Antagonist analogue of 6-[3′-(1-adamantyl)-4′-hydroxyphenyl]-2- naphthalenecarboxylic acid (AHPN) family of apoptosis inducers that effectively blocks AHPN-induced apoptosis but not cell-cycle arrest

3518

J . Med. Chem. 2004, 47, 3518-3536

An ta gon ist An a logu e of

6-[3′-(1-Ad a m a n tyl)-4′-h yd r oxyp h en yl]-2-n a p h th a len eca r boxylic Acid (AHP N)

F a m ily of Ap op tosis In d u cer s Th a t Effectively Block s AHP N-In d u ced Ap op tosis

bu t Not Cell-Cycle Ar r est

Marcia I. Dawson,*^,†Danni L. Harris,^§Gang Liu,^†Peter D. Hobbs,^‡Christopher W. Lange,^XLing J ong,^‡

Nathalie Bruey-Sedano,^†Sharon Y. J ames,^†Xiao-kun Zhang,^†Valerie J . Peterson,^#Mark Leid,^#Lulu Farhana,^|,∇

Arun K. Rishi,^|and J oseph A. Fontana^|,∇

The Burnham Institute, Cancer Center, 10901 North Torrey Pines Road, La J olla, California 92037;

Molecular Research Institute, 2495 Old Middlefield Road, Mountain View, California 94043; SRI International,

Retinoid Program, 333 Ravenswood Avenue, Menlo Park, California 94025; Oregon State University, College of Pharmacy,

Corvallis, Oregon 97331; and Wayne State University and J ohn D. Dingell Medical Center, Department of Veterans Affairs,

4646 J ohn R Street, Detroit, Michigan 48201

Received October 15, 2003

The retinoid 6-[3′-(1-adamantyl)-4′-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN) and

its active analogues induce cell-cycle arrest and programmed cell death (apoptosis) in cancer

cells independently of retinoic acid receptor (RAR) interaction. Its analogue, (E)-4-[3′-(1-

adamantyl)-4′-hydroxyphenyl]-3-(3′-acetamidopropyloxy)cinnamic acid (3-A-AHPC) selectively

antagonized cell apoptotic events (TR3/nur77/NGFI-B expression and nuclear-to-mitochondrial

translocation) but not the proliferative events (cell-cycle arrest and p21^WAF1/CIP1expression)

induced by proapoptotic AHPN and its analogues. The syntheses of 3-A-AHPC and proapoptotic

(E)-6-[3′-(1-adamantyl)-4′-hydroxyphenyl]-5-chloronaphthalenecarboxylic acid (5-Cl-AHPN) are

described. Computational studies on AHPN, AHPC, and three substituted analogues (5-Cl-

AHPN, 3-Cl-AHPC, and 3-A-AHPC) suggested reasons for their diametric effects on RAR

activation. Density functional theory studies indicated that the 1-adamantyl (1-Ad) groups of

the AHPN and AHPC configurations assumed positions that were nearly planar with the

aromatic rings of their polar termini. In contrast, in the configurations of the substituted

analogues having chloro and 3-acetamidopropyloxy groups, rather than a hydrogen, ortho to

the diaryl bonds, the diaryl bond torsion angles increased so that the 1-Ad groups were oriented

out of this plane. Docking and molecular dynamics of AHPN, AHPC, and these substituted

analogues in the RARγ ligand-binding domain illustrated how specific substituents on the

AHPN and AHPC scaffolds modulated the positions and dynamics of the 1-Ad groups. As a

result, the position of RARγ helix H12 in forming the coactivator-binding site was impacted in

a manner consistent with the experimental effect of each analogue on RARγ transcriptional

activation.

In tr od u ction

interact with RARR, whereas their inhibition of anchor-

age-independent (clonal) growth only correlated with

their transcriptional activation of RARR.⁷Fitzgerald and

co-workers extended these observations of the correla-

tion between growth inhibition by retinoids and RARR

activation to other breast cancer cell lines.⁸Our inability

to observe a correlation between the inhibition of

ovarian cancer cell growth and RAR subtype activation

by AHPN and its analogues (AHPNs)⁹underscored our

hypothesis that AHPN signaling in cancer cells occurred

through a pathway that did not involve the activation

of RARs by this new class of apoptosis-inducing agents.

Although the signaling pathway by which AHPN and

its analogues initiate apoptosis remains to be fully

elucidated, we found¹⁰that it involved the intrinsic

apoptotic pathway of mitochondrial membrane potential

change, mitochondrial cytochrome c release into the

cytosol, followed by the binding of cytochrome c to the

apaf-12 complex that, in turn, activated procaspase-9.

The activated enzyme initiated a caspase cascade of cell

degradation that included irreversible morphological

Previously, we established that the retinoid 6-[3′-(1-

adamantyl)-4′-hydroxyphenyl]-2-naphthalenecarboxylic

acid (CD437¹/AHPN, 1 in Figure 1) inhibited cancer

cell growth and induced cancer cell apoptosis (pro-

grammed cell death) by signaling pathways that did not

involve the direct interaction of AHPN with the retinoic

acid receptor subtypes (RARs) R, â, and γ,^2-5which

function as nuclear transcription factors.⁶AHPN was

first reported as an RARγ-selective retinoid.¹Earlier,

Dawson and co-workers had observed that the levels of

MCF-7 anchorage-dependent cell growth inhibition by

retinoids positively correlated with their abilities to

* To whom correspondence should be addressed. Phone: 858-646-

3165. Fax: 858-646-3197. E-mail: mdawson@burnham.org.

^†The Burnham Institute.

^§Molecular Research Institute.

^‡SRI International.

^||Wayne State University.

^∇Department of Veterans Affairs.

#

Oregon State University.

^XCurrent address: Discovery Partners International, Chem. RX

Division, 385 Oyster Point Blvd., South San Francisco, CA 94080.

Published on Web 06/05/2004

Blocker of AHPN-Induced Apoptosis

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3519

changes, DNA cleavage, nuclear fragmentation, and

other processes indicative of apoptosis.

Our efforts to define this pathway led to the further

discovery of a novel paradigm¹⁰for the extranuclear

behavior of the nuclear receptor TR3/nur77/NGFI-B

(human/mouse/rat TR3)^11-13and retinoid X receptor

(RXR)¹⁴that, like the RARs, functions in the nucleus

as a transcription factor. We established that AHPN and

its analogues induced the expression of TR3 and its

migration from the nucleus to mitochondria,¹⁰where

TR3 modulated the function of the anti-apoptotic protein

Bcl-2, which typically protects mitochondria from apo-

ptotic insults.¹⁵On interacting with TR3, Bcl-2 was no

longer able to protect the cell from apoptosis but

underwent a conformational change that promoted

apoptosis.¹⁶We also discovered that chemotherapeutic

drugs, such as etoposide, were able to induce prostate

cancer cell apoptosis through this pathway.¹⁰

An apoptotic signaling pathway for the AHPNs that

does not involve typical RAR signaling is actually

advantageous. The retinoids currently approved for the

treatment of cancer and other proliferative diseasess

all-trans-retinoic acid (trans-RA, 4), 9-cis-RA (5), 13-cis-

RA, and Targretin/bexarotene (6)shave adverse

effects.^17-29In retinoid-treated patients, these effects

have included hypertriglyceridemia leading to pancre-

atitis, pseudotumor cerebri, RA syndrome (respiratory

failure), hypothyroidism, and teratogenesis, all of which

limit effective dosage and discourage compliance. Tran-

scriptional activation of RARγ by retinoids has been

reported to correlate with retinoid toxicity.^30-33In

addition, transformed cells often lose sensitivity to

growth inhibition by retinoids through the loss of

functional RARs, particularly RARâ, which has been

thought to function as a tumor suppressor.^34-38

F igu r e 1. 6-[3′-(1-Adamantyl)-4′-hydroxyphenyl]-2-naphtha-

lenecarboxylic acid (AHPN, 1); AHPN analogues 5-chloro-

AHPN (5-Cl-AHPN, 2) and 5-methyl-AHPN (5-Me-AHPN, 3);

retinoic acid receptor (RAR)-selective agonist trans-retinoic

acid (trans-RA, 4); RAR and retinoid X receptor (RXR) pan-

agonist 9-cis-retinoic acid (9-cis-RA, 5); RXR-selective retinoids

Targretin (11247, 6), 11173 (7), and 11345 (8); (E)-4-[3′-(1-

adamantyl)-4′-hydroxyphenyl]cinnamic acid (AHPC, 11); AHPC

analogues 3-chloro-AHPC (3-Cl-AHPC, 9), 3-(3′-acetamido-

propyloxy)-AHPC (3-A-AHPC, 10), 3-Me-AHPC (12), 3-EtO-

AHPC (13), and 3-(3′-aminopropyloxy)-AHPC (14); RARâ,γ-

selective (E)-4-(5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetramethyl-2′-

naphthalenyl)cinnamic acid (15), 6-(5′,6′,7′,8′-tetrahydro-

5′,5′,8′,8′-tetramethyl-2′-naphthalenyl)-2-naphthalenecar-

boxylic acid (TTNN, 16), 5-Me-TTNN (17), 7-Me-TTNN (18),

3′-Me-TTNN (19); RARR-selective antagonist Ro41-5253 (20);

RAR-selective agonist 4-(5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetra-

methyl-2′-anthracenyl)benzoic acid (TTAB, 21); and RARγ-

selective antagonist 2-(5′,6′,7′,8′-tetrahydro-5′,5′,8′,8′-tetra-

methyl-2′-naphthalenyl)-2-(6′-carboxynaphthalene)-1,3-dithio-

lane (11253, 22).

Recently, we identified two new apoptotic AHPNs:

6-[3′-(1-adamantyl)-4′-hydroxyphenyl]-5-chloro-2-naph-

thalenecarboxylic acid (5-Cl-AHPN, 2)³⁹and (E)-4-[3′-

(1-adamantyl)-4′-hydroxyphenyl]-3-chlorocinnamic acid

(3-Cl-AHPC, 9).⁴⁰Herein, by using diverse computa-

tional chemistry approaches in combination with bio-

assay results, we present a rationale for the lack of

retinoid transcriptional activity displayed by these

substituted AHPN analogues. We also report the syn-

thesis of the related antagonist, 3-(3′-acetamidopropyl-

oxy)-AHPC (3-A-AHPC, 10) in addition to that of the

apoptosis inducer 5-Cl-AHPN. Our studies showed that

3-A-AHPN blocked the apoptotic signaling pathway

induced by AHPN (1), 5-Cl-AHPN, and 3-Cl-AHPC in

cancer cells and the binding of labeled AHPN to its

putative nuclear receptor⁴without impacting the ability

of AHPN and its proapoptotic analogues to induce cell-

cycle arrest²and the expression of the cyclin-dependent

namic acids were far more readily available than those

for similarly substituted naphthalenecarboxylic acids.

Second, we hypothesized that by replacing the naph-

thalenecarboxylic acid moiety of AHPN with a cinnamic

acid group we would reduce retinoid activity. Prior to

1990, we had synthesized the cinnamic acid 15⁴¹as an

analogue of the retinoid TTNN (16).⁴²This strategy was

not particularly useful for retinoid design as evidenced

by the two-log higher EC₅₀value determined for 15 in

reversing the keratinization of vitamin A-deficient

tracheal epithelium in organ culture (TOC assay) com-

pared to that of TTNN.⁴¹We subsequently found that

the activity of retinoids in the TOC assay^41,43correlated

with their ability to transcriptionally activate the RARs

bound to their RA-responsive elements (RAREs) in the

promoter regions of retinoid-responsive genes. There-

kinase inhibitor p21^{WAF1/CIP1 3}

.

Design /Syn th esis

Shortly after our observation of the apoptotic activity

of AHPN (1),²we undertook the synthesis of analogues,

including 5-Cl-AHPN (2), 3-Cl-AHPC (9), and AHPC

(11), with the goal of removing retinoid activity to

reduce any adverse effects that would be associated with

transcriptional activation of the RARs. We elected to

focus on the E-cinnamic acid scaffold of AHPC for two

reasons. First, synthetic precursors to substituted cin-

3520 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14

Dawson et al.

Ta ble 1. Cancer Cell Apoptosis and Growth Inhibition Activities Induced by AHPN, AHPC, and Analogues Compared to Their

Calculated Inter-Ring Dihedral Angles

apoptosis (%)^a

MDA-MB-231 (96 h)

growth inhibition (%)^b

H460 (144 h)

HL-60R (24 h)

analogue

AHPN (1)

5-Me-AHPN (3)

AHPC (11)

3-Me-AHPC (12)

3-EtO-AHPC (13)

5-Cl-AHPN (2)

3-Cl-AHPC (9)

3-A-AHPC (10)

0.1 × 10^-6

M

1.0 × 10^-6

M

0.5 × 10^-6

M

1.0 × 10^-6

M

0.1 × 10^-6

M

1.0 × 10^-6

M

angle^c(deg)

78

29

90

10

0

62

51

0

98

70

98

95

29

94

0

26

14

53

5

46

-

58

30

-

41

43

1

20

-

80

-

31

29

21

-

d

-

14

56

-

52

71

-

21

31

0

61

51

70

-

a

Average of triplicates ( e 10% for HL-60R leukemia and MDA-MB-231 breast cancer apoptosis as determined by acridine orange

staining. ^bAverage of triplicates ( e10% for H460 lung cancer apoptosis as determined by DAPI staining. AHPN scaffold 5-6-1′-2′ or

c

AHPC scaffold 3-4-1′-2′ dihedral angle determined by AM1. ^dNot determined.

fore, we rationalized that AHPC, the cinnamic acid

analogue of AHPN, would have comparably reduced

retinoid agonist activity. This hypothesis only held for

the activation of RARR by AHPC. Transcriptional acti-

vations of RAR subtypes R, â, and γ on the (TREpal)₂-

tk-CAT reporter induced by 1.0 × 10^-6M AHPC were

8%, 85%, and 103%, respectively, whereas those induced

by 1.0 × 10^-6M AHPN were 21%, 78%, and 88%.

Fortunately, because apoptosis-inducing activity was

retained by AHPC, we pursued further modifications

of the AHPC scaffold. The synthesis and apoptotic

activity of AHPC were recently reported by Cincinelli

et al.⁴⁴

Fortunately, further work indicated that introducing

an electronegative chloro group at a position ortho to

the diaryl bond on the AHPN and AHPC scaffolds

satisfactorily reduced RAR activation activity. Both

5-Cl-AHPN (2)³⁹and 3-Cl-AHPC (9)⁴⁰were efficient

inducers of apoptosis but had minimal ability to activate

the RARs.^39,40Therefore, a means of blocking the ability

of an ANPN analogue to activate the RARs was avail-

able. Moreover, while neither trans-RA (4) nor 9-cis-RA

(5) was able to displace labeled AHPN⁴from the

putative AHPN receptor in HL-60R cell extracts, non-

labeled AHPN (1) and 5-Cl-AHPN (2) were effective

competitive inhibitors.⁴These results provided ad-

ditional evidence that AHPN and analogues such

as 5-Cl-AHPN and 3-Cl-AHPC induced cancer cell

apoptosis through a pathway that did not involve RAR

signaling.

Introducing a methyl group at the 5- or 7-naphthalene

ring position of TTNN (16), which was ortho to the

central diaryl bond, also decreased retinoid agonist

activity, as was evidenced by the higher EC₅₀values for

5-Me-TTNN (17) and 7-Me-TTNN (18) in the TOC assay

compared to that of TTNN.⁴¹The ED₅₀values for both

analogues in inhibiting the induction of mouse epider-

mal ornithine decarboxylase (ODC) by the tumor pro-

moter 12-O-tetradecanoylphorbol-13-acetate were also

higher than that for TTNN.⁴⁵As in the TOC assay, the

inhibitory activities of retinoids in the ODC assay⁴⁵

correlated with their abilities to function as RAR trans-

criptional agonists on an RARE such as the TREpal,⁴⁶

a synthetic RE⁶used to evaluate the transcriptional

activation of both RAR and retinoid X receptor (RXR)

subtypes by retinoids.^47-52The 3′-methyl analogue (19)

of TTNN also had lower activity in the ODC assay.⁴⁵

Moreover, by introducing a 3′-methyl group onto the

tetrahydrotetramethylnaphthalene (TTN) ring of a re-

tinoid that bound RXRs to inhibit its interaction with

the homologous RARs, we successfully obtained highly

RXR-selective retinoids such as 6-8.^51-54The use of this

strategy was also reported by other groups.^55,56On the

basis of these findings, we elected to introduce methyl

groups ortho to the diaryl bridges of AHPN (1) and

AHPC (11). Replacing the TTN ring of TTNN (16) by a

3′-(1-adamantyl)-4′-hydroxyphenyl group was also found

to reduce retinoid agonist activity.¹We combined these

features in 5-Me-AHPN (3), which at 1.0 × 10^-7M had

growth inhibitory activity approaching that of AHPN

on MCF-7 and BT-20 breast cancer cells and was also

not able to activate RARR. However, its activations of

RARâ and RARγ were enhanced.⁵⁷Thus, this strategy

for designing AHPNs lacking RAR transcriptional ac-

tivation activity required modification.

Because access to an antagonist would facilitate

probing AHPN-signaling pathways, we also accom-

plished the design and synthesis of 3-A-AHPC (10). Its

design was based on our observation that the apoptotic

activities of AHPN analogues decreased as the length

of their substituents at the 3-cinnamyl ring and 5-naph-

thyl ring positions increased without comparable de-

creases in their abilities to compete with tritiated AHPN

for binding to the putative AHPN receptor in HL-60R

leukemia and MDA-MB-468 breast cancer cell nuclear

extracts.⁴HL-60R cell apoptosis induced by 24-h treat-

ments with 1.0 × 10^-6M AHPC (11), 3-Me-AHPC (12),

and 3-EtO-AHPC (13) was 98%, 95%, and 29% respec-

tively, of the vehicle-alone treated control (Table 1). The

Fontana group has found that this leukemia cell line

was particularly convenient for assessing apoptosis

induced by AHPN analogues without the interference

of possible interactions with RARs. Standard retinoids

such as trans-RA (4) and 9-cis-RA (5) were unable to

induce apoptosis at similar concentrations because the

RARâ and RARγ proteins were absent and the RARR

mutant present was dysfunctional in competently bind-

ing standard retinoids due to the deletion of its carboxyl

terminus.⁵⁸In MDA-MB-231 breast cancer cells, which

were also resistant to growth inhibition by RAR-selec-

tive retinoids²(data not shown), 96-h treatments with

1.0 × 10^-6M AHPC and 3-Me-AHPC produced 58% and

30% apoptosis, respectively. These results also support

a

retinoid-independent signaling pathway for the

AHPNs, as well as our findings about the impact of the

substitutents ortho to their diaryl bonds on apoptosis.

