An adamantyl-substituted retinoid-derived molecule that inhibits cancer cell growth and angiogenesis by inducing apoptosis and binds to small heterodimer partner nuclear receptor: Effects of modifying its carboxylate group on apoptosis, proliferation, and

2622

J. Med. Chem. 2007, 50, 2622-2639

An Adamantyl-Substituted Retinoid-Derived Molecule That Inhibits Cancer Cell Growth and

Angiogenesis by Inducing Apoptosis and Binds to Small Heterodimer Partner Nuclear

Receptor: Effects of Modifying Its Carboxylate Group on Apoptosis, Proliferation, and

Protein-Tyrosine Phosphatase Activity

Marcia I. Dawson,*^,†Zebin Xia,^†Gang Liu,^†Joseph A. Fontana,^‡Lulu Farhana,^‡Bhamik B. Patel,^‡Sankari Arumugarajah,^‡

Mohammad Bhuiyan,^‡Xiao-Kun Zhang,^†Young-Hoon Han,^†William B. Stallcup,^§Jun-ichi Fukushi,^§,|Tomas Mustelin,

Lutz Tautz, Ying Su,^†Danni L. Harris,^#Nahid Waleh,³Peter D. Hobbs,³Ling Jong,³Wan-ru Chao,³Leonard J. Schiff,^[and

Brahma P. Sani^∞

Cancer Center, Burnham Institute for Medical Research, La Jolla, California 92037, Wayne State, UniVersity School of Medicine and

Department of Veterans Affairs, Detroit, Michigan 48201, Molecular Research Institute, Mountain View, California 94043, Southern Research

Institute, Birmingham, Alabama 35025, IIT Research Institute, Chicago, Illinois 60616, and SRI International, Menlo Park, California 94025

ReceiVed NoVember 16, 2006

Apoptotic and antiproliferative activities of small heterodimer partner (SHP) nuclear receptor ligand (E)-

4-[3′-(1-adamantyl)-4′-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC), which was derived from 6-[3′-

(1-adamantyl)-4′-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN), and several carboxyl isosteric or

hydrogen bond-accepting analogues were examined. 3-Cl-AHPC continued to be the most effective apoptotic

agent, whereas tetrazole, thiazolidine-2,4-dione, methyldinitrile, hydroxamic acid, boronic acid, 2-oxoaldehyde,

and ethyl phosphonic acid hydrogen bond-acceptor analogues were inactive or less efficient inducers of

KG-1 acute myeloid leukemia and MDA-MB-231 breast, H292 lung, and DU-145 prostate cancer cell

apoptosis. Similarly, 3-Cl-AHPC was the most potent inhibitor of cell proliferation. 4-[3′-(1-Adamantyl)-

4′-hydroxyphenyl]-3-chlorophenyltetrazole, (2E)-5-{2-[3′-(1-adamantyl)-2-chloro-4′-hydroxy-4-biphenyl]-

ethenyl}-1H-tetrazole, 5-{4-[3′-(1-adamantyl)-4′-hydroxyphenyl]-3-chlorobenzylidene}thiazolidine-2,4-dione,

and (3E)-4-[3′-(1-adamantyl)-2-chloro-4′-hydroxy-4-biphenyl]-2-oxobut-3-enal were very modest inhibitors

of KG-1 proliferation. The other analogues were minimal inhibitors. Fragment-based QSAR analyses relating

the polar termini with cancer cell growth inhibition revealed that length and van der Waals electrostatic

surface potential were the most influential features on activity. 3-Cl-AHPC and the 3-chlorophenyltetrazole

and 3-chlorobenzylidenethiazolidine-2,4-dione analogues were also able to inhibit SHP-2 protein-tyrosine

phosphatase, which is elevated in some leukemias. 3-Cl-AHPC at 1.0 µM induced human microvascular

endothelial cell apoptosis but did not inhibit cell migration or tube formation.

Introduction

In investigating the effects of retinoids on cancer cell

proliferation, we first noticed that the retinoid 6-[3′-(1-adaman-

tyl)-4′-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN^a/

CD437, 1 in Figure 1)^1,2displayed the atypical functions of

* Address correspondence to Dr. Marcia I. Dawson, Burnham Institute

for Medical Research, 10901 North Torrey Pines Rd., La Jolla, California

92037. Phone: 858-646-3165. Fax: 858-646-3197. E-mail: mdawson@

burnham.org.

^†Cancer Center, Burnham Institute for Medical Research.

^‡Wayne State University School of Medicine and Department of

Veterans Affairs.

^§Neuroscience and Aging Center, Burnham Institute for Medical

Research.

^|Present address: Kyushu University Hospital, Fukuoka City, Japan.

Inflammatory and Infectious Disease Center, Burnham Institute for

Medical Research.

^#Molecular Research Institute.

³SRI International.

^[IIT Research Institute (retired).

Figure 1. Structures of AHPN (1), 5-Cl-AHPN (2), 5-Me-AHPN (3),

AHPC (4), 3-Cl-AHPC (5), 3-A-AHPC (6), trans-RA (7), TTNN (8),

^∞Southern Research Institute.

^aAbbreviations: 3-Cl-AHPC, (E)-4-[3′-(1-adamantyl)-4′-hydroxyphe-

5-Me-TTNN (9), and TTNC (10).

nyl]-3-chlorocinnamic acid; AHPN, 6-[3′-(1-adamantyl)-4′-hydroxyphenyl]-

2-naphthalenecarboxylic acid; AML, acute myelocytic leukemia; DR5-tk-

inducing the cell-cycle arrest and initiating the apoptosis of

CAT, synthetic gene of retinoic acid response element of two direct repeats

MCF-7 breast cancer and other cancer cell lines.^3,4Further

separated by five base pairs linked to the thymidine kinase promoter

followed by the base sequence for chloramphenicol acetyl transferase gene;

HMVE, human microvascular endothelial; RAR, retinoic acid receptor;

RXR, retinoid X receptor; TTNC, (E)-4-(5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-

tetramethyl-2′-naphthalenyl)cinnamic acid; TTNN, 6-(5′,6′,7′,8′-tetrahydro-

5′,5′,8′,8′-tetramethyl-2′-naphthalenyl)-2-naphthalenecarboxylic acid.

research led to our identification of (E)-4-[3′-(1-adamantyl)-4′-

hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC, 5)⁵that

retained these functions but not that exhibited by classical

retinoids, namely the transcriptional activation of the retinoic

Published on Web 05/10/2007

Effects of Isosteric Modifications of 3-Cl-AHPC Carboxylate

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2623

Table 1. Classical Retinoid Assays Used To Compare Activities of

Comformationally Restricted Retinoids with Those of trans-Retinoic

Acid^a

acid nuclear receptor (RAR) subtypes to induce the transcription

of genes regulated by trans-retinoic acid (trans-RA, 7) and its

synthetic retinoid analogues.^5-8By inducing apoptosis of

leukemia cells obtained from AML patients,⁵5 may have

potential as a therapeutic agent for treating acute myeloid

leukemia (AML). It also has the ability to induce apoptosis of

cells from a variety of human cancer cell lines, including those

derived from breast, lung, and prostate cancers.⁹Moreover,

apoptosis induced by 1 and 5 was found to be independent of

the sensitivity of the cancer cell line to growth regulation by

trans-RA or its p53 status,^3,4either of which is therapeutically

advantageous because during cancer progression the ability of

the trans-RA-RARR complex to regulate proliferation or induce

differentiation through induction of expression of the tumor

suppressor gene RARâ is lost^10,11and the tumor suppressor gene

p53 becomes dysfunctional or lost in about 50% of tumors.^12,13

Recently, we identified the first synthetic small-molecule

ligands, 3-Cl-AHPC (5) and several analogues, for the nuclear

receptor small heterodimer partner (SHP; NR0B2),¹⁴which

hitherto had only a putative ligand-binding domain and was

classified as an orphan receptor. SHP is an atypical member of

the steroid/thyroid hormone nuclear receptor family of transcrip-

tion factors because it lacks the amino terminal sequence (AB),

canonical DNA-binding domain (C), and hinge region (D) that

are typical of other nuclear receptors and predominately

functions as a transcriptional repressor through the binding of

one of its three NR box motifs (LXXLL) to the activation

function 2 (AF-2) site in the ligand-binding domain of its

dimeric nuclear receptor partner. SHP has been found to bind

such nuclear receptors as the androgen, aryl hydrocarbon

receptor nuclear translocator, constitutive activated, estrogen,

farnesoid, glucorticoid, liver X, pregnane X, peroxisome pro-

liferator-activated, retinoic acid, retinoid X, thyroid hormone,

TR3/nur77/NGFB-I, and vitamin D receptors.^15-21SHP modu-

lates gene transactivation or suppression induced by its het-

erodimeric partner by recruiting histone deacetylases 1, 3, or

6, G9a histone 3 K9 methyltransferase, and other members of

the Sin3a-Swi/Snf repressor complex.¹⁷

Tumor growth can be inhibited directly by inducing cancer

cell death or indirectly by inhibiting tumor neovascularization

(angiogenesis), a process that provides a conduit for delivery

of nutrients to and removal of metabolic byproducts from the

tumor.²²Tumor vasculature also provides a route for cancer cells

to escape from the tumor and move to secondary metastatic

sites.²³For these reasons, we explored the abilities of 3-Cl-

AHPC (5) and its analogues to function as anticancer and

antiangiogenic agents. We also undertook an analogue genera-

tion program to determine the pharmacophoric elements neces-

sary to confer apoptotic activity to 5. Here, we report the effects

of 5 and several analogues on the proliferation and functions

of human microvascular endothelial (HMVE) cells and the

impact that replacing the carboxylic acid group of 5 with other

hydrogen-bond acceptors or isosteric-like groups has on cancer

cell growth inhibition and induction of apoptosis.

IC₅₀value (nM)

ID₅₀value (nmol)

ODC^d

retinoid

TOC^b

F9^c

trans-RA (7)

TTNN (8)

TTNC (10)

AHPN (1)

0.01

0.007

0.32

nd^e

0.1

1.0

0.3

nd

0.19

2.0

7.5

3.1

5-Me-TTNN (9)

0.08

3.0

66

^aFor overview of these assays see Sporn and Roberts (1984). ^bTOC

assay, concentration required to reverse keratinization in 50% of vitamin

A-deficient hamster tracheas in organ culture after 10 days as determined

by analysis of stained cross-sections for the absence of keratin and

keratohyaline granules. ^cF9 assay, concentration required to induce terminal

differentiation in 50% of F9 murine embryonic teratocarcinoma cells in

culture after 3 days as measured by secreted plasminogen activator activity.

^dODC assay, topical dose of retinoid preapplied to mouse dorsal epidermis

at 1 h before TPA leading to 50% inhibition in level of ornithine

decarboxylase induced by TPA (7.5 nmol) at 4.5 h after TPA application

as measured by the release of labeled CO₂from [¹⁴C]ornithine by epidermal

homogenates. TPA, 12-O-tetradecanoylphorbol-13-acetate. ^end, not deter-

mined.

activity with those of the retinoid TTNN (8)²⁶from which

AHPN (1) was originally derived.²These assays, which histori-

cally have been widely used to assess retinoic acid-like activity,

were (i) induction of keratin granule formation in vitamin-A-

deficient hamster trachea in organ culture (TOC assay), which

was developed by Sporn and co-workers;²⁷(ii) induction of

differentiation in F9 embryonic teratocarcinoma cells (F9

assay);²⁸and (iii) inhibition of the induction of the proliferative

enzyme ornithine decarboxylase in mouse epidermis by the

tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA),

which was developed by Verma and Boutwell (ODC assay).²⁹

These assays have been reviewed by Sporn and Roberts.³⁰On

the basis of its lower activities in the TOC and ODC assays,

the cinnamic acid analogue (TTNC, 10)³¹of TTNN (8) was

deemed to have less retinoid activity (Table 1). Similarly, the

3′-(1-adamantyl)-4′-hydroxyphenyl ring of 1 conferred lower

retinoid activity than the 5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-

2-naphthalenyl ring of 8²⁶did in the ODC assay³²(Table 1),

just as it was subsequently found by Shroot and co-workers to

have lower activity in the RAR transcriptional activation assay.¹

Therefore, the two ring systems, 3-(1-adamantyl)-4-hydroxy-

phenyl and (E)-cinnamic acid, were joined at their 1- and 4-ring

positions, respectively, to produce AHPC (4).

AHPC (4) effectively induced cancer cell apoptosis and

exhibited lower activities in the TOC, F9, and ODC assays than

AHPN; however, we observed that mice injected intravenously

with AHPC displayed symptoms of retinoid-like toxicity. The

structure of 4 was later reported as ST1926, which was found

to have antileukemic activity.³³Recently, 4 was also found to

transactivate the RARs R, â, and γ on a DR5-tk-CAT reporter

construct in transfected COS-7 cells.³⁴Its half-maximal activa-

tion concentrations (AC₅₀s) were 8-, 2.8-, and 4.7-fold higher,

respectively, than those of trans-RA (7), thereby supporting our

design strategy.

Results and Discussion

As Table 1 indicates, introduction of a substituent at the

5-position of the naphthalene ring in TTNN (8), which is ortho

to the diaryl bond, reduced the retinoid activity of 5-Me-TTNN

(9) in the TOC, F9, and ODC assays. By adapting this

substitution strategy, namely introducing a chloro group adjacent

to the diaryl bond of AHPN (1) and AHPC (4), we achieved a

further reduction in RAR interaction by energetically hindering

the diaryl rings of 5-Cl-AHPN (2)³⁵and 3-Cl-AHPC (5)⁵from

assuming a small dihedral angle on binding to the RARs.⁶

Chemistry. Analogue Design. As a consequence of reports

that therapeutic retinoids and RAR ligands such as trans-RA

(7) and 9-cis-RA can cause adverse effects in cancer patients,^24,25

a major facet of our design strategy has been introducing groups

and scaffold modifications onto the AHPN scaffold that reduced

interaction with the RAR subtypes. This strategy was first

accomplished for the design of AHPC (4) by comparing the

activities of members of our retinoid library in assays for retinoid

2624 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11

Dawson et al.

Table 2. Comparison of Effects of 3-Cl-AHPC (5) and Its Analogues

on HMVE Cell Proliferation after 96 h of Treatment Compared to

trans-Retinoic Acid (7) and Synthetic RARγ-selective Analogues 13 and

14

^aConcentration inhibiting proliferation by 50%. ^bRelative to vehicle

alone control. ^cHighest concentration evaluated.

Figure 2. Pharmacophoric model showing essential components of

apoptosis-inducing activity used for the design of 3-Cl-AHPC and its

analogues. (A) Three-dimensional seven-point pharmacophore descrip-

tive of common elements in compounds in an AHPN analogue training

set of which 44% were active at inducing MDA-MB-231 breast cancer

cell apoptosis at 1.0 µM after a 96-h treatment. Components 1 and 2,

hydrogen-bond acceptor; 3, hydrogen-bond donor/acceptor; 4, hydro-

phobic ring; 5, hydrophobic group; 6, sterically accessible hydrophobic

region; and 7, sterically accessible polar-permissible region. Interpoint

distances (Å) ( SD: ∆_(1-2), 2.1 ( 0.01; ∆_(1-3), 12.5 ( 0.1; ∆_(1-4), 5.9

4′-hydroxyphenyl]-2-thienyl}propenoic acid were left out of the

training set and the CoMSIA model were used to predict their

ability at 1.0 µM to induce MDA-MB-231 apoptosis, the

predicted versus experimental results were: 92% versus 43%

for 5 and 0.8% versus 1% for the thienylpropenoic acid (Figure

2B). Thus, while not quantitative, even at this level the CoMSIA

model was able to make order of magnitude predictions,

underscoring the reasonableness of the model and the underlying

pharmacophore. In the study reported here, we focus on

identifying the character of the hydrogen-acceptor group(s) 1

and/or 2 required for induction of apoptosis.

( 0.1; ∆_(1-5), 12.4 ( 1.2; ∆_(2-3), 12.5 ( 0.2; ∆_(2-4), 5.7 ( 0.2; ∆_(3-5)

,

4.0 ( 0.1; ∆_(4-5), 7.5 ( 0.1; ∆_(4-6)g 2.3. (B) Comparative molecular

similarity index analysis on 19 analogues using the overlap rule/

pharmacophore defined in (A) for prediction of induction of apoptosis

of MDA-MB-231 breast cancer cells after treatment for 96 h at 1.0

µM by comparing natural logs of experimental and calculated values.

Predictive R²) 0.78; uncross-validated R²( SE ) 0.95 ( 0.45.

Replacement of the carboxylic acid group of 3-Cl-AHPC (5)

by isosteric and other groups having a similar pattern of polar

atoms that function as hydrogen-bond acceptors was explored

to determine whether pharmacologic properties could be im-

proved with retention of apoptotic activity. Our earlier studies

indicated that shifting the position of the carboxylate group

relative to the phenolic hydroxyl was not successful. No

apoptosis of trans-RA-resistant HL-60R leukemia cells³⁶was

observed after 24-h treatment with 1.0 µM 6-[3′-(1-adamantyl)-

4′-hydroxyphenyl]-3-naphthalenecarboxylic acid (11 in Table

2), compared to 98% apoptosis with 1.0 µM AHPN (1). The

96-h treatment of retinoid-resistant MDA-MB-231 breast cancer

cells with 2.0 µM 5-[3′-(1-adamantyl)-4′-hydroxyphenyl]-1-

naphthalenecarboxylic acid produced only 4% apoptosis, com-

pared to 46% apoptosis of MDA-MB-231 cells induced by 1.0

µM 1. Replacement of the 2-carboxylate group with phenolic

hydroxyl, carboxamide, and N-ethyl sulfonamide groups was

similarly unsuccessful. Only 15% inhibition of primary human

microvascular endothelial (HMVE) cell growth resulted on

treatment with 1.0 µM 6-[3′-(1-adamantyl)-4′-hydroxyphenyl]-

2-naphthol (12). The carboxamide derivative of 5-Me-AHPN

(3) at 1.0 µM or 5.0 µM was essentially inactive at inducing

retinoid-resistant KG-1 AML cell apoptosis at 48 h (3% and

5%, respectively, compared to 2% apoptosis in the vehicle-

treated control) although 3 at 1.0 µM and 5.0 µM induced 51%

and 73% apoptosis, respectively. After 48 h, KG-1 AML cell

growth inhibition by the carboxamide derivative of 3 at 5.0 µM

According to molecular dynamics calculations on 2, the

increased dihedral angle for the 1′,4-diaryl bond caused the

adamantyl group to interfere with the local dynamics of RARγ

helix H12 to prevent the formation of the AF-2 site with helices

H3 and H4 to which coactivator proteins bind to recruit the

transcriptional complex necessary for retinoid-induced gene

transcription.

We recently undertook quantitative structure-activity rela-

tionship (SAR) studies to identify the core recognition elements

on 55 analogues of AHPN (1) and AHPC (4) that were necessary

to induce the apoptosis of MDA-MB-231 breast cancer cells

after treatment at 1.0 µM for 96 h. The ‘overlap rule’ was used

to align the training set in SYBYL QSAR, and the comparative

molecular similarity index analysis (CoMSIA) electrostatic,

hydrophobic, and steric fields were computed on a grid

surrounding the overlapped ligands. The resulting CoMSIA

analysis for apoptosis induction in MDA-MB-231 breast cancer

cells [ln(% apoptosis)] resulted in a predictive R²of 0.78 and

an un-cross-validated R²of 0.95 with a standard error of 0.45.

