Inhibition of IκB kinase-β and IκB kinase-α by heterocyclic adamantyl arotinoids

Article abstract of DOI:10.1016/j.bmc.2014.01.006

We recently reported on a series of retinoid-related molecules containing an adamantyl group, a.k.a. adamantyl arotinoids (AdArs), that showed significant cancer cell growth inhibitory activity and activated RXRα (NR2B1) in transient transfection assays while devoid of RAR transactivation capacity. We have now explored whether these AdArs could also bind and inhibit IKKβ, a known target that mediates the induction of apoptosis and cancer cell growth inhibition by related AdArs containing a chalcone functional group. In addition, we have prepared and evaluated novel AdArs that incorporate a central heterocyclic ring connecting the adamantyl-phenol and the carboxylic acid at the polar termini. Our results indicate that the majority of the RXRα activating compounds lacked IKKβ inhibitory activity. In contrast, the novel heterocyclic AdArs containing a thiazole or pyrazine ring linked to a benzoic acid motif were potent inhibitors of both IKKα and IKKβ, which in most cases paralleled significant growth inhibitory and apoptosis inducing activities.

Full text of DOI:10.1016/j.bmc.2014.01.006

Bioorganic & Medicinal Chemistry 22 (2014) 1285–1302
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of IjB kinase-b and IjB kinase-a by heterocyclic
adamantyl arotinoids
José García-Rodríguez a, Santiago Pérez-Rodríguez a, María A. Ortiz b, Raquel Pereira a, Angel R. de Lera a,
,
⇑
F. Javier Piedrafita b,
⇑
a Departamento de Química Orgánica, CINBIO and Instituto de Investigación Biomédica de Vigo (IBIV), Universidade de Vigo, Lagoas-Marcosende, 36310 Vigo, Spain
b Torrey Pines Institute for Molecular Studies, 3550 General Atomics Ct, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently reported on a series of retinoid-related molecules containing an adamantyl group, a.k.a. ada-
mantyl arotinoids (AdArs), that showed significant cancer cell growth inhibitory activity and activated
Received 25 September 2013
Revised 26 November 2013
Accepted 3 January 2014
Available online 10 January 2014
RXRa (NR2B1) in transient transfection assays while devoid of RAR transactivation capacity. We have
now explored whether these AdArs could also bind and inhibit IKKb, a known target that mediates the
induction of apoptosis and cancer cell growth inhibition by related AdArs containing a chalcone func-
tional group. In addition, we have prepared and evaluated novel AdArs that incorporate a central hetero-
cyclic ring connecting the adamantyl-phenol and the carboxylic acid at the polar termini. Our results
Keywords:
Adamantyl arotinoids
Synthesis
Kinase activities
IKK
indicate that the majority of the RXR
a activating compounds lacked IKKb inhibitory activity. In contrast,
the novel heterocyclic AdArs containing a thiazole or pyrazine ring linked to a benzoic acid motif were
potent inhibitors of both IKK
and apoptosis inducing activities.
a
and IKKb, which in most cases paralleled significant growth inhibitory
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
1a, appears to be largely dependent on cell type. Evidences for
caspase-dependent and independent mechanisms14–16 via the
intrinsic17,18 and extrinsic pathways19 have been provided for
these compounds.
In addition to SHP, RRMs with pro-apoptotic activity can target
the IKK/NFjB signaling pathway via direct inhibition of IKKb,
The adamantyl arotinoids (AdArs)1,2 are included within the
group of atypical retinoids or retinoid-related molecules
(RRMs),3–5 a name that reflects the fact that they exert their anti-
cancer activities independently of the transactivation of the reti-
noid receptors (RARs, subtypes
a
, b, and
c
; and RXRs, subtypes
a
,
which has been validated as a cancer target in vitro and in preclin-
ical studies with very promising results, although clinical studies
have not yet produced positive outcomes.20,21 Thus, great efforts
b, and
c
).6,7 RARs and RXRs are members of the nuclear hormone
receptor superfamily of ligand-responsive transcription factors,8
which mediate the multifarious actions of natural and synthetic
retinoids in embryo and throughout life. Some AdArs, however,
are known to bind to RARs, such as 1a (6-[(3-adamant-1-yl)-4-hy-
droxy-phenyl]-2-naphthoic acid, CD437, also called AHPN), which
have been dedicated to develop IKK/NFjB inhibitors as potential
therapies for the treatment of cancer and inflammatory dis-
eases.22–24 We initially found that MX781 3 substantially inhibited
IKK isolated from TNFa-stimulated HeLa cells, and displayed effec-
is a RAR
tene, which also binds RAR
c
agonist.5 Other analogs of 1a, such as 2a (AHPC, Adaro-
and is a stronger apoptogenic agent
tive and consistent inhibition of IKK/NFj
B activity in various can-
c
cer cell lines, thus confirming IKK as an AdAr target.25 Furthermore,
a number of chalcone containing analogs with an additional substi-
tuent ortho to the carbonyl group were prepared and found to elicit
enhanced inhibition of recombinant IKKb in vitro, which paralleled
increased growth inhibitory and apoptosis inducing activities in
cancer cells.26 Most recently, substitution of the chalcone function-
ality by a heterocyclic group has demonstrated that AdArs can
inhibit IKK and induce apoptosis independently of a potential Mi-
than 1a), 5-Cl-AHPN 1b and 3-Cl-AHPC 2b (see Fig. 1), lack RAR
transactivation activity while preserving the induction of growth
arrest and apoptotic activity in a variety of cancer cell lines.9–11
Moreover, 3-Cl-AHPC 2b binds the nuclear hormone receptor small
heterodimer partner (SHP) and modulates SHP interaction with the
Sin3A repressor.12,13 While still unclear, the mechanism of
RRM-mediated cell death, in particular the apoptosis induced by
chael addition from a Cys residue to the
Contrasting with the inhibition of IKK/NF
and its apoptogenic analogs observed by us, activation of NF
CD437 1a is necessary for the induction of apoptosis in prostate
a
j
,b-unsaturated ketone.27
B signaling by MX781 3
B by
⇑
Corresponding authors. Tel.: +34 986 812316; fax: +34 986 818167 (A.R.L.); tel./
fax: +1 858 597 3884 (F.J.P.).
j
E-mail addresses: qolera@uvigo.es (A.R. de Lera), jpiedrafita@tpims.org (F.J.
Piedrafita).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2014.01.006

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J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
Figure 1. RRMs with adamantyl groups.
carcinoma cells.28,29 Cinnamic acid derivative 2b also activates
2. Synthesis
IKK
a and IKKb in breast carcinoma and leukemia cancer
cells.28,30,31 The exact mechanism of IKK/NF
jB activation by these
The synthesis of AdArs 4-6a,b has been reported before.32 An
improved preparation of 3-acetamidopropyloxy-AHPC, 3-A-AHPC
2c, an antagonist of 2a, and its methyl analog 5c has been devel-
oped, which differs from that of the original procedure in the order
of steps for the formation of the bromocinnamic ester 15,11 and
thus does not require protection/deprotection of the phenol. Bro-
mination of 3-hydroxybenzaldehyde 11 was followed by the con-
densation of 12 with triethylphosphonoacetate and DBU11 to
afford 13 in 94% yield. The phenoxide of 13 (generated from treat-
ment with K2CO3 in acetone) was treated with Boc-protected 3-
bromopropanamine 14 to give 15 (94%). This step was followed
by the Suzuki reaction with the previously described11,32 boronic
acids 16a11,32,33 and 16b32 to effect the coupling of the two frag-
ments and provide the AdAr skeleton of 17a and 17b. These steps
required optimization of the reaction conditions [Pd(PPh3)4, Na2-
CO3, dioxane, microwave irradiation, 100 °C, 30 min] and afforded
the desired biphenyl compounds in excellent yields (89 and 93%,
respectively). Sequential deprotection of the silyl ether (to 18a
and 18b) and tert-butoxycarbonyl groups afforded the intermedi-
ate aminophenols, which were treated with acetic anhydride and
pyridine to give the corresponding derivatives (19a and 19b,
respectively). Saponification of the esters in 19a and 19b produced
the desired AdArs 2c and 5c, respectively.