Blocker of AHPN-Induced Apoptosis

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3521

Sch em e 1^a

a

(a) EtOH, MeSO₃H, PhH, reflux. (b) Br₂, HOAc. (c) BnBr, Na₂CO₃, Me₂CO, Et₂O/THF, -78 °C, reflux. (d) DIBAL, THF, -78 °C;

H₃O⁺. (e) PCC, CH₂Cl₂. (f) [(EtO)₂P(O)CH₂CO₂Et, KN(SiMe₃)₂], -78° to 20 °C. (g) BBr₃, CH₂Cl₂, -78 °C; H₂O. (h) 33, K₂CO₃, Me₂CO,

reflux. (i) (t-Boc)₂O, Et₃N, CH₂Cl₂, 0-20 °C. (j) 1-AdOH, H₂SO₄, CH₂Cl₂. (k) [n-BuLi, THF]; B(Oi-Pr)₃; H₃O⁺. (l) Pd(PPh₃)₄, aq Na₂CO₃,

reflux. (m) H₃O⁺, EtOH, reflux. (n) Ac₂O, py, CH₂Cl₂. (o) NaOH, MeOH, reflux; H₃O⁺.

Sch em e 2^a

The synthesis of 3-Cl-AHPC (9) was previously re-

ported by our group.⁴⁰Antagonist 3-A-AHPC (10) was

synthesized by a similar route, which is shown in

Scheme 1. The challenge in the syntheses of 3-A-AHPC

and its free amine (14) was the presence of three

reactive groups (phenolic hydroxyl, carboxylic acid, and

amide) that required selective protection and deprotec-

tion so that both compounds would be accessible from

intermediate 38. For this reason, the tert-butylcarbonyl

group was used to protect the amine so that its removal

could be achieved in the presence of both benzyl and

ester protecting groups, but the amine group would

remain protected during the removal of the benzyl group

using boron tribromide at -78 °C. Briefly, the diaryl

a

bond of 3-A-AHPC was introduced using a palladium(0)-

catalyzed diaryl coupling of the aryl boronic acid 37³⁹

with the protected (E)-4-bromocinnamate 31. A three-

step sequence allowed the selective removal of the

protecting groups from 38, which was immediately

transformed to 10 and its related amine (14). The latter

will be tethered to a support for use in the affinity

purification of the AHPN receptor protein from HL-60R

nuclear extracts.⁴The E-cinnamyl double bond of in-

termediate 29, from which 31 was derived, was intro-

duced by the Horner-Emmons-Wadsworth olefination

of aldehyde 28 using the anion of triethyl phosphono-

acetate. Isomer 29 was readily separated from a small

amount (<5%) of the related Z-isomer by chromatogra-

phy. The overall yield of the 11-step convergent se-

quence from 23 to 10 was 12%.

(a) EtOH, H₂SO₄, reflux. (b) SO₂Cl₂, HOAc, 70 °C; H₂O. (c)

(CF₃SO₂)₂O, pyridine, CH₂Cl₂, 0-20 °C. (d) 37, Pd(PPh₃)₄, LiCl,

aq Na₂CO₃, DME. (e) BBr₃, CH₂Cl_2,-78 °C; H₂O. (f) aq NaOH,

EtOH, reflux; H₃O⁺.

reported to occur at the R-position.⁵⁹Palladium-cata-

lyzed diaryl coupling of triflate 47, which was derived

from 46, with arylboronic acid 37 and deprotection

provided 5-Cl-AHPN in 22% overall yield for the six

steps beginning with 44.

Com p u ta tion a l Stu d ies

Liga n d Deter m in a n ts of Tr a n scr ip tion a l Activa -

tion . Often, molecular determinants that affect nuclear

receptor transcriptional activation can be due to subtle

effects of the ligand on receptor conformation.^60-63Small

changes in the properties of a ligand group may signifi-

cantly alter either the binding mode of the ligand to the

receptor or the magnitude of the interaction of the

ligand with receptor binding-site residues so as to

profoundly affect conformation. Our docking and mo-

lecular dynamics studies suggested that its large 3-(3′-

The synthesis of 5-Cl-AHPN (2) is outlined in Scheme

2 and resembles that reported for 3-Cl-AHPC (9).⁴⁰The

5-chloro group of 46 was introduced specifically by

R-chlorination of ethyl 6-hydroxy-2-naphthalenecar-

boxylate (45) using sulfuryl chloride, as had been

expected because the chlorination of â-naphthol was

3522 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14

Dawson et al.

acetamidopropoxy) group rendered 3-A-AHPC (10) in-

capable of transcriptionally activating RARγ by prevent-

ing helix H12 from forming the coactivator-binding site.

Therefore, with the binding of the coactivator protein

blocked,⁶⁴the recruitment of the multiprotein complex

responsible for gene transcription could not occur.⁶⁵The

negative effect of the 3-(3′-acetamidopropoxy) group of

3-A-AHPC on RAR activation activity was not surpris-

ing because a methyl group at the 3′-position of the TTN

ring enhanced the RXR selectivity of retinoids, and

longer 3′-alkyl groups decreased RAR transcriptional

activation in analogues of TTNN (16) and other retin-

oids.^66,67For example, Ro-41-5253 (20) having a 3′-

heptyloxy substituent was a potent RARR antagonist,⁶⁶

and the 3′-butyl analogue of 11173 (7) antagonized

RXRR activation by 9-cis-RA (5).⁶⁰Further computa-

tional probing of AHPN and AHPC analogues having

similarly situated substitutents suggested that some

ortho groups prevented RAR agonism by a more subtle

means than by causing large changes in the LBD

conformation that were distinct from those caused by

AHPN (1). Our experimental results supported this

conclusion. The ortho Cl group prevented RARâ and

RARγ transcriptional activation by both 5-Cl-AHPN (2)

and 3-Cl-AHPC (9). In contrast, the similarly sized 5-

and 3-methyl groups of 5-Me-AHPN (3)⁵⁷and 3-Me-

AHPC (12), respectively, did not produce similar effects

on RARs â and γ.

To understand why chloro group substitution ortho

to the diaryl bond in AHPN analogues negatively

impacted RAR activation, whereas a methyl group did

not, computational studies were conducted using both

the structure reported for the crystalline RARγ LBD-

trans-RA (4) complex (Protein Data Bank (PDB) entry

3LBD) and small-molecule ab initio computational

methods. In these studies, electron-correlation effects

were included to accurately determine the torsional

potential surface of each compound, which depicted how

conformational energy varied as a function of torsion

angle size. Because the results of density functional

theory (DFT) calculations on the torsional energy bar-

riers of substituted biphenyls⁶⁸were in good agreement

with experimental values,⁶⁹nonlocal DFT low-energy

conformations and torsional potential surfaces were

determined for AHPN (1), 5-Cl-AHPN (2), 3-Cl-AHPC

(9), 3-A-AHPC (11), and AHPC (12). The torsional

potential surfaces were computed by parametrically

varying the diaryl bond torsion angles while fully

optimizing all other atomic positions for each step. CPK

models of the optimized lowest-energy configurations of

these AHPNs are illustrated in Figure 2A. The torsional

potential surface for each analogue was mapped as a

function of the change in the diaryl bond torsion angle

5-6-1′-2′ for AHPN and 5-Cl-AHPN and 3-4-1′-2′

for AHPC, 3-Cl-AHPC, and 3-A-AHPC, and is depicted

graphically in Figure 2B. The relaxed potential surface

scans indicated that the torsional potential surfaces of

AHPN and AHPC were significantly different from

those of the three substituted analogues. The geometry

optimization and the potential surface results implied

that the preferred shapes for low-energy conformations

of AHPN and AHPC were more planar. Therefore, we

hypothesized that if binding by a compact linear ligand

was required to induce the RARγ conformation neces-

F igu r e 2. Ortho substituents affect the inter-ring (diaryl)

bond twist in AHPN (1) and its analogues. A. Comparison of

DFT geometry-optimized structures of AHPN and 5-Cl-AHPN

(2) (top row), and AHPC (11), 3-Cl-AHPC (9), and 3-A-AHPC

(10) (bottom row). Differences in inter-ring twist angles and,

consequently, structure widths are depicted. Compounds are

shown in space-filling format with C and Cl colored in green,

O in red, N in blue, and H in white. B. Low-energy inter-ring

dihedral angles reflect differences in the torsional potential

surfaces of AHPN, AHPC, and their analogues that are

modulated by substitutents ortho to the diaryl bond. Total

energy calculated for each complex as a variable of the 5-6-

1′-2′ AHPN or 5-Cl-AHPN or the 3-4-1′-2′ 3-Cl-AHPC or

3-A-AHPC torsion angle was plotted. C. Impact of substituents

ortho to the diaryl bond on torsion angles of AHPN, AHPC,

and their analogues and on their RARs R, â, and γ and RXRR

transcriptional activation activities. Notice that on the AHPN

and AHPC scaffolds that are selective for transcriptional

activation of RARâ and RARγ, the chloro group had a far

greater negative impact on receptor activation than a methyl

group.

sary for coactivator binding and transcriptional activa-

tion, the diaryl torsion angles of 5-Cl-AHPN and 3-Cl-

AHPC would have been compressed to such an extent

Blocker of AHPN-Induced Apoptosis

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3523

that the potential energies of their complexes with the

RARγ LBD would have had to rise to energetically

disfavored levels. Consequently, any binding by 5-Cl-

AHPN and 3-Cl-AHPC to the RARγ LBD would favor

‘inactive’ or antagonist-like holo-receptor configurations.

In Figure 2C, the negative correlation between torsional

angle size and RARâ and RARγ transactivation is

presented graphically for six of the analogues listed in

Table 1. As shown, RXRR was not activated by these

analogues, and RARR activation was very low or not

existent.

LBD, as reflected in the relative calculated distances

between their 1-Ad carbons and the RARγ H12 iso-

leucine-412 backbone CR (Figure 3C) and side-chain

CD1 (Figure 3D) carbons during the periods of close

contacts of each ligand with helix H12, were quite

different. The time-dependence plots for the AHPN-

helix H12 contact distances showed only the small

fluctuations characteristic of tight or compact contacts,

whereas those for the 5-Cl-AHPN-helix H12 contact

distances showed the significant structural fluctuations

indicative of a larger range of contact distances. These

results suggested that the time-dependent dynamics of

the conformations of helix H12 in the RARγ LBD when

bound by AHPN or an analogue were significantly

affected by the identity of the substituent ortho to the

diaryl bond in AHPN and its analogues.

Initial docking of 3-A-AHPC (10) to the RARγ LBD

produced low-energy configurations similar to those

observed for 3-Cl-AHPC (9) for the interactions between

the 3-A-AHPC carboxylate and the RARγ LBD arginine-

278 and serine-289. In fact, one such docked configu-

ration showed that the 3′-acetamidopropoxy group (3-

A) of 3-A-AHPC contacted the same helix H12 residues

and that the 3-A acetamido group was oriented toward

the helix H11 arginine-396 (see Figure 3B). In addition

to producing the same contacts as those of AHPN

(shown in Figure 3A), the greatly accentuated twist in

the diaryl bond of 3-A-AHPC produced nonplanar

configurations in which the orientations of its 1-Ad

group permitted close contacts with residues near and

on helix H12, including leucine-416 (Figure 3E).

In Figure 3F are shown portions of the dynamics

trajectory analyses of solvated RARγ LBD complexes

with AHPC (11) and 3-A-AHPC (10), in which periodic

boundary conditions and particle-mesh Ewald summa-

tions of electrostatic interactions were employed to

provide realistic boundary forces, the damping of surface

fluctuations, and the elimination of electrostatic poten-

tial truncation artifacts. These simulations having a

realistic boundary force, which provided fluctuation

damping at the surface of the protein, revealed that the

3-A group of 3-A-AHPC caused larger local fluctuations

in residues proximate to helix H12 than the 3-chloro

group of 3-Cl-AHPC did on the basis the 1-Ad C-

leucine-416(CD2) C distances over time (Figure 3,

panels E and F). These results were clearly analogous

to those shown for AHPN (1) and 5-Cl-AHPN (2) in

Figure 3C. While the compact shape of AHPC allowed

intimate contacts between AHPC and residues 411-416

on helix H12, the twisted shape of 3-A-AHPC produced

perturbations in local dynamics on short subnanosecond

time scales that were significant enough to cause the

transient opening of the LBP to the LBD surface to

allow the entry of “bulk” water. The greater fluctuations

evident in the distance plot for 3-A-AHPC shown in

Figure 3F and the finding that LBP opening to the

surface enabled bulk water entry suggested that the 3-A

group had perturbed local dynamics.

We used molecular dynamics to explicitly test the

hypothesis that binding of 5-Cl-AHPN (2) or 3-Cl-AHPC

(9) would induce an RARγ antagonist conformation. Our

results are presented in Figures 2 and 3. Docking of

5-Cl-AHPN into the RARγ ligand-binding pocket (LBP),

which was obtained from the X-ray crystallographic

structure of the human RARγ LBD-9-cis-RA (5) com-

plex,⁶⁵using the energy-based Larmarkian Genetic

Algorithm (LGA) produced lowest-energy configurations

in which the 5-Cl-AHPN carboxylate group interacted

with the LBD helix H5 arginine-278 (Figure 3A), as had

the 9-cis-RA carboxylate,⁶⁵and with the hydroxyl side-

chain of serine-289, which was located between the two

â-sheet regions adjacent to the LBP (data not shown).

The 3′-(1-adamantyl) (1-Ad) group of docked 5-Cl-AHPN

nestled against the LBP hydrophobic groups of tryp-

tophan-227 on helix H3 and those of isoleucine-412 and

methionine-415 on helix H12 (Figure 3A). The 9-cis-RA

â-cyclogeranylidene ring made similar contacts.⁶⁵In the

5-Cl-AHPN-docking simulation, LBD helices H11 and

H12 formed portions of “the roof” of the LBP, as was

reported for the RARγ LBD-9-cis-RA complex.⁶⁵Dock-

ing of AHPN (1) into the LBP yielded a low-energy

binding configuration comparable to that observed for

5-Cl-AHPN (Figure 3A). These results suggested to us

that the effects of the substituent ortho to the diaryl

bond could not be explained by the initial configuration

of the ligand docked in the holo-RARγ LBD complex but

resulted from time-dependent receptor -ligand complex

conformational effects.

The superposition of the 300-ps molecular dynamics

configurations resulting from the simulations of the

RARγ LBD complexes with AHPN (1) and 5-Cl-AHPN

(2) is shown in Figure 3B. Because the docked configu-

rations of AHPN and 5-Cl-AHPN overlapped signifi-

cantly at their carboxylate termini, only 5-Cl-AHPN and

the nearby LBD helical backbones in both conformations

have been displayed so that backbone differences near

the docked compounds were visible. While the position

of the helix H10 backbone of the RARγ LBD was almost

invariant in the two conformations, portions of the

backbones of helices H11 (residues 393-403) and H12

(residues 410-415) adjacent to the docked compounds

differed appreciably. These differences were also evident

on comparing the distance-time series that tracked the

AHPN and 5-Cl-AHPN-helix contact distances (Figure

3 panels C and D, respectively). Compared to the

5-hydrogen, the 5-chloro group significantly impacted

the compound shape (Figure 2A) and the torsional

potential surface (Figure 2B), both of which, in turn,

impacted the forces on helix H12 (Figure 3A) and its

conformation (Figure 3B). As a result, the time-depend-

ent conformations of their complexes with the RARγ

Biologica l Resu lts

The binding of 3-Cl-AHPC (9) to the RAR subtypes

and RXRR was determined in competition radioligand-

binding experiments using the recombinant receptor

subtype LBDs and their panagonist ligand [³H]9-cis-RA.

3524 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14

Dawson et al.

F igu r e 3. A substituent ortho to AHPN inter-ring bond affects time-dependent contacts between the 3′-(1-Ad) group and RARγ

LBD helix H12. A. Energy-based docking of 5-Cl-AHPN (2) in the RARγ LBD. The lowest energy-based docked 5-Cl-AHPN

orientation (shown in ball-and-stick format without Hs) and residue contacts in the RARγ LBP pocket are shown. The RARγ

LBD-9-cis-RA (5) 3LBD crystal structure⁶⁵was used for docking. The contacts of the 1-Ad group, including those with the isoleucine

(Ile)-412 CD1 carbon of helix H12, are shown. Atom colorations are as in Figure 2A and S is in yellow; only Hs on side-chain

heteroatoms are depicted. B. AHPN (1) and 5-Cl-AHPN have differential effects on the local RARγ LBD backbone conformation.

The 300-ps time-points of MD simulations of AHPN (white) and 5-Cl-AHPN (magenta) in the RARγ LBD were superposed. To

highlight differences, only the backbone atoms of RARγ LBD residues 371-418 and 5-Cl-AHPN are shown. The 3′-(1-Ad) and

4′-OH groups of 5-Cl-AHPN are viewed head-on. AHPN with the exception of the 3′-(1-Ad)-4′-OH-phenyl group had the same

configuration as 5-Cl-AHPN in the LBP. The conformations of helix H10 (375-392) are similar, whereas those for helices H11

(393-403) and H12 (410-415) that are proximate to the 1-adamantyl (1-Ad) contacts are perturbed in the RARγ LBP-5-Cl-

AHPN complex (magenta arrows) compared to the AHPN-bound conformation (gray arrows). C and D. Time dependence of the

distance between the helix H12 isoleucine (Ile)-412 backbone CR (C) or the side-chain CD1 (D) carbon (labeled in panel A) and

the 1-Ad carbons of AHPN (solid line) and 5-Cl-AHPN (dashed line) in simulations of AHPN and 5-Cl-AHPN bound to the RARγ

LBD are depicted graphically. E. Docking demonstrates 3-A-AHPC (10)-RARγ LBD helix H12 contacts. As a result of the larger

diaryl bond dihedral angle, the 3-A side-chain and the 1-Ad group of 3-A-AHPC contact Ile-412 and leucine (Leu)-416, respectively.

Both amino acid residues and 3-A-AHPC are represented in matchstick format with hydrogens shown in gray and other atom

colorations as in Figure 2A. F. Results of molecular dynamics on AHPC (11) and 3-A-AHPC bound to the RARγ LBD. Time

dependence of the distance between the helix H12 Leu-416 side-chain CD2 carbon and the 1-Ad carbons of AHPC (solid line) and

3-A-AHPC (dashed line). In contrast to the small fluctuations caused by close contacts between the AHPC 1-Ad and the Leu-316

CD2 carbons, large structural fluctuations resulted from the contacts between the 3-A-AHPC 1-Ad and Leu-316 CD2 carbon

atoms.