This seven-point descriptive model is illustrated in Figure 2A.

Key polar points include two adjacent hydrogen-acceptor groups

1 and 2 and a hydrogen-donor/acceptor group 3. The predictivity

of this initial model was sufficient to score apoptosis induced

by analogues similar in character to those in the training set.

For example, if 3-Cl-AHPC (5) and (E)-3-{5-[3′-(1-adamantyl)-

Effects of Isosteric Modifications of 3-Cl-AHPC Carboxylate

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2625

was 3% compared to 2% in the control and 96% in 5.0 µM

3-treated cells. Similar results were obtained after 72 h of

treatment. Evidently, KG-1 cells were unable to cleave a primary

amide to the active carboxylate. Apoptosis induced in MDA-

MB-231 cells after a 96-h treatment with the 2-(N-ethyl

sulfonamide) analogue of 1 at 2.0 µM was only 2%, although

growth inhibition was 30%.

and 31 and the thiazolidendione-terminated target 39, the

hydroxyl group of 17 was protected as the benzyl ether so that

benzaldehyde 18 could be elongated to cinnamonitrile 19 and

ethyl cinnamate 32 by olefination chemistry or be derivatized

to the hydroxylimine 25 for dehydration to produce benzonitrile

26. The E isomer predominated in the olefination products 19

1

and 32 according to their H NMR spectra, which indicated

about 5% of the Z isomer at most. The unwanted isomer was

readily removed by chromatography. Low-temperature (-78 °C)

cleavage of the benzyl ether protecting groups from 19, 26, and

32 using boron tribromide afforded phenols 20, 27, and 33.

These phenols and 17 were converted to their respective triflates

21, 28, 34, and 40 using triflic anhydride and pyridine in

dichloromethane. Diaryl coupling of the triflates with 16

introduced the substituted 1,1′-biphenyl scaffolds of intermedi-

ates 22, 29, 35, and 41, respectively.

Because shifting the 2-carboxyl group of AHPN (1) to the

3-position and replacing the 2-carboxyl group by a hydroxyl

group (11 and 12 in Table 2) were unpromising, other

modifications were investigated using 3-Cl-AHPC (5) as the

scaffold. The tetrazole and thiazolidinedione termini were

investigated using analogues 24, 31, 39, and 43. These termini

were reported to reduce retinoid activity by the Dawson³²and

Shudo^37,38groups, respectively. The ability of nitriles (CdO

bioisostere)³⁹and hydroxamates to function as hydrogen-bond

acceptors led to the design of 45 and 48, respectively. The

boronic acid and phosphonic acid monoethyl ester analogues

(57 and 63, respectively) were also investigated. In the latter

case, the monoester rather than the free acid was prepared to

improve membrane permeability.

After removal of the benzyl protecting groups from 22 and

29, the nitrile groups of their parent phenols 23 and 30 were

allowed to undergo cycloaddition with trimethylsilyl azide in

the presence of di(n-butyl)tin oxide⁴⁷to introduce the 5-sub-

stituted tetrazole termini of targets 24 and 31, respectively. The

ethyl cinnamate, intermediate 35,³⁵was converted by hydride

reduction followed by Swern oxidation⁴⁸to (E)-cinnamaldehyde

37, which was subjected to condensation/elimination with 2,4-

thiazolidinedione to afford the 5-cinnamylidene-2,4-thiazo-

lidinedinone 38. The substituted benzaldehyde, intermediate 41,

was similarly transformed to the 5-benzylidene-2,4-thiazo-

lidinedione 42. Thiazolidinedione formation produced both

double-bond isomers. Their ¹H NMR spectra indicated that the

Z-double bond isomers 38 and 42 predominated as demonstrated

by the downfield positions (0.4 ppm) of their vinylic protons

trans to the sulfur atom (H-CdC-S) compared to those that

were cis. Chromatographic purification and debenzylation of

38 and 42 afforded the 5-cinnamylidene- and 5-benzylidene-

2,4-thiazolidinedione targets 39 and 43, respectively. Knoev-

enagel condensation of benzaldehyde 41 with malononitrile⁴⁹

and debenzylation produced the 2-benzylidenepropanedinitrile

45. However, if 17 were first converted to the 2-(3′-chloro-4′-

hydroxybenzylidene)propanedinitrile and then treated with triflic

anhydride, the triflate ester was not obtained.

On the basis of our finding that commonly used carboxylate

replacements (OH and CONH₂) resulted in loss of apoptotic

activity, we hypothesized that the carboxylate group of 3-Cl-

AHPC (5) was crucial for bioactivity. Retinoid carboxylates are

known to form strong salt bridges with the side-chain guani-

dinium groups of arginines in the ligand-binding pockets of

RARs and RXRs. For example, the crystallographic structure

of trans-RA (7) bound to the RARγ ligand-binding domain

(PDB 2LBD) reveals a strong salt bridge between the carboxy-

late of 7 and the guanidinium group of Arg-278 located at the

C-terminus of helix 5.^40,41We further hypothesized that a

putative arginine in the receptor protein for 5⁴²could interact

with the carboxylate of 5. If this were correct, an analogue

bearing a terminal 2-oxoaldehyde could undergo the Maillard

reaction with the arginine guanadinium group or at least

hydrogen-bond with this group. Methyl glyoxal and R-diones

undergo the Maillard reaction with the arginine guanidinium

group to form pyrimidines.^43-452-Oxoaldehyde 60 was designed

to test this hypothesis. The structures of these isosteric car-

boxylate analogues are shown in Table 3.

Syntheses of hydroxamic acid 48, boronic acid 57, 2-oxoal-

dehyde 60, and monoethyl phosphonate 63 are outlined in

Scheme 2. Cinnamyl ester 35 from Scheme 1 was hydrolyzed

to cinnamic acid 46,³⁵which was converted to the activated

ester by treatment with diisopropylcarbodiimide and 4-di-

methylaminopyridine. Reaction of this ester with the tetrahy-

dropyranyl ether of hydroxylamine⁵⁰produced the protected

hydroxamic acid 47. Concomitant low-temperature cleavage of

the benzyl and tetrahydropyranyl protecting groups with boron

tribromide afforded the hydroxamic acid target 48. Reaction of

the activated ester of 3-Cl-AHPC (5) with hydroxylamine failed

to produce 48 in the presence of the unprotected phenolic group.

Synthesis. Routes to the 3-Cl-AHPC (5) analogues are shown

in Schemes 1 and 2. Like 5, these analogues are characterized

by a tetrasubstituted 1,1′-biphenyl core that was introduced by

a Suzuki-Miyaura diaryl coupling reaction⁴⁶between the

arylboronic acid 16 and a 4-substituted 2-chlorophenyl triflate

(21, 28, 34, 40, or 50) using tetrakis(triphenylphosphine)-

palladium as the catalyst generally in the presence of LiCl and

an aqueous base in refluxing dimethoxyethane (Schemes 1 and

2). Coupling yields ranged from 51% to 91%. Intermediate 16⁶

was prepared in three steps (74% overall yield), namely (i)

Friedel-Crafts monoalkylation of 4-bromophenol (15) with

1-adamantanol catalyzed by methanesulfonic acid or concd

sulfuric acid, which gave comparable yields of 2-(1-adamantyl)-

4-bromophenol and no detectable diadamantylation product; (ii)

protection of the phenolic hydroxyl group of the adamantylation

product as the benzyl ether; and (iii) low-temperature (-78 °C)

lithium-halogen exchange of resultant 3-(1-adamantyl)-4-

benzyloxybromobenzene using n-butyllithium, conversion of the

aryllithium to the arylboronate ester by treatment with tri-

(isopropyl) borate, and acid hydrolysis during workup.

Construction of the 2-arylethenylboronic acid 57 was ac-

complished in eight steps from 2-chloro-4-nitrophenol (49).

Because aryl bromides and triflates typically undergo coupling

with boronic acids with equal efficiency and the ortho chloro

group in 49 was expected to hinder coupling by the aryl triflate,

the bromo group was masked as a nitro group in triflate 50 to

accomplish coupling with 16. The nitro group in the coupled

product, 51, was then transformed to the bromo group by

reduction to the amine and diazotization under hydrophobic

conditions using tert-butyl nitrite in the presence of cupric

bromide.⁵¹Aryl bromide 53 underwent Sonagashira coupling

with trimethylsilylacetylene⁵²to give the protected phenylacety-

4-Hydroxyl-3-chlorobenzaldehyde (17) was used to generate

four of the triflate intermediates (21, 28, 34, and 40) as shown

in Scheme 1. In the case of the tetrazole-terminated targets 24

2626 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11

Dawson et al.

Table 3. Effects of 3-Cl-AHPC (5) and Analogues on Leukemia Cell Apoptosis and Leukemia and Cancer Cell Proliferation

^aApoptosis after 48 h of treatment. ^bInhibition of proliferation relative to vehicle-alone control after 48 h of treatment for KG-1 leukemia cells. ^cInhibition

of proliferation relative to vehicle-alone control after 72 h for solid tumor cell lines, with one media ( compound change after 48 h. At 5.0 µM, inhibition

of KG-1 AML growth was as follows: 5, 67%; 24, 31%; 31, 21%; 39, 2%; 43, 41%; 45, 0%; 48, 0%; 57, 0%; 60, 38%; 63, 0%. ^dExtrapolated value;

highest concentration evaluated was 5.0 µM. ^eLow activity at highest concentration evaluated (5.0 µM or 10 µM) did not permit extrapolation. ^fExtrapolated

value; highest concentration evaluated was 10 µM.

lene 54. Desilylation of 54 with tetra(n-butyl)ammonium

fluoride⁵²provided phenylacetylene 55. Benzodioxaborole 56

was prepared by the cis-hydroboration of 55 with catecholbo-

rane.⁵³Treatment of 56 with boron tribromide (-78 °C) cleaved

both benzodioxa and benzyl protecting groups to produce the

(E)-2-phenylethenyl boronic acid 57.

To prepare the 2-oxoketoaldehyde 60, we introduced the (E)-

2-oxopropylidene group of 58 by olefination of benzaldehyde

41⁶with the ylid derived from (2-oxopropyl)triphenylphospho-

nium bromide.⁵⁴The aldehyde group of 59 was obtained by

selenous acid oxidation⁵⁵of the methyl group of 58. Debenzy-

lation of 59 afforded 60. Horner-Emmons-Wadsworth olefi-

nation of 41 using the anion of tetraethyl methylenediphospho-

nate⁵⁶produced diethyl phosphonate 61, which on acid

hydrolysis⁵⁷and debenzylation with boron tribromide produced

the monoethyl phosphonate 63.

inhibition of cell growth was comparable (44% and 67%,

respectively). After 96 h of treatment with 1.0 µM 5, apoptosis

rose to 66% compared to 7% in the Me₂SO-alone control.

Evidently, the region of the target protein with which the

carboxylate group of 5 interacts to mediate its apoptotic effects

is sufficiently constrained to prevent the other hydrogen-acceptor

groups in the analogues shown in Table 3 from efficient

interaction. However, similar constraints did not appear to

impact inhibition of proliferation as potently. Thus, tetrazoles

24 and 31 and thiazolidinedione 43 were modest inhibitors of

KG-1 cell proliferation as measured by cell counting but induced

minimal apoptosis (2%-10% compared to 2%-3% in the

control). On the basis of its efficient overlap with 5 and structural

similarity, we had predicted that tetrazole 24 would have

significant antiproliferative and apoptotic activities and that the

longer thiazolidine 39 would lack such activities. However, both

proved to be inactive in the apoptosis induction assays, and 39

lacked antiproliferative activity. 2-Oxoaldehyde 60 was also

unable to induce apoptosis but did show weak growth inhibition

Biological Activity. Of the compounds shown in Table 3,

only 3-Cl-AHPC (5) efficiently induced KG-1 cell apoptosis

after a 48-h treatment at 1.0 µM (30%) or 5.0 µM (55%). Its

Effects of Isosteric Modifications of 3-Cl-AHPC Carboxylate

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2627

Scheme 1^a

^aReagents and conditions: (a) 1-AdOH, concd H₂SO₄, CH₂Cl₂. (b) PhCH₂Br, K₂CO₃, acetone, reflux. (c) n-BuLi, -78 °C; B(Oi-Pr)₃, -78 °C to room

temperature; dil. HCl. (d) [(EtO)₂P(O)CH₂CN, KN(TMS)₂, THF, -78 °C], -78 °C to room temperature. (e) BBr₃, CH₂Cl₂, -78 °C; H₃O⁺. (f) Tf₂O,

pyridine, CH₂Cl₂, 0 °C to room temperature. (g) 16, Pd(PPh₃)₄, 2 M Na₂CO₃, LiCl, DME, reflux. (h) Me₃SiN₃, (n-Bu)₂SnO, PhMe, 90° to 95 °C. (i)

NH₂OH·HCl, MeOH, pyridine, 50 °C. (j) MeSO₂Cl, PhMe, pyridine. (k) [(EtO)₂P(O)CH₂CO₂Et, KN(TMS)₂, THF, -78 °C], -78 °C to room temperature.

(l) DIBAL, CH₂Cl₂, -78 °C; dil. HCl. (m) (COCl)₂, Me₂SO, NEt₃. (n) 2,4-Thiazolidinedione, HOAc, NHEt₂, PhMe, 50° to 60 °C. (o) CH₂(CN)₂, DMF,

reflux.

(38% at 5.0 µM). Recently, we demonstrated that 3-A-AHPC

(6) antagonized 5-induced apoptosis but retained antiproliferative

activity.⁶

(IC₅₀) 3.6 and 3.7 µM, respectively). Hydroxamic acid 48

displayed similar activity against prostate cancer cells (IC₅₀

)

3.5 µM) and had an IC₅₀of about 6.3 µM (extrapolated value)

against breast cancer cells. The other analogues were less active.

Most of the AHPN analogues in the original training set for

apoptosis induction had a carboxyl group at the 2-position of

the naphthalene ring. Modifying this group had a deleterious

impact on apoptosis induction but not on inhibition of prolifera-

tion (Table 3). We next addressed the impact on KG-1 cell

growth of variations in the position and character of the polar

hydrogen-bond acceptor region (descriptive points 1 and 2 in

Figure 2A) in an attempt to understand the biological data in

terms of fundamental changes in the polar region. Thus, a

3-Cl-AHPC (5) and its analogues were also assessed for their

abilities to inhibit the proliferation of retinoid-resistant MDA-

MB-231 breast, H292 lung, and DU-145 prostate cancer cells

grown in the presence of 10% fetal bovine serum. Dose-

response curves are shown in Figure 3. On the basis of their

IC₅₀values (Table 3), H292 and DU-145 cells were generally

more sensitive to inhibition under these growth conditions than

MDA-MB-231 cells (Figure 3). The most potent inhibitor in

the series was 5. Thiazolidinedione 43 displayed modest growth

inhibitory activity against the lung and prostate cancer cell lines

2628 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11

Dawson et al.

Scheme 2^a

^aReagents and conditions: (a) LiOH·H₂O, THF, H₂O; dil. HCl. (b) H₂NOTHP, DIC, DMAP, CHCl₃, 0 °C to room temperature. (c) BBr₃, CH₂Cl₂, -78

°C; H₃O⁺. (d) Tf₂O, pyridine, CH₂Cl₂, 0 °C to room temperature. (e) 16, Pd(PPh₃)₄, aq K₃PO₄, DME, reflux. (f) SnCl_2•2 H₂O, EtOH, reflux. (g) CuBr₂,

t-BuNO₂, MeCN, 0 °C to room temperature. (h) Pd(PPh)₃, CuI, Et₃N, Me₃Si-acetylene, reflux. (i) (n-Bu)₄NF, THF. (j) Catecholborane, THF, reflux. (k)

MeCOCH₂P(Ph)₃Br, 1,5,7-triazabicyclo[4.4.0]dec-5-ene, THF, room temperature to 70 °C. (l) H₂SeO₃, dioxane/H₂O (10:1). (m) Tetraethyl methylene-

diphosphonate, 50% aq NaOH, CH₂Cl₂. (n) 20% aq HCl, reflux.

fragment QSAR model was constructed using the isosteric polar/

hydrogen-bond acceptor replacements shown in Table 3 for the

carboxyl group of 3-Cl-AHPC (5). While the number of

analogues included in this portion of the SAR study was limited,

a predictive fragment QSAR, ln(IC₅₀KG-1) ) -2.65 +

0.121POLAR_V + 0.062 pK_a(raw), having an R²of 0.76 and

a standard error of 0.78, using two independent variables could

be developed and related to the ln(IC₅₀) values.⁵⁸Polar volume

(POLAR_V) and calculated (raw) pK_awere the most important

features influencing KG-1 growth inhibition. For the solid tumor

cell lines, modestly predictive QSAR models (R²) 0.5-0.8)

were developed using the three properties of the longest length

of the fragment (L), minimum in computed electrostatic potential

on the van der Waals surface of the fragments (MINIM_PS)

and polar volume. In Figure 4 is illustrated the quality of the

three-parameter QSAR model for MDA-MB-231 breast cancer

cell growth inhibition IC₅₀values in terms of these properties.

The resultant analysis had an R²of 0.75 and a standard error of

0.6. The QSAR equation ln(IC₅₀MBA-MB-231) ) 4.14 -

0.30L + 14.9MINIM_PS + 0.10POLAR_V with the ‘weights’

of the elements revealing their ‘relative contributions’. Analo-

gously, analysis of the results on the other cell lines yielded

similar equations: ln(IC₅₀H292) ) 3.33 + 0.28L + 11.78MIN-

IM_PS + 0.090POLAR_V with an R²of 0.44 and a standard

error of 1.1 and ln(IC₅₀DU-145) ) 3.87 - 0.073L +

19.3MINIM_PS + 0.10POLAR_V with an R²of 0.54 and a

standard error of 1.0. While the predictive R²values are only

modest, the analyses do indicate that both the amount of polar

atom (group) exposure and size are correlated with antiprolif-

erative activity, whereas the other computed properties are not

explanatory variables. Therefore, the analyses provide a pre-

liminary indication of molecular design features coupled to

growth inhibitory activity variations.

The NR4A1 nuclear receptor protein was found to interact

in the cytoplasm with the mitochondrial protein Bcl-2 to induce

cancer cell apoptosis.⁵⁹The translocation of NR4A1 from

nucleus to cytoplasm was observed to occur in several cancer

cell lines after their transfer to media lacking serum and

treatment with an analogue of AHPN (1). Once in the cytoplasm,

NR4A1 was able to induce a proapoptotic conformational

change in antiapoptotic Bcl-2 that led to mitochondrial cyto-

chrome c release followed by apoptosis. Therefore, we attempted

to correlate the sensitivity of MDA-MB-231 breast, H292 lung,

and DU-145 prostate cancer cells to apoptosis induction by 3-Cl-

Effects of Isosteric Modifications of 3-Cl-AHPC Carboxylate

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2629

Figure 4. Predictive fragment QSAR for isosteric replacements of

hydrogen-bond acceptor region (components 1 and 2) in Figure 2

performed on 3-Cl-AHPC carboxylate and other polar termini shown

in Table 3 and based on IC₅₀values for inhibition of MDA-MB-231

breast cancer proliferation after 96 h of treatment. The natural log of

the antiproliferative activity in terms of IC₅₀values would be expressed

as 4.14 - 0.30L + 14.9MINIM_PS + 0.10POLAR_V, where L is the

longest length of the polar fragment; MINIM_PS is the minimum in

the electrostatic potential on the van der Waals surface; and POLAR_V

is volume of polar group, R²( SE ) 0.75 ( 0.6.

pothesized that the effects of 1 as well as those of 5-Cl-AHPN

(2) and 3-Cl-AHPC (5) could be due to inhibition of an enzyme.