For the preparation of the heterocyclic derivatives, we have
chosen the dihalogenated heterocycles, namely dibromothiazole
20 (Scheme 2) and bromoiodopyrazine 26 (Scheme 3) as linchpin
units to which the other substituents could be incorporated by
exploiting the differential reactivity of the halogens in palladium-
catalyzed cross-coupling reactions.34,35 The premise that site-
selective reactions of dibromothiazole 20 and bromoiodopyrazine
26 at the most electron-deficient positions are feasible has previ-
ously been reported.36–39
The synthesis of 2,5-dibromothiazole 20 and the selective Pd-
catalyzed cross-coupling reaction at position C2 have been
described,40 but the functionalization of dibromothiazole by
sequential halogen replacement reactions with different organo-
metallics is unprecedented. For our purpose, the Suzuki coupling
AdArs is still undefined.13,30,31
In a previous study, we prepared a series of analogs of 2 that
incorporate an additional Me substituent at the adamantyl-phenol
end ortho to the biphenyl connection and naphthoic, cinnamic and
phenylacetic acids as polar end groups (compounds 4–6).32 The
compounds derived from cinnamic and naphthoic acid exhibited
potent antiproliferative activities in several cancer cell lines, and
this effect correlated in general with the induction of apoptosis
as measured by caspase activation. However, the strong deviation
of planarity of these AdArs due to the presence of the 2,20-disubsti-
tuted biphenyl connection altered their binding profile from RAR to
RXR. We were able to show that some of those analogs induced
RXR activity as efficiently as 9-cis-retinoic acid in transient trans-
fection assays, suggesting that RXR might be responsible for their
observed tumor cell growth inhibitory effects.32
We now report on the activities of these analogues in the inhi-
bition of IKKa, IKKb and IKKe in vitro and found that none of the
RXR ligands with the exception of the partial agonist 4b inhibited
IKKb by >75%. In contrast, potent growth inhibitory activity was
seen with 4a, which correlated well with strong inhibition of both
IKKa and IKKb in the absence of RAR/RXR activity. We also profiled
a novel series of AdArs (7–10) that incorporate a central heterocy-
clic ring that connects the adamantyl-phenol and the carboxylic
acid at the polar termini. The MEM derivative of the phenol ortho
to the adamantyl group was also included in order to reveal the
role of this substituent that is present in the parent compound
MX781 3, which is a potent inhibitor of IKK/NFj
B signaling.26,27
The new heterocyclic compounds elicited negligible RAR and RXR
transactivation activity and only the thiazole 8b functioned as
strong RAR antagonist. Furthermore, the benzoic-linked thiazoles
8a–d and methoxypyrazine 10a elicited an even greater inhibitory
activity against recombinant IKKb in vitro than compound 3, which
in some cases (8a and 10a) correlated with superior anticancer
activity. In general, the presence of a MEM group reduced IKKb
inhibitory activity compared to the hydroxyl group-containing
analog.

J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
1287
of 2,5-dibromothiazole 20 and aryl boronic acids 16a11,32,33 and
16b32 took place at 90 °C in the presence of Pd(PPh3)4 and aqueous
Na2CO3.38,39 Product 22a was obtained in moderate yield (50%) and
was accompanied by small amounts of the bis-coupled derivative
21a (5%). The mixture of mono- and bis-coupled products of 16b
could not be separated and was used in the fluoride-induced
deprotection to obtain 23b (30%, two steps) and bis-coupled 21b
(5%, two steps). After deprotection of 22a (Scheme 2), the adaman-
tylphenols 23a and 23b were treated with NaH and MEMCl in THF
at 0 °C to afford the MEM derivatives 23c and 23d, respectively.
Only by using the Jeffery modification of the Heck reaction,41 in
the presence of n-Bu4NCl and under strictly anhydrous conditions,
could 23 efficiently react with methyl acrylate to produce the de-
sired product (24a–d). Protection of phenols 24a–d with MEMCl
and hydrolysis (LiOHꢀH2O) of the esters afforded carboxylic acids
7a–d (Scheme 2). Due to the electron-withdrawing nature of the
methyl ester, the Suzuki coupling of bromothiazoles 23a–d with
4-(methoxycarbonyl)phenylboronic acid to afford arotinoids
25a–d required activation by microwave irradiation. Saponifica-
tion (LiOHꢀH2O) afforded carboxylic acids 8a–d in the yields shown
in Scheme 2.
A similar synthetic approach was employed for the pyrazine
derivatives (Scheme 3). The Suzuki coupling of dihalopyrazine 26
and arylboronic acids 16a and 16b (Scheme 1) afforded 27a and
27b in 76% and 30% yield, respectively. The Heck reaction of these
bromopyrazines with methyl acrylate also caused the silyl group
deprotection and produced 28a and 28b in 67% and 57% yield,
respectively. In the case of 27a, the starting pyrazine was partially
recovered (19%) in deprotected form (compound 29a, see below).
Treatment of the phenols with MEMCl gave derivatives 28c and
28d, respectively, in good yields. Saponification of 28a–d (LiOHꢀH2-
O) afforded carboxylic acids 9a–d (Scheme 3). Fluoride-promoted
deprotection of 27a and 27b gave rise to phenols 29a and 29b,
which were converted into the MEM derivatives 29c and 29d,
respectively, under the usual conditions. The coupling of these
bromopyrazines with 4-(methoxycarbonyl)phenylboronic acid
under the same Suzuki conditions was only successful for the
non-methylated analogs 29a and 29c, and produced the final AdAr
scaffold in moderate yields. However, similar treatment with
analogs having a methyl group (29b and 29d) led to their decom-
position. Finally saponification of esters 30a and 30c afforded
arotinoids 10a and 10c, respectively, in good yields (Scheme 3).
3. Biology
3.1. Inhibition of IKKb and IKKa by the novel AdArs
Following up on our previous reports that portrayed the inhibi-
tion of IKKb by MX781 3 and derivatives,25–27 we evaluated the ef-
fect of AdArs on recombinant IKKs using a LANCE TR-FRET kinase
assay. The naphthoic acid 20-Me-5-Cl-AHPN 4a was a strong inhib-
itor of both IKK
about 2.5 times more potent than MX781 3 (IC50 11.83
ble 1). The presence of a MEM chain in compound 4b substantially
reduced the activity against IKK , while preserving strong inhibi-
tion of IKKb, although to a lesser extent as illustrated by the higher
IC50 value (7.06 M). The presence of the naphthoic acid ring was
a
and IKKb with an IC50 of 4.75
lM against IKKb,
lM) (Ta-
a
l
crucial, as compounds 5a,b,c and 6a were inactive whereas 6b
elicited partial activity. Among the heterocyclic compounds, the
benzoic acid-linked thiazoles 8a–d were the strongest inhibitors
of IKKb and IKK
a, with the MEM chain and the Me substituent in
8d marginally reducing IKK
a
activity. The Me substituent at the
adamantyl-phenol end ortho to the biphenyl connection and the
MEM substitution did not interfere with anti-IKKb activity as all
four analogs elicited similar IC50 values, between 3.37
lM (8a)
and 5.16 M (8c). In contrast, substitution of the benzoic acid by
l
an acrylic acid in compounds 7a–d severely affected IKK activity
and partial activity was only detected with the methylated analog
7b. Likewise, the methoxypyrazine containing AdAr 10a was also a
potent inhibitor of both IKK
a and IKKb, but addition of the MEM
Scheme 1. Reagents and reaction conditions: (a) Br2, HOAc, 25 °C, 14 h, 31%; (b) triethylphosphonoacetate, DBU (3 equiv), CH2Cl2, 25 °C, 4 h, 72%; (c) K2CO3, acetone, 60 °C,
24 h, 94%; (d) Pd(PPh3)4, Na2CO3, DME, 100 °C, MW, 30 min (17a, 89%; 17b, 93%); (e) TBAF, THF, 0 °C, 2.5 h, 18b, 98%. (f) (i) EtOH, HCl, 85 °C, 1 h, (ii) Ac2O, Py, CH2Cl2, 25 °C,
20 h (19a, 58%; 19b, 37%); (g) MeOH, Na2CO3, 70 °C, 3 h (2c, 53%; 5c, 57%).

