Acyclic amides as estrogen receptor ligands: Synthesis, binding, activity and receptor interaction

Bioorganic & Medicinal Chemistry 8 (2000) 1293±1316

Acyclic Amides as Estrogen Receptor Ligands: Synthesis, Binding,

Activity and Receptor Interaction

Shaun R. Stauer, ^aJun Sun, ^bBenita S. Katzenellenbogen ^b,c

and John A. Katzenellenbogen ^a,*

^aDepartment of Chemistry, University of Illinois, 461 Roger Adams Laboratory, Box 37-5, 600 S. Mathews Avenue, Urbana,

IL 61801, USA

^bDepartment of Physiology, University of Illinois, 407 S. Goodwin Avenue, Urbana, IL 61801, USA

^cDepartment of Cell and Structural Biology, University of Illinois, 407 S. Goodwin Avenue, Urbana, IL 61801, USA

Received 7 November 1999; accepted 26 January 2000

AbstractÐWe have prepared a series of bisphenolic amides that mimic bibenzyl and homobibenzyl motifs commonly found as

substructures in ligands for the estrogen receptor (ER). Representative members were prepared from three classes: N-phenyl benz-

amides, N-phenyl acetamides, and N-benzyl benzamides; in some cases the corresponding thiocarboxamides and sulfonamides were

also prepared. Of these three classes, the N-phenyl benzamides had the highest anity for ER, the N-phenyl acetamides had lower,

and the N-benzyl benzamides were prone to fragmentation via a quinone methide intermediate. In the N-phenyl benzamide series,

the highest anity analogues had bulky N-substituents; a CF₃group, in particular, conferred high anity. The thiocarboxamides

bound better than the corresponding carboxamides and these bound better than the corresponding sulfonamides. Binding anity

comparisons suggest that the p-hydroxy group on the benzoate ring, which contributes most to the binding, is playing the role of

the phenolic hydroxyl of estradiol. Computational studies and NMR and X-ray crystallographic analysis indicate that the two

anilide systems studied have a strong preference for the s-cis or exo amide conformation, which places the two aromatic rings in a

syn orientation. We used this structural template, together with the X-ray structure of the ER ligand binding domain, to elaborate

an additional hydrogen bonding site on a benzamide system that elevated receptor binding further. When assayed on the individual

ER subtypes, ERa and ERb, these compounds show modest binding anity preference for ERa. In a reporter gene transfection

assay of transcriptional activity, the amides generally have full to nearly full agonist character on ERa, but have moderate to full

antagonist character on ERb. One high anity carboxamide is 500-fold more potent as an agonist on ERa than on ERb. This work

illustrates that ER ligands having simple amide core structures can be readily prepared, but that high anity binding requires an

appropriate distribution of bulk, polarity, and functionality. The strong conformational preference of the core anilide function in

all of these ligands de®nes a rather rigid geometry for further structural and functional expansion of these series. # 2000 Elsevier

Introduction

disorders, without adverse stimulation of the uterus and

breast.²SERMs comprise several structurally diverse

classes, including the triarylethylenes, triarylnaph-

thalenes, benzo[b]furans, benzopyrans, and various

other tetracyclic manifestations of these core structures.³

Collectively, SERMs display a wide range of tissue-

selective agonist and antagonist activities, but major

eorts continue to be directed toward optimizing ER

ligand structure in order to obtain desired tissue-speci®c

properties with minimal side-eects, which is an important

concern in maintaining ecacy and patient compliance.⁴

Recent advances in nuclear hormone receptor pharma-

cology and mechanism of action have rede®ned the

classi®cation of estrogen receptor (ER) ligands.¹The

selective estrogen receptor modulator class, or SERMs,

are considered to be very important because of the

potential of SERMs for maintaining bone mineral den-

sity and cardiovascular health, and for treating and

preventing breast cancer and other hormone-dependent

In connection with our interests in novel templates for

ER ligands, especially ones that might be suitable for

*Corresponding author. Tel.: +1-217-333-6310; fax: +1-217-333-

7325; e-mail: jkatzene@uiuc.edu

PII: S0968-0896(00)00075-4

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S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

combinatorial development,⁵we wondered whether

acyclic tertiary amides might be developed as mimics of

common structural motifs found at the core of many

ER ligands and thus support a combinatorial approach

to SERM discovery and optimization. In this report, we

describe the design and synthesis of three types of

amide-core ER ligands, and we investigate their molecular

structure, their structure binding±anity relationships,

and their activity in ERa and ERb transactivation

assays. A number of interesting trends and high anity

amides were discovered during this investigation. One

high anity amide in particular (16g) was found to be

an ERa-potency selective agonist.

From this analysis, model para-substituted bis-phenolic

carboxamides, sulfonamides and thioamides containing

N-alkyl and tri¯uoroalkyl substituents (R and R⁰) were

considered as potential templates for combinatorial

development (Fig. 3). Preparation for many of these

compounds is straightforward and thus not unreason-

able for adaptation to combinatorial library synthesis.

Results and Discussion

Rationale and design

Chemical syntheses

N-Phenyl benzamides and benzene sulfonamides. To

synthesize simple N-alkyl amide analogues in an expe-

ditious manner, we used catalytic phase transfer condi-

tions (Scheme 1). Thus, N-alkylation of the known

secondary benzene sulfonamide 3⁸proceeded under

mild conditions using n-Bu₄NSO₄H, NaOH, and excess

alkyl halide in CH₂Cl₂/H₂O.⁹The target N-alkylated

sulfonamides 5a±c were then obtained after deprotec-

tion. The less acidic carboxamide 6 was alkylated using

a solid±liquid two-phase system, consisting of powdered

NaOH and cat. n-Bu₄NBr in re¯uxing benzene, to aord

the tertiary amides 9a±e in good yield.^9,10Carboxamides

9f and 9g were conveniently prepared from the second-

ary anilines 7 and 8. Aniline 7 was prepared via reduc-

tive alkylation of the corresponding aldimine using a

modi®ed literature procedure.¹¹Aniline 8 was obtained

by reductive amination, using NaBH(OAc)₃, p-anisidine,

and 3-methyl-2-butanone. Thioamides were prepared in

relatively good yield by treating the carboxamide (9a±g)

with Lawesson's reagent.¹²Deprotection with BBr₃or

To the best of our knowledge, only two examples of ER

ligands containing, either partially or exclusively, an

amide core structure are known in the public literature.

Hartmann and co-workers studied a small series of bis-

phenolic carboxamides^6,7containing an invariant N-

(1,1,1-tri¯uoro-2-propyl) substituent (e.g., 1) and found

them to be anti-estrogens with weak to moderate

potency. An early example of an N-ethyl benzene sulfon-

amide (2)⁸was also reported and shown to be weakly

estrogenic in vivo. Neglecting amide bond stereoisomers

for the moment, these ligands represent potential

mimics (I) for either the anti- or syn-bibenzyl motifs A

and B, respectively (Fig. 1, right), that are found in

many common ER ligands (Fig. 1, left). In addition to

the bibenzyl structural template of these leads, we envis-

ioned two homologues (II and III, Fig. 2, right) which

mimic the homo-bibenzyl motif C (Fig. 2, II±III), a

structural element that is also well represented in high

anity ligands for the ER (Fig. 2, left).

Figure 1. Syn and anti-bibenzyl motifs and proposed acyclic amide analogues.

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1295

Figure 2. Homo-bibenzyl motif and proposed acylic amide analogues.

Figure 3. Potential amide ER ligand scaolds for combinatorial chemistry (X=O, S).

Scheme 1. Synthesis of N-phenyl benzamides.

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S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

ethanethiolate aorded the target benzamides 10a±f and

thiobenzamides 11a±g.

Amidation in warm toluene proceeded satisfactorily to

provide carboxamides 15a±e; however, unactivated ani-

lines 14c and 14d required 1,2-dichlorobenzene as sol-

vent and higher temperatures to aord benzamides 15f

and 15g in satisfactory yields. Subsequent deprotection

using BBr₃aorded the desired phenols 16a±g as before.

Thionation of the protected, CF₃-substituted carbox-

amides proceeded in signi®cantly lower yield (20±51%)

than did those with simple N-alkyl substitution

(>79%). Alternative thionation conditions, such as

P₂S₅and POCl₃/(TMS)₂S, did not give improved yields.

Nevertheless, the two thioamides 17a and 17b could be

obtained after deprotection with BBr₃.

CF₃-containing N-phenyl benzamides. To further explore

the eect that a tri¯uoromethyl group would have on

receptor binding anity, we prepared several carbox-

amide and thioamide analogues related to the weak anti-

estrogen (1) reported by Hartmann and co-workers.⁶The

synthetic route to CF₃-amide analogues, depicted in

Scheme 2, was adapted from that reported by Hartmann

and required introduction of the CF₃-alkyl substituent at

the aniline stage in two steps starting from the corre-

sponding iminophosphoranes. The starting tri¯uoro-

methylketones were commercially available, with the

exception of 2,2,2-tri¯uoro-l-(3-methoxyphenyl)ethanone,

which was prepared from 3-bromoanisole according to

the procedures described by Hatanaka et al. and references

therein.¹³Ylides 12a and 12b were readily obtained by

treatment of the corresponding azides with Ph₃P.^14,15Sub-

sequent Staudinger reaction to aord the CF₃-substituted

imines 13a±d, followed by LAH reduction, furnished the

desired secondary anilines 14a±d in good overall yield.

N-Phenyl acetamides and N-benzyl benzamides. Acet-

amides were attractive because they oer an additional

site for structural diversity that is not available in ben-

zamides (Scheme 3). N-Ethylation of amide 18 using

PTC conditions as before aorded 19 in excellent yield.

Subsequent a-alkylation using LDA at 78 ^ꢀC then

aorded the disubstituted carboxamides 20a±b in high

yield. Thionation and/or deprotection as before furnished

Scheme 2. Synthesis of CF₃-containing N-phenyl benzamides.

Scheme 3. Synthesis of N-phenyl-acetamides.

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1297

the desired bis-phenolic amides 21a±b and 22a±c with-

out complication.

attempts to thionate 26 with Lawesson's reagent failed;

however, treatment of the unsubstituted phenyl-

acetamide 25 with Lawesson's reagent did give thio-

amide 28, in albeit low yield. Repeated attempts using

other reagents (P₂S₅and POC₁₃/(TMS)₂S) or higher

temperatures failed to provide thioamide 26 or to

improve the yield of 28. Increased steric hindrance

about the CO bond is thought to be responsible for

the lack of reactivity of the CF₃-substituted N-phenyl

acetamides towards O±S replacement, as lower yields

for thionation were also observed for the CF₃-contain-

ing benzamides (Scheme 2).

The N-benzyl-N-alkylbenzamides 24 (Scheme 4), which

are essentially amide isomers of the acetamide class, and

related benzenesulfonamides (not shown), were also

investigated. These N-benzyl amides, protected as

methyl ethers, could be prepared by routes that are

analogous to those used in Schemes 1±3. However, these

systems proved to be unstable to various deprotection

conditions: BBr₃, ethanethiolate, HBr/AcOH and TMSI

all led to decomposition. The only isolatable products

were the corresponding secondary amides, presumed to

result from an elimination of the 4-hydroxy-N-benzyl

substituent via a quinone-methide intermediate (Scheme

4). Due to their propensity for elimination, additional

eorts were not made to prepare members of the N-

benzyl benzamide class.

Molecular conformation

As was noted in Figure 1, bibenzyl motifs in ER ligands

are well represented as both syn and anti conformations.

Since N-aryl-benzamides share a similar bibenzyl-like

two-atom connection between two aromatic rings, we

were interested in their conformational preference to

establish which structural motif they might be mimick-

CF₃-containing N-phenyl acetamides. Incorporation of

the CF₃-group was also explored in the acetamide

series, and the synthesis of a-substituted acetamides

containing an N-(1,1,1-tri¯uoro-2-propyl) substituent is

shown in Scheme 5. Thus, the N-(1,1,1-tri¯uoro-2-propyl)

substituted aniline 12a was treated with 4-methoxy-

phenylacetyl chloride to aord carboxamide 25. a-

Alkylation as before using LDA at 78 ^ꢀC and MeI

proceeded in excellent yield to give 26 as a ꢁ1:1 mixture

1

ing. From early H NMR studies of N-substituted ani-

lides, there is known to be a surprisingly strong

preference for the exo-isomer (N-phenyl group trans to

carbonyl oxygen).¹⁶Even simple N-methyl and N-

ethylformanilides, where steric considerations would

place the bulkier alkyl group next to the formyl hydro-

gen in an endo preference, have a 95% exo preference in

solution.¹⁷This so-called `exo-rule' is quite general, and

it has been stated that for N-substituted anilides other

than formyl, the exo-isomer dominates to the exclusion

1

of diastereomers as indicated by H NMR and HPLC

analysis. Demethylation then aorded the di-substituted

phenylacetamide 27 in good yield. Unfortunately,

Scheme 4. Attempted synthesis of N-benzyl-benzamides.

Scheme 5. Synthesis of CF₃-containing N-alkyl-phenylacetamides.

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S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

of the endo-isomer.¹⁶The endo-isomer is detectable only

when large ortho substituents are present on the N-

phenyl ring or when R⁰H.

addition, for comparison, a crystallographic structure

was determined on the less hindered N-(i-propyl)-car-

boxamide 10b. ORTEP representations of these mol-

ecules are shown in Figure 5.

As can be seen in Figure 5, both the i-propyl carbox-

amide and CF₃-substituted thioamide were found to

crystallize in the lower energy s-cis conformation. Prom-

inent torsion angles are indicated in Table 1. Com-

parison of the amide torsional bonds (torsion A) reveals

greater deviation of the N-aryl ring from the amide

plane in thiocarboxamide 17a compared to 10b (18^ꢀ

versus 9.7^ꢀ), consistent with the greater steric hindrance

anticipated in the amide plane of the thioamide system.

The greater distortion in the thioamide bond planarity

is also evident from its improper torsion (torsion E) and

its N-alkyl torsion B. Both structures also show a twist-

ing of the phenyl rings out of the amide plane (C and

D), with the thioamide being more planar. This dier-

ence appears in part to be due to additional nearby non-

bonding interactions with the CF₃-group in thioamide

17a.

Molecular modeling studies on both carboxamide 16a

and thiocarboxamide 17a that we have performed using

molecular mechanics (MM2) were in agreement with the

reported NMR observations, and indicated the exo or

s-cis conformer as being energetically more favorable

(Fig. 4). In the case of carboxamide 16a, a substantial

ÁH of 5.3 kcal/mol between the global minimum energy

cis and trans isomers was found, the major energetic

dierence being due to additional electrostatic con-

tributions in the s-trans conformation. Thiocarbox-

amide 17a has a smaller ÁH of 0.9 kcal/mol between the

global minimum energy cis and trans isomers (not

shown). This result is in accord with destabilization

of the s-cis (exo) thioamide conformer relative to the

s-trans (endo) conformer because of additional steric

repulsions between sulfur and the N-alkyl substituent

that arise from the larger covalent radii of sulfur versus

oxygen (1.4 A for sulfur, 0.74 A for oxygen) and its

longer bond length (CS 1.64 A; C0 1.24 A).

1

Solution VT H NMR experiments (Fig. 6) were also

conducted, and the results were found to be consistent

with a single amide conformer. Shown in Figure 6 is the

¹H NMR spectrum of the aromatic region for thio-

amide 17a at 60, +23, and +55 ^ꢀC in MeOD. The

spectra at 60 ^ꢀC and room temperature are very simi-

lar. Interestingly, there is a slow rotation about the N±C

bond of the N-phenyl group, which results in distinct

signals for the ortho and meta ring protons of the

N-phenyl ring. Assignments are based on COSY cross

couplings. Upon heating to +55 ^ꢀC, no new signals are

observed for either the CH₃group (not shown), which

remains a doublet at all three temperatures, or the aroyl

protons. However, the ortho and meta signals of

the N-phenyl ring coalesce. This type of slow N±C rota-

tion is well known in N-phenyl carboxamides and is

analogous to hindered bi-phenyl rotations.¹⁶The lack of

Direct structural studies of N-substituted thioanilides,

let alone hindered systems containing a CF₃-substituent,

have not been reported, as far as we have found. For

this reason and because of the relatively small energy

dierence found from molecular modeling for 17a, we

obtained an X-ray crystallographic structure of the

methyl ether derivative of 17a to ®rmly establish its

preferred stereochemistry about the amide bond. In

Figure 4. Capped-stick models of lowest energy c-cis and s-trans conformers of caboxamide 16a. This s-cis conformer (left) has lower energy

(ÁH=5.3 kcal/mol).

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1299

Figure 5. Molecular structures for thiocarboxamide 17a (left) and carboxamide 10b (right) (ORTEP; ellipsoids drawn at the 35% probability level).

