3.9 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H).

Ether Cleavage with BBr₃. 5,7-Dihydroxy-3-(4-hydroxyphen-

yl)-4(3H)-quinazolinone (1aa). To an ice cooled mixture of 5,7-

dimethoxy-3-(4-methoxyphenyl)-4(3H)-quinazolinone (8aa) (365

mg, 1.17 mmol) in 10 mL of CH₂Cl₂was added 3 mL (31.7 mmol)

of BBr₃. The mixture was stirred at room temperature for 2 days.

The solvent was evaporated, and the obtained residue was treated

with a cold NaHCO₃solution and extracted with EtOAc. The

organic layer was washed with water and brine, dried over MgSO₄,

and evaporated to yield a solid. Purification by flash chromatog-

raphy on silica gel (loaded with CH₂Cl₂and eluted with 66% EtOAc

in hexane) gave 165 mg of 5,7-dihydroxy-3- (4-hydroxyphenyl)-

4(3H)-quinazolinone 1aa (40%) as a white solid: LC/MS (ESI)

1

a yellow solid: LC/MS (ESI) (M + H)⁺) 321.1; H NMR (400

MHz, CDCl₃) δ 10.0 (s, 1H), 8.26 (t, 1H), 6.70 (m, 2H), 6.42 (b,

2H), 5.85 (d, 1H), 5.82 (d, 1H), 3.94 (s, 3H), 3.79 (s, 2H).

5,7-Dimethoxy-3-(4-methoxy-2-methylphenyl)-4(3H)-quinazoli-

none (8ab) A suspension of 7b (150 mg, 0.475 mmol) in

triethylorthoformate (3 mL) was heated to reflux for 5 h. The solvent

was removed in vacuo to give a pink color solid. Purification via

column chromatography on silica gel (70% EtOAc in hexane)

provided 140 mg (90%) of 5,7-dimethoxy-3-(4-methoxy-2-meth-

ylphenyl)-4(3H)-quinazolinone (8ab) as an off-white solid: LC/

MS (ESI) (M + H)⁺) 327.1; ¹H NMR (400 MHz, CDCl₃) δ 7.90

(s, 1H), 7.12 (d, J ) 8.8 Hz, 1H), 6.87 (d, J ) 2.6 Hz, 1H), 6.83

(dd, J ) 2.6, 8.8 Hz, 1H), 6.76 (d, J ) 2.2 Hz, 1H), 6.51 (d, J )

2.2 Hz, 1H), 3.94 (s, 6H), 3.84 (s, 3H) 2.1 (s, 3H).

1

(M + H)⁺) 271.1; mp 240-245 °C; H NMR (CDCl₃) δ 6.46

(1H, d, H6), 6.62 (1H, d, H8), 7.0 (2H, d, H3′, 5′), 7.26 (2H, d,

H2′, 6′), 8.09 (1H, s, H2).

Ether Cleavage with EtSNa. 5,7-Dihydroxy-3-(4-hydroxy-

phenyl)-4(3H)-quinazolinone (1aa). A mixture of 5,7-dimethoxy-

3- (4-methoxyphenyl)-4(3H)-quinazolinone (8aa) (100 mg, 0.32

mmol) and EtSNa (524 mg, 6.24 mmol) in 2 mL of DMF was

refluxed for 3.5 h. The DMF was evaporated and the residue was

dissolved in water. Concentrated HCl was added to adjust the pH

to 5. The precipitate was filtered, dried, and purified by chroma-

tography on silica gel (loaded with CH₂Cl₂and a slight amount of

MeOH, eluted with 70% EtOAc in hexane) to yield 51 mg of 5,7-

dihydroxy-3- (4-hydroxyphenyl)-4(3H)-quinazolinone (1aa) (59%)

as an off-white solid.

According to the general methods described above the following

compounds were obtained.

5-Hydroxy-3-(4-hydroxyphenyl)-7-methoxy-4(3H)-quinazoli-

none (1ab): C₁₅H₁₂N₂O₄) 284.27 g/mol; LC/MS (ESI) (M +

H)⁺) 285.2, (M - H)^-) 283.0; ¹H NMR (DMSO-d₆) δ 11.2 (s,

1H, OH), 9.95 (s, 1H, OH), 8.22 (s, 1H, H2), 7.38 (d, 2H, H2′,6′),

6.98 (d, 2H, H3′,5′), 6.72 (d, 1H, H8), 6.55 (d, 1H, H6), 3.90 (s,

3H, OCH₃).

Ether Cleavage with LiCl. 5-Hydroxy-7-methoxy-3-(4-meth-

oxyphenyl)-4(3H)-quinazolinone (1aj). A suspension of the 8aa

(2.40 g, 7.68 mmol) and anhydrous lithium chloride (6.51 g, 153.6

mmol) in anhydrous DMF (51.2 mL) was warmed to 135 °C. After

2.5 h, the reaction was cooled to room temperature, poured into

water (100 mL), and acidified with 1.0 N HCl to give a white

precipitate. The resulting mixture was extracted with CH₂Cl₂(3×).

The combined organic layers were washed with brine, dried over

MgSO₄, filtered, and concentrated to give a white suspension in

DMF. Filtration provided 1.10 g (48%) of 1aj as a white solid:

LC/MS (ESI) (M + H)⁺) 299.1; ¹H NMR (400 MHz, DMSO-d₆)

δ 11.77 (s, 1H), 8.26 (s, 1H), 7.48 (d, J ) 8.4 Hz, 2H), 7.10 (d,

J ) 8.8 Hz, 2H), 6.72 (d, J ) 2.2 Hz, 1H), 6.54 (d, J ) 1.3 Hz,

3-(4-Hydroxy-2-methylphenyl)-5,7-dihydroxy-4(3H)-quinazoli-

none (1ac): C₁₅H₁₂N₂O₄) 284 g/mol; LC/MS (ESI) (M + H)⁺

)

1

285.0; H NMR (DMSO-d₆) δ 11.7 (s, 1H), 10.7 (s, 1H), 9.79 (s,

1H), 8.09 (s, 1H), 7.20 (d, J ) 8.4 Hz, 1H), 6.78 (d, J ) 2.2 Hz,

1H), 6.72 (dd, J ) 2.6 Hz and J ) 8.8 Hz, 1H), 6.53 (d, J ) 1.8

Hz, 1H), 6.34 (d, J ) 2.2 Hz, 1H), 2.01 (s, 3H).

3-(3-Chloro-4-hydroxyphenyl)-5,7-dihydroxy-4(3H)-quinazoli-

none (1ad): C₁₄H₉ClN₂O₄) 304 g/mol; ESI/MS (M + H)⁺

)

1

304.8; H NMR (DMSO-d₆) δ 11.7 (s, 1H), 10.7 (s, 1H), 8.18 (s,

1H), 7.62 (d, 1H), 7.30 (d, 1H), 7.08 (d, 1H), 6.52 (d, 1H), 6.34

(d, 1H).

5,7-Dihydroxy-3-(3-Fluoro-4-hydroxyphenyl)-4(3H)-quinazoli-

none (1ae): C₁₄H₉FN₂O₄) 288 g/mol; LC/MS (ESI) (M + H)⁺

2448 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8

Gu¨ngo¨r et al.

1

) 288.9; H NMR (DMSO-d₆) δ 11.7 (s, 1H), 10.6 (b), 8.18 (s,

without further purification: ¹H NMR (400 MHz, CDCl₃) δ 8.15

(s, 1H), 7.29 (d, J ) 8.8 Hz, 2H), 7.01 (d, J ) 8.8 Hz, 2H), 6.55

(s, 1H), 4.05 (s, 3H), 4.01 (s, 3H), 3.86 (s, 3H).

1H), 7.47 (dd, 1H), 7.18 (d, 1H), 7.07 (t, 1H), 6.52 (d, 1H), 6.34

(d, 1H).

7-Hydroxy-3-(4-hydroxyphenyl)-5-methoxy-4(3H)-quinazoli-

A flame-dried flask containing 8al (0.299 g, 0.682 mmol),

methaneboronic acid (0.164 g, 2.74 mmol), K₃PO₄(0.5796 g, 2.73

mmol), and 1,1′-Bis(Diphenylphosphino)Ferrocene Palladium (II)

Chloride, Complex with Dichloromethane (1:1) [PdCl₂(dppf)]-

CH₂Cl₂(0.112 g, 0.137 mmol) was purged with argon. Next,

degassed THF (6.8 mL) was added and the reaction was placed in

a preheated oil bath (70 °C) for 10 h. The resulting yellow solution

was cooled to room temperature and diluted with dichloromethane

(50 mL). The organic layer was washed with water (25 mL) and

brine (25 mL) and filtered through a plug of Celite. The filtrate

was dried over MgSO₄, filtered, and concentrated to give an orange-

yellow solid weighing 0.479 g. Column chromatography on silica

gel (50% EtOAc/hexane) afforded 18.9 mg (8%) of 8am: ¹H NMR

(400 MHz, CDCl₃) δ 7.97 (s, 1H), 7.20 (d, J ) 8.4 Hz, 2H), 6.93

(d, J ) 8.4 Hz, 2H), 6.49 (s, 1H), 3.91 (s, 6H), 3.78 (s, 3H), 2.32

(s, 3H).

none (1af): C₁₅H₁₂N₂O₄) 284.27 g/mol; LC/MS (ESI) (M + H)⁺

1

) 285.2 and (M - H)^-) 283.0; H NMR (DMSO-d₆) δ 8.05 (s,

1H), 7.19 (d, J ) 8.8 Hz, 2H), 6.85 (d, J ) 8.4 Hz, 2H), 6.52 (s,

1H), 6.46 (s, 1H), 3.78 (s, 3H).

5,7-Dihydroxy-3-(2-fluoro-4-hydroxyphenyl)-4(3H)-quinazoli-

none (1ag): C₁₄H₉FN₂O₄) 288 g/mol; LC/MS (ESI) (M + H)⁺

1

) 288.8; H NMR (DMSO-d₆) δ 11.5 (s, 1H), 10.6 (b), 8.19 (s,

1H), 7.44 (t, 1H), 6.77 (dd, 1H), 6.77 (dd, 1H), 6.54 (d, 1H), 6.36

(s, 1H).

3-(2-Chloro-4-hydroxyphenyl)-5,7-dihydroxy-4(3H)-quinazoli-

none (1ah): C₁₄H₉ClN₂O₄) 304 g/mol; LC/MS (ESI) (M + H)⁺

1

) 304.8; H NMR (DMSO-d₆) δ 11.5 (s, 1H), 10.6 (b), 8.14 (s,

1H), 7.49 (d, 1H), 7.05 (d, 1H), 6.90 (dd, 1H), 6.55 (d, 1H), 6.36

(d, 1H).

5,7-Dihydroxy-3-(4-hydroxy-3-methylphenyl)-4(3H)-quinazoli-

none (1ai): C₁₅H₁₂N₂O₄) 284.27 g/mol; LC/MS (ESI) (M + H)⁺

To a cooled (0 °C) solution of 8am (0.0193 g, 0.058 mmol) in

dichloromethane (0.12 mL) was added boron tribromide (0.083 mL,

0.88 mmol). The reaction was allowed to warm to room temper-

ature. After 3 days, the reaction was added dropwise to a cooled

(0 °C), rapidly stirred biphasic mixture of saturated NaHCO₃and

ethyl acetate. The layers were separated, and the organic layer was

dried over MgSO₄, filtered, and concentrated to give 17.7 mg of

crude material. Column chromatography on silica gel (10%

methanol/dichloromethane) gave 9.6 mg (58%) of 1ao as an off-

white solid: C₁₅H₁₂N₂O₄; LC/MS (ESI) (M + H)⁺) 285.03; mp

1

) 284.8; LC/MS (ESI) (M - H)^-) 282.9; H NMR (400 MHz,

DMSO-d₆) δ 11.78 (s, 1H), 9.85 (s, 1H), 8.15 (s, 1H), 7.22 (s,

1H), 7.13 (d, J ) 8.4 Hz, 1H), 6.90 (d, J ) 8.8 Hz, 1H), 6.52 (s,

1H), 6.34 (s, 1H), 2.16 (s, 3H).