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J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
Scheme 2. Reagents and reaction conditions: (a) 20, Pd(PPh3)4, Na2CO3, MeOH, 1,4-dioxane, 90 °C, 6 h (22a, 50%; 21a, 5%); (b) n-Bu4NF, THF, 0 °C, 3 h (23a, 91%; 23b, 30% for
the two steps, 21b, 5%); (c) NaH, MEMCl, THF, 25 °C, 19 h (23c, 88%; 23d, 66%; 24c, 70%; 24d, 78%); (d) Methyl acrylate, Pd(OAc)2, PPh3, n-Bu4NCl, NaHCO3, 4 Å molecular
sieves, DMF, 70 °C, 17 h (24a, 68%; 24b, 56%); (e) LiOH, THF/H2O, 25 °C, 2 h (7a, 95%; 7b, 76%; 7c, 91%; 7d, 92%; 8a, 99%; 8b, 77%; 8c, 99%; 8d, 94%); (f) 4-
(methoxycarbonyl)phenylboronic acid, Pd(PPh3)4, Na2CO3, MeOH, DME, MW, 120 °C, 10 min (25a, 78%; 25b, 53%; 25c, 61%; 25d, 61%).
Scheme 3. Reagents and reaction conditions: (a) boronic acids 16a/16b, Pd(PPh3)4, Na2CO3, MeOH, 1,4-dioxane, 50 °C, 12 h (27a, 76%; 27b, 30%); (b) methyl acrylate,
Pd(OAc)2, PPh3, n-Bu4NCl, NaHCO3, 4 Å MS, DMF, 70 °C, 17 h (28a, 67%; 28b, 57%); (c) NaH, MEMCl, THF, 25 °C, 19 h (28c, 92%; 28d, 87%; 29c, 89%; 29d, 99%); (d) LiOH, THF/
H2O, 25 °C, 2 h (9a, 73%; 9b, 84%; 9c, 62%; 9d, 65%; 10a, 99%; 10c, 93%); (e) n-Bu4NF, THF, 0 °C, 3 h (29a, 83%; 29b, 63%); (f) 4-(methoxycarbonyl)phenylboronic acid,
Pd(PPh3)4, Na2CO3, MeOH, 1,4-dioxane, 50 °C, 12 h (30a, 58%; 30c, 66%).
chain in analog 10c greatly impaired IKK activity. As observed with
the thiazole containing AdArs, substitution of the benzoic acid by
acrylic acid in the pyrazines (compounds 9a–d) severely compro-
mised IKK activity and only the methylated analogs 9b and 9d
2b caused a partial inhibition of IKK
a
and IKKb activity in vitro,
B signaling reported by
which suggests that activation of IKK/NF
j
others in breast cancer and leukemia cells13,30,31 likely occurs up-
stream of the IKK complex.
caused partial inhibition of recombinant IKKa.
Interestingly, none of the previously reported RXR
a
agonists
3.2. Effect of AdArs on cancer cell growth
(4b, 5a, 5b, and 6b)32 were strong inhibitors of IKK, with the excep-
tion of 4b that inhibited IKKb by 80% and 6b, which partially inhib-
We have previously demonstrated that MX781 3 inhibits cancer
cell growth and induces apoptosis in an IKKb-dependent manner.25
This is further supported by our observations that several chalcone
ited both IKK
family member IKK
compound 4a elicited limited inhibition (41%). The cinnamic acid
a
and IKKb (<50%). On the other hand, the atypical IKK
e
was not affected by any of the AdArs, and only
analogs of MX781 3 with improved IKKa/IKKb inhibitory activity

J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
1289
Table 1
Effect of AdArs on recombinant IKKs, Jurkat cell viability, and DEVDase activity
AdAr
% Inhibition of IKK activitya
Cell viabilityb
% Viability
DEVDasec
IKKa
IKKb
IKK
e
IC50
(
lM)
IC50 (lM)
IKKb
2b
2c
3
41.3 3.8
5.0 4.9
40.6 1.1
0
24.1 6.9
14.4 4.1
15.8 5.6
41.5 14
13.7 5.2
11.9 9.5
2.9 4.8
5.2 8.0
8.5 6.8
0
6.6 0.7
42.7 8.6
28.5 5.5
7.7 4.8
74.3 16
6.1 1.7
20.7 7.8
55.8 8.0
12.4 2.0
0.8 0.9
1.3 1.2
6.3 0.9
1.8 0.8
2.6 0.5
0.6 1.8
1.5 0.8
0.6 0.3
1.6 1.2
81.4 8.6
12.1 2.9
9.8 2.8
4.9 0.5
3.1 1.3
2.6 0.4
0.2 0.9
0.3 1.0
42.6 3.2
2.5 0.3
56.3 4.4
87.3 5.5
33.9 3.2
23.7 4.5
13.5 2.9
2.3 4.0
26.9 2.9
42.3 4.4
32.8 3.6
46.6 5.0
4.5 5.8
29.2 1.6
85.3 2.2
87.9 9.5
82.9 3.7
73.3 5.7
17.9 4.9
50.1 5.1
4.2 4.3
43.9 4.7
96.4 2.7
79.2 6.0
4.9 2.4
4.3 3.6
0
11.83
4.75
7.06
3.33
2.19
5.62
4.04
4a
4b
5a
5b
5c
6a
6b
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
10a
10c
33.1 4.9
34.1 0.9
83.7 3.6
28.0 4.0
45.8 1.7
86.8 1.9
92.9 4.3
92.3 5.0
102.9 5.1
96.1 1.8
6.0 2.2
39.0 9.9
74.5 4.0
59.5 0.9
60.9 2.4
70.9 5.1
94.9 4.2
96.5 4.0
14.9 7.2
91.4 4.2
2.61
3.98
4.9 5.6
39.8 9.5
5.0 4.2
26.2 1.6
1.9 3.4
12.2 6.9
94.1 0.6
95.0 2.6
89.3 3.5
87.7 3.9
8.2 4.2
7.3 5.8
1.6 3.9
3.8 3.3
83.9 3.5
25.4 5.5
0
0
14.3 7.6
11.1 4.0
11.6 4.7
0
25.3 5.9
17.0 8.0
7.8 4.4
6.7 3.6
9.4 5.8
5.9 3.4
26.1 5.9
18.8 6.5
3.37
4.78
5.16
4.43
2.24
4.40
5.43
5.71
5.01
5.38
45.7 6.3
78.5 5.7
23.8 7.4
7.81
3.13
a
The effect of 20 lM AdArs on the activity of recombinant IKKs was determined by LANCE Ultra-kinase assay at a concentration of ATP near the apparent Km for each
kinase. Values indicate the percentage of inhibition with respect to solvent control samples standard deviation obtained in 3–4 experiments performed in triplicate. IC50
values (in M) were calculated over 8 point 1/2.5 serial dilution curve starting at 40 M.