Pfahl and Piedrafita subsequently reported that the IC₅₀value

obtained for 1 in inhibiting the dual-specificity mitogen-activated

protein kinase phosphatase (MKP)-1 in vitro was in the 6-µM

range.⁶¹Because of their report, we investigated the inhibitory

activity of several analogues of 5 on the protein-tyrosine

phosphatases (PTPs) SHP-2 and CD45, both of which are

implicated in the development of some forms of leukemia.

Somatic gain-of-function mutations were found to occur in the

PTPN11 gene for the cytoplasmic Src-homology 2 domain-

containing PTP (SHP-2) in juvenile myelomonocytic leukemia

and lead to hyperactivation of oncogenic Ras.^62,63Phosphory-

lated (activated) SHP-2 was reported to be overexpressed in 23

of 25 peripheral blood or bone marrow samples from adult

chronic myeloid myelocytic leukemia patients but was only

poorly or not expressed in samples from normal adults.⁶⁴In

addition, SHP-2 was observed to coimmunoprecipitate with

phosphatidylinositol-3 kinase (PI3-K) in BCR-ABL tyrosine

kinase-transformed cells.⁶⁵In earlier work, we had observed

that 5 was able to inhibit the PI3-K/Akt pathway in cancer

cells.⁶⁶The KG-1 leukemia cell line is reported to express SHP-

2.⁶⁴These observations suggested that inhibition of SHP-2

activity could affect KG-1 cell function and prompted us to

investigate the effects of 5, 31, and 43 on KG-1 cells. Both 31

and 43 were found to inhibit SHP-2 PTP activity (Figure 5A)

with IC₅₀values of 1.3 µM and 2.2 µM, respectively, and,

therefore, could serve as leads for the development of more

potent and selective inhibitors of this enzyme. The IC₅₀value

for 5 was found to be 2.1 µM. This small sample did not permit

us to correlate KG-1 growth inhibition with inhibition of enzyme

activity.

Figure 3. Effects of 3-Cl-AHPC (5) and analogues 24, 31, 39, 43, 45,

48, 57, 60, and 63 on proliferation of trans-retinoic acid-refractory

cancer cell lines after treatment for 72 h as described in the Experimental

Section. (A) MDA-MB-231 breast cancer; (B) H292 lung cancer; and

(C) DU-145 prostate cancer. Results shown are the averages of three

replicates. Standard errors were below 10%

AHPC (5) with their levels of NR4A1 (human TR3) protein

expression. In lysates obtained from these cancer cell lines that

had been both grown and treated with 1.0 µM 5 in media

containing 10% fetal bovine serum we were not able to detect

NR4A1 protein by Western blotting (data not shown). These

results suggest that serum constituents could influence NR4A1

expression.

CD45 PTP is found on the surface of cells of lymphohem-

atopoietic lineage, including leukemia cells,⁶⁷and the expression

of its isoforms is altered in acute myeloid leukemia.^67,68We

observed that AHPC (4), 31, and 43 at 10 µM inhibited CD45

activity by 84%, 46%, and 53%, respectively, when dimethyl-

formamide was used as the vehicle. 3-Cl-AHPC (5) and 60 were

not evaluated. However, because 45, 48, and 57 had similar

CD45 inhibitory activities (68%, 37%, and 54%, respectively)

under these same conditions but at 5 µM were not able to inhibit

KG-1 cell proliferation, the probability that 4, 31, and 43 inhibit

KG-1 proliferation by inhibiting CD45 activity appears to be

very low. As shown in the dose-response curve of Figure 5B,

After Zhang et al.⁶⁰found evidence to suggest that AHPN

(1) modulated enzyme activity on the basis of (i) its rapid

induction of cell-cycle arrest and apoptosis; (ii) its lack of a

requirement for gene transcription or protein synthesis as

evidenced by resistance to actinomycin D or cycloheximide

treatment, respectively, and (iii) its ability to inhibit the

phosphohatidylinositol-3-kinase (PI3-K)/Akt pathway, we hy-

2630 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11

Dawson et al.

Figure 6. 3-Cl-AHPC (5) inhibits HMVE cell proliferation, whereas

43 is a poor inhibitor and 24, 31, and 39 do not inhibit. Cells were

treated for 72 h at the indicated compound concentrations or with

vehicle alone (Me₂SO), detached by trypsinization and counted as

described in the Experimental Section. A representative experiment is

shown with proliferation expressed as the percent of vehicle alone-

treated control.

Figure 5. 3-Cl-AHPC analogues 31 and 43 inhibit the activity of

SHP-2 and CD45 protein tyrosine phosphatases in cleaving 6,8-difluoro-

4-methylumbelliferyl phosphate (100 µM) as measured by fluorescence

spectrometry of the cleavage product as described in the Experimental

Section. SHP-2 and CD45 concentrations were 5 nM and 1 nM,

respectively. Tetrazole 31, solid diamond; thiazolidinedione 43, solid

square. Results shown are the average of duplicates ( SE.

Figure 7. 3-Cl-AHPC (5) induces apoptosis of HMVE cells. Cells

were incubated for 20 h in the presence of Me₂SO (negative control),

positive control doxorubicin (1.0 µM), or 5 (10 nM to 500 nM) and

assayed for apoptosis using a cell death detection ELISA as described

in the the Experimental Section. Values represent the enrichment of

mono- and oligonucleosome levels in treated sample relative to Me₂-

SO control, and each bar represents the mean ( SD. Statistically

significant differences (Student’s t-test) compared with Me₂SO control

are indicated (*, p < 0.05; **, p < 0.001).

IC₅₀values for CD45 PTP activity inhibition by 31 and 43 were

1.2 µM and 2.3 µM, respectively, when dimethyl sulfoxide was

used as the vehicle.

Cancer cells stimulate angiogenesis to promote tumor growth

and metastasis. In the presence of growth factors released by

tumors and their related stroma, human microvascular endo-

thelial (HMVE) cells from the surrounding vasculature are

induced to proliferate, migrate into the tumor, and assemble

into microtubular vessels. Because cancer cells underwent both

cell-cycle arrest and apoptosis in response to 3-Cl-AHPC (5)

and 5-Cl-AHPN (2), we wondered whether proliferating primary

HMVE cells would behave similarly in culture. As shown in

Figure 6, 5 effectively reduced HMVE cell growth (IC₅₀) 0.3

µM). Thiazolidinedione 43 had low inhibitory activity (30%

inhibition at 1.0 µM), whereas 24, 31, and 39 had no significant

effect on HMVE cell growth compared to that of the vehicle-

treated control.

We next examined whether 3-Cl-AHPC (5) would induce the

apoptosis of proliferating HMVE cells in culture. Although no

obvious cell detachment or rounding was observed after a 20-h

incubation with 5, oligonucleosome levels were significantly

enhanced after treatment with 0.25 or 0.50 µM 5 in a

concentration-dependent manner, as shown in Figure 7. These

results demonstrate that 5 is able to induce HMVE cell

apoptosis. Apoptosis induced by 0.5 µM 5 was 64% of that

induced by 1.0 µM adriamycin. These results suggest that 5

has potential in vivo antiangiogenic activity.

3-Cl-AHPC (5) was further examined for its effects on HMVE

cell migration and tubule formation. As shown in Figure 8, 0.05

nM to 0.50 µM 5 had no statistically significant effect on cell

migration through Matrigel when cells were treated for 5-h

compared to cells treated with only vehicle. Moreover, 18-h

treatment with 0.25 µM or 1.0 µM 5 had no statistically

significant effects on either the level of tube formation or tube

length in Matrigel by cells obtained from 80% confluent cultures

(Figure 9). In contrast, trans-RA (7) and other classical retinoids

are reported to inhibit angiogenesis in chick chorioallantoic

membrane.⁶⁹To establish that these compounds were not

exerting their effects through any retinoid-mediated pathway,

the antiproliferative activity of AHPN (1) was compared to those

of trans-RA (7), RARγ-selective transcriptional agonist SR11254

(13),^70,71and RARγ-selective antagonist SR11253 (14)^70,71in

HMVE cells. The results shown in Table 2 demonstrate that 1

was the most potent of the four compounds, although it was

Effects of Isosteric Modifications of 3-Cl-AHPC Carboxylate

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2631

competitive displacement efficiencies of the tritiated label were

as follows: nonlabeled 1, 53%; 5, 87%; 3-A-AHPC (6), 72%;

tetrazole 39, 69%; thiazolidinedione 43, 37%; hydroxamic acid

48, 7%; and boronic acid 57, 0% at 50 µM.

Conclusion

Thus, 3-Cl-AHPC (5) appears to negatively impact cancer

cell growth through two signaling pathways, namely inhibiting

cell proliferation and inducing apoptosis. Our results suggest

that 5 also has antiangiogenic activity by modulating the same

pathways in tumor microvasculature. The present fragment-

based QSAR study on the hydrogen bond-accepting elements

1 and 2 of the pharmacophoric model shown in Figure 2A

indicates that the dimensions of the elements and their minima

in computed electrostatic potential on the van der Waals surface

of the fragments have a major role in determining growth

inhibitory activity. Defining the apoptotic activity properties of

these elements was not possible because the present isosteric

modifications of the carboxylate group abrogated apoptosis

induction. These results suggest that if interaction with SHP

regulates apoptotic activity, the region of SHP to which the

carboxylate group binds is sterically constrained. Of the three

hydrogen-acceptor analogues (5, 31, and 43) evaluated as

inhibitors of phosphatases, only 5 was a potent inhibitor of both

apoptosis and cell growth. Analogues 31 and 43 were only

modest inhibitors of cell proliferation. Thus, while the present

results suggest that inhibition of phosphatase activity may have

a role in inhibition of cell proliferation, further studies will be

necessary before robust conclusions can be drawn.

Figure 8. 3-Cl-AHPC (5) does not inhibit HMVE cell migration.

Migration through Matrigel was measured after 5-h incubation with

the indicated concentrations of 5 or with vehicle alone as described in

the Experimental Section. Results shown represent the mean number

of cells ( SD of three fields in three replicates examined microscopi-

cally.

As observed with 3-A-AHPC (6), tetrazole 39 was able to

compete with radiolabeled AHPN for binding to recombinantly

expressed small heterodimer partner protein¹⁴but was unable

to induce KG-1 AML cell apoptosis. Earlier, we found that 6

was able to attenuate apoptosis induced by 3-Cl-AHPC (5).⁶

These results suggest that both 6 and 39 can function as

antagonists of the apoptotic activity of 5. Thus, with identified

potent agonists (1 and 5) and antagonists (6 and 39) of SHP-

Sin3A-mediated apoptotic activity, we plan on pursuing our

studies on how these compounds regulate transcriptional signal-

ing through SHP. Because both 31 and 43 exhibited similar

modest antiproliferative activities but had very low apoptosis-

inducing activity, they may have application as probes of the

pathway by which 5 induces cell-cycle arrest independent of

apoptosis.

Figure 9. 3-Cl-AHPC (5) does not inhibit HMVE cell tubule formation

or affect tube length. Cells in medium were layered onto Matrigel

containing 0.25 µM or 1.0 µM 5, which was added as a Me₂SO solution,

or Me₂SO alone. Tube formation was examined microscopically as

described in the Experimental Section. (A) Results shown are repre-

sentative of one field of four in triplicate experiments. (B) Total tube

length was determined in four random fields for each triplicate

experiment. Bars represent the means ( SD.

Experimental Section

Chemistry. Chemicals and solvents from commercial sources

were used without further purification unless specified. Abbrevia-

tions for solvents and reagents are as follows: DIBAL, diisobu-

tylaluminum hydride; DIC, diisopropylcarbodiimide; DMAP, 4-(N,N-

dimethylamino)pyridine; DMF, dimethylformamide; DME, ethylene

glycol dimethyl ether; TBAF, tetra(n-butyl)ammonium fluoride;

THF, tetrahydrofuran; Tf₂O, trifluoromethanesulfonic anhydride.

Reactions were carried out under argon and monitored by thin-

layer chromatography on silica gel (mesh size 60, F₂₅₄) with

visualization under UV light. Organic extracts were dried over Na₂-

SO₄unless otherwise specified and concentrated at reduced pressure.

Standard and flash column chromatography employed silica gel

(Merck 60, 230-400 mesh). Experimental procedures were not

optimized. Melting points for samples were determined in capillaries

using a Mel-Temp II apparatus and are uncorrected. Infrared spectra

were obtained using an FT-IR Mason satellite spectrophotometer

less efficient as a transactivator of the RARs than 7.¹5-Cl-

AHPN (2), AHPC (4), and 5 were also able to inhibit the

proliferation of HMVE cells. In contrast, the 3-naphthalenecar-

boxylic acid and 2-naphthol analogues (11 and 12) of 1 were

poor inhibitors of HMVE cell proliferation, just as they were

of cancer cell growth.

Recently, we discovered that 3-Cl-AHPC (5) binds to the

small heterodimer partner (SHP) nuclear orphan receptor.¹⁴

Interestingly, prior to its inducing the intrinsic apoptosis cascade

in KG-1 AML and MDA-MB-231 breast cancer cells, 5 was

found to interact with SHP in the nuclear Sin3A repressor

complex. A recombinantly expressed glutathione S-transferase-

SHP chimeric protein (GST-SHP) also bound 5, as evidenced

by its ability to displace tritiated AHPN⁴²from GST-SHP

bound to glutathione-linked Sepharose beads, as did AHPN (1)

and the 3-chlorophenyltetrazole and 3-chlorobenzylidenethi-

azolidinedione analogues (39 and 43, respectively). The relative

1

on powdered samples. H NMR spectra were recorded on a 300-

MHz Varian Unity Inova spectrometer, and shift values are

expressed in ppm (δ) relative to CHCl₃as an internal standard.

Unless mentioned otherwise, compounds were dissolved in ²HCCl₃.

MALDI-FAB mass spectra were run on an Applied Biosystems

2632 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11

Dawson et al.

Voyager De-Pro MALDI-TOF instrument at the Burnham Institute.

High-resolution mass spectra were recorded on an Agilent ESI-

TOF mass spectrometer at The Scripps Research Institute (La Jolla,

CA). Electrospray mass spectrometry was performed on an ABI

EPI-3000 instrument.

(s, 6H, AdCH₂), 5.00 (s, 1H, OH), 5.90 (d, J ) 17.1 Hz, 1H, HCd

CHCN), 6.72 (d, J ) 7.8 Hz, 1H, 5′-ArH), 7.17 (dd, J ) 2.1, 8.1

Hz, 1H, 6′-ArH), 7.32 (d, J ) 9.3 Hz, 1H, 6-ArH), 7.33 (d, J )

18.0 Hz, 1H, CHdCHCN), 7.38 (s, 1H, 2′-Ar), 7.39 (d, J ) 7.8

Hz, 1H, 6-ArH), 7.55 ppm (d, J ) 1.8 Hz, 1H, 3-ArH).

3-Chloro-4-hydroxybenzonitrile (27). 26 (850 mg, 3.5 mmol)

yielded after chromatography (20% EtOAc/hexane) (470 mg, 88%)

of phenol 27 as a pale-yellow solid, which was converted directly

to triflate 28.

3-(1-Adamantyl)-4-benzyloxyphenylboronic Acid (16).³⁵Our

reported procedure⁸was adapted. To a solution of 3-(1-adamantyl)-

4-benzyloxyphenyl bromide⁸(2.00 g, 5.04 mmol) in THF (7 mL)

at -78 °C (dry ice/acetone bath) under argon was added in one

portion 1.6 M n-BuLi (7.56 mmol) in hexane (4.7 mL). The mixture

was stirred at -78 °C for 15 min, (i-PrO)₃B (3.5 mL, 15.1 mmol)

was added, and the resulting solution was stirred at -78 °C for 20

min and then at room temperature overnight. The mixture was

quenched with 0.1 N HCl (30 mL) and extracted with EtOAc (3 ×

190 mL). The extracts were washed (brine) and dried. The residue

after concentration was purified on silica gel (40% EtOAc/hexane)

4-[3′-(1-Adamantyl)-4′-hydroxyphenyl]-3-chlorobenzonitrile (30).

29 (300 mg, 0.66 mmol) yielded 0.45 g (87%) of 30 as a pale-

yellow solid, R_f0.37 (10% EtOAc/hexane); mp 220-

1

222 °C. IR (CHCl₃) 3410, 2966, 2237, 1278 cm^-1; H NMR δ

1.80 (s, 6H, AdCH₂), 2.10 (s, 3H, AdCH), 2.15 (s, 6H, AdCH₂),

5.02 (s, 1H, OH), 6.74 (d, J ) 8.4 Hz, 1H, 5′-ArH), 7.17 (dd, J )

8.1, 2.1 Hz, 1H, 6′-ArH), 7.30 (d, J ) 2.4 Hz, 1H, 2′-ArH), 7.44

(d, J ) 7.8 Hz, 1H, 5-ArH), 7.57 (dd, J ) 7.8, 1.5, 1H, 6-ArH),

7.75 ppm (d, J ) 1.5 Hz, 1H, 2-ArH). FTMS (HRMS) calcd C₂₃H₂₃-

ClNO [M + H]⁺expected 364.1463, found 364.1461.

1

to give 1.63 g (89%) of 16 as a white solid, mp 151-153 °C. H

NMR δ 1.78 (bs, 6H, AdCH₂), 2.09 (bs, 3H, AdCH), 2.27 (bs, 6H,

AdCH₂), 5.23 (s, 2H, ArCH₂), 7.07 (d, J ) 8.1 Hz, 1H, 5-ArH),

7.33-7.55 (m, 5H, ArH), 8.04 (dd, J ) 8.1, 1.5 Hz, 1H, 6-ArH),

8.2 ppm (d, J ) 1.2 Hz, 1H, 2-ArH).

Ethyl (E)-3-Chloro-4-hydroxycinnamate (33).⁸32⁷²yielded after

chromatography (24% EtOAc/hexane) 0.855 g (92%) of 33 as a

white solid, mp 106-108 °C. ¹H NMR δ 1.34 (t, J ) 7.2 Hz, 3H,

CH₂CH₃), 4.26 (q, J ) 7.2 Hz, 2H, CH₂CH₃), 5.84 (s, 1H, OH),

6.32 (d, J ) 16.2 Hz, 1H, CHdCHCO), 7.04 (d, J ) 8.4 Hz, 1H,

5-ArH), 7.37 (dd, J ) 8.4, 2.1 Hz, 1H, 6-ArH), 7.52 (d, J ) 1.8

Hz, 1H, 2-ArH), 7.58 ppm (d, J ) 16.2 Hz, 1H, CHdCHCO). The

product was used directly in the next step.