Table 1. Prominent torsion angles found in carboxamide 10b and

thiocarboxamide 17a

Torsion

Compound 17a

thioamide

1±4 atoms

Angle

Compound 10b

carboxamide

1±4 atoms

Angle

A

B

C

D

E

C(2)±C(9)

C(16)±S(1)

C(1)±C(10)

18^ꢀ

7.8^ꢀ

76^ꢀ

50^ꢀ

C(2)±C(8)

C(14)±O(1)

C(1)±C(9)

C(3)±N(1)

9.7^ꢀ

3.2^ꢀ

80^ꢀ

C(3)±N(1)

N-improper torsion

62^ꢀ

5.4^ꢀN-improper torsion

4.6^ꢀ

additional signals indicates that there is a single amide

conformer in solution, which we presume, on the basis of

the computational and X-ray structures, to be the s-cis.

Furthermore, for thioamides, it is worthy to note that

the barner for cis±trans inter-conversion is generally 2±

5 kcal greater than it is for the analogous carboxamides.

The traditional `resonance model' used to rationalize

the origin of the higher cis±trans barriers in thioamides

has recently been challenged¹⁸and is currently under

debate.^{19 22}Nevertheless, from these studies we believe

that it is reasonable to conclude that sterically hindered

N-aryl thiobenzamides also have a strongly preferred

s-cis conformation, similar to N-substituted oxoanilides.

Therefore, these systems can be considered as mimics of

the syn-bibenzyl substructural motif.

Receptor binding anities and structure±binding anity

relationships

Binding anities for the estrogen receptor and octanol±

water partition coecients (Log P^o/w) determinations of

the benzamides we have prepared are shown in Table 2

and are organized according to the substituent on the

amide nitrogen (either N-alkyl/aryl or N-CF₃contain-

ing alkyl/aryl). Binding anities and Log P^o/wdetermi-

nations for phenylacetamides are presented in Table 3.

Binding anities were obtained from a competitive

radiometric binding assay, using [³H]estradiol as tracer

and lamb uterine cytosol as a source of ER, and they

are expressed as relative binding anities (RBA), with

estradiol having an anity of 100%. Additional binding

Figure 6. VT-¹H NMR for 17a, aromatic region.

assays of select compounds for receptor subtypes ERa

and ERb, as well as ERa and ERb transcriptional acti-

vation pro®les in human endometrial cancer (HEC-1)

cells using an estrogen-responsive reporter gene con-

struct, are also shown in Table 4. Log P^o/wdetermina-

tions were obtained according to the method of Minick,

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S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

Table 2. Estrogen receptor relative binding anity RBA (estra-

diol=100) and log octanol±water partition coecient (log P^o/w) data

for N-phenyl-benzamides

Table 4. Estrogen receptor relative binding anity RBA (estra-

diol=100) and log octanol±water partition coecient (log P^o/w) data

for N-phenyl phenylacetamides

Compound

X

R¹

R²

RBA^a

Log P^o/w

21a

22a

27

O

CH₂CH₃

CH(CH₃)(CF₃)

H

0.006

0.004

0.010

2.68

3.52

Ð

CH₂CH₃

CH₃

RBA^a

Log P^o/w

28

S

CH(CH₃)(CF₃)

CH₂CH₃

H

0.66

0.20

0.089

0.34

Ð

3.40

4.27

Ð

Compound

Core structure

-CSO₂R-

R group

21b

22b

22c

5a

5b

5c

Et

n-Bu

Bn

0.23

0.13

0.053

2.76

3.66

4.62

CH₂CH₃

CH₂Ph

CH₂CH₃

^aRelative binding anities are the average of duplicate determinations

(CV 0.3), measured in lamb uterine cytosol. For estradiol, RBA=100.

For details, see Experimental.

10a

10b

10c

10d

10e

10f

-CONR-

Et

i-Pr

n-Bu

i-Bu

0.062

0.009

0.042

0.020

0.089

0.45

2.40

2.31

Ð

3.15

Ð

CH₂Ph

CH(C₆H₅)CH₃

3.96

thioamides (11a±g) bind with anity 2- to 90-fold

greater than their carboxamide counterparts, which is

presumably due to their greater hydrophobic character,

although there are not great dierences in the Log P^o/w

values, where comparisons can be made. A related trend

is also observed in other non-steroidal ligands contain-

ing heteroatoms, such as benzo[b]furans and benzo-

[b]thiophenes, with benzo[b]thiophenes displaying

higher anity than benzo[b]furans.^24,25

11a

11b

11c

11d

11e

11f

11g

-CSNR-

Et

i-Pr

n-Bu

i-Bu

0.62

0.83

1.3

1.5

0.15

2.5

2.80

2.97

3.99

Ð

3.98

4.31

CH₂Ph

CH(C₆H₅)CH₃

CH(CH₃)(i-Pr)

4.6

^aRelative binding anities are the average of duplicate determinations

(CV 0.3), measured in lamb uterine cytosol. For estradiol, RBA=100.

For details, see Experimental.

The bene®cial eect of placing more sterically encum-

bered groups near the amide core on receptor anity is

also evident in the thioamide series, because the same

relative anity increases which occurred for carbox-

amides (10a<10e<10f) are also re¯ected in the thio-

amides (11e<11a<11f). Replacement of the phenyl

substituent in 11f with an i-propyl group to aord

thioamide 11g results in even greater crowding near the

amide plane, enhancing the relative anity to 4.6%, the

highest observed for any simple N-phenyl benzamide.

Table 3. Estrogen receptor relative binding anity RBA (estra-

diol=100) and log octanol±water partition coecient (log P^o/w) data

for N-CF₃-substituted benzamides

Compound

X

R¹

R²

R³

RBA^a

Log P^o/w

16a

16b

16c

16d

16e

16f

16g

O

HO

H

CH₃

C₆H₅

p-HO

H

m-HO

o-HO

p-HO

0.49

9.0

0.063

0.45

0.068

8.9

3.29

4.51

4.46

Ð

4.97

4.63

C₆H₅

m-HOC₆H₄

Binding anity of CF₃-containing N-phenyl benzamides.

The RBA data for the CF₃-containing benzamides are

presented in Table 3. Immediately apparent is the

relatively high binding anity for thioamide 17b and

carboxamide 16g, and to a lesser extent amides 16b, 16f

and 17a. Compound 16a, reported by Hartmann and co-

workers, is included for comparison.⁶In general, all the

CF₃-substituted benzamides show anity enhancements

relative to their non-¯uorinated amide counterparts

(Table 2).

H

15

17a

17b

S

HO

CH₃

C₆H₅

p-HO

7.5

14

3.99

5.40

^aRelative binding anities are the average of duplicate determinations

(CV 0.3), measured in lamb uterine cytosol. For estradiol, RBA=100.

For details, see Experimental.

using a standardized Chromegabond MC8 reverse

phase HPLC system.²³

Shown in Figure 7 are four direct comparisons of RBA

and Log P^o/wdata for benzamides from Tables 2 and 3.

These four pairs of compounds clearly show the rela-

tionship between core structure (O and S substitution)

and peripheral group presentation (methyl to phenyl,

and methyl to tri¯uoromethyl) and how these compo-

nents alter ER anity.

Binding anity of N-phenyl benzamides. The receptor

binding anities for N-phenyl benzamides are shown in

Table 2. The sulfonamides and carboxamides all have

rather low anity, the highest being carboxamide 10f.

Within the carboxamide series (10a±f), it is interesting

to compare the eect of branching near the amide core.

For example, the ethyl (10a) and benzyl (10e) com-

pounds have similarly low anities, and likewise the

branched i-propyl amide 10b has very low anity.

However, amide 10f, which contains both an a-phenyl

and methyl substituent, shows a signi®cant, 5- to 7-fold

increase in anity compared to either monosubstituted

amide 10a or 10e. Overall, the analogous N-alkyl

Comparison of the relative RBAs for these four pairs of

amides, starting from the i-propyl pair (upper left)

to the CF₃-containing phenethyl pair (lower right),

shows a diminishing eect of sulfur±oxygen replacement

as binding anity increases and as larger substituents

are added near the amide core. The sulfur±oxygen

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1301

Figure 7. Comparison of the relative binding anities of the principal carboxamides and thiocarboxamides.

enhancement is greatest in the parent i-propyl system

(10b versus 11b), where the overall anity is lowest, and

is least when the substituents are phenyl and tri¯uoro-

methyl (16b versus 17b), where the overall anity is

highest. In addition, there is a general increase in bind-

ing anity with increasing lipophilicity. The observed

parallel anity±lipophilicity increase within the series is

not too surprising in light of what is known about the

hydrophobic character of the ER ligand binding

pocket.²⁶

the Log P^o/wvalues for 11g and 17a are quite similar,

with 11g being somewhat higher, suggests that the dif-

ference in binding anity is probably not due to a lipo-

philic component. Thus, based on this data it would

seem that the additional component has, in addition to

its steric eect, an electronic eect as well.

Optimization of binding orientation and hydroxylation

pattern. For unsymmetrical non-steroidal ER ligands, a

common approach to determining which phenol is

imitating the crucial A-ring phenol of estradiol is to

prepare and test the corresponding mono-phenolic

derivatives.²⁸The highest anity mono-phenolic ana-

logue is then presumed to be the A-ring mimic, because

the hydroxyl substituent at this position is known to

be very important in binding to the ER. Many non-

steroidal ER ligands bene®t from a second hydroxyl

hydrogen bonding partner and appear to do so in an

anti-bibenzyl motif, which places the hydroxyl oxygen±

oxygen interatomic distances approx. 10±12 A. Thus, it

was apparent to us that the rigid s-cis benzamide con-

formation (syn-bibenzyl motif) in our amides was likely

not bene®ting from a second hydrogen bonding inter-

action, because its hydroxyl oxygen±oxygen interatomic

distance was only 7.4 A. The importance for this second

hydrogen bond is evident in the recent non-steroidal

diethylstilbestrol and raloxifene ERa LBD crystal

structures.^26,29Both of these complexes have an expec-

ted A-ring phenol mimic and a second phenol imitating

the D-ring 17b-hydroxyl group of estradiol. In both

cases, the second phenol uses the same hydrogen bond

interaction with His₅₂₄as does the 17b-hydroxy1 group

of estradiol.

CF₃eect: lipophilic, steric or electrostatic eects? The

eect of adding a CF₃group in place of methyl on

the RBA may be the result of several factors, including

lipophilic, steric and/or electrostatic eects. In terms of

steric eects, a recent review by O'Hagan and Rzepa²⁷

cites several studies which place an upper size limit of a

CF₃-group being close to that of i-propyl. The greatest

increases in RBA for CH₃to CF₃substitution are found

in the carboxamide class (20- to 50-fold, versus 5- to 8-

fold for thioamide), with the highest being the i-propyl

compounds (Fig. 7, 16a versus 10b). In order to test

whether or not RBA increases associated with CF₃-

replacement are the result of its steric or its electronic

property, the branched CH(CH₃)(i-Pr) thioamide 11g

was prepared to compare with the CF₃analogue 17a.

Based on the above-mentioned studies, if steric eects

alone operate, compound 11g would be expected to

have an RBA close to that of the CF₃-substituted

thioamide 17a (7.5%). The observed RBA, however, for

compound 11g was only 4.6%. This indicates that an

additional eect may be contributing to the binding

energy, either lipophilic or electrostatic. The fact that

1302

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

To obtain an optimal hydroxylation pattern and discern

which ring phenol was imitating the A-ring phenol of

estradiol, we prepared benzamides 16c±g. The results of

these studies (Table 3) clearly show a pattern indicative

of a preferred A-ring phenol mimic. For example,

removal of the 4-hydroxyl phenol on the benzamide ring

and retention of the 4-hydroxyl of the N-phenyl in

compound 16c lead to a nearly complete loss in binding

anity compared to the parent di-hydroxy compound

16b (RBA=0.063% versus 9.0%). In contrast, removal

of N-phenyl hydroxyl and retention of the benzamide

4-hydroxyl result in an analogue 16f, which retains a

binding anity equivalent to that of the parent com-

pound. These ®ndings clearly implicate the benzoyl ring

phenol as the A-ring mimic of estradiol. In addition, as

might be expected, positioning the hydroxyls elsewhere

on the benzoyl ring is not well tolerated, as both the

ortho- and meta-hydroxy analogues 16d and 16e have

little or no anity for the receptor.

The choice of positioning the second OH at the meta

position on the phenethyl substituent seemed somewhat

obvious, since modeling showed that alternative OH

.

substitution on the N-phenyl ring fails to provide O O

interatomic distances much greater than in the para-

substituted system 16b. Furthermore, the binding mode

depicted in Figure 8 actually places the N-phenyl group

more closely in the 11b subpocket of the ligand binding

site, rather than the D-ring subpocket. In choosing the

ring-substitution for the hydroxyl on the phenethyl ring,

we considered both the para and meta isomers. How-

ever, in light of the problems encountered with quinone

methide elimination in the N-benzyl benzamide series

bearing a para-methoxy protecting group, we opted not

to prepare the para-hydroxy analogue of 16f, since the

electron withdrawing CF₃group would presumably

exacerbate quinone methide elimination.

With the recent release of the E₂ERa LBD crystal

structure coordinates, we performed molecular docking

studies of the proposed meta-hydroxybenzamide (16g)

using TRIPOS' Flexidock routine (see Experimental),

to see whether or not the proposed binding mode in

Figure 8 was reasonable and whether a meta-hydroxy

derivative could participate in a hydrogen bonding

Having discerned which phenol in the benzamide system

was important for binding, we wished to explore alter-

native positions for a second hydroxyl group in order

to bene®t from the second hydrogen bonding interac-

tion which is found in the raloxifene and DES ER

binding pockets. Depicted in Figure 8 is a schematic

representation of the predicted binding mode and phe-

nol interactions for benzamide 16f in the binding pocket

of ER. This rudimentary model was derived from the

interactions that were described to be present in the

estradiol (E₂) X-ray crystal structure in the ERa LBD,

the coordinates of which, at the time of our considera-

tion, were not released.²⁶Based on this view, we pro-

posed that a hydroxyl substituent on the phenethyl ring

might be positioned in the binding subpocket near

interaction with His₅₂₄

.

The ®nal docked and minimized model, using the S-

enantiomer of 16g, is depicted in crossed-stereo in Fig-

ure 9. The R-enantiomer (not shown) was also modeled

in the same manner but resulted in a higher energy

complex. The ®nal S-enantiomer model converged to

.

an RMS of <0.05 kcal/mol A with a reasonably favor-

able binding energy. In this conformation, the benzoyl

phenol is comfortably accommodated in the A-ring

pocket, and the N-phenyl group projects into what

His₅₂₄

.

Figure 8. Putative binding mode for N-phenyl benzamide template with A-ring mimic interactions and a proposed site for second HO-group.

Figure 9. Crossed-stereo view of 16g docked and minimized in ERa LBD binding pocket showing select residues within 3 A of the benzamide.

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1303

D-ring hydroxyl mimic, as in raloxifene, result in a

0.6 kcal/mol reduction in binding energy, which corre-

sponds to a 3-fold drop in anity.³⁰The fact that the

addition of the second hydroxyl does not give a full 3-

fold increase may be due to a somewhat unfavorable

hydrogen bond trajectory in the binding pocket. From

our model, the distance between the m-hydroxyl sub-

stituent and His₅₂₄(3.69 A) is suciently close for some

hydrogen bonding interaction, but is not ideal either in

terms of distance or geometry. A second more likely

explanation may have to do with stereochemical issues:

This compound (l6g) was tested as a racemate, so only

one enantiomer is likely to be able to bene®t from the

second hydrogen bonding interaction. The ERa and

ERb RBAs and the transcriptional activation pro®le for

rac-16g in HEC-l cells are discussed below.

N-Phenyl acetamides as ER ligands. The RBA and

Log P^o/wdata for N-alkyl- and CF₃-containing acet-

amides are shown in Table 4. Disappointingly, none of

these compounds were found to show appreciable a-

nity for the ER. Several direct comparisons can be made

to other series in this investigation, which suggest that

acetamides may not provide viable scaolds for further

combinatorial consideration, despite their potential to

support greater structural diversity than the benza-

mides. Even the CF₃-containing acetamide 27 and

thioacetamide 28 have anities less that 1%, which are

50- and 11-fold lower, respectively, than their benza-

mide counterparts 16a and 17a.

Figure 10. Depiction of solvent accessible surfaces for 16g in ERa

binding pocket.

would resemble the 11b pocket of the ERa-E₂structure.

Interestingly, the amide bond occupies a region of the

binding pocket similar to that of the isosteric ethylene

unit of DES in the recently solved ERa-DES crystal

structure.²⁹

Using the above model, we generated Connolly solvent

accessible surfaces for the ligand and binding pocket to

visualize close contacts (Fig. 10). This view reveals

mostly favorable interactions within acceptable van der

Waals radii. The ligand itself has a volume of 296 A³,

which is 51 A³greater than E₂, but is well under the

total volume of 450 A³for the binding pocket as repor-

ted in the ERa-E₂crystal structure.²⁶

One possible explanation for the low anity in the

acetamide class may lie in their conformation. Monte

Carlo conformational studies of compound 27 and the

unsubstituted analogue 27b, using molecular mechanics

methods, show a distinct preference for a syn con-

formation, as shown in Figure 11. The syn-conformer

for 27 is predicted to be 5 kcal/mol lower in energy than

the anti-conformer; even without the a-methyl sub-

stituent, which eliminates possible alkyl-N-phenyl steric

repulsions, the syn-conformer is predicted to be favored

by 2.5 kcal/mol (not shown). It is also noted that the

barrier to rotation about the acetyl bond was estimated

We were grateful to ®nd that the bis-(phenolic) benza-

mide 16g had an RBA of 15%, the highest of any car-

boxamide ER ligand. This value represents a 1.6-fold

increase over the anity of the corresponding mono-

phenolic benzamide 16f and is close to what one might

expect: in E₂and other non-steroidal ER ligand systems,

removal of the 17b hydroxyl, as in E₂, or the second

Figure 11. Conformational analysis of N-penyl-penylacetamide 27.