Effect of AdArs on cancer cell proliferation. The percentage of cell viability was calculated in Jurkat T cells treated with 4
were incubated with the same volume of solvent DMSO (0.1% v/v). Cell viability values below 60% identify cell-killing activity. The average standard deviation of three
independent experiments performed with triplicate data points is shown. IC50 values (in M) were calculated for active compounds over an 8 point dose response curve
starting at 10 M.
A DEVDase fluorometric assay was used as a measure of apoptosis in Jurkat cells treated with 5
l
l
b
l
M of the indicated AdArs for 24 h. Control cells
l
l
c
l
M of the indicated AdArs for 4 h, as described in Section 5. The values
indicate the fold induction of DEVDase activity with respect to control cells incubated in the presence of solvent DMSO. The average standard deviation of two independent
experiments performed in triplicate is shown. Values of 2 or below are not significant as they are within the standard error of control samples.
elicited enhanced growth inhibition and apoptosis inducing activ-
ities in Jurkat T cells.26,27 We therefore evaluated the effect of het-
erocyclic AdArs on the viability of Jurkat cells and DEVDase activity
as measures of cell proliferation and apoptosis inducing activity,
respectively. As shown in Table 1, the methylated analog of 5-Cl-
AHPN, compound 4a, and two heterocyclic AdArs, the thiazole 8a
and the pyrazine 10a, elicited the greatest inhibition of Jurkat cell
viability (>85%) and induction of apoptosis (>40 fold), similar to
that observed with the apoptogenic compound 3-Cl-AHPC 2b and
considerably higher than MX781 3. The corresponding derivatives
containing the MEM chain, which is characteristic of MX781 3, 4b
and 8c produced a much diminished growth inhibition and pro-
apoptotic effect, whereas 10c was inactive in both assays. Likewise,
the methylated compounds 8b and 8d also showed partial activity
in Jurkat cells even though they elicited significant inhibition of re-
independently of IKKa/b inhibition. This effect could be a conse-
quence of growth arrest, since 2c has been reported to block the
induction of apoptosis but not growth arrest by other AdArs.11
As reported by others, 3-Cl-AHPN 2b elicited robust cell killing
activity but limited inhibition of recombinant IKK
a/b in vitro,
which relates to the reported activation of IKK/NF B signaling in
j
breast cancer cells.13,30,31 The RXR agonists 4b and 5a had substan-
tial cancer cell killing activity, which appeared to be dependent
(4b) or independent (5a) of the activation of caspases (Table 1).
These results concur with our previous report on adamantyl rexi-
noids32 and the small differences observed here could be due to
different experimental conditions used in both studies.
3.3. RAR/RXR transactivation profile of AdArs
combinant IKK
a
and IKKb in vitro. Although we found clear differ-
The RAR/RXR profile of compounds 4, 5, and 6 has already been
reported, with compounds 4b, 5a,b and 6b functioning as RXR
agonists.32 Of the novel heterocyclic compounds described here,
ences in cell viability and DEVDase activity among the various
AdArs when tested at a single concentration, all active compounds
exhibited similar IC50 values against Jurkat cells, with compounds
none was able to induce significant RARa or RXRa-mediated
4a, 8a, and 10a having the lowest IC50s (2.19, 2.24, and 3.13
lM,
transcriptional activity on their own, with the exception of the
respectively). This agrees with the strongest induction of DEVDase
activity and robust inhibition of IKKs. While there is an overall
good correlation between the effect of AdArs on IKK activity and
MEMO-linked thiazole 8c, which induced weak transactivation of
Gal4-RXRa (3-fold induction over solvent control or 21% of the
maximum activity observed with the control rexinoid CD3254
Jurkat cell viability, the inhibition of IKK
a/b observed with some
33) (Table 2 and Fig. 2). In contrast, the 2-methylphenyl-thiazole
thiazole analogs was not sufficient to trigger cell killing and opti-
mal DEVDase activity (see compounds 8c and 8d), although they
still inhibited cell proliferation. Furthermore, it is worth mention-
ing that compound 2c and its methyl analog 5c exhibited growth
inhibition and weak but reproducible activation of caspases
8b inhibited luciferase activity of Gal4-RAR
mode (Table 2), and a subsequent dose response experiment
revealed an IC50 of 1.277 M. Similarly, the corresponding unme-
thylated positional isomer also functioned as an RAR antagonist
in this system, with an IC50 value of 2.65 M (data not shown).
a in the antagonistic
l
a
l

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J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
Table 2
RAR/RXR transactivation profile of AdArs
Ligand
RAR profile
RXR profile
RXR-coactivator interactionc
SRC-1
Agonista
Antagonistb
Agonista
Antagonistb
D22
None
31, atRA
32, UVI2024
33, CD3254
1
1.2 0.2
100
7.1 2.9
n.t.
1
n.t.
n.t.
7.1 2.9
n.t.
n.t.
100
n.t.
n.t.
4
148
n.t.
n.t.
7
110.8 12
1.3 0.3
n.t.
14.3 3.4
1.0 0.2
0.9 0.1
9.8 2.8
13.3 1.9
19.0 3.8
0.9 0.1
1.4 0.2
16.0 4.4
1.7 0.4
1.3 0.2
0.9 0.1
1.5 0.2
0.9 0.1
1.9 0.5
3.1 1.3
0.9 0.2
1.7 0.3
1.0 0.1
0.9 0.1
1.1 0.1
1.0 0.1
1.1 0.1
100
416 15
561 12
34, UVI3003
n.t.
n.t.
23.3 2.4
69.9 2.5
84.7 5.4
96.2 7.2
120.6 11
73.6 3.7
96.1 5.0
96.3 8.1
78.7 2.6
85.7 9.8
83.9 6.1
91.7 5.5
67.2 6.6
91.7 5.5
83.2 5.9
91.4 7.6
119.4 4.8
89.4 2.7
94.9 4.4
86.3 4.7
83.7 7.7
87.7 4.0
ꢁ4
4
3
6
ꢁ2
3
6
7
4a
4b
5a
5b
5c
6a
6b
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
1.1 0.2
1.1 0.1
1.1 0.2
1.8 0.5
1.0 0.4
1.4 0.4
1.3 0.4
1.1 0.1
1.0 0.1
1.0 0.1
1.9 2.0
4.6 1.2
1.4 0.1
1.2 0.2
0.9 0.1
1.1 0.2
1.0 0.3
1.1 0.2
0.8 0.1
1.9 0.4
1.1 0.3
90.0 4.2
82.7 5.9
67.1 1.4
61.5 6.2
70.0 2.7
79.7 5.2
74.1 6.0
92.4 5.0
87.3 8.4
94.2 7.8
75.3 8.4
66.2 2.4
33.2 4.4
92.4 9.4
61.6 4.0
93.2 5.0
89.0 6.2
78.7 5.4
99.5 9.9
95.4 12
108.7 12
29
53
159
487 36
654 81
113
28
214 12
156
77
127 23
88
94 13
113
133
93
92
93
119 15
87
102 11
170
187
373 18
437 16
152
81 24
422 11
162
82
130
133
183 17
117 10
159
115
116
106
122
106
4
1
7
3
5
9
8
9
3
3
1
5
5
6
4
2
7
4
9
6
3
9d
10a
10c
3
111 16
172
9
5
n.t.: not tested.
a
Values indicate the fold induction of RAR
M atRA 31, whereas RXR -expressing cells were treated with 1
of two independent experiments with triplicate data points is shown.
a
or RXR
a
activity with respect to solvent control cells (non-stimulated). As positive control, RAR
a-transfected cells were treated
with 1
l
a
l
M of the synthetic rexinoid CD3254 33. AdArs were used at 4 M. The average standard deviation
l
b
To determine the antagonist profile of AdAr analogs, RAR/RXR-expressing cells were incubated with 100 nM atRA 31/CD3254 33 in the presence of a 40 fold molar excess
of the indicated AdArs. The values indicate the percentage of activity with respect to control cells stimulated with agonist ligand (atRA 31 or CD3254 33) in the absence of
AdAr (100% activity). The percentage of activity of non-stimulated cells is also shown. As positive controls, RAR and RXR-transfected cells were treated with 1
lM of the well-
known antagonists UVI2024 32 and UVI3003 34, respectively. The average standard deviation of 2 or 3 experiments performed with triplicate data points is shown.
c
Two separate TR-FRET-based homogeneous assays were used to measure the interaction of GST-RXRa-LBD with two different coactivator-derived peptides, D22 and
SRC1. The value indicates the percentage of change compared to samples that contained no ligand (Delta F%, see experimental section for definition). The experiment was
repeated twice with triplicate data points and the average data standard deviation (n = 6) is shown.