4-Benzyloxy-3-chlorobenzaldehyde (18).⁷²To a suspension of 17

(4.68 g, 30 mmol) and K₂CO₃(6.90 g, 50 mmol) in THF (50 mL)

and DMF (10 mL) under argon was added benzyl bromide (6.84

g, 40 mmol). The mixture was heated at reflux for overnight,

concentrated, diluted with CH₂Cl₂(100 mL), washed (water, 1 N

HCl and brine), and dried. Concentration and chromatography (15%

EtOAc/hexane) afforded 6.22 g (84%) of 18 as a white solid; mp

92-94 °C. IR (CHCl₃) 2966, 1682 cm^-1; ¹H NMR δ 5.23 (s, 2H,

ArCH₂), 7.08 (d, J ) 8.4 Hz, 1H, 5-ArH), 7.35-7.46 (m, 5H, ArH),

7.74 (d, J ) 6.6 Hz, 1H, 6-ArH), 7.94 (d, J ) 1.5 Hz, 1H, 2-ArH),

9.85 ppm (s, 1H, CHO).

5-[3-(3′-(1-Adamantyl)-2-chloro-4′-hydroxy-4-biphenyl)-(2E)-2-

propenylidene]-2,4-thiazolidinedione (39). 38 (35 mg, 0.06 mmol)

yielded 25 mg (85%) of 39 as an orange powder, R_f0.27 (30%

EtOAc/hexane); mp 162-164 °C (dec). IR (CHCl₃) 3394, 2912,

1695, 1601, 1278 cm^-^{1 1}H NMR (C²H₃O²H) δ 1.78 (s, 6H,

;

(E)-4-Benzyloxy-3-chlorocinnamonitrile (19). To diethyl (cya-

nomethyl)phosphonate (885 mg, 5.0 mmol) in THF (5 mL) stirred

with cooling in a dry ice/acetone bath was added under argon 10

mL of 0.5 M KN(SiMe₃)₂(5.0 mmol) in toluene. Stirring was

continued for 0.5 h before 18⁷²(738 mg, 3.0 mmol) in THF (5.0

mL) was slowly added over a 0.5-h period. After being stirred for

1 h more, the mixture was allowed to warm to room temperature,

stirred overnight, poured into 1 M NH₄Cl (10 mL) and water (20

mL), and extracted with hexane (100 mL). The extract was washed

(water and brine) and dried. Concentration and chromatography

on silica gel (EtOAc/hexane) afforded 710 mg (91%) of 19 as a

white powder, R_f0.47 (EtOAc/hexane); mp 110-114 °C. IR

AdCH₂). 2.07 (s, 3H, AdCH), 2.17 (s, 6H, AdCH₂), 6.80 (d, J )

8.1 Hz, 1H, 5′-ArH), 6.91 (dd, J ) 15.9, 11.4 Hz, 1H, ArCHd

CHCHdC), 7.12 (dd, J ) 7.8, 2.1 Hz, 1H, 6′-ArH), 7.16 (d, J )

15 Hz, 1H, ArCHdCHCHdC), 7.26 (d, J ) 2.1 Hz, 1H, 2′-ArH),

7.38 (d, J ) 8.1 Hz, 1H, 5-ArH), 7.52 (d, J ) 11.4 Hz, 1H, ArCHd

CHCHdC), 7.58 (dd, J ) 8.1, 1.8 Hz, 1H, 6-ArH), 7.70 ppm (d,

J ) 1.8 Hz, 1H, 2-ArH). HRMS calcd C₂₈H₂₆ClNO₃S [M - H]^-

490.1249, found 490.1245.

5-{4-[3′-(1-Adamantyl)-4′-hydroxyphenyl]-3-chlorobenzylidene}-

2,4-thiazolidinedione (43). 42 (12 mg, 0.02 mmol) yielded after

chromatography (20% EtOAc/hexane) 8.3 mg (91%) of 43 as a

yellow powder, R_f0.36 (20% EtOAc/hexane); mp 280-284 °C.

IR (CHCl₃) 3372, 2902, 1655, 1577 cm^-1; ¹H NMR (C²H₃O²H) δ

1.85 (s, 6H, AdCH₂), 2.08 (s, 3H, AdCH), 2.22 (d, J ) 2.4 Hz,

6H, AdCH₂), 6.82 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.16 (dd, J ) 8.4,

1.8 Hz, 1H, 6′-ArH), 7.28 (d, J ) 2.4 Hz, 1H, 2′-ArH), 7.50 (d, J

) 8.1 Hz, 1H, 5-ArH), 7.55 (d, J ) 8.1 Hz, 1H, 6-ArH), 7.68 (s,

1H, 2-ArH), 7.80 ppm (s, 1H, ArCHdC). HRMS calcd C₂₆H₂₄-

ClNO₃S [M - H]^-464.1093, found 464.1107.

1

(CHCl₃) 2966, 2215 cm^-1; H NMR δ 5.21 (s, 2H, ArCH₂), 5.73

(d, J ) 15.9 Hz, 1H, HCdCCN), 6.97 (d, J ) 8.4 Hz, 1H, 5-ArH),

7.26 (d, J ) 16.5 Hz, 1H, CdCHCN), 7.36-7.45 (m, 6H, ArH,

6-ArH), 7.51 ppm (d, J ) 2.1 Hz, 1H, 2-ArH). The crude nitrile

was converted directly to 20.

General Procedure for Debenzylation of 19, 22, 26, 29, 32, 38,

42, 44, 47, 56, 59, and 61. To a stirred mixture of the benzyl ether

(2.5 mmol) in CH₂Cl₂(10 mL) at -78 °C under argon was added

slowly over a 10-min period 6.0 mL of 1.0 M BBr₃(6.0 mmol) in

CH₂Cl₂. The mixture was stirred for 2 h at -78 °C. Water (10

mL) was added, and the mixture was stirred for 10 min and then

extracted (EtOAc). The extract was washed (water and brine), dried,

and concentrated. Flash chromatography of the residue on silica

gel (10% EtOAc/hexane) yielded the phenol.

2-{[3′-(1-Adamantyl)-2-chloro-4′-hydroxy-4-biphenyl]methylene}-

propanedinitrile (45). 44 (74 mg, 0.15 mmol) yielded after

chromatography (20% EtOAc/hexane) 42.5 mg (70%) of 45 as a

yellow solid, mp 225-226 °C. IR 3450, 2901, 2848, 2230, 1276

1

cm^-1; H NMR δ 1.80 (bs, 6H, AdCH₂), 2.11 (bs, 3H, AdCH),

2.15 (bs, 6H, AdCH₂), 5.08 (s, 1H, OH), 6.76 (d, J ) 8.4 Hz, 1H,

5′-ArH), 7.24 (dd, J ) 7.8, 2.4 Hz, 1H, 6′-ArH), 7.36 (d, J ) 1.8

Hz, 1H, 5-ArH), 7.53 (d, J ) 7.8 Hz, 1H, 2 ′-ArH), 7.74 (s, 1H,

CHdC(CN)₂), 7.90-7.94 ppm (m, 2H, 3-ArH, 6-ArH). HRMS

calcd C₂₆H₂₃ClN₂O [M - H]^-413.1426, found 413.1418.

(E)-3-Chloro-4-hydroxycinnamonitrile (20). 19 (672 mg, 2.5

mmol) yielded 420 mg (94%) of 20 as a pale-yellow powder, R_f

0.12 (10% EtOAc/hexane); mp 165-169 °C. IR (powder) 3416,

1

2966, 2213 cm^-1; H NMR δ 6.32 (d, J ) 16.2 Hz, 1H, HCd

N-(Hydroxy) (E)-4-[3′-(1-Adamantyl)-4′-hydroxyphenyl]-3-chlo-

rocinnamamide (48). 47 (70 mg, 0.117 mmol) yielded after

chromatography (HOAc/EtOAc/hexane, 0.3:10:10) 22 mg (45%)

of 48 as a pale-tan solid, mp 208 °C (dec). IR 3334, 2899, 1652,

CHCN), 7.02 (dd, J ) 3.0, 8.7 Hz, 1H, 5-ArH), 7.48 (d, J ) 10.2

Hz, 1H, 6-ArH), 7.54 (d, J ) 16.2 Hz, 1H, HCdCHCN), 7.75 (s,

1H, 2-ArH), 10.88 ppm (s, 1H, OH). The crude product was

converted directly to the triflate 21.

1

1382 cm^-1; H NMR [(C²H₃)₂SO] δ 1.75 (bs, 6H, AdCH₂), 2.04

(E)-4-[3′-(1-Adamantyl)-4′-hydroxyphenyl]-3-chlorocinnamoni-

trile (23). 22 (420 mg, 87 mmol) yielded 310 mg (87%) of 23 as

a pale-yellow solid, R_f0.15 (5% EtOAc/hexane); mp 142-148 °C,

which was used to prepare 24. IR (CHCl₃) 3394, 2906, 2226, 1284

cm^-1; ¹H NMR δ 1.80 (s, 6H, AdCH₂), 2.10 (s, 3H, AdCH), 2.16

(bs, 3H, AdCH), 2.16 (bs, 6H, AdCH₂), 6.65 (d, J ) 15.9 Hz, 1H,

CHdCHCO), 6.88 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.12 (dd, J ) 8.1,

1.8 Hz, 1H, 6′-ArH), 7.16 (d, J ) 1.8 Hz, 1H, 2′-ArH), 7.41 (s,

1H, 3-ArH), 7.50 (d, J ) 15.9 Hz, 1H, CHdCHCO), 7.55 (d, J )

8.4 Hz, 1H, 5-ArH), 7.71 (d, J ) 8.4 Hz, 1H, 6-ArH), 9.63 (bs,

Effects of Isosteric Modifications of 3-Cl-AHPC Carboxylate

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2633

1H, OH), 9.69 (bs, 1H, N HOH), 10.81 ppm (bs, 1H, NHO H).

HRMS calcd C₂₅H₂₆ClNO₃[M + H]⁺424.1674, found 424.1669.

(E)-2-[3′-(1-Adamantyl)-2-chloro-4′-hydroxy-4-biphenyl] -

ethenylboronic Acid (57). 56 (56 mg, 0.097 mmol) yielded after

chromatography (50% EtOAc/hexane) 31 mg (67%) of 57 as a pale-

Hz, 1H, 6ArH), 8.11 (d, J ) 1.8 Hz, 1H, 2-ArH), 10.07 ppm (s,

1H, CHO). FTMS (HRMS) calcd C₈H₅ClF₃O₄S [M + H]⁺expected

288.9544, found 288.9542.

2-Chloro-4-nitrophenyl Trifluoromethanesulfonate (50). 49 (1.58

g, 9.12 mmol) gave after chromatography (12.5% EtOAc/hexane)

2.73 g (97%) of 50 as a yellow oil, which was used to prepare 51.

¹H NMR δ 7.59 (d, J ) 9 Hz, 1H, 6-ArH), 8.26 (dd, J ) 9, 2.7

Hz, 1H, 5-ArH), 8.45 ppm (d, J ) 2.7 Hz, 1H, 3-ArH).

General Procedure for the Coupling between Aryl Boronic Acid

16 and Aryl Triflates 21, 28, 34, 40, and 50. To a stirred

suspension of the aryl triflate (1.6 mmol), 16 (651 mg, 1.8 mmol),

Pd(PPh₃)₄(115 mg, 0.1 mmol), and LiCl (242 mg, 2.8 mmol) in

DME (8 mL) was added under argon 1.3 mL of 2.0 M aq Na₂CO₃

(2.6 mmol). The mixture was heated at reflux for 24 h, cooled,

and extracted (EtOAc). The extract was washed (water and brine),

dried, and concentrated. Flash chromatography on silica gel (5%

EtOAc/hexane) yielded the diaryl-coupling product.

1

tan solid, mp 186-189 °C. IR 3350, 2901, 1621, 1217 cm^-1; H

NMR δ 1.81 (bs, 6H, AdCH₂), 2.11 (bs, 3H, AdCH), 2.18 (m, 6H,

AdCH₂), 4.92 (s, 1H, OH), 6.38 (d, J ) 18 Hz, 1H, CHdCHB),

6.74 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.22 (dd, J ) 8.4, 2.1 Hz, 1H,

6′-ArH), 7.36 (d, J ) 2.1 Hz, 1H, 2′-ArH), 7.39 (d, J ) 8.1 Hz,

1H, 6-ArH), 7.55 (dd, J ) 8.1, 1.5 Hz, 1H, 5-ArH), 7.73 (d, J )

1.5 Hz, 1H, 3-ArH), 7.76 ppm (d, J ) 18 Hz, 1H, CHdCHB).

HRMS calcd C₂₄H₂₆BClO₃[M + H]⁺409.1736, found 409.1730.

(3E)-4-[3′-(1-Adamantyl)-2-chloro-4′-hydroxy-4-biphenyl]-2-ox-

obut-3-enal (60). 59 (35 mg, 0.068 mmol) yielded after chroma-

tography (25% to 33% EtOAc/hexane) 22 mg (77%) of 60 as a

yellow wax, mp 121-123 °C. IR 3383, 2902, 1695, 1678, 1603,

1251 cm^-1; ¹H NMR δ 1.80 (bs, 6H, AdCH₂), 2.11 (bs, 3H, AdCH),

2.16 (bs, 6H, AdCH₂), 5.10 (s, 1H, OH), 6.74 (d, J ) 8.1 Hz, 1H,

5′-ArH), 6.97 (d, J ) 16.2 Hz, 1H, CHdCHCO), 7.20 (dd, J )

8.4, 2.1 Hz, 1H, 6′-ArH), 7.34 (d, J ) 2.1 Hz, 1H, 2′-ArH), 7.41

(d, J ) 8.1 Hz, 1H, 6-ArH), 7.52 (dd, J ) 8.1, 2.1 Hz, 1H, 5-ArH),

7.71 (d, J ) 1.5 Hz, 1H, 3-ArH), 7.80 (d, J ) 16.2 Hz, 1H, CHd

CHCO), 9.47 ppm (s, 1H, CHO). HRMS calcd C₂₆H₂₅ClO₃[M +

H]⁺421.1565, found 421.1573.

(E)-3-[3′-(1-Adamantyl)-4′-benzyloxyphenyl]-3-chlorocinnamoni-

trile (22). 21 (498 mg, 1.6 mmol) yielded 510 mg (64%) of 22 as

a white solid, R_f0.55 (5% EtOAc/hexane); mp 72-74 °C. IR

1

(CHCl₃) 2906, 2221 cm^-1; H NMR δ 1.75 (s, 6H, AdCH₂), 2.06

(s, 3H, AdCH), 2.20 (s, 6H, AdCH₂), 5.18 (s, 2H, ArCH₂), 5.91

(d, J ) 16.8 Hz, 1H, CHdCHCN), 7.02 (d, J ) 8.7 Hz, 1H, 5′-

ArH), 7.28 (dd, J ) 8.4, 2.1 Hz, 1H, 6′-ArH), 7.45-7.35 (m, 7H,

ArH, 5-ArH, 2′-ArH), 7.48 (d, J ) 16.8 Hz, 1H, CHdCHCN),

7.53 (d, J ) 8.4 Hz, 1H, 6-ArH), 7.56 ppm (d, J ) 1.5 Hz, 1H,

3-ArH). The crude product was used to prepare 23.

Diethyl

(E)-2-[3′-(1-Adamantyl)-2-chloro-4′-hydroxy-4-bi-

phenyl]ethenylphosphonate (62). 61 (0.14 mmol, 82 mg) yielded

after chromatography (50% to 67% EtOAc/hexane) 61 mg (87%)

of 62 as an off-white solid, mp 110-112 °C. IR 3414, 2932, 1028,

4-[3′-(1-Adamantyl)-4′-benzyloxyphenyl]-3-chlorobenzonitrile (29).

28 (428 mg, 1.5 mmol) yielded 390 mg (57%) of 29 as a white

solid, R_f0.55 (5% EtOAc/hexane); mp 60-62 °C, which was used

1

989 cm^-1; H NMR δ 1.38 (t, J ) 6.9 Hz, 6H, OCH₂CH₃), 1.79

1

(bs, 6H, AdCH₂), 2.09 (bs, 3H, AdCH), 2.17 (bs, 6H, AdCH₂),

4.18 (m, 4H, OCH₂CH₃), 6.29 (t, J ) 17.4 Hz, 1H, CHdCHP),

6.82 (d, J ) 8.4 Hz, 1H, 5′-ArH), 7.17 (dd, J ) 8.4, 2.1 Hz, 1H,

6′-ArH), 7.31 (d, J ) 2.1 Hz, 1H, 2′-ArH), 7.33-7.41 (m, 3H,

5-ArH, 6-ArH, OH), 7.48 (dd, J ) 17.4, 22.5 Hz, 1H, CHdCHP),

7.59 ppm (s, 1H, 3-ArH). HRMS calcd C₂₈H₃₄ClO₄P [M + H]⁺

501.1956, found 501.1958.

to prepare 30. IR (CHCl₃) 2906, 2232 cm^-1; H NMR δ 1.74 (s,

6H, AdCH₂), 2 (s, 3H, AdCH), 2.18 (s, 6H, AdCH₂), 5.18 (s, 2H,

ArCH₂), 7.02 (d, J ) 8.4 Hz, 1H, 5′-ArH), 7.27 (dd, J ) 8.4, 2.1

Hz, 1H, 6′-ArH), 7.35-7.47 (m, 6H, 2′-ArH, ArH), 7.51 (d, J )

7.8 Hz, 1H, 5-ArH), 7.58 (dd, J ) 8.1, 1.5 Hz, 1H, 6-ArH), 7.76

ppm (d, J ) 1.5 Hz, 1H, 2-ArH).

Ethyl (E)-4-[3′-(1-Adamantyl)-4′-benzyloxyphenyl]-3-chlorocin-

namate (35).⁸34 (500 mg, 1.40 mmol) yielded 1.33 g (91%) of

35 as a white solid, which was reduced to 36. R_f0.51 (10% EtOAc/

General Procedure for Converting Phenols 20, 27, 33, 17, and

49 to Their Triflates. To a stirred solution of the phenol (2.0 mmol)

in pyridine (5 mL) at 0 °C (ice-bath) under argon was slowly added

Tf₂O (846 mg, 3.0 mmol) over a 0.5-h period. The reaction mixture

was stirred at room temperature overnight and then extracted

(EtOAc). The extract was washed (10% HCl, 5% NaHCO₃, brine

and water), dried, and concentrated to afford an oil, which was

purified by chromatography (5% EtOAc/hexane) to give the triflate.

(E)-3-(3-Chloro-4-trifluoromethanesulfonyloxy)cinnamonitrile

(21). 20 (358 mg, 2.0 mmol) gave 565 mg (91%) of 21 as a white

1

hexane); mp 72-74 °C. IR (CHCl₃) 2906, 1713, 1640 cm^-1; H

NMR δ 1.35 (t, J ) 7.2 Hz, 3H, CH₃), 1.72 (s, 6H, AdCH₂), 2.04

(s, 3H, AdCH), 2.17 (s, 6H, AdCH₂), 4.27 (q, J ) 6.9 Hz, 2H,

OCH₂), 5.17 (s, 2H, ArCH₂), 6.46 (d, J ) 15.2 Hz, 1H, HCd

CHCO), 7.00 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.29 (d, J ) 8.1 Hz,

1H, 6′-ArH), 7.35-7.42 (m, 5H, ArH), 7.43 (d J ) 6.9 Hz, 1H,

5-ArH), 7.50 (s, 1H, 2′-ArH), 7.52 (d, J ) 7.2 Hz, 1H, 6-ArH),

7.62 (s, 1H, 2-ArH), 7.64 ppm (d, J ) 15.3 Hz, 1H, CHdCHCO).