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J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3525

Compared to nonlabeled 9-cis-RA (5), 3-Cl-AHPC (both

at 1.0 × 10^-6M) only marginally displaced [³H]9-cis-

RA bound to RARR, RARâ, and RXRR (31%, 17%, and

16% displacement, respectively; Figure 4A), but quite

effectively displaced [³H]9-cis-RA bound to RARγ (83%

displacement; Figure 4A). These results suggested that

3-Cl-AHPC efficiently and selectively competed with

[³H]9-cis-RA for binding to RARγ.

The binding of a coactivator protein to its site on the

surface of a nuclear receptor permits the recruitment

of the multiprotein complex that initiates gene tran-

scription. X-ray crystallographic studies demonstrated

that on binding a ligand capable of inducing transcrip-

tional activation, the RARγ LBD underwent an ap-

preciable conformational change so that its helix H12

moved from an extended conformation to one that

formed the coactivator binding site with helices H3 and

H4 on the LBD surface.⁶⁵Thus, an RARγ agonist

induced a change in the apo-RARγ LBD conformation

to one that was more compact and protease-resistant,

which was detectable by using the differential protease

sensitivity assay (DPSA) coupled with mass spectral

identification of the peptide fragments resulting from

limited proteolysis.⁷⁰The ability of 3-Cl-AHPC (9) to

induce a conformational change in the [³⁵S]methionine-

labeled RARγ ([³⁵S]RARγ) LBD that was indicative of

agonist activity was evaluated by DPSA to determine

if the peptide fragments produced would be the same

as those from the LBD bound by the agonist 9-cis-RA

(5). Incubation of the LBD with 9-cis-RA (1.0 × 10^-6

M) followed by partial tryptic digestion produced two

protease-resistant fragments, PF30 and PF31 (compare

Figure 4B lanes 2-6 showing the proteolysis profile of

the apo-RARγ LBD with lanes 12-16 showing that of

the holo-LBD-9-cis-RA complex). PF30 and PF31 cor-

responded to the RARγ LBD Glu¹⁷³-Lys⁴⁵¹and Glu¹⁷⁰

-

Lys⁴⁵¹sequences, respectively.⁷⁰3-Cl-AHPC (5.0 × 10^-6

M) also induced the formation of PF30 and PF31

(compare Figure 4B lanes 2-6 with lanes 7-11 showing

the proteolytic profile of the holo-LBD-3-Cl-AHPC

complex). Interestingly, the fragments induced by 3-Cl-

AHPC binding appeared to be somewhat less stable

to proteolytic digestion than the fragments induced by

the binding of 9-cis-RA (compare lanes 10 and 11 with

lanes 15 and 16). Nonetheless, the 3-Cl-AHPC-bound

[³⁵S]RARγ LBD was clearly more resistant to tryptic

digestion than the apo-[³⁵S]RARγ. Thus, the results of

the DPSA studies suggested that binding by 3-Cl-AHPC

and 9-cis-RA induced grossly similar conformational

changes within the LDB of RARγ.

The RARγ comformational change that occurred on

binding by a retinoid agonist was found to lead to the

dissociation of an apo-receptor-associated corepressor

protein such as NCoR, followed by the recruitment of a

transcriptional coactivator protein such as p300.⁷⁰There-

fore, corepressor dissociation and coactivator recruit-

ment studies were conducted to determine whether

binding by 3-Cl-AHPC (9) promoted and/or stabilized a

RARγ conformation that disfavored corepressor binding

but favored coactivator binding in a manner similar to

that of the classical RAR transcriptional agonists such

as TTAB (21).⁷¹Both corepressor (NCoR 2110-2453)

and coactivator (p300 1-450) proteins were expressed

as glutathione S-transferase (GST) fusions in bacteria,

F igu r e 4. 3-Cl-AHPC (9) lacks RARγ agonist activity in vitro.

A. Competition radioligand-binding experiments. Recombi-

nant, bacterially expressed RAR subtype and RXRR LBDs

(approximately 1.0 × 10^-8M) were incubated with 1.0 × 10^-9

M [11,12-³H₂]9-cis-RA (43 Ci/mmol) with or without 1.0 × 10^-9

M nonlabeled 9-cis-RA (5) or 3-Cl-AHPC as indicated. Bound

and free radioligand were separated by gel filtration using

Sephadex G-50. B. 3-Cl-AHPC-bound [³⁵S]RARγ LBD is less

susceptible to protolytic degradation than the apo-LBD. DPSAs

were conducted using [³⁵S]RARγ LBD preincubated with either

vehicle (DMSO, 0.1% v/v), 9-cis-RA (1.0 × 10^-6M), or 3-Cl-

AHPC (5.0 × 10^-6M) as indicated. The receptor was then

digested with increasing concentrations of trypsin-TPCK (10,

100, 250, 500, and 1000 µg/mL) for 15 min at 22 °C. Trypsin

was inactivated, and the digest was subjected to electrophore-

sis on a denaturing gel and autoradiography. C. 3-Cl-AHPC-

induced conformational change is not sufficient to induce either

the dissociation of corepressor from or the recruitment of

coactivator to the [³⁵S]RARγ LBD. GST-pulldown experiments

were conducted using [³⁵S]RARγ LBD and either GST-NCoR

2110-2453 or GST-p300 1-450 as indicated in the presence

of Me₂SO (0.1% v/v), TTAB (21) (1.0 × 10^-6M), or 3-Cl-AHPC

(5.0 × 10^-6M) as indicated. [³⁵S]RARγ bound to GST-fusion

proteins was visualized by gel electrophoresis and auto-

radiography. The radioligand binding data shown in A repre-

sents the mean ( SEM of three independent experiments. The

autoradiographs shown in B and C are representative of three

and four independent experiments, respectively.

purified, and used as affinity matrixes for the binding

of the [³⁵S]RARγ LBD in standard GST-pulldown ex-

periments, as we previously described.⁷⁰TTAB strongly

3526 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14

Dawson et al.

F igu r e 6. 3-A-AHPC (10) does not block 3-Cl-AHPC (9)-

mediated inhibition of MDA-MB-468 breast cancer cell pro-

liferation. Cells (1.0 × 10⁶) were incubated for 24 h before the

indicated concentrations of 3-A-AHPC were added. After 2 h,

0.5 × 10^-6M 3-Cl-AHPC was added, and incubation was

continued for 72 h. Cells were then harvested, and proliferation

in the presence of 3-A-AHPC and 3-Cl-AHPC was assessed

using a colorimetric assay (see Experimental Section) and

expressed relative to that of the 3-Cl-AHPC alone-treated

control.

activate RARγ and RXRR (Figure 5 panels A and B,

respectively).

As cancers progress to more aggressive phenotypes,

the regulatory controls that hormones and retinoids

exert on their proliferation can be lost. For example,

unlike the MCF-7 breast cancer cell line, the growth of

MDA-MB-231 and MDA-MB-468 breast cancer cell lines

is neither regulated by estrogen⁷²nor inhibited by

standard retinoids.^2,34Thus, the latter two cancer cell

lines represent more aggressive tumors and have been

used to evaluate compounds for apoptotic activity that

would not be mediated by retinoid-signaling pathways.

F igu r e 5. Transcriptional activation of RARγ by 5-Cl-AHPN

(2) and 3-A-AHPC (10) compared to trans-RA (4) and that of

RXRR compared to 9-cis-RA (5) on the (TREpal)₂-tk-CAT

reporter in transfected CV-1 cells. Cells were treated with 1.0

× 10^-7M or 1.0 × 10^-6M 5-Cl-AHPC, 3-A-AHPC or trans-

RA; or 1.0 × 10^-8M or 1.0 × 10^-7M 9-cis-RA. Reporter gene

activity was normalized to that of cotransfected â-galactosidase

gene activity and expressed relative to that of 1.0 × 10^-6

M

trans-RA on RARγ or 1.0 × 10^-7M 9-cis-RA on RXRR as 100%

as described in Methods. See Experimental Section for meth-

odology.

In addition, both cell lines lack functional p53,^2,72

a

tumor suppressor protein involved in regulating the G₁-

checkpoint of the cell cycle. p53 has been found to induce

cell-cycle arrest in part through its induction of the

expression of cell-cycle inhibitor p21^WAF1/CIP1, thereby

permitting a cell to undergo repair in response to a

stress event or to commit apoptosis. The induction of

p21^WAF1/CIP1that is independent of p53 status⁷³is

therapeutically advantageous because in approximately

50% of tumor samples, including those from breast

cancer patients, p53 is absent or present as a nonfunc-

tional mutant.^72,74,75In the MDA-MB-231 and MDA-

MB-468 breast cancer cell lines, AHPN (1)²and 3-Cl-

AHPC (9) were found to induce cell-cycle arrest in G₀/

G₁through their posttranscriptional stabilization of

p21^WAF1/CIP1message and protein,³followed by apopto-

sis.^2,3

In both breast cancer cell lines, the antagonist 3-A-

AHPC (10) alone had no effects on cell-cycle arrest and

apoptosis induction, whereas both processes were readily

induced by AHPN (1) and its apoptotic analogues such

as 5-Cl-AHPN (2) and 3-Cl-AHPC (9). Even at a

concentration as high as 4.0 × 10^-6M, 3-A-AHPC after

48 or 72 h had only a minimal effect on the MDA-MB-

468 cell cycle as determined by counting cell numbers

(data not shown). Moreover, 3-A-AHPC at an 8-fold

higher concentration was not able to block the ability

of 0.5 × 10^-6M 3-Cl-AHPC to inhibit MDA-MB-468 cell

proliferation (Figure 6), as evidenced by its inability to

induced both the dissociation of GST-NCoR from

[³⁵S]RARγ (compare lanes 1 and 2 in Figure 4C) and

the binding of GST-p300 to [³⁵S]RARγ (compare lanes

5 and 6). In sharp contrast, 3-Cl-AHPC neither induced

the dissociation of GST-NCoR from the [³⁵S]RARγ LBD

(compare lanes 1 and 3) nor the binding of GST-p300

to [³⁵S]RARγ (compare lanes 5 and 7). These findings

indicate that, although 3-Cl-AHPC bound to RARγ and

enhanced its proteolytic stability, the type of RARγ LBD

conformational change caused by 3-Cl-AHPC was not

sufficient to induce either NCoR dissociation or p300

recruitment. In this respect, the behavioral profile of

3-Cl-AHPC resembled that observed for 2-(5′,6′,7′,8′-

tetrahydro-5′,5′,8′,8′-tetramethyl-2′-naphthalenyl)-2-(6′-

carboxy-2′-naphthalenyl)-1,3-dithiolane (22), which we

previously characterized as an RARγ-selective retinoid

that effectively antagonized the transactivation of RARγ

by such agonists as trans-RA (4).⁷⁰Subsequently, we

found that the lack of RAR transcriptional agonist

activity in 3-Cl-AHPC was supported by the results of

reporter assays in CV-1 cells transfected with the

(TREpal)₂-tk-CAT construct and a vector for RARR, â,

or γ or RXRR. 3-Cl-APHC at 1.0 × 10^-7M and 1.0 ×

10^-6M was transcriptionally inactive (data not shown).

Similarly, both 5-Cl-AHPN (2) and 3-A-AHPC (10) at

the same concentrations were unable to effectively

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J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3527

cancer cells (Figure 8C). Even after 72 h, 3-A-AHPC

alone had no apoptotic activity in HL-60R cells and had

only about 6% of the apoptotic activity of AHPN against

MDA-MB-231 cells, a level which we have found was

within background limits in these cells. These results

substantiate our earlier findings that AHPN and its

analogues function through two signaling pathways in

cancer cellsscell-cycle arrest and apoptosis,^{2,10,73,76,77}of

which only apoptosis is inhibited by the antagonist 3-A-

AHPC.

F igu r e 7. 3-A-AHPC (10) does not block the induction of

p21^WAF1/CIP1by 3-Cl-AHPC (9) in MDA-MB-468 breast cancer

cells. Cells were grown in the presence or absence of 0.5 ×

10^-6M 3-Cl-AHPC or 2.0 × 10^-6M 3-A-AHPC for 72 h, then

harvested. Western blots using anti-p21^WAF1/CIP1antibody were

performed³(see experimental methods). â-Actin was used as

the loading control.

We also evaluated the effects of 5-Cl-AHPN (2), 3-Cl-

AHPC (9), and 3-A-AHPC (10) on H460 lung cancer and

LNCaP prostate cancer cell lines, both of which are

retinoid-sensitive and express wild-type p53 (Figure 9).

5-Cl-AHPN at 1.0 × 10^-6M induced their apoptosis at

36 h, which was partially suppressed (34% to 9%, and

31% to 15%, respectively) by an equal concentration of

antagonist 3-A-AHPC, as is indicated by staining for

nuclear fragmentation (Figure 9A). A 5-fold excess of

3-A-AHPC was able to decrease 1.0 × 10^-6M 3-Cl-

AHPC-induced apoptosis in H460 cells from 77% to 5%

(Figure 9B). AHPN (1), 5-Cl-AHPN, and 3-Cl-AHPC at

1.0 × 10^-6M were comparably effective at inducing

halt cell-cycle arrest. Next, we examined the effect of

3-A-AHPC on the abilities of AHPN and 3-Cl-AHPC to

induce the expression of p21^WAF1/CIP1protein in MDA-

MB-468 cells (Figure 7). Alone, 3-A-AHPC had no

detectable effect on p21^WAF1/CIP1protein levels and did

not block the induction of p21^WAF1/CIP1by 3-Cl-AHPC

(Figure 7). However, 3-A-AHPC markedly inhibited

MDA-MB-468 cell apoptosis induced by 3-Cl-AHPC or

AHPN (Figure 8A). Antagonist 3-A-AHPC also inhibited

apoptosis induced by AHPN in retinoid-resistant HL-

60R leukemia cells (Figure 8B) and MDA-MB-231 breast

F igu r e 8. 3-A-AHPC (10) blocks 3-Cl-AHPC (9) and AHPN (1)-induced apoptosis in (A) MDA-MB-468 breast cancer, (B) HL-60R

leukemia, and (C) MDA-MB-231 breast cancer cell lines. Cells were grown in the presence or absence of 0.5 × 10^-6M 3-Cl-AHPC

or 0.5 × 10^-6M AHPN alone, with or without added 2.0 × 10^-6M 3-A-AHPC. Cells were harvested at 24, 48, or 72 h and acridine

orange-stained.³Apoptotic cell numbers were determined (see methods).

F igu r e 9. 3-A-AHPC (10) inhibits cancer cell apoptosis induced by AHPN analogues. A. H460 lung cancer and LNCaP prostate

cancer cells were treated for 36 h with 1.0 × 10^-65-Cl-AHPN (2) or 1.0 × 10^-6M 3-A-AHPC alone or combined, then DAPI-

stained (see methods) for visualization of nuclear morphology by fluorescence microscopy. Apoptosis levels were determined in

200-cell samples in three different fields. B. H460 cells were treated with 3-Cl-AHPC (9) (1.0 × 10^-6M) alone or combined with

3-A-AHPC (1.0 × 10^-6M, 2.5 × 10^-6M, or 5.0 × 10^-6M) or 3-A-AHPC (5.0 × 10^-6) alone or vehicle alone for 36 h, then DAPI-

stained (see methods) and examined as in A to determine apoptosis.

3528 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14

Dawson et al.

compete with AHPN (1) for binding, and 9-cis-RA (5)

did not effectively displace bound [5,5′-³H₂]AHPN.⁴We

next examined whether 3-A-AHPC (10) could inhibit the

binding of [5,5′-³H₂]AHPN to HL-60R nuclear extracts.

As shown in Figure 11, 3-A-AHPC had efficacy similar

to that shown by AHPN in displacing the tritiated label.

Similar results were observed using 3-Cl-AHPC (9)

(data not shown). Thus, 3-A-AHPC is capable of inter-

acting with the same protein as apoptotic AHPN and

3-Cl-AHPC.

F igu r e 10. 3-A-AHPC (10) inhibits TR3 mRNA expression

induced by 5-Cl-AHPN (2) in H460 lung cancer cells. Cells

were treated for 4 h with 5-Cl-AHPN (1.0 × 10^-6M) or 3-A-

AHPC (1.0 × 10^-6or 5.0 × 10^-6M) alone or combined. Total

mRNAs were prepared and analyzed for TR3 expression by

Northern blotting (see methods). Ethidium bromide-stained

ribosomal RNA was used as the loading control.

Discu ssion

While the configurations derived from dynamics in

model simulations of AHPN (1) and 5-Cl-AHPN (2)

docked in the RARγ LBP⁶⁵could not be characterized

as “holo” (bound) or “apo” (nonbound) in character, they

did suggest to us that dramatic differences in local

dynamics affected the recognition of a coactivator or

corepressor protein by the receptor-ligand complex. Our

computational results also suggested that small changes

in the nature of substituents ortho to the diaryl bond

in AHPN and its analogues impacted the positions of

their 1-Ad groups relative to their polar termini (Figure

2A). Thus, in contrast to hydrogen and methyl groups,

chloro groups significantly affected the inter-aromatic

ring torsional potential surfaces so that they, in turn,

altered the positions and dynamics of the 3′-(1-Ad)

groups. In contrast, the carboxylate groups in these

compounds remained anchored to the RARγ LBD helix

H5 arginine-278.

DFT energy-minimized configurations of AHPN (1),

5-Cl-AHPN (2), 3-Cl-AHPC (9), 3-A-AHPC (10), and

AHPC (11) are shown in Figure 2A. Our docking studies

suggested that (i) 5-Cl-AHPN as initially docked was

tightly packed into the LBP with close contacts to RARγ

helix H12 residues; (ii) a chloro group ortho to the diaryl

bond in an AHPN analogue produced a larger twist

angle between the aryl rings than did an ortho hydrogen

or methyl group; and (iii) the larger angle caused the

3′-(1-Ad) group to more frequently occupy positions that

resulted in unfavorable interactions with the RARγ LBD

helix H12. These in silico results suggested that both

the position of the 3′-(1-Ad) group and the effective

compound width were significantly affected by the

identity of the substituent ortho to the diaryl bond on

an AHPN or AHPC scaffold, which, in turn, influenced

the dynamics of regions in the RAR LBD that were

responsible for transcriptional response recognition.