1304

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

to be between 4 and 5 kcal/mol for 27, based on MM2

dihedral drive calculations.

As in the benzamides, in the acetyl conformations of 27

the exo-rule is upheld; this places the N-phenyl group

exo with respect to the amide CO, making the amide

con®guration s-cis. This exo-eect may also be opera-

tive for the a-phenyl substituent as well, since it also

prefers to be essentially exo to the amide CO rather

.

than endo. Since the hydroxyl O O interatomic distances

for many estrogens requires a minimum of approxi-

mately 10.5 A, the syn-conformation is excluded as a

structure that is likely to ®t in the ER binding pocket.

Based on these studies and the RBA data available at

this time, further investigations of the acetamide series

did not appear to be warranted.

Figure 12. Transcription activation by ERa and ERb in response to

benzamide 16g. Human endometrial cancer (HEC-1) cells were trans-

fected with expression vectors for ERa or ERb and an (ERE)₃-pS2-

CAT reporter gene and were treated with the indicated concentrations

of benzamide 16g for 24 h. CAT activity was normalized for b-galac-

tosidase activity from an internal control plasmid. Values are the mean

ÆSD for three or more separate experiments, and are expressed as a

percent of the ERa or ERb response with 1 nM E₂.

ERꢀ/ERꢁ binding anity, transcriptional activity and

enantioselectivity

Shown in Table 5 are the RBA values of select benza-

mides for puri®ed ERa and ERb, along with their tran-

scriptional activity in HEC-1 cells using an estrogen-

responsive reporter gene construct ((ERE)₃-pS2-CAT)

with expression vectors for either ERa or ERb and

whereas some were eective partial antagonists through

ERb. Compound 16g was a full agonist on ERa and

ERb.

6

benzamide at 10 M. It is apparent that binding an-

ities show selectivity for ERa, mostly in the range of

2- to 8-fold; compound 16g has the highest selectivity

for ERa of 21-fold (27% versus 1.3%). The enantio-

mers of 17b, which were separated using chiral HPLC

(ChiralPak AD column), also showed interesting RBA

dierences between the two receptor subtypes. For

example, enantiomer ent₂-17b was found to have greater

selectivity for ERa than enantiomer ent₁-17b by more

than 5-fold, indicating that the individual binding

pockets for ERa and ERb are uniquely sensitive to

subtle changes in ligand shape.

6

Because of the high activity of amide 16g at 10 M on

both ERa and ERb, a full dose±response curve was

determined to see whether any potency selectivity exis-

ted for either ERa or ERb. As shown in Figure 12,

benzamide 16g behaves as an ERa potency selective

agonist, displaying a 500-fold higher potency for acti-

vation of ERa over ERb. It is of note that this high

potency selectivity for ERa is greater than the 21-fold

ERa anity selectivity of this compound (cf. Table 5).

In the transcription assays,³¹agonism is measured with

1 mM of compound alone and is expressed as the percent

of transcriptional activity with 1 nM E₂(high values

indicate agonist activity); antagonism is measured with

1 mM compound together with l nM E₂(low values

indicate antagonist activity). All of the benzamides

tested were full or nearly full agonists through ERa,

Conclusions

Through an analysis of potent non-steroidal estrogens,

we have identi®ed common substructural bibenzyl and

homobibenzyl motifs in the structure of ER ligands.

Based on this simple pharmacophore model, we have

Table 5. Benzamide ERa and ERb binding anities and transciptional activity

Compound

RBA (%) (uterine)

RBA (%)^a

Agonism^b% CAT Act.

Antagonism^b% CAT Act.+E₂

ERa

ERb

a/b ratio

ERa

ERb

ERa

ERb

11g

17a

16b

rac-17b

ent₁-17b

ent₂-17b

16g

4.6

7.5

9.0

14

10

22

14

23

17

25

18

40

27

2.4

15

2.7

8.9

11

5.8

1.5

6.3

2.8

1.6

8.3

21

65

60

75

80

85

80

99

20

15

30

10

99

90

65

90

83

80

90

100

40

45

35

48

40

97

4.8

1.3

15

^aRelative binding anities are the average of duplicate determinations (CV 0.3), determined with purifed RDa and ERb preparations. For estradiol,

RBA=100. For details, see Experimental.

^bHEC-1 cells were transfected with either REa or ERb expression vectors and an (ERE)₃-pS2-CAT reporter gene. CAT activity was normalized for

b-galactosidase activity from an internal control plasmid and is expressed as a percent of the ERa or ERb response with 1 nM E₂. Benzamides were

tested at 10 M concentration to test for agonism and at 10 M with 10 M E₂to test for antagonism. These assays are generally reproductive

6

9

with a CV of 30% relative.

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1305

made a substantial exploration of the amide function-

ality as a core replacement that might support com-

pound libraries for developing novel SERMs. Using this

approach, we prepared and tested representative exam-

ples of bis-phenolic amides which displayed both simple

N-alkyl substituents and CF₃-containing N-alkyl sub-

stituents. Of the two classes examined, benzamides and

acetamides, the benzamides displayed the highest an-

ity, in particular the CF₃-containing benzamides. The

recent X-ray crystal structure of E₂bound to ERa and

structure±binding anity relationships have allowed us

to design a bis-phenolic benzamide 16g, which has the

highest anity for any carboxamide ligand for the ER.

This compound is also an ERa potency-selective agonist

in cell-based assay of ER-mediated transcriptional

activity. This work illustrates that ER ligands based on

simple amide core structures can be readily prepared.

High anity ER binding, however, requires an appro-

priate distribution of bulk and functionality. The strong

conformational preference of the basic anilide function

in all of these ligands de®nes the scope of further struc-

tural and functional expansion of these series.

resolution electron impact mass spectra were obtained

on Finnigan MAT CH-5 or 70-VSE spectrometers. Ele-

mental analyses were performed by the Microanalytical

Service Laboratory of the University of Illinois.

Unless otherwise stated, a standard procedure for

product isolation was used; this involved quenching by

addition of water or an aqueous solution, exhaustive

extraction with an organic solvent, washing the extracts,

drying over Na₂SO₄, and solvent evaporation under

reduced pressure. Quenching media, extraction solvents,

and aqueous washes used are noted parenthetically after

the phrase `product isolation'.

Biological procedures

Relative binding anity assay. Ligand binding anities

(RBAs) using lamb uterine cytosol as a receptor source

were determined by a competitive radiometric binding

assay using 10 nM [³H]estradiol as tracer and dextran-

coated charcoal as an adsorbant for free ligand.³²Puri-

®ed ERa and ERb binding anities were determined

using a competitive radiometric binding assay using

10 nM [³H]estradiol as tracer, commercially available

ERa and ERb preparations (PanVera Inc., Madison,

WI), and hydroxylapatite (HAP) to adsorb bound

receptor±ligand complex.³³HAP was prepared follow-

ing the recommendations of Williams and Gorski.³⁴All

incubations were done at 0 ^ꢀC for 18±24 h. Binding

anities are expressed relative to estradiol on a percent

scale (i.e. for estradiol, RBA=100%). All essays were

run in separate, duplicate experiments which were

reproducible with a coecient of variation of less than

30% relative.

Experimental

General

Melting points were determined on a Thomas-Hoover

UniMelt capillary apparatus and are uncorrected. All

reagents and solvents were obtained from Aldrich,

Fisher or Mallinckrodt. Tetrahydrofuran was distilled

from sodium/benzophenone. Dimethylformamide was

vacuum-distilled prior to use, and stored over 4 A

molecular sieves. n-Butyllithium was titrated with N-

pivaloyl-o-toluidine. All reactions were performed

under a dry N₂atmosphere unless otherwise speci®ed.

Reaction progress was monitored by analytical thin-

layer chromatography using GF silica plates purchased

from Analtech. Visualization was achieved by short-

wave UV light (254nm) or potassium permanganate.

Radial preparative-layer chromatography was performed

on a Chromatotron instrument (Harrison Research, Inc.,

Palo Alto, CA) using EM Science silica gel Kieselgel 60

PF₂₅₄as adsorbent. Flash column chromatography was

performed using Woelm 32±63 mm silica gel packing.

Transcriptional activation assay. Human endometrial

cancer (HEC-l) cells were maintained in culture and

transfected as described previously.³¹Transfection of

HEC-1 cells in 60 mm dishes used 0.4 mL of a calcium

phosphate precipitate containing 2.5 mg of pCMVbGal

as internal control, 2 mg of the reporter gene plasmid,

100 ng of the ER expression vector, and carrier DNA to

a total of 5 mg DNA. CAT activity, normalized for the

internal control b-galactosidase activity, was assayed as

previously described.³¹

Molecular modeling

Logarithms of octanol±water partition coecients (Log

P

^o/w) were determined using a standardized reverse

Small molecule modeling. Dihedral drive and Monte

Carlo conformational searches for N-phenyl benza-

mides 16a and 17a and N-phenyl acetamide 27 were

conducted using the MM2 force ®eld as implemented in

Macromodel v.5.5 with CHCl₃as a solvent model. All

generated conformers from Monte Carlo searches

underwent full matrix assisted minimization using the

phase HPLC Chromegabond MC 8 column.²³HPLC

separation of the enantiomers for 17b was performed on

an analytical ChiralPak AD (4.6Â25 cm) column from

Chiral Technologies using 10% i-propanol/hexane as

solvent. ¹H and ¹³C NMR spectra were recorded on

either a General Electric QE-300 (300 MHz), Varian

Unity 400 or 500 MHz spectrometers using CDCl₃,

MeOD or (CD₃)₂SO as solvent. Chemical shifts were

reported as parts per million down®eld from an internal

PRCG function with

a convergence criteria of

0.001 kcal/mol. Dihedral drives were conducted in 5^ꢀ

steps with a convergence criteria of 0.05 kcal/mol using

1000 minimization steps. Analysis of the energy versus

torsion angle provided the estimated rotational barrier.

1

tetramethylsilane standard (d=0.0 for H) or from sol-

vent references. NMR coupling constants are reported

in Hertz. ¹³C NMR were determined using either the

Attached Proton Test (APT) experiment or standard

¹³C pulse sequence parameters. Low resolution and high

Receptor docking studies. The starting conformation

used for docking studies for 16g was obtained from a

1306

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

random conformational search using the TRIPOS' force

®eld (MAXIMIN) as implemented in the program

sybyl, version 6.5.2 (Tripos Inc., St. Louis, MO). The

cis global minima obtained for benzamide 16g was then

overlaid with E₂in the E₂-ERa LBD crystal structure²⁶

using a least squares multi®tting of ®ve atoms: atoms

C(3), C(10), C(9), C(11), and C(12) of E₂were ®tted

with the 1 and 4 carbons of the benzamide phenyl ring,

the CO carbon, the nitrogen and the C(2) carbon of

the phenethyl group, respectively. The pre-positioned

benzamide was then optimally docked in the ERa bind-

ing pocket using TRIPOS' Flexidock. Both hydrogen-

bond donors and acceptors within the pocket sur-

rounding the ligand (Glu₃₅₃, Arg₃₉₄and His₅₂₄) and

the ligand itself, in addition to select torsional bonds

were de®ned. The best docked receptor ligand complex

from Flexidock then underwent a three-step minimiza-

tion: ®rst non-ring torsional bonds of the ligand were

minimized in the context of the receptor using the tors-

min command, followed by minimization of the side-

chain residues within 8 A of the ligand while holding the

backbone and residues Glu₃₅₃and Arg₃₉₄®xed. A ®nal

third minimization of both the ligand and receptor was

conducted using the anneal function (hot radius 8 A,

interesting radius 16 A) while holding residues Glu₃₅₃

and Arg₃₉₄®xed to aord the ®nal model. Minimiza-

tions were done using the TRIPOS' Force®eld (MAXI-

MIN) with the Powell gradient method and default

Data were collected with an area detector by using the

measurement parameters listed in Table 6. No systematic

absences are noted for the P-1 space group. The measured

intensities were reduced to structure factor amplitudes and

their esds by correction for background, scan speed, and

Lorentz and polarization eects. While corrections for

crystal decay were unnecessary (the data were corrected

for crystal decay), a É-scan absorption correction was

applied, the maximum and minimum transmission factors

being 0.991 and 0.876. Systematically absent re¯ections

were deleted and symmetry equivalent re¯ections were

averaged to yield the set of unique data. All 5344 data

were used in the least squares re®nement.

The structure was solved using direct methods

(shelxtl). The correct positions for the C, N, O, S, and

F atoms were deduced from an E-map. Subsequent

least-squares re®nement and dierence Fourier calcu-

lations revealed the positions of the remaining non-

hydrogen atoms. The quantity minimized by the least-

squares program was Æw(F²₀F_c²)², where w=

{[ꢀ(F²₀)]²+(0.883P)²} ¹and P=(F₀²⁺2F²_c)/3. The analy-

tical approximations to the scattering factors were used,

and all structure factors were corrected for both real

and imaginary components of anomalous dispersion. In

the ®nal cycle of least squares, independent anisotropic

displacement factors were re®ned for the non-hydrogen

atoms and the aromatic, methyl, and methine hydrogen

atoms were ®xed in `idealized' positions with C±

H0.95 A for the aromatic hydrogens, C±H0.98 A for

the methyl hydrogens, and C±H1.00 A for the methine

hydrogen. Successful convergence was indicated by the

maximum shift/error of 0.001 for the last cycle. Final

re®nement parameters are given in Table 6. The largest

peak in the ®nal Fourier dierence map (0.795 eA ³) was

located 1.64 A from C(18). A ®nal analysis of variance

between observed and calculated structure factors

showed no apparent errors.

.

settings (®nal RMS <0.05 kcal/mol A).

X-Ray crystallography

Cystallography details for 17a and 10b. Crystals of

17a were obtained by slow evaporation from ether at

0 ^ꢀC. Crystals were mounted on glass ®bers with Para-

tone-N oil (Exxon) and immediately cooled to 75 ^ꢀC

in a cold nitrogen gas stream on the diractometer.

Standard peak search and indexing procedures gave

rough cell dimensions, and least squares re®nement

using 5344 re¯ections yielded the cell dimensions as

given in Table 6.

Single crystals of 10b were grown from MeOH by slow

evaporation at 23 ^ꢀC in a partially sealed scintillation

Table 6. Crystal data and structure re®nement for thiocarboxamide 17a and carboxamide 10b

Complex

Empirical formula

fw

17a

₁₈H₁₈F₃NO₂S

369.39

10b

C₁₆H₁₇NO₃

271.31

C

Temperature

Wavelength

Cryst syst

Space group

Unit cell dimensions

198(2) K

0.71073 A

triclinic

P-1

198(2) K

1.54178 A

monoclinic

P2₁/n

a=18.248(4) A

b=9.054(2) A

c= 18.248(4) A

2916.4(10) A³

8

1.236 Mg/m³

0.695 mm¹

a=9.5099(7) A

b=10.5351(8) A

c= 11.1127(8) A

868.07(11) A³

2

Volume

Z

Density, calcd

Abs coe

1.413 Mg/m³

1

0.228 mm

No. of indep re®ns

Re®nement method

No. of data/restraints/params

Goodness-of-®t (GooF) on F²

Final R indices (I> 2ꢀ(I))

R indices (all data)

5344 [R(int)=0.0293]

full matrix least-squares on F²

3727/0/226

0.949

R1=0.0502, wR2=0.1393

R1=0.0892, wR2=0.1533

4796 (R(int)=0.0556)

full matrix least-squares on F²

2727/0/362

1.062

R 1=0.0624, wR2=0.1662

R1=0.0767, wR2=0.1881

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1307

vial. Crystals were mounted on glass ®bers with Para-

tone-N oil (Exxon) and immediately cooled to 75 ^ꢀC in

a cold nitrogen gas stream on the diractometer. Stan-

dard peak search and indexing procedures gave rough

cell dimensions. Initial inspection of the data suggested

a C-centered orthorhombic cell, but no successful solu-

tion could be found in any orthorhombic space group.

Eventually, a successful solution was found in a primi-

tive monoclinic cell of half the volume in the space

group P2₁/n. The re®nement stalled at wR₂=0.63,

despite having located all atoms and re®ning the non-

hydrogen atoms anisotropically. The unusual equality

in the lengths of the a and c axes, combined with the

observation that F_obsꢂ F_calcfor all the re¯ections in the

`most disagreeable re¯ections' list, suggested that the

crystal was twinned. A twin law involving mirror re¯ec-

tion through the (101) plane was assumed, and a re®n-

able parameter describing the relative volume fractions

of the two twin individuals was added to the model. The

intensities were calculated from the relation F²_TOT=f

F²_hkl+(1 f ) F_h²⁰_k₀_l₀, where f is the volume fraction of

twin individual one and F²_hkland F_h²⁰_k₀₁₀are the con-

tributions from the two twins (the twin law speci®es that

h⁰k⁰l⁰=lkh). The scale factor re®ned to 0.500(3), and the

wR₂factor dropped to an acceptible value of 0.185.