4-[3′-(1-Adamantyl)-4′-benzyloxyphenyl]-3-chlorobenzalde-

hyde (41). 40 (289 mg, 1.00 mmol) yielded after chromatography

(10% EtOAc/hexane) 215 mg (78%) of 41 as a white solid, mp

powder,

R_f

0.68

(10%

EtOAc/hexane);

mp

93-

99 °C. IR (CHCl₃) 2966, 2226 cm^-1; ¹H NMR δ 5.93 (d, J ) 16.5

Hz, 1H, HCdCHCN), 7.34 (d, J ) 16.8 Hz, 1H, HCdCHCN),

7.42 (s, 2H, 5,6-ArH), 7.62 ppm (s, 1H, 2-ArH). The crude product

was used to prepare 22.

1

115-116 °C. IR 2902, 1699, 1596, 1262 cm^-1; H NMR δ 1.75

(bs, 6H, AdCH₂), 2.06 (bs, 3H, AdCH), 2.19 (bs, 6H, AdCH₂),

5.19 (s, 2H, ArCH₂), 7.04 (d, J ) 8.4 Hz, 1H, 5′-ArH), 7.31-7.56

(m, 8H, 6-ArH, 2′-ArH, 6′-ArH, ArH), 7.81 (dd, J ) 7.8, 1.5 Hz,

1H, 5-ArH), 7.99 (d, J ) 1.5 Hz, 1H, 2-ArH), 10.01 ppm (s, 1H,

CHO). HRMS calcd C₃₀H₂₉ClO₂[M + Na]⁺479.1748, found

479.1763.

3-Chloro-4-(trifluoromethanesulfonyloxy)benzonitrile (28). Crude

27 (306 mg, 2.0 mmol) gave 530 mg (93%) of 28 as a white

powder, R_f0.80 (20% EtOAc/hexane); mp 55-56 °C, which was

used directly for coupling to produce 29. IR (CHCl₃) 2961, 2243

cm^-1; ¹H NMR δ 7.50 (d, J ) 8.7 Hz, 1H, 5-ArH), 7.69 (dd, J )

8.1, 1.8 Hz, 1H, 6-ArH), 7.86 ppm (s, 1H, 2-ArH).

Ethyl (E)-3-Chloro-4-(trifluoromethanesulfonyloxy)cinnamate

(34).⁸33 (800 mg, 3.54 mmol) gave 1.13 g (93%) of 34 as a white

solid, R_f0.62 (10% EtOAc/hexane); mp 78-80 °C. IR (CHCl₃)

2961, 1706, 1651, 1432 cm^-1; ¹H NMR δ 1.35 (t, J ) 6.9 Hz, 3H,

CH₃), 4.29 (q, J ) 7.2 Hz, 2H, OCH₂), 6.44 (d, J ) 15.9 Hz, 1H,

HCdCHCO), 7.39 (d, J ) 8.4 Hz, 1H, 5ArH), 7.48 (dd, J ) 8.4,

1.8 Hz, 1H, 6-ArH), 7.60 (d, J ) 16.2 Hz, 1H, CHdCHCO), 7.68

ppm (s, 1H, 2-ArH). HRMS calcd C₁₂H₁₀ClF₃O₅S [M + H]⁺

358.9962, found 358.9951.

3′-(1-Adamantyl)-4′-benzyloxy-2-chloro-4-nitrobiphenyl (51). 50

(721 mg, 2.36 mmol) yielded after chromatography (7% EtOAc/

hexane) 908 mg (81%) of 51 as a yellow solid, mp 52-54 °C,

1

which was reduced to 52. H NMR δ 1.77 (bs, 6H, AdCH₂), 2.09

(bs, 3H, AdCH), 2.21 (m, 6H, AdCH₂), 5.21 (s, 2H, ArCH₂), 7.06

(d, J ) 8.7 Hz, 1H, 5′-ArH), 7.32 (dd, J ) 8.7, 2.1 Hz, 1H, 6′-

ArH), 7.35-7.56 (m, 7H, 2′-ArH, 6-ArH, ArH), 8.16 (dd, J ) 8.7,

2.4 Hz, 1H, 5-ArH), 8.37 ppm (d, J ) 2.4 Hz, 1H, 3-ArH).

(E)-5-{2-[3′-(1-Adamantyl)-2-chloro-4′-hydroxy-4-biphenyl]ethe-

nyl}-1H-tetrazole (24). A reported procedure⁷³was applied. To

23 (78 mg, 0.2 mmol) in toluene (2 mL) were added (n-Bu)₂SnO

(5.0 mg, 0.02 mmol) and azidotrimethylsilane (58 mg, 1.0 mmol).

The mixture was stirred at 90-95 °C for 48 h. Additional

azidotrimethylsilane (34 mg, 0.3 mmol) was added, and stirring

3-Chloro-4-(trifluoromethanesulfonyloxy)benzaldehyde (40). 17

(1.56 g, 10.0 mmol) gave 2.30 g (79%) of 40 as a pale-yellow oil,

R_f0.65 (20% EtOAc/hexane). IR (CHCl₃) 2961, 1717, 1223 cm^-1

;

¹H NMR δ 7.61 (d, J ) 8.4 Hz, 1H, 5-ArH), 7.94 (dd, J ) 8.6, 1.8

2634 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11

Dawson et al.

was continued for 20 h. The mixture was concentrated, diluted with

EtOAc (10 mL) and 10% HCl (3 mL), and stirred for 0.5 h. The

organic layer was washed (water and brine) and dried. Concentration

and crystallization (EtOAc/CH₂Cl₂/hexane) gave 55 mg (64%) of

24 as a white powder, R_f0.28 (50% EtOAc/hexane); mp 260-

262 °C. IR (CHCl₃) 3405, 2966, 2237 cm^-1; ¹H NMR (C²H₃O²H)

δ 1.72 (s, 6H, AdCH₂), 2.01 (s, 3H, AdCH), 2.12 (s, 6H, AdCH₂),

7.09 (d, J ) 16.5 Hz, 1H, CHdCH), 7.11 (d, J ) 10.5 Hz, 1H,

5′-ArH), 7.22 (s, 1H, 2′-ArH), 7.28 (d, J ) 8.1 Hz, 1H, 6′-ArH),

7.32 (d, J ) 7.8 Hz, 1H, 5-ArH), 7.40 (d, J ) 8.7 Hz, 1H, 6-ArH),

7.56 (s, 1H, 3-ArH), 7.61 ppm (d, J ) 16.5 Hz, 1H, CHdCH).

FTMS (HRMS) calcd C₂₅H₂₅ClN₄O (M + H)⁺expected 433.1790,

found 433.1801.

4-Benzyloxy-3-chlorobenzonitrile (26). To a solution of 18 (1.23

g, 5 mmol) in MeOH (10 mL) and pyridine (2 mL) was added

NH₂OH·HCl (695 mg, 10 mmol). The mixture was stirred at 50 °C

under argon for 5 h and then poured into ice water to give a solid,

which was collected by filtration, washed with water, and dried.

Chromatography (9% EtOAc/hexane) gave 1.21 g (94%) of oxime

25 as a white solid, which was used for the elimination step.

To 25 (1.1 g, 4.2 mmol) in toluene (15 mL) and pyridine (2 mL)

cooled in an ice bath was slowly added methanesulfonyl chloride

(798 mg, 7 mmol) over a 10-min period. The mixture was stirred

at reflux for 3 h, cooled to room temperature, and diluted with

toluene (30 mL). The organic phase was washed (1 N HCl, 5%

NaHCO₃, water, and brine) and dried. Concentration and chroma-

tography (10% EtOAc/hexane) gave 910 mg (91%) of 26 as a white

solid, R_f0.60 (20% EtOAc/hexane); mp 120-122 °C. IR (CHCl₃)

2966, 2232 cm^-1; ¹H NMR δ 5.23 (s, 2H, ArCH₂), 7.0 (d, J ) 8.7

Hz, 1H, 5-ArH), 7.35-7.45 (m, 5H, ArH), 7.50 (dd, J ) 8.7, 2.1

Hz, 1H, 6-ArH), 7.68 ppm (d, J ) 2.1 Hz, 1H, 2-ArH). The crude

product was used directly in the next step.

Chromatography (10% EtOAc/hexane) gave 220 mg (91%) of 36

as a white powder, R_f0.28 (20% EtOAc/hexane); mp 72-74 °C.

IR (CHCl₃) 3399, 2906, 1601 cm^-^{1 1}H NMR δ 1.73 (s, 6H,

;

AdCH₂), 2.04 (s, 3H, AdCH), 2.17 (s, 6H, AdCH₂), 4.35 (d, J )

4.2 Hz, 2H, OCH₂), 5.16 (s, 2H, ArCH₂), 7.64 (td, J ) 5.4, 16.2

Hz, 1H, CHdCHCH₂OH), 6.61 (d, J ) 15.2 Hz, 1H, CHdCHCH₂-

OH), 7.00 (d, J ) 8.7 Hz, 1H, 5′-ArH), 7.30-7.42 (m, 5H, ArH),

7.40 (d, J ) 7.5 Hz, 1H, 6′-ArH), 7.42 (s, 1H, 2′-ArH), 7.50 (d, J

) 8.1 Hz, 2H, 5,6-ArH), 7.53 ppm (s, 1H, 2-ArH). HRMS calcd

C₃₂H₃₃ClO₂[M + Na]⁺507.2061, found 507.2066.

(E)-4-[3′-(1-Adamantyl)-4′-benzyloxyphenyl]-3-chlorocinnama-

ldehyde (37). To a stirred solution of oxalyl chloride (127 mg, 1

mmol) in CH₂Cl₂(2 mL) at -78 °C under argon was slowly added

Me₂SO (156 mg, 2 mmol) in CH₂Cl₂(1 mL) according to the

method of Swern.⁴⁸The solution was stirred at -78 °C for 10 min,

36 (170 mg, 0.35 mmol) in CH₂Cl₂(2 mL) was slowly added, and

stirring was continued at -78 °C for 20 min before Et₃N (303 mg,

3.0 mmol) was added. The mixture was allowed to warm to room

temperature and diluted with CH₂Cl₂(20 mL). The organic layer

was washed (water and brine), dried, and concentrated. Chroma-

tography (5% EtOAc/hexane) gave 122 mg (72%) of 37 as a white

1

powder, mp 56-58 °C. IR (CHCl₃) 2906, 1681, 1602 cm^-1; H

NMR δ 1.73 (s, 6H, AdCH₂), 2.05 (s, 3H, AdCH), 2.17 (s, 6H,

AdCH₂), 5.17 (s, 2H, ArCH₂), 6.73 (dd, J ) 15.2, 7.5 Hz, 1H,

CHdCHCHO), 7.02 (d, J ) 8.7 Hz, 1H, 5′-ArH), 7.29 (dd, J )

8.1, 2.1 Hz, 1H, 6′-ArH), 7.35-7.44 (m, 5H, ArH), 7.41 (d, J )

6.9 Hz, 1H, 5-ArH), 7.49 (d, J ) 6.9 Hz, 1H, 6-ArH), 7.50 (s, 1H,

2′-ArH), 7.51 (d, J ) 15.9 Hz, 1H, CHdCHCHO), 7.66 (s, 1H,

2-ArH), 9.73 ppm (d, J ) 7.5 Hz, 1H, CHO). FTMS (HRMS) calcd

C₃₂H₃₂ClO₂[M + H]⁺expected 501.2200, found 501.2276.

General Procedure for Converting Aldehydes 37 and 41 to 2,4-

Thiazolidinediones. To a stirred solution of aldehyde (0.1 mmol)

and 2,4-thiazolidinedione (23 mg, 0.2 mmol) in toluene (1 mL)

were added NHEt₂(0.1 mL) and HOAc (0.05 mL). The mixture

was stirred at reflux for 2 h, cooled, and diluted with CH₂Cl₂(10

mL). The organic layer was washed (water and brine) and dried.

Concentration and chromatography gave the thiazolidinedione.

5-{3-[3′-(1-Adamantyl)-4′-benzyloxy-2-chloro-4-biphenyl]-(2E)-

2-propenylidene}-2,4-thiazolidinedione (38). 37 (46 mg, 0.1

mmol) gave 34 mg (62%) of 38 as a yellow powder, R_f0.31 (30%

EtOAc/hexane); mp 212-215 °C. IR (CHCl₃) 3186, 1733, 1692

cm^-1; ¹H NMR δ 1.73 (s, 6H, AdCH₂), 2.04 (s, 3H, AdCH), 2.17

(s, 6H, AdCH₂), 5.17 (s, 2H, ArCH₂), 6.70 (dd, J ) 15.6, 8.7 Hz,

1H, ArCHdCHCHdC), 7.00 (d, J ) 8.4 Hz, 1H, 5′-ArH), 7.02

(d, J ) 14.2 Hz, 1H, ArCHdCHCHdC), 7.27-7.46 (m, 7H,

5-ArH, 6′-ArH, Ar), 7.51 (d, J ) 1.2 Hz, 1H, 2′-ArH), 7.52 (d, J

) 6.6 Hz, 1H, 6-ArH), 7.54 (d, J ) 15.1 Hz, 1H, ArCHdCHCHd

C), 7.60 (s, 1H, 3-ArH), 8.39 ppm (s, 1H, NH). HRMS calcd C₃₅H₃₂-

ClNO₃S [M - H]^-580.1719, found 580.1714.

5-[3′-(1-Adamantyl)-2-chloro-4′-hydroxy-4-biphenyl]-1H-tetra-

zole (31). A reported procedure was applied.⁷³To 30 (73 mg, 0.2

mmol) in toluene (2 mL) was added (n-Bu)₂SnO (5.0 mg, 0.02

mmol) and azidotrimethylsilane (115 mg, 1.0 mmol). This mixture

was stirred at 90-95 °C for 48 h and then concentrated. EtOAc

(10 mL) and 10% HCl (3 mL) were added. The mixture was stirred

for 0.5 h, washed (water and brine), and dried. Concentration and

crystallization (EtOAc/CH₂Cl₂/hexane) produced 58 mg (72%) of

31 as a white powder, R_f0.27 (50% EtOAc/hexane); mp 235-

240 °C. IR (powder) 3377, 2906, 1486, 1256 cm^-^{1 1}H NMR

;

(C²H₃O²H) δ 1.85 (s, 6H, AdCH₂), 2.08 (s, 3H, AdCH), 2.22 (s,

6H, AdCH₂), 6.83 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.18 (dd, J ) 8.1,

2.1 Hz, 1H, 6′-ArH), 7.29 (d, J ) 2.4 Hz, 1H, 2′-ArH), 7.57 (d, J

) 7.8 Hz, 1H, 5-ArH), 8.01 (dd, J ) 7.8, 1.5 Hz, 1H, 6-ArH),

8.18 ppm (d, J ) 1.5 Hz, 1H, 2-ArH). FTMS (HRMS) calcd C₂₃H₂₃-

ClN₄O [M + H]⁺expected 407.1633, found 407.1630.

Ethyl (E)-4-Benzyloxy-3-chlorocinnamate (32).^8,72To triethyl

phosphonoacetate (2.24 g, 10 mmol) in Et₂O (20 mL) stirred under

argon in a dry ice-acetone bath was added 0.91 M KN(SiMe₃)₂

(10.0 mmol) in THF (11.0 mL). The mixture was stirred for 0.5 h

before 4-benzyloxy-3-chlorobenzaldehyde (18) (2.00 g, 8.1 mmol)

in THF (40 mL) was added dropwise over a 0.5-h period, stirred

for 1 h more, allowed to warm to room temperature, stirred

overnight, poured into water (50 mL) containing HOAc (2 mL),

and extracted (Et₂O, 100 mL). The extract was washed (water and

brine), dried, and concentrated. Chromatography (EtOAC/hexane)

afforded 2.3 g (91%) of 32 as a white solid, R_f0.41 (10% EtOAc/

5-{4-[3′-(1-Adamantyl)-4′-benzyloxyphenyl]-3-chlorobenzylidene}-

2,4-thiazolidinedione (42). 41 (46 mg, 0.1 mmol) gave 34 mg

(62%) of 42 as a yellow powder, R_f(10% EtOAc/hexane); mp 200-

1

203 °C. IR (CHCl₃) 3173, 2904, 1739, 1689 cm^-1; H NMR δ

1.74 (s, 6H, AdCH₂), 2.05 (s, 3H, AdCH), 2.18 (d, J ) 2.4 Hz,

6H, AdCH₂), 5.18 (s, 2H, ArCH₂), 7.02 (d, J ) 8.4 Hz, 1H, 5′-

ArH), 7.30 (dd, J ) 8.4, 2.1 Hz, 1H, 6′-ArH), 7.35-7.43 (m, 6H,

2′-ArH, ArH), 7.48 (d, J ) 7.8 Hz, 1H, 6-ArH), 7.52 (d, J ) 6.6

Hz, 1H, 5-ArH), 7.60 (d, J ) Hz, 1H, 2-ArH), 7.81 (s, 1H, ArCHd

C), 8.14 ppm (bs, 1H, NH). HRMS calcd C₃₃H₃₀ClNO₃S [M -

H]^-554.1562, found 554.1552.

hexane); mp 72-74 °C. IR (CHCl₃) 2961, 1706, 1634, 1508 cm^-1

;

¹H NMR δ 1.33 (t, J ) 7.2 Hz, 3H, CH₃), 4.25 (q, J ) 7.2 Hz, 2H,

OCH₂), 5.20 (s, 2H, ArCH₂), 6.31 (d, J ) 15.9 Hz, 1H, CHd

CHCO), 6.95 (d, J ) 8.7 Hz, 1H, 5-ArH), 7.36-7.45 (m, 6H,

6-ArH, ArH), 7.56 (d, J ) 16.2 Hz, 1H, CHdCHCO), 7.59 ppm

(s, 1H, 2-ArH). This product was debenzylated to 33.

2-{[3′-(1-Adamantyl)-4′-benzyloxy-2-chloro-4-biphenyl]-

methylene}propanedinitrile (44). A reported procedure⁴⁹was

adapted. A mixture of 41 (102 mg, 0.22 mmol) and malononitrile

(18 mg, 0.27 mmol) in anhydrous DMF (0.6 mL) was stirred in a

103 °C oil-bath for 7 h, cooled to room temperature, and diluted

with H₂O (8 mL) and EtOAc (80 mL). The organic layer was

washed (brine) and dried. The residue obtained on concentration

was purified by chromatography (9% EtOAc/hexane) to give 78

mg (70%) of 44 as a yellow solid, mp 170-171 °C, which was

(E)-4-[3′-(1-Adamantyl)-4′-benzyloxyphenyl]-3-chlorocin-

namyl Alcohol (36). To a stirred solution of 35 (264 mg, 0.5 mmol)

in CH₂Cl₂(5 mL) at -78 °C under argon was added 1.5 mL of 1.0

M DIBAL (1.5 mmol) in hexane. The mixture was stirred at

-78 °C for 3 h, diluted with 1 N HCl, and then extracted (EtOAc).

The extract was washed (water and brine), dried, and concentrated.