Our experimental results supported the molecular

dynamics results. Earlier, we observed that transcrip-

tional agonist binding to the RARγ LBD produced a

more compact tertiary structure that was more resistant

to limited proteolytic digestion than that of the apo-

receptor,⁵whereas binding by an antagonist produced

a more open configuration that was more sensitive to

proteolysis. The chloro group ortho to the AHPN diaryl

bond reduced the ability of the AHPN analogue 3-Cl-

AHPC (9) to interact with the RARγ LBD in terms of

both its binding affinity to RARγ and the proteolytic

stability of the resulting complex (Figure 4) compared

to that formed by a standard retinoid agonist such as

9-cis-RA (5). Although the same peptide fragments were

produced from both complexes, those from the 3-Cl-

AHPC-containing complex were found to be less stable.

F igu r e 11. 3-A-AHPC (10) inhibits [5,5′-³H₂]AHPN binding

to the AHPN receptor. Nuclear extracts were prepared from

HL-60R cells as we described.⁴[³H₂]AHPN (1.0 × 10^-8M,

150 000 dpm) was added to the nuclear extracts in the

presence or absence of nonlabeled AHPN (1) or 3-A-AHPC (2.0

× 10^-5M) or vehicle alone. Binding was assessed as described

in the methods.

apoptosis in retinoid-resistant, p53-deficient H292 hu-

man lung cancer cells after a 40-h treatment (data not

shown). Thus, the apoptotic activity found for these

compounds in retinoid-refractory, p53-nonfunctional

breast cancer cells extended to similarly deficient lung

cancer cells, in addition to retinoid-sensitive breast,

lung, and prostate cancer cells.

Earlier, we established¹⁰a novel pathway by which

prostate and lung cancer cells underwent apoptosis

induced by AHPN (1) and its apoptotic analogues,

namely induction of the expression of the nuclear

transcription factor TR3 followed by its translocation

from the nucleus to the mitochondrial membrane. The

association between TR3 and mitochondria was followed

by mitochondrial outer membrane permeabilization and

the release of mitochondrial cytochrome c into the

cytosol,^15,78leading to caspase activation and apopto-

sis.⁷⁹As indicated in Figure 10, antagonist 3-A-AHPC

(10) alone had no effect on the expression of TR3 mRNA

in H460 cells but was able to inhibit the induction of

TR3 expression by 5-Cl-AHPN (2).

By using binding assays and Scatchard analysis, we

previously demonstrated that [5,5′-³H₂]AHPN, which we

had synthesized,⁴bound to a novel protein(s) contained

in both HL-60R and MDA-MB-231 cell nuclear extracts.⁴

Our results strongly suggested that this protein was not

an RAR or RXR subtype because the RAR/RXR panago-

nist [10,11-³H₂]9-cis-RA was not able to effectively

Blocker of AHPN-Induced Apoptosis

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3529

This result suggested that the peptides had different

tertiary structures despite their originating from the

cleavage of the same lysine-451 amide bond in the

C-terminus of RARγ (domain F). Evidently, the subtle

differences in the positions of the RARγ domain E helix

H12 that occurred on binding these ligands, which were

suggested by our molecular dynamics studies, did not

sufficiently impact the tertiary structure of domain F

so as to affect the enzymatic cleavage point but appar-

ently made a less dramatic impact. The affinity of 3-Cl-

AHPC (9) for the RARγ LBD was apparently lower than

that of 9-cis-RA because a higher concentration of 3-Cl-

AHPC was required to produce the same level of

proteolysis obtained using 9-cis-RA. Moreover, 3-Cl-

AHPC was not able to induce the dissociation of co-

repressor NCoR from the RARγ LBD or the recruitment

of coactivator p300. Thus, subtle differences in the ortho

substituent were able to control the degree of retinoid

agonist character of the AHPNs bound in the RAR LBP,

as was also evidenced by the contrasting impacts of the

ortho methyl and chloro groups on RARγ transcriptional

activation as reported by Dawson et al.⁵⁷and shown in

Figure 4, respectively, despite their similar sizes. Short

time-scale dynamics studies (Figure 3, panels C and D)

suggested that the configuration of helix H12 in RARγ

bound to 5-Cl-AHPN (2) was less compact and produced

the larger fluctuations more characteristic of those

found in the apo-RARγ LBD than that of H12 in the

RARγ LBD-AHPN (1) complex. The effect of the 3-(3′-

acetamidopropoxy) group of 3-A-AHPC on the inter-ring

torsion angle was even more pronounced (Figure 2,

panels A and B), as was its impact on the local

fluctuations of residues near and on helix H12 (Figure

3, panels E and F). Thus, these computational results

provide a distinct structure-activity linkage for our

substituted AHPN/AHPC analogues that may be har-

nessed to hamper the formation of the binding site for

RAR transcriptional coactivators and minimize RAR-

related toxic adverse effects while retaining apoptotic

activity.

of TR3,^10,16,77,98which has an essential role in the

apoptosis-signaling pathway induced by AHPN ana-

logues, was also inhibited by 3-A-AHPC. However, 3-A-

AHPC did not suppress AHPN or 3-Cl-AHPC-induced

p21^WAF1/CIP1protein expression and cell-cycle arrest.

These results suggest to us that the inhibition of cell

proliferation, as measured by the blockade of cell-cycle

progression, and the induction of apoptosis, as measured

by cell morphological changes, that are induced by active

members of the AHPN family represent two separate

events or perhaps even separate signaling pathways.

Other evidence supporting our premise of dual path-

ways exists in reports of cancer cell line mutants in

which only one of these AHPN-sensitive pathways

operates.^98,102

The identification of an antagonist of AHPN-induced

apoptosis, 3-A-AHPC (10), that is structurally and

configurationally similar to the apoptotic analogues

5-Cl-AHPN (2) and 3-Cl-AHPC (9) strongly suggests

that several earlier hypotheses on the mechanism by

which AHPN initiates apoptosis may not be valid. First,

because both the inducers and the inhibitor of apoptosis

have similarly situated phenolic hydroxyl groups, the

premise that AHPN-induced apoptosis occurs solely as

the result of nonspecific free-radical generation^103,104

appears unlikely. Nonspecific mitochondrial membrane

permeabilization has also been postulated as an AHPN

signaling pathway.^105,106Our findings that 3-A-AHPC

blocks both 5-Cl-AHPN-induced apoptosis and TR3

expression are strong evidence that the apoptosis-

initiating event probably occurs upstream of any direct

effects on the mitochondrial membrane. With 3-A-AHPC

available, we now have an excellent opportunity to

dissect these separate AHPN-signaling pathways: (i)

the 3-A-AHPC-independent suppression of cell prolif-

eration by the induction of cell-cycle arrest; and (ii) the

induction of TR3-mediated apoptosis, which is blocked

by 3-A-AHPC.

Exp er im en ta l Section

Ch em istr y. Gen er a l. Starting materials were obtained

from commercial sources. Unless otherwise mentioned, during

workup procedures organic layers were washed with water and

sat. brine, dried (anhydrous Na₂SO₄), filtered, and concen-

trated at reduced pressure. Standard column chromatography

employed silica gel (Merck 60) as did flash chromatography

(Merck, grade 9385, 230-400 mesh). Experimental procedures

were not optimized and were typically conducted only twice.

Melting point temperatures were determined in capillary tubes

using a Mel-Temp II apparatus and are uncorrected. Fourier

transform IR spectra were obtained on powdered samples,

unless otherwise specified, using an FT-IR Mason satellite

infrared spectrophotometer. ¹H NMR spectra were recorded

on a 300-MHz Varian Unity Inova spectrometer unless oth-

erwise indicated, whereas 400 MHz spectra were recorded on

a Bruker instrument. Shift values are expressed in ppm (δ)

relative to Me₄Si as the internal standard. Unless otherwise

mentioned, spectra were run on compounds dissolved in

²HCCl₃. MALDI-FAB mass spectra were obtained by using

an Applied Biosystems Voyager De-Pro MALDI-TOF instru-

ment at the Institute, and MALDI-FTMS high-resolution

mass spectra on an IonSpec Ultima instrument at The Scripps

Research Institute (La J olla, CA).

In addition to their apoptotic activity in breast

cancer,^2,3,80-82lung cancer,^5,77,83-86and leukemia cell

lines,^{4,5,39,40,87-89}AHPN (1) and its analogues in-

hibited the growth and/or induced the apoptosis of other

cancer cell lines, including those derived from cervical,⁹⁰

esophageal,⁹¹lymphoma,⁹²melanoma,^93,94neuroblas-

toma,⁹⁵ovarian,^9,96-98and prostate cancers.^10,99-101

AHPN-induced apoptosis in breast cancer cells was

preceded by the expression of the cyclin kinase inhibitor

p21^WAF1/CIP1and G₀/G₁cell-cycle arrest and was inde-

pendent of the functional status of p53,^2,3which has

been found to be mutated or lost in approximately half

of all tumors.^74,75Thus, AHPN represents an important

progenitor for identifying more effective agents that

induce cancer cell apoptosis. This therapeutic strategy

is supported by a recent finding by the Lotan group that

lung cancer cells were far more sensitive to AHPN than

the normal cells from which they originated⁸⁶and our

own promising in vivo findings using 3-Cl-AHPC (9) in

a murine acute myelogenous leukemia model.^39,40

Eth yl 4-Br om o-3-h yd r oxyben zoa te (25). According to a

reported procedure,¹⁰⁷MeSO₃H (2.0 mL) was added to 23 (5.0

g, 0.036 mol) in EtOH (10 mL) and benzene (100 mL). The

reaction mixture was heated at reflux (10 h, Dean-Stark trap),

cooled, washed (water, 5% NaHCO₃, and sat. brine), dried, and

We found that 3-A-AHPC (10) inhibited the induction

of apoptosis by AHPN (1) and 3-Cl-AHPC (9) and the

binding of both to the putative AHPN receptor in HL-

60R and MDA-MB-231 nuclear extracts. The expression

3530 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14

Dawson et al.

concentrated to a solid, which was chromatographed (10%

EtOAc/hexane) to give ethyl ester 24 (5.65 g, 94%) as a white

powder, mp 71-73 °C, which was used without further

purification.

(1.5 mmol) in CH₂Cl₂(1.5 mL) was stirred at -78 °C under

Ar for 2 h, then diluted with water (10 mL) and CH₂Cl₂(20

mL). The organic phase was washed, dried, and concentrated.

Flash chromatography (10% EtOAc/hexane) gave 30 as a white

powder (180 mg, 92%), mp 135-137 °C. TLC (9% EtOAc/

A reported procedure for 4-bromo-3-hydroxybenzoic acid¹⁰⁸

was modified to substantially improve the yield of 25. To a

stirred solution of 24 (332 mg, 2.0 mmol) in glacial HOAc (2.0

mL) was slowly added Br₂(320 mg, 2 mmol) in HOAc (1.0 mL)

with stirring at room temperature. After stirring for 6 h,

additional Br₂(90 mg, 0.56 mmol) in HOAc (0.3 mL) was

added, and stirring was continued (14 h). Ether (50 mL) was

added, and the organic layer was washed (water, 5% NaHCO₃,

and sat. brine), dried, and concentrated to an oil. Chromatog-

raphy (8% EtOAc/8% CH₂Cl₂/hexane) gave 25 as a white

powder (260 mg, 53%), mp 90-92 °C. TLC (17% EtOAc/

1

hexanes) R_f0.25. FT-IR (CHCl₃) 3328, 1678 cm^-1. H NMR δ

1.34 (t, J ) 6.9 Hz, 3H, CH₃), 4.27 (q, J ) 7.2 Hz, 2H, OCH₂),

5.62 (s, 1H, OH), 6.42 (d, J ) 15.9 Hz, 1H, ArCHdC), 6.98 (d,

J ) 8.4 Hz, 1H, 6-ArH), 7.18 (s, 1H, 2-ArH), 7.48 (d, J ) 8.1

Hz, 1H, 5-ArH), 7.58 ppm (d, J ) 15.9 Hz, 1H, CdCHCO₂).

MALDI-FTMS (HRMS) calcd C₁₁H₁₂BrO₃[MH⁺] 270.9970,

found 270.9971.

Eth yl (E)-4-Br om o-3-(3′-ter t-bu toxyca r boxa m id op r o-

p oxy)cin n a m a te (31). A suspension of 30 (320 mg, 1.18

mmol) and K₂CO₃(500 mg, 3.62 mmol) in acetone (50 mL)

containing 3-(tert-butoxycarboxamido)propyl bromide (33) (480

mg, 1.98 mmol) was heated at reflux under Ar for 20 h, then

concentrated. The residue was extracted with CH₂Cl₂, and the

extract was washed (water, 1 N HCl, and brine) and dried.

Concentration and chromatography (10% EtOAc/hexane) of the

residue afforded 410 mg (81%) of 31 as a white powder, mp

65-67 °C. TLC (17% EtOAc/hexane) R_f0.30. FT-IR 3425, 2980,

1

hexanes) R_f0.47. FT-IR (CHCl₃) 3388, 1695, cm^-1. H NMR δ

1.39 (t, J ) 7.2 Hz, 3H, CH₃), 4.37 (q, J ) 7.2 Hz, 2H, OCH₂),

5.75 (s, 1H, OH), 7.48 (d, J ) 8.1 Hz, 1H, 6-ArH), 7.55 (d, J )

8.1 Hz, 1H, 5-ArH), 7.68 ppm (s, 1H, 2-ArH).

Eth yl 4-Br om o-3-ben zyloxyben zoa te (26). To a suspen-

sion of 25 (4.5 g, 18.4 mmol) and K₂CO₃(4.1 g, 30 mmol) in

acetone (150 mL) under Ar was added benzyl bromide (3.4 g,

20 mmol). The mixture was heated at reflux for 15 h,

concentrated, then diluted with CH₂Cl₂(100 mL), washed

(water, 1 N HCl, and brine), and dried. Concentration and

chromatography (5% EtOAc/hexane) afforded 5.2 g (84%) of

26 as a white powder, mp 50-52 °C. TLC (20% CH₂Cl₂/hexane)

1

1706 cm^-1. H NMR δ 1.34 (t, J ) 7.2 Hz, 3H, CH₃), 1.44 (s,

9H, OC(CH₃)₃), 2.07 (t, J ) 6.0 Hz, 2H, 2′-CH₂), 3.40 (q, J )

5.7 Hz, 2H, 3′-CH₂N), 4.13 (t, J ) 5.7 Hz, 2H, 1′-CH₂O), 4.26

(q, J ) 7.2 Hz, 2H, OCH₂), 5.22 (s, 1H, NH), 6.47 (d, J ) 15.9

Hz, 1H, ArCHdC), 7.00 (s, 1H, 2-ArH), 7.01 (d, J ) 6.3 Hz,

1H, 6-ArH), 7.54 (d, J ) 8.7 Hz, 1H, 5-ArH), 7.60 ppm (d, J )

15.9 Hz, 1H, CdCHCO₂). MALDI-FTMS (HRMS) calcd

1

R_f0.35. FT-IR (CHCl₃) 1722 cm^-1. H NMR δ 1.39 (t, J ) 7.2

Hz, 3H, CH₃), 4.37 (q, J ) 7.2 Hz, 2H, OCH₂), 5.21 (s, 2H,

OCH₂Ph), 7.39 (m, 3H, ArH), 7.50 (m, 3H, ArH), 7.63 ppm (m,

2H, ArH). MALDI-FTMS (HRMS) calcd C₁₆H₁₅BrNaO₃[MNa⁺]

357.0097, found 357.0093.

C

₁₉H₂₆BrNNaO₅[MNa⁺] 450.0886, found 450.0889.

3-Br om o-1-(ter t-bu toxyca r bon yl)p r op yla m in e (33). A

reported procedure was applied¹⁰⁹to 3-bromopropylamine

hydrobromide (32) (4.4 g, 20 mmol), Et₃N (3.0 mL, 20 mmol),

and di-(tert-butyl)dicarbonate (5.5 g, 25 mmol) in CH₂Cl₂(20

mL) to give after workup and chromatography (5% EtOAc/

hexane) 3.95 g (83%) of 33 as a colorless liquid. TLC (17%

EtOAc/hexane) R_f0.61. FT-IR (film) 3399, 1678 cm^-1. ¹H NMR

δ 1.45 (s, 9H, OC(CH₃)₃), 2.05 (m, 2H, 2-CH₂), 3.28 (q, J ) 6.3

Hz, 2H, 1-CH₂N), 3.45 (t, J ) 6.3 Hz, 2H, 3-CH₂Br), 4.66 ppm

(s, 1H, NH).

4-Br om o-3-ben zyloxyben zyl Alcoh ol (27). To 26 (3.35 g,

10 mmol) dissolved in CH₂Cl₂(25 mL) and cooled under Ar in

a dry ice-acetone bath was slowly added 20 mL of 1.0 M

DIBAL (20 mmol) in hexanes with stirring. After 2 h, the

stirred reaction mixture was diluted with 1 N HCl (20 mL)

and CH₂Cl₂(50 mL), and stirring was continued for 0.5 h. The

organic layer was washed and dried. Concentration and

chromatography (9% EtOAc/hexane) afforded 2.78 g (91%) of

27 as a white solid, mp 73-75 °C. TLC (9% EtOAc/hexane) R_f

Eth yl (E)-4-[3′-(1-Ad a m a n tyl)-4′-ben zyloxyp h en yl]-3-

[(3′-ter t-bu toxyca r boxa m id o)p r op oxy]cin n a m a te (38). To

31 (321 mg, 0.75 mmol), 3-(1-adamantyl)-4-benzyloxyphenyl-

boronic acid⁵(37) (362 mg, 1.0 mmol), and Pd(PPh₃)₄(60 mg,

0.052 mmol) in DME (5 mL) was added under Ar with stirring

2.0 M aq Na₂CO₃(1.0 mL). The reaction mixture was heated

at reflux for 20 h, cooled to room temperature, then extracted

(EtOAc). The extract was washed, dried, and concentrated.

Flash chromatography (EtOAc/hexane) of the residue gave 38

as a yellow powder (410 mg, 83%), mp 73-75 °C, which was

used without further purification in the deprotection steps to

give 10 and 14. TLC (20% EtOAc/hexane) R_f0.30. FT-IR 3366,

0.17. FT-IR (powder) 3383 cm^-^{1 1}H NMR δ 4.63 (d, J ) 5.7

.

Hz, 2H, ArCH₂O), 5.16 (s, 2H, OCH₂Ph), 6.83 (d, J ) 8.1 Hz,

1H, 6-ArH), 7.00 (s, 1H, 2-ArH), 7.33-7.54 (m, 5H, ArH), 7.53

ppm (d, 1H, 5-ArH). MALDI-FTMS (HRMS) calcd C₁₄H₁₃

-

BrNaO₂[MNa⁺] 314.9991, found 314.9998.