Although not as powerful as the inverse agonist UVI2024 32
(IC50 0.114
antagonists of RAR
(IC50 4.474 M).
l
M),27 both thiazole containing AdArs were stronger
a
than the parental compound MX781 3
l
To confirm that the RXR transactivating AdArs are indeed able
to directly bind to the RXR LBD, we performed cell free coactivator
recruitment assays with purified recombinant GST-RXRa LBD and
two coactivator derived peptides: the synthetic peptide D22 la-
beled with fluorescein and biotin-tagged SRC-1-676-700 peptide
containing the second LXXLL motif. Concurring with the cell-based
luciferase transactivation assays, compounds 5a and 5b induced a
robust binding of RXRa LBD to both D22 and SRC-1 peptides, to a
level comparable to that observed with the rexinoid agonist
CD3254 33. The full agonist 6b32 also induced strong interactions
with SRC-1 peptide, but behaved as a partial agonist with D22 pep-
tide (see Table 2 and Fig. S1). As expected, the naphthoic acid
Figure 2. Structure of selected RAR and RXR modulators.
derivative 4b produced a modest increase of RXRa-peptide interac-
tions above basal controls in the absence of ligand, which is in
accordance with the partial agonist activity observed in transient
transfection assays (see Table 2), as we have recently reported.32
The heterocyclic AdArs induced no or very limited (<2 fold) recruit-
suggesting that 4a and 6a might function as weak inverse agonists.
When used in combination with a low concentration of CD3254
ment of coactivator peptides by RXR
The rexinoid antagonist UVI3003 3442 completely inhibited
RXR -D22/SRC-1 interactions, even to the level of non-protein
a
LBD.
agonist 33, both 4a and 6a inhibited the interaction of RXRa LBD
with D22 coactivator peptide stimulated by CD3254 (Fig. S2). Even
though this effect was not comparable to that of UVI3003 34 (25%
a
control (Table 2). Likewise, the adamantyl-phenol derivatives
(lacking the MEM chain) of the two rexinoids 4b and 6b, 4a and
6a respectively, also inhibited the recruitment of both D22 and
inhibition by 4a and 6a versus complete inhibition of RXR
binding by UVI3003 34), it concurred with a weak but consistent
effect elicited by 4a in a cell based Gal4-RXR transactivation assay
in the antagonist mode (30% inhibition) (see Table 2).
a/D22
a
SRC-1 peptides by RXRa LBD significantly (Table 2 and Fig. S2),

J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
1291
compounds 8 and 10 by an acrylic acid (compounds 7 and 9,
respectively), greatly impaired both IKK inhibition and anticancer
activity. Furthermore, while this correlation between the inhibition
of IKK and cancer cell growth, and induction of apoptosis is remi-
niscent of the activity observed with chalcone containing
AdArs,26,27 inhibition of recombinant IKK in vitro does not always
correlate with potent anticancer activity among the heterocyclic
AdArs, in particular with those containing a MEM chain. Thus,
addition of a Me group and/or a MEM chain into thiazole 8a (com-
pounds 8b, 8c, and 8d) diminished growth inhibitory activity and
greatly impaired apoptosis inducing capability in Jurkat cells with-
4. Discussion
Adamantyl arotinoids (AdArs) have been recognized as promis-
ing anticancer therapeutic agents due to their growth inhibitory
and apoptosis inducing activities. Although originally derived from
RARc/b selective retinoid-related molecules, AdArs are often classi-
fied as atypical retinoids as they exert their cancer cell growth
inhibitory and apoptogenic activities independently of RAR trans-
activation. While certain AdArs have been shown to target IKKb
as a likely mediator of their anticancer activity, others have been
shown to function as RXR agonists. Yet, other AdArs have been
reported by others to bind the nuclear receptor SHP while activat-
out affecting the inhibition of IKKa/b. This reduced cellular activity
could be the consequence of impaired cellular uptake, metabolism,
and/or off target activities unrelated to cancer cell growth and
apoptosis.
Other than IKK and RXR, the orphan receptor SHP has also been
shown to be the target of AdArs, in particular 3-Cl-AHPC 2b and
analogs.12,43 3-Cl-AHPC 2b has been reported by others to activate
ing IKK/NFjB signaling in order to induce apoptosis.
Here we report on the IKK inhibitory activity of rexinoid AdArs
and a novel series of heterocyclic containing AdArs with OH or
OMEM substitutions at the adamantyl phenyl ring. None of the no-
vel heterocyclic AdArs described has any significant RAR or RXR
transactivating effect. Only the thiazole analog 8b functioned as a
IKK/NF
the induction of apoptosis.28 The mechanism of IKK/NF
tion has not been delineated, but is in conflict with our observed
modest but consistent inhibition of recombinant IKK /b activity
in our cell-free assay. This suggests that 3-Cl-AHPC 2b has pleiotro-
pic effects in a cellular environment, including but not limited to
inhibition of protein phosphatases as possible mediators of IKK
activation.
j
B signaling pathway, an activity that seems necessary for
RAR
a antagonist, whereas the unmethylated MEMO-linked thia-
jB activa-
zole 8c produced a weak activation of RXR (ꢂ20% of control). We
demonstrate that inhibition of IKK and activation of RXR do not
overlap, but they both correlate with robust tumor cell growth
inhibitory activity. Thus, compound 4a was shown previously to
exhibit strong antiproliferative activity but not RXR activity;32
we now demonstrate that 4a is the most potent inhibitor of Jurkat
a
cell proliferation (IC50 2.19
lM) coinciding with strong inhibition
The recent resolution of the IKKb crystal structure44,45 will cer-
tainly have a positive impact in the future lead optimization efforts
to improve the potency and selectivity of AdArs as IKK inhibitors.
Until now, we have relied on the synthesis and evaluation of
numerous compounds, including chalcones and heterocyclic AdArs
in order to withdraw some structural requirements for optimal
inhibition. Whereas our lead compound MX781 3 had a chalcone
functional group and a MEM chain in the adamantyl phenolic ring,
the results presented here and elsewhere clearly demonstrate that
eliminating the MEM chain has generally a beneficial effect on the
overall IKK/growth inhibitory activity of the AdAr (compare com-
pounds 4a and 4b). Moreover, the chalcone function is not critical
for IKK inhibition by AdArs, as substitution by certain heterocyclic
groups also improves activity (see for example thiazole 8a and pyr-
azine 10a). The inclusion of a heterocyclic thiazole or pyrazine
group within the arotinoid structure could have advantages over
classical arotinoids for the inhibition of protein kinases that we
are trying to achieve with these AdARs. Thiazole and pyrazine ring
systems are important structural elements found in kinase inhibi-
tors, even in FDA approved drugs (i.e., dasatinib). Pyrazine is less
basic than pyridine or pyrimidine, which are commonly found in
ATP-competitive kinase inhibitors.
of IKK and IKKb (IC50 4.75
a
l
M), which suggests that inhibition
of IKKb might be responsible for the growth inhibitory activity of
this AdAr. This contrasts with the limited or no anti-IKK activity
found with rexinoid AdArs that show proven cancer cell growth
inhibitory effects (5a,b and 6b). One exception to this rule is the
MEM analog of 4a, naphthoic acid 4b, which has strong growth
inhibitory activity, functions as a partial RXR agonist, and also
inhibits IKKb with an IC50 of 7.06 lM.
