Adamantyl arotinoids that inhibit IκB Kinaseα and IκB Kinaseβ

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DOI: 10.1002/cmdc.201300100

Adamantyl Arotinoids That Inhibit IkB Kinase a and IkB

Kinase b

Paula Lorenzo,^[a]Marꢀa A. Ortiz,^[b]Rosana ꢁlvarez,*^[a]F. Javier Piedrafita,*^[b]and

ꢁngel R. de Lera*^[a]

A series of analogues of the adamantyl arotinoid (AdAr) chal-

cone MX781 with halogenated benzyloxy substituents at C2’

and heterocyclic derivatives replacing the chalcone group

were found to inhibit IkBa kinase a (IKKa) and IkBa kinase b

(IKKb) activities. The growth inhibitory capacity of some ana-

logues against Jurkat Tcells as well as prostate carcinoma (PC-

3) and chronic myelogenous leukemia (K562) cells, which con-

tain elevated basal IKK activity, correlates with the induction of

apoptosis and increased inhibition of recombinant IKKa and

IKKb in vitro, pointing toward inhibition of IKK/NFkB signaling

as the most likely target of the anticancer activities of these

AdArs. While the chalcone functional group present in many

dietary compounds has been shown to mediate interactions

with IKKb via Michael addition with cysteine residues, AdArs

containing a five-membered heterocyclic ring (isoxazoles and

pyrazoles) in place of the chalcone of the parent system are

potent inhibitors of IKKs as well, which suggests that other

mechanisms for inhibition exist that do not depend on the

presence of a reactive a,b-unsaturated ketone.

Introduction

The RAR antagonist MX781 (5) is part of the fairly heterogene-

ous group of atypical retinoids or retinoid related molecules

(RRMs), which includes N-hydroxyphenylretinamide (4-HPR)

and anhydroretinol and adamantyl arotinoids (AdArs), among

others.^[1]RRMs have been shown to inhibit cell growth by cas-

pase-dependent and -independent mechanisms^[2]and to

induce apoptosis via the intrinsic^[3]and extrinsic pathways.^[4]

Some of the apoptogenic AdArs, namely CD437 (1, AHPN), 5-

Cl-AHPN (2), AHPC (3, Adarotene), 3-Cl-AHPC (4), and MX781

(5),^[5]are shown in Figure 1. Although binding to the retinoid

receptors (RARs, subtypes a, b, and g)^[6]and retinoid X recep-

tors (RXRs, subtypes a, b, and g),^[7]which are members of the

nuclear receptor superfamily,^[8]has been documented in some

cases (i.e., MX781 is a RAR antagonist), their apoptogenic and

cell growth inhibitory activities appear to be unrelated to the

transactivation of these receptors.

Figure 1. Structures of selected RRMs with adamantyl groups.

lines, thus validating IKKb as a potential AdAr target.^[9]In con-

trast, CD437 (1) and its analogue 2 induce NFkB activity in

prostate carcinoma cells, and this NFkB activation is necessary

for apoptosis induction,^[10]whereas the cinnamic acid deriva-

tive 4 seems to activate IKKa and IKKb in several cancer

cells.^[10a,11][12]The exact mechanism of IKK/NFkB activation by

these AdArs remains ill-defined.^[11–13]

The activity of RRMs on IkB kinase (IKK) inhibition has been

the subject of recent interest. We initially found that MX781 (5)

substantially inhibits IKK isolated from tumor necrosis factor

(TNF)a-stimulated HeLa cells, and displays effective and consis-

tent inhibition of IKK/NFkB signals in a number of cancer cell

We synthesized a series of derivatives of 5 that preserve the

chalcone unit and incorporate an additional substituent ortho

to the carbonyl group (i.e., OR¹in 6, Scheme 1). We have ex-

amined their activities as IKK inhibitors to gain information on

the structural determinants of this novel anti-kinase activity of

AdArs.^[14]Some of these AdArs 6 surpassed the lead compound

in the growth inhibition of several cancer cell lines, including

leukemia, prostate, and estrogen-dependent and -independent

breast cancer cell lines. Because RAR transactivation was not

detected, the antiproliferative activity of the novel AdArs 6 is

likely due to inhibition of IKKb.^[14]

[a] Dr. P. Lorenzo, Prof. Dr. R. ꢀlvarez, Prof. Dr. ꢀ. R. de Lera

Departamento de Quꢁmica Orgꢂnica e Instituto de

Investigaciones Biomꢃdicas de Vigo (IBIV)

Universidade de Vigo, Lagoas-Marcosende, 36310 Vigo (Spain)

E-mail: rar@uvigo.es

qolera@uvigo.es

[b] Dr. M. A. Ortiz, Dr. F. J. Piedrafita

Torrey Pines Institute for Molecular Studies

3550 General Stomics Ct., San Diego, CA 92121 (USA)

E-mail: jpiedrafita@tpims.org

What is unique to MX781 (5) and analogues 6 that distin-

guish them from other RRMs is the presence of a chalcone

(1,3-diarylprop-2-en-one) functional group. Chalcones are natu-

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201300100.

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tivation.^[18]Isoliquiritigenin (7b) inhibits the translocation and

activation of NFkB by blocking the phosphorylation-dependent

degradation of IkBa.^[19,20]Direct inhibition of IKK has also been

demonstrated for butein (7c).^[21]Likewise cardamonin (7d)

causes a dose-dependent inhibition of p65 NFkB nuclear trans-

location.^[22]Whereas kawain and its derivatives flavokawains A

(7e) and B (7 f) inhibit TNF-mediated IkB degradation and sub-

sequent translocation of p50 and p65 NFkB subunits into the

nucleus, only flavokawain A 7e inhibits IKK and other kinases

as well.^[23]Moreover, xanthohumol (9) potentiates TNFa-in-

duced apoptosis in leukemia and myeloma cells and directly

inhibits TNF-induced IKK activation,^[24]as does licochalcone A

(10),^[25]but not the retrochalcone echinatin (11).^[25]

The a,b-unsaturated ketone of the chalcones is deemed re-

sponsible for some of the reported biological actions of this

compound class, due to its reactivity with nucleophilic residues

in target proteins. Thus, a Michael-type addition of a thiol

group of IKKb to the a,b-unsaturated ketones has been sug-

gested to explain the NFkB inhibitory profile of a series of syn-

thetic 4,3’,4’,5’-substituted chalcones such as 8.^[26]Consistent

with this view is the lack of activity toward IKK in analogous li-

gands with allyl alcohols.^[26,27]In addition, it has been shown

that the cysteine residue at position 179 of IKKb is essential for

licochalcone A-induced IKK inhibition, because licochalcone A

(10) fails to affect the kinase activity of the IKKb (C179A)

mutant.^[25]Butein (7c) and xanthohumol (9) also inhibit IKKb

via direct interaction with the Cys179 residue.^[21,24]Further-

more, curcumin, which contains a double a,b-unsaturated

ketone, inhibits constitutive NFkB activity in mantle cell lym-

phoma (MCL), possibly through the same mechanism.^[28]

Figure 2. Structures of chalcones with reported IKK inhibitory activities.

ral products and biogenetic precursors of the flavonoids and

isoflavonoids present in edible plants, some of which are con-

sidered responsible for the health benefits of this food

group.^[15]Several biological activities have been reported for

compounds with this privileged scaffold^[15]with direct inhibi-

tion of IKK as a viable mechanism of action. The parent unsub-

stituted chalcone arrests cell-cycle progression and induces mi-

tochondria-dependent apoptosis via inhibition of NFkB signal-

ing in human bladder cancer cells.^[16]2’-Hydroxychalcone

(7a)^[17]and close natural relatives (Figure 2) inhibit LPS-mediat-

ed induction of NO and TNFa by preventing NFkB and AP-1 ac-

Contrasting with those observations, we have found that

MX781 (5) does inhibit the activity of IKKb (C179A) mutant,^[9]

suggesting a Michael-addition-independent, but rather an ATP-

competitive mechanism of IKK inhibition. Because several ana-

logues of MX781 (compounds 6) inhibit recombinant IKKb

with higher potency than MX781 (5), and they also feature the

chalcone functional group, we synthesized a new series of

AdArs in which the unsaturated ketone has been converted

into heterocyclic derivatives in an attempt to define the struc-

tural requirements of the chalcone functional group for AdAr–

IKK interactions. The skeletons of the heterocyclic series pre-

serve both the benzoic acid at the polar terminus and the

OMEM-protected 4-adamantylphenol at the hydrophobic end.

Interestingly, some of these derivatives lacking the chalcone

functionality showed an even greater inhibitory activity against

recombinant IKKb in vitro than compounds 6, which further

demonstrates that AdArs inhibit IKK in a Michael-addition-inde-

pendent manner. We also found that AdArs inhibit IKKa, but

not the atypical IKK family member IKKe.

Results and Discussion

Synthesis

Two additional chalcones 6k,l were added to the series of sa-

turated and unsaturated analogues described earlier. Their syn-

thesis followed the sequence described previously,^[14]based on

Scheme 1. Reagents and conditions: a) LDA, THF, ꢀ788C; then 13k,l, 258C

(14k, 89%; 14l, 73%); b) LDA, THF, ꢀ788C; then 15, ꢀ78!258C (16k, 31%;

16l, 39%); c) NaOH, MeOH, 708C (6k, 73%; 6l, 80%).

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the Claisen–Schmidt condensation of methyl 4-formylbenzoate

(15) and the ortho-substituted C2’-(alkyl saturated and unsatu-

rated) alkoxy acetophenone derivatives 14 (Scheme 1). Chal-

cones 16k and 16l were obtained in two steps from the previ-

ously described ketone 12.^[14]Alkylation of the phenoxide de-

rived from 12 (HNa, DMF, 08C) with 4-bromobenzyl- or 3,5-di-

fluorobenzylbromide (13) provided 14k and 14l, respectively.

The classical Claisen–Schmidt condensation of 14 with methyl

4-formylbenzoate (15) under the conditions described for the

parent system (NaOH in MeOH at 708C),^[29]also produced the

saponification of the ester,^[14]but afforded the products 6k

and 6l in very low yields. However, prior generation of the lith-

ium enolate of 14k or 14l using LDA at ꢀ788C followed by

addition of 15 produced the a,b-unsaturated ketones 16k and

16l, respectively, in higher yields (31 and 39%). Saponification

of the benzoates provided the desired analogues 6k and 6l in

good yield.

provide propargylic ketone 20 in 89% yield.^[31]Selective depro-

tection of the less hindered MEM acetal ortho to the ketone^[32]

led to phenol 21 in 69% yield. Isoxazole 23 was acquired in

77% yield by the addition of hydroxylamine to the propargylic

ketone 20, in a MeOH/CH₂Cl₂solvent mixture due to poor solu-

bility, followed by saponification of 22 (83% yield).

The regioselectivity of the synthesis of pyrazoles from the

condensation of propargylic ketones and hydrazines depends

on the structure and substituents of the ketone, but can be

fine-tuned by the substituents of the reagent (alkyl or arylhy-

drazines).^[33]Accordingly, treatment of 20 with methyl- and

phenylhydrazine afforded regioisomeric structures 24 and 26,

respectively. Saponification gave the respective pyrazoles 25

and 27 (Scheme 2). To confirm the regioselectivity, the signals

1

for the adamantyl group in the H NMR spectra of phenylpyra-

zole 27 showed non-equivalence due to the proximity of the

phenyl substituent.^[33b]For the N-methyl analogue 25, NOE

studies showed the correlation of the N-methyl substituent

with the H3/H5 protons of the benzoate, thus confirming the

anticipated structure.

The inherent reactivity of a,b-unsaturated ketones was ex-

ploited for the incorporation of heterocycles at the central

region of the AdAr. Isoxazoles, pyrazoles, and pyrimidines were

easily accessed by reaction of propargylic ketone 21 derived

from aldehyde 17^[14](Scheme 2) with hydroxylamines, hydra-

zines and amidines, respectively. In addition, ketone 21 might

allow the preparation of an analogue of MX781 with the bio-

genetically related flavone structure via a 6-endo-dig cycliza-

tion.

Generation of the alkynyl anion of 18^[30]obtained upon

treatment with LDA at ꢀ788C followed by addition of alde-

hyde 17^[14]afforded the propargylic alcohol 19, which under-

went oxidation using CrO₃and pyridine in CH₂Cl₂at 258C to

For the construction of pyrimidines we first attempted the

microwave-assisted reaction of 21 with the amidinium salts.^[34]

However, the use of Na₂CO₃as base promoted a 5-exo-dig cyc-

lization of the ortho-hydroxyaryl phenylethynyl ketone to yield

the aurone 33 (Scheme 3). The b-methoxyethoxymethyl (MEM)

ether-protected analogues uneventfully provided pyrimidines

28a and 28b. Treatment of these intermediates with BCl₃at

ꢀ788C led to deprotection of the MEM group, and final sapo-

nification of 29 (1m NaOH in MeOH at 708C) provided the car-

boxylic acids 30.

Previous studies on the

based-induced cyclization of

ortho-hydroxyaryl phenylethynyl

ketones demonstrated the criti-

cal role of the reaction condi-

tions,^[35]which can determine

the kinetic/thermodynamic pref-

erence of the intermediate carb-

anion corresponding to the 5-

exo-dig or 6-endo-dig reactions

to afford the aurones and/or fla-

vones, respectively. Regardless

of the experimental conditions,

aurone 33 was always the major

product obtained in this case,

a bias presumably due to the

effect of the electron-withdraw-

ing ester. The structures of the

isomeric products were secured

by NOE correlations and by the

characteristic UV spectra exhibit-

ed by these skeletons (aurone:

l_max~342 nm; flavone: l_max

~

315 nm). Basic hydrolysis

(Scheme 3) yielded carboxylic

Scheme 2. Reagents and conditions: a) LDA, THF, ꢀ78!258C (97%); b) CrO₃, Py, CH₂Cl₂, 258C (89%); c) BCl₃,

CH₂Cl₂, ꢀ788C (69%); d) NH₂OH·HCl, NaOAc, MeOH/CH₂Cl₂, 808C (77%); e) NaOH, MeOH, 50–708C (23, 92%; 25,

99%; 27, 99%); f) MeNHNH₂, EtOH, 258C (81%); g) PhNHNH₂, EtOH, 808C (69%).

acids 32 and 34.

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IKKb by 6d and 6h correlates

well with the strongest induc-

tion of apoptosis observed in

Jurkat cells.^[14]More interesting-

ly, compounds 6 f and 6i exhib-

ited improved anti-IKKb and

growth inhibitory activities and

have lost their RAR-dependent

transactivation function.^[14]

We have now re-assayed the

series 6a–j as well as the new

chalcones 6k,l together with

the heterocyclic derivatives for

determination of their IKK inhib-

Scheme 3. Reagents and conditions: a) Acetamidine·HCl or benzamidine·HCl, Na₂CO₃, CH₃CN, MW, 1008C (28a,

90%; 28b, 56%); b) BCl₃, CH₂Cl₂, ꢀ788C (29a, 54%; 29b, 46%); c) 1m NaOH, MeOH, 708C (30a, 68%; 30b, 72%;

32, 75%; 34, 85%); d) K₂CO₃, acetone, 708C (31, 24%; 33, 76%), or K₂CO₃, EtOH, 808C (31, 20%; 33, 67%).

itory activity using a homogene-

ous LANCE Ultra kinase assay

with recombinant kinases and

Ulight–IkBa peptide as sub-

Biological evaluation

strate. The values obtained for the inhibition of IKKb with

AdArs at 20 mm are generally higher in this TR-FRET-based

assay than from a previous kinase assay that measured the

amount of ³²P incorporated into GST–IkBa substrate, with the

exception of 6a, which is a poor inhibitor of IKK; 6b, which

shows decreased potency; and 6j, which elicits stronger inhibi-

tion (~57%) in this TR-FRET assay. Nevertheless, the results

listed in Table 1 confirm the previously reported inhibition of

IKKb by C2’-substituted chalcones 6^[14]and demonstrate that

AdArs function as dual IKKa/b inhibitors in vitro

We have shown that MX781 (5) is a strong inhibitor of IKKb ac-

tivity in vitro as well as in cell-based assays.^[9]Most of the ana-

logues 6a–j were previously shown to exhibit enhanced inhibi-

tion of the kinase relative to parent compound 5, and similar

to that observed with two well-characterized inhibitors of IKK

(BMS345541 and SC-514).^[14]Moreover, the highest inhibition of

Table 1. Effect of AdAr analogues of 5 (MX781) on IKK activity, cell growth, and apoptosis.