1

deprotected to give 45. H NMR δ 1.76 (bs, 6H, AdCH₂), 2.08

(bs, 3H, AdCH), 2.20 (bs, 6H, AdCH₂), 5.20 (s, 2H, ArCH₂), 7.05

Effects of Isosteric Modifications of 3-Cl-AHPC Carboxylate

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2635

(d, J ) 8.4 Hz, 1H, 5′-ArH), 7.32-7.57 (m, 8H, 5-ArH, 2′-ArH,

6′-ArH, ArH), 7.74 (s, 1H, CHdC(CN)₂), 7.92-7.96 ppm (m, 2H,

3-ArH, 6-ArH).

of 53 (340 mg, 0.67 mmol), trimethylsilylacetylene (186 µL, 1.34

mmol), Pd(PPh₃)₄(92 mg, 0.08 mmol), and CuI (5 mg, 0.026 mmol)

was heated at 102 °C (oil-bath) under argon for 3.5 h, cooled to

room temperature, diluted with EtOAc (90 mL), and filtered through

silica gel. The filtrate was concentrated, and the residue was

chromatographed (1% EtOAc/hexane) to give 350 mg (99%) of

(E)-4-[3′-(1-Adamantyl)-4′-benzyloxyphenyl]-3-chlorocinnam-

ic Acid (46).⁵A mixture of 35⁵(174 mg, 0.34 mmol) and LiOH·

H₂O (47 mg, 3.3 mmol) in CH₃OH/THF/H₂O (0.6:1.6:0.55) was

stirred for 6 h. After removal of solvent, the residue suspended in

H₂O (7 mL) and 2 N HCl (5 mL) was extracted with EtOAc (100

mL). The organic layer was washed (brine) and dried. Removal of

solvent gave 157 mg (93%) of 46 as a pale-tan solid, which was

54 as a pale-yellow solid, mp 55-57 °C. IR 2902, 2221, 1286 cm^-1

;

¹H NMR δ 0.27 (s, 9H, Si(CH₃)₃), 1.74 (bs, 6H, AdCH₂), 2.05 (bs,

3H, AdCH), 2.18 (m, 6H, AdCH₂), 5.17 (s, 2H, ArCH₂), 7.01 (d,

J ) 8.7 Hz, 1H, 5′-ArH), 7.27 (dd, J ) 8.7, 2.1 Hz, 1H, 6′-ArH),

7.29 (d, J ) 8.4 Hz, 1H, 6-ArH), 7.30-7.46 (m, 6H, 2′-ArH, ArH),

7.53 (dd, J ) 8.4, 1.5 Hz, 1H, 5-ArH), 7.58 ppm (d, J ) 1.5 Hz,

1H, 3-ArH). HRMS calcd C₃₄H₃₇ClOSi [M + H]⁺525.2375, found

525.2379.

1

used directly in the next step. H NMR δ 1.74 (bs, 6H, AdCH₂),

2.05 (bs, 3H, AdCH), 2.19 (bs, 6H, AdCH₂), 5.18 (s, 2H, ArCH₂),

6.48 (d, J ) 15 Hz, 1H, CHdCHCO), 7.01 (m, 1H, 5′-ArH), 7.28-

7.55 (m, 9H, 6′-ArH, 2-ArH, 5-ArH, 6-ArH, ArH), 7.66 (s, 1H,

2′-ArH), 7.75 ppm (d, J ) 15 Hz, 1H, CHdCHCO).

3′-(1-Adamantyl)-4′-benzyloxy-2-chloro-4-ethynylbiphenyl (55).

To a solution of 54 (350 mg, 0.67 mmol) in THF (15 mL) was

added 0.9 mL of 1.0 M (n-Bu)₄NF (0.88 mmol) in THF. The

mixture was stirred for 1 h, diluted with H₂O (20 mL), and extracted

with EtOAc (3 × 200 mL). The extract was washed (sat. brine)

and dried. After concentration, the residue was chromatographed

(1% EtOAc/hexane) to give 295 mg (96%) of 55 as a white solid,

N-(Tetrahydro-2H-pyran-2-yloxy) (E)-4-[3′-(1-Adamantyl)-4′-

benzyloxyphenyl]-3-chlorocinnamamide (47). A reported proce-

dure⁵⁰was followed. To a stirred solution of 46 (102 mg, 0.205

mmol) in CHCl₃(4 mL) at 0 °C (ice-bath) were added DMAP (47

mg, 0.39 mmol) and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine

(36 mg, 0.31 mmol). Stirring was continued at 0 °C for 20 min

more, before DIC (48 µL, 0.31 mmol) was added. After 1 h, the

mixture was warmed to room temperature, stirred for 48 h,

quenched with saturated NH₄Cl (8 mL), and extracted with EtOAc

(90 mL). The extract was washed (sat. NH₄Cl and brine) and dried.

Solvent was removed at reduced pressure and the residue chro-

matographed (33% EtOAc/hexane) to give 56 mg (46%) of 47 as

1

mp 94-96 °C. H NMR δ 1.74 (bs, 6H, AdCH₂), 2.06 (bs, 3H,

AdCH), 2.18 (m, 6H, AdCH₂), 3.14 (s, 1H, CtCH), 5.17 (s, 2H,

ArCH₂), 7.01 (d, J ) 8.4 Hz, 1H, 5′-ArH), 7.28 (dd, J ) 8.1, 2.4

Hz, 1H, 6′-ArH), 7.31 (d, J ) 8.1 Hz, 1H, 6-ArH), 7.35 (d, J )

2.4 Hz, 1H, 2′-ArH), 7.32-7.55 (m, 6H, 5ArH, ArH), 7.6 ppm (d,

J ) 1.5 Hz, 1H, 3-ArH). HRMS calcd C₃₁H₂₉ClO [M + H]⁺

453.1980, found 453.1978.

1

a pale-tan solid, 91-93 °C. H NMR δ 1.64 (m, 4H, 4-THPH,

5-THPH), 1.74 (bs, 6H AdCH₂), 1.87 (m, 2H, 3-THPH), 2.06 (bs,

3H, AdCH), 2.18 (bs, 6H, AdCH₂), 3.64 (m, 1H, 6-THPH), 3.98

(m, 1H, 6-THPH), 5.06 (bs, 1H, 2-THPH), 5.18 (s, 2H, ArCH₂),

7.01 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.29 (dd, J ) 8.4, 1.5 Hz, 1H,

6′-ArH), 7.33-7.46 (m, 7H, CHdCHCO, 5-ArH, ArH), 7.51-7.63

(m, 3H, 2-ArH, 6-ArH, 2′-ArH), 7.72 ppm (d, J ) 15.3 Hz, 1H,

CHdCHCO).

(E)-2-[3′-(1-Adamantyl)-4′-benzyloxy-2-chloro-4-biphenyl]ethe-

nyl-1,3,2-benzodioxaborole (56). The procedure of Brown et al.⁵³

was adapted. A mixture of 55 (295 mg, 0.65 mmol) and 0.67 mL

of 1.0 M catecholborane (0.67 mmol) in THF was stirred under

argon at 91 °C (oil-bath) for 5 h, cooled to room temperature, and

diluted with THF (3 mL) for transfer. After concentration, the

residue was chromatographed (1% to 40% EtOAc/hexane) to give

1

3′-(1-Adamantyl)-4′-benzyloxyphenyl)-3-chloroaniline (52). A

mixture of 51 (2.08 g, 4.4 mmol) and SnCl₂‚2H₂O (4.96 g, 44

mmol) in EtOH (20 mL) was heated at 88 °C (oil-bath) for 2.5 h,

cooled to room temperature, diluted with H₂O (10 mL), and adjusted

to pH 7-8 by addition of 2 N NaOH (21 mL) and 5% NaHCO₃

(19 mL). This mixture was stirred for 1 h and extracted (3 × 350

mL EtOAc). The organic layer was washed (sat. brine) and dried.

The concentrated residue was chromatographed (28% EtOAc/

hexane) to give 1.766 g (90%) of 52 as a white solid, mp 154-

59 mg (26%) of 56 as a pale-yellow solid, mp 158-161 °C. H

NMR δ 1.75 (bs, 6H, AdCH₂), 2.07 (bs, 3H, AdCH), 2.21 (m, 6H,

AdCH₂), 5.19 (s, 2H, ArCH₂), 6.42 (d, J ) 18 Hz, 1H, CHdCHB),

7.01-7.07 (m, 3H, ArH), 7.27-7.49 (m, 8H, ArH), 7.52-7.60 (m,

3H, ArH), 7.75 (s, 1H, ArH), 7.79 ppm (d, J ) 18 Hz, 1H, CHd

CHB).

(3E)-4-[3′-(1-Adamantyl)-4′-benzyloxy-2-chloro-4-biphenyl]but-

3-en-2-one (58). A reported procedure⁵⁴was modified. A mixture

of 4-[3′-(1-adamantyl)-4′-benzyloxyphenyl]-3-chlorobenzaldehyde

(41) (303 mg, 0.66 mmol), (2-oxopropyl)triphenylphosphonium

bromide (397 mg, 0.99 mmol) and 1,5,7-triazabicyclo[4.4.0]dec-

5-ene (157 mg, 1.13 mmol) in THF (3 mL) was stirred for 23 h at

room temperature and then at 70 °C for 2 h. The mixture was cooled

to room temperature and diluted with H₂O (10 mL) and EtOAc

(150 mL). The organic layer was washed (sat. brine) and dried.

The residue obtained on concentration was chromatographed (12%

EtOAc/hexane) to give 124 mg (38%) of 58 as a yellow wax, mp

155 °C. IR 3391, 2905, 1189 cm^-^{1 1}H NMR δ 1.73 (m, 6H,

;

AdCH₂), 2.05 (m, 3H, AdCH), 2.18 (m, 6H, AdCH₂), 3.73 (bs,

2H, NH₂), 5.16 (s, 2H, ArCH₂), 6.62 (dd, J ) 8.1, 2.4 Hz, 1H,

5-ArH), 6.8 (d, J ) 2.4 Hz, 1H, 2-ArH), 6.97 (d, J ) 8.1 Hz, 1H,

6-ArH), 7.15 (d, J ) 8.4 Hz, 1H, 5′-ArH), 7.35 (dd, J ) 8.4, 2.4

Hz, 1H, 6′-ArH), 7.32 (d, J ) 8.4 Hz, 1H, 2′-ArH), 7.33-7.55

ppm (m, 5H, ArH). HRMS calcd C₂₉H₃₀ClNO [M + H]⁺444.2089,

found 444.2096.

1

3′-(1-Adamantyl)-4′-benzyloxy-4-bromo-2-chlorobiphenyl (53).

To CuBr₂(1.19 g, 5.32 mmol) and tert-butyl nitrite (899 µL, 6.81

mmol) in anhydrous MeCN (5 mL) at 0 °C were slowly added

under argon 52 (1.89 g, 4.25 mmol) and MeCN (11 mL), and

stirring was continued for 30 min. The mixture was warmed to

room temperature, stirred for 2 h, at which time no insoluble 52

remained, quenched with 2 N HCl (100 mL), and extracted (3 ×

300 mL Et₂O). The organic layer was washed (sat. brine) and dried.

After concentration, the residue was chromatographed (hexane) to

give 973 mg (45%) of 53 as a pale-yellow solid, mp 58-60 °C. IR

2902, 1220 cm^-1; ¹H NMR δ 1.74 (bs, 6H, AdCH₂), 2.05 (bs, 3H,

AdCH), 2.18 (m, 6H, AdCH₂), 5.17 (s, 2H, ArCH₂), 7.01 (d, J )

8.1 Hz, 1H, 5′-ArH), 7.23 (d, J ) 8.4 Hz, 1H, 6-ArH), 7.25 (dd, J

) 8.4, 2.1 Hz, 1H, 6′-ArH), 7.32 (d, J ) 2.1 Hz, 1H, 2′-ArH),

7.33-7.55 (m, 6H, 5-ArH, ArH), 7.64 ppm (d, J ) 2.1 Hz, 1H,

3-ArH). HRMS calcd C₂₉H₂₈BrClO [M + H]⁺507.1085, found

507.1068.

73-75 °C. IR 2902, 2849, 1683 cm^-1; H NMR δ 1.74 (bs, 6H,

AdCH₂), 2.06 (bs, 3H, AdCH), 2.19 (bs, 6H, AdCH₂), 2.42 (s, 3H,

CH₃CO), 5.18 (s, 2H, ArCH₂), 6.75 (d, J ) 16.2 Hz, 1H, CHd

CHCO), 7.02 (d, J ) 8.4 Hz, 1H, 5′-ArH), 7.30 (dd, J ) 8.4, 2.1

Hz, 1H, 6′-ArH), 7.34-7.56 (m, 9H, 2′-ArH, 5-ArH, 6-ArH, CHd

CHCO, ArH), 7.66 ppm (d, J ) 1.2 Hz, 1H, 3-ArH). HRMS calcd

C₃₃H₃₃ClO₂[M + H]⁺497.2242, found 497.2240.

(3E)-4-[3′-(1-Adamantyl)-4′-benzyloxy-2-chloro-4-biphenyl]-2-

oxobut-3-enal (59). A reported procedure⁵⁵was applied. A mixture

of 58 (124 mg, 0.25 mmol) and H₂SeO₃(35 mg, 0.27 mmol) in

1.0 mL of dioxane and 0.1 mL of H₂O was heated at reflux for 1.5

h under argon, cooled to room temperature, and diluted with EtOAc

(120 mL). The organic layer was washed (sat. brine) and dried.

The residue obtained on concentration was chromatographed (33%

EtOAc/hexane) to give 35 mg (27%) of 59 as a yellow wax, mp

84-86 °C. IR 2904, 2853, 1713, 1690 cm^-1; ¹H NMR δ 1.74 (bs,

6H, AdCH₂), 2.06 (bs, 3H, AdCH), 2.19 (bs, 6H, AdCH₂), 5.18 (s,

2H, ArCH₂), 6.93 (d, J ) 16.2 Hz, 1H, CHdCHCO), 7.02 (dd, J

) 8.1, 2.1 Hz, 1H, 6′-ArH), 7.28-7.56 (m, 9H, 5-ArH, 6-ArH,

3′-(1-Adamantyl)-4′-benzyloxy-2-chloro-4-(2-trimethylsilylethy-

nyl)biphenyl (54). A reported procedure⁵²was adapted. A mixture

2636 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11

Dawson et al.

2′-ArH, 5′-ArH, ArH), 7.76 (d, J ) 1.5 Hz, 1H, 3-ArH), 7.97 (d,

J ) 16.2 Hz, 1H, CHdCHCO), 9.47 ppm (s, 1H, CHO). HRMS

calcd C₃₃H₃₁ClO₃[M + H]⁺511.2034, found 511.2038.

field descriptors employed in Tripos QSAR (St. Louis, MO). The

validity of the MOLMOD pharmacophore developed from the

training set was assessed by using the overlap rule in 3DQSAR

(CoMSIA). Models predictive of the percentage of apoptosis of

MDA-MB-231 breast cancer cells were developed and used to

predict the percentage of apoptosis of MDA-MB-231 breast cancer

cells of analogues not included in building the CoMSIA models.

A fragment QSAR model was constructed using the isosteric polar/

H-bond acceptor replacements shown in Table 3 for the 3-Cl-AHPC

carboxyl group. In an effort to understand the SAR of these

substitutions we computed: (i) frontier orbital energetics, (ii)

Sterimol parameters, (iii) group hydrophobicities, (iv) volumes, (v)

areas, (vi) solvent-accessible surface areas, (vii) polar and nonpolar

volumes, (viii) globularities, (ix) electrostatic potentials on the van

der Waals surface using MOPAC-AM1-derived properties devel-

oped by the in-house program GRAPHA and (x) solvation energies

and raw pK_avalues using density functional theory evaluation. The

raw pK_avalues were computed from unscaled/uncorrected gas-phase

protonation energies.⁵⁸Combinations of 19 properties were used

in several SAR models to interrogate the data for 50% proliferation

inhibition (IC₅₀value) of KG-1, H292, MDA-MB-231, and Du-

145 cancer cells in terms of fundamental changes in the polar region.

We were able to obtain predictive models for all of the data sets.

Biology. Cell Growth. Cancer cell lines were obtained from ATCC

(Manassas, VA) and grown in DMEM (MDA-MB-231 breast

cancer) or RPMI-1640 medium (H292 lung cancer and Du-145

prostate cancer) containing 10% fetal bovine serum (FBS; Tissue

Culture Biologicals, Tulare, CA) at 37 °C under 6% CO₂. MDA-

MB-231 cells were also grown and cultured in DMEM/F-12

medium supplemented with 5% heat-inactivated FBS, as we have

previously described.⁶KG-1 cells were obtained from Dr. H. Phillip

Koeffler (UCLA, Cedars-Sinai Medical Center, Los Angeles, CA)

and were grown in RPMI-1640 medium supplemented with 5%

heat-inactivated FBS and gentamycin (10 µg/mL). Retinoid-resistant

HL-60R leukemia cells were obtained from Dr. Steve Collins

(University of Washington, Seattle) and grown under the same

conditions.

Diethyl (E)-2-[3′-(1-Adamantyl)-4′-benzyloxy-2-chloro-4-biphe-

nyl]ethenylphosphonate (61). A reported method⁵⁶was modified.

To a solution of 41 (0.14 mmol, 65 mg) and tetraethyl methyl-

enebisphosphonate (0.14 mmol, 36 µL) in CH₂Cl₂(0.8 mL) was

added 50% aq NaOH (0.8 mL). The mixture was stirred for 10

min, diluted with H₂O (8 mL), and extracted (60 mL EtOAc). The

extract was washed (2 N HCl and brine) and dried. The residue

obtained on concentration was purified by chromatography (50%

to 60% EtOAc/hexane) to give 82 mg (98%) of 61 as a colorless

gel. ¹H NMR δ 1.40 (t, J ) 6.9 Hz, 6H, OCH₂CH₃), 1.76 (bs, 6H,

AdCH₂), 2.07 (bs, 3H, AdCH), 2.20 (bs, 6H, AdCH₂), 4.18 (m,

4H, OCH₂CH₃), 5.20 (s, 2H, ArCH₂), 6.32 (t, J ) 17.4 Hz, 1H,

CHdCHP), 7.03 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.31 (dd, J ) 8.1,

2.1 Hz, 1H, 6′-ArH), 7.28-7.57 (m, 9 H, 2′-ArH, 5-ArH, 6-ArH,

ArH, CHdCHP), 7.64 (s, 1H, 3-ArH). HRMS calcd C₃₅H₄₀ClO₄P

[M + H]⁺591.2425, found 591.2429.