Eth yl (E)-4-Br om o-3-ben zyloxycin n a m a te (29). To a

stirred solution of 27 (2.77 g, 10 mmol) in CH₂Cl₂(30 mL)

cooled in an ice bath was slowly added PCC (3.23 g, 15 mmol).

This mixture was stirred for 5 h at room temperature before

Et₂O (50 mL) was added. Filtration and concentration gave

the 3-benzyloxy-4-bromobenzaldehyde (28) as a white powder,

which was used in the next step without further purification.

1

1711 cm^-1. H NMR δ 1.35 (t, J ) 6.9 Hz, 3H, CH₃), 1.43 (s,

9H, OC(CH₃)₃), 1.72 (s, 6H, AdCH₂), 1.95 (m, 2H, 2′-CH₂), 2.04

(s, 3H, AdCH), 2.17 (s, 6H, AdCH₂), 3.24 (d, J ) 5.4 Hz, 2H,

3′-CH₂N), 4.03 (t, J ) 5.7 Hz, 2H, 1′-CH₂O), 4.28 (q, J ) 6.6

Hz, 2H, OCH₂), 4.55 (s, 1H, NH), 5.16 (s, 2H, OCH₂Ph), 6.45

(d, J ) 15.9 Hz, 1H, ArCHdC), 7.00 (d, J ) 8.7 Hz, 1H, 5′-

ArH), 7.11 (s, 1H, 2-ArH), 7.20 (d, J ) 7.8 Hz, 1H, 6′-ArH),

7.34-7.45 (m, 5H, ArH), 7.51 (s, 1H, 2′-ArH), 7.53 (d, J ) 7.2

Hz, 2H, 5,6-ArH), 7.68 ppm (d, J ) 15.9 Hz, 1H, CdCHCO₂).

MALDI-FAB calcd C₄₂H₅₂NO₆[MH⁺] 664.8, found 664.8.

Eth yl (E)-4-[3′-(1-Ad a m a n tyl)-4′-ben zyloxyp h en yl]-3-

(3′-a ceta m id op r op oxy)cin n a m a te (40). To a mixture of 38

(400 mg, 0.6 mmol) in EtOH (10 mL) was added concentrated

HCl (1.0 mL). This mixture was heated at reflux for 1 h, then

concentrated. The residue containing the primary amine 39

was sequentially treated with CH₂Cl₂(20 mL), pyridine (1.0

mL), and Ac₂O (1.0 mL) and stirred overnight at room

temperature before being washed, dried (MgSO₄), and con-

centrated. Flash chromatography (EtOAc/hexane) gave 40 as

a white powder (280 mg, 77%), mp 80-82 °C. TLC (67%

EtOAc/hexane) R_f0.28. FT-IR 3293, 1716 cm^-1. ¹H NMR δ 1.35

(t, J ) 7.2 Hz, 3H, CH₃), 1.45 (s, 2H, 2′-CH₂), 1.72 (s, 6H,

To triethyl phosphonoacetate (0.33 g, 1.5 mmol) dissolved

in anhydrous Et₂O (10 mL) then cooled in a dry ice-acetone

bath was added 1.5 mL of 0.91 M KN(SiMe₃)₂(1.35 mmol) in

THF under Ar with stirring, which was continued for 0.5 h.

Next, 28 (0.24 g, 0.83 mmol) in Et₂O (10 mL) was slowly added

to the solution with cooling in the dry ice-acetone bath. After

stirring for 1 h more, the mixture was allowed to warm to

ambient temperature, stirred overnight, poured into water (50

mL) containing HOAc (1.0 mL), and extracted into Et₂O (20

mL). The extract was washed, dried, and concentrated. Chro-

matography (5% EtOAc/hexane) of the residue afforded 0.28

g (91%) of 29 as a white solid, mp 45-48 °C. TLC (9% EtOAc/

hexane) R_f0.67. FT-IR (CHCl₃) 1711, 1640 cm^-^{1 1}H NMR δ

.

1.33 (t, J ) 7.2 Hz, 3H, CH₃), 4.26 (q, J ) 6.9 Hz, 2H, CH₂),

5.18 (s, 2H, CH₂Ph), 6.39 (d, J ) 15.9 Hz, 1H, ArCHdC), 7.01

(d, J ) 8.4 Hz, 1H, 6-ArH), 7.06 (s, 1H, 2-ArH), 7.33-7.50 (m,

5H, ArH), 7.57 (d, J ) 8.4 Hz, 1H, 5-ArH), 7.58 ppm (d, J )

15.9 Hz, 1H, CdCHCO₂). MALDI-FTMS (HRMS) calcd

C

₁₈H₁₈BrO₃[MH⁺] 361.0434, found 361.0437.

Eth yl (E)-4-Br om o-3-h yd r oxycin n a m a te (30). A solution

of 29 (260 mg, 0.72 mmol) in CH₂Cl₂(5 mL) and 1.0 M BBr₃

Blocker of AHPN-Induced Apoptosis

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3531

AdCH₂), 2.04 (s, 6H, AdCH and COCH₃), 2.15 (s, 6H, AdCH₂),

3.37 (q, J ) 5.4 Hz, 2H, 3′-CH₂N), 4.12 (t, J ) 5.1 Hz, 2H,

1′-CH₂O), 4.28 (q, J ) 7.2 Hz, 2H, OCH₂), 5.15 (s, 2H,

OCH₂Ph), 5.67 (s, 1H, NH), 6.45 (d, J ) 16.2 Hz, 1H,

ArCHdC), 7.00 (d, J ) 7.8 Hz, 1H, 5′-ArH), 7.09 (s, 1H, 2′-

ArH), 7.22 (d, J ) 7.8 Hz, 1H, ArH), 7.26-7.42 (m, 5H, ArH),

7.45 (d, 1H, J ) 6.9 Hz, 1H, ArH), 7.49 (s, 1H, 2-ArH), 7.50

(d, J ) 8.1 Hz, 1H, ArH), 7.69 ppm (d, J ) 16.2 Hz, 1H,

CdCHCO₂). MALDI-FTMS (HRMS) calcd C₃₉H₄₅NNaO₅

(MNa⁺) 630.3190, found 630.3172.

mmol) in water (1.0 mL). The reaction mixture was stirred at

reflux temperature under Ar for 1 h, cooled to room temper-

ature, acidified (1 N HCl), and concentrated. The resultant

solid was diluted with water (5 mL) and Et₂O (10 mL), then

stirred for 1 h, filtered, and dried under vacuum to give 14 as

an off-white powder (236 mg, 89%), mp 238-240 °C. FT-IR

3381, 1686 cm^-1. ¹H NMR (CD₃OD) δ 1.87 (s, 6H, AdCH₂), 2.11

(s, 3H, AdCH), 2.13 (m, 2H, 2′-CH₂), 2.24 (s, 6H, AdCH₂), 3.08

(t, J ) 7.5 Hz, 2H, 3′-CH₂N), 4.20 (t, J ) 6.0 Hz, 2H, 1′-CH₂O),

6.56 (d, J ) 16.2 Hz, 1H, ArCHdC), 6.81 (d, J ) 8.4 Hz, 1H,

5′-ArH), 7.23 (d, J ) 8.4 Hz, 1H, 6′-ArH), 7.30 (d, J ) 8.4 Hz,

2H, ArH), 7.33 (s, 2H, 2,2′-ArH), 7.73 ppm (d, J ) 16.2 Hz,

1H, CdCHCO₂). MALDI-FTMS (HRMS) calcd C₂₈H₃₄NO₄

(MH⁺) 448.2482, found 448.2488.

Eth yl 5-Ch lor o-6-h yd r oxy-2-n a p h th a len eca r boxyla te

(46). A suspension of 44 (4.90 g, 26.0 mmol), in EtOH (25 mL)

was treated with concentrated H₂SO₄(1.0 g), stirred for 5 days,

then heated at reflux for 1 h to complete esterification. After

removal of EtOH at reduced pressure, the ethyl ester 45 was

isolated by extraction (EtOAc), followed by washing (2 × 5%

NaHCO₃and sat. brine) and drying (MgSO₄). Concentration

and crystallization afforded 45 (4.81 g, 85%), mp 109-110 °C.

TLC (EtOAc) R_f0.83. ¹H NMR (400 MHz) δ 1.47 (t, J ) 7.0

Hz, 3H, CH₃), 4.47 (q, J ) 7.0 Hz, 2H, CH₂), 6.13 (s, 1H, OH),

7.19 (dd, J ) 8.8 Hz, J ) 2.4 Hz, 1H, 7-NapH), 7.20 (s, 1H,

5-NapH), 7.70 (d, J ) 8.5 Hz, 1H, 8-NapH), 7.87 (d, J ) 8.3

Hz, 1H, 4-NapH), 8.01 (dd, J ) 8.6 Hz, J ) 1.8 Hz, 1H,

3-NapH), 8.54 ppm (d, J ) 0.8 Hz, 1H, 1-NapH). Anal.

(C₁₃H₁₂O₃) C, H. MALDI-FTMS (HRMS) calcd C₁₃H₁₃O₃

(MH⁺) 217.0859, found 217.0861.

Eth yl (E)-4-[3′-(1-Ad a m a n tyl)-4′-h yd r oxyp h en yl]-3-(3′-

a ceta m id op r op oxy)cin n a m a te (41). A solution of 40 (303

mg, 0.5 mmol) and 1.0 M BBr₃(1.5 mmol) in CH₂Cl₂(1.5 mL)

and CH₂Cl₂(5 mL) was stirred at -78 °C under Ar for 2 h,

then diluted with water (10 mL) and CH₂Cl₂(20 mL). The

organic phase was washed, dried, and concentrated. Flash

chromatography (EtOAc/hexane) gave 41 as an off-white

powder (226 mg, 77%), mp 110-113 °C. TLC (67% EtOAc/

hexane) R_f0.20. FT-IR 3408, 1706, 1637 cm^-1. ¹H NMR δ 1.35

(t, J ) 6.9 Hz, 3H, CH₃), 1.44 (s, 2H, 2′-CH₂), 1.77 (s, 6H,

AdCH₂), 2.04 (s, 3H, AdCH). 2.08 (s, 3H, COCH₃), 2.14 (s, 6H,

AdCH₂), 3.38 (q, J ) 5.4 Hz, 2H, 3′-CH₂N), 4.13 (t, J ) 6.3

Hz, 2H, 1′-CH₂O), 4.27 (q, J ) 7.2 Hz, 2H, OCH₂), 5.81 (s, 1H,

NH), 6.30 (s, 1H, OH), 6.44 (d, J ) 15.9 Hz, 1H, ArCHdC),

6.75 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.09 (s, 1H, 2′-ArH), 7.20 (d,

J ) 7.5 Hz, 1H, ArH), 7.26 (d, J ) 8.1 Hz, 1H, ArH), 7.33 (d,

J ) 8.1 Hz, 1H, ArH), 7.40 (s, 1H, 2-ArH), 7.68 ppm (d, J )

15.9 Hz, 1H, CdCHCO₂). MALDI-TFMS (HRMS) calcd

C

₃₂H₄₀NO₅(MH⁺) 518.2906, found 518.2892.

(E)-4-[3′-(1-Ad a m a n t yl)-4′-h yd r oxyp h en yl]-3-(3′-a cet -

a m id op r op oxy)cin n a m ic Acid (10). To a suspension of 41

(190 mg, 0.387 mmol) in MeOH (10 mL) was added NaOH (100

mg, 2.5 mmol). The mixture was stirred at reflux temperature

under Ar for 1 h, cooled to room temperature, acidified (1 N

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

(MgSO₄), and concentrated to afford 10 as an off-white powder

(170 mg, 94%), mp 195-199 °C. FT-IR (powder) 3400, 1691,

R-Chlorination of 45 was conducted by modifying a reported

procedure.⁵⁹To a solution of 45 (3.62 g, 16.7 mmol) in glacial

HOAc (32 mL) was added sulfuryl chloride (2.27 g, 16.8 mmol).

The mixture heated for 2.5 h at 70 °C, then stirred at room

temperature for 36 h. Concentration, ice-bath cooling, and

filtration afforded 46 as white crystals (2.60 g, 62%), mp 139-

142 °C. TLC (20% EtOAc/benzene) R_f0.61. ¹H NMR (400 MHz)

δ 1.45 (t, J ) 5.4 Hz, 3H, CH₃), 4.44 (q, J ) 5.4 Hz, 2H, CH₂),

6.16 (s, 1H, OH), 7.32 (d, J ) 8.8 Hz, 1H, 7-NapH), 7.82 (d, J

) 8.8 Hz, 1H, 8-NapH), 8.07 (d, J ) 8.8 Hz, 1H, 4-NapH), 8.15

(dd, J ) 8.8 Hz, J ) 1.6 Hz, 1H, 3-NapH), 8.54 ppm (s, 1H,

1-NapH). Anal. (C₁₃H₁₁ClO₃) C, H. MALDI-FTMS (HRMS)

calcd C₁₃H₁₂ClO₃(MH⁺) 251.0469, found 251.0473.

Eth yl 6-[3′-(1-Adam an tyl)-4′-ben zyloxyph en yl]-5-ch lor o-

2-n a p h th a len eca r boxyla te (48). To 46 (2.05 g, 8.18 mmol)

and pyridine (2.0 mL, 25 mmol) dissolved in CH₂Cl₂(55 mL)

and cooled in an ice bath was added over a 5-min period

trifluoromethanesulfonic anhydride (1.7 mL, 10.1 mmol). The

reaction mixture was allowed to warm to room temperature,

stirred for 18 h, washed (2 × H₂O, 10% HCl, and sat. brine),

then dried (MgSO₄). Concentration afforded ethyl 5-chloro-6-

trifluoromethanesulfonyloxy-2-naphthalenecarboxylate (47) as

white crystals (2.43 g, 78%), mp 99-100 °C. TLC (25% EtOAc/

hexane) R_f0.80, which was used without further purification

in the following step. ¹H NMR δ 1.47 (t, J ) 6.9 Hz, 3H, CH₃),

4.47 (d, J ) 6.8 Hz, 2H, CH₂), 7.52 (d, J ) 8.9 Hz, 1H, 7-NapH),

7.97 (d, J ) 9.0 Hz, 1H, 8-NapH), 8.27 (d, J ) 8.7 Hz, 1H,

4-NapH), 8.37 (d, J ) 8.8 Hz, 1H, 3-NapH), 8.64 ppm (s, 1H,

1-NapH).

To a solution of arylboronic acid 37 (500 mg, 1.38 mmol),

triflate 47 (524 mg, 1.37 mmol), Pd(PPh₃)₄(165 mg, 0.143

mmol), and LiCl (114 mg, 2.69 mmol) in dimethoxyethane (12

mL) under Ar was added 2.0 M aq Na₂CO₃(1.4 mL). The

reaction mixture was heated at reflux (18 h), diluted (EtOAc

and sat. brine), and extracted (CH₂Cl₂). The organic extract

was dried (MgSO₄) and concentrated. Flash chromatography

(5% EtOAc/hexane) of the residue and concentration provided

48 as white crystals (420 mg, 56%), mp 177-179 °C. TLC (10%

EtOAc/hexane) R_f0.37. FT-IR (KBr) 1716 cm^-1. ¹H NMR (400

MHz) δ 1.47 (t, J ) 6.0 Hz, 3H, CH₃), 1.74 (s, 6, AdCH₂), 2.05

(s, 3H, AdH), 2.20 (s, 6H, AdCH₂), 4.46 (q, J ) 7.0 Hz, 2H,

OCH₂), 5.20 (s, 2, CHPh₂), 7.06 (d, J ) 8.8 Hz, 1H, 5-ArH),

7.3-7.4 (m, 2, ArH), 7.4-7.5 (m, 3, ArH), 7.5-7.6 (m, 3, ArH

and 7-NapH), 7.90 (d, J ) 8.0 Hz, 1H, 8-NapH), 8.20 (dd, J )

1

1632 cm^-1. H NMR (DMSO-²H₆) δ 1.73 (s, 6H, AdCH₂), 1.77

(s, 3H, COCH₃), 1.78 (m, 2H, 2′-CH₂), 2.03 (s, 3H, AdCH), 2.10

(s, 6H, AdCH₂), 3.17 (d, J ) 5.4 Hz, 2H, 3′-CH₂N), 4.06 (broad

s, 2H, 3′-CH₂O), 6.63 (d, J ) 15.9 Hz, 1H, ArCHdC), 6.80 (d,

J ) 8.1 Hz, 1H, 5′-ArH), 7.21 (d, J ) 8.7 Hz, 1H, ArH), 7.29

(s, 3H, ArH and 2′-ArH), 7.37 (s, 1H, 2-ArH), 7.61 (d, J ) 15.9

Hz, 1H, CdCHCO₂), 7.86 (s, 1H, NH), 9.41 ppm (s, 1H, OH).

MALDI-FTMS (HRMS) calcd C₃₀H₃₅NNaO₅[MNa⁺] 512.2407,

found 512.2402.

Eth yl (E)-4-[3′-(1-Ad a m a n tyl)-4′-h yd r oxyp h en yl]-3-(3′-

a m in op r op oxy)cin n a m a te (43). A solution of 38 (500 mg,

0.75 mmol) and 1.0 M BBr₃(1.5 mmol) in CH₂Cl₂(1.5 mL)

and CH₂Cl₂(20 mL) was stirred at -78 °C under Ar for 2 h,

then diluted with water (10 mL) and CH₂Cl₂(20 mL). The

organic phase was washed, dried, and concentrated to give

ethyl

(E)-4-[3′-(1-adamantyl)-4′-hydroxyphenyl]-3-(3′-tert-

butoxycarboxamido)propoxycinnamate (42) as a pale-yellow

solid. The solid in EtOH (20 mL) containing concentrated HCl

(1.5 mL) was heated at reflux under Ar for 2 h. The solution

was concentrated before dilution with MeOH (20 mL). NaHCO₃

(200 mg) was added, and this mixture was stirred for 1 h under

Ar, then concentrated. Flash chromatography (9% MeOH/

CH₂Cl₂) gave 43 as a white solid (278 mg, 78%), mp 181-183

°C. TLC (9% MeOH/CH₂Cl₂) R_f0.62. FT-IR 3244, 1706, 1632

cm^-^{1 1}H NMR δ 1.34 (t, J ) 7.2 Hz, 3H, CH₃), 1.76 (s, 6H,

.