Compd

IKKa

IKKb

IKKe

Inh. [%]^[a]

Cell Viability

Apoptosis

DEVDase^[e]

Inh. [%]^[a]

IC₅₀[mm]^[b]

Inh. [%]^[a]

IC₅₀[mm]^[b]

Via. [%]^[c]

IC₅₀[mm]^[d]

5

69.2ꢁ13

38.7ꢁ9.9

72.6ꢁ14

77.5ꢁ8.3

98.0ꢁ0.5

96.1ꢁ1.4

81.1ꢁ5.2

86.2ꢁ9.2

76.9ꢁ8.7

91.8ꢁ4.6

50.4ꢁ17

91.2ꢁ1.2

97.4ꢁ0.2

86.9ꢁ6.0

30.4ꢁ4.0

83.3ꢁ10

1.6ꢁ8.3

8.29

50.0ꢁ6.8

21.9ꢁ13

46.3ꢁ5.6

86.2ꢁ6.5

99.0ꢁ0.3

97.9ꢁ1.6

96.9ꢁ0.7

92.0ꢁ8.7

73.9ꢁ8.6

94.6ꢁ3.3

57.1ꢁ3.4

93.7ꢁ5.5

96.8ꢁ2.2

86.3ꢁ11

35.3ꢁ4.5

95.3ꢁ3.4

4.6ꢁ4.5

16.69

15.8ꢁ5.6

17.5ꢁ7.6

0

28.5ꢁ5.5

34.0ꢁ5.5

17.4ꢁ7.8

8.4ꢁ0.4

4.6ꢁ1.2

4.5ꢁ1.6

4.5ꢁ0.5

10.3ꢁ4.6

9.5ꢁ3.0

3.2ꢁ1.3

97.6ꢁ1.2

2.1ꢁ0.4

2.6ꢁ1.3

1.3ꢁ0.2

31.0ꢁ2.9

2.4ꢁ0.5

92.3ꢁ4.9

97.3ꢁ2.1

96.5ꢁ1.3

70.2ꢁ3.9

1.80

20.7ꢁ7.8

1.2ꢁ0.6

6a

6b

6c

6d

6e

6 f

6g

6h

6i

6j

6k

6l

23

25

27

30a

30b

32

34

3.72

4.98

2.86

3.73

2.48

5.30

5.90

2.28

10.25

6.83

4.17

3.48

3.33

5.85

6.62

3.35

2.38

2.21

1.44

1.48

1.78

2.17

2.23

0.97

9.6ꢁ2.2

15.6ꢁ4.5

39.2ꢁ12

39.3ꢁ12

34.2ꢁ6.8

20.6ꢁ6.0

17.5ꢁ6.8

34.7ꢁ6.4

31.2ꢁ8.4

45.5ꢁ7.7

30.7ꢁ6.7

31.5ꢁ4.2

15.0ꢁ9.1

40.1ꢁ4.1

7.5ꢁ9.0

18.6ꢁ8.6

5.2ꢁ6.8

39.1ꢁ6.8

49.0ꢁ11

65.0ꢁ3.7

65.0ꢁ1.0

48.5ꢁ2.7

36.4ꢁ4.4

40.2ꢁ8.1

43.4ꢁ0.8

1.1ꢁ0.7

2.66

2.16

2.73

1.83

0.84

0.93

1.95

4.73

2.54

44.5ꢁ2.2

48.3ꢁ5.7

105.9ꢁ20

10.4ꢁ1.4

93.2ꢁ14

2.0ꢁ1.2

17.7ꢁ13

65.6ꢁ5.2

75.4ꢁ7.1

13.3ꢁ8.0

24.3ꢁ4.3

86.4ꢁ6.1

1.6ꢁ2.8

1.2ꢁ0.2

7.5ꢁ1.9

[a] The effect of 20 mm AdArs on the activity of recombinant IKKs was determined by LANCE Ultra kinase assay at an ATP concentration near the apparent

K_Mvalue for each kinase. Values indicate percent inhibition with respect to solvent control samples ꢁSD obtained in 3–4 experiments performed in tripli-

cate. [b] Where indicated, IC₅₀values were calculated using half-log eight-point titration curves, with 100 mm as the maximum concentration. All experi-

ments were performed twice with triplicate data points. [c] Percent cell viability was calculated with Jurkat Tcells treated with the indicated AdArs at 4 mm

for 24 h. Control cells were incubated with the same volume of solvent DMSO (0.1% v/v). The average ꢁSD of three independent experiments performed

with triplicate data points is shown. [d] Detailed titration experiments were performed in Jurkat cells treated for 24 h with eight different concentrations

(1:2.5 serial dilutions starting at 10 mm) of the most active AdArs. Cell viability was measured as described in the Experimental Section. [e] A DEVDase fluori-

metric assay was used as a measure of apoptosis in Jurkat cells treated with the indicated AdArs at 5 mm for 4 h, as described in the Experimental Section.

Values indicate the fold induction of DEVDase activity with respect to control cells incubated in the presence of solvent DMSO. The average ꢁSD of two

independent experiments performed in triplicate is shown.

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bromo (6k) and difluoro (6l) substitutions in 6i preserve

strong IKKb interactions, with IC₅₀values of 2.73 and 1.83 mm,

respectively. Moreover, all these compounds were also strong

inhibitors of IKKa, but not of the atypical IKK family member

IKKe. Only AdArs 6d,e,k, 27, and 34 showed a weak but repro-

ducible inhibition of IKKe, <50%. In general, the IC₅₀values

obtained for all chalcone analogues were similar with both

IKKa and IKKb, with the exception of the parental compound 5

and 6b, which elicited stronger activity toward IKKa. Among

the heterocyclic analogues, aurone 34 also showed activity

against IKKa and IKKb, but flavone 32 proved inactive toward

IKKb. With the exception of pyrimidines 30a and 30b, all other

heterocyclic derivatives were highly active at inhibiting both

IKKa and IKKb, in particular isoxazole 23 and phenylpyrazole

27.

Figure 3. AdArs inhibit the viability of cancer cells. Cells were treated with

the indicated AdArs at 4 mm for 48 h with PC-3 and K562 cells; Jurkat cells

were treated for 24 h. Cell viability was measured by luminescence as de-

scribed in the Experimental Section.

proliferative activity of 6k and 6l correlates well with increased

inhibition of IKKa/b.

AdArs inhibit growth and induce apoptosis in cancer cells

RAR/RXR transactivation profile of novel AdArs

The effect of AdArs on cancer cell proliferation was first evalu-

ated in Jurkat Tcells. As previously reported, chalcones 6c–

i and the new chalcones 6k,l elicited very strong inhibition of

cell viability, which paralleled strong induction of apoptosis, as

demonstrated by elevated DEVDase activity (Table 1). Only

chalcone 6j, the pyrimidines 30a,b, and the heterocyclic ana-

logue 32 proved inactive in this assay, with aurone 34 showing

weak activity. With the exception of chalcone 6a and pyrazole

25, all active analogues elicited IC₅₀values similar to or lower

than that of the parental compound MX781 (5). Furthermore,

inhibition of cell viability paralleled induction of DEVDase activ-

ity as a measure of apoptosis (Table 1). In particular, the heter-

ocyclic AdArs 23 and 27 produced the highest levels of DEV-

Dase activity after only 4h of exposure in Jurkat cells, with

about a 100-fold induction over untreated cells and five times

higher than parental compound MX781 (5). This superior stim-

ulation of DEVDase activity by 23 and 27 over other chalcones,

despite having similar IC₅₀values in the cell proliferation assay,

is likely due to a more rapid activation of caspases by those

heterocyclic compounds, as determined in a time-course ex-

periment (data not shown). In contrast, decreased DEVDase ac-

tivity induced by chalcones MX781 5 and 6b, as well as pyra-

zole 25, did not correlate well with the substantial inhibition of

cell viability observed (Table 1). This is in agreement with the

slower kinetics of DEVDase activation detected with MX781

(5),^[2c]although cytostatic effects cannot be ruled out. The

fused heterocyclic analogue with aurone skeleton 34 elicited

limited activity, whereas flavone 32 was inactive, which coin-

cides with the failure to inhibit cell viability and IKK activity. In

agreement with the IKK inhibition profile, the halogenated

benzylethers 6k and 6l, together with isoxazole 23 and phe-

nylpyrazole 27, were also strong inhibitors of cell proliferation

and inducers of DEVDase activity. Moreover, the growth inhibi-

tory activities of 6k and 6l against prostate carcinoma (PC-3)

and chronic myelogenous leumekia (K562) cells were substan-

tially higher than those observed with the non-halogenated

analogue 6i or MX781 5 (Figure 3). Both PC-3 and K562 cells

have elevated basal IKK/NFkB activity, and this enhanced anti-

The RAR/RXR transactivation profile was next determined. As il-

lustrated in Table 2, none of the compounds was capable of in-

ducing transactivation activity of the Gal4–RARa fusion protein

in HEK-293 cells; in contrast, the natural ligand all-trans-retinoic

acid (atRA) activated the receptor over 100-fold. Likewise, all

AdArs were inactive as RXRa agonists. To test the activity of

AdArs as potential RAR/RXR antagonists, Gal4–RAR/RXR trans-

fected cells were stimulated with their corresponding agonist

control (100 nm) in the absence or presence of a 40-fold molar

excess of the AdArs (4 mm). Table 2 shows that the AdArs were

not active against RXRa, but some of them elicited significant

antagonistic activity against RARa. As originally reported, chal-

cone 6a (IC₅₀: 1.063 mm) was stronger as antagonist than the

parental compound MX781 5 (IC₅₀: 4.474 mm). Other chalcones

6b–g showed partial inhibition of atRA-driven RARa transacti-

vation, whereas derivatives 6i–l were inactive as antagonists.

Chalcone 6h and the fused heterocyclic analogues 32 and 34

showed similar activity as MX781 (5) when tested at a single

concentration, but subsequent IC₅₀determinations demon-

strated that 6h and flavone 32 are indeed stronger antago-

nists, with respective IC₅₀values of 1.656 and 0.858 mm. The N-

methylpyrazole 25 also elicited strong antagonist activity, with

an IC₅₀value of 0.772 mm. Other chalcones that had partial ac-

tivity in the preliminary single-concentration assay (6c and

6g), elicited IC₅₀values lower than that of MX781 (2.859 and

1.598 mm, respectively).

In summary, although some of the chalcones (6g, 6h) and

heterocyclic AdArs (25, 32) were observed to be stronger an-

tagonists than MX781 (5), they were still weak RAR antagonists

compared with the inverse agonist UVI2024, which shows an

IC₅₀value of 0.114 mm. Most notably, the halogenated benzy-

lether chalcones 6k and 6l and the isoxazole 23 have no RAR/

RXR activity whatsoever, while eliciting the strongest IKK/

growth inhibitory and apoptosis-inducing effects, which indi-

cates a clear separation of these biological activities.

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also been found to link inflammation with cancer,^[40]

whereas IKKa seems to play a role in cancer meta-

stasis via inhibition of maspin expression levels.^[41]

Moreover, the atypical IKK family members IKKe and

TBK1 have recently been found to play a role in tu-

morigenesis. IKKe functions as a breast and ovarian

proto-oncogene; it is overexpressed in breast and

ovarian cancer cell lines and in primary tumors, par-

ticularly in hormone-resistant breast tumors.^[42]TBK1

is essential for the survival of K-Ras-dependent lung

cancer cell lines.^[43]It is therefore clear that inhibitors

of the IKK/NFkB pathway can offer exceptional op-

portunities to develop novel anticancer strategies.^[44]

Numerous inhibitors of IKK/NFkB activity have

been described that act at different levels, including

inhibitors that function upstream of the IKK com-

plex, inhibitors of IKK activity, or inhibitors of NFkB

nuclear functions.^[45]As IKKb is the catalytic subunit

essential for activation of the classical NFkB pathway

and is required for the decreased apoptosis rates ob-

served in cancer cells,^[39a,46]the development of IKKb

inhibitors has been heavily explored, and several

IKKb-selective as well as dual IKKa/b inhibitors have

indeed been described and are under clinical evalua-

tion.^[44b,47]This search will certainly be accelerated by

the structural insight gained with the recently de-

scribed crystal structure of IKKb from Xenopus laevis

in complex with an inhibitor at resolutions of 4 and

3.6 ꢃ.^[48]

Table 2. Activation of RAR and RXR by novel AdArs.

Compd

RAR Profile

RXR Profile

Agonist^[a]Antagonist [%]^[b]IC₅₀[mm]^[c]Agonist^[a]Antagonist [%]^[b]

non-stimulated

1.0 1.0

1.2ꢁ0.2

7.1ꢁ2.9

atRA (or CD3254) 110.8ꢁ12 100

14.3ꢁ3.4 100

no AdAr

5

6a

6b

6c

6d

6e

6 f

6g

6h

6i

6j

6k

6l

23

25

27

30a

30b

32

0.9ꢁ0.2 37.1ꢁ5.3

1.1ꢁ0.2 5.8ꢁ2.2

4.474

1.063

7.212

2.859

5.896

0.8ꢁ0.1 90.8ꢁ4.7

1.4ꢁ0.6 94.9ꢁ3.4

0.9ꢁ0.2 105.1ꢁ5.3

0.9ꢁ0.1 91.4ꢁ3.5

0.9ꢁ0.1 81.0ꢁ7.6

1.1ꢁ0.2 99.9ꢁ10

1.2ꢁ0.1 111.4ꢁ9.2

1.0ꢁ0.2 108.6ꢁ8.0

1.1ꢁ0.2 108.5ꢁ9.6

1.3ꢁ0.3 117.4ꢁ3.1

0.9ꢁ0.1 93.4ꢁ3.2

0.9ꢁ0.2 125.5ꢁ3.6

1.3ꢁ0.2 129.2ꢁ8.0

1.0ꢁ0.1 109.8ꢁ9.8

0.9ꢁ0.1 86.5ꢁ3.4

1.4ꢁ0.2 67.0ꢁ3.9

0.8ꢁ0.1 93.7ꢁ5.8

1.2ꢁ0.1 106.3ꢁ4.7

1.3ꢁ0.1 134.3ꢁ12

0.9ꢁ0.2 109.3ꢁ5.5

1.1ꢁ0.2 61.6ꢁ2.1

1.2ꢁ0.3 45.8ꢁ4.9

1.2ꢁ0.2 51.6ꢁ5.1

1.3ꢁ0.3 56.8ꢁ6.1

1.4ꢁ0.5 54.9ꢁ4.1

1.3ꢁ0.4 44.7ꢁ3.0

1.4ꢁ0.4 39.0ꢁ3.2

1.1ꢁ0.1 84.4ꢁ5.4

0.9ꢁ0.2 90.6ꢁ7.6

1.3ꢁ0.4 81.9ꢁ4.3

1.1ꢁ0.1 79.6ꢁ5.4

1.4ꢁ0.3 81.7ꢁ4.3

0.9ꢁ0.2 14.7ꢁ3.4

1.6ꢁ0.4 67.7ꢁ5.7

1.1ꢁ0.1 80.2ꢁ3.1

1.1ꢁ0.1 106.3ꢁ11

2.5ꢁ1.2 38.1ꢁ7.1

2.5ꢁ1.7 50.9ꢁ6.7

3.0ꢁ0.4

1.598

1.656

0.772

0.858

4.417

0.114

34

UVI2024

UVI3003

23.3ꢁ2.5

[a] Values indicate the fold induction of RAR/RXR activity with respect to solvent con-

trol cells (non-stimulated). As positive control, RARa-transfected cells were treated

with 1 mm atRA, whereas RXRa-expressing cells were treated with the synthetic rexi-

noid CD3254 at 1 mm. AdArs were used at 4 mm. The average ꢁSD of two independ-

ent experiments with triplicate data points is shown. [b] To determine the antagonist

profile of AdAr analogues, RAR/RXR-expressing cells were incubated with 100 nm

atRA/CD3254 in the presence of the indicated AdArs at 40-fold molar excess. The

values indicate the percent activity with respect to control cells incubated with atRA/

CD3254 and no AdAr (100% activity). The percent activity of non-stimulated cells is

also shown. As positive controls, RAR- and RXR-transfected cells were treated with the

well-known antagonists UVI2024 and UVI3003, respectively (1 mm in each case). The

average ꢁSD of two or three experiments performed with triplicate data points is

shown. [c] IC₅₀values were calculated following an eight-point titration (1:2.5 serial di-

lutions) of the most active compounds in an antagonist setting.