N-(4-Methoxyphenyl)-N-(ethyl)-4-methoxybenzene sulfo-

namide (4a).⁸Puri®cation by ¯ash chromatography

(20% EtOAc:hexane) aorded o-white powder (86%):

mp 95±7 ^ꢀC, mp⁸100±104 ^ꢀC; H NMR (CDCl₃) d 1.06

1

(t, 311, J=7.1 Hz), 3.52 (q, 2H, J=7.0 Hz), 3.81 (s, 3H),

3.84 (s, 3H), 6.82 (d, 211, J=8.8 Hz), 6.94 (d, 2H, J=

8.0 Hz), 6.96 (d, 2H, J=8.0 Hz), 7.54 (d, 2H, J=8.8 Hz).

N-(4-Methoxyphenyl)-N-(butyl)-4-methoxybenzene sulfo-

namide (4b). Puri®cation by ¯ash chromatography

(20%EtOAc:hexane) aorded transparent oil (81%):

¹H NMR (CDCl₃) d 0.85 (t, 3H, J=7.2 Hz), 1.34 (m,

4H), 3.46 (t, 2H, J=6.8 Hz), 3.80 (s, 3H), 3.86 (s, 3H),

6.81 (d, 2H, J=9.0 Hz), 6.92 (app t, 4H, J=9.1 Hz), 7.51

(d, 2H, J=9.0 Hz); MS (EI, 70eV) m/z 349 (M⁺); HRMS

calcd for (C₁₈H₂₃NO₄S, 349.1347, found 349.1348).

N-(4-Methoxyphenyl)-N-(benzyl)-4-methoxybenzene sul-

fonamide (4c). Recrystallization from 20% EtOAc/hex-

ane aorded ¯occulent white crystals (93%): mp 141±

142 ^ꢀC; H NMR (CDCl₃) d 3.72 (s, 3H), 3.87 (s, 3H),

1

4.67 (s, 2H), 6.89 (d, 2H, J=11 Hz), 6.86 (d, 2H,

J=11.5 Hz), 6.94 (d, 2H, J=11 Hz), 7.21 (m, 5H), 7.59

(d, 2H, J=11 Hz); ¹³C NMR (CDCl₃) d 55.1, 55.4, 55.8,

114.2, 127.7, 128.5, 128.7, 129.9, 130.4, 130.4, 131.7,

136.3, 158.9, 163.0; HRMS calcd for C₂₁H₂₁NSO₄,

383.1191, found 383.118.

In the ®nal cycle of least squares, independent aniso-

tropic displacement factors were re®ned for the non-

hydrogen atoms and the aromatic, methyl, and methine

hydrogen atoms were ®xed in `idealized' positions with

C±H0.95 A for the aromatic hydrogens, C±H0.98 A

for the methyl hydrogens, and C±H=1.00 A for the

methine hydrogen. Successful convergence was indicated

by the maximum shift/error of 0.001 for the last cycle.

Final re®nement parameters are given in Table 6. The

largest peak in the ®nal Fourier dierence map

(0.795 eA ³) was located 1.64 A from C(18). A ®nal

analysis of variance between observed and calculated

structure factors showed no apparent errors.

General demethylating procedure using BBr₃. To a solu-

tion of methyl ether compound in CH₂Cl₂at 78 ^ꢀC

was added a 1.0 M BBr₃in CH₂Cl₂(3 equiv) dropwise

over 15 mm. The reaction was allowed to reach rt and

stir overnight or until disappearance of starting material

as indicated by TLC. The mixture was then cooled to

0 ^ꢀC. Product isolation (H₂O, Et₂O, brine) and puri-

®cation via radial or ¯ash chromatography or recrys-

tallization from an appropriate solvent aorded the

desired phenolic compounds.

General demethylating procedure using EtSH. To a stir-

red DMF solution of NaH (6.2 equiv of a 60% w/w

dispersion) at 0 ^ꢀC was added dropwise 6.0 equiv of

EtSH. After 30 min the mixture was warmed to rt and a

solution of the methyl ether protected compound in

5 mL DMF was added dropwise. The reaction mixture

was heated to re¯ux for 4 h; then cooled in an ice-bath

and acidi®ed with 2 N HCl. Remaining product isola-

tion (EtOAc, brine) and puri®cation aorded the phe-

nolic compounds.

Chemical syntheses

4-Methoxyphenyl-4-methoxybenzene sulfonamide (3).⁸

To a mixture of 4-methoxybenzenesulfonylchloride

(1.0 equiv) and pyridine (1.1 equiv) in CH₂Cl₂(0.5 M) at

0 ^ꢀC was added dropwise to a solution of p-anisidine

(1.5±5.0 equiv) in CH₂Cl₂(1.5 M) over 30 min. The

reaction mixture was warmed to rt and stirred until

completion of reaction as indicated by TLC. Product

isolation (H₂O, 2 N HCl) and recrystallized from EtOH

aor_ꢀded product as light red crystals (91%): mp 91±

92.5 C, mp⁸93 ^ꢀC.

N-(4-Hydroxyphenyl)-N-(ethyl)-4-hydroxybenzene sulfo-

namide (5a). Deprotected according to BBr₃procedure.

SiO₂plug (30% EtOAc/hexane) to aord o-white

crystals (62%): ¹H NMR ((CD₃)₂SO) 6 0.98 (t, 3H,

J=7.2 Hz), 3.43 (q, 2H, J=6.8 Hz), 6.67 (d, 2H, J=

8.7 Hz), 6.76 (d, 2H, J=8.4 Hz), 6.86 (d, 2H, J=

8.5 Hz), 7.34 (d, 2H, J=8.7 Hz); ¹³C NMR ((CD₃)₂SO)

d 14.1 (CH₃), 45.2 (CH₂), 115.7 (ArCH), 115.8 (ArCH),

122.6 (ArC), 128.1 (ArC), 129.9 (ArCH), 130.1 (ArCH),

157.1 (ArC), 161.6 (ArC); MS (EI, 70 eV) m/z 293

(M⁺); HRMS calcd for C₁₄H₁₅NSO₄, 293.0721, found

293.0722.

General procedure for phase transfer N-alkylation of sul-

fonamides.⁹To a CH₂Cl₂(0.06 M) solution of sulfona-

mide (1.0 equiv) was added an equal volume solution of

NaOH (5 equiv) and (n-Bu)₄NSO₄H (0.1 equiv) in H₂O.

Alkylhalide (2.0 equiv) was then added directly to the

bi-phasic solution and stirred at rt. After 12 h the layers

were separated. Product isolation (Na₂CO₃, brine)

followed by ¯ash chromatography aorded the pure

N-alkyl benzene sulfonamide.

1308

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

N-(4-Hydroxyphenyl)-N-(butyl)-4-hydroxybenzene sulfo-

namide (5b). Deprotected according to BBr₃procedure

to aord tan crystals, recrystallized from EtOAc/hexane

(71%): mp 137±138 ^ꢀC; ¹H NMR (CDCl₃) d 0.86 (t, 3H,

J=7.0 Hz), 1.35 (m, 4H), 3.48 (t, 2H, J=7.0 Hz), 6.42

(d, 2H, J=8.8 Hz), 6.87 (app t, 4H, J=8.9 Hz), 7.47

(d, 2H, J=9.0 Hz) ¹³C NMR (CD₃)₂SO) d 13.8 (CH₃),

19.2 (CH₂), 29.9 (CH₂), 49.7 (CH₂), 115.6 (ArCH),

115.7 (ArCH), 122.6 (ArCH), 128.0 (ArC), 129.9

(ArCH), 130.0 (ArC), 157.0 (ArC), 161.6 (ArC); MS

(EI, 70 eV) m/z 321 (M⁺); HRMS calcd for C₁₆H₁₉

79.26; H, 7.54; N, 6.16. Found: C, 79.31; H, 7.54; N,

6.27.

N-[2-(3-Methyl)butane]-4-methoxyaniline (8). To a THF

solution at 0 ^ꢀC containing p-anisidine (1 equiv), 3-

methyl-2-butanone (1.0 equiv), and acetic acid (1.0

equiv) was added solid NaHB(OAc)₃. The mixture was

allowed to reach rt and stirred for 15 h. The mixture

was re-cooled to 0 ^ꢀC; careful workup (H₂O, EtOAc,

NaHCO₃, brine, MgSO₄) and concentration in vacuo

aorded crude amine. Kugelrohr distillation provided

pure product as light yellow oil (71%): bp 100 ^ꢀC

.

NSO₄, 321.1034, found 321.1035. Anal. (C₁₆H₁₉N 0.3

H₂O): C, 58.81; H, 6.05; N, 4.29. Found: C, 58.60; H,

6.03; N, 4.13.

1

(0.3 mm); H NMR (CDCl₃) d 0.93 (d, 3H, J=6.5 Hz),

1.00 (d, 3H, J=6.5 Hz), 1.10 (d, 3H, J=6.0 Hz), 1.86

(m, 1H, J=6.5 Hz), 3.23 (br s, 1H, ArNH), 3.28 (dq,

1H, J=7.0, 6.0 Hz), 3.77 (s, 3H), 6.58 (d, 2H, J=

7.0 Hz), 6.80 (d, 211, J=7.0 Hz); ¹³C NMR (CDCl₃) d

16.5 (CH₃), 17.3 (CH₃), 19.3 (CH₃), 32.1 (CH), 54.5

(CH), 55.8 (CH₃), 114.6 (ArCH), 114.9 (ArCH), 142.1

(ArC), 151.7 (ArC); MS (EI, 70 eV) m/z 193 (M⁺); anal.

(C₁₂H₁₉NO): C, 74.57; H, 9.91; N, 7.25. Found: C,

74.46; H, 9.76; N, 7.32.

N-(4-Hydroxyphenyl)-N-(benzyl)-4-hydroxybenzene sulf-

onamide (5c). Deprotected according to BBr₃procedure

to aord title compound after puri®cation by radial

1

chromatography (10%): H NMR (CDCl₃) d 4.65 (s,

2H), 6.66 (d, 2H, J=8.5 Hz), 6.80 (d, 2H, J=8.5 Hz),

6.96 (d, 2H, 8.8), 7.21 (br s, 5H), 7.59 (d, 2H, J=

8.9 Hz); MS (EI, 70 eV) m/z 355 (M⁺); HRMS calcd for

C₁₉H₁₇NSO₄, 355.0878, found 355.0881.

General procedure for phase transfer N-alkylation of

carboxamides.^9,10Alkylhalide (3 equiv) in benzene was

added dropwise to a stirring mixture of carboxamide

(1 equiv), ®nely powdered potassium hydroxide (2

equiv), and n-tetrabutylammonium bromide (0.05 equiv)

in benzene (0.2 M) at rt. Reaction mixture was heated to

80 ^ꢀC and maintained at this temperature until dis-

appearance of starting material was observed by TLC

(1±3 h). The reaction mixture was then cooled to rt and

the salts removed by ®ltration. Product isolation (H₂O,

brine), followed by ¯ash chromatography (10±25%

EtOAc/hexane) aorded product, usually as an oil.

N-(4-Methoxyphenyl)-4-methoxybenzamide (6).³⁵A CH₂

Cl₂solution of p-anisidine (1.3 equiv) was added drop-

wise to a stirring CH₂Cl₂solution (2±5 mL) of pyridine

(1.1 equiv) and p-anisoyl chloride (1.0 equiv) at 0 ^ꢀC.

Upon complete addition of amine (30 mm) mixture was

allowed to reach rt, then re-cooled and vacuum ®lter to

aord the crude carboxamide. Recrystallization from

EtOH/benzene aorded the pure carboxamide as o-

white crystals (72%): mp 199±200.5 ^ꢀC, mp³²202±

203 ^ꢀC.

N-(1-Phenethyl)-4-methoxyaniline (7).⁷To a 0 ^ꢀC EtOH

solution containing p-anisidine (1.0 equiv) and catalytic

.

p-toluene sulfonic acid H₂O (0.05 equiv) was added

General amidation procedures for anilines 7±8 and CF₃-

containing anilines 14a±d. A 0.18 M benzene or toluene

solution containing 1 equiv of aniline (14a, 14b, 7±8) in

addition to 3 equiv of dry powdered K₂CO₃and 1 equiv

of pyridine was treated with 1.5 equiv of the corre-

sponding acid chloride and heated between 80 and

110 ^ꢀC. The reaction was monitored by TLC and the

reaction cooled to rt upon completion. The crude mix-

ture was ®ltered, concentrated in vacuo and taken up in

Et₂O. Product isolation (H₂O, brine) and chromato-

graphic puri®cation aorded the protected carboxamide

(15a±e and 9f and 9g).

freshly distilled benzaldehyde dropwise. The mixture

was allowed to reach rt and complete imine formation

occurred within 0.5 h. The crude mixture was con-

centrated in vacuo, and product isolation followed

(EtOAc, brine) to aord a dark brown powder. ¹H

NMR con®rmed imine which was directly used in the

next step without further puri®cation. CH₃addition:

Crude imine was dissolved in toluene and cooled to

78 ^ꢀC. To this was added MeLi (1.0 M toluene solu-

tion, 3 equiv) dropwise over 15 mm. After 60 min at

78 ^ꢀC, the crude mixture was warmed to rt and stirred

for an additional 2 h. The reaction was then cooled in

an ice-bath and quenched with cold MeOH followed by

addition of satd NH₄Cl until neutral pH. Concentration

in vacuo followed by remaining workup (EtOAc, H₂O,

brine) aorded amine as a red oil. Kugelrohr distillation

provided pure product, which crystallized on standing

Benzamides 15f and l5g were prepared from anilines 14c

and 14d and the corresponding acid chloride (1.5 equiv)

using l,2-dichlorobenzene as solvent, 3 equiv of dry

powdered K₂CO₃and heated to 120±150 ^ꢀC for 16 h or

until disappearance of starting aniline. Reaction mix-

ture was cooled to rt and eluted over silica with hexane

to remove 1,2-dichlorobenzene. Gradient chromato-

graphy with 10, 20, and 30% EtOAc/hexane aorded

the pure carboxamides 15f and 15g upon concentration.

1

as a red±orange powder (75%): H NMR (CDCl₃) d

1.50 (d, 3H, J=7.0 Hz), 3.69 (s, 3H), 4.41 (q, 1H,

J=7.0 Hz), 6.47 (AA⁰BB⁰, 2H, J=9.0, 3.0 Hz), 6.69

(AA⁰BB⁰, 2H, J=9.0, 3.5 Hz), 7.29 (m, 5H); ¹³C NMR

(CDCl₃) d 25.4 (CH₃), 54.5 (CH), 55.9 (CH₃), 114.7

(ArCH), 114.9 (ArCH), 126.1 (ArCH), 127.0 (ArCH),

128.8 (ArCH), 141.7 (ArC), 145.7 (ArC), 152.1 (ArC);

MS (El, 70 eV) m/z 227 (M⁺); anal. (C₁₅H₁₇NO): C,

N-(4-Methoxyphenyl)-N-(ethyl)-4-methoxybenzamide (9a).

Yellow oil (72%): ¹H NMR (CDCl₃) d 1.12 (t, 3H,

J=7.2 Hz, CH₃), 3.62 (s, 3H, OCH₃), 3.65 (s, 3H,

OCH₃), 3.84 (q, 2H, J=7.0 Hz, CH₂), 6.57 (d, 2H,

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1309

J=8.9 Hz, ArH ortho OCH₃, meta RNCO), 6.68 (d, 2H,

J=8.9 Hz, ArH ortho OCH₃, meta CONR), 6.87 (d, 2H,

J=8.9 Hz, ArH ortho RNCO), 7.20 (d, 2H, J=8.7 Hz,

ArH ortho CONR); ¹³C NMR (CDCl₃) d 12.7 (CH₃),

45.5 (CH₂), 55.0 (OCH₃), 55.2 (OCH₃), 112.8 (ArCH),

114.2 (ArCH), 128.4 (ArC), 128.9 (ArCH), 130.7

(ArCH), 136.3 (ArC), 157.8 (ArC), 160.2 (ArC), 169.6

(CO); MS (EI, 70 eV) m/z 285 (M⁺); HRMS calcd for

C₁₇H₁₉NO₃, 285.1365, found 285.1364.

(ArC), 158.3 (ArC), 160.2 (ArC), 170.6 (CO); MS (EI,

70 eV) m/z 361 (M⁺).

N-(4-Methoxyphenyl)-N-(1-methyl-2-methyl-1-propyl)-4-

methoxybenzamide (9g). Prepared according to general

procedure above using aniline 8 and p-anisoyl chloride.