5,7-Dihydroxy-3-(5-hydroxy-2-pyridyl)-4(3H)-quinazoli-

none (1ak): C₁₃H₉N₃O₄) 271.06 g/mol; LC/MS (ESI) (M + H)⁺)

1

271.8 and (M - H)^-) 269.8; H NMR (400 MHz, DMSO-d₆) δ

11.64 (s, 1H), 10.65 (s, 1H), 8.3 (s, 1H), 8.13 (d, J ) 2.6 Hz, 1H),

7.59 (d, J ) 8.4 Hz, 1H), 7.4 (q, J ) 2.6 and 8.4 Hz, 1H), 6.56 (d,

J ) 1.8 Hz, 1H), 6.36 (d, J ) 1.8 Hz, 1H).

1

266-268 °C (dec); H NMR (400 MHz, MeOD₄) δ 8.04 (s, 1H),

7.26 (d, J ) 8.8 Hz, 2H), 6.92 (d, J ) 8.8 Hz, 2H), 6.39 (s, 1H),

5,7-Dihydroxy-3-(6-hydroxy-3-pyridinyl)-4(3H)-quinazoli-

none (1al): C₁₃H₉N₃O₄; LC/MS (ESI) (M + H)⁺) 272; mp >360

2.28 (s, 3H).

1

°C; H NMR (400 MHz, DMSO-d₆) δ 11.6 (s, 1H, OH), 10.7 (s,

5,7-Dihydroxy-3-(4-hydroxyphenyl)-6-iodo-3H-quinazolin-4-

one (1ap). To a solution of 5-hydroxy-7,4′-dimethoxy derivative

1aj (0.454 g, 1.52 mmol) in ethanol (7.6 mL) was added a solution

of iodine (0.300 g, 1.18 mmol) and periodic acid (0.064 g, 0.281

mmol) in ethanol (5.7 mL). The resulting orange suspension was

stirred at room temperature. After 24 h, additional periodic acid

(0.060 g, 0.263 mmol) was added to the reaction. After 18 h, the

reaction was concentrated, redissolved in dichloromethane (100

mL), washed with saturated Na₂SO₃(2 × 50 mL) and brine, dried

over MgSO₄, filtered, and concentrated to give 0.593 g of the crude

material. Column chromatography on silica gel (20% EtOAc/

hexane) gave 0.0232 g (3.6%) of 5-hydroxy-6-iodo-7-methoxy-3-

(4-methoxyphenyl)-3H-quinazolin-4-one: ¹H NMR (400 MHz,

CDCl₃) δ 12.5 (s, 1H), 7.98 (s, 1H), 7.26 (d, J ) 8.8 Hz, 2H), 6.99

(d, J ) 8.8 Hz, 2H), 6.66 (s, 1H), 3.95 (s, 3H), 3.80 (s, 3H).

To a cooled (0 °C) solution of the above compound (0.0232 g,

0.055 mmol) in dichloromethane (0.10 mL) was added boron

tribromide (0.051 mL, 0.54 mmol). The reaction was allowed to

warm to room temperature. After 24 h, the reaction was added

dropwise to a cooled (0 °C), rapidly stirred biphasic mixture of

saturated NaHCO₃and ethyl acetate. The layers were separated,

and the organic layer was dried over MgSO₄, filtered, and

concentrated to give 0.0202 g. Column chromatography on silica

gel (10% methanol/dichloromethane) gave 0.0010 g (4.6%) of 1ap

as a white solid: C₁₄H₉IN₂O₄; LC/MS (ESI) (M + H)⁺) 396.98;

1H, OH) 8.18 (s, 1H, H2), 7.77 (s, 1H, H6′), 7.59 (d, 1H, H4′),

6.51 (d, 1H, H8), 6.61 (d, 1H, H3′) 6.34 (d, 1H, H6).

7-Hydroxy-3-(4-hydroxyphenyl)-3H-quinazolin-4-one (1as):

C₁₄H₁₀N₂O₃) 254.24 g/mol; LC/MS (ESI) (M - H)^-) 255.1;

¹H NMR (CD₃OD) δ 6.93 (2H, d, J ) 8.8 Hz), 7.03 (1H, d, J )

2.6 Hz), 7.07 (1H, q, J ) 2.6 and 8.8 Hz), 7.28 (2H, d, J ) 8.8

Hz).

5,7-Dihydroxy-3-(3-hydroxyphenyl)-3H-quinazolin-4-one

1

(1at): C₁₄H₁₀N₂O₄; LC/MS (ESI) (M + H)⁺) 271.06; H NMR

(400 MHz, DMSO-d₆) δ 11.73 (s, 1H), 10.71 (bs, 1H), 9.92 (bs,

1H), 8.19 (s, 1H), 7.32 (m, 1H), 6.92 (m, 3H), 6.53 (s, 1H), 6.35

(s, 1H).

5,7-Dihydroxy-3-phenyl-3H-quinazolin-4-one (1au): C₁₄H₁₀N₂O₃;

1

LC/MS (ESI) (M + H)⁺) 255.07; H NMR (400 MHz, DMSO-

d₆) δ 11.75 (s, 1H), 10.77 (s, 1H), 8.28 (s, 1H), 7.61 (s, 5H), 6.6

(s, 1H), 6.41 (s, 1H).

Preparation of Compounds 1am-1ar According to Scheme

2. 5,7-Dihydroxy-3-(4-hydroxyphenyl)-8-iodo-3H-quinazolin-4-

one (1am) and 5,7-Dihydroxy-3-(4-hydroxyphenyl)-6,8-diiodo-

3H-quinazolin-4-one (1an). To a solution of 1aa (0.0603 g, 0.223

mmol) in ethanol (2 mL) was added a solution of iodine (0.0295

g, 0.116 mmol) and periodic acid (0.0173 g, 0.076 mmol) in ethanol

(2 mL). The reaction was stirred at room temperature overnight.

Concentration gave 0.103 g of the crude material. Purification by

preparative HPLC gave the 8-iodo derivative 1am and the 6,8-

diiodo derivative 1an. 1am: ¹H NMR (400 MHz, MeOD₄) δ 8.12

(s, 1H), 7.28 (d, J ) 8.8 Hz, 2H), 6.93 (d, J ) 8.8 Hz, 2H), 6.50

(s, 1H). 1an: ¹H NMR (400 MHz, MeOD₄) 8.19 (s, 1H), 7.29 (d,

J ) 8.8 Hz, 2H), 6.93 (d, J ) 8.8 Hz, 2H).

5,7-Dihydroxy-3-(4-hydroxyphenyl)-8-methyl-3H-quinazolin-

4-one (1ao). To a solution of 8aa (2.0 g, 6.41 mmol) in ethanol

(80 mL) was added a solution of iodine (1.62 g, 6.38 mmol) and

periodic acid (0.45 g, 1.974 mmol) in ethanol (80 mL). The reaction

was stirred at 50 °C overnight and then cooled to room temperature

and concentrated to give a solid residue. The residue was dissolved

in dichloromethane, washed with saturated sodium sulfite and brine,

dried over MgSO₄, filtered, and concentrated to give 2.73 g (97%)

of the 8-iodo derivative 8al as a yellow solid, which was used

1

mp 225-227 °C (dec); H NMR (400 MHz, MeOD₄) δ 8.15 (s,

1H), 7.28 (d, J ) 8.8 Hz, 2H), 6.93 (d, J ) 8.8 Hz, 2H), 6.68 (s,

1H).

5,7-Dihydroxy-3-(4-hydroxyphenyl)-6-propyl-3H-quinazolin-

4-one (1aq). To a cooled (0 °C) solution of 5-hydroxy-7,4′-

dimethoxyquinazolinone (1aj) (1.45 g, 4.85 mmol) in DMF (16

mL) was added NaH (0.250 g, 10.4 mmol). The reaction was

allowed to warm to room temperature. After 2 h, the reaction was

cooled to 0 °C and allyl bromide (0.46 mL, 5.3 mmol) was added.

The reaction was allowed to warm to room temperature and stir

for 18 h. The reaction was poured into water (200 mL) and extracted

with ethyl acetate (200 mL). The organic layer was washed with

water (3 × 100 mL) and brine (1 × 100 mL), dried over MgSO₄,

filtered, and concentrated to give 2.04 g. Column chromatography

ERâ Modulators Based on Genistein

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2449

on silica gel (50% EtOAc/hexane) gave 1.15 g (70%) of 8ao: ¹H

NMR (400 MHz, CDCl₃) δ 8.01 (s, 1H), 7.30 (d, J ) 8.8 Hz, 2H),

7.01 (d, J ) 8.8 Hz, 2H), 6.75 (d, J ) 2.6 Hz, 1H), 6.49 (d, J )

2.2 Hz, 1H), 6.10 (tdd, J ) 17.6, 10.1. 4.8 Hz, 1H), 5.64 (dq, J )

17.6, 1.8 Hz, 1H), 5.31 (dq, J ) 10.1, 1.4 Hz, 1H), 4.64 (dt, J )

4.8, 1.3 Hz, 2H), 3.92 (s, 3H), 3.85 (s, 3H).

A mixture of 8ao (0.654 g, 1.91 mmol) in diphenyl ether (1.9

mL) was heated to 150 °C for 24 h. The reaction was cooled and

purified directly. Column chromatography on silica gel (50%

EtOAc/hexane) gave 0.552 g (86%) of the 6-allyl derivative 1ar

as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 11.77 (s, 1H),

7.98 (s, 1H), 7.32 (d, J ) 8.8 Hz, 2H), 7.05 (d, J ) 8.8 Hz, 2H),

6.75 (s, 1H), 5.98 (tdd, J ) 17.2, 9.9, 6.2 Hz, 1H), 5.03 (dq, J )

17.2, 1.8 Hz, 1H), 4.98 (dq, J ) 9.9, 1.8 Hz, 1H), 3.95 (s, 3H),

3.87 (s, 3H), 3.48 (dt, J ) 6.2, 1.4 Hz, 2H).

) 236.09; ¹H NMR (CDCl₃, 400 MHz) δ 1.33 (3H, t, J ) 7.5 Hz),

2.66 (2H, q, J ) 7.5 Hz), 3.91 (3H, s), 3.97 (3H, s), 6.46 (1H, d,

J ) 2.2 Hz), 6.61 (1H, d, J ) 2.2 Hz).

5,7-Dimethoxy-3-(4-methoxyphenyl)-4(3H)-quinazolinone

(10aa). A mixture of 200 mg (0.97 mmol) of the benzoxazinone

9a, 119 mg (0.97 mmol) of p-anisidine, and 5 mL of xylene was

heated to reflux for 4 h. The reaction was concentrated. Column

chromatography on silica gel (40% EtOAc/hexane) gave 120 mg

of 5,7-dimethoxy-3-(4-methoxyphenyl)-4(3H)-quinazolinone (10aa),

which is identical to 8aa (40%), as a white solid: LC/MS (ESI)

1

(M + H)⁺) 313; H NMR (400 MHz, DMSO-d₆) δ 8.17 (s, 1H,

H2), 7.36 (d, 1H, H2′), 7.06 (d, 1H, H3′), 6.73 (d, 1H, H8), 6.61

(d, 1H, H6), 3.9 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H).