We previously established a correlation between

inhibition of IKK/NFkB activity by AdArs such as 5

and non-retinoidal small molecules, as well as by ge-

netic approaches, with induction of apoptosis in

lung and prostate carcinoma cells.^[9]Furthermore,

the RAR antagonist MX781 (5) inhibited the in vitro

activity of IKK immunopurified from TNFa-stimulated

HeLa cells,^[9]and by using recombinant IKKa and

IKKb proteins, we have now demonstrated that 5 in-

hibits both IKKa and IKKb.^[9,14]In striking contrast,

Discussion and Conclusions

other AdArs such as 2 and its analogue 4 induce NFkB activity

in prostate carcinoma cells, and this NFkB activation seems to

be necessary for apoptosis induction.^[10]Compound 4 activates

IKKa and IKKb in prostate and breast carcinoma cells,^[10a]and

acute leukemia cells.^[12]In contrast to the inhibition of IKK/

NFkB signaling by 5, cinnamic acid 4 activates the canonical

and non-canonical NFkB pathways in the human breast carci-

noma and leukemia cell lines.^[12]Besides IKK, a different target

has been reported for 4 and related compounds, which bind

the nuclear hormone receptor small heterodimer partner (SHP)

and modulate SHP interactions with the Sin3A repressor.^[13,49]

Binding of 4 to SHP seems to mediate apoptosis, as SHP-defi-

cient mouse embryo fibroblasts and breast carcinoma cells ex-

pressing decreased levels of SHP exhibit significantly lower de-

grees of apoptosis in response to this AdAr.^[13]In addition to

IKK and SHP, some AdArs can also target certain protein phos-

phatases in vitro, including MKP-1 and the protein tyrosine

phosphatases CD45 and SHP-2.^[50]Inhibition of protein phos-

Overwhelming evidence supports a critical role for IKK/NFkB

activity in tumor initiation and progression, cancer cell survival,

and protection from TNFa-induced apoptosis.^[36]High levels of

NFkB activity have been detected in numerous tumor cell

types, including melanoma, breast, prostate, and ovarian can-

cers, among others, and NFkB levels increase in breast cancers

as the tumor progresses.^[37]Constitutive NFkB activation con-

tributes significantly to cell survival in human colon carcinoma

cells as well, rendering them resistant to TRAIL as well as to

other chemotherapeutic agents, including a cisplatin analogue.

Using quinacrine as an NFkB signaling inhibitor, a highly syner-

gistic potentiation of the cytotoxic effect of TRAIL and the cis-

platin analogue was observed, supporting the use of NFkB in-

hibitors for the treatment of colon cancer.^[38]Genetic studies

originally pointed toward IKKb as the principal mediator of

cancer cell survival and resistance to apoptosis.^[39]IKKb has

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MA, USA). All other reagents were commercial compounds of the

highest purity available. Unless otherwise indicated, all reactions in-

volving air- and moisture-sensitive materials were carried out

under argon atmosphere, and those not involving aqueous re-

agents were carried out in oven-dried glassware. Analytical thin-

layer chromatography (TLC) was performed on aluminum plates

with Merck Kieselgel 60 F₂₅₄and visualized by UV irradiation

(l 254 nm) or by staining with an ethanolic solution of phospho-

molybdic acid. Flash column chromatography was carried out with

Merck Kieselgel 60 (230–400 mesh) under pressure. Electron impact

(EI) mass spectra were obtained on a Hewlett–Packard HP59970 in-

strument operating at 70 eV. Chemical ionization mass spectra

were acquired on an Agilent 6890N gas chromatograph coupled to

a mass spectrometer with a Micromass GCT Time of Flight (TOF)

analyzer. Alternatively an APEX III FT-ICR MS (Bruker Daltonics, Bill-

erica, MA, USA) equipped with a 7 T actively shielded magnet was

used, and ions were generated using an Apollo API electrospray

ionization (ESI) source, with a voltage between 1800 and 2200 V

(to optimize ionization efficiency) applied to the needle, and

a counter voltage of 450 V applied to the capillary. For ESI spectra

samples were prepared by adding a spray solution of 70:29.9:0.1

(v/v/v) CH₃OH/H₂O/formic acid to a solution of the sample at a v/v

ratio of 1 to 5% to give the best signal-to-noise ratio. High-resolu-

tion mass spectra were taken on a VG Autospec instrument.

¹H NMR spectra were recorded in CDCl₃, (CD₃)₂CO, CD₃CO₂D, and

[D₆]DMSO at ambient temperature on Bruker AV-300 or AMX-400

spectrometers at 300 or 400 MHz, with residual protic solvent as

the internal reference [CDCl₃, d_H=7.26 ppm; (CD₃)₂CO, d_H=

2.05 ppm; CD₃CO₂D, d_H=2.10 ppm; and [D₆]DMSO, d_H=2.50 ppm];

chemical shifts (d) are given in parts per million (ppm), and cou-

pling constants (J) are given in Hertz (Hz). The proton spectra are

reported as follows: d (multiplicity, coupling constant J, number of

protons, assignment). ¹³C NMR spectra were recorded at ambient

temperature on the same spectrometers at 75.4 or 100 MHz, with

the central peak of the solvent [CDCl₃, d_C=77.0 ppm; (CD₃)₂CO,

d_C=30.83 ppm; CD₃CO₂D, d_C=175.99 ppm; and [D₆]DMSO, d_C=

39.52 ppm] as the internal reference. The DEPT sequence was rou-

tinely used for ¹³C multiplicity assignment. Additional HSQC, HMBC,

and NOE spectra were recorded in particular cases to enable struc-

tural and stereochemical assignment. IR spectra were obtained on

a JASCO FT/IR-4200 infrared spectrometer, from a thin film deposit-

ed onto a NaCl glass or as a KBr disc; data include only characteris-

tic absorptions. Peaks are quoted in wave numbers (cm^ꢀ1), and

their relative intensities are reported as follows: s=strong, m=

medium, w=weak. UV spectra were recorded on a HP5989A spec-

trophotometer using MeOH as solvent. Absorption maxima are re-

ported in nm. Melting points were measured in a Stuart Scientific

apparatus (Staffordshire, UK). Elemental analyses were carried out

in a Fisons EA-1108 elemental analyzer. Microwave irradiation was

performed on a CEM Explorer model with Discover accessory,

Class 1, continuous operation, maximum power 300 W. All tested

compounds gave correct analytical data or were purified by RP-

HPLC (as indicated in each case) and shown to have >95% purity.

phatases has not yet been demonstrated in cell-based assays,

and their relevance to AdAr anticancer activity needs to be ad-

dressed.

Herein we report on the effect of a series of analogues of 5

on recombinant IKKs and cancer cell proliferation. Although in-

itially thought to be selective for IKKb, the present study dem-

onstrates that AdArs actually inhibit IKKa with slightly higher

potency than IKKb and thereby function as dual IKKa/b inhibi-

tors. A hydroxy group at C2’ combined with a five-membered

heterocycle (isoxazole, pyrazole) or an alkoxy group on the

ring adjacent to the chalcone unit (6, with n-butyloxy or n-pen-

tyloxy group, and halogenated benzyloxy groups) retain or im-

prove the activity of the parent system with the strongest in-

hibition of IKKb and IKKa concurrent with the strongest

growth inhibitory activity against Jurkat cells. Enhanced

growth inhibitory activity by halogenated chalcones 6k and 6l

was also observed in PC-3 and K562 cells, which contain ele-

vated basal IKK activity. These data confirm preliminary reports

that IKK inhibition could mediate the anticancer activity of

AdArs,^[9,14]although alternative mechanisms cannot be ruled

out, including the targeting of protein phosphatases, the nu-

clear receptor SHP, or possibly other kinases. Moreover, where-

as the anticancer activity of most of these AdArs is completely

independent of RAR transactivation activity (neither activation

nor antagonism; see chalcones 6i, 6k, and 6l), the a,b-unsatu-

rated ketone of the chalcones could be replaced by a five-

membered heterocycle without loss of activity (i.e., com-

pounds 23 and 27). This suggests that the adamantyl and ben-

zoic acid termini, which are not present in the natural chal-

cones of Figure 2, are also important determinants of the in-

hibitory activities of these AdArs. This also demonstrates a Mi-

chael-addition-independent mechanism of IKKb inhibition by

the chalcone-containing AdArs, which contrasts with the

mechanism of action of natural chalcones. In support of this,

we previously reported that the parent compound MX781 (5)

inhibits the mutant IKKb (C179A),^[3b]which is resistant to the

inhibitory activity of other natural chalcones.^[21,24,25]

Heterocyclic derivatives of the parent chalcone MX781 (5)

can be considered conformationally restrained congeners. Pyr-

azoles/isoxazoles lock the s-cis conformation of the enone,

whereas the flavones/aurones are conformationally constrained

s-trans-locked enone analogues (they also lock the CArꢀC(=O)

bond). From the above results it appears that further develop-

ment on more diverse s-cis-locked adamantyl arotinoids is war-

ranted in order to get a more comprehensive view of their IKK

inhibitory profile and the structural determinants of the com-

pound class for enzyme binding. In addition, the role of the

OMEM chain in IKK activity within the chalcone containing

AdAr series needs to be investigated.

1-[5-(Adamant-1-yl)-2-(4-bromophenylmethoxy)-4-(2-methoxye-

thoxymethoxy)phenyl]ethan-2-one 14k: General procedure for

the alkylation of phenols: A solution of 1-[5-(adamant-1-yl)-2-hy-

droxy-4-(2-methoxyethoxymethoxy)phenyl]ethan-2-one 12 (0.15 g,

0.40 mmol) in DMF (2.6 mL) was added to a cooled (08C) disper-

sion of NaH (0.018 g, 60% in mineral oil, 0.44 mmol) in DMF

(1.2 mL), and the reaction mixture was stirred for 0.5 h. After reach-

ing 258C, 1-bromo-4-(bromomethyl)benzene 13k (0.2 mL,

0.8 mmol) was added, and the reaction was stirred for 16 h. The re-

sulting mixture was poured into ice-water and the layers were sep-

Experimental Section

General procedures

Solvents were dried according to published methods and distilled

before use. Recently they were dispensed from a Puresolv solvent

purification system from Innovative Technology, Inc. (Amesbury,

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arated. The aqueous layer was extracted with Et₂O (3ꢄ), the com-

bined organic layers were dried (Na₂SO₄), and the solvent was

evaporated. The residue was purified by column chromatography

(silica gel, 90:10 hexane/EtOAc) to afford 0.193 g (89%) of a white

solid identified as 1-[5-(adamant-1-yl)-2-(4-bromophenylmethoxy)-

4-(2-methoxyethoxymethoxy)phenyl]ethan-2-one 14k; mp: 1128C

(hexane/acetone); Anal. calcd for C₂₉H₃₅BrO₅·H₂O: C 62.03, H 6.64,

found: C 62.22, H 6.65; ¹H NMR (400.13 MHz, CDCl₃): d=7.74 (s,

1H, H3’ or H6’), 7.51 (dd, J=6.7, 1.8 Hz, 2H, ArH), 7.32 (d, J=

8.3 Hz, 2H, ArH), 6.84 (s, 1H, H3’ or H6’), 5.32 (s, 2H, OCH₂O), 5.07

(s, 2H, OCH₂), 3.9–3.8 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H,

OCH₂O(CH₂)₂OCH₃), 3.39 (s, 3H, OCH₃), 2.56 (s, 3H, C(O)CH₃), 2.1–2.0

(m, 9H, Ad), 1.8–1.7 ppm (m, 6H, Ad); ¹³C NMR (100.62 MHz,

CDCl₃): d=198.0 (s), 161.0 (s), 157.8 (s), 135.3 (s), 131.8 (d), 131.5

(s), 129.5 (d, 2ꢄ), 129.4 (d, 2ꢄ), 122.2 (s), 121.0 (s), 99.9 (d), 93.3 (t),

71.4 (t), 70.0 (t), 67.8 (t), 59.0 (q), 40.8 (t, 3ꢄ), 37.0 (t, 3ꢄ), 36.6 (s),

32.2 (q), 29.0 ppm (d, 3ꢄ); MS (FAB⁺): m/z (%) 545 ([M+H]⁺, 20),

544 ([M]⁺, 14), 543 ([M+H]⁺, 21), 542 ([M]⁺, 8), 308 (10), 307 (38),

289 (17), 171(10), 169 (11), 155 (31), 154 (100), 151 (11); HRMS

(FAB⁺): calcd for C₂₉H₃₆⁷⁹BrO₅[M+H]⁺: 543.1746, found: 543.1746;

IR (NaCl): n˜ =2904 (s, CꢀH), 2850 (m, CꢀH), 1667 (s, C=O), 1598 (s,

(2 mL) was added, and the mixture was vigorously stirred for

10 min. The mixture was extracted with Et₂O (3ꢄ), the combined

organic layers were dried (Na₂SO₄) and the solvent was evaporated.

The residue was purified by column chromatography (silica gel,

85:15 hexane/EtOAc) to afford 0.036 g (31%) of a yellow oil identi-

fied as methyl (E)-4-[1-(5-adamant-1-yl)-2-(4-bromophenylme-

thoxy)-4-(((2-methoxyethoxy)methoxy)phenyl)-1-oxoprop-2-en-3-

yl]benzoate 16k; mp: 1288C (EtOAc/hexane); Anal. calcd for

C₃₈H₄₁BrO₇: C 66.18, H 5.99, found: C 65.66, H 6.21; ¹H NMR

(400.13 MHz, (CD₃)₂CO): d=7.98 (d, J=8.4 Hz, 2H, ArH), 7.78 (d, J=

16.0 Hz, 1H, H3’ or H2’), 7.77 (s, 1H, H3’’ or H6’’), 7.58 (d, J=

15.4 Hz, 1H, H2’ or H3’), 7.6–7.5 (m, 6H, ArH), 7.06 (s, 1H, H3’’ or

H6’’), 5.48 (s, 2H, OCH₂O), 5.24 (s, 2H, OCH₂), 3.93 (s, 3H, CO₂CH₃),

3.9–3.8 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O-

(CH₂)₂OCH₃), 3.32 (s, 3H, OCH₃), 2.1–2.0 (m, 9H, Ad), 1.8–1.7 ppm

(m, 6H, Ad); ¹³C NMR (100.62 MHz, CDCl₃): d=189.7 (s), 166.5 (s),

161.3 (s), 157.7 (s), 139.7 (d), 135.0 (s), 132.1 (s), 131.9 (d, 2ꢄ), 130.8

(s), 130.2 (d), 129.9 (d, 2ꢄ), 129.8 (d), 129.6 (d, 2ꢄ), 127.9 (d, 2ꢄ),

126.4 (s), 122.3 (s), 121.4 (s), 100.0 (d), 93.3 (t), 71.5 (t), 70.3 (d), 67.9

(t), 59.0 (q), 52.2 (q), 40.8 (t, 3ꢄ), 37.0 (t, 3ꢄ), 36.7 (s), 29.0 ppm (d,

3ꢄ); HRMS (ESI⁺): calcd for C₃₈H₄₂⁸¹BrO₇[M+H]⁺: 689.2108, found:

689.2098; IR (NaCl): n˜ =2904 (s, CꢀH), 2850 (w, CꢀH), 1720 (s, C=O),

1652 (m, C=O), 1607 (s, C=C), 1279 (s), 1107 cm^ꢀ1(s); UV (MeOH):

l_max=307 nm.