Puri®cation by ¯ash chromatography (25% EtOAc/

1

hexane) aorded product as light yellow oil (90%): H

NMR (CDCl₃) d 0.93 (d, 6H, J=6.7 Hz), 1.90 (sept,

1H, J=7.0 Hz), 3.69 (s, 3H), 3.71 (s, 3H), 3.73 (d,

2H, J=7.0 Hz), 6.62 (d, 2H, J=8.5 Hz), 6.71 (d, 2H,

J=9.0 Hz), 6.93 (d, 2H, J=8.6 Hz), 7.20 (d, 2H,

J=8.9 Hz); ¹³C NMR (CDCl₃) d 20.3 (CH₃), 27.0 (CH),

55.2 (CH₃), 55.4 (CH₃), 57.2 (CH₂), 113.0 (ArCH),

114.3 (ArCH), 128.7 (ArCH), 129.0 (ArC), 130.7

(ArCH), 137.1 (ArC), 157.8 (ArC), 160.3 (ArC), 170.3

(CO); MS (EI, 70 eV) m/z 313 (M⁺).

N-(4-Methoxyphenyl)-N-(2-propyl)-4-methoxybenzamide

(9b). Amber oil (43%): ¹H NMR (CDCl₃) d 1.16 (d, 6H,

J=6.5 Hz), 3.72 (s, 3H), 3.75 (s, 3H), 5.09 (br s, 1H),

6.63 (d, 2H, J=8.5 Hz), 6.74 (d, 2H, J=9.0 Hz), 6.93 (d,

2H, J=8.5 Hz), 7.21 (d, 2H, J=8.5 Hz); ¹³C NMR

(CDCl₃) d 21.2 (CH₃), 55.3 (CH₃), 55.5 (CH₃), 113.0

(ArCH), 113.9 (ArCH), 129.1 (ArC), 130.4 (ArCH),

131.8 (ArCH), 132.5 (ArC), 158.6 (ArC), 160.1 (ArC),

170.5 (CO); MS (EI, 70 eV) m/z 299 (M⁺); HRMS

calcd for C₁₈H₂₁NO₃299.1521, found 299.1521.

N-(4-Hydroxyphenyl)-N-(ethyl)-4-hydroxybenzamide (10a).

Deprotected according to BBr₃procedure to aord o-

white powder, recrystallized from MeOH (70%): mp

208±209 ^ꢀC; H NMR ((CD₃)₂SO) d 1.03 (t, 3H, J=

1

N-(4-Methoxyphenyl)-N-(butyl)-4-methoxybenzamide (9c).

Yellow oil (81%): ¹H NMR (CDCl₃) d 0.84 (t, 3H,

J=7.3 Hz, CH₃), 1.29 (sext, 2H, J=7.0 Hz, CH₃CH₂),

1.53 (quint, 2H, J=7.8 Hz, CH₂CH₂CH₂), 3.62 (s, 3H,

OCH₃ortho RNCO), 3.66 (s, 3H, OCH₃ortho CONR),

3.78 (t, 2H, J=7.7 Hz, CH₂CH₂N), 6.58 (d, 2H, J=

8.8 Hz, ArH ortho OCH₃, meta RNCO), 6.68 (d, 2H,

J=8.9 Hz, ArH ortho OCH₃, and meta CONR), 6.88 (d,

2H, J=8.9 Hz, ArH ortho RNCO), 7.19 (d, 2H, J=

8.9 Hz, ArH ortho CONR); ¹³C NMR (CDCl₃) d 13.7

(CH₃), 20.0 (CH₂), 29.5 (CH₂), 50.3 (CH₂), 54.9

(OCH₃), 55.1 (OCH₃), 112.7 (ArCH), 114.1 (ArCH),

128.4 (ArC), 128.6 (ArCH), 130.5 (ArCH), 136.5 (ArC),

157.6 (ArC), 160.0 (ArC), 169.6 (ArC); MS (EI, 70 eV)

m/z 313 (M⁺); HRMS calcd for C₁₉H₂₃NO₃, 313.7794,

found 313.7879.

6.9 Hz, CH₃), 3.72 (q, 2H, J=7.1 Hz, CH₂), 6.52 (d, 2H,

J=8.6 Hz, ArH meta RNCO), 6.62 (d, 2H, J=8.5 Hz,

ArH meta CONR), 6.86 (d, 2H, J=8.6 Hz, ortho

RNCO), 7.06 (d, 2H, J=8.5 Hz, ortho CONR), 9.48 (s,

1H, OH), 9.71 (s, 1H, OH); ¹³C NMR ((CD₃)₂SO) d

12.6 (CH₃), 39.1 (CH₂), 114.3 (ArCH), 115.7 (ArCH),

127.1 (ArC), 129.1 (ArC), 130.5 (ArCH), 134.7 (ArC),

155.7 (ArC), 158.3 (ArC), 168.9 (ArC); MS (EI, 70 eV)

m/z 257 (M⁺); HRMS calcd for C₁₅H₁₅NO₃, 257.1052,

found 257.1051.

N-(4-Hydroxyphenyl)-N-(2-propyl)-4-hydroxybenzamide

(l0b). Deprotected according to BBr₃procedure to

aord powder, recrystallized from MeOH (77%): mp

210±212 ^ꢀC; ¹H NMR (MeOD) d 1.15 (d, 6H, J=

6.8 Hz), 4.94 (br s, 1H), 6.56 (br s, 2H), 6.67 (d, 2H,

J=8.4 Hz), 6.88; MS (EI, 70 eV) 271 (M⁺); anal.

N-(4-Methoxyphenyl)-N-(benzyl)-4-methoxybenzamide

(9e). White ¯akes (82%): mp 104±106 ^ꢀC; ¹H NMR

(CDCl₃) d 3.72 (s, 3H, OCH₃para RNCO), 3.74 (s, 3H,

OCH₃para CONR), 5.09 (s, 2H, CH₂Ph), 6.68 (d, 2H,

J=9.2 Hz, ArH ortho OCH₃, and meta RNCO), 7.31

(m, 9H, Ph and ArH); ¹³C NMR (CDCl₃) d 54.3

(PhCH₂), 55.3 (OCH₃), 55.5 (OCH₃), 113.1 (ArCH),

114.3 (ArCH), 127.4 (ArCH), 128.6 (ArCH), 128.3

(ArC), 128.7 (ArCH), 128.9 (ArCH), 131.1 (ArCH),

137.0 (ArC), 138.0 (ArC), 158.0 (ArC), 160.6 (ArC),

170.2 (ArC); MS (EI, 70 eV) m/z 347 (M⁺); HRMS

calcd for C₂₂H₂₁NO₃, 347.1521, found 347.1522.

.

(C₁₆H₁₇NO₃0.1 H₂O): C, 70.36; H, 6.36; N, 5.13.

Found: C, 70.35; H, 6.37; N, 4.90.

N-(4-Hydroxyphenyl)-N-(butyl)-4-hydroxybenzamide (10c).

Deprotected according to BBr₃procedure and puri®ed

by ¯ash chromatography (25% EtOAc/hexane) to

aord o-white crystals (95%): mp 174±175 ^ꢀC; ¹H NMR

((CD₃)₂SO) d 0.84 (s, 3H), 1.25 (sext, 2H, J=7.6 Hz),

1.43 (quint, 2H, J=7.6 Hz), 3.69 (t, 2H, J=7.5 Hz), 6.51

(d, 2H, J=8.6 Hz), 6.61 (d, 2H, J=8.7 Hz), 6.85 (d,

2H, J=8.7 Hz), 7.03 (d, 2H, J=8.5 Hz); ¹³C NMR

((CD₃)₂SO) d 14.1 (CH₃), 19.9 (CH₂), 40.2 (CH₂), 114.5

(ArCH), 115.9 (ArCH), 127.4 (ArC), 129.2 (ArCH),

130.6 (ArCH), 135.1 (ArC), 155.8 (ArC), 158.4 (ArC);

MS (EI, 70 eV) m/z 285 (M⁺); HRMS (EI) calcd for

C,₇H₁₉NO₃, 285.1365, found 285.1368; anal. (C₁₇H₁₉

N-(4-Methoxyphenyl)-N-(1-phenethyl)-4-methoxybenz-

amide (9f). Prepared according to general procedure

above using aniline 7 and p-anisoyl chloride. Puri®ca-

tion by ¯ash chromatography (2% (CH₃)₂CO/CH₂Cl₂)

1

.

aorded product as clear oil (70%): H NMR (CDCl₃)

NO₃0.5 H₂O): C, 69.37; H, 6.85; N, 4.76. Found: C,

69.30; H, 6.65; N, 4.57.

d 1.48 (d, 3H, J=6.8 Hz), 3.67 (s, 3H), 3.69 (s, 3H), 6.38

(br q, 1H, J=6.8 Hz), 6.56 (br d overlapping br s, 4H),

6.62 (d, 2H, J=8.8 Hz), 7.23 (d, 2H, J=8.5 Hz), 7.29

(m, 5H); ¹³C NMR (CDCl₃) d 16.9 (CH₃), 53.3 (CH),

55.2 (CH₃), 55.3 (CH₃), 112.9 (ArCH), 113.6 (ArCH),

127.5 (ArCH), 128.2 (ArCH), 128.3 (ArCH), 129.2

(ArC), 130.6 (ArCH), 131.5 (ArCH), 132.7 (ArC), 141.7

N-(4-Hydroxyphenyl)-N-(2-methyl-1-propyl)-4-hydroxy-

benzamide (10d). Deprotected according to BBr₃

procedure and puri®ed by ¯ash chromatography (25%

EtOAc/hexane) to aord white foam (quant): mp 166 ^ꢀC

1

dec.; H NMR ((CD₃)₂SO) d 0.93 (d, 6H, J=6.5 Hz),

1310

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1.82 (m, 1H, J=6.5 Hz), 3.66 (d, 2H, J=7.5 Hz), 6.59

(d, 2H, J=8.5 Hz), 6.68 (d, 2H, J=9 Hz), 6.94 (d, 2H,

J=8.5 Hz), 7.10 (d, 2H, J=8.5 Hz); MS (EI, 70 eV) m/z

285 (M ); anal. (C₁₇H₁₉NO₃l.0 H₂O): C, 67.31; H,

6.98; N, 4.62. Found: C, 67.45; H, 6.74; N, 4.41.

128.5 (ArH), 129.8 (ArH), 135.3 (ArC), 136.4 (ArC),

156.3 (ArC), 157.6 (ArC), 201.0 (CS); MS (EI, 70eV) m/z

273 (M⁺); HRMS (EI) calcd for C₁₅H₁₅NSO₂, 273.0824,

found 273.0824; anal. (C₁₅H₅NSO₂0.3 H₂O): C, 64.63;

H, 5.64; N, 5.02. Found: C, 64.51; H, 5.93; N, 4.64.

+

.

N-(4-Hydroxyphenyl)-N-(benzyl)-4-hydroxybenzamide

(l0e). Deprotected according to EtSH procedure and

puri®ed by ¯ash chromatography (25% EtOAc/hexane),

which upon solvent concentration and recrystallization

from CHCl₃aorded title compound as o-white ¯akes

(60%): mp 103±105 ^ꢀC; ¹H NMR ((CD₃)₂SO) d 4.95

(s, 2H, CH₂), 6.53 (d, 2H, J=6.3 Hz, ArH meta

RNCO), 6.56 (d, 211, J=6.2 Hz, ArH ortho RNCO),

6.77 (d, 2H, J=8.7 Hz, ArH meta CONR), 7.13 (d, 2H,

J=8.7 Hz, AsH ortho CONR), 7.25 (m, 5H, Ph), 9.67

(broad s, 2H, OH); ¹³C NMR ((CD₃)₂SO) d 53.1 (CH₂),

114.2 (ArCH), 115.5 (ArCH), 122.3 (ArC), 126.6 (ArC),

126.9 (ArCH), 127.9 (ArCH), 128.3 (ArCH), 128.7

(ArCH), 130.6 (ArCH), 134.9 (ArC), 137.9 (ArC), 155.5

(ArC), 158.5 (ArC), 169.4 (ArC); MS (EI, 70 eV) m/z

319 (M⁺); HRMS (EI) calcd for C₂₀H₁₇N0₃, 319.1208,

N-(4-Hydroxyphenyl)-N-(i-propyl)-4-hydroxythiobenz-

amide (11b). Deprotected according to BBr₃procedure

and puri®ed by ¯ash chromatography (25% EtOAc/

hexane) to aord yellow foam (98%): ¹H NMR (MeOD,

i-propyl rotamers 1:4:1 ratio) d 1.15 (d, 1H, J=6.5 Hz),

1.20 (d, 4H, J=6.5 Hz), 1.27 (d, 1H, J=6.5 Hz), 6.00

(sept, 1H, J=6.5 Hz), 6.46 (d, 2H, J=8.5 Hz), 6.61 (d,

2H, J=9 Hz), 6.81 (d, 2H, J=8.5 Hz), 6.96 (d, 2H,

J=8.5 Hz) additional minor ArCH resonances not lis-

ted; ¹³C NMR (MeOD) d 20.6 (CH₃), 21.9 (CH₃minor),

54.8 (CH), 114.8 (ArCH), 115.9 (ArCH), 130.1 (ArCH),

131.7 (ArCH), 133.4 (ArC), 137.8 (ArC), 157.9 (ArC),

158.0 (ArC), 204.4 (CS); MS (EI, 70 eV) m/z 287

(M⁺); HRMS, C₁₆H₁₇NSO₂, 287.0980; found, 287.0971.

N-(4-Hydroxyphenyl)-N-(n-butyl)-4-hydroxythiobenzamide

(11c). Deprotected according to BBr₃procedure and

puri®ed by radial chromatography (5% MeOH/

.

found 319.1206; anal. (C₂₀H₁₇NO₃2.5 H₂O): C, 65.92;

H, 6.09; N, 3.84. Found: C, 65.58; H, 5.81; N, 3.53.

1

CH₂Cl₂) to aord yellow oil (10%): H NMR ((CD₃)₂

N-4-Hydroxyphenyl-N-(1-phenethyl)-4-hydroxybenzamide

(10f). Deprotected according to BBr₃procedure recrys-

tallized from MeOH/CH₂Cl₂to aord white powder

(95%): mp 204±206 ^ꢀC; ¹H NMR (MeOD) d 1.50 (d, 3H,

J=6.8 Hz), 4.95 (s, under MeOH), 6.24 (br s, 2H), 6.49

(br s, 2H), 6.54 (br s, 2H), 6.54 (d, 2H, J=8 Hz), 7.10 (d,

2H, J=8 Hz), 7.28 (m, 5H); ¹³C NMR (MeOD) d 17.4

(CH₃), 49.0 (CH), 115.3 (ArCH), 115.9 (ArCH), 124.9

(ArCH), 128.6 (ArCH), 128.9 (ArC), 129.1 (ArCH),

129.3 (ArCH), 131.4 (ArCH), 132.4 (ArC), 132.7 (ArCH),

142.5 (ArC), 157.9 (ArC), 159.8 (CO); MS (EI, 70 eV)

m/z 333 (M⁺); HRMS (EI) calcd for C₂₁H₁₉NO₃,

333.1365, found 333.1360; anal. (C₂₁H₁₉NO₃): C, 75.66;

H, 5.74; N, 4.20. Found: C, 75.51; H, 5.80; N, 4.21.

SO) d 0.84 (s, 3H, J=7.5 Hz, CH₃), 1.27 (sext, 2H,

J=6.8 Hz, CH₃CH₂), 1.61 (quint, 2H, J=8.0 Hz, CH₃

CH₂CH₂), 4.30 (t, 2H, J=7.7 Hz, CH₂NCS), 6.43 (d,

2H, J=8.5 Hz, ArH meta RNCS), 6.58 (d, 2H, J=

8.2 Hz, ArH meta CSNR), 6.87 (d, 2H, J=8.2 Hz, ArH

ortho RNCS), 6.97 (d, 2H, J=8.5 Hz, ArH ortho

CSNR); ¹³C NMR ((CD₃)₂SO) d 14.2 (CH₃), 19.7

(CH₂), 27.9 (CH₂), 56.8 (CH₂), 114.1 (ArCH), 115.9

(ArCH), 128.5 (ArCH), 129.9 (ArCH), 135.2 (ArC),

136.8 (ArC), 156.2 (ArC), 157.7 (ArC), 201.0 (CS);

MS (EI, 70 eV), m/z 301 (M⁺); HRMS calcd for

C₁₇H₁₉NO₂S, 301.1136; found 301.1133.

N-(4-Hydroxyphenyl)-N-(i-butyl)-4-hydroxythiobenzamide

(11d). Deprotected according to BBr₃procedure and

puri®ed by ¯ash chromatography (35% EtOAc/hexane)

General thionation procedure¹²

1

to aord yellow foam (86%): H NMR (CDCl₃) d 0.98

The carboxamide (1.01 mmol) and Lawesson's reagent

(0.51 mmol) in 3 mL of HMPA were heated to 80±

100 ^ꢀC until the carboxamide had been consumed. After

disappearance of carboxamide, the reaction mixture was

allowed to cool to rt and then poured onto 5 mL of

water. Product was extracted repeatedly with Et₂O,

dried over Na₂SO₄, rotary evaporated in vacuo and

puri®ed via ¯ash chromatography. The protected

thioamides were then directly used in the deprotection

step with minimal puri®cation.