5,7-Dimethoxy-2-methyl-3-(4-methoxyphenyl)-4(3H)-quinazoli-

none (10ab). A mixture of 135.5 mg (0.612 mmol) of benzoxazi-

none 9b and p-anisidine (0.735 mmol) in glacial acetic acid (2 mL)

was heated to 60 °C. After 24 h, the reaction mixture was

concentrated in vacuo and then dissolved in ethyl acetate. The

organic layer was washed with saturated NaHCO₃, water, and brine;

dried over MgSO₄; filtered; and concentrated to give 23.7 mg of a

A suspension of 6-allyl derivative 1ar (0.075 g, 0.222 mmol)

and 10% Pd/C (0.0118 g) in a mixture of methanol (4.5 mL)/ethyl

acetate (2 mL) was purged with hydrogen and then stirred under a

hydrogen atmosphere. After 2 h, the reaction was filtered through

Celite and the filtrate was concentrated to give 0.065 g (91%) of

5-hydroxy-7-methoxy-3-(4-methoxyphenyl)-6-propyl-3H-quinazo-

lin-4-one as a gray solid, which was used without further purifica-

1

yellow oil (13.7%): LC/MS (ESI) (M + H)⁺) 327.0; H NMR

(CDCl₃) δ 2.19 (s, 3H, methyl), 3.86 (s, 3H, methoxy), 3.90 (s,

3H, methoxy), 3.91 (s, 3H, methoxy), 6.43 (d, 1H, H8), 6.68 (d,

1H, H6), 7.01 (d, 2H, H3′,5′), 7.13 (d, 2H, H2′,6′).

1

tion: C₁₉H₂₀N₂O₄; LC/MS (ESI) (M + H)⁺) 341.08; H NMR

(400 MHz, CDCl₃) δ 11.72 (s, 1H), 7.97 (s, 1H), 7.32 (d, J ) 8.8

Hz, 2H), 7.05 (d, J ) 8.8 Hz, 2H), 6.73 (s, 1H), 3.94 (s, 3H), 3.87

(s, 3H), 2.71-2.67 (m, 2H), 1.60-1.52 (m, 2H), 0.96 (t, J ) 7.3

Hz, 3H).

5,7-Dihydroxy-3-(4-hydroxyphenyl)-2-methyl-3H-quinazolin-

4-one (1av): C₁₅H₁₂N₂O₄) 284.27 g/mol; LC/MS (ESI) (M +

H)⁺) 285.04; LC/MS (ESI) (M - H)^-) 283.02; ¹H NMR (CD₃-

OD) δ 2.17 (s, 3H), 6.29 (d, J ) 2.2 Hz, 1H), 6.47 (d, J ) 2.2 Hz,

1H), 6.94 (d, J ) 8.8 Hz, 2H), 7.14 (d, J ) 8.8 Hz, 2H).

5,7-Dihydroxy-2-ethyl-3-(4-hydroxyphenyl)-3H-quinazolin-4-

one (1aw): C₁₆H₁₄N₂O₄) 298.30 g/mol; LC/MS (ESI) (M - H)^-

) 297.17; ¹H NMR (CD₃OD) δ 1.07 (t, J ) 7.5 Hz, 3H), 2.35 (q,

J ) 7.5 Hz, 2H), 6.20 (d, J ) 2.2 Hz, 1H), 6.44 (d, J ) 2.2 Hz,

1H), 6.85 (d, J ) 8.8 Hz, 2H), 7.05 (d, J ) 8.8 Hz, 2H).

5,7-Dihydroxy-3-(4-hydroxyphenyl)-2-propyl-3H-quinazolin-

4-one (1ax): C₁₇H₁₆N₂O₄) 312.g/mol; LC/MS (ESI) (M + H)⁺

) 313.1; LC/MS (ESI) (M - H)^-) 311.0; ¹H NMR (CD₃OD) δ

0.77 (t, J ) 7.46 Hz, 3H), 1.57 (m, 2H), 2.37 (t, J ) 7.7 Hz, 2H),

6.24 (d, J ) 2 Hz, 1H), 6.44 (d, J ) 2 Hz, 1H), 6.86 (d, J ) 8.8

Hz, 2H), 7.07 (d, J ) 8.8 Hz, 2H).

To a cooled (0 °C) solution of 5-hydroxy-7-methoxy-3-(4-

methoxyphenyl)-6-propyl-3H-quinazolin-4-one (0.0684 g, 0.201

mmol) in dichloromethane (0.40 mL) was added boron tribromide

(0.190 mL, 2.0 mmol). The reaction was allowed to warm to room

temperature. Additional boron tribromide (0.76 mL, 8.0 mmol) was

added over the course of several days. After 7 days, the reaction

was added dropwise to a cooled (0 °C), rapidly stirred biphasic

mixture of saturated NaHCO₃and ethyl acetate. The layers were

separated, and the organic layer was dried over MgSO₄, filtered,

and concentrated to give a yellow solid. Column chromatography

on silica gel (5% methanol/dichloromethane) gave 0.0010 g (1.5%)

of 1aq as a white solid: C₁₇H₁₆N₂O₄; LC/MS (ESI) (M + H)⁺

)

313.09; ¹H NMR (400 MHz, MeOD₄) δ 8.04 (s, 1H), 7.26 (d, J )

8.8 Hz, 2H), 6.92 (d, J ) 8.8 Hz, 2H), 6.59 (s, 1H), 2.67-2.63 (m,

2H), 1.58-1.53 (m, 2H), 0.94 (t, J ) 7.2 Hz, 3H).

2-Butyl-5,7-dihydroxy-3-(4-hydroxyphenyl)-3H-quinazolin-4-

one (1ay): C₁₈H₁₈N₂O₄) 326 g/mol; LC/MS (ESI) (M + H)⁺

)

1

General Procedure for the Synthesis of Compounds 1av-

1aaa according to Scheme 3. 5,7-Dimethoxy-3,1-benzoxazine-

4-one (9a). 4,6-Dimethoxyanthranilic acid (4) (1.0 g, 5.07 mmol)

and 15 mL of triethylorthoformate (90.2 mmol) were heated to 140

°C for 4 h. The reaction mixture was concentrated to give 0.8 g of

5,7-dimethoxy-3,1-benzoxazine-4-one (9a) as a yellow solid (76%

yield), which was used without any further purification: ¹H NMR

(400 MHz, CDCl₃) δ 7.74 (s, 1H), 6.66 (d, J ) 2.2 Hz, 1H), 6.52

(d, J ) 2.2 Hz, 1H), 3.98 (s, 3H), 3.93 (s, 3H).

327.13; LC/MS (ESI) (M - H)^-) 325.09; H NMR (CD₃OD) δ

0.71 (t, J ) 7.5 Hz, 3H), 1.14 (m, 2H), 1.50 (m, 2H), 2.32 (t, J )

7.9 Hz, 2H), 6.19 (d, J ) 2.2 Hz, 1H), 6.41 (d, J ) 2.2 Hz, 1H),

6.85 (d, J ) 8.6 Hz, 2H), 7.05 (d, J ) 8.6 Hz, 2H).

5,7-Dihydroxy-3-(4-hydroxyphenyl)-2-isopropyl-3H-quinazo-

lin-4-one (1az): C₁₇H₁₆N₂O₄) 312.33 g/mol; LC/MS (ESI)

1

(M + H)⁺) 313.0; LC/MS (ESI) (M - H)^-) 311.0; H NMR

(CD₃OD) δ 1.08 (d, J ) 6.6 Hz, 6H), 2.61 (m, 1H), 6.08 (d, J )

2.2 Hz, 1H), 6.34 (d, J ) 2.2 Hz, 1H), 6.83 (d, J ) 8.8 Hz, 2H),

7.01 (d, J ) 8.8 Hz, 2H).

5,7-Dimethoxy-2-methyl-3,1-benzoxazine-4-one (9b). To an-

thranilic acid 4 (388 mg, 1.97 mmol) in methylene chloride (10

mL) were added sequentially TEA (2.36 mmol) and acetic

anhydride (2.36 mmol). The reaction mixture was heated to 40 °C.

After 18 h, the reaction was poured into water and extracted with

methylene chloride. The organic layer was washed with saturated

NaHCO₃and brine, dried over MgSO₄, and concentrated in vacuo

to afford 5,7-dimethoxy-2-methyl-3,1-benzoxazine-4-one (9b)

5,7-Dihydroxy-3-(4-hydroxyphenyl)-2-isobutyl-3H-quinazolin-

4-one (1aaa): C₁₈H₁₈N₂O₄) 326 g/mol; LC/MS (ESI) (M + H)⁺

) 327.17; LC/MS (ESI) (M - H)^-) 325.12; ¹H NMR (CD₃OD)

δ 0.76 (d, J ) 6.6 Hz, 6H), 1.94 (m, 1H), 2.24 (d, J ) 7 Hz, 2H),

6.21 (d, J ) 2.2 Hz, 1H), 6.43 (d, J ) 2.2 Hz, 1H), 6.85 (d, J )

8.8 Hz, 2H), 7.03 (d, J ) 8.8 Hz, 2H).

General Procedure for the Synthesis of Compounds 1aab-

1aae According to Scheme 4. 5,7-Dimethoxy-3-(4-methoxyphen-

yl)-2,4-(1H,3H)-quinazolinedione (10ak). A mixture of anthranilic

acid 4 (627 mg, 3.18 mmol) and trimethylsilyldiazomethane (8 mL)

in MeOH (7 mL) and THF (7 mL) was stirred at room temperature

overnight. The reaction mixture was concentrated to yield 0.9 g of

methyl 2-amino-4,6-dimethoxybenzoate (11), which was used in

the next step without further purification.

1

(51%): LC/MS (ESI) (M + H)⁺) 222.07; H NMR (400 MHz,

DMSO-d₆) δ 6.58 (d, J ) 1.76 Hz, 1H), 6.46 (d, J ) 1.76 Hz,

1H), 3.84 (s, 3H), 3.80 (s, 3H), 2.24 (s, 3H).

5,7-Dimethoxy-2-ethyl-3,1-benzoxazine-4-one (9c). To a cooled

(0 °C) mixture of anthranilic acid 4 (207 mg, 1.05 mmol) and TEA

(2.73 mmol) in methylene chloride (3 mL) was added dropwise

propionyl chloride (2.52 mmol). The resulting solution was allowed

to warm to ambient temperature over 2 h. The reaction mixture

was quenched with water and extracted methylene chloride (2×).

The combined organic layers were washed with brine, dried over

MgSO₄, filtered, and concentrated in vacuo to afford 5,7-dimethoxy-

2-ethyl-3,1-benzoxazine-4-one (5c) (83%): LC/MS (ESI) (M + H)⁺

Compound 11 (489 mg, 2.48 mmol) was dissolved in anhydrous

ethyl acetate and stirred over 4 Å molecular sieves. 4-Methoxy-

benzoic isocyanate (2.48 mmol) was added and the reaction mixture

was heated to reflux for 1 h. The reaction mixture was cooled to

ambient temperature. The reaction mixture was filtered to remove

2450 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8

Gu¨ngo¨r et al.

the molecular sieves. The solvent was removed in vacuo to afford

a mixture of uncyclized product and cyclized product. The crude

mixture was taken up in ethanol and saturated with hydrochloric

acid. The reaction mixture was heated to reflux for 2 h and then

allowed to cool to ambient temperature and concentrated. Column

chromatography on silica gel (50% EtOAc/hexane) provided 479.9

mg (59%) of 10ak: LC/MS (ESI) (M + H)⁺) 329.13; LC/MS

J ) 8.8 Hz, 2H) 6.83 (d, J ) 2.2 Hz, 1H), 6.59 (d, J ) 2.2 Hz,

1H), 5.18 (s, 2H), 5.12 (s, 2H) 3.93 (s, 3H).

To a solution of 5-methoxydibenzyloxy compound (0.200 g,

0.430 mmol) in 1:1 EtOAc/ethanol (12.9 mL) was added 10%

palladium on carbon (0.100 g). Hydrogen (balloon) was bubbled

through the reaction for 5 min to purge the vessel and then the

reaction was stirred vigorously under a hydrogen atmosphere for 4

h. The reaction was filtered through Celite and the filtrate was

concentrated to give a white solid weighing 0.122 g. The crude

material was absorbed on silica gel (600 mg). Column chromatog-

raphy (7.5% MeOH in CH₂Cl₂) gave 0.015 g (12%) of 1aak as a

white solid: LC/MS (ESI) (M + H)⁺) 285; mp 240-360 °C

(dec); ¹H NMR (400 MHz, DMSO-d₆) δ 8.05 (s, 1H), 7.19 (d, J )

8.8 Hz, 2H), 6.85 (d, J ) 8.4 Hz, 2H), 6.52 (s, 1H), 6.46 (s, 1H),

3.78 (s, 3H).