C=C), 1501 (s), 1260 (s), 1162 (s), 1105 cm^ꢀ1(s); UV (MeOH): l_max

=

310, 267, 231 nm.

1-[5-(Adamant-1-yl)-2-(3,5-difluorophenylmethoxy)-4-(2-methox-

yethoxymethoxy)phenyl]ethan-2-one 14l: Following the general

procedure for alkylation of phenols, the reaction of 1-[5-(adamant-

1-yl)-2-hydroxy-4-(2-methoxyethoxymethoxy)phenyl]ethan-2-one

12 (0.15 g, 0.4 mmol), NaH (0.018 g, 60% in mineral oil, 0.44 mmol)

(E)-4-[1-(5-Adamant-1-yl)-2-(4-bromobenzyloxy)-4-(((2-methoxye-

thoxy)methoxy)phenyl)-1-oxoprop-2-en-3-yl]benzoic acid 6k:

General procedure for the hydrolysis of esters: To a solution of

methyl (E)-4-[1-(5-adamant-1-yl)-2-(4-bromophenylmethoxy)-4-(((2-

methoxyethoxy)methoxy)phenyl)-1-oxoprop-2-en-3-yl]benzoate

16k (0.016 g, 0.023 mmol) in MeOH (0.84 mL) was added a 1m

aqueous NaOH solution (0.14 mL, 0.139 mmol) and the mixture

was heated at 708C for 20 h. The reaction was cooled to ambient

temperature, H₂O and a 10% aqueous solution of HCl were se-

quentially added until acidic pH. The mixture was extracted with

EtOAc (3ꢄ)), the combined organic layers were dried (Na₂SO₄) and

the solvent was evaporated. The residue was purified by recrystalli-

zation (hexane), 0.0095 g (73%) of a yellow solid identified as (E)-4-

[1-(5-adamant-1-yl)-2-(4-bromobenzyloxy)-4-(((2-methoxyethoxy)-

methoxy)phenyl)-1-oxoprop-2-en-3-yl]benzoic acid 6k; mp: 2088C

(hexane); ¹H NMR (400.13 MHz, (CD₃)₂CO): d=8.03 (d, J=8.3 Hz,

2H, ArH), 7.78 (d, J=15.8 Hz, 1H, H3’), 7.75 (s, 1H, H3’’ or H6’’),

7.59 (d, J=15.9 Hz, 1H, H2’), 7.6–7.5 (m, 6H, ArH), 7.07 (s, 1H, H3’’

or H6’’), 5.49 (s, 2H, OCH₂O), 5.26 (s, 2H, OCH₂), 3.9–3.8 (m, 2H,

OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.32 (s, 3H,

OCH₃), 2.1–2.0 (m, 9H, Ad), 1.9–1.8 ppm (m, 6H, Ad); ¹³C NMR

(100.62 MHz, CDCl₃): d=189.7 (s), 170.0 (s), 161.4 (s), 157.7 (s),

140.6 (s), 139.6 (d), 135.0 (s), 132.1 (s), 131.9 (d, 2ꢄ), 130.6 (d, 2ꢄ),

130.2 (d), 130.1 (d), 129.8 (s), 129.7 (d, 2ꢄ), 128.0 (d, 2ꢄ), 122.4 (s),

121.3 (s), 100.0 (d), 93.3 (t), 71.5 (t), 70.4 (d), 67.9 (t), 59.1 (q), 40.8

(t, 3ꢄ), 37.0 (t, 3ꢄ), 36.7 (s), 29.0 ppm (d, 3ꢄ); HRMS (ESI⁺): calcd

for C₃₇H₄₀⁷⁹BrO₇([M+1]⁺), 675.1952, found: 675.1945; IR (NaCl): n˜ =

2903 (s, CꢀH), 2849 (m, CꢀH), 1717 (m, C=O), 1690 (m, C=O), 1652

and

1-(bromomethyl)-3,5-difluorobenzene

13l

(0.104 mL,

0.8 mmol) in DMF (2.8 mL) afforded, after purification by column

chromatography (silica gel, 98:2 CH₂Cl₂/MeOH), 0.146 g (73%) of

a white solid identified as 1-[5-(adamant-1-yl)-2-(3,5-difluorophenyl-

methoxy)-4-(2-methoxyethoxymethoxy)phenyl]ethan-2-one

14l;

mp: 968C (hexane/acetone); Anal. calcd for C₂₉H₃₄F₂O₅: C 69.58, H

1

6.85, found: C 69.31, H 6.78; H NMR (400.13 MHz, CDCl₃): d=7.74

3

(s, 1H, H3’ or H6’), 6.97 (d, J_H-F=7.7 Hz, 2H, ArH), 6.83 (s, 1H, H3’

or H6’), 6.77 (tt, ³J_H-F=8.9 Hz, ⁴J_H-H=2.2 Hz, 1H, ArH), 5.31 (s, 2H,

OCH₂O), 5.10 (s, 2H, OCH₂), 3.9–3.8 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.6–

3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.38 (s, 3H, OCH₃), 2.57 (s, 3H,

C(O)CH₃), 2.1–2.0 (m, 9H, Ad), 1.8–1.7 ppm (m, 6H, Ad); ¹³C NMR

1

2

(100.62 MHz, CDCl₃): d=198.9 (s), 163.2 (s)(s, J_C-F=249.0 Hz, J_C-F

=

3

12.0 Hz, 2ꢄ), 161.1 (s), 157.4 (s), 140.3 (s)(s, J_C-F=9.1 Hz), 131.8 (s),

2

129.6 (d), 121.1 (s), 110.2 (d)(d, J_C-F=23.1 Hz, 2ꢄ), 103.5 (d)(d, J_C-

_F=25.1 Hz), 100.0 (d), 93.4 (t), 71.4 (t), 69.5 (t), 67.8 (t), 59.0 (q), 40.8

(t, 3ꢄ), 37.0 (t, 3ꢄ), 36.6 (s), 32.1 (q), 29.0 ppm (d, 3ꢄ); MS (FAB⁺):

m/z (%) 501 ([M+H]⁺, 100), 500 ([M]⁺, 24), 391 (26), 307 (26), 289

(14), 155 (26), 154 (85); HRMS (FAB⁺): calcd for C₂₉H₃₅F₂O₅[M+H]⁺:

501.2453, found: 501.2452; IR (NaCl): n˜ =2905 (s, CꢀH), 2851 (s, Cꢀ

H), 1668 (s, C=O), 1598 (s, C=C), 1500 (s), 1457 (s), 1246 (s), 1119 (s),

1105 cm^ꢀ1(s); UV (MeOH): l_max=309, 267, 228 nm.

Methyl (E)-4-[1-(5-Adamant-1-yl)-2-(4-bromophenylmethoxy)-4-

(((2-methoxyethoxy)methoxy)phenyl)-1-oxoprop-2-en-3-yl]ben-

zoate 16k: General procedure for the aldol condensation: To

a cooled (08C) solution of iPr₂NH (0.035 mL, 0.25 mmol) in THF

(0.4 mL) was added nBuLi (0.13 mL, 1.37m in hexane, 0.18 mmol)

and the mixture was stirred for 30 min. The reaction was cooled to

(m, C=O), 1604 (s, C=C), 1249 (s), 1152 cm^ꢀ1(m); UV (MeOH): l_max

=

307, 229 nm; purity: 92% (HPLC-UV, Sunfire C₁₈, 1 mLmin^ꢀ1, 95:5

CH₃CN/H₂O, t_R=16 min).

Methyl (E)-4-[1-(5-adamant-1-yl)-2-(3,5-difluorophenylmethoxy)-

4-(((2-methoxyethoxy)methoxy)phenyl)-1-oxoprop-2-en-3-yl]-

benzoate 16l: Following the general procedure for the aldol con-

densation, the reaction of 1-(5-bromo-2-(3,5-difluorophenylme-

thoxy)-4-(((2-methoxyethoxy)methoxy)phenyl)ethanone 14l (0.09 g,

0.18 mmol), iPr₂NH (0.038 mL, 0.27 mmol), nBuLi (0.14 mL, 1.37m in

hexane, 0.2 mmol) and methyl 4-formylbenzoate 15 (0.038 g,

ꢀ788C,

a solution of methyl 4-formylbenzoate 15 (0.035 g,

0.22 mmol) in THF (0.4 mL) was added and the mixture was stirred

for 1 h. Then, a solution of 1-[5-(adamant-1-yl)-2-(4-bromophenyl-

methoxy)-4-(2-methoxyethoxymethoxy)phenyl]ethan-2-one

14k

(0.09 g, 0.17 mmol) in THF (0.85 mL) was added, and the reaction

was stirred for 1 h at ꢀ788C and then for 18 h at 258C. Brine

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0.23 mmol) in THF (1.8 mL) afforded, after purification by chroma-

tography (silica gel, 85:15 hexane/EtOAc), 0.037 g (39%) of

a yellow solid identified as methyl (E)-4-[1-(5-adamant-1-yl)-2-(3,5-

difluorophenylmethoxy)-4-(((2-methoxyethoxy)methoxy)phenyl)-1-

oxoprop-2-en-3-yl]benzoate 16l; mp: 1088C (EtOAc/hexane);

¹H NMR (400.13 MHz, (CD₃)₂CO): d=7.98 (d, J=8.2 Hz, 2H, ArH),

7.74 (s, 1H, H3’’ or H6’’), 7.68 (d, J=15.8 Hz, 1H, H3’ or H2’), 7.60

tion mixture was stirred for 1 h at ꢀ788C and for 18 h at 258C.

Brine (2 mL) was added and the mixture was vigorously stirred for

10 min and then was extracted with Et₂O (3ꢄ). The combined or-

ganic layers were dried (Na₂SO₄) and the solvent was evaporated.

The residue was purified by column chromatography (silica gel,

98:2 CH₂Cl₂/MeOH) to afford 0.268 g (97%) of a yellow oil identi-

fied as ethyl 4 ethyl 4-[1-(5-(adamant-1-yl)-2,4-(2-methoxyethoxy-

(d, J=15.8 Hz, 1H, H2’ or H3’), 7.47 (d, J=8.2 Hz, 2H, ArH), 6.96 (d,

methoxy)phenyl)-prop-2-yn-1-ol-3-yl]benzoate

19;

¹H NMR

3

³J_H-F=6.0 Hz, 2H, ArH), 6.91 (s, 1H, H3’’ or H6’’), 6.7–6.8 (tt, J_H-F

=

(400.13 MHz, CDCl₃): d=7.96 (d, J=8.3 Hz, 2H, ArH), 7.50 (s, 1H,

H3’’ or H6’’), 7.49 (d, J=8.3 Hz, 2H, ArH), 6.95 (s, 1H, H3’’ or H6’’),

5.87 (s, 1H, OH), 5.3–5.2 (m, 4H, 2ꢄOCH₂O), 4.35 (q, J=7.1 Hz, 2H,

CO₂CH₂CH₃), 3.9–3.8 (m, 4H, 2ꢄO(CH₂)₂OCH₃), 3.6–3.5 (m, 4H, 2ꢄ

O(CH₂)₂OCH₃), 3.37 (s, 3H, OCH₃), 3.33 (s, 3H, OCH₃), 2.1–2.0 (s, 9H,

Ad), 1.8–1.7 (s, 6H, Ad), 1.36 ppm (t, J=7.1 Hz, 3H, CO₂CH₂CH₃);

¹³C NMR (100.62 MHz, CDCl₃): d=165.9 (s), 157.3 (s), 153.1 (s), 132.6

(s), 131.5 (d, 2ꢄ), 129.9 (s), 129.3 (d, 2ꢄ), 127.5 (s), 126.4 (d), 122.3

(s), 102.9 (d), 94.3 (t), 93.4 (t), 92.2 (s), 84.8 (s), 71.5 (t, 2ꢄ), 68.2 (t),

67.8 (t), 61.1 (q), 61.0 (t), 59.0 (d), 58.9 (q), 40.8 (t, 3ꢄ), 37.0 (t, 3ꢄ),

36.6 (s), 29.0 (d, 3ꢄ), 14.2 ppm (q); MS (FAB⁺): m/z (%) 605

([MꢀOH]⁺, 100), 517 (18), 516 (16), 515 (10), 442 (10), 441 (24), 440

(16), 428 (11), 427 (14), 185 (17); HRMS (FAB⁺): calcd for C₃₆H₄₅O₈

([MꢀOH]⁺), 605.3114, found: 605.3122; IR (NaCl): n˜ =3600–3200

(br, O-H), 2904 (s, CꢀH), 2850 (m, CꢀH), 1717 (m, C=O), 1632 (s, C=

9.1 Hz, ⁴J_H-H=2.3 Hz, 1H, ArH), 5.38 (s, 2H, OCH₂O), 5.19 (s, 2H,

OCH₂), 3.93 (s, 3H, CO₂CH₃), 3.9–3.8 (m, 2H, OCH₂O(CH₂)₂OCH₃),

3.6–3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.41 (s, 3H, OCH₃), 2.1–2.0 (m,

9H, Ad), 1.8–1.7 ppm (m, 6H, Ad); ¹³C NMR (100.62 MHz, CDCl₃):

d=190.0 (s), 166.6 (s), 163.1 (s)(dd, ¹J_C-F=250.0 Hz, ³J_C-F=12.0 Hz,

3

2ꢄ), 161.2 (s), 157.0 (s), 140.2 (d), 140.0 (s)(t, J_C-F=9.2 Hz), 139.6 (s),

132.4 (s), 130.9 (s), 130.1 (d), 130.0 (d, 2ꢄ), 127.9 (d, 2ꢄ), 121.8 (s),

2

110.2 (d)(dd, ²J_C-F=18.6 Hz, ⁴J_C-F=7.2 Hz, 2ꢄ), 103.5 (d)(d, J_C-

_F=24.6 Hz), 100.2 (d), 93.4 (t), 71.5 (t), 69.7 (d), 67.9 (t), 59.0 (q), 52.2

(q), 40.8 (t, 3ꢄ), 37.0 (t, 3ꢄ), 36.7 (s), 29.0 ppm (d, 3ꢄ); HRMS (ESI⁺

): calcd for C₃₈H₄₁F₂O₇[M+H]⁺: 647.2815, found: 647.2803; IR

(NaCl): n˜ =2904 (s, CꢀH), 2849 (w, CꢀH), 1720 (s, C=O), 1651 (w, C=

O), 1606 (s, C=C), 1278 (s), 1116 cm^ꢀ1(s); UV (MeOH): l_max

=

306 nm.

C), 1493 (m), 1366 (s), 1272 (s), 1104 cm^ꢀ1(s); UV (MeOH): l_max

=

268 nm.