(d, 6H, J=6.8 Hz), 2.16 (m, 1H, J=6.8 Hz), 4.34 (d,

2H, J=7.6 Hz), 6.41 (d, 2H, J=8.8 Hz), 6.60 (d, 2H,

J=8.8 Hz), 6.79 (d, 2H, J=8.8 Hz), 6.96 (d, 2H,

J=8.8 Hz); ¹³C NMR (CDCl₃) d 20.3, 26.9, 27.2, 50.8,

63.9, 114.5, 116.1, 127.9, 129.5, 136.4, 137.7, 155.4,

156.5, 203.0; MS (EI, 70 eV) m/z 301 (M⁺); HRMS

calcd for C₁₇H₁₉NO₂S, 301.1136; found, 301.1144.

N-(4-Hydroxyphenyl)-N-(benzyl)-4-hydroxythiobenzamide

(11e). Deprotected according to EtSH procedure and

puri®ed by ¯ash chromatography (35% EtOAc/hexane)

to aord yellow foam (16%): ¹H NMR ((CDCl₃)) d 5.76

(s, 2H, CH₂), 6.59 (d, 2H, J=8.4 Hz, ArH meta RNCS),

6.61 (d, 211, J=8.3 Hz, ArH meta CSNR), 6.77 (d, 2H,

J=8.8 Hz, ArH ortho RNCS), 7.27 (m, 5H, Ph), 7.38 (d,

2H, J=6.4 Hz, ArH ortho CSNR); MS (EI, 70 eV) m/z

335 (M⁺); HRMS calcd for C₂₀H₁₇NO₂S, 335.0980;

found, 335.0984.

N-(4-Hydroxyphenyl)-N-(ethyl)-4-hydroxythiobenzamide

(11a). Deprotected according to BBr₃procedure and

puri®ed by ¯ash chromatography (25% EtOAc/hexane)

to aord yellow foam (63%): ¹H NMR ((CD₃)₂SO) d 1.16

(t, 3H, J=6.8 Hz, CH₃), 4.34 (q, 2H, J=6.8 Hz, CH₂),

6.44 (d, 2h, J=8.4 Hz, ArH meta RNCS), 6.59 (d, 2H,

J=8.9 Hz, ArH meta CSNR), 6.87 (d, 2H, J=8.7 Hz,

ArH ortho RNCS), 6.99 (d, 2H, J=8.6 Hz, ArH ortho

CSNR); ¹³C NMR ((CD₃)₂SO) d 11.1 (CH₃), 52.1 (CH₂),

114.1 (ArH), 115.6 (ArH), 115.7 (ArH), 122.6 (ArH),

N-(4-hydroxyphenyl)-N-(1-phenethyl)-4-hydroxythiobenz-

amide (11f). Deprotected according to BBr₃procedure

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1311

and recrystallized from CHCl₃to aord light yellow

reduced pressure to aord product as light yellow oil

powder (quant): mp 174±175 ^ꢀC; H NMR (CDCl₃) d

(92%): bp 66 ^ꢀC (0.1 mm); H NMR (CDCl₃) d 2.06 (s,

1

1.55 (d, 3H, J=7.2 Hz); 5.89 (br s, 1H); 6.27 (br s, 1H)

6.44 (d, 2H, J=8.8 Hz), 6.5 (br s, 1H), 6.71 (br s, 1H),

6.96 (d, 2H, J=8.8 Hz), 7.32 (m, 5H), 7.52 (q, 1H,

J=6.8 Hz); MS (EI, 70 eV) m/z 349 (M⁺); anal.

3H), 3.81 (s, 3H), 6.79, 6.92 (AA⁰BB⁰, 4H, J=8.8,

4.0 Hz); ¹³C NMR d 14.6 (CH₃), 55.6 (CH₃), 114.5

(ArCH), 120.5 (q, CF₃, J=273 Hz), 121.1 (ArCH),

121.5 (ArCH), 140.5 (ArC), 157.1 (q, CCF₃, J=33 Hz),

157.6 (ArC); MS (EI, 70 eV) m/z 217 (M⁺); anal.

(C₁₀H₁₀NF₃O): C, 55.30; H, 4.64; N, 6.45. Found: C,

55.59; H, 4.99; N, 6.64.

.

(C₂₁H₁₉NO₂S 0.4 H₂O): C, 70.72; H, 5.60; N, 3.93.

Found: C, 70.88; H, 5.42; N, 3.98.

N-(4-Hydroxyphenyl)-N-[2-(3-methyl)-butyl]-4-hydroxy-

thiobenzamide (11g). Deprotected according to BBr₃

procedure and puri®ed by ¯ash chromatography (25%

EtOAc/hexane) to aord foam (37%, two steps from

N-(1,1,1-Tri¯uoro-2-propyl)-4-methoxyaniline (14a).⁶To

a Et₂O solution of LiAlH₄(0.5 equiv) at 0 ^ꢀC was added

a Et₂O solution of propylidene 13a dropwise over

30 min. Upon complete addition the mixture was

re¯uxed for 2 h. The mixture was then cooled to 0 ^ꢀC

and quenched using a 1:1:3 work up (H₂O:3M

NaOH:H₂O). The resulting mixture was decanted into a

separatory funnel, the aqueous layer separated and

ether layer washed with brine, dried over MgSO₄and

concentrated. Puri®cation by Kugelrohr distillation

provided pure amine as light red oil (64%): bp 80 ^ꢀC

1

carboxamide): H NMR (CDCl₃) d (major methyl rota-

mer) 0.95 (d, 3H, J=6.6 Hz), 1.14 (d, 3H, J=6.9 Hz),

1.21 (d, 3H, J=6.3 Hz), 1.82 (br m, 1H), 5.82 (quin, 1H,

J=7.4 Hz), 6.30 (d, 2H, J=8.5 Hz), 6.60 (br m, 4H),

6.83 (d, 2H, J=8.5 Hz); ¹³C NMR (CDCl₃) d 17.1

(CH₃), 19.8 (CH₃), 20.9 (CH), 32.5 (CH), 114.7 (ArCH),

115.6 (ArCH), 128.8 (ArCH), 129.5 (ArCH), 133.2

(ArC), 137.2 (ArC), 155.2 (ArC), 155.3 (ArC), 204.4

(CS); MS (EI, 70 eV) m/z 315 (M⁺); anal.

1

(0.2 mm); H NMR (CDCl₃) d 1.34 (d, 3H, J=7.0 Hz),

(C₁₈H₂₁NO₂S 0.8 H₂O): C, 65.54; H, 6.91; N, 4.35.

3.32 (br s, 1H), 3.72 (s, 3H), 3.87 (m, 1H), 6.61 (AA⁰BB⁰,

2H, J=8.8, 4.0 Hz), 6.77 (AA⁰BB⁰, 2H, J=8.8, 3.5 Hz);

¹³C NMR (CDCl₃) d 15.2 (CH₃), 52.7 (q, CHCF₃,

J=30 Hz), 55.8 (CH₃), 115.1 (ArCH), 115.4 (ArCH),

126.5 (q, CF₃, J=280 Hz), 140.2 (ArC), 153.2 (ArC);

MS (EI, 70 eV) m/z 219 (M⁺).

.

Found: C, 65.15; H, 6.67; N, 3.91.

Triphenylphosphine-4-methoxyphenylimine (12a).³⁶p-

Anisidine was diazotized in 50% H₂SO₄and then added

to a NaN₃±sodium acetate buered solution.¹⁵The

azide was extracted with diethyl ether and the phenolic

side-product removed by Na₂CO₃wash. Concentration

in vacuo aorded the crude azide which was directly

used in the next step. Ylide formation: to an etheral

solution of azide was added an equimolar solution of

PPh₃at rt. After the solution was heated under re¯ux

for 2 h and N₂evolution ceased, the solvent was con-

centrated and resulting Ph₃PO oxide removed. The

crude imine was passed over SiO₂plug (ether) to remove

additional oxide. Final concentration and tituration

with hexanes aorded pure imine as orange crystals

(53% from p-anisidine): mp 116±117.5 ^ꢀC; mp³³

119±120 ^ꢀC.

N-(1,1,1-Tri¯uoro-2-phenethyl)-4-methoxyaniline (14b).

Prepared according to procedures outlined above from

ylide 12a and tri¯uoroacetophenone. The imine was

isolated then used directly in reduction step to aord the

title compound as a light yellow oil upon distillation

(69%): bp 110 ^ꢀC (0.1 mm); ¹H NMR (CDCl₃) d 3.72 (s,

3H), 4.10 (br s, 1H), 4.81 (q, 1H, J=7.0 Hz), 6.61

(AA⁰BB⁰, 2H, J=8.8, 4.0 Hz), 6.75 (AA⁰BB⁰, 2H,

J=8.8, 3.5 Hz), 7.40 (m, 5H); ¹³C NMR (CDCl₃) d 55.8

(CH₃), 62.0 (CHCF₃, q, J=30 Hz), 115.0 (ArCH), 115.9

(ArCH), 125.5 (q, CF₃, J=280 Hz), 128.2 (ArCH),

129.1 (ArCH), 129.3 (ArCH), 134.5 (ArC), 139.7 (ArC),

153.5 (ArC); MS (EI, 70 eV) m/z 281 (M⁺); anal.

(C₁₅H₁₄NF₃O): C, 64.05; H, 5.02; N, 4.98. Found: C,

63.90; H, 4.84; N, 5.05.

Triphenylphosphine-phenylimine (12b).^14,37A cold aqu-

eous solution of NaNO₂(1.2 equiv) was added dropwise

to a solution of aniline in 10% HCl at 0±5 ^ꢀC with vig-

orous stirring. The mixture was kept below 5 ^ꢀC for

30 min, and then a solution of NaN₃in water was added

dropwise while the temperature was kept below 5 ^ꢀC.

After 1 h the reaction was warmed to rt and extracted

with diethyl ether. The extracts were dried over Na₂SO₄

and concentrated to aord the crude azide¹⁴as an oil,

which was directly used in the next step without addi-

tional puri®cation. Ylide formation: same as described

for 12a to aord light yellow crystals upon solvent con-

centration (64% from aniline): mp 128±130 ^ꢀC (mp³⁴

131±132 ^ꢀC).

N-(1,1,1-Tri¯uoro-2-phenethyl)-aniline (14c). Prepared

according to procedures outlined above using ylide 12b

and tri¯uoroacetophenone. The imine was isolated then

used directly in reduction to aord the title compound

as a clear oil upon distillation (59%): bp 100 ^ꢀC

1

(0.5 mm); H NMR (CDCl₃) d 4.36 (d, 1H, J=6.5 Hz,

NH), 4.96 (quint, 1H, J=7.0 Hz), 6.68 (d, 2H, J=

8.0 Hz), 6.82 (t, 1H, J=7.0 Hz), 7.20 (d, 2H, J=7.0 Hz),

7.42 (m, 3H), 7.50 (d, 2H, J=6.5 Hz), ¹³C NMR

(CDCl₃) d 60.7 (q, CHCF₃, J=29 Hz), 114.1 (ArCH),

119.5 (ArCH), 122.5 (q, CF₃, J=278 Hz), 128.1

(ArCH), 129.2 (ArCH), 129.4 (ArCH), 129.6 (ArCH),

134.3 (ArC), 145.8 (ArC); MS (EI, 70 eV) m/z 251

(M⁺); anal. (C₁₄H₁₂F₃N): C, 66.93; H, 4.81; N, 5.57.

Found: C, 66.79; H, 4.80; N, 5.54.

N-(1,1,1-Tri¯uoro-2-propylidene)-4-methoxyaniline (13a).⁶

Ylide 12a and tri¯uoroacetone were heated to re¯ux in

C₆H₆for 12 h. The reaction was cooled to rt and con-

centrated in vacuo. The residue was triturated with

Et₂O to remove Ph₃PO and the resulting ®ltrate con-

centrated followed by Kugelrohr distillation under

N-[1,1,1-Tri¯uoro-2-(3-methoxy)-phenethyl]-aniline (14d).

2,2,2-Tri¯uoro-1-(3-methoxyphenyl)-ethanone precursor).¹³

1312

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

Mg turnings (1.25 g, 50mmol), 3-methoxyphenyl bromide

(9.35 g, 50 mmol), and anhydrous THF (50 mL) were

gingerly heated until a vigorous reaction took place.

When all the Mg turnings were dissolved the reaction

mixture was cooled to 0 ^ꢀC. A solution of N-tri-

¯uoroacetylpiperidine³⁵(7.52 g, 45 mmol) in anhydrous

THF (10 mL) was added to the Grignard reagent drop-

wise over 0.5 h with stirring at 0 ^ꢀC. Upon complete

addition, ice-bath was removed and the mixture stirred

for 2 h. The reaction was quenched by the addition of

satd aq NH₄Cl (5 mL), and the precipitates romoved by

®ltration. The ®ltrate was dried over MgSO₄, evapor-

ated in vacuo, and the crude ketone distilled to give

6.6 g (72%) of a colorless liquid: bp 50 ^ꢀC (0.5 mm) (bp⁹

84±85 ^ꢀC (12 mm).

N-(4-Methoxyphenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-ben-

zamide (15c). Prepared according to general procedure

for CF₃-containing anilines and puri®ed by ¯ash chro-

matography (10% EtOAc/hexane) to aord product as

light yellow oil (73%): bp 125 ^ꢀC (0.3 mm); H NMR

1

(CDCl₃) d 3.61 (s, 3H), 6.01 (br s, 1H), 6.52 (2 over-

lapping br s, 3H), 7.12 (m, 4H), 7.26 (m, 6H); ¹³C NMR

(CDCl₃) d 55.4 (CH₃), 58.1 (CHCF₃), 113.6 (ArCH),

125.1 (q, CF₃, J=282 Hz), 127.9 (ArCH), 128.7

(ArCH), 129.2 (ArCH), 129.6 (ArCH), 130.2 (ArCH),

131.4 (ArC), 131.9 (ArC), 132.4 (ArCH), 135.7 (ArC),

159 (ArC), 172.1 (CO); MS (EI, 70 eV) m/z 385 (M⁺);

anal. (C₂₂H₁₈F₃NO₂): C, 68.57; H, 4.71; N, 3.68.

Found: C, 68.22; H, 5.03; N, 3.68.

N-(4-Methoxyphenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-3-

methoxybenzamide (15d). Prepared according to general

procedure for CF₃-containing anilines and puri®ed by

¯ash chromatography (10% EtOAc/hexane) to aord

The above prepared 2,2,2-tri¯uoro-1-(3-methoxyphenyl)-

ethanone was then reacted with ylide 12a as described

previously. Imine isolation and LAH reduction aorded

the title compound as a light green oil upon distillation

(80%): bp 125 ^ꢀC (0.8 mm); ¹H NMR (CDCl₃) d 3.79 (s,

3H), 4.31 (br s, 1H), 4.87 (q, 1H, J=7.2 Hz), 6.63 (d,

2H, J=7.6 Hz), 6.76 (td, 1H, J=7.6, 0.8 Hz), 6.89 (dd,

1H, J=8.5, 2.4 Hz), 6.99 (br s, 1H), 7.03 (d, 1H,

J=7.6 Hz), 7.15 (m, 2H), 7.28 (t, 1H, J=8.0 Hz); ¹³C

1

product as transparent oil (79%): H NMR (CDCl₃) d

3.66 (2-s, 3H, ꢁ1.2/1 ratio benzoyl rotamers), 3.88 (s,

3H), 6.78 (br d, 1H, J=8.5 Hz), 6.8 (br s, 1H), 7.03 (q,

1H, J=6.5 Hz, CHCF₃), 7.25 (m, 5H), 7.65 (dd, 1H,

J=8.5, 2.3 Hz), 7.75 (app dt, 1H, J=7.6, 0.8 Hz); MS

(EI, 70 eV) m/z 415 (M⁺).

NMR (CDCl₃)

d 55.1 (CH₃), 60.3 (q, CHCF₃,

J=30 Hz), 113.8 (ArCH), 114.1 (ArCH), 119.1 (ArCH),

120.1 (ArCH), 124.9 (q, CF₃, J=281 Hz), 128.7

(ArCH), 129.2 (ArCH), 129.8 (ArCH), 135.5 (ArC),

145.4 (ArC), 159.8 (ArC); MS (EI, 70 eV) m/z 281

(M⁺); anal. (C₁₅H₁₄F₃NO): C, 64.05; H, 5.02; N, 4.98.

Found: C, 64.15; H, 4.99; N, 4.84.

N-(4-Methoxyphenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-2-

methoxybenzamide (15e). Prepared according to general

procedure for CF₃-containing anilines and puri®ed by

¯ash chromatography (10% EtOAc/hexane) to aord

product as transparent oil (36%, 84% corrected for

1

recovered aniline); H NMR (CDCl₃) d 3.63 (s, 3H),

3.68 (s, 3H), 6.10 (br s, 1H), 6.43 (br s, 2H), 6.58 (d, 2H,

J=8.5 Hz), 6.72 (app t, 2H, J=8.5 Hz), 7.21 (m, 7H);

MS (EI, 70 eV) m/z 415 (M⁺).