AdCH₂), 1.94 (m, 2H, 2′-CH₂), 2.05 (s, 3H, AdCH), 2.15 (s, 6H,

AdCH₂), 2.85 (m, 2H, 3′-CH₂N), 3.74 (s, 2H, NH₂), 4.09 (s, 2H,

3′-CH₂O), 4.28 (q, J ) 7.5 Hz, 2H, OCH₂), 5.71 (s, 1H, OH),

6.43 (d, J ) 15.9 Hz, 1H, ArCHdC), 6.62 (d, J ) 7.8 Hz, 1H,

5′-ArH), 7.08 (s, 1H, 2-ArH), 7.17 (d, J ) 7.2 Hz, 1H, ArH),

7.20 (d, J ) 8.4 Hz, 1H, ArH), 7.30 (s, 1H, 2′-ArH), 7.32 (d, J

) 7.5 Hz, 1H, ArH), 7.68 ppm (d, J ) 15.9 Hz, 1H, CdCHCO₂).

MALDI-FTMS (HRMS) calcd C₃₀H₃₈NO₄[MH⁺] 476.2795,

found 476.2790.

(E)-4-[3′-(1-Ad a m a n tyl)-4′-h yd r oxyp h en yl]-3-(3′-a m in o-

p r op oxy)cin n a m ic Acid (14). To a suspension of 43 (260 mg,

0.547 mmol) in MeOH (10 mL) was added NaOH (100 mg, 2.5

3532 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14

Dawson et al.

8.8 Hz, J ) 1.6 Hz, 1H, 3-NapH), 8.44 (d, J ) 8.8 Hz, 1H,

4-NapH), 8.62 ppm (d, J ) 1.6 Hz, 1H, 1-NapH). MALDI-

FTMS (HRMS) calcd C₃₆H₃₆ClO₂(MH⁺) 550.2275, found

550.2285.

employing SHAKE. Coordinates were saved every 0.250 ps for

subsequent analysis. The nonbonded pair list was updated

every 10 steps. A radial screened dielectric constant (D ) r)

was employed. Production dynamics was performed for 300

ps to make initial probes of the different potential effects of

ligand groups on receptor conformation in the neighborhood

of the LBP. To permit a critical comparison of the dynamics

of AHPC and 3-A-AHPC in the RARγ LBD with reasonable

damping of surface fluctuations, following the identification

of low-energy docking modes, the docked lowest-energy con-

formations of the RARγ LBD-AHPC and 3-A-AHPC com-

plexes were solvated within a large box comprised of 4014

waters and having the initial dimensions of 70 Å × 60 Å × 60

Å. After an initial short 50-ps equilibration at constant volume

and a 100-ps equilibration at constant pressure, the system

was subjected to 300 ps of production dynamics at 300 K.

Periodic boundary conditions were employed with a particle-

mesh Ewald summation to provide realistic boundary forces

and eliminate the distorting effects of potential truncation.

Den sit y F u n ct ion a l Th eor y (DF T) E xp lor a t ion of

In ter -r in g Tor sion a l P oten tia l Su r fa ces. AHPN (1), 5-Cl-

AHPN (2), 3-Cl-AHPC (9), 3-A-AHPC (10), and AHPC (11)

torsional relaxed potential surfaces were computed using the

nonlocal B3LYP DFT functional and a 6-31G** basis set.

Optimizations employed J aguar 4.1 (Schrodinger), default self-

consistent field (SCF), and geometric convergence criteria.

Unconstrained geometry optimization was performed at each

of the parametrically varied torsional angles.

Biology. Rea gen ts. trans-RA (4) and tryspin-TPCK were

purchased from Sigma, and [11,12-³H₂]9-cis-retinoic acid (43

Ci/mmol) from Amersham. 4-(5′,6′,7′,8′-Tetrahydro-5′,5′,8′,8′-

tetramethyl-2′-anthracenyl)benzoic acid (TTAB, 21)⁷¹and the

2-substituted 1,3-dithiolane 22⁷⁰were synthesized as pre-

viously described, as were [5,5′-³H₂]6-[3′-(1-adamantyl)-4′-

hydroxyphenyl]-2-naphthalenecarboxylic acid, AHPN (1), (E)-

4-[3′-(1-adamantyl)-4′-hydroxyphenyl]-3-chlorocinnamic acid

(3-Cl-AHPC, 9), and 9-cis-RA (5).^{4,39,40,116,117}

AHP N An a logu es. Both agonists and antagonists were

dissolved in Me₂SO prior to addition to media. The final

concentration of Me₂SO was 0.1% for cancer and leukemia

cells. All experiments were conducted in triplicate.

Eth yl 6-[3′-(1-Ad a m a n tyl)-4′-h yd r oxyp h en yl]-5-ch lor o-

2-n a p h th a len eca r boxyla te (49). To 48 (170 mg, 0.31 mmol)

dissolved in CH₂Cl₂(30 mL) and cooled in a dry ice-EtOH

bath was added 1.0 M BBr₃(900 mmol) in CH₂Cl₂(0.90 mL)

over a 2-min period. After being stirred at this temperature

for 1 h, the mixture was diluted with cold H₂O (10 mL) and

allowed to warm to room temperature. The organic layer was

separated, dried (MgSO₄), and concentrated to give 49 as white

crystals (101 mg, 71%), mp 256-260 °C. TLC (toluene) R_f0.26.

¹H NMR (300 MHz, CDCl₃) δ 1.47 (t, J ) 7.0 Hz, 3H, CH₃),

1.80 (s, 6H, AdCH₂), 2.10 (s, 3H, AdCH), 2.18 (s, 6H, AdCH₂),

4.47 (q, J ) 7 Hz, 2H, CH₂), 4.91 (s, 1H, OH), 5.30 (s, 2H,

CH₂), 6.77 (d, J ) 7.3 Hz, 1H, 5′-ArH), 7.38 (s, 1H, 2′-ArH),

7.54 (d, J ) 8.1 Hz, 2H, 7-NapH and 6′-ArH), 7.90 (d, J ) 9.6

Hz, 1H, 8-NapH), 8.19 (d, J ) 9.0 Hz, 1H, 4-NapH), 8.44 (d, J

) 9.0 Hz, 1H, 3-NapH), 8.62 ppm (s, 1H, 1-NapH). MALDI-

FTMS (HRMS) calcd C₂₉H₃₀ClO₂(MH⁺) 460.1805, found

460.1798.

6-[3′-(1-Adam an tyl)-4′-h ydr oxyph en yl]-5-ch lor o-2-n aph -

th a len eca r boxylic Acid (2). A suspension of 49 (230 mg, 0.50

mmol) in 1.0 M NaOH in 75% aq EtOH (5 mL) was heated at

reflux for 40 min, cooled, acidified (10% HCl), then extracted

(EtOAc). The extract was washed (sat. brine), dried (MgSO₄),

and concentrated to give 2 as a white powder (205 mg, 95%),

mp 282-284 °C (dec). TLC (5% MeOH/CHCl₃) R_f0.26. FT-IR

2983, 1640 cm^-1. ¹H NMR (300 MHz, d₆-DMSO) δ 1.74 (s, 6H,

AdCH₂), 2.05 (s, 3H, AdCH), 2.16 (s, 6H, AdCH₂), 6.91 (d, J )

8.1 Hz, 1H, 5′-ArH), 7.24 (d, J ) 8.5 Hz, 1H, 6′-ArH), 7.27 (s,

1H, 2′-ArH), 7.62 (d, J ) 8.4 Hz, 1H, 7-NapH), 8.16 (d, J )

8.5 Hz, 2H, 4,8-NapH), 8.37 (d, J ) 9.1 Hz, 1H, 3-NapH), 8.68

(s, 1H, 1-NapH), 9.62 ppm (s, 1H, OH). MALDI-FTMS

(HRMS) calcd C₂₇H₂₆ClO₂(MH⁺) 432.1492, found 432.1502.

Com p u ta tion a l Stu d ies. Gen er a tion of a n RARγ LBD

Mod el for Dock in g a n d Sim u la tion s. An all-H representa-

tion of the RARγ LBD was generated using the XLEAP module

of AMBER6¹¹⁰and the 9-cis-RA (5)-bound holo-RARγ LBD

crystal structure⁶⁵(PDB entry 3LBD). The initial all-atom

model was energy-minimized with a small number of steps

(100 steepest descents plus 1900 conjugate-gradient) to remove

large initial repulsions in atomic positions as evaluated by the

AMBER force-field compared to crystal structure contacts.

Ra d ioliga n d Bin d in g a n d DP SA. DPSA studies using

[³⁵S]RARγ and LBD competition radioligand-binding experi-

ments were conducted using bacterially expressed RARs (as

GST-fusion proteins) and (His)₆-RXRR as described.⁷⁰

Short molecular dynamics (MD) equilibration of the model

at constant temperature was performed to provide additional

structure relaxation and establish reasonable hydrogen-bond-

ing patterns in the vicinity of the LBP. The model was

equilibrated at 300 K with decreasing harmonic constraints

over a 20-ps time interval using a 1-femtosecond integration

time-step followed by a 70-ps unconstrained equilibration. All

bonds involving hydrogens were constrained using SHAKE.¹¹¹

The model was then converted to a polar-H representation

using AMBER41 for energy-based docking using AUTODOCK

3.0.¹¹²

En er gy-Ba sed Dock in g. Energy-based docking of flexible

AHPN (1), 5-Cl-AHPN (2), 3-Cl-AHPC (9), 3-A-AHPC (10), and

AHPC (11) in the rigid RARγ model was performed using the

LGA¹¹³approach embodied in AUTODOCK 3.0. The LGA

docking parameters were: mutation rate, 0.02; crossover rate,

0.80; maximum number of generations, 2.7 × 10⁴; elitism, 1;

and local search frequency, 0.06. The lowest energy-binding

mode was investigated in short exploratory dynamics employ-

ing AMBER6 and an all-atom representation to examine

differential ligand effects on RARγ.¹¹⁴Charge parametrization

for all ligands was derived from restrained electrostatic

potential fits derived from DFT computations at ligand

optimized geometries.¹¹⁵

GST-P u lld ow n Assa ys. Experiments were performed as

previously described using GST-p300 1-450 or GST-NCoR

2110-2453 fusion proteins and [³⁵S]methionine-labeled hRARγ

LBD prepared by in vitro translation.⁷⁰

P la sm id s. Expression vectors for RARγ, RXRR, and the

reporter gene (TREpal)₂-tk-CAT have been described.¹¹⁸

Recep tor Tr a n scr ip tion a l Activa tion in Cotr a n fected

Cells. CV-1 cells were routinely maintained in DMEM,

supplemented with 10% fetal calf serum (FCS), 100 units/mL

of penicillin, and 100 µg/mL of streptomycin. For transfection

assays, cells were seeded at 1.0 × 10⁵cells/mL in 24-well plates

for 16-24 h before transfection. Cells were then transfected

using the calcium chloride precipitation method¹¹⁹with either

(TREpal)₂-tk-CAT (200 µg) alone or with the RARγ vector (100

µg) or RXRR vector (20 µg). Cells were cotransfected with

â-galactosidase (â-gal) expression vector (pCH 110, Amersham

Biosciences) and carrier DNA (pBluescript, Stratagene) to a

final concentration of 1000 µg/well. Following transfection for

20 h, the medium was changed to DMEM containing 5%

charcoal-stripped FCS, and cells were treated for 24 h with

an AHPN analogue or retinoid. Chloramphenicol acetyl trans-

ferase (CAT) activity was normalized to â-gal activity to

standardize for transfection efficiency.

MD Mod el Equ ilibr a tion . MD equilibrations of the all-

atom model representations of AHPN (1), 5-Cl-AHPN (2), 3-A-

AHPC (10), and AHPC (11) docked to the RARγ LBD at

constant temperature were performed using a 1-fs integration

time-step with constraint of all bonds involving hydrogens

Cell Lin es. Retinoid-resistant MDA-MB-231 and MDA-MB-

468 breast cancer and H292 lung cancer and retinoid-sensitive

H460 lung and LNCaP prostate cancer cell lines were obtained

from the American Type Cell Collection. Retinoid-resistant

HR-60R leukemia cells were a gift from Dr. Stephen Collins

Blocker of AHPN-Induced Apoptosis

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3533

(University of Washington, Seattle, WA). Cells were cultured

as described below.

Ap p en d ix

Cell P r olifer a tion . MDA-MB-468 cells (1.0 × 10⁵cells)

were seeded in DMEM/F12 medium (Invitrogen Life Technolo-

gies) supplemented with 5% fetal bovine serum and 25 µg per

mL of gentamicin in Petri dishes or 6-well plates. After 24 h,

3-A-AHPC (10) (2.0 × 10^-6M final concentration) and/or 3-Cl-

AHPC (9) (0.5 × 10^-6M) or vehicle alone were added. Cells

were harvested at the times indicated in the figures, and living

cell numbers were assessed using a commercial colorimetric

assay for the mitochondrial dehydrogenase cleavage of water-

soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (Sigma-Aldrich) according to the manufacturer’s direc-

tions. H460 and H292 lung cancer and LNCaP prostate cancer

cell lines were grown in RPMI 1640 supplemented with 10%

FCS.

Acromyns and/or code numbers for 6-[3′-(1-adaman-

tyl)-4′-hydroxyphenyl)]-2-naphthalenecarboxylic acid, its

analogues, and retinoids are listed in the legend for

Figure 1. Other abbreviations are 1-Ad, 1-adamantyl;

3-A, 3-(3′-acetamidopropyloxy); Bcl-2, diffuse B-cell lym-

phoma cytoprotective protein; CAT, chloramphenicol

acetyl transferase; CPK, Corey-Pauling-Kortum; CV-

1, African green monkey kidney cell line; DAPI, 4,6-

diamidino-2-phenylindole; DPSA, differential protease

sensitivity assay; DTF, density functional theory; FCS,

fetal calf serum; GST, glutathione S-transferase; LBD,

ligand-binding domain; LGA, Larmarkian Genetic Al-

gorithm; LBP, ligand-binding pocket; NCoR, nuclear

receptor corepressor protein; p21^WAF/CIP1, 21-kDa wild-

type p53-activated fragment 1 or cyclin-dependent ki-

nase-2-interacting protein; p300, an acetyl transferase

and transcriptional coactivator of nuclear receptors;

PBS, phosphate-buffered saline, pH 7.4; RA, retinoic

acid; RAR, retinoic acid receptor; RXR, retinoid X

receptor; TPCK-trypsin, bovine pancreas trypsin treated

with N-(p-tosyl)-L-phenylanlanine chloromethyl ketone;

TREpal, palindromic RAR- and RXR-responsive ele-

ment; tk, thymidine kinase promoter; TTN, 5,6,7,8-

tetrahydro-5,5,8,8-tetramethyl-2-naphthalene.

Ap op tosis. At 24 h after seeding with 1.0 × 10⁵MDA-MB-

468 cells, agonist (0.5 × 10^-6M final concentration) or

antagonist (2.0 × 10^-6M) alone or the combination was added,

and incubations were continued for 48 or 72 h. Cells were then

harvested, and the relative number of apoptotic cells was

assessed after fixing and acridine orange staining by counting

200-cell fields for the presence of nuclear fragmentation as

previously described.³

H460 cells (5 × 10⁵) were treated with 1.0 × 10^-6M 5-Cl-

AHPN (2) or 3-Cl-AHPC (9) alone or combined with 1.0 × 10^-,⁶

2.5 × 10^-,⁶or 5.0 × 10^-6M 3-A-AHPC (10). After 36 h, cells

were trypsinized, then washed with phosphate-buffered saline

(PBS, pH 7.4). After fixation with 3.7% paraformaldehyde, cells

were washed (PBS) and stained with 4,6-diamidino-2-phenyl-

indole (DAPI, 50 µg/mL) to visualize nuclei by fluorescent

microscopy.³⁸Cells displaying nuclear morphology character-

istic of apoptosis were scored in samples of at least 600 cells.

Apoptosis (%) was expressed as the ratio of apoptotic cells to

total cells × 100.

Su p p or tin g In for m a tion Ava ila ble: Elemental analyses

for compounds 45 and 46. This material is available free of

charge via the Internet at http://pubs.acs.org.

Wester n Blottin g. MDA-MB-468 cells (1.0 × 10⁵) were

treated for 72 h with a proapoptotic AHPN (0.5 × 10^-6M final

concentration) or antagonist 3 (2.0 × 10^-6M) alone or

combined. Cells were lysed, and Western blots were performed

on the lysates using anti-p21^WAF1/CIP1antibody (Transduction

Laboratories) and â-actin as the loading control as previously

described.⁴

Refer en ces

(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) 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.

(3) Li, X.-S.; Rishi, A. K.; Shao, Z. M.; Dawson, M. I.; J ong, L.;

Shroot, B.; Reichert, U.; Ordonez, J .; Fontana, J . A. Posttrans-

criptional regulation of p21^WAF1/CIP1expression in human breast

carcinoma cells. Cancer Res. 1996, 56, 5055-5062.

(4) Fontana, J . A.; Dawson, M. I.; Leid, M.; Rishi, A. K.; Zhang, Y.;

Hsu, C.-A.; Lu, J . S.; Peterson, V. J .; J ong, 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.

(5) 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-

naphthalenecarboxylic acid lacking retinoid receptor transcrip-

tional activation activity. Cancer Res. 2001, 61, 4723-4730.

(6) Mangelsdorf, D. J .; Umesono, K.; Evans, R. M. The retinoid

receptors. In The Retinoids: Biology, Chemistry, and Medicine;

Sporn, M. B., Roberts, A. B., Goodman, D. S., Eds.; Raven

Press: New York, 1994; pp 319-349.

RNA P r ep a r a tion a n d Nor th er n Blottin g. Total RNA

samples were prepared by the guanidine hydrochloride/ultra-

centrifugation method.¹¹⁹Total RNA (30 µg) from cancer cells

treated with or without 1.0 × 10^-6M 5-Cl-AHPN (2) alone or

combined with 1.0 × 10^-6M or 5.0 × 10^-6M 3-A-AHPC (10)

for 4 h was fractionated on 1% agarose gels. The fractionated

RNA was transferred to nylon filters and probed with ³²P-

labeled TR3 LBD cDNA.¹¹⁸Ethidium bromide-stained riboso-

mal RNA was used as the loading control.

Liga n d Bin d in g. Nuclear extracts were prepared from HL-

60R cells (1.0 × 10⁵) as we previously described.⁴[5,5′-³H₂]-

AHPN⁴(10 × 10^-9M, 150 000 dpm) was added to the nuclear

extracts in the presence and absence of 20 × 10^-6M nonlabeled

AHPN (1) or 3-A-AHPC (10) or vehicle alone. After a 90-min

incubation at 22 °C, free ligand was separated from bound

ligand on a 0.5-cm × 4.0-cm column of fine-mesh G25 Sepha-

dex (Pharmacia) by elution with binding buffer.⁴The bound

fraction was counted as we described.⁴

Ack n ow led gm en t. These studies were supported by

NCI grant P01 CA51993 (M.I.D., M.L., J .A.F., and X.Z.),

California Breast Cancer Research Program grant 8WB-

017 (M.I.D., D.H.L., X.Z.), and California Tobacco-

Related Diseases Research Program grants 6RT-0212A

(M.I.D.) and 11RT-0081 (X.Z., M.I.D.). N.B.-S. was

supported by a postdoctoral fellowship from the Cali-

fornia Breast Cancer Research Program. D.L.H. ac-

knowledges the NSF supercomputer grant award

MCB030032 at the Pittsburgh Computer Center. Spe-

cial thanks go to Cynthia Cook and Helen Hansen for

their help with preparing this manuscript.