1

(ESI) (M - H)^-) 327.07; H NMR (DMSO-d₆) δ 3.78 (3H, s,

methoxy), 3.79 (3H, s, methoxy), 3.83 (3H, s, methoxy), 6.28 (1H,

d, H8), 6.31 (1H, d, H6), 6.97 (2H, d, H3′,5′), 7.12 (2H, d, H2′,6′).

2-Mercapto-5,7-dimethoxy-3-(4-methoxyphenyl)-4(3H)-quin-

azolinone (10ag). A mixture of compound 11 (0.675 g, 3.19 mmol)

and 4-methoxyphenylisothiocyanate (0.525 g, 3.2 mmol) in toluene

(20 mL) was heated to reflux for 15 h. The solvent was evaporated

and the residue triturated with MeOH/CH₂Cl₂to yield 0.537 g (49%)

of 10ag as an off-white solid: LC/MS (ESI) (M + H)⁺) 345.2;

5-Ethoxy-7-hydroxy-3-(4-hydroxyphenyl)-4(3H)-quinazoli-

none (1aah). To a suspension of 5-hydroxy-7-methoxy-3-(4-

methoxyphenyl)-4(3H)-quinazolinone (1aj) (0.0.15 g, 0.0503 mmol)

in dimethylformamide (0.26 mL) were added potassium carbonate

(0.069 g, 0.503 mmol) and iodoethane (0.040 mL, 0.503 mmol).

The reaction vessel was placed in a preheated oil bath (120 °C).

After 3 h, the reaction was cooled to room temperature and excess

ethyl iodide was removed via rotary evaporator. The reaction

mixture was diluted with water (0.75 mL) and extracted with EtOAc

(3×). The combined organic layers were washed with brine (1×),

dried over MgSO₄, filtered, and concentrated to give a yellow solid

weighing 0.010 g. Absorption onto silica gel (60 mg) and column

chromatography (1:1 hexane/EtOAc) provided 0.0080 g (49%) of

5-ethoxy-7-methoxy-3-(4-methoxyphenyl)-4(3H)-quinazolinone as

1

mp 322-326 °C; H NMR (CDCl₃) δ 3.85 (3H, s, OCH₃), 3.9

(3H, s, OCH₃), 3.91 (3H, s, OCH₃) 6.06 (1H, d, H5), 6.08 (1H, d,

H7), 7.02 (1H, d, H3′,5′) 7.16 (2H, d, H2′,6′), 9.38 (1H, s, SH).

5,7-Dimethoxy-3-(4-methoxyphenyl)-2-(methylthio)-4(3H)-

quinazolinone (10ah). A mixture of 171 mg (0.497 mmol) of 10ag,

15 mL of MeOH, and 1 mL of 1 N NaOH was heated gently to

complete the dissolution. Next, 295 mg (2 mmol) of iodomethane

was then added and the reaction was stirred at room temperature

for 3 days. The precipitate was filtered to yield 89.9 mg (50%)

of 5,7-dimethoxy-2-methylthio-3-(4-methoxyphenyl)quinazolin-4-

one as an off-white solid: LC/MS (ESI) (M + H)⁺) 358.9; mp

212-215 °C; ¹H NMR (CDCl₃) δ 2.49 (3H, s, SCH₃), 3.86 (3H, s,

OCH₃), 3.9 (3H, s, OCH₃), 3.92 (3H, s, OCH₃) 6.38 (1H, d, H5),

6.64 (1H, d, H7), 7.00 (1H, d, H3′,5′) 7.17 (2H, d, H2′,6′).

5-Hydroxy-3-(4-hydroxyphenyl)-2-mercapto-7-methoxy-3H-

quinazolin-4-one (1aab): LC/MS (ESI) (M + H)⁺) 316.8; LC/

MS (ESI) (M - H)^-)314.8; mp 325-328 °C; ¹H NMR (CD₃OD)

δ 7.75 (1H, s), 7.03 (2H, d, J ) 9.1 Hz), 6.91 (2H, d, J ) 9.1 Hz),

6.28 (2H, s), 3.88 (3H, s).

1

a flaky white solid: LC/MS (ESI) (M + H)⁺) 327.2; H NMR

(400 MHz, CDCl₃) δ 8.0 (s, 1H), 7.29 (d, J ) 8.8 Hz, 2H), 7.00

(d, J ) 8.8 Hz, 2H), 6.74 (d, J ) 2.6 Hz, 1H), 6.48 (d, J ) 2.6,

1H),4.12 (q, J ) 7.0, 2H), 3.92 (s, 3H), 3.85 (s, 3H), 1.52 (t, J )

7.0 Hz, 3H).

To a solution of 5-ethoxy-7-methoxy-3-(4-methoxyphenyl)-

4(3H)-quinazolinone (0.0080 g, 0.0245 mmol) in dimethylforma-

mide (0.25 mL) was added sodium ethanethiolate (0.0047 g, 0.0563

mmol). The resulting suspension was warmed to 120 °C, at which

time a solution formed. After each hour (for 7 h) additional sodium

ethanethiolate (0.0047, 0.0563 mmol) was added. The cloudy orange

reaction was cooled to room temperature and diluted with water

(0.75 mL). The resulting yellow solution was acidified with 1.0 N

HCl (pH 3) and the reaction became cloudy. The reaction was

extracted with EtOAc (3×) and dichloromethane (3×). The

combined organic layers were washed with brine (1×), dried over

MgSO₄, filtered, and concentrated. Column chromatography on

silica gel (5% MeOH in CH₂Cl₂) gave 0.0017 g (23%) of 5-ethoxy-

7-hydroxy-3-(4-hydroxyphenyl)quinazolin-4-one (1aah), as a white

solid: LC/MS (ESI) (M + H)⁺) 299.1; ¹H NMR (400 MHz, CD₃-

OD) δ 8.09 (s, 1H), 7.22 (d, J ) 8.8 Hz, 2H), 6.90 (d, J ) 8.2 Hz,

2H), 6.61 (d, J ) 2.2 Hz, 1H), 6.52 (d, J ) 2.2 Hz, 1H), 4.13 (q,

J ) 7.0 Hz, 2H), 1.44 (t, J ) 7.0 Hz, 3H).

5,7-Dihydroxy-3-(4-hydroxyphenyl)-2-mercapto-3H-quinazo-

lin-4-one (1aac): LC/MS (ESI) (M + H)⁺) 303; mp 320-326

°C; ¹H NMR (400 MHz, CD₃OD) δ 7.01 (2H, d, J ) 8.8 Hz), 6.86

(2H, d, J ) 8.8 Hz), 6.16 (1H, d, J ) 1.9 Hz), 6.11 (1H, d, J ) 1.9

Hz).

2-Chloro-5,7-dihydroxy-3-(4-hydroxyphenyl)-3H-quinazolin-

4-one (1aad): LC/MS (ESI) (M + H)⁺) 304.9; LC/MS (ESI)

1

(M - H)^-) 302.96; H NMR (CD₃OD) δ 7.16 (2H, m), 6.91

(2H, m), 6.47 (1H, d, J ) 2.2 Hz), 6.35 (1H, d, J ) 2.2 Hz).

5,7-Dihydroxy-3-(4-hydroxyphenyl)-1H-quinazoline-2,4-di-

one (1aae): LC/MS (ESI) (M + H)⁺) 287.02; LC/MS (ESI)

1

(M - H)^-) 284.99; H NMR (CD₃OD) δ 5.98 (1H, d, J ) 2.2

Hz), 6.09 (1H, d, J ) 2.2 Hz), 6.68 (2H, d, J ) 8.8 Hz), 7.33 (2H,

d, J ) 8.8 Hz).

General Procedure for the Synthesis of Compounds 1aah-

1aas According to Schemes 5 and 6. 7-Hydroxy-5-methoxy-3-

(4-hydroxyphenyl)-4(3H)-quinazolinone (1aak). To a cooled (0

°C), clear, colorless solution of 5,7-dihydroxy-3-(4-hydroxyphenyl)-

4(3H)-quinazolinone (1aa) (0.330 g, 1.22 mmol) in anhydrous

dimethylacetamide (18.5 mL) was added sodium hydride (60%

dispersion, 0.274 g, 6.85 mmol) in five portions over a 25 min

period. Following the final addition, the reaction mixture was stirred

for an additional 30 min at 0 °C and then warmed to room

temperature for 1.5 h. Upon recooling to 0 °C, benzyl bromide

(0.29 mL, 2.44 mmol) was added. The reaction stirred at 0 °C for

1 h, and over this time, the reaction became a clear, orange solution.

Next, iodomethane (0.14 mL, 2.22 mmol) was added. After 1 h at

0 °C, the reaction was allowed to warm to room temperature and

stir for 1 h. The reaction was diluted with water (45 mL) and

extracted with EtOAc (2 × 10 mL). The combined organic layers

were washed with brine (1 × 5 mL), dried over MgSO₄, filtered,

and concentrated to give an orange residue weighing 0.67 g. Column

chromatography on silica gel (1:1 hexane/EtOAc) provided 0.25 g

(44%) 5-methoxydibenzyloxy compound, as a white foam: LC/

MS (ESI) (M + H)⁺) 465.2; ¹H NMR (400 MHz, CDCl₃) δ 8.02

(s, 1H), 7.48-7.32 (m, 10H), 7.29 (d, J ) 9.2 Hz, 2H), 7.07 (d,

Trifluoromethanesulfonic Acid 7-Hydroxy-3-(4-hydroxyphen-

yl)-4-oxo-3,4-dihydroquinazolin-5-yl Ester (1aam). To a cooled

(0 °C) solution of 1aj (5.83 g, 19.54 mmol) in DMF (65 mL) was

added in portions 60% sodium hydride in mineral oil (1.01 g, 25.41

mmol). Gas evolution was observed and after 30 min at 0 °C, the

clear, slightly yellow solution was warmed to room temperature.

After 30 min, the reaction was cooled to 0 °C and N-phenyltri-

fluoromethanesulfonimide (7.33 g, 20.52 mmol) was added. The

reaction was stirred at 0 °C for 30 min and then warmed to room

temperature. After 30 min, the reaction was quenched with saturated

NH₄Cl and diluted with water (150 mL). The reaction was extracted

with EtOAc (3 × 60 mL). The combined organic layers were

washed with brine, dried over MgSO₄, filtered, and concentrated

to give an off-white solid. Column chromatography on silica gel

(1:1 hexane/EtOAc) provided 7.13 g (85%) of 8ah as a white

1

solid: LC/MS (ESI) (M + H)⁺) 430.9; H NMR (400 MHz,

CDCl₃) δ 8.09 (s, 1H), 7.32 (d, J ) 8.8 Hz, 2H), 7.18 (d, J ) 2.2

Hz, 1H), 7.03 (d, J ) 8.8 Hz, 2H), 6.92 (d, J ) 2.2 Hz, 1H), 3.96

(s, 3H), 3.85 (s, 3H).

ERâ Modulators Based on Genistein

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2451

To a cooled (0 °C), clear, yellow solution of 8ah (1.88 g, 4.36

mmol) in CH₂Cl₂(43.6 mL) was added dropwise over 5 min boron

tribromide (6.20 mL, 65.5 mmol). The reaction was stirred at 0 °C

for 1 h and then warmed to room temperature. After 7 days, the

reaction was added dropwise to a vigorously stirred, cold (ice bath)

mixture of ethyl acetate (150 mL) and saturated NaHCO₃(100 mL).

The mixture was stirred for 10 min, and then the layers were

separated. The aqueous layer was extracted with EtOAc (2 × 20

mL). The combined organic layers were washed with brine, dried

over MgSO₄, filtered, and concentrated to give an orange foam

weighing 2.47 g. Column chromatography on silica gel (5% MeOH

in CH₂Cl₂) gave 1.63 g (93%) of 1aam as a white foam: LC/MS

(ESI) (M + H)⁺) 402.9; ¹H NMR (400 MHz, DMSO-d₆) δ 11.39

(s, 1H), 9.86 (s, 1H), 8.29 (s, 1H), 7.28 (d, J ) 8.6 Hz, 2H), 7.08

(bs, 1H), 6.95 (bs, 1H), 6.90 (d, J ) 8.6 Hz, 2H).