(E)-4-[1-(5-Adamant-1-yl)-2-(3,5-difluorobenzyloxy)-4-(((2-me-

thoxyethoxy)methoxy)phenyl)-1-oxoprop-2-em-3-yl]benzoic acid

6l: Following the general procedure for the hydrolysis of esters,

the reaction of methyl (E)-4-[1-(5-adamant-1-yl)-2-(3,5-difluorophe-

nylmethoxy)-4-(((2-methoxyethoxy)methoxy)phenyl)-1-oxoprop-2-

en-3-yl]benzoate 16l (0.017 g, 0.026 mmol) and a 1m aqueous

NaOH solution (0.16 mL, 0.158 mmol) in MeOH (0.9 mL) at 708C af-

forded, after purification by recrystallization (hexane), 0.015 g

(80%) of a yellow solid identified as (E)-4-[1-(5-adamant-1-yl)-2-(3,5-

difluorobenzyloxy)-4-(((2-methoxyethoxy)methoxy)phenyl)-1-oxo-

prop-2-em-3-yl]benzoic acid 6l; mp: 1988C (hexane/EtOAc);

¹H NMR (400.13 MHz, (CD₃)₂CO): d=8.01 (d, J=8.3 Hz, 2H, ArH),

7.80 (d, J=15.8 Hz, 1H, H3’ or H2’), 7.72 (s, 1H, H3’’ or H6’’), 7.66

Ethyl

1-[3-(5-(adamant-1-yl)-2,4-(2-methoxyethoxymethoxy)-

phenyl)-1-oxoprop-2-yn-3-yl]benzoate 20: To a cooled (08C) solu-

tion of pyridine (0.86 mL, 10.71 mmol) in CH₂Cl₂(16 mL) was added

CrO₃(0.54 g, 5.35 mmol) and the mixture was stirred for 10 min. A

solution of ethyl 4-[1-(5-(adamant-1-yl)-2,4-(2-methoxyethoxyme-

thoxy)phenyl)-prop-2-yn-1-ol-3-yl]benzoate 19 (0.56 g, 0.18 mmol)

in CH₂Cl₂(3.3 mL) was added and the reaction was stirred for 4 h

at 258C. The mixture was washed with a 5% aqueous solution of

NaOH (3ꢄ) and the combined organic layers were dried (Na₂SO₄)

and the solvent was evaporated. The residue was purified by

column chromatography (silica gel, 99:1 CH₂Cl₂/MeOH) to afford

0.49 g (89%) of a yellow oil identified as ethyl 1-[3-(5-(adamant-1-

yl)-2,4-(2-methoxyethoxymethoxy)phenyl)-1-oxoprop-2-yn-3-yl]ben-

(d, J=8.1 Hz, 2H, ArH), 7.64 (d, J=16 Hz, 1H, H2’ or H3’), 7.25 (m,

4

2H, ArH), 7.05 (s, 1H, H3’’ or H6’’), 6.98 (tt, ³J_H-F=9.1 Hz, J_H-H

=

1

2.3 Hz, 1H, ArH), 5.48 (s, 2H, OCH₂O), 5.32 (s, 2H, OCH₂), 3.9–3.8

(m, 2H, OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.32

(s, 3H, OCH₃), 2.1–2.0 (m, 9H, Ad), 1.9–1.8 ppm (m, 6H, Ad);

¹³C NMR (100.62 MHz, CDCl₃): d=190.0 (s), 170.8 (s), 163.1 (s)(dd,

¹J_C-F=249.7 Hz, ⁴J_C-F=12.7 Hz, 2ꢄ), 161.2 (s), 157.1 (s), 140.4 (d),

140.0 (s)(t, ³J_C-F=9.2 Hz), 139.9 (s), 132.4 (s), 130.9 (s), 130.6 (d),

130.2 (d, 2ꢄ), 130.0 (s), 129.8 (d), 128.0 (d, 2ꢄ), 121.7 (s), 110.3

(d)(dd, ²J_C-F=18.6 Hz, ⁴J_C-F=7.1 Hz, 2ꢄ), 103.5 (d)(d, ²J_C-F=25.1 Hz),

100.1 (d), 93.4 (t), 71.5 (t), 69.7 (d), 67.9 (t), 59.0 (q), 40.8 (t, 3ꢄ),

37.0 (t, 3ꢄ), 36.7 (s), 29.0 ppm (d, 3ꢄ); HRMS (ESI⁺): calcd for

C₃₇H₃₉F₂O₇[M+H]⁺: 633.2658, found: 633.2649; IR (NaCl): n˜ =2905

(s, CꢀH), 2850 (m, CꢀH), 1691 (m, C=O), 1604 (s, C=C), 1319 (m),

1245 (m), 1120 cm^ꢀ1(m); UV (MeOH): l_max=309, 230 nm; purity:

92% (HPLC-UV, Sunfire C₁₈, 1 mLmin^ꢀ1, 95:5 CH₃CN/H₂O, t_R=

15 min).

zoate 20; H NMR (400.13 MHz, CDCl₃): d=8.05 (d, J=8.2 Hz, 2H,

ArH), 8.01 (s, 1H, H3’’ or H6’’), 7.66 (d, J=8.2 Hz, 2H, ArH), 6.96 (s,

1H, H3’’ or H6’’), 5.37 (s, 2H, OCH₂O), 5.36 (s, 2H, OCH₂O), 4.38 (q,

J=7.1 Hz, 2H, CO₂CH₂CH₃), 3.9–3.8 (m, 4H, 2ꢄOCH₂O(CH₂)₂OCH₃),

3.6–3.5 (m, 4H, 2ꢄOCH₂O(CH₂)₂OCH₃), 3.38 (s, 3H, OCH₃), 3.36 (s,

3H, OCH₃), 2.1–2.0 (m, 9H, Ad), 1.8–1.7 (m, 6H, Ad), 1.39 ppm (t,

J=7.1 Hz, 3H, CO₂CH₂CH₃); ¹³C NMR (100.62 MHz, CDCl₃): d=175.1

(s), 165.7 (s), 161.9 (s), 157.2 (s), 132.6 (s), 132.5 (d, 2ꢄ), 131.6 (s),

131.3 (d), 129.6 (d, 2ꢄ), 125.4 (s), 120.5 (s), 102.8 (d), 94.4 (t), 93.2

(t), 91.2 (s), 89.3 (s), 71.5 (t, 2ꢄ), 68.2 (t, 2ꢄ), 61.3 (t), 59.0 (q), 40.7

(t, 3ꢄ), 37.0 (t, 3ꢄ), 36.7 (s), 29.0 (d, 3ꢄ), 14.3 ppm (q); MS (FAB⁺):

m/z (%) 621 ([M+H]⁺, 48), 620 ([M]⁺, 16), 561 (16), 546 (19), 545

(50), 544 (15), 469 (20), 457 (37), 456 (20), 455 (31), 447 (29), 411

(28), 360 (24), 359 (100), 313 (16), 283 (25), 281 (19), 271 (16), 269

(29), 201 (30), 177 (16), 155 (23), 154 (67); HRMS (FAB⁺): calcd for

C₃₆H₄₅O₉[M+H]⁺: 621.3064, found: 621.3063; IR (NaCl): n˜ =2905 (s,

CꢀH), 2851 (m, CꢀH), 2201 (w, CꢂC), 1719 (s), 1624 (m, C=O), 1245

(s), 1104 cm^ꢀ1(s); UV (MeOH): l_max=301, 243 nm.

Ethyl

4-[1-(5-(Adamant-1-yl)-2,4-(2-methoxyethoxymethoxy)-

phenyl)-prop-2-yn-1-ol-3-yl]benzoate 19: To a cooled (08C) solu-

tion of iPr₂NH (0.1 mL, 0.71 mmol) in THF (0.45 mL) was added

nBuLi (0.49 mL, 1.47m in hexane, 0.71 mmol) and the mixture was

stirred for 30 min. The reaction was cooled to ꢀ788C, a solution of

ethyl 4-ethynylbenzoate 18 (0.12 g, 0.71 mmol) in THF (0.45 mL)

was added and the mixture was stirred for 1 h. Then, a solution of

5-(adamant-1-yl)-2,4-(2-methoxyethoxymethoxy)benzaldehyde

17^[14](0.2 g, 0.45 mmol) in THF (0.45 mL) was added and the reac-

Ethyl 4-[1-(5-(Adamant-1-yl)-2-hydroxy-4-(2-methoxyethoxyme-

thoxy)phenyl)-1-oxoprop-2-yn-3-yl]benzoate 21: General proce-

dure for the deprotection of methoxyethoxymethoxy ethers:

BCl₃(0.7 mL, 1m in hexane, 0.69 mmol) was added to a cooled

(ꢀ788C) solution of ethyl 4-[1-(5-(adamant-1-yl)-2,4-(2-methoxye-

thoxymethoxy)phenyl)-1-oxoprop-2-yn-3-yl]benzoate 20 (0.21 g,

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0.34 mmol) in CH₂Cl₂(3.1 mL), and the reaction mixture was stirred

for 18 h. The resulting mixture was poured into ice-water and ex-

tracted with CH₂Cl₂(3ꢄ). The combined organic layers were

washed with H₂O (5ꢄ), dried (Na₂SO₄) and the solvent was evapo-

rated. The residue was purified by column chromatography (silica

gel, 90:10 CH₂Cl₂/MeOH) to afford 0.125 g (69%) of a yellow solid

identified as ethyl 4-[1-(5-(adamant-1-yl)-2-hydroxy-4-(2-methoxye-

zoic acid 23; mp: 2248C (hexane/acetone); Anal. calcd for

C₃₀H₃₃NO₇·H₂O: C 67.02, H 6.56, N 2.61, found: C 66.50, H 6.41, N

2.50; ¹H NMR (400.13 MHz, (CD₃)₂CO): d=8.18 (d, J=8.2 Hz, 2H,

ArH), 8.09 (d, J=8.2 Hz, 2H, ArH), 7.76 (s, 1H, H5’), 7.28 (s, 1H, H3’’

or H6’’), 6.90 (s, 1H, H3’’ or H6’’), 5.37 (s, 2H, OCH₂O), 3.9–3.8 (m,

2H, OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.31 (s,

3H, OCH₃), 2.1–2.0 (m, 9H, Ad), 1.9–1.8 ppm (s, 6H, Ad); ¹³C NMR

(100.62 MHz, (CD₃)₂CO): d=169.8 (s), 168.2 (s), 163.8 (s), 160.9 (s),

155.7 (s), 135.9 (s), 133.6 (s), 132.6 (s), 132.1 (d, 2ꢄ), 128.6 (d, 2ꢄ),

126.8 (d), 109.3 (s), 104.9 (d), 101.2 (d), 95.1 (t), 73.3 (t), 70.0 (t),

59.8 (q), 42.8 (t, 3ꢄ), 38.7 (t, 3ꢄ), 38.4 (s), 31.0 ppm (d, 3ꢄ); MS

(FAB⁺): m/z (%) 520 ([M+H] ⁺, 100), 519 ([M]⁺, 37), 307 (28), 289

(15), 155 (26), 154 (86); HRMS (FAB⁺): calcd for C₃₀H₃₄NO₇[M+H]⁺:

520.2335, found: 520.2338; IR (NaCl): n˜ =2901 (s, CꢀH), 2848 (s, Cꢀ

H), 2659 (w), 1687 (s, C=O), 1615 (s, C=C), 1389 (s), 1261 (s), 1102

(s), 1018 cm^ꢀ1(s); UV (MeOH): l_max=319, 266 nm.

thoxymethoxy)phenyl)-1-oxoprop-2-yn-3-yl]benzoate

21;

mp:

1

1218C (hexane/acetone); H NMR (400.13 MHz, CDCl₃): d=8.13 (d,

J=8.2 Hz, 2H, ArH), 7.96 (s, 1H, H3’’ or H6’’), 7.74 (d, J=8.2 Hz, 2H,

ArH), 7.28 (s, 1H, H3’’ or H6’’), 6.69 (s, 1H, OH), 5.40 (s, 2H, OCH₂O),

4.43 (q, J=7.1 Hz, 2H, CO₂CH₂CH₃), 3.9–3.8 (m, 2H, OCH₂O-

(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.42 (s, 3H, OCH₃),

2.2–2.1 (m, 9H, Ad), 1.9–1.8 (m, 6H, Ad), 1.44 ppm (t, J=7.1 Hz,

3H, CO₂CH₂CH₃); ¹³C NMR (100.62 MHz, CDCl₃): d=180.3 (s), 165.6

(s), 164.0 (s), 163.6 (s), 132.8 (d, 2ꢄ), 132.2 (s), 131.5 (d), 131.3 (s),

129.8 (d, 2ꢄ), 124.5 (s), 115.2 (s), 102.5 (d), 93.6 (s), 93.2 (t), 87.5 (s),

71.5 (t), 68.6 (t), 61.5 (t), 59.1 (q), 40.9 (t, 3ꢄ), 37.0 (t, 3ꢄ), 36.7 (s),

29.0 (d, 3ꢄ), 14.3 ppm (q); MS (FAB⁺): m/z (%) 533 ([M+H]⁺, 100),

532 ([M]⁺, 37), 479 (20), 457 (36), 456 (26), 443 (19), 307 (15), 271

(15), 155 (20), 154 (59); HRMS (FAB⁺): calcd for C₃₂H₃₇O₇[M+H]⁺:

533.2539, found: 533.2523; IR (NaCl): n˜ =2904 (m, CꢀH), 2849 (w,

CꢀH), 2205 (w, CꢂC), 1720 (m, C=O), 1627 (m, C=O), 1588 (m, C=C),

1357 (s), 1271 (s), 1104 cm^ꢀ1(s); UV (MeOH): l_max=293 nm.

Ethyl 4-[3-(5-Adamantyl-2-hydroxy-4-(2-methoxyethoxymethox-

y)phenyl)-1-methyl-1H-pyrazol-5-yl]benzoate 24: To a solution of

ethyl 4-[1-(5-(adamant-1-yl)-2-hydroxy-4-(2-methoxyethoxymethox-

y)phenyl)-1-oxoprop-2-yn-3-yl]benzoate 21 (0.06 g, 0.11 mmol) in

EtOH (0.8 mL) was added methylhydrazine (0.012 mL, 0.23 mmol)

at 258C. The reaction was stirred for 6 h and the solvent was re-

moved. The residue was purified by column chromatography (silica

gel, 80:20 hexane/EtOAc) to afford 0.051 g (81%) of a white solid

identified as ethyl 4-(3-(5-adamantyl-2-hydroxy-4-(2-methoxyethox-

ymethoxy)phenyl)-1-methyl-1H-pyrazol-5-yl)-benzoate 24; mp:

1558C (hexane/EtOAc); ¹H NMR (400.13 MHz, CDCl₃): d=8.15 (d,

J=8.0 Hz, 2H, ArH), 7.56 (d, J=8.0 Hz, 2H, ArH), 7.38 (s, 1H, H5’),

6.81 (s, 1H, H3’’ or H6’’), 6.65 (s, 1H, H3’’ or H6’’), 5.33 (s, 2H,

OCH₂O), 4.42 (q, J=7.1 Hz, 2H, CO₂CH₂CH₃), 3.91 (s, 3H, CH₃), 3.9–

3.8 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃),

3.41 (s, 3H, OCH₃), 2.1–2.0 (m, 9H, Ad), 1.9–1.8 (m, 6H, Ad),

1.43 ppm (t, J=7.1 Hz, 3H, CO₂CH₂CH₃); ¹³C NMR (100.62 MHz,

CDCl₃): d=166.0 (s), 157.1 (s), 155.0 (s), 151.0 (s), 143.4 (s), 134.3 (s),

130.7 (s), 130.0 (s), 129.9 (d, 2ꢄ), 128.6 (d, 2ꢄ), 124.1 (d), 109.3 (s),

103.3 (d), 102.3 (d), 93.3 (t), 71.6 (t), 68.0 (t), 61.3 (t), 59.1 (q), 41.1

(t, 3ꢄ), 37.6 (q), 37.1 (t, 3ꢄ), 36.4 (s), 29.1 (d, 3ꢄ), 14.4 ppm (q);

HRMS (ESI⁺): calcd for C₃₃H₄₁N₂O₆[M+H]⁺: 561.2959, found:

561.2975; IR (NaCl): n˜ =3300–3000 (br, O-H), 2903 (s, CꢀH), 2847

(w, CꢀH), 1717 (s, C=O), 1274 (s), 1103 (s), 1018 cm^ꢀ1(s); UV

(MeOH): l_max=267 nm.