N-(4-Methoxyphenyl)-N-(1,1,1-tri¯uoro-2-propyl)-4-meth-

oxybenzamide (15a).⁶Prepared according to general

procedure for CF₃-containing anilines and puri®ed by

¯ash chromatography (25% EtOAc/hexane) to aord

product as white powder (68%): mp 85±86 ^ꢀC; ¹H NMR

(CDCl₃) d 1.21 (d, 3H, J=7.0 Hz), 3.69 (s, 3H), 3.73 (s,

3H), 5.79 (sept, 1H, J=6.5 Hz), 6.62 (d, 2H, J=8.5 Hz),

6.75 (br s, 2H), 7.12 (br s, 2H), 7.22 (d, 2H, J=8.5 Hz);

¹³C NMR (CDCl₃) d 12.5 (CH₃), 55.2 (CH₃), 55.4

(CH₃), 113.1 (ArCH), 114.1 (ArCH), 125.2 (q, CF₃,

J=282 Hz), 128.0 (ArC), 130.6 (ArCH), 131.7 (ArC),

159.0 (ArC), 160.6 (ArC), 171.6 (CO); MS (EI, 70 eV)

m/z 353 (M⁺).

N-(Phenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-4-methoxy-

benzamide (15f). Prepared according to the general

procedure for CF₃-containing anilines using 1,2-

dichlorobenzene as solvent and puri®ed by ¯ash chro-

matography (10% EtOAc/hexane) to aord product as

1

a transparent oil (54%): H NMR (CDCl₃) d 3.69 (s,

3H), 6.62 (d, 2H, J=8.5 Hz), 7.11 (m, 3H), 7.25 (m,

9H); ¹³C NMR (CDCl₃) d 55.1 (CH₃), 58.5 (CHCF₃),

113.0 (ArCH), 125.2 (q, CF₃, J=282 Hz), 127.3 (ArC),

127.9 (ArCH), 128.6 (ArCH), 129.0 (ArCH), 129.9

(ArCH), 130.7 (ArCH), 130.9 (ArCH), 131.9 (ArC),

N-(4-Methoxyphenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-4-

methoxybenzamide (l5b). Prepared according to general

procedure for CF₃-containing anilines and puri®ed by

¯ash chromatography (25% EtOAc/hexane) to aord

139.5 (ArC), 161.0 (ArC), 171.2 (CO); MS (EI, 70 eV)

+

.

m/z 385 (M ); anal. (C₂₃H₂₀NO₃F₃1.1 CHCl₃): C,

52.95; H, 3.89; N, 2.56. Found: C, 52.84; H, 3.90; N,

2.65.

1

product as transparent oil (77%): H NMR (CDCl₃) d

3.68 (s, 3H), 3.69 (s, 3H), 6.05 (br s, 1H), 6.42 (br s,

1H), 6.61 (d overlapping br s, 3H, J=8.5 Hz) 7.04 (br

q, 1H, J_HF=6.5 Hz), 7.25 (m, 8H); ¹³C NMR (CDCl₃)

d 55.1 (CH₃), 55.2 (CH₃), 58.8 (ArCHCF₃), 113.0

(ArCH), 113.6 (ArCH), 125.1 (q, CF₃, J=281 Hz),

127.5 (ArC), 128.5 (ArCH), 129.0 (ArCH), 130.0

(ArCH), 130.7 (ArCH), 131.9 (ArC), 132.0 (ArCH),

132.9 (ArCH), 158.8 (ArC), 160.5 (ArC), 171.4 (CO);

MS (EI, 70 eV) m/z 415 (M⁺); HRMS calcd for

C₂₃H₂₀F₃NO₃, 415.1395. Found, 415.1387.

N-(Phenyl)-N-[1,1,1-tri¯uoro-2-(3-methoxy)-phenethyl]-

4-methoxybenzamide (15g). Prepared according to gen-

eral procedure for CF₃-containing anilines using 1,2-

dichlorobenzene as solvent and puri®ed by ¯ash chro-

matography (10±25% EtOAc/hexane) to aord product

1

as transparent oil (51%): H NMR (CDCl₃) d 3.68 (s,

3H), 3.69 (s, 3H), 6.61 (d, 2H, J=8.5 Hz), 6.77 (s, 1H),

6.88 (app td, 2H, J=8.5, 3.5 Hz), 7.10 (m, 5H), 7.18

(app t, J=8.0 Hz), 7.26 (d, 2H, J=8.5 Hz); ¹³C NMR

(CDCl₃) d 55.3 (CH₃), 55.4 (CH₃), 58.7 (CHCF₃), 113.1

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1313

(ArCH), 115.0 (ArCH), 115.5 (ArCH), 122.5 (ArCH),

125.4 (q, CF₃, J=281 Hz), 127.5 (ArC), 128.1 (ArCH),

128.7 (ArCH), 129.7 (ArCH), 130.9 (ArCH), 131.1

(ArCH), 133.3 (ArC), 139.7 (ArC), 159.6 (ArC), 160.9

(ArC), 171.4 (CO); MS (EI, 70 eV) m/z 415 (M⁺).

N-(Phenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-4-hydroxybenz-

amide (16f). Deprotected according to BBr₃procedure

and recrystallized from EtOAc/hexane to aord o-

white powder (quant): mp 114±116 ^ꢀC; ¹H NMR

(CDCl₃) d 6.48 (d, 2H, J=8.4 Hz), 7.0 (m, 4H) 7.15 (d,

2H, J=8.4 Hz), 7.26 (d, 6H), 7.72 (s, 2H, exchange with

D₂O); ¹³C NMR (MeOD) d 50.8 (CH), 114.9 (ArCH),

125.0 (q, CF₃, J_CF=281 Hz), 126.3 (ArC), 128.2

(ArCH), 128.6 (ArCH), 129.3 (ArCH), 130.0 (ArCH),

130.8 (ArCH), 131.0 (ArCH), 131.6 (ArC), 139.2 (ArC),

N-(4-Hydroxyphenyl)-N-(1,1,1-tri¯uoro-2-propyl)-4-

hydroxybenzamide (16a).⁶Deprotected according to

BBr₃procedure and recrystallized from EtOAc/hexane

to aord white powder (62%): mp 203±205 ^ꢀC (mp⁶

207±207.5 ^ꢀC); ¹H NMR (acetone-d₆) d 1.24 (d, 3H,

J=6.5 Hz), 5.78 (q, 1H, J=6.5 Hz), 6.62 (d, 2H,

J=8.8 Hz), 6.78 (br s, 2H), 7.05 (br s, 2H), 7.18 (d, 2H,

J=8.8 Hz), 8.61 (s, 1H), 8.70 (s, 1H); ¹³C NMR (ace-

tone-d₆) d 12.6 (CH₃), 51.3 (q, CH, ³J_H-F=30 Hz), 114.9

(ArCH), 115.0 (ArCH), 116.2 (ArCH), 126.8 (q, CF₃,

¹J_CF=282 Hz), 128.2 (ArC), 131.4 (ArCH), 131.6

(ArC), 157.7 (ArC), 159.3 (ArC), 171.8 (CO); MS (EI,

70 eV) m/z 325 (M⁺); anal. (C₁₆H₁₄NO₃F₃): C, 59.08; H

4.34; N, 4.31. Found: C, 59.14; H, 4.37; N, 3.96.

158.2 (ArC), 172.6 (CO); MS (EI, 70 eV) m/z 371

+

.

(M ); anal. (C₂₁H₁₆F₃NO₂0.5 H₂O): C, 66.31; H, 4.50;

N, 3.68. Found: C, 66.19; H, 4.27; N, 3.57.

N-(Phenyl)-N-(1,1,1-tri¯uoro-2-(3-hydroxyphenethyl))-4-

hydroxybenzamide (16g). Deprotected according to

BBr₃procedure and recrystallized from CHCl₃to aord

small o-white crystals (95%): mp 163±165 ^ꢀC; ¹H NMR

(CDCl₃) d 6.43 (d, 2H, J=8.5 Hz), 6.67 (d, 1H, J= 8.0

Hz), 6.82 (dd, 1H, J=8.5, 2 Hz), 6.91 (s, 1H), 7.08 (m,

8H), 7.62 (s, 1H); ¹³C NMR (CDCl₃) 114.9 (ArCH),

116.6 (ArCH), 117.0 (ArCH), 121.2 (ArCH), 124.6 (q,

CF₃, J=226 Hz), 126.1 (ArC), 128.3 (ArCH), 128.6

(ArCH), 129.8 (ArCH), 130.6 (ArCH), 132.6 (ArC),

138.8 (ArC), 156.3 (ArC), 157.8 (ArC), 173.0 (CO); MS

(EI, 70 eV) m/z 387 (M⁺); anal. (C₂₁H₁₆F₃NO₃): C,

65.12; H, 4.16; N, 3.62. Found: C, 64.78; H, 4.18; N, 3.38.

N-(4-Hydroxyphenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-4-

hydroxybenzamide (16b). Deprotected according to

BBr₃procedure and puri®ed by radial chromatography

(25% EtOAc/hexane) to aord product as thick oil

1

(53%): H NMR (MeOD) d 5.91 (br s, 1H) 6.32 (br s,

1H), 6.54 (d, 2H, J=8.8 Hz), 6.62 (br s, 1H), 6.94 (q,

1H, J_H-F=9.2 Hz), 7.13 (d, 2H, J=8.8 Hz), 7.23 (m,

6H); ¹³C NMR (MeOD) d 60.2 (CH), 113.9 (ArCH),

114.5 (ArCH), 125.0 (q, CF₃, J=282 Hz), 125.8 (ArC),

128.0 (ArCH), 128.7 (ArCH), 129.6 (ArCH) 130.0

(ArCH), 130.2 (ArC), 131.3 (ArC), 131.8 (ArC), 156.9

N-(4-Hydroxyphenyl)-N-(1,1,1-tri¯uoro-2-propyl)-4-

hydroxythiobenzamide (17a). Deprotected according to

BBr₃procedure and recrystallized from ether to aord

yellow powder (93%): ¹H NMR (MeOD) d 1.33 (d, 3H,

J=7.5 Hz), 6.47 (d, 2H, J=8 Hz), 6.58 (d, 1H, J=

6.5 Hz), 6.64 (d, 1H, J=6 Hz), 6.79 (d, 1H, J=8.5 Hz),

6.96 (d, 1H, J=8.0 Hz), 7.01 (d, 3H, J=8.5 Hz; ArCH

ortho CO and CHCF₃); MS (EI, 70 eV) m/z 341 (M⁺);

(ArC), 158.8 (ArC), 172.7 (CO); MS (EI, 70 eV) m/z

+

.

387 (M ); anal. (C₂₁H₁₆F₃NO₃0.1 H₂O): C, 64.81; H,

4.20; N, 3.60. Found: C, 64.68; H, 4.30; N, 3.45

.

N-(4-Hydroxyphenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-

benzamide (16c). Deprotected according to BBr₃pro-

cedure and recrystallized from EtOAc/hexane to aord

white powder (83%): mp 158±159 ^ꢀC, ¹H NMR (CDCl₃)

d 5.91 (br s, 1H), 6.30 (br s, 1H), 6.61 (br s, 1H), 6.74 (s,

1H), 6.95 (q, 1H, J=8.8 Hz), 7.24 (m, 9H); MS (EI,

70 eV) m/z 371 (M⁺); anal. (C₂₁H₁₆F₃NO₂): C, 67.92;

H, 4.34; N, 3.77. Found: C, 68.05; H, 4.35; N, 3.63.

anal. (C₁₆H₁₄F₃NO₂S 0.1 H₂O): C, 56.00; H, 4.17; N,

4.08. Found: C, 55.76; H, 4.13; N, 3.74.

N-(4-Hydroxyphenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-4-

hydroxythiobenzamide (17b). Deprotected according to

BBr₃procedure and puri®ed by chromatography (5%

MeOH/CH₂Cl₂) to aord title compound as yellow

1

foam (74%): H NMR (CDCl₃) d 5.47 (br s, 1H, OH),

5.58 (br s, 1H, OH), 5.86 (d, 1H, J=5.5 Hz, ArH ortho

to OH on aniline ring), 6.25 (d, 1H, J=5.5 Hz, ArH

ortho to OH on aniline ring), 6.36 (d, 2H, J=8 Hz, ArH

ortho to OH on benzamide ring), 6.57 (d, 1H, J=6 Hz,

ArH ortho to N±CS), 6.93 (d, 2H, J=9 Hz, ArH ortho

to CS), 6.98 (d, 1H, J=6 Hz, ArH ortho to N±CS),

8.39 (q, 1H, J_HF=9 Hz); ¹³C NMR (CDCl₃) d 63.8

(CHCF₃), 114.6 (ArCH), 115.0 (ArCH), 115.5 (ArCH),

125.1 (q, J=163 Hz, CF₃), 129.0 (ArCH), 129.3

(ArCH), 129.8 (ArCH), 129.9 (ArCH), 131.4 (ArC),

133.4 (ArC), 136.3 (ArC), 155.3 (ArC), 155.7 (ArC),

208.3 (CS); MS (EI, 70 eV) m/z 403 (M⁺); HRMS

calcd for C₂₁H₁₆F₃NO₂S, 403.0853, found 403.0858;

N-(4-Hydroxyphenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-3-

hydroxybenzamide (16d). Deprotected according to

BBr₃procedure and recrystallized from EtOAc/_ꢀhexane

1

to aord o-white powder (77%): mp 163±164 C; H

NMR (CDCl₃) d 5.92 (br s, 1H), 6.28 (br s, 1H), 6.64

(m, 2H), 6.96 (m, 3H), 7.31 (m, 7H); MS (EI, 70 eV) m/z

+

.

387 (M ); anal. (C₂₁H₁₆NO₃F₃H₂O): C, 62.52; H, 4.48;

N, 3.46. Found: C, 62.52; H, 4.17; N, 3.27.

N-(4-Hydroxyphenyl)-N-(1,1,1-tri¯uoro-2-phenethyl)-2-

hydroxybenzamide (16e). Deprotected according to

BBr₃procedure and recrystallized from EtOAc/hexane

to aord o-white powder (59%): mp 191.5±193 ^ꢀC; H

anal. (C₂₁H₁₆F₃NO₂S 0.3 H₂O): C, 61.70; H, 4.09; N,

3.43. Found: C, 61.68; H, 3.92; N, 3.25.

1

.

NMR (MeOD) d 6.10 (br s, 2H), 6.59 (app q, 2H,

J=7.6, 7.2 Hz), 6.96 (m, 3H), 7.32 (m, 6H); MS (EI,

+

.

70 eV) m/z 387 (M ); anal. (C₂₁H₁₆F₃NO₃0.7 H₂O): C,

N-(4-Methoxyphenyl)-4-methoxyphenylacetamide (18).

To a 0 ^ꢀC CH₂Cl₂solution of p-anisidine (1.3 equiv)

and pyridine (1.1 equiv) was added dropwise a CH₂Cl₂

63.06; H, 4.38; N, 3.50. Found: C, 63.04; H, 4.08; N,

3.50.

1314

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

solution (2±5 mL) of 4-methoxyphenylacetyl chloride

(1.0 equiv). Upon complete addition (15 min) mixture

was allowed to reach rt. Upon completion of the reac-

tion, the mixture was diluted with CH₂Cl₂folowed by

product isolation (H₂O, CuSO₄, brine). Recrystalliza-

tion from EtOAc/hexane aorded the title carboxamide

as small white crystals (82%): mp 127±128 ^ꢀC; ¹H NMR

(CDCl₃) d 3.65 (s, 2H), 3.76 (s, 3H), 3.82 (s, 3H), 6.80

(d, 2H, J=9.0 Hz), 6.92 (d, 2H, J=8.5 Hz), 7.07 (s, 1H),

7.23 (d, 2H, J=9.0 Hz), 7.30 (d, 2H, J=8.5 Hz); ¹³C

NMR (CDCl₃) d 43.7 (CH₂), 55.3 (CH₃), 55.5 (CH₃),

114.1 (ArCH), 114.6 (ArCH), 121.8 (ArCH), 126.5

(ArC), 130.7 (ArCH), 130.8 (ArC), 156.5 (ArC), 159.0

(ArC), 169.7 (CO); MS (EI, 70 eV) m/z 271 (M⁺);

anal. (C_l6H₁₇NO₃): C, 70.83; H, 6.32; N, 5.16. Found:

C, 70.87; H, 6.29; N, 5.29.

NMR (CDCl₃) d 0.94 (t, 3H, J=7.2 Hz, NCH₂CH₃),

2.75 (dd, 1H, J=12.8, 4.8 Hz, methine H), 3.43 (m, 2H,

NCH₂CH₃), 3.58 (dd, 1H, J=10.2, 5.1 Hz), 3.70 (dd,

1H, J=13.3, 7.1 Hz) 3.76 (s, 3H), 3.79 (s, 3H), 6.15 (br

s, 1H), 6.75 (d, 2H, J=8.3 Hz), 6.78 (d, 2H, J=6.7 Hz),

7.09 (m, 4H), 7.24 (m, 3H); ¹³C NMR (CDCl₃) d 12.8

(CH₃), 41.5 (CH₂), 44.2 (CH₂), 50.3 (CH), 55.2 (OCH₃),

55.4 (OCH₃), 113.7 (ArCH), 114.3 (ArCH), 126.2

(ArCH), 128.2 (ArCH), 129.1 (ArCH), 129.4 (ArCH),

130.0 (ArCH), 132.4 (ArC), 134.6 (ArC), 140.1 (ArC),

158.5 (ArC), 158.9 (ArC), 172.5 (CO); MS (EI, 70 eV)

m/z 389 (M⁺).