Our present studies further confirm the rexinoid activity of
these AdArs using a cell free biochemical assay in which we mea-
sure the interaction of recombinant GST-RXRa LBD with coactiva-
tor derived peptides. This assay proved more sensitive than cellular
assays in detecting weak inverse agonists. As a result, we could
demonstrate that removal of the MEM chain from the adamantyl
phenol ring converts two of the rexinoid agonists (4b and 6b) into
weak RXRa inverse agonists (4a and 6a). Compounds 4a and 6a
elicited strong inhibition of RXR-peptide interaction in vitro, but
had no effect on RXR-driven luciferase activity when used alone.
When assayed in the antagonist mode (in the presence of subopti-
mal concentrations of a rexinoid agonist, CD3254 33)42 we found
that 4a behaved as a weak antagonist in both cellular and
biochemical assays, whereas 6a was only active in the biochemical
assay with D22 peptide. It is very likely that AdArs function in a
coactivator specific fashion and therefore affect RXR
transactivation in a cell type dependent manner.
a-driven
5. Experimental
5.1. General
In general, there is a good correlation between the inhibition of
IKKb activity and the inhibition of cancer cell viability, which in
turn is associated with induction of apoptosis. Thus, a correlation
analysis between the inhibition of IKKb and inhibition of cell via-
bility by the heterocyclic AdArs (7-9) and including the parent
compound 3 as well as the naphtoic acid derivatives 4a and 4b,
produced a correlation coefficient of 0.8039 with a P value of
0.0002 (data not shown). The strongest correlation is seen with
4a, thiazole 8a, and pyrazine 10a. Thiazole 8a displays the greatest
Solvents were dried according to published methods and dis-
tilled before use. All other reagents were commercial compounds
of the highest purity available. All reactions were carried out under
argon atmosphere, and those not involving aqueous reagents were
carried out in oven-dried glassware. Analytical thin layer chroma-
tography (TLC) was performed on aluminium plates with Merck
Kieselgel 60F254 and visualized by UV irradiation (254 nm) or by
staining with a solution of phosphomolibdic acid. Flash column
chromatography was carried out using Merck Kieselgel 60
(230–400 mesh) under pressure. Infrared spectra were obtained
on JASCO FTIR 4200 spectrophotometer, from a thin film deposited
onto a NaCl glass. 1H NMR spectra were recorded in CDCl3, CD3OD,
inhibition of IKKb (IC50 3.37
DEVDase activity (>80 fold stimulation), whereas pyrazine 10a also
elicits robust inhibition of IKKb (IC50 7.81 M) and induction of
lM) and the maximum induction of
l
apoptosis in terms of DEVDase activity (43 fold increase). To
strengthen this correlation, substitution of the benzoic acid in

1292
J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
CD3CN and DMSO-d6 at ambient temperature on a Bruker AMX-
400 spectrometer at 400 MHz with residual protic solvent as the
internal reference (CDCl3, dH = 7.26 ppm; CD3CN, dH = 1.94 ppm;
CD3OD, dH = 3.31 ppm; DMSO-d6, dH = 2.50 ppm); chemical shifts
(d) are given in parts per million (ppm), and coupling constants
(J) are given in Hertz (Hz). The proton spectra are reported as fol-
lows: d (multiplicity, coupling constant J, number of protons,
assignment). 13C NMR spectra were recorded in CD3Cl3, CD3OD,
DMSO-d6 and CD3CN at ambient temperature on the same
spectrometer at 100 MHz, with the central peak of CDCl3
(dc = 77.0 ppm), CD3OD (dc = 49.0 ppm), DMSO-d6 (dc = 39.4 ppm)
or CD3CN (dc = 118.3, 1.3 ppm) as the internal reference. The
DEPT135 sequence was used to aid in the assignment of signals
on the 13C NMR spectra. Melting points were determined on a Stu-
art SMP10 apparatus. Elemental analyses were determined on a
Carlo ErbaEA 1108 analyzer. MS experiments were performed on
an APEX III FT-ICR MS (Bruker Daltonics, Billerica, MA), equipped
with a 7T actively shielded magnet. Ions were generated using an
Apollo API electrospray ionization (ESI) source (Bruker Daltonics,
Billerica, MA), with a voltage between 1800 and 2200 V (to opti-
mize ionisation efficiency) applied to the needle, and a counter
voltage of 450 V applied to the capillary. Samples were prepared
by adding a spray solution of 70:29.9:0.1 (v/v/v) methanol/water/
formic acid to a solution of the sample at a v/v ratio of 1–5% to give
the best signal-to-noise ratio. Data acquisition and data processing
were performed using the XMASS software, version 6.1.2 (Bruker
Daltonics). FAB experiments were performed on a VG AutoSpec
instrument, using 3-nitrobenzylalcohol or glycerol as matrix.
solvent was evaporated in vacuo. The residue was purified by
column chromatography on silica gel as indicated.
5.5. General procedure for the protection of phenols with
MEMCl
Sodium hydride (60% as a dispersion in oil, 1.1 mmol) was
added to a solution of the phenol (1.0 mmol) in THF (10 mL). After
stirring for 30 min at room temperature, 2-methoxyethoxymethyl
chloride (1.1 mmol) was added and the mixture was stirred at
room temperature for 19 h. The reaction was quenched with water
and extracted with EtOAc (3ꢃ). The combined organic extracts
were dried over Na2SO4, filtered and the solvent was evaporated
in vacuo. The residue was purified by column chromatography
and crystallized as indicated.
5.6. General procedure for the Heck reaction
Tetrabutylammonium chloride (1.0 mmol) was added to a solu-
tion of sodium hydrogencarbonate (2.5 mmol) and 4 Å molecular
sieves (0.4 g/mmol) in DMF (8 mL) and stirring was maintained
for 15 min at room temperature. Then, a solution containing
triphenylphosphine (0.08 mmol), methyl acrylate (2 mmol) and
the haloheterocycle (1.0 mmol) in DMF (0.13 mL) was added via
cannula. The mixture was stirred for 15 min and Pd(OAc)2
(0.08 mmol) was added. After stirring the reaction mixture at
70 °C for 17 h, the solid was removed via filtration through CeliteÒ
and the solvent was evaporated in vacuo. The residue was treated
with CH2Cl2 and washed with NaHCO3 (3ꢃ). The combined organic
layers were dried over Na2SO4, filtered and the solvent was evapo-
rated in vacuo. The residue was purified by column chromatogra-
phy on silica gel and crystallized as indicated.
5.2. General procedure for the Suzuki coupling
In a Schlenk flask, a solution containing the haloheterocycle
(1 mmol), the boronic acid (1.3 mmol), methanol (5 mL) and a
2 M aqueous sodium carbonate solution (3.35 mmol) in benzene
(25 mL) was deoxygenated by bubbling a stream of argon through
it. Pd(PPh3)4 (0.14 mmol) was then added and the flask was evac-
uated and purged with argon. The reaction mixture was heated
for the time indicated and the reaction was quenched with water
and extracted with CH2Cl2 (3ꢃ). The combined organic extracts
were dried over Na2SO4, filtered and the solvent was evaporated
in vacuo. The residue was purified by column chromatography
on silica gel as indicated.