(7) Dawson, M. I.; Chao, W. R.; Pine, P.; J ong, L.; Hobbs, P. D.;

Rudd, C. K.; Quick, T. C.; Niles, R. M.; Zhang, X.-K.; Lombardo,

A.; Ely, K. R.; Shroot, B.; Fontana, J . A. Correlation of retinoid

binding affinity to RARR with retinoid inhibition of growth of

estrogen receptor-positive MCF-7 mammary carcinoma cells.

Cancer Res. 1995, 55, 4446-4451.

(8) Fitzgerald, P.; Teng, M.; Chandraratna, R. A. S.; Heyman, R.

A.; Allegretto, E. A. Retinoic acid receptor R expression correlates

with retinoid-induced growth inhibition of human breast cancer

cells regardless of estrogen receptor status. Cancer Res. 1997,

57, 2642-2650.

(9) Chao, W. R.; Hobbs, P. D.; J ong, L.; Zhang, X.-K.; Zheng, Y.;

Wu, Q.; Shroot, B.; Dawson, M. I. Effects of receptor class- and

subtype-selective retinoids and an apoptosis-inducing retinoid

on the adherent growth of the NIH:OVCAR-3 ovarian cancer

cell line in culture. Cancer Lett. 1997, 115, 1-7.

3534 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14

Dawson et al.

(10) 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.

(11) Milbrandt, J . Nerve growth factor induces a gene homologous

to the glucocorticoid receptor gene. Neuron 1988, 1, 183-188.

(12) Chang, C.; Kokontis, J . Identification of a new member of the

steroid receptor super-family by cloning and sequence analysis.

Biochem. Biophys. Res. Commun. 1988, 155, 971-977.

(13) Hazel, T. G.; Nathans, D.; Lau, L. F. A gene inducible by serum

growth factors encodes a member of the steroid and thyroid

hormone receptor superfamily. Proc. Natl. Acad. Sci. U.S.A.

1988, 85, 8444-8448.

(14) Leid, M.; Kastner, P.; Lyons, R.; Nakshatri, H.; Saunders: M.;

Zacharewski, T.; Chen, J .-Y.; Staub, A.; Garnier, J .-M.; Mader,

S.; Chambon, P. Purification, cloning, and RXR identity of the

HeLa cell factor with which RAR or TR heterodimerizes to bind

target sequences efficiently. Cell 1992, 68, 377-395.

(15) Green, D. R.; Reed, J . C. Mitochondria and apoptosis. Science

1998, 281, 1309-1312.

(16) Lin, B.; Kolluri, S. K.; Lin, F.; Liu, W.; Han, Y. H.; Cao, X.;

Dawson, M. I.; Reed, J . C.; Zhang, X.-K. Conversion of Bcl-2 from

protector to killer by interaction with nuclear orphan receptor

Nur77/TR3. Cell 2004, 116, 527-540.

(17) Armstrong, R. B.; Ashenfelter, K. O.; Eckhoff, C.; Levin, A. A.;

Shapiro, S. S. General and reproductive toxicology of retinoids.

In The Retinoids: Biology, Chemistry, and Medicine; Sporn, M.

B., Roberts, A. B., Goodman, D. S., Eds.; Raven Press: New

York, 1994; pp 545-572.

(18) Graf, N.; Riesinger, P.; Reinhard, H. Retinoids in the treatment

of acute promyelocytic leukemia. Review of the literature. Klin.

Padiatr. 1995, 207, 43-47.

(19) Selleri, C.; Pane, F.; Notaro, R.; Catalano, L.; Santoro, L. E.;

Luciano, L.; Frigeri, F.; Salvatore, F.; Rotoli, B. All-trans-retinoic

acid (ATRA) responsive skin relapses of acute promyelocytic

leukaemia followed by ATRA-induced pseudotumour cerebri. Br.

J . Haematol. 1996, 92, 937-940.

(29) Shin, D. M.; Glisson, B. S.; Khuri, F. R.; Clifford, J . L.; Clayman,

G.; Benner, S. E.; Forastiere, A. A.; Ginsberg, L.; Liu, D.; Lee,

J . J .; Myers, J .; Goepfert, H.; Lotan, R.; Hong, W. K.; Lippman,

S. M. Phase II and biologic study of interferon R, retinoic acid,

and cisplatin in advanced squamous skin cancer. J . Clin. Oncol.

2002, 20, 364-370.

(30) Standeven, A. M.; Teng, M.; Chandraratna, R. A. S. Lack of

involvement of retinoic acid receptor R in retinoid-induced skin

irritation in hairless mice. Toxicol. Lett. 1997, 92, 231-240.

(31) Iulianella, A.; Lohnes, D. Contribution of retinoic acid receptor

γ to retinoid-induced craniofacial and axial defects. Dev. Dyn.

1997, 209, 92-104.

(32) Look, J .; Landwehr, J .; Bauer, F.; Hoffmann, A. S.; Bluethmann,

H.; LeMotte, P. Marked resistance of RARγ-deficient mice to the

toxic effects of retinoic acid. Am. J . Physiol. 1995, 269, E91-E98.

(33) Reczek, P. R.; Ostrowski, J .; Yu, K. L.; Chen, S.; Hammer, L.;

Roalsvig, T.; Starrett, J . E., J r.; Driscoll, J . P.; Whiting, G.;

Spinazze, P. G. Role of retinoic acid receptor γ in the Rhino

mouse and rabbit irritation models of retinoid activity. Skin

Pharmacol. 1995, 8, 292-299.

(34) Sheikh, M. S.; Shao, Z.-M.; Li, X.-S.; Dawson, M. I.; J etten, A.

M.; Wu, S.; Conley, B. A.; Garcia, M.; Rochefort, H.; Fontana, J .

A. Retinoid-resistant estrogen receptor-negative human breast

carcinoma cells transfected with retinoic acid receptor-R acquire

sensitivity to growth inhibition by retinoids. J . Biol. Chem. 1994,

269, 21440-21447.

(35) Swisshelm, K.; Ryan, K.; Lee, X.; Tsou, H. C.; Peacocke, M.;

Sager, R. Down-regulation of retinoic acid receptor â in mam-

mary carcinoma cell lines and its up-regulation in senescing

normal mammary epithelial cells. Cell Growth Differ. 1994, 5,

133-141.

(36) Li, X.-S.; Shao, Z.-M.; Sheikh, M. S.; Eiseman, J . L.; Sentz, D.;

J etten, A. M.; Chen, J .-C.; Dawson, M. I.; Aisner, S.; Rishi, A.

K.; Fontana, J . A. Retinoic acid nuclear receptor â (RARâ)

inhibits breast carcinoma anchorage independent growth. J . Cell.

Physiol. 1995, 165, 449-458.

(37) Lotan, R.; Xu, X. C.; Lippman, S. M.; Ro, J . Y.; Lee, J . S.; Lee,

J . J .; Hong, W.-K. Suppression of retinoic acid receptor-â in

premalignant oral lesions and its up-regulation by isotretinoin.

New Engl. J . Med. 1995, 332, 1405-1410.

(20) De Botton, S.; Dombret, H.; Sanz, M.; Miguel, J . S.; Caillot, D.;

Zittoun, R.; Gardembas, M.; Stamatoulas, A.; Conde, E.; Guerci,

A.; Gardin, C.; Geiser, K.; Makhoul, D. C.; Reman, O.; de la

Serna, J .; Lefrere, F.; Chomienne, C.; Chastang, C.; Degos, L.;

Fenaux, P. Incidence, clinical features, and outcome of all trans-

retinoic acid syndrome in 413 cases of newly diagnosed acute

promyelocytic leukemia. The European APL Group. Blood 1998,

92, 2712-2718.

(21) Levi, I.; Raanani, P.; Shalmon, B.; Schiby-Brilliant, R.; Ben-

Bassat, I. Acute neutrophilic dermatosis induced by all-trans-

retinoic acid treatment for acute promyelocytic leukemia. Leuk.

Lymphoma 1999, 34, 401-404.

(22) Wu, W.; Sun, G.; Zhou, R.; Li, X.; Shen, Z.; Wang, Z. The

relationship between the levels of granulocyte colony-stimulating

factor and leukocytosis induced by all-trans retinoic acid in acute

promyelocytic leukemia. Chin. Med. J . (Engl.) 1999, 112, 1085-

1087.

(23) Tommasino, C.; De Felice, L.; Colombo, S.; Salaris, D.; Capocasa,

T.; Giudici, D. Retinoic acid syndrome. Severe respiratory

insufficiency treated with CPAP. Minerva Anestesiol. 2000, 66,

555-559.

(24) Duvic, M.; Hymes, K.; Heald, P.; Breneman, D.; Martin, A. G.;

Myskowski, P.; Crowley, C.; Yocum, R. C. Bexarotene is effective

and safe for treatment of refractory advanced-stage cutaneous

T-cell lymphoma: Multinational phase II-III trial results. J .

Clin. Oncol. 2001, 19, 2456-2471.

(25) Mart´ın del Pozo, M.; Cisneros de la Fuente, E.; Solano, F.;

Mart´ın, M. L.; de la Serna, J . The retinoic acid syndrome, a

complication of acute promyelocytic leukemia therapy. An. Med.

Intern. 2001, 18, 195-200.

(26) Goncalves, A.; Camerlo, J .; Bun, H.; Gravis, G.; Genre, D.;

Bertucci, F.; Resbeut, M.; Pech-Gourg, F.; Durand, A.; Maran-

inchi, D.; Viens, P. Phase II study of a combination of cisplatin,

all-trans-retinoic acid and interferon-R in squamous cell carci-

noma: Clinical results and pharmacokinetics. Anticancer Res.

2001, 21, 1431-1437.

(27) Khuri, F. R.; Rigas, J . R.; Figlin, R. A.; Gralla, R. J .; Shin, D.

M.; Munden, R.; Fox, N.; Huyghe, M. R.; Kean, Y.; Reich, S. D.;

Hong, W.-K. Multi-institutional Phase I/II trial of oral bexaro-

tene in combination with cisplatin and vinorelbine in previously

untreated patients with advanced nonsmall-cell lung cancer. J .

Clin. Oncol. 2001, 19, 2626-2637.

(38) Liu, Y.; Lee, M.-O.; Wang, H.-G.; Li, Y.; Hashimoto, Y.; Klaus,

M.; Reed, J . C.; Zhang, X. RARâ mediates the growth-inhibitory

effect of retinoic acid by promoting apoptosis in human breast

cancer cells. Mol. Cell. Biol. 1996, 16, 1138-1149.

(39) 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 analogue. Blood 2002, 100,

2917-2925.

(40) 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.; Moham-

mad, 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 analogue. Blood 2003, 102, 3743-3752.

(41) Schiff, L. J .; Okamura, W. H.; Dawson, M. I.; Hobbs, P. D.

Structure-biological activity relationships of new synthetic ret-

inoids on epithelial differentiation of cultured hamster trachea.

In Chemistry and Biology of Synthetic Retinoids; Dawson, M.

I., Okamura, W. H., Eds.; CRC Press: Boca Rotan, 1990; pp

302-363.

(42) Dawson, M. I.; Chan, R. L.-S.; 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)ethen-1-yl]-and6-(1,2,3,4-

tetrahydro-1,1,4,4-tetramethyl-6-naphthyl)-2-naphthalenecar-

boxylic acids. J . Med. Chem. 1983, 26, 1653-1656.

(43) Schiff, L. J .; Moore, S. J .; Dawson, M. I.; Hobbs, P. D.; Chan, R.

L.-S.; Derdzinski, K. Biological activity of aromatic retinoid acid

analogues in hamster tracheal organ culture. In In Vitro Models

of Respiratory Epithelium; Schiff, L. J ., Ed.; CRC Press: Boca

Raton, FL, 1986; pp 51-83.

(44) Cincinelli, R.; Dallavalle, S.; Merlini, L.; Penco, S.; Pisano, C.;

Carminati, P.; Giannini, G.; Vesci, L.; Gaetano, C.; Illy, B.; Zuco,

V.; Supino, R.; Zunino, F. A novel atypical retinoid endowed with

proapoptotic and antitumor activity. J . Med. Chem. 2003, 46,

909-912.

(45) Dawson, M. I.; Chao, W.-R.; Hobbs, P. D.; Delair, T. The

inihibitory 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 Rotan, 1990; pp

385-466.

(28) Park, C. J .; Bae, Y. D.; Choi, J . Y.; Heo, P. S.; Lee, K. S.; Park,

Y. S.; Lee, J . A. Sweet’s syndrome during the treatment of acute

promyelocytic leukemia with all-trans retinoic acid. Korean J .

Intern. Med. 2001, 16, 218-221.

(46) Dawson, M. I.; Park, J .-H.; Chen, G.; Chao, W.; Dousman, L.;

Waleh, N.; Hobbs, P. D.; J ong, L.; Toll, L.; Zhang, X.; Gu, J .;

Agadir, A.; Merchant, J . L.; Bai, L.; Verma, A. K.; Thacher, S.

M.; Chandraratna, R. A. S.; Shroot, B.; Hill, D. L. Retinoic acid

Blocker of AHPN-Induced Apoptosis

J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3535

(RA) receptor transcriptional activation correlates with inhibi-

tion of 12-O-tetradecanoylphorbol-13-acetate-induced ornithine

decarboxylase (ODC) activity by retinoids: A potential role for

trans-RA-induced ZBP-89 in ODC inhibition. Int. J . Cancer 2001,

91, 8-21.

(66) Apfel, C.; Bauer, F.; Crettaz, M.; Forni, L.; Kamber, M.;

Kaufmann, F.; LeMotte, P.; Pirson, W.; Klaus, M. A retinoic acid

receptor R antagonist selectively counteracts retinoic acid effects.

Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7129-7133.

(67) Lala, D. S.; Mukherjee, R.; Schulman, I. G.; Koch, S. S.;

Dardashti, L. J .; Nadzan, A. M.; Croston, G. E.; Evans, R. M.;

Heyman, R. A. Activation of specific RXR heterodimers by an

antagonist of RXR homodimers. Nature 1996, 383, 450-453.

(68) Arulmozhiraja, S.; Selvin, P. C.; Fujii, T. Structures, potential

energy curves, and torsional barrier heights for selected poly-

chlorinated biphenyls: A density functional theory study. J .

Phys. Chem. A 2002, 106, 1765-1769.

(69) Arulmozhiraja, S.; Fujii, T. Torsional barrier, ionization poten-

tial, and electron affinity of biphenylsA theoretical study. J .

Chem. Phys. 2001, 115, 10589-10594.

(70) Peterson, V. J .; Barofsky, E.; Deinzer, M. L.; Dawson, M. I.; Feng,

K. C.; Zhang, X.-K.; Madduru, M. R.; Leid, M. Mass-spectro-

(47) Dawson, M. I.; Hobbs, P. D.; Stein, R. B.; Berger, T. S.; Heyman,

R. A. Interaction of retinoids with retinoic nuclear receptor

isoforms. In Retinoids: New Trends in Research and Clinical

Applications; Livrea, M. A., Packer, L., Eds.; Marcel Dekker:

New York, 1992; pp 205-221.

(48) Lehmann, J . M.; Dawson, M. I.; Hobbs, P. D.; Husmann, M.;

Pfahl, M. Identification of retinoids with nuclear receptor

subtype-selective activities. Cancer Res. 1991, 51, 4804-4809.

(49) Dawson, M. I.; Zhang, X.; Hobbs, P. D.; J ong, L. Synthetic

retinoids and their usefulness in biology and medicine. In

Vitamin A and Retinoids: An Update of Biological Aspects and

Clinical Applications; Livrea, M. A., Ed.; Birkha¨user Verlag:

Basel, Switzerland, 2000; pp 161-196.

(50) Lehmann, J . M.; J ong, L.; Fanjul, A.; Cameron, J . F.; Liu, X. P.;

Haefner, P.; Dawson, M. I.; Pfahl, M. A novel class of retinoids,

selective for retinoid X receptor response pathways. Science

1992, 258, 1944-1946.

(51) Dawson, M. I.; J ong, L.; Hobbs, P. D.; Cameron, J . F.; Chao,

W.-R.; Pfahl, M.; Lee, M.-O.; Shroot, B. Conformational effects

on retinoid receptor selectivity. 2. Effects of retinoid bridging

group on retinoid X receptor activity and selectivity. J . Med.

Chem. 1995, 38, 3368-3383.

(52) Dawson, M. I.; Cameron, J . F.; Hobbs, P. D.; J ong, L.; Pfahl,

M.; Zhang, X.; Lehmann, J . M. (SRI International and The

Burnham Institute, formerly La J olla Cancer Research Founda-

tion). Bridged bicyclic aromatic compounds and their use in

modulating gene expression of retinoid receptors. U.S. Patent

5,837,725, 1998.

(53) Dawson, M. I.; Hobbs, P. D.; J ong, L.; Xiao, D.; Chao, W.-R.;

Pan, C.; Zhang, X.-K. sp2-bridged diaryl retinoids: Effects of

bridge-region substitution on retinoid X receptor (RXR) selectiv-

ity. Bioorg. Med. Chem. Lett. 2000, 10, 1307-1310.

(54) Dawson, M. I.; Zhang, X.-K. Discovery and design of retinoic acid

receptor and retinoid X receptor class- and subtype-selective

synthetic analogues of all-trans-retinoic acid and 9-cis-retinoic

acid. Curr. Med. Chem. 2002, 9, 623-637.

(55) Boehm, M. F.; Zhang, L.; Zhi, L.; McClurg, M. R.; Berger, E.;

Wagoner, M.; Mais, D. E.; Suto, C. M.; Davies, J . A.; Heyman,

R. A.; Nadzan, A. M. Design and synthesis of potent retinoid X

receptor selective ligands that induce apoptosis in leukemia cells.

J . Med. Chem. 1995, 38, 3146-3155.

metric analysis of agonist-induced retinoic acid receptor

conformational change. Biochem. J . 2002, 362, 173-181.