7-Hydroxy-3-(4-hydroxyphenyl)-5-methyl-3H-quinazolin-4-

one (1aaj). A flame-dried flask containing 8ah (0.300 g, 0.697

mmol), [PdCl₂(dppf)]CH₂Cl₂(0.114 g, 0.139 mmol), potassium

phosphate tribasic (0.592 g, 2.78 mmol), and methylboronic acid

(0.166 g, 2.78 mmol) was purged with argon for 10 min, and then

degassed THF (4.65 mL) was added. The resulting dark red-purple

mixture was placed in a preheated oil bath (73 °C). After 5 h, the

reaction was cooled to room temperature, diluted with methylene

chloride (20 mL), washed with water (5 mL) and brine (5 mL),

and filtered through a plug of Celite. The filtrate was dried over

MgSO₄, filtered, and concentrated to give an off-white solid

weighing 0.282 g. Column chromatography on silica gel (1:1

hexane/EtOAc) gave 0.174 g (84%) of 8ar as a white solid: ¹H

NMR (500 MHz, CDCl₃) δ 8.03 (s, 1H), 7.31 (d, J ) 8.8 Hz, 2H),

7.03 (d, J ) 8.8 Hz, 2H), 7.00 (d, J ) 2.5 Hz, 1H), 6.86 (d, J )

2.5 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 2.82 (s, 3H).

were added. The reaction was purged with carbon monoxide

(balloon) as described above. The reaction was placed in a preheated

oil bath (70 °C). After 2 h, the dark orange reaction was cooled to

room temperature. The reaction was diluted with water (6 mL) and

extracted with methylene chloride (3 × 5 mL). The combined

organic layers were washed with brine, dried over MgSO₄, filtered,

and concentrated to give an orange solid. Column chromatography

on silica gel (1:1 hexane/EtOAc) provided 0.166 g (84%) of 8av

as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 7.32

(d, J ) 9.0 Hz, 2H), 7.19 (d, J ) 2.2 Hz, 1H), 7.05 (d, J ) 2.2 Hz,

1H), 7.02 (d, J ) 9.0 Hz, 2H), 3.96 (s, 3H), 3.95 (s, 3H), 3.85 (s,

3H).

To a cooled (0 °C) suspension of compound 8av (0.083 g, 0.243

mmol) in THF (1.21 mL) was added dropwise 1.0 M lithium

aluminum hydride in THF (0.24 mL). The resulting clear, yellow-

brown solution was stirred at 0 °C. Over the course of the reaction

a precipitate formed. After 4 h, the reaction was quenched with

saturated ammonium chloride and diluted with ethyl acetate. The

layers were separated, and the aqueous layer was extracted with

ethyl acetate (2×). The combined organic layers were washed with

brine, dried over MgSO₄, filtered, and concentrated to give a yellow

residue weighing 0.063 g. The crude material was dissolved in

ethanol (1.0 mL) to give a clear, yellow solution. Next, 2,3-dichloro-

5,6-dicyano-1,4-benzoquinone (0.048 g, 0.213 mmol) was added.

The resulting clear, orange solution was stirred at room temperature.

A precipitate formed and additional ethanol (2 mL) was added to

facilitate stirring. After 4 h, the excess solvent was removed and

the reaction was purified directly. Column chromatography on silica

gel (5% MeOH in CH₂Cl₂) gave 0.070 g (92%) of 8ax as an off-

white solid: LC/MS (ESI) (M + H)⁺) 312.9; ¹H NMR (400 MHz,

CDCl₃) δ 8.10 (s, 1H), 7.33 (d, J ) 9.2 Hz, 2H), 7.12 (d, J ) 2.6

Hz, 1H), 7.10-7.04 (m, 3H), 4.88 (d, J ) 7.9 Hz, 2H), 4.72 (t,

J ) 7.9 Hz, 1H), 3.95 (s, 3H), 3.88 (s, 3H).

Compound 8ar was converted to 1aaj according to the depro-

tection procedure described for 1aam: LC/MS (ESI) (M + H)⁺)

1

269.0; H NMR (500 MHz, DMSO-d₆) δ 10.44 (s, 1H), 9.79 (s,

To a cooled (0 °C) suspension of compound 8ax (0.040 g, 0.128

mmol) in methylene chloride (1.28 mL) was added dropwise boron

tribromide (0.24 mL, 2.56 mmol). The suspension was stirred at 0

°C for 30 min and then warmed to room temperature to give a

clear, orange solution. After 24 h, the reaction was then added

dropwise to a vigorously stirred cold (ice bath) mixture of ethyl

acetate/saturated NaHCO₃. The mixture was stirred for 10 min, and

then the layers were separated. The aqueous layer was extracted

with ethyl acetate (2×). The combined organic layers were washed

with brine, dried over MgSO₄, filtered, and concentrated to give

5-bromomethyl-7-hydroxy-3-(4-hydroxyphenyl)-3H-quinazolin-4-

one, as an off-white solid weighing 0.0525 g.

1H), 8.10 (s, 1H), 7.23 (d, J ) 8.8 Hz, 2H), 6.87 (d, J ) 8.8 Hz,

2H), 6.81 (d, J ) 2.2 Hz, 1H), 6.76 (d, J ) 2.2 Hz, 1H), 2.67 (s,

3H).

5-Ethyl-7-hydroxy-3-(4-hydroxyphenyl)-3H-quinazolin-4-

one (1aan). A flame-dried flask containing compound 8ah (0.300

g, 0.697 mmol), [PdCl₂(dppf)]Cl₂CH₂Cl₂(0.114 g, 0.139 mmol),

and potassium phosphate tribasic (0.592 g, 2.78 mmol) was purged

with argon for 10 min, and then degassed THF (6.97 mL) and 1.0

M triethylborane in THF (2.78 mL, 2.78 mmol) were added. The

resulting rust-colored mixture was placed in a preheated oil bath

(75 °C). After 5.5 h, the black reaction was cooled to room

temperature, diluted with methylene chloride (15 mL), washed with

water and brine, dried over MgSO₄, filtered, and concentrated to

give a black solid. Column chromatography on silica gel (1:1

hexane/EtOAc) gave 0.168 g (78%) of 8at as an off-white solid:

¹H NMR (400 MHz, CDCl₃) δ 8.05 (s, 1H), 7.32 (d, J ) 9.0 Hz,

2H), 7.04 (d, J ) 9.0 Hz, 2H), 7.01 (d, J ) 2.4 Hz, 1H), 6.90 (d,

J ) 2.4 Hz, 1H), 3.93 (s, 3H), 3.86 (s, 3H), 3.27 (q, J ) 7.5 Hz,

2H), 1.26 (t, J ) 7.5 Hz, 3H).

To a clear, slightly brown solution of the crude 5-bromomethyl

compound in acetonitrile (1.5 mL) was added potassium acetate

(0.0444 g, 0.453 mmol) and 18-crown-6 (0.120 g, 0.453 mmol).

The reaction was warmed to 50 °C. After 15 h, the clear, colorless

reaction was cooled to room temperature and 1.0 M sodium

hydroxide (0.60 mL, 0.604 mmol) was added. The biphasic mixture

was stirred vigorously for 5 h. The reaction was acidified to pH 4

with 1.0 M hydrogen chloride. The reaction was extracted with

ethyl acetate (2×). The combined organic layers were washed with

brine, dried over MgSO₄, filtered, and concentrated to a slightly

purple solid weighing 0.153 g. Column chromatography on silica

gel (5% MeOH in CH₂Cl₂) gave 0.017 g as an off-white solid,

which was 93% pure. Purification by preparative HPLC (method

1: 30-90% B, 10 min gradient) (method 1: YMC S5 ODS 30 ×

100 mm column, solvent A ) 10% MeOH/H₂O containing 0.1%

TFA, solvent B ) 90% MeOH/H₂O containing 0.1% TFA, flow

rate 40 mL/min, UV detection at 220 nm) gave 0.0123 g (34%) of

Compound 8at was converted to 1aan according to the depro-

tection procedure described for 1aam: LC/MS (ESI) (M + H)⁺)

1

283.0; H NMR (400 MHz, DMSO-d₆) δ 8.11 (s, 1H), 7.24 (d,

J ) 8.8 Hz, 2H), 6.87 (d, J ) 8.8 Hz, 2H), 6.83 (d, J ) 2.4 Hz,

1H), 6.78 (d, J ) 2.4 Hz, 1H), 3.13 (q. J ) 7.3 Hz, 2H), 1.14 (q,

J ) 7.3 Hz, 3H).

7-Hydroxy-5-hydroxymethyl-3-(4-hydroxyphenyl)-3H-quinazo-

lin-4-one (1aar). Into a flame-dried flask were placed compound

8ah (0.250 g, 0.581 mmol), palladium acetate (0.0130 g, 0.058

mmol), and diphenylphosphinopropane (0.0240 g, 0.058 mmol).

Next, dimethyl sulfoxide (1.81 mL), methanol (1.16 mL), and

triethylamine (0.17 mL) were added. Carbon monoxide (balloon)

was bubbled through the cloudy, yellow mixture for 5 min, and

the vessel was maintained under a carbon monoxide atmosphere.

The reaction was placed in a preheated oil bath (70 °C) and a clear,

slightly yellow solution formed. After 15 h, additional palladium

acetate (0.0130 g, 0.058 mmol), diphenylphosphinopropane (0.0240

g, 0.058 mmol), methanol (1.16 mL), and triethylamine (0.17 mL)

1

Iaar as white solid: LC/MS (ESI) (M + H)⁺) 284.9; H NMR

(400 MHz, DMSO-d₆) δ 10.50 (s, 1H), 9.80 (s, 1H), 8.11 (s, 1H),

7.29 (d, J ) 2.5 Hz, 1H), 7.23 (dd, J ) 8.8, 2.2 Hz, 2H), 6.87 (dd,

J ) 8.8, 2.2 Hz, 2H), 6.83 (d, J ) 2.5 Hz, 1H), 5.26 (t, J ) 5.6

Hz, 1H), 4.98 (d, J ) 5.6 Hz, 2H).

7-Hydroxy-3-(4-hydroxyphenyl)-5-isopropenyl-3H-quinazo-

lin-4-one (1aas). Isopropenylmagnesium bromide (0.5 M in tet-

rahydrofuran, 0.93 mL, 0.464 mmol) was added dropwise to a 0.5

M solution of zinc chloride in THF (0.93 mL, 0.464 mmol). A

2452 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8

Gu¨ngo¨r et al.

mild exotherm was observed and a white precipitate formed. The

resulting suspension was stirred at room temperature for 1.5 h. In

a separate flamed-dried flask was placed compound 8ah (0.100 g,

0.232 mmol) and tetrakis(triphenylphosphine)palladium (0.0404 g,

0.035 mmol). The vessel was purged with argon for 10 min, and

then degassed THF (1.16 mL) was added. To the clear, yellow

solution was added the isopropenyl zinc halide suspension prepared

above. The resulting bright yellow suspension was placed in a

preheated oil bath (75 °C) for 2 h. The reaction was cooled to room

temperature, quenched with saturated ammonium chloride, and

extracted with EtOAc (3×). The combined organic layers were

washed with brine, dried over MgSO₄, filtered, and concentrated

to give a yellow foam weighing 0.120 g. Column chromatography

on silica gel (1:1 hexane/EtOAc) provided 0.052 g (69%) of 8ay

as a pale yellow foam: ¹H NMR (400 MHz, CDCl₃) δ 8.08 (s,

1H), 7.32 (d, J ) 9.0 Hz, 2H), 7.08 (d, J ) 2.6 Hz, 1H), 7.01 (d,

J ) 9.0 Hz, 2H), 6.86 (d, J ) 2.6 Hz, 1H), 5.09 (d, J ) 1.3 Hz,

1H), 4.85 (d, J ) 1.3 Hz, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 2.10 (s,

3H).

mmol) in dimethylformamide (1.4 mL) was added in portions (0.2

mL every 15 min). Following the addition, the reaction was

continued for 3 h. The reaction was cooled to room temperature

and diluted with water (6 mL), giving an orangish precipitate, which

was extracted with methylene chloride (4 × 10 mL). The combined

organic layers were washed with brine, dried over MgSO₄, filtered,

and concentrated to give an orange solid. Column chromatography

on silica gel (2:1 EtOAc/hexane) provided 0.148 g (83%) of 8as

as a faint orange solid: LC/MS (ESI) (M + H)⁺) 308.1; ¹H NMR

(400 MHz, CDCl₃) δ 8.14 (s, 1H), 7.48 (d, J ) 2.6 Hz, 1H), 7.35-

7.33 (m, 3H), 7.04 (d, J ) 8.8 Hz, 2H), 3.98 (s, 3H), 3.87 (s, 3H).