Ethyl 4-[5-(5-(Adamant-1-yl)-2-hydroxy-4-(2-methoxyethoxyme-

thoxy)phenyl)-isoxazol-3-yl]benzoate 22: To a solution of ethyl 4-

[1-(5-(adamant-1-yl)-2-hydroxy-4-(2-methoxyethoxymethoxy)phen-

yl)-1-oxoprop-2-yn-3-yl]benzoate 21 (0.032 g, 0.059 mmol) in

MeOH (1 mL) and CH₂Cl₂(0.25 mL) was added NH₂OH·HCl (0.006 g,

0.089 mmol) and NaOAc (0.014 g, 0.18 mmol) and the mixture was

stirred for 18 h at 808C. The reaction mixture was poured into H₂O,

the layers were separated and the aqueous layer was extracted

with EtOAc (3ꢄ). The combined organic layers were washed with

brine and dried (Na₂SO₄) and the solvent was evaporated. The resi-

due was purified by column chromatography (silica gel, 80:20

hexane/EtOAc) to afford 0.025 g (77%) of a yellow oil identified as

ethyl 4-[5-(5-(adamant-1-yl)-2-hydroxy-4-(2-methoxyethoxymethox-

y)phenyl)-isoxazol-3-yl]benzoate 22; ¹H NMR (400.13 MHz, CDCl₃):

d=8.13 (d, J=8.3 Hz, 2H, ArH), 7.94 (d, J=8.3 Hz, 2H, ArH), 7.62 (s,

1H, H5’), 6.94 (s, 1H, H3’’ or H6’’), 6.83 (s, 1H, H3’’ or H6’’), 5.31 (s,

2H, OCH₂O), 4.40 (q, J=7.1 Hz, 2H, CO₂CH₂CH₃), 3.9–3.8 (m, 2H,

OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.39 (s, 3H,

OCH₃), 2.1–2.0 (m, 9H, Ad), 1.8–1.7 (m, 6H, Ad), 1.41 ppm (t, J=

7.1 Hz, 3H, CO₂CH₂CH₃); ¹³C NMR (100.62 MHz, CDCl₃): d=169.0 (s),

166.2 (s), 162.0 (s), 159.1 (s), 153.0 (s), 133.4 (s), 131.9 (s), 131.6 (s),

130.1 (d, 2ꢄ), 126.8 (d, 2ꢄ), 125.8 (d), 107.1 (s), 103.6 (d), 98.6 (d),

93.3 (t), 71.5 (t), 67.9 (t), 61.2 (t), 58.9 (q), 41.0 (t, 3ꢄ), 37.0 (t, 3ꢄ),

36.7 (s), 29.0 (d, 3ꢄ), 14.3 ppm (q); MS (FAB⁺): m/z (%) 548 ([M+

H]⁺, 100), 547 ([M]⁺, 31), 471 (16), 155 (11), 154 (35); HRMS (FAB⁺):

calcd for C₃₂H₃₈NO₇[M+H]⁺: 548.2648, found: 548.2648; IR (NaCl):

n˜ =2904 (m, CꢀH), 2850 (w, CꢀH), 1716 (m), 1617 (m), 1504 (m),

1438 (m), 1273 (s), 1105 (s), 1019 cm^ꢀ1(s); UV (MeOH): l_max=319,

268 nm.

4-[3-(5-Adamantyl-2-hydroxy-4-(2-methoxyethoxymethoxy)-

phenyl)-1-methyl-1H-pyrazol-5-yl]benzoic acid 25: Following the

general procedure for the hydrolysis of esters, the reaction of 4-[3-

(5-adamantyl-2-hydroxy-4-(2-methoxyethoxymethoxy)phenyl)1-

methyl-1H-pyrazol-5-yl]benzoate 24 (0.025 g, 0.045 mmol) and

a 1m aqueous solution of NaOH (0.17 mL, 0.18 mmol) in MeOH

(0.8 mL) at 708C afforded, after purification by column chromatog-

raphy (silica gel, 95:5 CH₂Cl₂/MeOH), 0.025 g (99%) of a yellow

solid identified as 4-[3-(5-adamantyl-2-hydroxy-4-(2-methoxyethox-

ymethoxy)phenyl)-1-methyl-1H-pyrazol-5-yl]benzoic acid 25; mp:

254–2558C (hexane/EtOAc); Anal. calcd for C₃₁H₃₆N₂O₆·H₂O:

C

67.62, H 6.96, N 5.09, found: C 68.21, H 6.98, N 5.07; ¹H NMR

(400.13 MHz, CDCl₃): d=8.23 (d, J=8.0 Hz, 2H, ArH), 7.62 (d, J=

8.2 Hz, 2H, ArH), 7.39 (s, 1H, H5’), 6.82 (s, 1H, H3’’ or H6’’), 6.68 (s,

1H, H3’’ or H6’’), 5.33 (s, 2H, OCH₂O), 3.95 (s, 3H, CH₃), 3.9–3.8 (m,

2H, OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.42 (s,

3H, OCH₃), 2.1–2.0 (m, 9H, Ad), 1.9–1.8 ppm (m, 6H, Ad); ¹³C NMR

(100.62 MHz, CDCl₃): d=170.2 (s), 157.2 (s), 155.0 (s), 151.1 (s),

143.3 (s), 135.3 (s), 130.7 (d, 2ꢄ), 130.1 (s), 129.3 (s), 128.8 (d, 2ꢄ),

124.2 (d), 109.3 (s), 103.4 (d), 102.5 (d), 93.4 (t), 71.6 (t), 68.0 (t),

59.1 (q), 41.1 (t, 3ꢄ), 37.7 (q), 37.1 (t, 3ꢄ), 36.5 (s), 29.1 ppm (d,

4-[5-(5-(Adamant-1-yl)-2-hydroxy-4-(2-methoxyethoxymethoxy)-

phenyl)-isoxazol-3-yl]benzoic acid 23: Following the general pro-

cedure for the hydrolysis of esters, the reaction of ethyl 4-[5-(5-

(adamant-1-yl)-2-hydroxy-4-(2-methoxyethoxymethoxy)phenyl)-iso-

xazol-3-yl]benzoate 22 (0.019 g, 0.035 mmol) and a 1m aqueous

solution of NaOH (0.14 mL, 0.14 mmol) in MeOH (0.6 mL) at 508C

afforded, after purification by recrystallization (hexane/EtOAc),

0.015 g (83%) of a white solid identified as 4-[5-(5-(adamant-1-yl)-

2-hydroxy-4-(2-methoxyethoxymethoxy)phenyl)-isoxazol-3-yl]ben-

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3ꢄ); MS (FAB⁺): m/z (%) 533 ([M+H]⁺, 100); HRMS (FAB⁺): calcd

for C₃₁H₃₇N₂O₆[M+H]⁺: 533.2646, found: 533.2653; IR (NaCl): n˜ =

2880 (s, CꢀH), 1688 (s, C=O), 1292 (s), 1016 cm^ꢀ1(m); UV (MeOH):

120 min). The mixture was filtered through a Celite pad washing

with CH₃CN and the solvent was evaporated. The residue was puri-

fied by column chromatography (C₁₈-SiO₂, 80:10 CH₃CN/H₂O) to

afford 0.134 g (90%) of a yellow oil identified as ethyl 4-[3-(5-(ada-

mant-1-yl)-2,4-(2-methoxyethoxymethoxy)phenyl)-2-methylpyrimi-

l_max=297, 265 nm; purity: 100% (HPLC-UV, Sunfire C₁₈, 1 mLmin^ꢀ1

90:10 CH₃CN/H₂O, t_R=11 min).

,

1

din-4-yl]benzoate 28a; H NMR (400.13 MHz, CDCl₃): d=8.2–8.1 (m,

Ethyl 4-[3-(5-Adamantyl-2-hydroxy-4-(2-methoxyethoxymethox-

y)phenyl)-1-phenyl-1H-pyrazol-5-yl]benzoate 26: To a solution of

ethyl 4-[1-(5-(adamant-1-yl)-2-hydroxy-4-(2-methoxyethoxymethox-

y)phenyl)-1-oxoprop-2-yn-3-yl]benzoate 21 (0.015 g, 0.028 mmol)

in EtOH (0.4 mL) was added phenylhydrazine (0.006 mL,

0.056 mmol). The solution was stirred at 808C for 8 h and the sol-

vent was evaporated. The residue was purified by column chroma-

tography (silica gel, 70:30 hexane/EtOAc) to afford 0.012 g (69%)

of a colorless oil identified as ethyl 4-[3-(5-adamantyl-2-hydroxy-4-

(2-methoxyethoxymethoxy)phenyl)-1-phenyl-1H-pyrazol-5-yl]ben-

4H, ArH), 8.11 (s, 1H, H3’’ or H6’’), 7.88 (s, 1H, H6’), 7.03 (s, 1H, H3’’

or H6’’), 5.36 (s, 2H, OCH₂O), 5.30 (s, 2H, OCH₂O), 4.41 (q, J=

7.1 Hz, 2H, CO₂CH₂CH₃), 3.9–3.8 (m, 2H, 2ꢄO(CH₂)₂OCH₃), 3.8–3.7

(m, 2H, 2ꢄO(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, 2ꢄO(CH₂)₂OCH₃), 3.5–3.4

(m, 2H, 2ꢄO(CH₂)₂OCH₃), 3.39 (s, 3H, OCH₃), 3.31 (s, 3H, OCH₃),

2.85 (s, 3H, CH₃), 2.1–2.0 (m, 9H, Ad), 1.8–1.7 (m, 6H, Ad),

1.42 ppm (t, J=7.1 Hz, 3H, CO₂CH₂CH₃); ¹³C NMR (100.62 MHz,

CDCl₃): d=168.2 (s), 166.3 (s), 164.0 (s), 162.3 (s), 158.8 (s), 154.5 (s),

142.0 (s), 133.1 (s), 131.8 (s), 130.0 (d, 2ꢄ), 129.1 (d, 2ꢄ), 127.2 (d),

120.2 (s), 115.0 (d), 102.7 (d), 94.4 (t), 93.2 (t), 71.5 (t), 71.4 (t), 68.1

(t), 68.0 (t), 61.2 (t), 59.1 (q), 59.0 (q), 40.6 (t, 3ꢄ), 37.0 (t, 3ꢄ), 36.7

(s), 29.0 (d, 3ꢄ), 26.6 (q), 14.3 ppm (q); HRMS (ESI⁺): calcd for

C₃₈H₄₉N₂O₈[M+H]⁺: 661.3483, found: 661.3476; IR (NaCl): n˜ =2903

(s, CꢀH), 2849 (m, CꢀH), 1718 (s, C=O), 1581 (s), 1568 (s), 1274 (s),

1105 cm^ꢀ1(s); UV (MeOH): l_max=328, 276 nm

1

zoate 26; H NMR (400.13 MHz, CDCl₃): d=8.12 (d, J=8.5 Hz, 2H,

ArH), 8.00 (d, J=8.0 Hz, 2H, ArH), 7.4–7.3 (m, 5H, ArH), 6.93 (s, 1H,

H5’), 6.79 (s, 1H, H3’’ or H6’’), 6.73 (s, 1H, H3’’ or H6’’), 5.41 (s, 2H,

OCH₂O), 4.41 (q, J=7.1 Hz, 2H, CO₂CH₂CH₃), 3.9–3.8 (m, 2H,

OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.42 (s, 3H,

OCH₃), 2.0–1.9 (m, 3H, Ad), 1.8–1.7 (m, 6H, Ad), 1.7–1.6 (m, 6H,

Ad), 1.43 ppm (t, J=7.1 Hz, 3H, CO₂CH₂CH₃); ¹³C NMR (100.62 MHz,

CDCl₃): d=166.5 (s), 158.1 (s), 152.1 (s), 151.1 (s), 139.9 (s, 2ꢄ),

137.2 (s), 131.3 (s), 130.0 (d, 2ꢄ), 129.8 (s), 129.3 (d), 128.9 (d, 2ꢄ),

127.4 (d), 125.5 (d, 2ꢄ), 124.8 (d, 2ꢄ), 108.8 (s), 105.6 (d), 102.8 (d),

93.4 (t), 71.6 (t), 68.0 (t), 61.0 (t), 59.1 (q), 40.8 (t, 3ꢄ), 37.0 (t, 3ꢄ),

36.3 (s), 28.9 (d, 3ꢄ), 14.4 ppm (q); MS (ESI⁺): m/z (%) 623 ([M+

H]⁺, 100); HRMS (ESI⁺): calcd for C₃₈H₄₃N₂O₆[M+H]⁺: 623.3116,

found: 623.3121; IR (NaCl): n˜ =3500–3100 (br, O-H), 2904 (s, CꢀH),

2849 (m, CꢀH), 1712 (s, C=O), 1611 (s), 1499 (s, C=C), 1274 (s), 1104

(s), 1020 cm^ꢀ1(s); UV (MeOH): l_max=293 nm.

Ethyl 4-[3-(5-(Adamant-1-yl)-4-(2-methoxyethoxymethoxy)phen-

yl)-2-methylpyrimidin-4-yl]benzoate 29a: Following the general

procedure for the deprotection of methoxyethoxymethoxy ethers,

the reaction of ethyl 4-[3-(5-(adamant-1-yl)-2,4-(2-methoxyethoxy-

methoxy)phenyl)-2-methylpyrimidin-4-yl]benzoate

28a

(0.1 g,

0.15 mmol), BCl₃(0.45 mL, 1m in hexane, 0.454 mmol) in CH₂Cl₂

(3.5 mL) afforded, after purification by chromatography (silica gel,

75:25 hexane/EtOAc), 0.047 g (54%) of a yellow solid identified as

ethyl 4-[3-(5-(adamant-1-yl)-4-(2-methoxyethoxymethoxy)phenyl)-2-

methylpyrimidin-4-yl]benzoate 29a; mp: 185–1868C (EtOAc/

hexane); Anal. calcd for C₃₄H₄₀N₂O₆: C 71.31, H 7.04, N 4.89, found:

C 70.94, H 7.42, N 4.78; ¹H NMR (400.13 MHz, CDCl₃): d=8.2–8.1

(m, 4H, ArH), 7.87 (s, 1H, H3’’ or H6’’), 7.69 (s, 1H, H6’), 6.76 (s, 1H,

H3’’ or H6’’), 5.38 (s, 2H, OCH₂O), 4.43 (q, J=7.1 Hz, 2H,

CO₂CH₂CH₃), 3.9–3.8 (m, 2H, O(CH₂)₂OCH₃), 3.7–3.6 (m, 2H,

O(CH₂)₂OCH₃), 3.42 (s, 3H, OCH₃), 2.81 (s, 3H, CH₃), 2.1–2.0 (m, 9H,

Ad), 1.8–1.7 (m, 6H, Ad), 1.43 ppm (t, J=7.0 Hz, 3H, CO₂CH₂CH₃);

¹³C NMR (100.62 MHz, CDCl₃): d=166.1 (s), 165.8 (s), 165.4 (s), 163.5

(s), 161.2 (s), 160.8 (s), 141.3 (s), 132.4 (s), 130.6 (s), 130.1 (d, 2ꢄ),

127.4 (d, 2ꢄ), 124.8 (d), 109.9 (s), 107.6 (d), 104.0 (d), 93.2 (t), 71.6

(t), 68.4 (t), 61.3 (t), 59.1 (q), 41.1 (t, 3ꢄ), 37.1 (t, 3ꢄ), 36.7 (s), 29.1

(d, 3ꢄ), 25.9 (q), 14.3 ppm (q); HRMS (ESI⁺): calcd for C₃₄H₄₁N₂O₆

[M+H]⁺: 573.2959, found: 573.2953; IR (NaCl): n˜ =2904 (s, CꢀH),

2850 (m, CꢀH), 1718 (s, C=O), 1577 (s), 1273 (s), 1105 cm^ꢀ1(s); UV

(MeOH): l_max=348, 284 nm.