N-(4-Hydroxyphenyl)-N-(ethyl)-4-hydroxyphenylacetamide

(21a). Deprotected according to BBr₃procedure and

puri®ed by chromatography (50% EtOAc/hexane) to

aord tan foam (quant): mp 75 ^ꢀC dec.; ¹H NMR

((CD₃)₂SO) d 0.96 (t, 3H, J=7.5 Hz), 3.16 (s, 2H), 3.36

(q, 2H, J=7.5 Hz), 6.59 (d, 2H, J=8.5 Hz), 6.78 (m,

4H), 6.97 (d, 2H, J=8.5 Hz), 9.18 (br s, 1H), 9.62 (br s,

1H); MS (EI, 70 eV) m/z 271 (M⁺); anal. (C₁₆H₁₇

N-(4-Methoxyphenyl)-N-(ethyl)-4-methoxyphenylacetamide

(19). Prepared according to phase transfer catalysis

conditions described above for carboxamides. Puri®ca-

tion by either Kugelrohr distillation or ¯ash chromato-

graphy (25% EtOAc/hexane) aorded product as red±

orange oil (quant): bp 150 ^ꢀC (0.2 mm); ¹H NMR

(CDCl₃) d 1.08 (t, 3H, J=7.0 Hz), 3.33 (s, 2H), 3.70 (q,

2H, J=7.1 Hz), 3.78 (s, 3H), 3.84 (s, 3H), 6.77 (d, 2H,

J=8.5 Hz), 6.79 (d, 2H, J=8.5 Hz), 6.97 (d overlapping,

2H, J=8.5 Hz), 6.98 (d overlapping, 2H, J=8.5 Hz);

¹³C NMR (CDCl₃) d 13.2 (CH₃), 40.5 (CH₂), 44.4

(CH₂), 55.4 (CH₃O), 55.7 (CH₃O), 113.9 (ArCH), 114.8

(ArCH), 127.9 (ArC), 129.9 (ArCH), 130.2 (ArCH),

135.2 (ArC), 158.4 (ArC), 159.2 (ArC), 171.2 (CO);

.

NO₃0.9 H₂O): C, 66.84; H, 6.59; N, 4.87. Found: C,

66.95; H, 6.20; N, 4.77.

N-(4-Hydroxyphenyl)-N-(ethyl)-4-hydroxyphenylthioace-

tamide (21b). Deprotected according to BBr₃procedure

and puri®ed by chromatography (50% EtOAc/hexane)

to aord tan powder (84%): mp 156±157 ^ꢀC; H NMR

1

((CD₃)₂SO) d 1.08 (t, 3H, J=7.0 Hz), 3.67 (s, 2H), 4.14

(q, 2H, J=2 Hz), 6.54 (d, 2H, J=8.5 Hz), 6.75 (app t,

4H, J=8.0 Hz), 6.87 (d, 2H, J=8.5 Hz), 9.17 (s, OH),

9.78 (s, OH); MS (EI, 70 eV) m/z 287 (M⁺); HRMS

calcd for C₁₆H₁₇NO₂S, 287.0980; found, 287.0977; anal.

+

.

MS (EI, 70 eV) m/z 299 (M ); anal. (C₁₈H₂₁NO₃0.1

CHCl₃): C, 69.83; H, 6.83; N, 4.50. Found: C, 69.82; H,

6.98; N, 3.98.

.

(C₁₆H₁₇NO₂S 0.2H₂O): C, 66.04; H, 6.03; N, 4.81.

Found: C, 65.97; H, 6.03; N, 4.78.

N-(4-Methoxyphenyl)-N-(ethyl)-ꢀ-ethyl-4-methoxyphenyl-

acetamide (20a). n-BuLi in hexane (1.56 M, 2.0 equiv)

was added dropwise to a 78 ^ꢀC THF (i-propyl)₂NH

(2.2 equiv) solution, then warmed to 0 ^ꢀC. After 0.5 h at

0 ^ꢀC the mixture was re-cooled to 78 ^ꢀC and a THF

solution of acetamide 19 (1.0 equiv) added dropwise.

After 20 min EtI (1.3 equiv) was added in one portion

and the mixture allowed to reach rt. Product isolation

(H₂O, Et₂O, Na₂SO₄) and puri®cation by ¯ash chro-

matography (25% EtOAc/hexane) as light yellow oil

(95%): ¹H NMR (CDCl₃) d 0.78 (t, 3H, J=7.5 Hz, CH₃

a-ethylacetamide), 1.05 (t, 3H, J=7.0 Hz, CH₃N-ethyl),

1.60 (dquint, 1H, J=14.0, 7.0 Hz, a-CH₂), 2.04 (dquint,

1H, J=14.0, 7.0 Hz, a-CH₂), 3.27 (dd, 1H, J=8.5,

7.0 Hz, CH a-benzylic), 3.67 (m, 2H, -NCH₂CH₃), 3.77

(s, 3H), 3.85 (s, 3H), 6.75 (d, 2H, J=8.8 Hz), 6.87 (br s,

4H), 6.98 (d, 2H, J=8.8 Hz); ¹³C NMR (CDCl₃) d 12.5

(CH₃), 13.1 (CH₃), 28.5 (CH₂), 44.3 (CH₂), 55.2 (CH₃),

55.6 (CH₃), 113.7 (ArCH), 114.5 (ArCH), 129.2 (ArCH),

132.9 (ArC), 135.0 (ArC), 158.4 (ArC), 159.0 (ArC),

173.5 (CO); MS (EI, 70 eV) m/z 327 (M⁺); anal.

(C₂₀H₂₅NO₃): C, 73.37; H, 7.70; N, 4.28. Found: C,

72.84; H, 7.56; N, 3.59.

N-(4-Hydroxyphenyl)-N-(ethyl)-ꢀ-ethyl-4-hydroxyphenyl-

acetamide (22a). Deprotected according to BBr₃pro-

cedure and puri®ed by chromatography (50% EtOAc/

hexane) to aord white foam (70%): ¹H NMR (CDCl₃)

d 0.76 (t, 3H, J=7.5 Hz), 1.04 (t, 3H, J=7.0 Hz), 1.54

(m, 1H, CH₂b-amide), 1.94 (m, 1H, CH₂b-amide),

3.27 (t, 1H, J=7.0 Hz, CH a-amide), 3.62 (m, 2H,

-NCH₂CH₃), 6.63 (d, 2H, J=8.8 Hz), 6.79 (d, over-

lapping br s, 2H, J=8.8 Hz), 7.05 (br s, 4H); ¹³C NMR

(CDCl₃) d 12.5 (CH₃), 13.0 (CH₃), 28.2 (CH₂), 44.8

(CH₂), 50.7 (CH), 116.1 (ArCH), 128.6 (ArCH), 129.3

(ArCH), 131.7 (ArCH), 133.7 (ArC), 155.4 (ArC), 156.8

(ArC), 174.7 (CO); MS (EI, 70 eV) m/z 299 (M⁺);

HRMS calcd for C₁₈H₂₁NO₃, 299.1521, found

299.1520.

N-(4-Hydroxyphenyl)-N-(ethyl)-ꢀ-ethyl-4-hydroxyphenyl-

thioacetamide (22b). Deprotected according to BBr₃

procedure and puri®ed by chromatography (25%

EtOAc/hexane) to aord yellow residue (95%): ¹H

NMR (MeOD) d 0.73 (t, 3H, J=7.3 Hz), 1.14 (t, 3H,

J=7.2 Hz), 1.76 (m, 1H, CH₂b-amide), 2.17 (m, 1H,

CH₂b-amide), 3.62 (dd, 1H, J=7.8, 7.0 Hz, CH a-

amide), 4.16 (m, 1H, CH₂a-NCO), 4.30 (m, 1H, CH₂a-

NCO), 6.56 (dd, 1H, J=8.4, 2.6 Hz), 6.56 (AA⁰BB⁰, 2H,

J=8.7, 2.3 Hz), 6.75 (dd, 1H, J=8.4, 2.8 Hz), 6.91 (dd,

1H, J=8.6, 2.7 Hz), 6.94 (AA⁰BB⁰, 2H, J=8.5, 1.9 Hz),

N-(4-Methoxyphenyl)-N-(ethyl)-ꢀ-benzyl-4-methoxy-

phenylacetamide (20b). Preparation as described in 20a

and puri®cation by ¯ash chromatography (15% EtOAc/

1

hexane) aorded title compound as an oil (86%): H

S. R. Stauer et al. / Bioorg. Med. Chem. 8 (2000) 1293±1316

1315

7.0 (dd, 1H, J=8.7, 2.8 Hz); ¹³C NMR (CDCl₃) d 11.4

(CH₃), 12.6 (CH₃), 13.9 (CH₂), 52.3 (CH₂), 56.2 (CH),

115.0 (ArCH), 116.3 (ArCH), 128.5 (ArCH), 129.1

(ArCH), 129.9 (ArCH), 133.5 (ArC), 136.5 (ArC), 154.6

(ArC), 155.9 (ArC), 207.5 (CS); MS (EI, 70 eV) m/z

315 (M⁺); HRMS calcd for C₁₈H₂₁NO₂S, 315.1293;

found, 315.1301.

N-(4-Hydroxyphenyl)-N-(1,1,1-tri¯uoro-2-propyl)-ꢀ-methyl-

4-hydroxyphenylacetamide (27). Deprotection using

BBr₃procedure and chromatography (35% EtOAc/

hexane) aorded title compound as a white powder

1

(95%) with a 1:1 ratio of diastereomers as found by H

NMR: mp 165±170 ^ꢀC; ¹H NMR (MeOD) d 1.10

(overlapping ds, 3H, J=7.5 Hz), 1.25 (overlapping ds,

3H, J=7.5 Hz), 3.49 (overlapping qs, 1H, J=7.5 Hz),

5.52 (m, 1H, CHCF₃), 6.43 (2-dds, 1H, J=8.8, 2.8 Hz),

6.57 (dd, 0.5H, J=8.8, 2.8 Hz), 6.64 (m, 2.5H), 6.72 (m,

2.5H), 6.86 (app td, 1H, J=8.4, 2.3 Hz), 7.07 (2-dds, 1H,

J=8.4, 2.3 Hz); MS (EI, 70 eV) m/z 353 (M⁺); HRMS

calcd for C₁₈H₁₈NF₃O₃, 353.1238; found, 353.1235.

N-(4-Hydroxyphenyl)-N-(ethyl)-ꢀ-benzyl-4-hydroxyphenyl-

thioacetamide (22c). Deprotected according to BBr₃

procedure and puri®ed by chromatography (15%

EtOAc/hexane) to aord yellow residue (47%): ¹H

NMR (MeOD) d 1.05 (t, 3H, J=7.2 Hz), 2.90 (dd, 1H,

J=12.9, 4.9 Hz, PhCH₂), 3.66 (dd, 1H, J=12.9, 9.5 Hz,

PhCH₂), 3.95 (m, 1H, CH₃CH₂), 4.05 (dd, 1H, J=9.3,

5.2 Hz, PhCH₂CH-), 4.30 (m, 1H, CH₃CH₂), 6.02 (dd,

1H, J=8.4, 2.7 Hz), 6.56 (d, 2H, J=8.3 Hz), 6.66

(overlapping dds, 2H, J=9.2, 3.1 Hz), 6.75 (dd, 1H,

J=8.8, 3.0 Hz), 7.00 (d, 2H, J=8.7 Hz), 7.10 (m, 5H);

HRMS calcd for C₂₃H₂₃NSO₂, 377.1449; found,

377.1443.

N-(4-Hydroxyphenyl)-N-(1,1,1-tri¯uoro-2-propyl)-4-hy-

droxyphenylthioacetamide (28). Carboxamide 25 (0.3

mmol, 100 mg) was treated with Lawesson's reagent as

outlined in general procedure. Product isolation (H₂O,

Et₂O, Na₂SO₄) and radial chromatography (20%

EtOAc/hexane) aorded 5 mg of protected thiocarbox-

amide in low yield (5%). Subsequent deprotection using

BBr₃procedure aorded the title compound as a light

yellow residue upon SiO₂puri®cation (30% EtOAc/

N-(4-Methoxyphenyl)-N-(1,1,1-tri¯uoro-2-propyl)-4-meth-

oxyphenylacetamide (25). Prepared according to gen-

eral amidation procedure for CF₃-containing anilines;

product isolation and puri®cation via ¯ash chromato-

graphy (25% EtOAc/hexane) aorded title compound

as light yellow oil (40%): ¹H NMR (CDCl₃) d 1.16

(d, 3H, J=7 Hz), 3.32 (s, 2H), 3.76 (s, 3H), 3.84 (s,

3H), 5.61 (sept, 1H, J=7.6 Hz), 6.77 (d, 2H,

J=8.9 Hz), 6.90 (m, 3H), 6.92 (d, 2H, J=8.9 Hz), 7.06

(m, 1H); ¹³C NMR (CDCl₃) d 12.5 (CH₃), 40.7

(CH₂), 50.1 (q, CHCF₃, J=31 Hz), 55.3 (CH₃O), 55.6

(CH₃O), 113.9 (ArCH), 114.3 (ArCH), 114.6 (ArCH),

125.1 (q, CF₃. J=283 Hz), 127.0 (ArC), 129.9 (ArC),

130.2 (ArCH), 132.0 (ArCH), 158.6 (ArC), 159.9

(ArC), 172.8 (CO); HRMS calcd for C₁₉H₂₀NF₃O₃,

367.1395, found, 367.1394; anal. (C₁₉H₂₀NF₃O₃): C,

62.12; H, 5.49; N, 3.81. Found: C, 61.76; H, 5.58; N,

3.66.

1

hexane, quant): H NMR (MeOD) d 1.24 (d and m,

3H, J=7.8 Hz), 3.85 (ABq, 2H, J=13 Hz), 5.56 (sept,

1H, J=7.8 Hz), 6.72 (m, 4H), 6.85 (m, 4H); HPLC:

Whatman C18 column, 90%MeOH:10%H₂O R_t=

3.123>98% pure at 254 nm.

Acknowledgements

We are grateful for support of this research through grants

from the US Army Breast Cancer Research Program

(DAMD17-97-1-7076) and the National Institutes of

Health (HHS 5R37 DK15556 and CA18119). We thank

Kathryn E. Carlson for performing binding assays and

Scott Wilson, Richard Cesati and Professor Greg

Girolami for valuable assistance with X-ray data acqui-

sition, structure determination and re®nement. NMR

spectra were obtained in the Varian Oxford Instrument

Center for Excellence in NMR Laboratory. Funding for

this instrumentation was provided in part from the

W. M. Keck Foundation and the National Science

Foundation (NSF CHE 96-10502). Mass spectra were

obtained on instruments supported by grants from the

National Institute of General Medical Sciences (GM

27029), the National Institute of Health (RR 01575),

and the National Science Foundation (PCM 8121494).

N-(4-Methoxyphenyl)-N-(1,1,1-tri¯uoro-2-propyl)-ꢀ-methyl-

4-methoxyphenylacetamide (26). n-BuLi in hexane

(1.56 M, 2.0 equiv) was added dropwise to a 78 ^ꢀC

THF (i-propyl)₂NH (2.2 equiv) solution, then warmed

to 0 ^ꢀC. After 0.5 h at 0 ^ꢀC the mixture was re-cooled to

78 ^ꢀC and a THF solution of N-(1,1,1-tri¯uoro-2-pro-

pyl) substituted acetamide 25 (1.0 equiv) was added

dropwise. After 20 min MeI (1.3 equiv) was added in

one portion and the mixture allowed to reach rt. Crude

product isolation (H₂O, Et₂O, Na₂SO₄) aorded an

amber oil (83%) which was determined by both ¹H

NMR and HPLC to be an approx. 1:1 ratio of diastereo-

mers for the expected product: ¹H NMR (CDCl₃) d

1.05 (2-ds, 3H, J=7.5 Hz), 1.28 (2-ds, 3H, J=7.5 Hz),

3.42 (overlapping qs, 1H, J=7.5 Hz), 5.58 (m, 1H,

CHCF₃), 6.44 (2-dds, 1H, J=8.8, 2.8 Hz), 6.68 (dd, 0.5 H,

J=8.8, 2.8 Hz), 6.70 (m, 2.5H), 6.81 (m, 2H), 7.01

(2-dds, 1H, J=8.4, 2.3 Hz), 7.20 (2-dds, 1H, J=8.4,

2.3 Hz); HPLC (254 nm) 25% EtOAc/hexane, Micropac

SiO₂column: 5.82 min. (46.2%), 7.19 min. (53.7%),

sdÆ5%.

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