A suspension of compound 8as (0.120 g, 0.393 mmol) and

sodium ethanethiolate (0.662 g, 7.87 mmol) in dimethylformamide

(4.0 mL) was warmed to 120 °C to give a clear, yellow solution.

After 6.5 h, the reaction was cooled to 0 °C and diluted with water

(12 mL). The reaction was acidified to pH 5 with the dropwise

addition of 1 N hydrochloric acid. The reaction was extracted with

methylene chloride (3 × 10 mL). The combined organic layers were

washed with brine (1 × 5 mL), dried over MgSO₄, filtered, and

concentrated to give a yellow solid weighing 0.264 g. The crude

material was adsorbed onto silica gel (1.0 g). Column chromatog-

raphy (7.5% MeOH in CH₂Cl₂) gave 0.027 g as yellow solid, which

was further purified by preparative HPLC (method 2: 0-100% B,

30 min gradient). The fractions were combined and neutralized with

saturated NaHCO₃, and the volume was reduced by approximately

80%. A few drops of 1 N hydrochloric acid were added and the

volumne was concentrated to give a residue. Extraction with

methanol and concentration gave 0.0047 g (4%) of 1aal as a faint

Compound 8ay was converted to 1aas according to the depro-

tection procedure described for 1aam: LC/MS (ESI) (M + H)⁺)

1

295.0; H NMR (400 MHz, CDCl₃) δ 8.16 (s, 1H); 7.23 (d, J )

8.8 Hz, 2H); 6.96 (d, J ) 2.8 Hz, 1H), 6.92 (d, J ) 8.8 Hz, 2H),

6.75 (d, J ) 2.8 Hz, 1H), 5.01 (d, J ) 1.4 Hz, 1H), 4.77 (d, J )

1.4 Hz, 1H), 2.05 (s, 3H).

5-Amino-7-hydroxy-3-(4-hydroxyphenyl)-3H-quinazolin-4-

one (1aai). A flame-dried vessel containing compound 8ah (0.933

g, 2.17 mmol), palladium acetate (0.0487 g, 0.217 mmol), (S)-

BINAP (0.203 g, 0.326 mmol), and cesium carbonate (0.990 g,

3.04 mmol) was purged with argon for 10 min. Next, degassed

THF (10.8 mL) was added, followed by benzophenone imine (0.44

mL, 2.60 mmol). The resulting dark orange solution was placed in

a preheated oil bath (75 °C). After 24 h, the cloudy dark orange

mixture was cooled to room temperature, diluted with diethyl ether

(10 mL), and filtered through Celite. Concentration gave a burgundy

foam. The burgundy foam was dissolved in THF (10.8 mL) and 1

N hydrochloric acid (4.33 mL) was added. The reaction was stirred

at room temperature for 3 h. The resulting clear orange solution

was diluted with methylene chloride (50 mL) and quenched with

saturated NaHCO₃. The layers were separated, and the aqueous layer

was extracted with methylene chloride (15 mL). The combined

organic layers were washed with brine, dried over MgSO₄, filtered,

and concentrated to give a yellow solid weighing 1.34 g. Column

chromatography (1:1 hexane/EtOAc) gave 0.471 g (73%) of 8aq

as an off-white solid: LC/MS (ESI) (M + H)⁺) 298.1; ¹H NMR

(500 MHz, CDCl₃) δ 7.92 (s, 1H), 7.30 (d, J ) 8.8 Hz, 2H), 7.03

(d, J ) 8.8 Hz, 2H), 6.49 (d, J ) 2.2 Hz, 1H), 6.22 (bs, 2H), 6.15

(d, J ) 2.2 Hz, 1H), 3.86 (s, 3H).

A clear, slightly yellow solution of 8aq (0.0245 g, 0.0.082 mmol)

and sodium ethanethiolate (0.0693 g, 0.82 mmol) in dimethylform-

amide (0.82 mL) was warmed to 120 °C. After 5 h, the reaction

was cooled to 0 °C and diluted with water (2 mL). The reaction

was acidified to pH 5 with the dropwise addition of 1 N

hydrochloric acid. The reaction was extracted with methylene

chloride (3×) and ethyl acetate (3×). The combined organic layers

were washed with brine, dried over MgSO₄, filtered, and concen-

trated to give a brown solid weighing 0.026 g. The crude material

was absorbed onto silica gel (0.250 g). Column chromatography

(5% MeOH in CH₂Cl₂) gave 0.0086 g (39%) of 1aai as an off-

white solid: LC/MS (ESI) (M + H)⁺) 270.1; ¹H NMR (400 MHz,

DMSO-d₆) δ 10.0 (bs, 1H), 9.80 (s, 1H), 7.94 (s, 1H), 7.21 (d, J )

8.8 Hz, 2H), 7.17 (bs, 2H), 6.86 (d, J ) 8.8 Hz, 2H), 6.12 (d, J )

2.2 Hz, 1H), 6.08 (d, J ) 2.2 Hz, 1H).

1

yellow solid: LC/MS (ESI) (M + H)⁺) 277.8 H NMR (400

MHz, DMSO-d₆) δ 8.24 (s, 1H), 7.34 (bs, 1H), 7.29 (d, J ) 8.8

Hz, 2H), 7.10 (bs, 1H), 6.88 (d, J ) 8.8 Hz, 2H).

7-Hydroxy-3-(4-hydroxyphenyl)-5-vinyl-3H-quinazolin-4-

one (1aao). A flame-dried flask containing compound 1aam (0.200

g, 0.497 mmol), bis(triphenylphosphine)palladium dichloride (0.035

g, 0.049 mmol), triphenylphosphine (0.052 g, 0.199 mmol), lithium

chloride (0.084 g, 1.99 mmol), and 2,6-di-tert-butyl-4-methylphenol

(a crystal) was purged with argon for 10 min. Next, dimethylform-

amide (degassed, 4.97 mmol) and tributylvinylstannane (0.29 mL,

0.99 mmol) were added. The reaction was placed in a preheated

oil bath (120 °C). After 4 h, the cloudy, yellow reaction was cooled

to room temperature, diluted with water (15 mL), and extracted

with ethyl acetate (3×) and methylene chloride (3×). The combined

organic layers were washed with brine, dried over MgSO₄, filtered,

and concentrated to give a viscous oil weighing 0.447 g. Column

chromatography on silica gel (5% MeOH in CH₂Cl₂) gave 0.069 g

(50%) of 1aao as an off-white solid: LC/MS (ESI) (M + H)⁺

)

1

278.8 H NMR (500 MHz, DMSO-d₆) δ 10.60 (s, 1H), 9.81 (s,

1H), 8.14 (s, 1H), 7.91 (dd, J ) 17.4, 11.0 Hz, 1H), 7.24 (d, J )

8.5 Hz, 2H), 7.05 (d, J ) 2.5 Hz, 1H), 6.93 (d, J ) 2.5 Hz, 1H),

6.87 (d, J ) 8.5 Hz, 2H), 5.58 (dd, J ) 17.4, 1.6 Hz, 1H), 5.28 (d,

J ) 11.0 Hz, 1H).

7-Hydroxy-3-(4-hydroxyphenyl)-4-oxo-3,4-dihydroquinazoline-

5-carboxylic Acid Methyl Ester (1aap). Into a flame-dried flask

were placed compound 1aam (0.300 g, 0.746 mmol), palladium

acetate (0.0168 g, 0.075 mmol), and diphenylphosphinopropane

(0.0309 g, 0.075 mmol). Next, dimethyl sulfoxide (2.3 mL),

methanol (3.0 mL), and triethylamine (0.44 mL) were added.

Carbon monoxide (balloon) was bubbled through the clear, yellow

solution for 10 min, and the vessel was maintained under a carbon

monoxide atmosphere. The reaction was placed in a preheated oil

bath (73 °C). After 5 h, the dark brown reaction was cooled to

room temperature. The reaction was diluted with water (10 mL),

acidified (pH 3) with 1.0 M hydrochloric acid, and extracted with

ethyl acetate (3 × 15 mL). The combined organic layers were

washed with brine, dried over MgSO₄, filtered, and concentrated

to give an orange residue weighing 0.400 g. Column chromatog-

raphy on silica gel (5% MeOH in CH₂Cl₂) provided 0.0580 g (25%)

7-Hydroxy-3-(4-hydroxyphenyl)-4-oxo-3,4-dihydroquinazoline-

5-carbonitrile (1aal). A flame-dried test tube containing compound

8ah (0.250 g, 0.581 mmol), tris(dibenzylideneacetone)dipalladium

(0.0531 g, 0.058 mmol), and 1,1′-bis(diphenylphosphino)ferrocene

(0.128 g, 0.232 mmol) was purged with argon for 10 min. Next,

degassed dimethylformamide (1.9 mL) was added and the reaction

was placed in a preheated oil bath (90 °C) to give a clear, orange-

brown solution. A suspension of zinc cyanide (0.0818 g, 0.697

1

of 1aap as a white solid: LC/MS (ESI) (M + H)⁺) 312.8; H

NMR (400 MHz, DMSO-d₆) δ 10.98 (s, 1H), 9.85 (s, 1H), 8.23 (s,

1H), 7.26 (d, J ) 8.8 Hz, 2H), 7.04 (d, J ) 2.2 Hz, 1H), 6.92 (d,

J ) 2.2 Hz, 1H), 6.88 (d, J ) 8.8 Hz, 2H), 3.76 (s, 3H).

ERâ Modulators Based on Genistein

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2453

7-Hydroxy-3-(4-hydroxyphenyl)-5-phenyl-3H-quinazolin-4-

one (1aaq). A flame-dried flask containing compound 8ah (0.300

g, 0.697 mmol), [PdCl₂(dppf)]CH₂Cl₂(0.0853 g, 0.104 mmol),

potassium phosphate tribasic (0.592 g, 2.78 mmol), and phenyl-

boronic acid (0.340 g, 2.78 mmol) was purged with argon for 10

min. Next, degassed THF (7.0 mL) was added. The resulting dark

orange suspension was placed in a preheated oil bath (73 °C). After

14 h, the dark red reaction was cooled to room temperature and

diluted with ethyl acetate (20 mL). The reaction was washed with

water (5 mL), saturated NaHCO₃(5 mL), and brine (5 mL); dried

over MgSO₄; filtered; and concentrated to give a burgundy residue

weighing 0.667 g. Column chromatography on silica gel (1:1

hexane/EtOAc) gave 0.216 g (86%) of 8aw as a white solid: LC/

was warmed to 145 °C. After 44 h, additional Lawesson’s reagent

(0.590 g, 1.46 mmol) was added and the reaction was stirred at

145 °C for an additional 26 h. The reaction was cooled to room

temperature, diluted with methylene chloride, filtered, and concen-

trated to give an orange solid weighing 0.865 g. Column chroma-

tography on silica gel (1.5:1 hexane/EtOAc) gave 0.0599 g (46%)

of 8bd as a yellow solid: ¹H NMR (400 MHz, CDCl₃) δ 8.13 (s,

1H), 7.23 (d, J ) 8.8 Hz, 2H), 7.04 (d, J ) 8.8 Hz, 2H), 7.02 (d,

J ) 2.2 Hz, 1H), 6.97 (d, J ) 2.2 Hz, 1H), 3.93 (s, 3H), 3.88 (s,

3H), 3.07 (s, 3H).