4-[3-(5-Adamantyl-2-hydroxy-4-(2-methoxyethoxymethoxy)-

phenyl)-1-phenyl-1H-pyrazol-5-yl]benzoic acid 27: Following the

general procedure for the hydrolysis of esters, the reaction of 4-[3-

(5-adamantyl-2-hydroxy-4-(2-methoxyethoxymethoxy)phenyl)1-

phenyl-1H-pyrazol-5-yl]benzoate 26 (0.029 g, 0.047 mmol) and

a 1m aqueous NaOH solution (0.23 mL, 0.233 mmol) in MeOH

(0.8 mL) afforded, after crystallization (hexane), 0.026 g (99%) of

a white solid identified as 4-[3-(5-adamantyl-2-hydroxy-4-(2-me-

thoxyethoxymethoxy)phenyl)-1-phenyl-1H-pyrazol-5-yl]benzoic acid

27; ¹H NMR (400.13 MHz, CDCl₃): d=8.15 (d, J=8 Hz, 2H, ArH),

8.02 (d, J=8.4 Hz, 2H, ArH), 7.4–7.3 (m, 5H, ArH), 6.94 (s, 1H, H5’),

6.78 (s, 1H, H3’’ or H6’’), 6.73 (s, 1H, H3’’ or H6’’), 5.29 (s, 2H,

OCH₂O), 3.9–3.8 (m, 2H, OCH₂O(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, OCH₂O-

(CH₂)₂OCH₃), 3.40 (s, 3H, OCH₃), 2.0–1.9 (m, 3H, Ad), 1.8–1.7 (m, 6H,

Ad), 1.7–1.6 ppm (m, 6H, Ad); ¹³C NMR (100.62 MHz, CDCl₃): d=

171.4 (s), 158.0 (s), 152.3 (s), 150.8 (s), 140.3 (s), 139.9 (s), 138.1 (s),

131.1 (s), 130.7 (d, 2ꢄ), 129.3 (d), 128.9 (d, 2ꢄ), 128.5 (s), 127.5 (d),

125.7 (d, 2ꢄ), 124.9 (d, 2ꢄ), 108.9 (s), 105.9 (d), 103.0 (d), 93.4 (t),

71.6 (t), 67.8 (t), 59.0 (q), 40.8 (t, 3ꢄ), 37.0 (t, 3ꢄ), 36.3 (s), 28.9 ppm

(d, 3ꢄ); HRMS (ESI⁺): calcd for C₃₆H₃₉N₂O₆[M+H]⁺: 595.2803,

found: 595.2819; IR (NaCl): n˜ =3500–3100 (br, O-H), 2905 (s, CꢀH),

2851 (m, CꢀH), 1690 (s, C=O), 1607 (s), 1498 (s), 1252 (m), 1103 (m),

1020 cm^ꢀ1(m); UV (MeOH): l_max=284 nm; purity: 99% (HPLC-UV,

Sunfire C₁₈, 1 mLmin^ꢀ1, 90:10 CH₃CN/H₂O, t_R=12 min).

4-[3-(5-(Adamant-1-yl)-4-(2-methoxyethoxymethoxy)phenyl)-2-

methylpyrimidin-4-yl]benzoic acid 30a: Following the general

procedure for the hydrolysis of esters, the reaction of ethyl 4-[3-(5-

(adamant-1-yl)-4-(2-methoxyethoxymethoxy)phenyl)-2-methylpyri-

midin-4-yl]benzoate 29a (0.046 g, 0.08 mmol) and a 1m aqueous

solution of NaOH (0.4 mL, 0.40 mmol) in MeOH (1.5 mL) at 708C af-

forded, after purification by recrystallization (EtOH), 0.03 g (80%) of

a yellow solid identified as 4-[3-(5-(adamant-1-yl)-4-(2-methoxye-

thoxymethoxy)phenyl)-2-methylpyrimidin-4-yl]benzoic acid 30a;

mp: 2518C (EtOH); Anal. calcd for C₃₂H₃₆N₂O₆·H₂O: C 68.31, H 6.81,

N 4.98, found: C 68.42, H 7.14, N 4.61; ¹H NMR (400.13 MHz,

[D₆]DMSO, 323 K): d=8.34 (d, J=8.4 Hz, 2H, ArH), 8.28 (s, 1H, H3’’

or H6’’), 8.12 (d, J=8.2 Hz, 2H, ArH), 7.83 (s, 1H, H6’), 6.64 (s, 1H,

H3’’ or H6’’), 5.36 (s, 2H, OCH₂O), 3.9–3.8 (m, 2H, O(CH₂)₂OCH₃),

3.6–3.5 (m, 2H, O(CH₂)₂OCH₃), 3.18 (s, 3H, OCH₃), 2.75 (s, 3H, CH₃),

2.1–2.0 (m, 9H, Ad), 1.8–1.7 ppm (m, 6H, Ad); ¹³C NMR

Ethyl

4-[3-(5-(Adamant-1-yl)-2,4-(2-methoxyethoxymethoxy)-

phenyl)-2-methylpyrimidin-4-yl]benzoate 28a: General proce-

dure for the synthesis of pyrimidines: A mixture of ethyl 4-[1-(5-

(adamant-1-yl)-2,4-(2-methoxyethoxymethoxy)phenyl)-1-oxoprop-2-

yn-3-yl]benzoate 20 (0.14 g, 0.23 mmol), acetimidamide hydrochlo-

ride (0.052 g, 0.9 mmol) and Na₂CO₃(0.096 g, 0.9 mmol) in CH₃CN

(3 mL) was heated under microwave irradiation (250 W, 1208C,

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(100.62 MHz, [D₆]DMSO): d=166.5 (s), 165.0 (s), 164.4 (s), 162.5 (s),

159.7 (s), 159.6 (s), 140.2 (s), 132.5 (s), 129.7 (s), 129.4 (d, 2ꢄ), 127.3

(d, 2ꢄ), 125.5 (d), 110.1 (s), 108.4 (d), 103.1 (d), 92.7 (t), 70.7 (t), 67.8

(t), 57.7 (q), 41.2 (t, 3ꢄ), 36.3 (t, 3ꢄ), 36.0 (s), 28.2 (d, 3ꢄ), 25.3 ppm

(q); HRMS (ESI⁺): calcd for C₃₂H₃₇N₂O₆[M+H]⁺: 545.2646, found:

545.2639; IR (NaCl): n˜ =2900 (w, CꢀH), 2848 (w, CꢀH), 1690 (m, C=

O), 1571 (s), 1118 cm^ꢀ1(m); UV (MeOH): l_max=350, 285 nm; purity:

100% (HPLC-UV, Sunfire C₁₈, 1 mLmin^ꢀ1, 95:5 CH₃CN/H₂O, t_R=

13 min).

4-[3-(5-(Adamant-1-yl)-4-(2-methoxyethoxymethoxy)phenyl)-2-

phenylpyrimidin-4-yl]benzoic acid 30b: Following the general

procedure for the hydrolysis of esters, the reaction of ethyl 4-[3-(5-

(adamant-1-yl)-4-(2-methoxyethoxymethoxy)phenyl)-2-phenylpyri-

midin-4-yl]benzoate 29b (0.028 g, 0.04 mmol) and a 1m aqueous

NaOH solution (0.2 mL, 0.22 mmol) in MeOH (1.8 mL) at 708C af-

forded, after purification by recrystallization (EtOH), 0.019 g (72%)

of a yellow solid identified as 4-[3-(5-(adamant-1-yl)-4-(2-methoxye-

thoxymethoxy)phenyl)-2-phenylpyrimidin-4-yl]benzoic acid 30b;

1

mp: 2488C (EtOH); H NMR (400.13 MHz, CDCl₃/CD₃CO₂D): d=8.5–

8.4 (m, 2H, ArH), 8.34 (d, J=8.0 Hz, 2H, ArH), 8.26 (d, J=8.0 Hz,

2H, ArH), 7.98 (s, 1H, H3’’ or H6’’), 7.74 (s, 1H, H6’), 7.6–7.5 (m, 3H,

ArH), 6.81 (s, 1H, H3’’ or H6’’), 5.38 (s, 2H, OCH₂O), 3.9–3.8 (m, 2H,

O(CH₂)₂OCH₃), 3.7–3.6 (m, 2H, O(CH₂)₂OCH₃), 3.42 (s, 3H, OCH₃),

2.1–2.0 (m, 9H, Ad), 1.8–1.7 ppm (m, 6H, Ad); ¹³C NMR

(100.62 MHz, CDCl₃/CD₃CO₂D): d=171.4 (s), 165.6 (s), 163.3 (s),

162.6 (s), 160.8 (s), 160.7 (s), 142.2 (s), 136.5 (s), 131.3 (d), 131.2 (s),

130.8 (s), 130.7 (d, 2ꢄ), 128.8 (d, 2ꢄ), 128.2 (d, 2ꢄ), 127.5 (d, 2ꢄ),

124.9 (d), 110.2 (s), 108.3 (d), 103.9 (d), 93.1 (t), 71.5 (t), 68.3 (t), 59.0

(q), 41.0 (t, 3ꢄ), 37.0 (t, 3ꢄ), 36.7 (s), 29.0 ppm (d, 3ꢄ); HRMS (ESI⁺

): calcd for C₃₇H₃₉N₂O₆[M+H]⁺: 607.2803, found: 607.2792; IR

(NaCl): n˜ =2900 (s, CꢀH), 2849 (w, CꢀH), 1701 (s, C=O), 1569 (s),

1526 (w), 1296 (w), 1103 (w), 1019 cm^ꢀ1(w); UV (MeOH): l_max=348,

256 nm; purity: 96% (HPLC-UV, Sunfire C₁₈, 1 mLmin^ꢀ1, CH₃CN, t_R=

20 min).

Ethyl

4-[3-(5-(Adamant-1-yl)-2,4-(2-methoxyethoxymethoxy)-

phenyl)-2-phenylpyrimidin-4-yl]benzoate 28b: Following the

general procedure for the synthesis of pyrimidines, the reaction of

ethyl 4-[3-(5-(adamant-1-yl)-2,4-(2-methoxyethoxymethoxy)phenyl)-

3-oxoprop-1-yn-1-yl]benzoate 20 (0.078 g, 0.126 mmol), benzimida-

mide hydrochloride (0.079 g, 0.5 mmol) and Na₂CO₃(0.053 g,

0.5 mmol) in CH₃CN (2 mL) afforded, after purification by column

chromatography (C₁₈-SiO₂, 80:10 CH₃CN/H₂O), 0.052 g (56%) of

a yellow oil identified as ethyl 4-[3-(5-(adamant-1-yl)-2,4-(2-methox-

yethoxymethoxy)phenyl)-2-phenylpyrimidin-4-yl]benzoate

28b;

¹H NMR (400.13 MHz, CDCl₃): d=8.70 (d, J=8.4 Hz, 2H, ArH), 8.32

(d, J=8.0 Hz, 2H, ArH), 8.31 (s, 1H, H3’’ or H6’’), 8.23 (s, 1H, H6’),

8.18 (d, J=8.0 Hz, 2H, ArH), 7.6–7.5 (m, 3H, ArH), 7.08 (s, 1H, H3’’

or H6’’), 5.39 (s, 2H, OCH₂O), 5.37 (s, 2H, OCH₂O), 4.44 (q, J=

7.1 Hz, 2H, CO₂CH₂CH₃), 3.9–3.8 (m, 2H, 2ꢄO(CH₂)₂OCH₃), 3.8–3.7

(m, 2H, 2ꢄO(CH₂)₂OCH₃), 3.6–3.5 (m, 2H, 2ꢄO(CH₂)₂OCH₃), 3.5–3.4

(m, 2H, 2ꢄO(CH₂)₂OCH₃), 3.42 (s, 3H, OCH₃), 3.33 (s, 3H, OCH₃),

2.1–2.0 (m, 9H, Ad), 1.8–1.7 (m, 6H, Ad), 1.44 ppm (t, J=7.1 Hz,

3H, CO₂CH₂CH₃); ¹³C NMR (100.62 MHz, CDCl₃): d=166.3 (s), 164.2

(s), 164.0 (s), 162.4 (s), 159.1 (s), 155.0 (s), 142.1 (s), 138.3 (s), 133.2

(s), 132.0 (s), 130.5 (d), 130.1 (d, 2ꢄ), 129.4 (d), 128.5 (d, 2ꢄ), 128.3

(d, 2ꢄ), 127.2 (d, 2ꢄ), 120.3 (s), 115.4 (d), 103.0 (d), 94.7 (t), 93.4 (t),

71.6 (t), 71.5 (t), 68.2 (t), 68.1 (t), 61.2 (t), 59.1 (q), 59.0 (q), 40.9 (t,

3ꢄ), 37.1 (t, 3ꢄ), 36.9 (s), 29.1 (d, 3ꢄ), 14.4 ppm (q); HRMS (ESI⁺):

calcd for C₄₃H₅₁N₂O₈[M+H]⁺: 723.3640, found: 723.3657; IR (NaCl):

n˜ =2904 (s, CꢀH), 2851 (m, CꢀH), 1717 (s, C=O), 1566 (s), 1517 (s),

1273 (s), 1105 cm^ꢀ1(s); UV (MeOH): l_max=264 nm.

Ethyl 4-[6-(adamant-1-yl)-7-(2-methoxyethoxymethoxy)-4-oxo-4-

chromen-2-yl]benzoate 31 and ethyl (Z)-4-[5-(adamant-1-yl)-6-

(2-methoxyethoxymethoxy)-3-(oxobenzofuran-2-ylidene)me-

thyl]benzoate 33: Method A: A mixture of 4-[1-(5-(adamant-1-yl)-2-

hydroxy-4-(2-methoxyethoxymethoxy)phenyl)-1-oxoprop-2-yn-3-yl]-

benzoate 21 (0.075 g, 0.14 mmol) and K₂CO₃(0.011 g, 0.077 mmol)

in acetone (0.5 mL) was stirred for 22 h at 608C. The mixture was

filtered, the solvent was evaporated and the residue was diluted

with CH₂Cl₂/H₂O (1:1, v/v). The layers were separated and the aque-

ous layer was extracted with CH₂Cl₂(3ꢄ). The combined organic

layers were dried (Na₂SO₄) and the solvent was evaporated. The

residue was purified by column chromatography (silica gel, 99:1

CH₂Cl₂/MeOH) to afford 0.018 g (24%) of a yellow solid identified

as ethyl 4-[6-(adamant-1-yl)-7-(2-methoxyethoxymethoxy)-4-oxo-4-

chromen-2-yl]benzoate 31 and 0.057 g (76%) of a yellow oil identi-

fied as ethyl (Z)-4-[5-(adamant-1-yl)-6-(2-methoxyethoxymethoxy)-

3-(oxobenzofuran-2-ylidene)-methyl]benzoate 33.