(E)-2-[3′-(1-Adamantyl)-2-chloro-4′-hydroxy-4-biphenyl]ethen-

ylphosphonic Acid Monoethyl Ester (63). A literature method⁵⁷

was adapted. A suspension of 62 (0.12 mmol, 61 mg) in aq 20%

HCl (5 mL) was heated at reflux under argon for 6 h. After removal

of some water at reduced pressure from the resultant solution, the

residue was extracted (3 × 40 mL EtOAc). The organic extract

was washed (brine) and dried. The residue obtained on concentration

was chromatographed (EtOAc/hexane/MeOH, 2:2:1, to EtOAc/

MeOH/HOAc, 5:3:0.01) to give 45 mg (80%) of 63 as an off-white

solid, mp 260 °C (dec). IR 3328, 2902, 1617, 1603, 1035 cm^-1

;

¹H NMR δ 1.27 (t, J ) 6.9 Hz, 3H, OCH₂CH₃), 1.81 (bs, 6H,

AdCH₂), 2.05 (bs, 3H, AdCH), 2.17 (bs, 6H, AdCH₂), 3.93 (m, J

) 6.9 Hz, 2H, OCH₂CH₃), 6.46 (dd, J ) 17.4, 15.0 Hz, 1H, CHd

CHP), 6.76 (d, J ) 7.8 Hz, 1H, 5′-ArH), 7.06 (dd, J ) 7.8, 2.1 Hz,

1H, 6′-ArH), 7.18 (d, J ) 2.1 Hz, 1H, 2′-ArH), 7.23 (dd, J ) 17.4,

15.0 Hz, 1H, CHdCHP), 7.29 (d, J ) 7.8 Hz, 1H, 6-ArH), 7.43

(d, J ) 7.8 Hz, 1H, 5-ArH), 7.57 ppm (s, 1H, 3-ArH). HRMS calcd

C₂₆H₃₀ClO₄P [M- H]^-471.1492, found 471.1487.

Computational Studies. Conformational libraries were prepared

for a training set of 55 AHPN (1) analogues including 24 active

analogues using 30° increment grid rotations for all rotatable bonds

employing the CHARMm forcefield in Accelerys Quanta, a

continuum dielectric constant of 80 and no potential truncation for

correlation with their apoptotic activity at 1.0 µM against MDA-

MB-231 breast cancer cells after treatment for 96 h. Most of the

analogues had a carboxylate moiety and a hydroxyl group adjacent

to a bulky hydrophobic group as in AHPN (1 in Figure 1) and

variable hydrophobic groups and ring systems. While not all of

the analogues with these features were apoptosis inducers, these

moieties appeared to be core-recognition elements inasmuch as their

removal eliminated apoptotic acitivity. An initial pharmacophore/

overlap rule was developed using the in-house program MOL-

MOD,^74,75which took as input trial pharmacophore hypotheses in

the form of (i) either ligand or ‘receptor-based’ sites of hydrogen-

bond acceptors/donors, hydrophobic moieties, centroids, or user-

defined criteria; (ii) an energy window for the low-energy confor-

mations to employ in pharmacophore testing; and (iii) a distance

criterion (cutoff) for pharmacophoric distances in order to determine

whether a conformation of each ligand had a 3D-distance matrix

between the pharmacophore points in common with at least one

conformation of each of the other ligands in the training set. Any

pharmacophore found within the energy and distance criteria was

reported along with all conformers of each analogue complying

with the pharmacophore. The program also output a superposition

of the analogues at their pharmacophore points for use in 3D-QSAR.

Following development of the initial apoptosis pharmacophore for

these compounds, the properties of all analogues in their conforma-

tions complying with the pharmacophore definition were evaluated

using a combination of semiempirical quantum mechanics, a

MOPAC-7 AM1 Hamiltonian,⁷⁶density functional theory as

incorporated in Gaussian⁷⁷and Jaguar (Schro¨dinger, Portland, OR),

and the comparative molecular similarity index analysis (CoMSIA)

Cryopreserved, pooled primary human microvascular endothelial

(HMVE) cells (Clonetics, San Diego, CA) were grown in endo-

thelial cell growth medium EGM-MV (Clonetics) or in MEV

growth medium containing 5% fetal calf serum plus the growth

factors and antimicrobials in the accompanying kit (Clonetics).

Human dermal microvascular endothelial (HMVE) cells (Cambrex

Bioscience, Walkersville, MD) were grown and maintained in

EGM-MV medium (Cambrex).

Growth Inhibition. Cells (4 × 10³/well in 96-well plates for MDA-

MB-231 or 2 × 10³/well for H292 and Du-145) were allowed to

attach for 24 h and then treated for 72 h with each analogue

dissolved in Me₂SO or Me₂SO alone (0.1% Me₂SO final concentra-

tion). Media and compounds were replaced after 48 h. The number

of viable cells was determined using the 3-(4,5-dimethylthiazol-2-

yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoli-

um inner salt (MTS) reduction assay (Cell Titer 96 AQueous Non-

radioactive Cell Proliferation Assay, Promega, Madison, WI). Three

replicates were done at each concentration.

KG-1, HL-60R, and MDA-MB-231 cells were seeded in six-well

plates containing 3 mL of media plus 5% FBS per well and

incubated overnight. Compounds were then added in Me₂SO (0.1%

final Me₂SO concentration), and cells were incubated at various

concentrations for the cited times and harvested. Cell numbers were

determined using a hemocytometer for counting as we described.⁶

HMVE cells (3 × 10³in 200-µL medium/well in 96-well plates)

were allowed to attach for 24 h. Compounds dissolved in Me₂SO

were added to give 100, 125, 250, and 500-nM final concentrations

(final Me₂SO concentration e0.1%). Medium and test compound

were replaced every 48 h. After 7 days of treatment, cell growth

was determined by Alamar blue staining (BioSource, Camarillo,

CA) and measuring emission at 590 nm after excitation at 530 nm

by using a fluorimeter (Cytoflor, ABI, Foster City, CA). Results

shown in Table 2 are averages of five replicates. HMVE cells (1

× 10⁴in 1.0 mL of EGM-MV medium/well) were plated into 24-

Effects of Isosteric Modifications of 3-Cl-AHPC Carboxylate

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2637

well plates and incubated for 1 h at 37 °C. Compounds in Me₂SO

or Me₂SO alone (0.5% final concentration) were added. After 72-h

incubation at 37 °C, cells were detached using trypsin/EDTA and

counted using a hemocytometer. Three replicates were performed

for the experiments shown in Figure 6.

Cell Apoptosis. KG-1 and HL-60R leukemia and MDA-MB-231

breast cancer cells were seeded and incubated as described in the

growth inhibition experiments. Apoptotic cells were identified using

acridine orange staining as we have described.⁶

a FLx800 microplate reader (Bio-Tek Instruments, Winooski, VT).

Nonenzymatic hydrolysis of the substrate was corrected by measur-

ing the Me₂SO control without enzyme. IC₅₀values were deter-

mined by plotting the relative activity versus inhibitor concentration

using Prism software (GraphPad Software, San Diego, CA) and

fitting to the equation V_i/V₀)IC₅₀/(IC₅₀+ [I]), where V_iwas reaction

velocity at inhibitor concentration [I], V₀was the reaction velocity

without inhibitor and IC₅₀) K_i+ K_i[S]/K_m, where K_iwas the

dissociation constant for binding of inhibitor to enzyme, [S] was

the substrate concentration, and K_mwas the Michaelis-Menten

constant.

HMVE cells (6 × 10⁴/well) were seeded in EGM-MV medium

into 24-well plates. After a 6-h incubation at 37 °C, cells were

further incubated with medium containing various concentrations

of 3-Cl-AHPC or 1.0 µM doxorubicin (Sigma, St. Louis, MO) in

Me₂SO, or Me₂SO alone (control) (0.5% final Me₂SO concentra-

tion) for 20 h more. Cells were lysed, lysates were centrifuged,

and the supernate was analyzed for cytoplasmic histone-associated

DNA fragments indicative of apoptosis-induced DNA laddering

using the Cell Death Detection ELISA^PLUSkit (Roche Diagnostics,

Indianapolis, IN) according to the manufacturer’s protocol and a

microtiter plate reader (Fisher Scientific, Pittsburgh, PA). Experi-

ments were done in triplicate. This protocol was used to obtain

data shown in Figure 7.

HMVE Cell Migration. Migration was assayed using 6.5-mm

Transwell Chambers having 8 µm-diameter pores (Corning Costar,

Cambridge, MA). Their membrane undersides were coated over-

night at 4 °C with Matrigel (Becton Dickinson Labware, Bedford,

MA) (30 µg/mL) in EBM medium. Chambers were blocked for 2

h with a solution of 1% bovine serum albumin in phosphate-

buffered saline, pH 7.2, and then transferred to wells in 24-well

plates filled with EGM-MV medium (500 µL/well). HMVE cells

(80% confluent) were released with enzyme-free cell dissociation

buffer (Gibco, Long Island, NY), washed, and resuspended in

medium containing 3-Cl-AHPC (5) (0.05, 0.5, 5.0, 50, and 500

nM final concentration) in Me₂SO or Me₂SO alone (0.01% final

Me₂SO concentration). Cells (40 × 10⁴/well) in 200 µL of medium

were placed in the Transwells, and migration to the underside of

the precoated filter was examined after 5-h incubation at 37 °C.

Membranes were fixed and stained with Diff-Quick (Dade Diag-

nostics, Aguada, PR). Three high-power microscope fields were

counted in each replicate well, and results were expressed as mean

number of cells per field ( SD of three replicates.

HMVE Tubule Formation. Tube formation was assayed as

described by Nangia-Makker et al.⁷⁸with some modification.

Matrigel (100 µL) liquified on ice was added to 96-well plates and

then heated at 37 °C for 1 h to induce gelation. Trypsinized HMVE

cells (5 × 10⁴in 150 µL EGM-MV medium/well) were plated in

each well. After a 1-h incubation at 37 °C, cells were incubated at

37 °C with 0.25 µM or 1.0 µM 3-Cl-AHPC (5) in Me₂SO or Me₂-

SO alone (0.05% final concentration) for 18 h. Tubule formation

was assessed using an Optiphot microscope (Nikon, Melville, KN)

attached to a camera (Coolsnap Pro) having 1392 × 1040 resolution

(Media Cybernetics, Silver Spring, MD). Phase-contrast images

were captured using Image-Pro Express software (Media Cybernet-

ics) and a PlanApo 10× objective (Nikon) with a 0.5 numerical

aperture. Total tube length was determined using NIH Image

software (NIMH, Rockville, MD). Four random fields were

measured for each well of three replicates.

Assay for NR4A1 Expression by Western Blotting. MDA-MB-

231, H292, and DU145 cells were grown for 24 h in medium

containing 10% FBS and then treated with 1.0 µM 3-Cl-AHPC (5)

for 3 or 6 h. Cell lysates were prepared and boiled in sodium

dodecyl sulfate (SDS) sample buffer and resolved by SDS-8%

polyacrylamide gel electrophoresis. PAGE-separated proteins were

transferred to nitrocellulose membranes, which were then blocked

in 5% milk in TBST buffer (10 mM Tris·HCl, pH 8.0, containing

150 mM NaCl and 0.05% Tween 20) for 30 min, incubated with

monoclonal anti-NGFI-B (rat NR4A1) antibody (1:1000 dilution)

(R&D, Minneapolis, MN) in 5% milk in TBST for 2 h at room

temperature, washed (3 × TBST), incubated in TBST containing

horseradish peroxidase-linked anti-mouse immunoglobulin G for

1 h at room temperature, and washed (3 × TBST). Immunoreactive

products were detected by chemiluminescence using an enhanced

chemiluminescence system (Amersham, Piscataway, NJ).

Receptor Binding Assay. Expression of recombinant glutathione

S-transferase (GST)-human SHP chimeric protein (GST-SHP),

cell lysis, isolation of GST-SHP using glutathione-Sepharose

beads, incubation of beads with [5,5′-³H₂]AHPN⁴²in the presence

or absence of 3-Cl-AHPC (5), an analogue, or vehicle alone, and

separation of bound from nonbound label were performed as we

described.¹⁴Briefly, to GST-SHP (150-500 ng)-bound beads was

added nonlabeled 5 or an analogue at a final concentration of 50

µM in binding buffer (0.4 mL). This mixture was incubated on ice

for 45 min before [5,5′-³H₂]AHPN (5-nM final concentration) was

added. Incubation was continued for 2 h at 4 °C. Beads were then

washed (binding buffer) and either added directly to the scintillation

liquid or first eluted with 50 mM sodium bicarbonate and 1%

sodium dodecyl sulfate and the eluate added to scintillation fluid.

Both methods gave similar results.

Acknowledgment. We are grateful for support of this

research by the following grants P01 CA51993 (M.I.D., J.A.F.,

X.-K. Z.), R01 CA109370 (M.I.D., J.A.F.), California Breast

Cancer Research Program Grant 6XB-0018 (M.I.D., D.L.H.,

G.H.L.) and California Tobacco-Related Diseases Research

Grant 11RT-0081 (M.I.D., X.-K. Z.).

Supporting Information Available: Tables S1 and S2 showing

data and calculations illustrated by graph in Figure 4, Table S3

listing HPLC analyses of target compounds using two different

solvent systems, and Table S4 listing data on competitive inhibition

of binding to SHP. This material is available free of charge via the

Internet at http://pubs.acs.org.

Inhibition of PTP Activity. The phosphatase-catalyzed hydrolysis

of 6,8-difluoro-4-methylumbelliferyl phosphate (Invitrogen, Carls-

bad, CA) in the presence of each compound in Me₂SO or Me₂SO

alone (5% final concentration) was assayed at 30 °C in 60 µL of

0.15 M Bis-Tris buffer, pH 6.0, having 150-mM ionic strength

(adjusted with NaCl) and containing 1.0 mM dithiothreitol per well

in 96-well plates. SHP-2 (gift of Dr. Gen-Sheng Feng, Burnham

Institute) was at 5 nM and CD45 (BioMol, Plymouth Meeting, PA)

at 1 nM. Compounds were assayed at 0, 0.032, 0.16, 0.8, 4.0, 20.0,

100, and 500 µM. The initial reaction rate at a fixed substrate

concentration, which was equal to the corresponding K_mvalue of

100 µM for SHP-2 and CD45, was determined by measuring the

460-nm emission after excitation of the fluorescent cleavage

product, 6,8-difluoro-7-hydroxy-4-methylcoumarin, at 360 nm using

References

(1) Bernard, B. A.; Bernardon, J. M.; Delescluse, C.; Martin, B.; Lenoir,

M. C.; Maignan, J.; Charpentier, B.; Pilgrim, W. R.; Reichert, U.;

Shroot, B. Identification of synthetic retinoids with selectivity for

human nuclear retinoic acid receptor gamma. Biochem. Biophys. Res.

Commun. 1992, 186, 977-983.

(2) Charpentier, B.; Bernardon, J. M.; Eustache, J.; Millois, C.; Martin,

B.; Michel, S.; Shroot, B. Synthesis, structure-affinity relationships,

and biological activities of ligands binding to retinoic acid receptor

subtypes. J. Med. Chem. 1995, 38, 4993-5006.

(3) Shao, Z. M.; Dawson, M. I.; Li, X. S.; Rishi, A. K.; Sheikh, M. S.;

Han, Q. X.; Ordonez, J. V.; Shroot, B.; Fontana, J. A. p53

independent G₀/G₁arrest and apoptosis induced by a novel retinoid

in human breast cancer cells. Oncogene 1995, 11, 493-504.

2638 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11

Dawson et al.

(4) Li, X. S.; Rishi, A. K.; Shao, Z. M.; Dawson, M. I.; Jong, L.; Shroot,

B.; Reichert, U.; Ordonez, J.; Fontana, J. A. Posttranscriptional

regulation of p21^WAF1/CIP1expression in human breast carcinoma cells.

Cancer Res. 1996, 56, 5055-5062.

(26) Dawson, M. I.; Chan, R. L.; Derdzinski, K.; Hobbs, P. D.; Chao, W.

R.; Schiff, L. J. Synthesis and pharmacological activity of 6-[(E)-

2-(2,6,6-trimethyl-1-cyclohexen-1-yl)ethen1-yl]- and 6-(1,2,3,4-tet-

rahydro-1,1,4,4-tetramethyl-6-naphthyl)-2-naphthalenecarboxylic ac-

ids. J. Med. Chem. 1983, 26, 1653-1656.

(27) Newton, D. L.; Henderson, W. R.; Sporn, M. B. Structure-activity

relationships of retinoids in hamster tracheal organ culture. Cancer

Res. 1980, 40, 3413-3425.

(28) Strickland, S.; Breitman, T. R.; Frickel, F.; Nurrenbach, A.; Hadicke,

E.; Sporn, M. B. Structure-activity relationships of a new series of

retinoidal benzoic acid derivatives as measured by induction of

differentiation of murine F9 teratocarcinoma cells and human HL60

promyelocytic leukemia cells. Cancer Res. 1983, 43, 5268-5272.

(29) Verma, A. K.; Shapas, B. G.; Rice, H. M.; Boutwell, R. K. Correlation

of the inhibition by retinoids of tumor promoter-induced mouse

epidermal ornithine decarboxylase activity and of skin tumor promo-

tion. Cancer Res. 1979, 39, 419-425.

(30) Sporn, M. B.; Roberts, A. B. Biological methods for analysis and

assay of retinoids: Relationships between structure and activity. In

The Retinoids; Sporn, M. B.; Roberts, A. B.; Goodman, D. S., Eds.;

Academic Press, Inc.: Orlando, FL, 1984; Chapter 5, Vol. 1, pp 236-

279.

(31) Schiff, L. J.; Okamura, W. H.; Dawson, M. I.; Hobbs, P. D. Structure-

biological activity relationships of new synthetic retinoids on

epithelial differentiation of cultured hamster trachea. In Chemistry

and Biology of Synthetic Retinoids; Dawson, M. I.; Okamura, W.

H., Eds.; CRC Press: Boca Raton, FL, 1990; pp 307-363.

(32) Dawson, M. I.; Chao, W.-R.; Hobbs, P. D.; Delair, T. The inhibitory

effects of retinoids on the induction of ornithine decarboxylase and

the promotion of tumors in mouse epidermis. In Chemistry and

Biology of Synthetic Retinoids; Dawson, M. I.; Okamura, W. H., Eds.;

CRC Press: Boca Raton, FL, 1990; pp 385-466.

(33) Cincinelli, R.; Dallavalle, S.; Nannei, R.; Carella, S.; De Zani, D.;

Merlini, L.; Penco, S.; Garattini, E.; Giannini, G.; Pisano, C.; Vesci,

L.; Carminati, P.; Zuco, V.; Zanchi, C.; Zunino, F. Synthesis and

structure-activity relationships of a new series of retinoid-related

biphenyl-4-ylacrylic acids endowed with antiproliferative and proap-

optotic activity. J. Med. Chem. 2005, 48, 4931-4946.

(34) Parrella, E.; Gianni, M.; Fratelli, M.; Barzago, M. M.; Raska, I. J.;

Diomede, L.; Kurosaki, M.; Pisano, C.; Carminati, P.; Merlini, L.;

Dallavalle, S.; Tavecchio, M.; Rochette-Egly, C.; Terao, M.; Garattini,

E. Antitumor activity of the retinoid related molecules, ST1926 and

CD437, in F9 teratocarcinoma: Role of retinoic acid receptor γ and

retinoid-independent pathways. Mol. Pharmacol. 2006, 70, 909-

924.

(5) Zhang, Y.; Dawson, M. I.; Mohammad, R.; Rishi, A. K.; Farhana,

L.; Feng, K. C.; Leid, M.; Peterson, V.; Zhang, X. K.; Edelstein,

M.; Eilander, D.; Biggar, S.; Wall, N.; Reichert, U.; Fontana, J. A.

Induction of apoptosis of human B-CLL and ALL cells by a novel

retinoid and its nonretinoidal analog. Blood 2002, 100, 2917-2925.