γ

(71) Dawson, M. I.; Hobbs, P. D.; Derdzinski, K. A.; Chao, W.-R.;

Frenking, G.; Loew, G. H.; J etten, A. M.; Napoli, J . L.; Williams,

J . B.; Sani, B. P.; Wille, J . J ., J r.; Schiff, L. J . Effect of structural

modifications in the C7-C11 region of the retinoid skeleton on

biological activity in a series of aromatic retinoids. J . Med. Chem.

1989, 32, 1504-1517.

(72) Nielsen, L. L.; Dell, J .; Maxwell, E.; Armstrong, L.; Maneval,

D.; Catino, J . J . Efficacy of p53 adenovirus-mediated gene

therapy against human breast cancer xenografts. Cancer Gene

Ther. 1997, 4, 129-138.

(73) Sheikh, M. S.; Li, X.-S.; Chen, J . C.; Shao, Z. M.; Ordonez, J . V.;

Fontana, J . A. Mechanisms of regulation of WAF1/Cip1 gene

expression in human breast carcinoma: Role of p53-dependent

and independent signal transduction pathways. Oncogene 1994,

9, 3407-3415.

(74) Xinarianos, G.; Liloglou, T.; Prime, W.; Sourvinos, G.; Karach-

ristos, A.; Gosney, J . R.; Spandidos, D. A.; Field, J . K. p53 status

correlates with the differential expression of the DNA mismatch

repair protein MSH2 in nonsmall cell lung carcinoma. Int. J .

Cancer 2002, 101, 248-252.

(75) Campling, B. G.; el-Deiry, W. S. Clinical implications of p53

mutations in lung cancer. Methods Mol. Med. 2003, 75, 53-77.

(76) Zhang, Y.; Huang, Y.; Rishi, A. K.; Sheikh, M. S.; Shroot, B.;

Reichert, U.; Dawson, M. I.; Poirer, G.; Fontana, J . A. Activation

of the p38 and J NK/SAPK mitogen-activated protein kinase

pathways during apoptosis is mediated by a novel retinoid. Exp.

Cell Res. 1999, 247, 233-240.

(56) Beard, R. L.; Colon, D. F.; Song, T. K.; Davies, P. J .; Kochhar,

D. M.; Chandraratna, R. A. S. Synthesis and structure-activity

relationships of retinoid X receptor selective diaryl sulfide

analogues of retinoic acid. J . Med. Chem. 1996, 39, 3556-3563.

(57) Dawson, M. I.; Chao, W. R.; Hobbs, P. D.; Zhang, X.-K. Effects

of trans-retinoic acid, 9-cis-retinoic acid, 1R,25-(dihydroxy)-

vitamin D₃and a novel apoptosis-inducing retinoid on breast

cancer and endothelial cell growth. Cancer Lett. 1998, 133, 1-8.

(58) Robertson, K. A.; Emami, B.; Collins, S. J . Retinoic acid-resistant

(77) Li, Y.; Lin, B.; Agadir, A.; Liu, R.; Dawson, M. I.; Reed, J . C.;

Fontana, J . A.; Bost, F.; Hobbs, P. D.; Zheng, Y.; Chen, G.-Q.;

Shroot, B.; Mercola, D.; Zhang, X.-K. Molecular determinants

of AHPN (CD437)-induced growth arrest and apoptosis in

human lung cancer cell lines. Mol. Cell. Biol. 1998, 18, 4719-

4731.

(78) White, E. Life, death, and the pursuit of apoptosis. Genes Dev.

1996, 10, 1-15.

(79) Green, D.; Kroemer, G. The central executioners of apoptosis:

Caspases or mitochondria? Trends Cell. Biol. 1998, 8, 267-271.

(80) Hsu, C. K. A.; Rishi, A. K.; Li, X.-S.; Dawson, M. I.; Reichert,

U.; Shroot, B.; Fontana, J . A. Bcl-X_Lexpression and its down-

regulation by a novel retinoid in breast carcinoma cells. Exp.

Cell Res. 1997, 232, 17-24.

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.

(59) Ota, E.; Shimozawa, J . T. Substitution reactions of phenanthrols.

XII. Conversion of phenanthrols to the corresponding chloro- and

bromophenanthrenes and their dipole moment measurements.

Nippon Kagaku Kaishi 1987, 4, 757-761.

(81) Fanjul, A. N.; Piedrafita, F. J .; Al-Shamma, H.; Pfahl, M.

Apoptosis induction and potent antiestrogen receptor-negative

breast cancer activity in vivo by a retinoid antagonist. Cancer

Res. 1998, 58, 4607-4610.

(82) Rishi, A. K.; Sun, R. J .; Gao, Y.; Hsu, C. K.; Gerald, T. M.; Saeed

Sheikh, M.; Dawson, M. I.; Reichert, U.; Shroot, B.; Fornace, A.

J ., J r.; Brewer, G.; Fontana, J . A. Posttranscriptional regulation

of the DNA damage-inducible gadd45 gene in human breast

carcinoma cells exposed to a novel retinoid CD437. Nucleic Acids

Res. 1999, 27, 3111-3119.

(83) Lu, X. P.; Fanjul, A.; Picard, N.; Pfahl, M.; Rungta, D.; Nared-

Hood, K.; Carter, B.; Piedrafita, J .; Tang, S.; Fabbrizio, E. Novel

retinoid-related molecules as apoptosis inducers and effective

inhibitors of human lung cancer cells in vivo. Nat. Med. 1997,

3, 686-690.

(84) Sun, S.-Y.; Yue, P.; Shroot, B.; Hong, W. K.; Lotan, R. Implication

of c-Myc in apoptosis induced by the retinoid CD437 in human

lung carcinoma cells. Oncogene 1999, 18, 3894-3901.

(85) Sun, S.-Y.; Yue, P.; Wu, G. S.; El-Deiry, W. S.; Shroot, B.; Hong,

W. K.; Lotan, R. Implication of p53 in growth arrest and

apoptosis induced by the synthetic retinoid CD437 in human

lung cancer cells. Cancer Res. 1999, 59, 2829-2833.

(86) Sun, S.-Y.; Yue, P.; Chen, X.; Hong, W. K.; Lotan, R. The

synthetic retinoid CD437 selectively induces apoptosis in human

lung cancer cells while sparing normal human lung epithelial

cells. Cancer Res. 2002, 62, 2430-2436.

(60) Dawson, M. I.; Cavasotto, C. N.; Hobbs, P. D.; J ong, L.; Feng,

K.-C.; Lui, H.; Abagyan, R.; Zhang, X. Synthesis and biological

activity of an RXR antagonist. Presented at the 93rd Annual

AACR National Meeting, San Francisco, CA, 2002.

(61) Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R. E.; Bonn,

T.; Engstrom, O.; Ohman, L.; Greene, G. L.; Gustafsson, J .-A.;

Carlquist, M. Molecular basis of agonism and antagonism in the

oestrogen receptor. Nature 1997, 389, 753-758.

(62) Pike, A. C.; Brzozowski, A. M.; Hubbard, R. E.; Bonn, T.;

Thorsell, A. G.; Engstrom, O.; Ljunggren, J .; Gustafsson, J . A.;

Carlquist, M. Structure of the ligand-binding domain of oestro-

gen receptor â in the presence of a partial agonist and a full

antagonist. EMBO J . 1999, 18, 4608-4618.

(63) Delabre, K.; Guiochon-Mantel, A.; Milgrom, E. In vivo evidence

against the existence of antiprogestins disrupting receptor

binding to DNA. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 4421-

4425.

(64) Brand, C.; Se´gard, P.; Plouvier, P.; Formstecher, P.; Danze´, P.

M.; Lefebvre, P. Selective alteration of gene expression in

response to natural and synthetic retinoids. BMC Pharmacol.

2002, 2, 13.

(65) Klaholz, B. P.; Renaud, J . P.; Mitschler, A.; Zusi, C.; Chambon,

P.; Gronemeyer, H.; Moras, D. Conformational adaptation of

agonists to the human nuclear receptor RARγ. Nat. Struct. Biol.

1998, 5, 199-202.

3536 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14

Dawson et al.

(87) Hsu, C. A.; Rishi, A. K.; Li, X.-S.; Gerald, T. M.; Dawson, M. I.;

Schiffer, C.; Reichert, U.; Shroot, B.; Poirer, G. C.; Fontana, J .

(103) Lovat, P. E.; Ranalli, M.; Bernassola, F.; Tilby, M.; Malcolm, A.

J .; Pearson, A. D.; Piacentini, M.; Melino, G.; Redfern, C. P.

Synergistic induction of apoptosis of neuroblastoma by fen-

retinide or CD437 in combination with chemotherapeutic drugs.

Int. J . Cancer 2000, 88, 977-985.

(104) Lovat, P. E.; Ranalli, M.; Bernassola, F.; Tilby, M.; Malcolm, A.

J .; Pearson, A. D.; Piacentini, M.; Melino, G.; Redfern, C. P.

Distinct properties of fenretinide and CD437 lead to synergistic

responses with chemotherapeutic reagents. Med. Pediatr. Oncol.

2000, 35, 663-668.

(105) Costantini, P.; J acotot, E.; Decaudin, D.; Kroemer, G. Mitochon-

drion as a novel target of anticancer chemotherapy. J . Natl.

Cancer Inst. 2000, 92, 1042-1053.

(106) Marchetti, P.; Mortier, L.; Beauvillain, V.; Formstecher, P. Are

mitochondria targets of anticancer drugs responsible for

apoptosis? Ann. Biol. Clin. (Paris) 2002, 60, 391-403.

(107) Buehler, C. A.; Harris, J . O.; Shacklett, C.; Block, B. P. The action

of formaldehyde on m-hydroxybenzoic acid. II. J . Am. Chem. Soc.

1946, 68, 574-577.

(108) Kelly, K. G.; Ellenbogen, L. Histidine decarboxylase inhibiting

4-bromo-3-hydroxy-hippuric acid. Ger. Patent 2,062,611, 1971.

(109) Barrow, J . C.; Nantermet, P. G.; Selnick, H. G.; Glass, K. L.;

Rittle, K. E.; Gilbert, K. F.; Steele, T. G.; Homnick, C. F.;

Freidinger, R. M.; Ransom, R. W.; Kling, P.; Reiss, D.; Broten,

T. P.; Schorn, T. W.; Chang, R. S.; O’Malley, S. S.; Olah, T. V.;

Ellis, J . D.; Barrish, A.; Kassahun, K.; Leppert, P.; Nagarath-

nam, D.; Forray, C. In vitro and in vivo evaluation of dihydro-

pyrimidinone C-5 amides as potent and selective R_1Areceptor

antagonists for the treatment of benign prostatic hyperplasia.

J . Med. Chem. 2000, 43, 2703-2718.

A. Retinoid induced apoptosis in leukemia cells through

a

retinoic acid nuclear receptor-independent pathway. Blood 1997,

89, 4470-4479.

(88) Giann`ı, M.; de The´, H. In acute promyelocytic leukemia NB4

cells, the synthetic retinoid CD437 induces contemporaneously

apoptosis,

a caspase-3-mediated degradation of PML/RARR

protein and the PML retargeting on PML-nuclear bodies.

Leukemia 1999, 13, 739-749.

(89) Mologni, L.; Ponzanelli, I.; Bresciani, F.; Sardiello, G.; Berga-

maschi, D.; Gianni, M.; Reichert, U.; Rambaldi, A.; Terao, M.;

Garattini, E. The novel synthetic retinoid 6-[3-adamantyl-4-

hydroxyphenyl]-2-naphthalenecarboxylic acid (CD437) causes

apoptosis in acute promyelocytic leukemia cells through rapid

activation of caspases. Blood 1999, 93, 1045-1061.

(90) Oridate, N.; Higuchi, M.; Suzuki, S.; Shroot, B.; Hong, W.-K.;

Lotan, R. Rapid induction of apoptosis in human C33A cervical

carcinoma cells by the synthetic retinoid 6-[3-(1-adamantyl)-

hydroxyphenyl]-2-naphthalenecarboxylic acid (CD437). Int. J .

Cancer 1997, 70, 484-487.

(91) Wan, X.; Duncan, M. D.; Nass, P.; Harmon, J . W. Synthetic

retinoid CD437 induces apoptosis of esophageal squamous HET-

1A cells through the caspase-3-dependent pathway. Anticancer

Res. 2001, 21, 2657-2663.

(92) Adachi, H.; Adams, A.; Hughes, F. M.; Zhang, J .; Cidlowski, J .

A.; J etten, A. M. Induction of apoptosis by the novel retinoid

AHPN in human T-cell lymphoma cells involves caspase-

dependent and independent pathways. Cell Death Differ. 1998,

5, 973-983.

(93) Schadendorf, D.; Worm, M.; J urgovsky, K.; Dippel, E.; Reichert,

U.; Czarnetzki, B. M. Effects of various synthetic retinoids on

proliferation and immunophenotype of human melanoma cells

in vitro. Recent Results Cancer Res. 1995, 139, 183-193.

(94) Schadendorf, D.; Kern, M. A.; Artuc, M.; Pahl, H. L.; Rosenbach,

T.; Fichtner, I.; Nurnberg, W.; Stuting, S.; von Stebut, E.; Worm,

M.; Makki, A.; J urgovsky, K.; Kolde, G.; Henz, B. M. Treatment

of melanoma cells with the synthetic retinoid CD437 induces

apoptosis via activation of AP-1 in vitro, and causes growth

inhibition in xenografts in vivo. J . Cell Biol. 1996, 135, 1889-

1898.

(95) Meister, B.; Fink, F. M.; Hittmair, A.; Marth, C.; Widschwendter,

M. Antiproliferative activity and apoptosis induced by retinoic

acid receptor-γ selectively binding retinoids in neuroblastoma.

Anticancer Res. 1998, 18, 1777-1786.

(96) Langdon, S. P.; Rabiasz, G. J .; Ritchie, A. A.; Reichert, U.;

Buchan, P.; Miller, W. R.; Smyth, J . F. Growth-inhibitory effects

of the synthetic retinoid CD437 against ovarian carcinoma

models in vitro and in vivo. Cancer Chemother. Pharmacol. 1998,

42, 429-432.

(110) Case, D. A.; Pearlman, D. A.; Caldwell, J . W.; Cheatham, T. E.,

III.; Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.;

Stanton, R. V.; Cheng, A. L.; Vicent, J . J .; Crowley, M.; Tsui, V.;

Radmer, R. J .; Duan, Y.; Pitera, J .; Massova, I.; Seibel, G. L.;

Singh, U. C.; Weiner, P. K.; Kollman, P. A. AMBER6. University

of California, San Francisco, 1999.

(111) Ciccotti, G.; Ryckaert, J . P. Molecular dynamics simulation of

rigid molecules. Comput. Phys. Rep. 1986, 3, 345.

(112) Goodsell, D. S.; Morris, G. M.; Olson, A. J . Automated docking

of flexible ligands: Applications of AutoDock. J . Mol. Recognit.

1996, 9, 1-5.

(113) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart,

W. E.; Belew, R. K.; Olson, A. J . Automated docking using a

Lamarckian genetic algorithm and an empirical binding free

energy function. J . Comput. Chem. 1998, 19, 1639-1662.

(114) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K.

M., J r.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell,

J . W.; Kollman, P. A. A second generation force field for the

simulation of proteins, nucleic acids, and organic molecules. J .

Am. Chem. Soc. 1995, 117, 5179-5197.

(97) Holmes, W. F.; Soprano, D. R.; Soprano, K. J . Comparison of

the mechanism of induction of apoptosis in ovarian carcinoma

cells by the conformationally restricted synthetic retinoids

CD437 and 4-HPR. J . Cell Biochem. 2003, 89, 262-278.

(98) Holmes, W. F.; Soprano, D. R.; Soprano, K. J . Elucidation of

molecular events mediating induction of apoptosis by synthetic

retinoids using a CD437-resistant ovarian carcinoma cell line.

J . Biol. Chem. 2002, 277, 45408-45419.

(115) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A well-

behaved electrostatic potential based method using charge

restraints for deriving atomic charges: The RESP model. J .

Phys. Chem. 1993, 97, 10269-10280.

(116) 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.

(117) Sakashita, A.; Kizaki, M.; Pakkala, S.; Schiller, G.; Tsuruoka,

N.; Tomosaki, R.; Cameron, J . F.; Dawson, M. I.; Koeffler, H. P.

9-cis-retinoic acid: Effects on normal and leukemic hemato-

poiesis in vitro. Blood 1993, 81, 1009-1016.

(118) Zhang, X.-K.; Hoffmann, B.; Tran, P. B.; Graupner, G.; Pfahl,

M. Retinoid X receptor is an auxiliary protein for thyroid

hormone and retinoic acid receptors. Nature 1992, 355, 441-

446.

(99) Hsu, J . Y.; Pfahl, M. ET-1 expression and growth inhibition of

prostate cancer cells: A retinoid target with novel specificity.

Cancer Res. 1998, 58, 4817-4822.

(100) Liang, J . Y.; Fontana, J . A.; Rao, J . N.; Ordonez, J . V.; Dawson,

M. I.; Shroot, B.; Wilber, J . F.; Feng, P. Synthetic retinoid CD437

induces S-phase arrest and apoptosis in human prostate cancer

cells LNCaP and PC-3. Prostate 1999, 38, 228-236.

(101) Lu, X. P.; Fanjul, A.; Picard, N.; Shroot, B.; Pfahl, M. A selective

retinoid with high activity against an androgen-resistant pros-

tate cancer cell type. Int. J . Cancer 1999, 80, 272-278.

(102) Ponzanelli, I.; Gianni, M.; Giavazzi, R.; Garofalo, A.; Nicoletti,

I.; Reichert, U.; Erba, E.; Rambaldi, A.; Terao, M.; Garattini, E.

Isolation and characterization of an acute promyelocytic leuke-

mia cell line selectively resistant to the novel antileukemic and

apoptogenic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naph-

thalenecarboxylic acid. Blood 2000, 95, 2672-2682.

(119) Wu, Q.; Dawson, M. I.; Zheng, Y.; Hobbs, P. D.; Agadir, A.; J ong,

L.; Li, Y.; Liu, R.; Lin, B.; Zhang, X.-K. Inhibition of trans-

retinoic acid-resistant human breast cancer cell growth by

retinoid X receptor-selective retinoids. Mol. Cell. Biol. 1997, 17,

6598-6608.

J M030524K

Article Doi

Antagonist analogue of 6-[3′-(1-adamantyl)-4′-hydroxyphenyl]-2- naphthalenecarboxylic acid (AHPN) family of apoptosis inducers that effectively blocks AHPN-induced apoptosis but not cell-cycle arrest

DOI: 10.1021/jm030524k

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