Ethyl 4-[3-(5-(Adamant-1-yl)-4-(2-methoxyethoxymethoxy)phen-

yl)-2-phenylpyrimidin-4-yl]benzoate 29b: Following the general

procedure for the deprotection of methoxyethoxymethoxy ethers,

the reaction of ethyl 4-[3-(5-(adamant-1-yl)-2,4-(2-methoxyethoxy-

methoxy)phenyl)-2-phenylpyrimidin-4-yl)]benzoate 28b (0.05 g,

0.07 mmol), BCl₃(0.2 mL, 1m in hexane, 0.21 mmol) in CH₂Cl₂

(1.6 mL) afforded, after purification by column chromatography

(silica gel, 70:30 hexane/EtOAc), 0.021 g (46%) of a yellow solid

identified as ethyl 4-[3-(5-(adamant-1-yl)-4-(2-methoxyethoxyme-

Ethyl 4-[6-(adamant-1-yl)-7-(2-methoxyethoxymethoxy)-4-oxo-4-

chromen-2-yl]benzoate 31: ¹H NMR (400.13 MHz, (CD₃)₂CO): d=

8.20 (d, J=8.6 Hz, 2H, ArH), 8.09 (s, 1H, H5’), 8.01 (d, J=8.6 Hz, 2H,

ArH), 7.34 (s, 1H, H8’), 6.90 (s, 1H, H2’), 5.49 (s, 2H, OCH₂O), 4.45

(q, J=7.1 Hz, 2H, CO₂CH₂CH₃), 3.9–3.8 (m, 2H, O(CH₂)₂OCH₃), 3.7–

3.6 (m, 2H, O(CH₂)₂OCH₃), 3.44 (s, 3H, OCH₃), 2.2–2.1 (m, 9H, Ad),

1.8–1.7 (s, 6H, Ad), 1.46 ppm (t, J=7.1 Hz, 3H, CO₂CH₂CH₃);

¹³C NMR (100.62 MHz, CDCl₃): d=177.9 (s), 165.8 (s), 161.5 (s), 161.3

(s), 156.1 (s), 137.9 (s), 135.9 (s), 132.8 (s), 130.1 (d, 2ꢄ), 126.0 (d,

2ꢄ), 123.7 (d), 117.8 (s), 108.6 (d), 102.5 (d), 93.4 (t), 71.5 (t), 68.4 (t),

61.4 (t), 59.1 (q), 40.7 (t, 3ꢄ), 37.5 (s), 37.0 (t, 3ꢄ), 28.9 (d, 3ꢄ),

14.3 ppm (q); MS (FAB⁺): m/z (%) 533 ([M+H]⁺, 100), 532 ([M]⁺, 7),

457 (12), 443 (11), 347 (27), 281 (23), 207 (13); HRMS (FAB⁺): calcd

for C₃₂H₃₇O₇[M+H]⁺: 533.2539, found: 533.2536; IR (NaCl): n˜ =

2906 (s, CꢀH), 2851 (s, CꢀH), 1718 (s, C=O), 1646 (s, C=O), 1605

(m), 1450 (m), 1277 (s), 1107 cm^ꢀ1(s); UV (MeOH): l_max=315,

265 nm.

thoxy)phenyl)-2-phenylpyrimidin-4-yl]benzoate

29b;

¹H NMR

(400.13 MHz, CDCl₃): d=8.5–8.4 (m, 2H, ArH), 8.34 (d, J=8.0 Hz,

2H, ArH), 8.25 (d, J=8.4 Hz, 2H, ArH), 7.99 (s, 1H, H3’’ or H6’’), 7.75

(s, 1H, H6’), 7.6–7.5 (m, 3H, ArH), 6.82 (s, 1H, H3’’ or H6’’), 5.40 (s,

2H, OCH₂O), 4.45 (q, J=7.1 Hz, 2H, CO₂CH₂CH₃), 3.9–3.8 (m, 2H,

O(CH₂)₂OCH₃), 3.7–3.6 (m, 2H, O(CH₂)₂OCH₃), 3.42 (s, 3H, OCH₃),

2.1–2.0 (m, 9H, Ad), 1.8–1.7 (m, 6H, Ad), 1.45 ppm (t, J=7.1 Hz,

3H, CO₂CH₂CH₃); ¹³C NMR (100.62 MHz, CDCl₃): d=166.1 (s), 165.6

(s), 163.5 (s), 162.6 (s), 161.1 (s), 160.8 (s), 141.3 (s), 136.6 (s), 132.5

(s), 131.3 (d), 130.7 (s), 130.1 (d, 2ꢄ), 128.9 (d, 2ꢄ), 128.2 (d, 2ꢄ),

127.4 (d, 2ꢄ), 124.9 (d), 110.2 (s), 108.2 (d), 104.0 (d), 93.2 (t), 71.6

(t), 68.4 (t), 61.3 (t), 59.1 (q), 41.1 (t, 3ꢄ), 37.1 (t, 3ꢄ), 36.8 (s), 29.1

(d, 3ꢄ), 14.3 ppm (q); HRMS (ESI⁺): calcd for C₃₈H₄₃N₂O₆[M+H]⁺:

635.3115, found: 635.3112; IR (NaCl): n˜ =2900 (m, CꢀH), 2848 (w,

CꢀH), 1713 (m, C=O), 1589 (m), 1569 (s), 1524 (m), 1276 (s), 1104

(s), 1017 cm^ꢀ1(s); UV (MeOH): l_max=356, 268 nm.

Ethyl

(Z)-4-[5-(Adamant-1-yl)-6-(2-methoxyethoxymethoxy)-3-

(oxobenzofuran-2-ylidene)-methyl]benzoate 33: mp: 117–1188C

(hexane/EtOAc); ¹H NMR (400.13 MHz, (CD₃)₂CO): d=8.09 (d, J=

ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2013, 8, 1184 – 1198 1195

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8.4 Hz, 2H, ArH), 7.93 (d, J=8.4 Hz, 2H, ArH), 7.66 (s, 1H, H4’’), 7.09

(s, 1H, H7’’), 6.78 (s, 1H, H1’), 5.45 (s, 2H, OCH₂O), 4.39 (q, J=

7.1 Hz, 2H, CO₂CH₂CH₃), 3.9–3.8 (m, 2H, O(CH₂)₂OCH₃), 3.6–3.5 (m,

2H, O(CH₂)₂OCH₃), 3.40 (s, 3H, OCH₃), 2.0–1.9 (m, 9H, Ad), 1.8–1.7

(s, 6H, Ad), 1.41 ppm (t, J=7.1 Hz, 3H, CO₂CH₂CH₃); ¹³C NMR

(100.62 MHz, CDCl₃): d=182.5 (s), 166.0 (s), 165.1 (s), 163.5 (s),

148.0 (s), 135.8 (s), 134.8 (s), 130.0 (d, 2ꢄ), 129.7 (s), 128.9 (d, 2ꢄ),

122.0 (d), 113.4 (s), 109.0 (d), 97.4 (d), 92.5 (t), 70.5 (t), 67.5 (t), 60.1

(t), 58.1 (q), 39.8 (t, 3ꢄ), 36.3 (s), 36.0 (t, 3ꢄ), 28.0 (d, 3ꢄ), 13.3 ppm

(q); MS (FAB⁺): m/z (%) 533 ([M+H]⁺, 100), 532 ([M]⁺, 14), 457

(24), 443 (21), 307 (12), 281 (11) 166 (13), 155 (24), 154 (76); HRMS

(FAB⁺): calcd for C₃₂H₃₇O₇[M+H]⁺: 533.2539, found: 533.2549; IR

(NaCl): n˜ =2904 (s, CꢀH), 2850 (m, CꢀH), 1702 (s, C=O), 1656 (w, C=

508C afforded, after purification by recrystallization (MeOH),

0.016 g (85%) of a yellow solid identified as (Z)-4-[5-(adamant-1-yl)-

6-(2-methoxyethoxymethoxy)-3-(oxobenzofuran-2-ylidene)-methyl]-

benzoic acid 34; mp: 1208C (MeOH); ¹H NMR (400.13 MHz,

CD₃CO₂D): d=8.26 (d, J=8.4 Hz, 2H, ArH), 8.16 (d, J=8.4 Hz, 2H,

ArH), 7.84 (s, 1H, H4’), 7.26 (s, 1H, H₁), 7.08 (s, 1H, H7’), 5.64 (s, 2H,

OCH₂O), 4.1–4.0 (m, 2H, O(CH₂)₂OCH₃), 3.8–3.7 (m, 2H,

O(CH₂)₂OCH₃), 3.50 (s, 3H, OCH₃), 2.2–2.1 (m, 9H, Ad), 2.0–1.9 ppm

(s, 6H, Ad); ¹³C NMR (100.62 MHz, CD₃CO₂D): d=185.3 (s), 171.6 (s),

168.5 (s), 166.3 (s), 150.4 (s), 138.7 (s), 137.3 (s), 132.4 (d, 2ꢄ), 131.4

(d, 2ꢄ), 131.0 (s), 124.1 (d), 115.0 (s), 112.1 (d), 99.3 (d), 94.6 (t), 72.4

(t), 69.6 (t), 59.0 (q), 41.6 (t, 3ꢄ), 38.3 (s), 37.8 (t, 3ꢄ), 30.2 ppm (d,

3ꢄ); MS (FAB⁺): m/z (%) 505 ([M+H]⁺, 9), 429 (12), 329 (17), 178

(10), 177 (20), 176 (100), 167 (9), 166 (16), 165 (19), 155 (15), 154

(69), 152 (15); HRMS (FAB⁺): calcd for C₃₀H₃₃O₇[M+H]⁺: 505.2226,

found: 505.2232; IR (NaCl): n˜ =2898 (s, CꢀH), 1702 (m, C=O), 1609

O), 1608 (s, C=C), 1470 (m), 1275 cm^ꢀ1(s); UV (MeOH): l_max

=

342 nm.

(s, C=O), 1538 (s), 1414 (s), 1135 cm^ꢀ1(s); UV (MeOH): l_max

=

Ethyl 4-[6-(adamant-1-yl)-7-(2-methoxyethoxymethoxy)-4-oxo-4-

chromen-2-yl]benzoate 31 and ethyl (Z)-4-[5-(adamant-1-yl)-6-

(2-methoxyethoxymethoxy)-3-(oxobenzofuran-2-ylidene)me-

thyl]benzoate 33: Method B: A mixture of 4-[3-(5-(adamant-1-yl)-2-

hydroxy-4-(2-methoxyethoxymethoxy)phenyl)-3-oxoprop-1-yn-1-yl]-

benzoate 21 (0.027 g, 0.05 mmol) and K₂CO₃(0.027 g, 0.05 mmol)

in EtOH (0.6 mL) was stirred for 22 h at 808C. The mixture was fil-

tered, the solvent was evaporated and the residue was diluted

with CH₂Cl₂/H₂O (1:1, v/v). The layers were separated and the aque-

ous layer was extracted with CH₂Cl₂(3ꢄ), the combined organic

layers were dried (Na₂SO₄) and the solvent was evaporated. The

residue was purified by column chromatography (silica gel, 90:10

hexane/EtOAc) to afford 0.005 g (20%) of a yellow solid identified

as ethyl 4-[6-(adamant-1-yl)-7-(2-methoxyethoxymethoxy)-4-oxo-4-

chromen-2-yl]benzoate 31 and 0.018 g (67%) of a yellow oil identi-

fied as ethyl (Z)-4-[5-(adamant-1-yl)-6-(2-methoxyethoxymethoxy)-

3-(oxobenzofuran-2-ylidene)methyl]benzoate 33.

344 nm.

Biology

Kinase assays: A LANCE Ultra time-resolved fluorescence resonance

energy transfer (TR-FRET) assay was performed to measure the

effect of test compounds on the activity of purified recombinant

IKKs. Assays were carried out in white 384-well OptiPlates (Perki-

nElmer) in 10 mL total volume. Recombinant kinases were pur-

chased from Carna Biosciences and were diluted in kinase buffer

(50 mm HEPES pH 7.4, 10 mm MgCl₂, 1 mm EGTA, 2 mm DTT, and

0.01% Tween-20) to a final concentration of 4 nm (IKKa), 1 nm

(IKKb), or 2 nm (IKKe). 50 nm Ulight-IkBa and Ulight-rpS6 (Perki-

nElmer) were used as peptide substrates for IKKa/b and IKKe, re-

spectively. All assays were performed with an ATP concentration

close to the apparent K_Mfor each enzyme (1.25 mm for IKKa/b and

5 mm for IKKe). After 2 h incubation at room temperature, the reac-

tion was stopped by addition of 20 mm EDTA in LANCE detection

buffer, containing 2 nm europium-labeled phospho-specific anti-

body (PerkinElmer). Two hours later, the TR-FRET signals at 615 and

665 nm were measured upon excitation at 340 nm with a 50 ms

delay in a Victor V multilabel reader. LANCE counts were normal-

ized following the manufacturer’s instructions. IC₅₀values for active

compounds were determined using an eight-point titration experi-

ment and GraphPad Prism software.

4-[6-(Adamant-1-yl)-7-(2-methoxyethoxymethoxy)-4-oxo-4-chro-

men-2-yl]benzoic acid 32: Following the general procedure for

the hydrolysis of esters, the reaction of ethyl 4-[6-(adamant-1-yl)-7-

(2-methoxyethoxymethoxy)-4-oxo-4-cromen-2-yl]benzoate

31

(0.012 g, 0.023 mmol) and 1m aqueous solution of NaOH

a

(0.14 mL, 0.136 mmol) in MeOH (0.8 mL) afforded, after purification

by recrystallization (EtOAc/hexane), 0.082 g (75%) of a yellow solid

identified as 4-[6-(adamant-1-yl)-7-(2-methoxyethoxymethoxy)-4-

oxo-4-chromen-2-yl]benzoic acid 32; mp: 1258C (EtOAc/hexane);

Anal. calcd for C₃₀H₃₂O₇·H₂O: C 69.95, H 6.56, found: C 69.90, H

1

6.27; H NMR (400.13 MHz, CDCl₃/CD₃CO₂D): d=8.21 (d, J=7.6 Hz,

Cell proliferation assay: We used a luminescence-based assay (Cell-

Titer-GLO, Promega) to determine the ATP levels as a measure of

cell viability. Jurkat and K562 cells were grown in RPMI supple-

mented with 10% heat-inactivated FBS; PC-3 cells were cultured in

RPMI with 10% FBS. Cells were seeded the day before treatment in

medium containing 0.5% FBS in a 384-well CulturPlate (PerkinElm-

er) at the following densities: 2000 cells per well (PC-3), 5000 cells

per well (K562), or 10000 cells per well (Jurkat). Cells were treated

with increasing concentrations of the compounds in triplicate

points. Control cells were treated with the same amount of sol-

vent, DMSO, up to 0.1% v/v. After 24 h (Jurkat) or 48 h (K562, PC-3)

of treatment, a CellTiter-GLO assay was carried out following the

manufacturer’s instructions.

2H, ArH), 8.07 (s, 1H, H5’), 8.03 (d, J=7.9 Hz, 2H, ArH), 7.33 (s, 1H,

H8’), 7.00 (s, 1H, H2’), 5.47 (s, 2H, OCH₂O), 3.9–3.8 (m, 2H,

O(CH₂)₂OCH₃), 3.7–3.6 (m, 2H, O(CH₂)₂OCH₃), 3.41 (s, 3H, OCH₃),

2.1–2.0 (m, 9H, Ad), 1.8–1.7 ppm (s, 6H, Ad); ¹³C NMR (100.62 MHz,

CDCl₃): d=170.9 (s), 161.7 (s), 161.6 (s), 161.5 (s), 156.3 (s), 138.3 (s),

136.6 (s), 131.7 (s), 130.7 (d, 2ꢄ), 126.3 (d, 2ꢄ), 123.8 (d), 117.4 (s),

108.5 (d), 102.4 (d), 93.4 (t), 71.4 (t), 68.4 (t), 59.0 (q), 40.7 (t, 3ꢄ),

37.5 (s), 36.9 (t, 3ꢄ), 28.9 ppm (d, 3ꢄ); HRMS (ESI⁺): calcd for

C₃₀H₃₃O₇[M+H]⁺: 505.2221, found: 505.2205; IR (NaCl): n˜ =2923 (s,

CꢀH), 2851 (s, CꢀH), 1717 (m, C=O), 1636 (m, C=O), 1458 (m), 1261

(w), 1104 (w), 1020 cm^ꢀ1(w); UV (MeOH): l_max=316, 263 nm;

purity: 100% (HPLC-UV, Sunfire C₁₈, 1 mLmin^ꢀ1, 88:12 CH₃CN/H₂O,

t_R=8 min).

DEVDase assay: Jurkat cells (20000 per well) were seeded in 0.5%

FBS-RPMI in a 384-well black OptiPlate. Following a 4 h incubation

with 5 mm test compounds, cells were lysed for 15 min (25 mm

PIPES pH 7, 25 mm KCl, 5 mm EGTA, 1 mm DTT, 10 mm cytochala-

sin B, 0.5% NP-40, and a mixture of protease inhibitors consisting

of 1 mm PMSF, 1 mgmL^ꢀ1leupeptin, and 1 mgmL^ꢀ1aprotinin) and

DEVDase activity was measured following the addition of caspase

(Z)-4-[5-(Adamant-1-yl)-6-(2-methoxyethoxymethoxy)-3-(oxoben-

zofuran-2-ylidene)methyl]benzoic acid 34: Following the general

procedure for the hydrolysis of esters, the reaction of ethyl (Z)-4-[5-

(adamant-1-yl)-6-(2-methoxyethoxymethoxy)-3-(oxobenzofuran-2-

ylidene)-methyl]benzoate 33 (0.019 g, 0.035 mmol) and a 1m aque-

ous solution of NaOH (0.2 mL, 0.18 mmol) in MeOH (0.8 mL) at

ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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[2] a) H. Adachi, A. Adams, F. M. Hughes, J. Zhang, J. A. Cidlowski, A. M.

Jetten, Cell Death Differ. 1998 , 5 , 97 3 –9 83; b) Y. Zhang, M. I. Dawson, Y.

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Keywords: adamantyl arotinoids · chalcones · IkB kinases ·

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Published online on May 7, 2013

Please note: Scheme 1 was incorrectly printed and has been corrected in

this version. The Editor.

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Adamantyl arotinoids that inhibit IκB Kinaseα and IκB Kinaseβ

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