To a cooled (0 °C), clear, yellow solution of 8bd compound

(0.0599 g, 0.191 mmol) in methylene chloride (1.91 mL) was added

dropwise boron tribromide (0.09 mL, 0.958 mmol). The resulting

clear, dark orange solution was stirred at 0 °C for 30 min and then

warmed to room temperature. After 9 h, the reaction was then added

dropwise to a vigorously stirred cold (ice bath) mixture of ethyl

acetate/saturated NaHCO₃. The mixture was stirred for 10 min, and

then the layers were separated. The aqueous layer was extracted

with ethyl acetate (2×). The combined organic layers were washed

with brine, dried over MgSO₄, filtered, and concentrated to give a

orange solid weighing 0.063 g. The crude material was adsorbed

onto silica gel (0.200 g). Column chromatography (5% MeOH in

CH₂Cl₂) gave 0.0252 g (46%) of 1bd as a yellow solid: LC/MS

(ESI) (M + H)⁺) 284.9; ¹H NMR (500 MHz, MeOD₄) δ 8.20 (s,

1H); 7.11 (d, J ) 8.8 Hz, 2H); 6.91-6.87 (m, 4H), 2.99 (s, 3H);

HRMS m/z calcd for C₁₅H₁₃N₂O₂S (M + H)⁺285.0698, found

285.0696.

5-Ethyl-7-hydroxy-3-(4-hydroxyphenyl)-3H-quinazoline-4-

thione (1be). A yellow suspension of 8at (0.116 g, 0.373 mmol)

and Lawesson’s reagent (0.755 g, 1.87 mmol) in m-xylene (2.0

mL) was warmed to 145 °C. After 72 h, additional Lawesson’s

reagent (1.51 g, 3.74 mmol) was added and the reaction was stirred

at 145 °C for an additional 24 h. The reaction was cooled to room

temperature, diluted with methylene chloride (40 mL), filtered, and

concentrated to give a dark brown solid weighing 0.773 g. Column

chromatography on silica gel (2:1 hexane/EtOAc) gave 0.0211 g

(17%) of 8be as a yellow solid: LC/MS (ESI) (M + H)⁺) 327.1;

¹H NMR (500 MHz, CDCl₃) δ 8.17 (s, 1H), 7.23 (d, J ) 8.8 Hz,

2H), 7.04-6.95 (m, 4H), 3.94 (s, 3H), 3.88 (s, 3H), 3.65 (q, J )

7.4 Hz, 2H), 1.29 (t, J ) 7.4 Hz, 3H).

1

MS (ESI) (M + H)⁺) 359.15; H NMR (400 MHz, CDCl₃) δ

8.07 (s, 1H), 7.35-7.29 (m, 5H), 7.24 (d, J ) 8.9 Hz, 2H), 7.18

(d, J ) 2.6 Hz, 1H), 6.94 (d, J ) 8.9 Hz, 2H), 6.91 (d, J ) 2.6 Hz,

1H), 3.96 (s, 3H), 3.80 (s, 3H).

To a cooled (0 °C), clear, colorless solution of compound 8aw

(0.095 g, 0.265 mmol) in methylene chloride (2.60 mL) was added

dropwise boron tribromide (0.50 mL, 5.30 mmol). The resulting

bright yellow solution was stirred at 0 °C for 30 min and then

warmed to room temperature. After 72 h, the reaction was added

dropwise to a vigorously stirred cold (ice bath) mixture of ethyl

acetate/saturated NaHCO₃. The layers were separated, and the

aqueous layer was extracted with ethyl acetate (2×). The combined

organic layers were washed with brine, dried over MgSO₄, filtered,

and concentrated to give an off-white solid. Trituration with ethyl

acetate and filtration gave 0.0547 g (62%) of 1aaq as a white

1

solid: LC/MS (ESI) (M + H)⁺) 331.2; H NMR (400 MHz,

DMSO-d₆) δ 10.69 (s, 1H), 9.76 (s, 1H), 8.22 (s, 1H), 7.33-7.22

(m, 5H), 7.18 (d, J ) 8.8 Hz, 2H), 6.99 (d, J ) 2.6 Hz, 1H), 6.82

(d, J ) 8.8 Hz, 2H), 6.70 (d, J ) 2.6 Hz, 1H).

General Procedure for the Synthesis of “Thio” Derivatives

1ba-1bi According to Scheme 7. 5,7-Dimethoxy-3-(4-methoxy-

phenyl)-4(3H)-quinazolinethione (8ba): The mixture of 5,7-

dimethoxy-3-(4-methoxyphenyl)quinazolin-4-one (8aa) (240 mg,

0.76 mmol) and Lawesson’s reagent (310 mg, 0.76 mmol) in toluene

(4 mL) was refluxed overnight. The mixture was evaporated to

dryness. EtOAc was added and the mixture was washed with water

and brine, dried over MgSO₄, and concentrated. Column chroma-

tography on silica gel (45% EtOAc in hexane) gave 180 mg (72%)

To a cooled (0 °C), clear, yellow solution of 8be (0.0211 g,

0.0646 mmol) in methylene chloride (0.65 mL) was added dropwise

boron tribromide (0.09 mL, 0.97 mmol). The resulting orange

solution was stirred at 0 °C for 30 min and then warmed to room

temperature. After 8 h, the reaction was then added dropwise to a

vigorously stirred cold (ice bath) mixture of ethyl acetate/saturated

NaHCO₃. The mixture was stirred for 10 min, and then the layers

were separated. The aqueous layer was extracted with ethyl acetate

(2×). The combined organic layers were washed with brine, dried

over MgSO₄, filtered, and concentrated to give an orange solid

weighing 0.0192 g. Column chromatography (5% MeOH in CH₂-

Cl₂) gave 0.0117 g (60%) of 1be as a yellow foam: LC/MS (ESI)

(M + H)⁺) 298.9; ¹H NMR (500 MHz, MeOD₄) δ 8.22 (s, 1H);

7.12 (d, J ) 8.2 Hz, 2H); 6.92-6.88 (m, 4H), 3.61 (q, J ) 7.2 Hz,

2H), 1.25 (t, J ) 7.2 Hz, 3H).

1

of 8ba as a yellow foam: LC/MS (ESI) (M + H)⁺) 329; H

NMR (400 MHz, DMSO-d₆) δ 8.08 (s, 1H, H2), 7.20 (d, 1H, H2′),

7.00 (d, 1H, H3′), 6.71 (d, 1H, H8), 6.52 (d, 1H, H6), 3.9 (s, 3H,

OCH₃), 3.83 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃).

Ether Cleavage. 5,7-Dihydroxy-3-(4-hydroxyphenyl)-4(3H)-

quinazolinethione (1ba). 5,7-Dimethoxy-3-(4-methoxyphenyl)-

4(3H)-quinazolinethione (8ba) (100 mg, 0.30 mmol) was mixed

with pyridine hydrochloride salt (701 mg, 6.1 mmol). The mixture

was heated to 189 °C for 2.5 h. The reaction was cooled to room

temperature. The resulting residue was purified by preparative

HPLC to give 35 mg (40%) of 1ba as a yellow solid: LC/MS

1

(ESI) (M + H)⁺) 287; mp 275-280 °C; H NMR (400 MHz,

CD₃OD) δ 8.28 (s, 1H, H2), 7.18 (d, 2H, H2′,6′), 6.93 (d, 2H,

H3′,5′), 6.52 (d, 1H, H8), 6.42 (d, 1H, H6).

5,7-Dihydroxy-3-(3-fluoro-4-hydroxyphenyl)-4(3H)-quinazo-

linethione (1bb): C₁₄H₉FN₂O₃S ) 304.3 g/mol; LC/MS (ESI)

5,7-Dimethoxy-3-(5-methoxypyridin-2-yl)quinazoline-4(3H)-

1

thione (8bf): LC/MS (ESI) (M + H)⁺) 330.1; H NMR (400

1

(M + H)⁺) 305.8; H NMR (400 MHz, DMSO-d₆) δ 13.4 (1H,

MHz, CDCl₃) δ 8.17 (s, 1H), 7.23 (d, J ) 8.8 Hz, 2H), 7.04-6.95

(m, 4H), 3.94 (s, 3H), 3.88 (s, 3H), 3.65 (q, J ) 7.4 Hz, 2H), 1.29

(t, J ) 7.4 Hz, 3H).

s), 10.51 (1H, bs), 8.35 (1H, s), 7.42 (1H, dd, J ) 11.9 and 2.2

Hz), 7.11 (2H, m), 6.56 (1H, d, J ) 2.2 Hz), 6.45 (1H, d, J ) 2.2

Hz).

5,7-Dihydroxy-3-(5-hydroxypyridin-2-yl)quinazoline-4(3H)-

1

5,7-Dihydroxy-3-(4-hydroxy-3-methylphenyl)-4(3H)-quinazo-

linethione (1bc): C₁₅H₁₂N₂O₃S ) 300.8 g/mol; LC/MS (ESI)

thione (1bf): LC/MS (ESI) (M + H)⁺) 287.04; H NMR (400

MHz, CDCl₃) δ 8.17 (s, 1H), 7.23 (d, J ) 8.8 Hz, 2H), 7.04-6.95

(m, 4H), 3.94 (s, 3H), 3.88 (s, 3H), 3.65 (q, J ) 7.4 Hz, 2H), 1.29

(t, J ) 7.4 Hz, 3H).

1

(M + H)⁺) 300.8; LC/MS (ESI) (M - H)^-) 298.8; H NMR

(400 MHz, DMSO-d₆) δ 13.52 (1H, s), 11.03 (1H, s), 9.86 (1H, s),

8.34 (1H, s), 7.15 (1H, s), 7.07 (1H, d, J ) 8.8 Hz), 6.90 (1H, d,

J ) 8.8 Hz), 6.58 (1H, d, J ) 1.8 Hz), 6.46 (1H, d, J ) 1.8 Hz),

2.15 (3H, s, Me).

5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-chromene-4-thione (13).

To a solution of biocanin A (500 mg, 1.89 mmol) in 5 mL of

pyridine were added 2 mg (0.016 mmol) of DMAP and 357 µL of

Ac₂O (3.78 mmol). The mixture was stirred at room temperature

for 5 h. The volatiles were evaporated, the residue was dissolved

in 5 mL of EtOAc, and the solution was washed with water (2 ×

7-Hydroxy-3-(4-hydroxyphenyl)-5-methyl-3H-quinazoline-4-

thione (1bd). A yellow suspension of 8ar (0.124 g, 0.42 mmol)

and Lawesson’s reagent (1.18 g, 2.93 mmol) in m-xylene (2.0 mL)

2454 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8

Gu¨ngo¨r et al.

3 mL) and then with 1 N HCl (3 mL), dried, and evaporated to

yield 650 mg (93%) of the acetic acid 7-acetoxy-3-(4-methoxy-

phenyl)-4-oxo-4H-chromen-5-yl ester, which was used in the next

step without further purification: ¹H NMR (400 MHz, CDCl₃) δ

7.86 (s, 1H), 7.4 (d, J ) 8.8 Hz, 2H), 7.24 (d, J ) 2.2 Hz, 1H)

6.96 (d, J ) 8.8 Hz, 2H), 6.85 (d, J ) 2.2 Hz, 1H), 3.83 (s, 3H),

2.42 (s, 3H), 2.35 (s, 3H).

tion of compound required to achieve 50% of transactivation caused

by 10 nM estradiol) was calculated using XL-fit one-site dose

response.

Acknowledgment. The authors thank Dr. Petia Shipkova,

Robert Langish; Marie Ellen Salyan, Jeannette S. Manello,

Yajun Liu, Yi-Xin Li, and Feng Qiu for technical assistance.