(6) Dawson, M. I.; Harris, D. L.; Liu, G.; Hobbs, P. D.; Lange, C. W.;

Jong, L.; Bruey-Sedano, N.; James, S. Y.; Zhang, X. K.; Peterson,

V. J.; Leid, M.; Farhana, L.; Rishi, A. K.; Fontana, J. A. Antagonist

analogue of 6-[3′-(1-adamantyl)-4′-hydroxyphenyl]-2-naphthalene-

carboxylic acid (AHPN) family of apoptosis inducers that effectively

blocks AHPN-induced apoptosis but not cell-cycle arrest. J. Med.

Chem. 2004, 47, 3518-3536.

(7) Farhana, L.; Dawson, M. I.; Fontana, J. A. Apoptosis induction by

a novel retinoid-related molecule requires nuclear factor-κΒ activa-

tion. Cancer Res. 2005, 65, 4909-4917.

(8) Zhang, Y.; Dawson, M. I.; Ning, Y.; Polin, L.; Parchment, R. E.;

Corbett, T.; Mohamed, A. N.; Feng, K. C.; Farhana, L.; Rishi, A.

K.; Hogge, D.; Leid, M.; Peterson, V. J.; Zhang, X. K.; Mohammad,

R.; Lu, J. S.; Willman, C.; VanBuren, E.; Biggar, S.; Edelstein, M.;

Eilender, D.; Fontana, J. A. Induction of apoptosis in retinoid-

refractory acute myelogenous leukemia by a novel AHPN analog.

Blood 2003, 102, 3743-3752.

(9) Han, Y. H.; Cao, X.; Lin, B.; Lin, F.; Kolluri, S. K.; Stebbins, J.;

Reed, J. C.; Dawson, M. I.; Zhang, X.-K. Regulation of Nur77 nuclear

export by c-Jun N-terminal kinase and Akt. Oncogene 2006, 25,

2974-2986.

(10) Widschwendter, M.; Berger, J.; Muller, H. M.; Zeimet, A. G.; Marth,

C. Epigenetic downregulation of the retinoic acid receptor-beta2 gene

in breast cancer. J. Mammary Gland Biol. Neoplasia 2001, 6, 193-

201.

(11) Lotan, R. Retinoids and their receptors in modulation of differentia-

tion, development, and prevention of head and neck cancers.

Anticancer Res. 1996, 16, 2415-2419.

(12) Lynch, C. J.; Milner, J. Loss of one p53 allele results in four-fold

reduction of p53 mRNA and protein: A basis for p53 haplo-

insufficiency. Oncogene 2006, 25, 3463-3470.

(13) Hollstein, M.; Sidransky, D.; Vogelstein, B.; Harris, C. C. p53

mutations in human cancers. Science 1991, 253, 49-53.

(14) Farhana, L.; Dawson, M. I.; Leid, M.; Wang, L.; Moore, D. D.; Liu,

G.; Xia, Z.; Fontana, J. A. Adamantyl-substituted retinoid-related

molecules bind small heterodimer partner and modulate the Sin3A

repressor. Cancer Res. 2007, 67, 318-325.

(15) Borgius, L. J.; Steffensen, K. R.; Gustafsson, J. A.; Treuter, E.

Glucocorticoid signaling is perturbed by the atypical orphan receptor

and corepressor SHP. J. Biol. Chem. 2002, 277, 49761-49766.

(16) Brendel, C.; Schoonjans, K.; Botrugno, O. A.; Treuter, E.; Auwerx,

J. The small heterodimer partner interacts with the liver X receptor

alpha and represses its transcriptional activity. Mol. Endocrinol. 2002,

16, 2065-2076.

(17) Gobinet, J.; Carascossa, S.; Cavailles, V.; Vignon, F.; Nicolas, J.

C.; Jalaguier, S. SHP represses transcriptional activity via recruitment

of histone deacetylases. Biochemistry 2005, 44, 6312-6320.

(18) Kassam, A.; Capone, J. P.; Rachubinski, R. A. The short heterodimer

partner receptor differentially modulates peroxisome proliferator-

activated receptor alpha-mediated transcription from the peroxisome

proliferator-response elements of the genes encoding the peroxisomal

beta-oxidation enzymes acyl-CoA oxidase and hydratase-dehydro-

genase. Mol. Cell. Endocrinol. 2001, 176, 49-56.

(19) Klinge, C. M.; Jernigan, S. C.; Risinger, K. E.; Lee, J. E.;

Tyulmenkov, V. V.; Falkner, K. C.; Prough, R. A. Short heterodimer

partner (SHP) orphan nuclear receptor inhibits the transcriptional

activity of aryl hydrocarbon receptor (AHR)/AHR nuclear translocator

(ARNT). Arch. Biochem. Biophys. 2001, 390, 64-70.

(20) Seol, W.; Chung, M.; Moore, D. D. Novel receptor interaction and

repression domains in the orphan receptor SHP. Mol. Cell. Biol. 1997,

17, 7126-7131.

(21) Yeo, M. G.; Yoo, Y. G.; Choi, H. S.; Pak, Y. K.; Lee, M. O. Negative

cross-talk between Nur77 and small heterodimer partner and its role

in apoptotic cell death of hepatoma cells. Mol. Endocrinol. 2005,

19, 950-63.

(22) Dhanabal, M.; Jeffers, M.; Larochelle, W. J. Anti-angiogenic therapy

as a cancer treatment paradigm. Curr. Med. Chem. Anticancer Agents

2005, 5, 115-130.

(23) Blagosklonny, M. V. Prospective strategies to enforce selectively cell

death in cancer cells. Oncogene 2004, 23, 2967-2975.

(24) Fenaux, P.; De Botton, S. Retinoic acid syndrome. Recognition,

prevention and management. Drug Saf. 1998, 18, 273-279.

(25) Cheer, S. M.; Foster, R. H. Alitretinoin. Am. J. Clin. Dermatol. 2000,

1, 307-314; discussion 315-316.

(35) Dawson, M. I.; Hobbs, P. D.; Peterson, V. J.; Leid, M.; Lange, C.

W.; Feng, K. C.; Chen, G.; Gu, J.; Li, H.; Kolluri, S. K.; Zhang, X.;

Zhang, Y.; Fontana, J. A. Apoptosis induction in cancer cells by a

novel analogue of 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtha-

lene carboxylic acid lacking retinoid receptor transcriptional activation

activity. Cancer Res. 2001, 61, 4723-4730.

(36) Robertson, K. A.; Emami, B.; Collins, S. J. Retinoic acid-resistant

HL-60R cells harbor a point mutation in the retinoic acid receptor

ligand-binding domain that confers dominant negative activity. Blood

1992, 80, 1885-1889.

(37) Tashima, T.; Kagechika, H.; Tsuji, M.; Fukasawa, H.; Kawachi, E.;

Hashimoto, Y.; Shudo, K. Polyenylidene thiazolidinedione derivatives

with retinoidal activities. Chem. Pharm. Bull. (Tokyo) 1997, 45,

1805-1813.

(38) Ebisawa, M.; Kawachi, E.; Fukasawa, H.; Hashimoto, Y.; Itai, A.;

Shudo, K.; Kagechika, H. Novel thiazolidinedione derivatives with

retinoid synergistic activity. Biol. Pharm. Bull. 1998, 21, 547-549.

(39) Yang, D. Y. 4-Hydroxyphenylpyruvate dioxygenase as a drug

discovery target. Drug News Perspect. 2003, 16, 493-496.

(40) Renaud, J. P.; Rochel, N.; Ruff, M.; Vivat, V.; Chambon, P.;

Gronemeyer, H.; Moras, D. Crystal structure of the RAR-R ligand-

binding domain bound to all-trans retinoic acid. Nature 1995, 378,

681-689.

(41) Klaholz, B. P.; Mitschler, A.; Belema, M.; Zusi, C.; Moras, D.

Enantiomer discrimination illustrated by high-resolution crystal

structures of the human nuclear receptor hRARR. Proc. Natl. Acad.

Sci. U.S.A. 2000, 97, 6322-6327.

(42) Fontana, J. A.; Dawson, M. I.; Leid, M.; Rishi, A. K.; Zhang, Y.;

Hsu, C. A.; Lu, J. S.; Peterson, V. J.; Jong, L.; Hobbs, P.; Chao, W.

R.; Shroot, B.; Reichert, U. Identification of a unique binding protein

specific for a novel retinoid inducing cellular apoptosis. Int. J. Cancer

2000, 86, 474-479.

(43) Shipanova, I. N.; Glomb, M. A.; Nagaraj, R. H. Protein modification

by methylglyoxal: Chemical nature and synthetic mechanism of a

major fluorescent adduct. Arch. Biochem. Biophys. 1997, 344, 29-

36.

Effects of Isosteric Modifications of 3-Cl-AHPC Carboxylate

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2639

(44) Richardson, R. M.; Pares, X.; Cuchillo, C. M. Chemical modification

by pyridoxal 5′-phosphate and cyclohexane-1,2-dione indicates that

Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite

of bovine pancreatic ribonuclease A. Biochem. J. 1990, 267, 593-

599.

(45) Jiang, Z. Y.; Thorpe, C. Modification of an arginine residue in pig

kidney general acyl-coenzyme A dehydrogenase by cyclohexane-

1,2-dione. Biochem. J. 1982, 207, 415-419.

(46) Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling reactions

of organoboron compounds. Chem. ReV. 1995, 95, 2457-2483.

(47) Ek, F.; Manner, S.; Wistrand, L. G.; Frejd, T. Synthesis of fused

tetrazole derivatives via a tandem cycloaddition and N-allylation

reaction and parallel synthesis of fused tetrazole amines. J. Org.

Chem. 2004, 69, 1346-1352.

(48) Mancuso, A. J.; Huang, S.-L.; Swern, D. Oxidation of long-chain

and related alcohols to carbonyls by dimethyl sulfoxide “activated”

by oxalyl chloride. J. Org. Chem. 1978, 43, 2480-2482.

(49) Cho, B. R.; Chajara, K.; Oh, H. J.; Son, K. H.; Jeon, S. J. Synthesis

and nonlinear optical properties of 1,3,5-methoxy-2,4,6-tris(styryl)-

benzene derivatives. Org. Lett. 2002, 4, 1703-1706.

(50) Bouchain, G.; Leit, S.; Frechette, S.; Khalil, E. A.; Lavoie, R.;

Moradei, O.; Woo, S. H.; Fournel, M.; Yan, P. T.; Kalita, A.; Trachy-

Bourget, M. C.; Beaulieu, C.; Li, Z.; Robert, M. F.; MacLeod, A.

R.; Besterman, J. M.; Delorme, D. Development of potential

antitumor agents. Synthesis and biological evaluation of a new set

of sulfonamide derivatives as histone deacetylase inhibitors. J. Med.

Chem. 2003, 46, 820-830.

(51) Doyle, M. P.; Siegfried, B.; Dellaria, J. F., Jr. Alkyl nitrile-metal

halide deamination reactions. 2. Substitutive deamination of aryl-

amines by alkyl nitrites and copper(II) halides. A direct and

remarkably efficient conversion of arylamines to aryl halides. J. Org.

Chem. 1977, 42, 2426-2430.

(52) Musso, D. L.; Clarke, M. J.; Kelley, J. L.; Boswell, G. E.; Chen, G.

Novel 3-phenylprop-2-ynylamines as inhibitors of mammalian squalene

epoxidase. Org. Biomol. Chem. 2003, 1, 498-506.

(53) Brown, H. C.; Subrahmanyam, C.; Hamaoka, T.; Ravindran, N.

Vinylic organoboranes. 13. A convenient stereospecific synthesis of

(Z)-1-halo-1-alkenes from 1-alkynes via (E)-1-alkenylborane deriva-

tives with halogens. J. Org. Chem. 1989, 54, 6068-6075.

(54) Simoni, D.; Rossi, M.; Rondanin, R.; Mazzali, A.; Baruchello, R.;

Malagutti, C.; Roberti, M.; Invidiata, F. P. Strong bicyclic guanidine

base-promoted Wittig and Horner-Wadsworth-Emmons reactions.

Org. Lett. 2000, 2, 3765-3768.

(55) Miyano, M.; Dorn, C. R.; Mueller, R. A. Prostaglandins. IV. A

synthesis of F-type prostaglandins. A total synthesis of prostaglandin

F₁. J. Org. Chem. 1972, 37, 1810-1818.

(56) Zabell, A. P.; Corden, S.; Helquist, P.; Stauffacher, C. V.; Wiest, O.

Inhibition studies with rationally designed inhibitors of the human

low molecular weight protein tyrosine phosphatase. Bioorg. Med.

Chem. 2004, 12, 1867-1880.

(57) Palacios, F.; Aparicio, D.; Vicario, J. Synthesis of quinolinylphos-

phane oxides and -phosphonates from N-arylimines derived from the

phosphane oxides and phosphonates. Eur. J. Org. Chem. 2002, 4131-

4136.

(58) Klicic, J.; Friesner, F.; Liu, S.-Y.; Guida, W. Accurate prediction of

acidity constants in aqueous solution via density functional theory

and self-consistent reaction field methods. J. Phys. Chem. A 2002,

106, 1327-1335.

(59) Li, H.; Kolluri, S. K.; Gu, J.; Dawson, M. I.; Cao, X.; Hobbs, P. D.;

Lin, B.; Chen, G.; Lu, J.; Lin, F.; Xie, Z.; Fontana, J. A.; Reed, J.

C.; Zhang, X. Cytochrome c release and apoptosis induced by

mitochondrial targeting of nuclear orphan receptor TR3. Science 2000,

289, 1159-1164.

(64) Zhu, X. Z.; Yu, Y. Z.; Fang, Y. M.; Liang, Y.; Lu, Q. H.; Xu, R. Z.

[Overexpression of Shp-2 is associated with the unlimited growth

and apoptosis resistance of p210 bcr-abl-mediated chronic myeloid

leukemia]. Zhonghua Yi Xue Za Zhi 2005, 85, 1903-1906.

(65) Tauchi, T.; Miyazawa, K.; Feng, G. S.; Broxmeyer, H. E.; Toyama,

K. A coiled-coil tetramerization domain of BCR-ABL is essential

for the interactions of SH2-containing signal transduction molecules.

J. Biol. Chem. 1997, 272, 1389-1394.

(66) Farhana, L.; Dawson, M. I.; Huang, Y.; Zhang, Y.; Rishi, A. K.;

Reddy, K. B.; Freeman, R. S.; Fontana, J. A. Apoptosis signaling by

the novel compound 3-Cl-AHPC involves increased EGFR proteoly-

sis and accompanying decreased phosphatidylinositol 3-kinase and

AKT kinase activities. Oncogene 2004, 23, 1874-1884.

(67) Tchilian, E. Z.; Beverley, P. C. Altered CD45 expression and disease.

Trends Immunol. 2006, 27, 146-153.

(68) Miyachi, H.; Tanaka, Y.; Gondo, K.; Kawada, T.; Kato, S.; Sasao,

T.; Hotta, T.; Oshima, S.; Ando, Y. Altered expression of CD45

isoforms in differentiation of acute myeloid leukemia. Am. J.

Hematol. 1999, 62, 159-164.

(69) Arsenou, E. S.; Papadimitriou, E. P.; Kliafa, E.; Hountala, M.;

Nikolaropoulos, S. S. Effects of retinoic acid steroidal analogs on

human leukemic HL60 cell proliferation in vitro and on angiogenesis

in vivo. Anticancer Drugs 2005, 16, 151-158.

(70) Dawson, M. I.; Zhang, X.-K. Discovery and design of retinoic acid

receptor and retinoid X receptor class-and subtype-selective synthetic

analogs of all-trans-retinoic acid and 9-cis-retinoic acid. Curr. Med.

Chem. 2002, 9, 623-637.

(71) Peterson, V. J.; Barofsky, E.; Deinzer, M. L.; Dawson, M. I.; Feng,

K. C.; Zhang, X.-K.; Madduru, M. R.; Leid, M. Mass-spectrometric

analysis of agonist-induced retinoic acid receptor γ conformational

change. Biochem. J. 2002, 362, 173-181.

(72) Rao, A. V. R.; Chakraborty, T. K.; Reddy, K. L.; Rao, A. S. An

expeditious approach for the synthesis of â-hydroxylaryl R-amino

acids present in vancomycin. Tetrahedron Lett. 1994, 35, 5043-

5046.

(73) Wittenberger, S. J.; Donner, B. G. Dialkyltin oxide mediated addition

of trimethylsilyl azide to nitriles. A novel preparation of 5-substituted

tetrazoles. J. Org. Chem. 1993, 58, 4139-4141.

(74) Harris, D. L.; DeLorey, T. M. Determinants of recognition of ligands

binding to benzodiazepine receptor/GABA_Areceptors initiating

sedation. Eur. J. Pharmacol. 2000, 401, 271-287.

(75) Harris, D. L.; Loew, G. H. Development and assessment of a 3D

pharmacophore for ligand recognition of BDZR/GABA_Areceptors

initiating the anxiolytic response. Bioorg. Med. Chem. 2000, 8, 2527-

2538.

(76) Stewart, J. J. P. A semiempirical molecular orbital program. MOPAC.

J. Comput.-Aided Mol. Des. 1990, 4, 1-105.

(77) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,

M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin,

K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone,

V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G.

A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;

Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai,

H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.;

Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R.

E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J.

W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.;

Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;

Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghava-

chari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.;

Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.;

Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-

Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill,

P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople,

J. A. Gaussian 03, ReVision C.02, Gaussian, Inc., Wallingford, CT,

2004.

(60) Zhang, Y.; Huang, Y.; Rishi, A. K.; Sheikh, M. S.; Shroot, B.;

Reichert, U.; Dawson, M.; Poirer, G.; Fontana, J. A. Activation of

the p38 and JNK/SAPK mitogen-activated protein kinase pathways

during apoptosis is mediated by a novel retinoid. Exp. Cell. Res. 1999,

247, 233-240.

(61) Pfahl, M.; Piedrafita, F. J. Retinoid targets for apoptosis induction.

Oncogene 2003, 22, 9058-9062.

(62) Schubbert, S.; Lieuw, K.; Rowe, S. L.; Lee, C. M.; Li, X.; Loh, M.

L.; Clapp, D. W.; Shannon, K. M. Functional analysis of leukemia-

associated PTPN11 mutations in primary hematopoietic cells. Blood

2005, 106, 311-317.

(63) Yu, W. M.; Daino, H.; Chen, J.; Bunting, K. D.; Qu, C. K. Effects

of a leukemia-associated gain-of-function mutation of SHP-2 phos-

phatase on interleukin-3 signaling. J. Biol. Chem. 2006, 281, 5426-

5434.

(78) Nangia-Makker, P.; Honjo, Y.; Sarvis, R.; Akahani, S.; Hogan, V.;

Pienta, K. J.; Raz, A. Galectin-3 induces endothelial cell morpho-

genesis and angiogenesis. Am. J. Pathol. 2000, 156, 899-909.

JM0613323

Article Doi

An adamantyl-substituted retinoid-derived molecule that inhibits cancer cell growth and angiogenesis by inducing apoptosis and binds to small heterodimer partner nuclear receptor: Effects of modifying its carboxylate group on apoptosis, proliferation, and

DOI: 10.1021/jm0613323

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm0613323

Full text of DOI:10.1021/jm0613323

Products guided by the article

R&D Labs maybe for 73088-08-1

Relevant to this article

Hot Product