5.7. General procedure for the hydrolysis of esters
Lithium hydroxide (15 mmol) was added to a solution of ester
(1 mmol) in a 1:1 THF/H2O (17 mL) mixture. The solution was stir-
red at room temperature for 2 h, neutralized with 10% HCl and
extracted with EtOAc (4ꢃ). The combined organic layers were
washed with brine, dried over Na2SO4, filtered and the solvent
was evaporated in vacuo. Crystallization of the residue provided
the desired acid as indicated.
5.7.1. Ethyl (E)-3-[3-(adamant-1-yl)-(2-(3-(tert-butoxycarbon
ylamino)propoxy)-4-(tert-butyldimethylsilyloxy)biphenyl-4-
yl]acrylate (17a)
5.3. General procedure for the microwave-assisted Suzuki
coupling
According to the general procedure for the microwave-assisted
Suzuki coupling, the reaction of 1511 (0.15 g, 0.35 mmol) and aryl-
boronic acid 16a11 (0.017 g, 0.46 mmol) gave, after purification of
column chromatography on silica gel (70:30 hexane/EtOAc),
In a reaction flask, a solution containing the haloheterocycle
(1.0 mmol), the boronic acid (1.1 mmol) and a 2 M aqueous sodium
carbonate solution (4.0 mmol) in DME (10 mL) was deoxygenated
by bubbling a stream of argon through it. Pd(PPh3)4 (0.10 mmol)
was then added and the flask was evacuated and purged with ar-
gon. The reaction mixture was heated in a microwave reactor for
the time indicated and the reaction was quenched with water
and extracted with CH2Cl2 (3ꢃ). The combined organic extracts
were dried over Na2SO4, filtered and the solvent was evaporated
in vacuo. The residue was purified by column chromatography
on silica gel as indicated.
0.12 g (55%) of
a
white solid identified as 17a. 1H NMR
(400.13 MHz, CDCl3): d 7.74 (d, J = 16.0 Hz, 1H, H3), 7.30 (d,
J = 8.4 Hz, 1H, ArH), 7.14 (d, J = 2.4 Hz, 1H, H200 or H20), 7.1 (d,
J = 2.0 Hz, 1H, H20 or H200), 6.99 (dd, J = 8.0, 2.0 Hz, 1H, H600 or
H60), 6.96 (dd, J = 8.4, 2.4 Hz, 1H, H60 or H600), 6.82 (d, J = 8.0 Hz,
1H, ArH), 6.34 (d, J = 16.0 Hz, 1H, ArCHCH), 4.75 (br, 1H, NH),
4.20 (q, J = 7.1 Hz, 2H, COOCH2CH3), 4.07 (t, J = 5.8 Hz, 2H, H2),
3.34 (q, J = 5.6 Hz, 2H, CH2NHAc), 1.9–2.1 (m, 11H, 3 ꢃ AdCH2 + -
OCH2CH2CH2N + 3 ꢃ AdCH), 1.75 (s, 6H, 3 ꢃ AdCH2), 1.44 (s, 9H,
CO-t-Bu), 1.28 (t, J = 7.1 Hz, 3H, COOCH2CH3), 1.06 (s, 9H, SiC(CH3)),
0.36 (s, 6H, 2 ꢃ SiCH3) ppm. 13C NMR (100.62 MHz, CDCl3): d 166.9
(s), 157.7 (s), 156.0 (s), 154.1 (s), 144.4 (d), 139.0 (s), 136.3 (s),
133.6 (s), 133.5 (s), 131.5 (d), 131.4 (s), 129.4 (d), 127.5 (d),
119.0 (d), 118.7 (d), 116.7 (d), 111.8 (d), 66.0 (t, ArOCH2), 60.3
(t, COOCH2CH3), 40.4 (t, 3 ꢃ AdCH2), 38.0 (t, CH2N), 37.1
(t, 3 ꢃ AdCH2), 37.0 (s, AdC), 29.6 (t, OCH2CH2CH2N), 29.0
5.4. General procedure for the deprotection of silyl ethers
Tetrabutylammonium fluoride (1.5 mmol) was added to a solu-
tion of the silyl ether (1 mmol) in THF (1 mL) at 0 °C. After the solu-
tion was stirred for 2 h at this temperature, water was added and
the reaction was extracted with EtOAc (3ꢃ). The combined organic
layers were washed with brine, dried over Na2SO4, filtered and the

J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
1293
(d, 3 ꢃ AdCH), 28.9 (q, 3 x, HNCOC(CH3)3), 26.4 (q, 3ꢃ, SiC(CH3)3),
18.9 (s, SiC + COCH3), 14.3 (q, COOCH2CH3), ꢁ3.4 (q, 2 ꢃ Si(CH3))
3 ꢃ AdCH2 + AdC), 28.9 (t, OCH2CH2CH2NHAc), 28.8 (d, 3 ꢃ AdCH),
23.2 (q), 21.8 (q), 14.3 (q, COOCH2CH3) ppm. IR (NaCl): 3293 (w,
m
ppm. IR (NaCl):
m
3380 (w, N–H), 2928 (s, C–H), 2906 (s, C–H),
N–H), 2903 (m, C–H), 2849 (m, C–H), 1753 (m), 1705 (m, C@O),
1651 (m, C@O), 1633 (m, C@O), 1476 (m), 1367 (m), 1203 (s),
1173 (s), 1038 (m), 750 (s) cmꢁ1. EM (EI): m/z (%) 559 (M+, 5),
418 (12), 135 (11), 100 (100). HRMS (EI): calcd for C34H41NO6,
559.2934; found, 559.2928.
2855 (s, C–H), 1713 (s, C@O), 1631 (m), 1602 (m), 1480 (s), 1259
(s), 1172 (s) cmꢁ1. MS (FAB+): m/z (%) 689 (M+, 100), 633 (18),
632 (10), 591 (18), 590 (41), 588 (20), 545 (34), 544 (76), 542
(36), 397 (20), 341 (32).