A mixture of 370 mg (1.09 mmol) of acetic acid 7-acetoxy-3-

(4-methoxyphenyl)-4-oxo-4H-chromen-5-yl ester and 370 mg (1.66

mmol) of P₂S₅in 5 mL of toluene was refluxed for 16 h and then

concentrated in vacuo. The residue was purified by flash chroma-

tography on silica gel (60% EtOAc/hexane) to give 650 mg (93%)

of a yellow solid.

Supporting Information Available: Analytical and spectral

data of new compounds. This material is available free of charge

via the Internet at http://pubs.acs.org.

References

(1) Estrogens and antiestrogens. Handbook of Experimental Pharmacol-

ogy; Springer-Verlag: Berlin, 1999; vol. 135 I and II.

(2) Kuiper, G. G. J. M.; Enmark, E.; Pelto-Huikko, M.; Nilsson, S.;

Gustafsson, J.-A. Cloning of a novel estrogen receptor expressed in

rat prostate and ovary. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5925-

5930.

(3) Mosselman, S.; Polman, J.; Dijkema, R. ERâ: Identification and

characterization of a novel human estrogen receptor. FEBS Lett. 1996,

392, 49-53.

(4) Kuiper, G. G. J. M.; Gustafsson, J.-A. The novel estrogen receptor

â subtype: Potential role in the cell- and promoter specific actions

of estrogens and anti estrogens. FEBS Lett. 1997, 410, 87-90.

(5) Kuiper, G. G. J. M.; Carlsson, B.; Grandien, K.; Enmark, E.;

Haeggblad, J.; et al. Comparison of the ligand binding specificity

and transcript tissue distribution of estrogen receptors R and â.

Endocrinology 1997, 138, 863-870.

(6) Dechering, K.; Boersma, Ch.; Mosselman, S. Estogen receptors R

and â: Two receptors of a kind? Curr. Med. Chem. 2000, 7, 561-

576.

(7) Greene, G. L.; Gilna, P.; Waterfield, M.; Baker, A.; Hort, Y.; Shine,

J. Sequence and expression of human estrogen receptor complemen-

tary DNA. Science 1986, 231, 1150-1154.

(8) Pike, A. C. W.; Brzozowski, A. M.; Hubbard, R. E.; Bonn, T.;

Thorsell, A.-G.; et al. Structure of the ligand-binding domain of

estrogen receptor beta in the presence of a partial agonist and a full

antagonist. EMBO J. 1999, 18, 4608-4618.

(9) Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard, R. E.; Bonn,

T.; et al. Molecular basis of agonism and antagonism in the estrogen

receptor. Nature 1997, 389, 753-758.

(10) Gao, H.; Katzenellenbogen, J. A.; Garg, R.; Hansch, C. Comparative

QSAR analysis of estrogen receptor ligands. Chem. ReV. 1999, 99,

723-744.

(11) Anstead, G., M.; Carlson, K., E.; Katzenellenbogen, J. A. The

estradiol pharmacophore: Ligand structure-estrogen receptor binding

affinity relationships and a model for the receptor binding site.

Steroids 1997, 62, 268-304.

(12) Grese, T., A.; Dodge, J. A. Selective estrogen receptor modulators

(SERMs). Curr. Pharm. Des. 1998, 4, 1, 71-92.

(13) (a) Sun, J.; Meyers, M. J.; Fink, B. E.; Rajendran, R.; Katzenellen-

bogen, J. A.; Katzenellenbogen B. S. Novel ligands that function as

selective estrogens or antiestrogens for estrogen receptor-R or

estrogen receptor-â. Endocrinology 1999, 140, 800-804. (b) Stauffer,

S. R.; Coletta, C. J.; Tedesco, R.; Nishiguchi, G.; Carlson, K.; Sun,

J.; Katzenellenbogen, B. S.; Katzenellenbogen, J. A. Pyrazole

ligands: Structure-affinity/activity relationships of estrogen receptor-R

selective agonists. J. Med. Chem. 2000, 43, 4934-4947.

(14) Kuiper, G. G. J. M.; Lemmen, J. G.; Carlsson, B.; Corton, J. C.;

Safe, S. H.; van der Saag, P. T.; van der Burg, B.; Gustafsson, J. A.

Interaction of estrogenic chemicals and phytoestrogens with estrogen

receptor b. Endocrinology 1998, 139, 4252-4263.

(15) (a) Schopfer, U.; Schoeffter, P.; Bischoff, S. F.; Nozulak, J.;

Feuerbach, D.; Floersheim, P. Toward selective ERâ agonists for

central nervous system disorders: Synthesis and characterization of

aryl benzthiophenes. J. Med. Chem. 2002, 45, 1399-1401. (b) Henke,

B. R.; Consler, T. G.; Go, N.; Hale, R. L.; Hohman, D. R.; Jones, S.

A.; Lu, A. T.; Moore, L. B.; Moore, J. T.; Orband-Miller, L. A.;

Robinett, R. G.; Shearin, J.; Spearing, P. K.; Stewart, E. L.; Turnbull,

P. S.; Weaver, S. L.; Williams, S. P.; Wisely, G. B.; Lambert, M. H.

A new series of estrogen receptor modulators that display selectivity

for estrogen receptor â. J. Med. Chem. 2002, 45, 5492-5505.

(16) (a) Manas, E. S.; Unwalla, R. J.; Xu, Z. B.; Malamas, M. S.; Miller,

C. P.; Harris, H. A.; Hsiao, C.; Akopian, T.; Hum, W.-T.; Malakian,

K.; Wolfrom, S.; Bapat, A.; Bhat, R. A.; Stahl, M. L.; Somers, W.

S.; Alvarez, J. C. Structure-based design of estrogen receptor â

selective ligands. J. Am. Chem. Soc. 2004, 126, 15106-15119. (b)

Malamas, M. S.; Manas, E. S.; McDevitt, R. E.; Gunawan, I.; Xu,

Z. B.; Collini, M. D.; Miller, C. P.; Dinh, T.; Henderson, R. A.;

Keith, J. C., Jr.; Harris, H. A. Design and synthesis of aryl diphenolic

To a cooled solution (0 °C) of the above compound (50 mg,

0.166 mmol) in 1 mL of CH₂Cl₂was added 0.5 mL of BBr₃. The

reaction mixture was stirred at room temperature overnight, cooled

to -70 °C, and quenched with 1 mL of MeOH. The mixture was

then evaporated to dryness, 2 mL of AcOEt was added, and the

solution was washed with saturated NaHCO₃solution and brine,

dried, and evaporated. Further purification by preparative HPLC

yielded a yellow solid: C₁₅H₁₀O₄S; LC/MS (ESI) (M + H)⁺

)

286.03; mp 245-248 °C; ¹H NMR (500 MHz, MeOD₄) δ 7.75 (s,

1H), 7.17 (d, J ) 8.8 Hz, 2H), 7.31 (d, J ) 8.8 Hz, 2H), 6.36 (d,

J ) 2.6 Hz, 1H), 6.29 (d, J ) 2.6 Hz, 1H).

Biological Assays. Estrogen Receptor Binding Assay. A fusion

protein, expressed in Escherichia coli, consisted of maltose binding

protein (MBP), a specific biotinylation sequence (BioP) that is a

substrate for biotin ligase, an enterokinase cleavage site (EK), and

the ligand binding domain of ERâ (ERâ-LBD) or ERR (ERR-LBD).

ERâ-LBD and ERR-LBD were purified by affinity chromatography

on an amylose column and biotinylated with biotin ligase. For

competition binding studies, purified biotinylated ERR-LBD (bio-

tin-ERR-LBD) and ERâ-LBD (biotin-ERâ-LBD) were used. The

assay was performed in 100 µL of 25 mM Tris-HCl buffer, pH

8.0, containing 250 mM NaCl, 1 mM EDTA, and 1 mM dithio-

theitol in a 96-well white OptiPlate (Packard).

Biotin-ERâ-LBD (40 nM) was incubated with 22 nM [³H]-

estrone and 75 µg of streptavidin SPA bead (Amersham), or biotin-

ERR-LBD (25 nM) was incubated with 11 nM [³H]estrone and 50

µg of streptavidin SPA bead (Amersham), and to both were added

increasing concentrations of compounds for 2 h at room temperature

with gentle shaking. Total binding in the absence of compounds

and nonspecific binding in the presence of 1 µM estradiol were

determined. After 2 h, the plate was counted in a TopCount (Perkin-

Elmer). All the compounds tested were dissolved in DMSO to 10

mM concentration, diluted in 100% DMSO, with 10× intermediate

dilution in assay buffer. The final concentration of DMSO in the

reaction was 1%. The binding IC₅₀value (the concentration of

compound required for 50% inhibition of [³H]estrone binding to

ERâ-LBD or ERR-LBD) was calculated using XL-fit one-site dose

response.

Transactivation Assay. To determine the agonist activity of the

compounds on ERR and ERâ receptors, the full length receptors

were stably expressed under ERE promoter with luciferase as

reporter in Hela cells. The clones were screened and the best

responders with estradiol were selected. Cells were cultured in

DMEM [medium with high glucose containing nonessential amino

acids, sodium pyruvate, antibiotic antimycotic solution, 5% heat-

inactivated FBS, 0.3 µg/mL of puromycin, and 1 mg/mL geneticin

(GIBCO)] to 75% confluency. Cells (20 000 cells/well) were plated

into tissue culture (TC) treated 96-well white plates (Perkin-Elmer)

in phenol-red-free medium, without antibiotics, and returned to the

TC incubator. After overnight incubation, the medium was aspirated,

and increasing concentrations of compounds were added in PBS

supplemented with 0.5% BSA and 10 mM glucose to the cells,

which were incubated at 37 °C in the TC incubator overnight. The

medium was aspirated and the luciferase induced was measured

using LucScreen assay kit (Tropix). The signal was read in a

TopCount (Perkin-Elmer). The transactivation EC₅₀(the concentra-

ERâ Modulators Based on Genistein

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2455

azoles as potent and selective estrogen receptor â ligands. J. Med.

Chem. 2004, 47, 5021-5040. (c) Chesworth, R.; Wessel, M. D.;

Heyden, L.; Mangano, F. M.; Zawistoski, M.; Gegnas, L.; Galluzzo,

D.; Lefker, B.; Cameron, K. O.; Tickner, J.; Lu, B.; Castleberry, T.

A.; Petersen, D. N.; Brault, A.; Perry, P.; Ng, O.; Owen, T. A.; Pan,

L.; Ke, H.-Z.; Brown, T. A.; Thompson, D. D.; DaSilva-Jardine, P.

Estrogen receptor beta selective ligands: Discovery and SAR of novel

heterocyclic ligands. Bioorg. Med. Chem. Lett. 2005, 15, 5562-5566.

(d) De Angelis, M.; Stossi, F.; Carlson, K. A.; Katzenellenbogen, B.

S.; Katzenellenbogen, J. A. Indazole estrogens: Highly selective

ligands for the estrogen receptor â. J. Med. Chem. 2005, 48, 1132-

1144.

(17) Gu¨ngo¨r, T.; Corte, J. R. US Patent 2003/0220227, Nov. 27, 2003.

(18) Newman, H.; Angier, R. B. Synthesis of the ring B sulfur analog of

dehydrogriseofulvin. J. Org. Chem. 1969, 34, 11, 3484-3491.

(19) (a) McMurry, J. E.; Scott, W. J. A method for the regiospecific

synthesis of enol triflates by enolate trapping. Tetrahedron Lett. 1983,

24, 10, 979-982. (b) Comins, D. L.; Dehghani, A. Pyridine-derived

triflating reagents: An improved preparation of vinyl triflates from

metallo enolates. Tetrahedron Lett. 1992, 33, 11, 6299-6302.

JM0509389

Article Doi

Synthesis and characterization of 3-arylquinazolinone and 3-arylquinazolinethione derivatives as selective estrogen receptor beta modulators

DOI: 10.1021/jm0509389

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Article abstract of DOI:10.1021/jm0509389

Full text of DOI:10.1021/jm0509389

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