5.7.2. Ethyl (E)-3-[5-(adamant-1-yl)-2-(3-(tert-butoxycarbon
ylamino)propoxy)-4-(tert-butyldimethylsilyloxy)-2-meth
ylbiphenyl-4-yl]acrylate (17b)
5.7.4. Ethyl (E)-3-[5-(adamant-1-yl)-2-(3-(tert-butoxycarbon
ylamino)propoxy)-4-hydroxy-2-methylbiphenyl-4-yl]acrylate
(18b)
According to the general procedure for the microwave-assisted
Suzuki coupling, the reaction of 1511 (0.15 g, 0.35 mmol) and aryl-
boronic acid 16b32 (0.18 g, 0.46 mmol) gave, after purification of
column chromatography on silica gel (85:15 hexane/EtOAc),
According to the general procedure for the cleavage of silyl
ethers, compound 17b (0.17 g, 0.25 mmol) was treated with a solu-
tion of TBAF (0.27 mL, 1 M in THF, 0.27 mmol) to afford, after puri-
fication by column chromatography on C18 silica gel (100%
acetonitrile), 0.16 g (98%) of a yellow solid identified as 18b. 1H
NMR (400.13 MHz, CDCl3): d 7.42 (d, J = 16.0 Hz, 1H, H3), 7.19 (d,
J = 2.6 Hz, 1H, H3), 7.15 (d, J = 8.4 Hz, 1H, H6), 6.95 (dd, J = 8.4,
2.6 Hz, 1H, H5), 6.88 (s, 1H, H60), 6.55 (s, 1H, H30), 6.30 (d,
J = 16.0 Hz, 1H, H2), 4.80 (s, 1H, NH or OH), 4.18 (q, J = 7.0 Hz,
2H, COOCH2CH3), 4.09 (t, J = 5.6 Hz, 2H, ArOCH2), 3.30 ꢁ3.40
(m, 2H, CH2NHBoc), 1.9–2.1 (m, 14 H, 3 ꢃ AdCH + ArCH3 +
3 ꢃ AdCH2 + OCH2CH2CH2N), 1.76 (br, 6H, 2 ꢃ AdCH2), 1.46 (s, 9H,
C(CH3)3), 1.27 (t, J = 7.0 Hz, 3H, COOCH2CH3) ppm. 13C NMR
(100.62 MHz, CDCl3): d 166.9 (s), 157.8 (s), 156.0 (s), 153.8 (s),
143.5 (d), 136.0 (s), 134.8 (s), 134.3 (s), 133.5 (s), 132.1 (d), 131.3
(s), 129.4 (d), 118.8 (d), 118.3 (d), 116.4 (d), 111.1 (d), 79.4 (s,
C(CH3)3), 65.9 (t, OCH2), 60.3 (q, COOCH2CH3), 40.7 (t, 3 ꢃ AdCH2),
38.0 (t, CH2N), 37.1 (t, 3 ꢃ AdCH2), 36.4 (s, AdC), 29.6 (t, CH2CH2-
CH2N), 29.0 (d, 3 ꢃ AdCH or 3 ꢃ C(CH3)3), 28.4 (q or d, 3 ꢃ C(CH3)3
or 3 ꢃ AdCH), 19.4 (q, ArCH3), 14.3 (q, COOCH2CH3) ppm. IR (NaCl):
0.22 g (91%) of
a
white solid identified as 17b. 1H NMR
(400.13 MHz, CDCl3): d 7.43 (d, J = 16.0 Hz, 1H, H3), 7.17 (d,
J = 2.7 Hz, 1H, H30), 7.16 (d, J = 8.5 Hz, 1H, H60), 6.94 (dd, J = 8.5,
2.7 Hz, 1H, H50), 6.87 (s, 1H, ArH), 6.66 (s,1H, ArH), 6.28 (d,
J = 16.0 Hz, 1H, H2), 4.76 (br, 1H, NH), 4.16 (q, J = 7.2 Hz, 2H,
COOCH2CH3), 4.07 (t, J = 6.0 Hz, 2H, ArOCH2), 3.34 (t, J = 6.0 Hz,
2H, CH2NHtBoc), 1.9–2.1 (m, 14H, 3 ꢃ AdCH2 + OCH2CH2CH2-
N + 3 ꢃ AdCH + ArCH3), 1.73 (s, 6H, 3 ꢃ AdCH2), 1.44 (s, 9H, COtBu),
1.25 (t, J = 7.2 Hz, 3H, COOCH2CH3), 1.06 (s, 9H, SiC(CH3)3), 0.37 (s,
3H, SiCH3), 0.36 (s, 3H, SiCH3) ppm. 13C NMR (100.62 MHz, CDCl3):
d 166.9 (s), 157.7 (s), 156.0 (s), 153.9 (s), 143.6 (d), 136.6 (s), 136.2
(s), 134.2 (s), 134.1 (s), 132.1 (d), 130.9 (s), 129.6 (d), 120.5 (d),
118.7 (d), 116.4 (d), 111.1 (d), 79.2 (s, OC(CH3)3), 65.9 (t, OCH2),
60.2 (q, COOCH2CH3), 40.5 (t, 3 ꢃ AdCH2), 38.0 (t, CH2N), 37.0 (t,
3 ꢃ AdCH2), 36.5 (s, AdC), 29.6 (t, CH2CH2CH2N), 29.0 (q or d,
3 ꢃ AdCH or SiC(CH3)3 or 3ꢃ C(CH3)3), 28.4 (q or d, 3 ꢃ C(CH3)3
or 3 ꢃ AdCH or SiC(CH3)3), 26.4 (q or d, SiC(CH3)3 or 3 ꢃ C(CH3)3
or 3 ꢃ AdCH), 19.7 (q, ArCH3), 18.9 (s, SiC(CH3)3), 14.2 (q, COOCH2-
m
3315 (m, N–H), 3400-3100 (br, O–H), 2976 (m, C–H), 2903 (m, C–
H), 2849 (m, C–H), 1700 (s, C@O), 1683 (s, C@O), 1537 (m), 1395
(m), 1366 (m), 1315 (m), 1272 (m), 1227 (s), 1170 (s), 1040 (m),
858 (m), 727 (s) cmꢁ1. MS (EI): m/z (%) 589 (M+, 86), 534 (10),
533 (30), 516 (32), 515 (84), 490 (13), 489 (39), 444 (13), 433
(25), 432 (79), 427 (10), 344 (21), 287 (13), 136 (12), 135 (100),
102 (86), 93 (12). HRMS (EI): calcd for C36H47NO6, 589.3403; found,
589.3401.
CH3), ꢁ3.4 (q, SiCH3), ꢁ3.3 (q, SiCH3) ppm. IR (NaCl):
m 3377 (m, N–
H), 2959 (m, C–H), 2903 (m, C–H), 2853 (s, C–H), 1702 (s, C@O),
1505 (m), 1486 (m), 1454 (m), 1256 (s), 1230 (s), 1168 (s), 1019
(s), 872 (s), 837 (s), 782 (s), 731 (s) cmꢁ1. MS (FAB+): m/z (%) 705
([M+1]+, 19), 704 (M+, 100), 703 ([M-1]+, 54), 648 (11), 647 (11),
646 (11), 605 (10), 604 (22), 604 (14), 559 (15), 558 (32). HRMS
(FAB+): calcd for C42H61NO6Si, 703.4223; found, 703.4243.
5.7.5. Ethyl (E)-3-[4-acetoxy-(2-(3-acetamidopropoxy)-5-
5.7.3. Ethyl (E)-3-(2-(3-acetamidopropoxy)-4-acetoxy-3-
(adamant-1-yl)-2-methylbiphenyl-4-yl]acrylate (19b)
(adamant-1-yl)-biphenyl-4-yl)acrylate (19a)
To a mixture of 18b (0.16 g, 0.28 mmol) in EtOH (4.6 mL) was
added concentrated HCl (0.46 mL). This mixture was heated at
85 °C for 1 h and then concentrated. The residue containing the pri-
mary amine was sequentially treated with CH2Cl2 (9.2 mL), pyri-
dine (0.49 mL, 6.07 mmol) and Ac2O (0.07 mL, 0.72 mmol) and
stirred overnight at room temperature before addition of H2O
and extraction with EtOAc (3ꢃ), followed by washing with H2O,
brine and dried (Na2SO4). The solvent was removed in vacuo and
the residue was crystallized from CH2Cl2/MeOH/hexane afford
the corresponding acid 19b (0.06 g, 37%) as white solid (mp
215 °C, CH2Cl2/MeOH/hexane). 1H NMR (400.13 MHz, CD3OD): d
7.41 (d, J = 16.0 Hz, 1H, H3), 7.27 (d, J = 2.4 Hz, 1H, H30), 7.11 (d,
J = 8.6 Hz, 1H, H60), 7.00 (dd, J = 8.6, 2.4 Hz, 1H, H50), 6.75 (s, 1H,
H60), 6.62 (s, 1H, H30), 6.34 (d, J = 16.0 Hz, 1H, H2), 4.14 (q,
J = 7.1 Hz, 2H, COOCH2CH3), 4.09 (t, J = 5.5 Hz, 2H, ArOCH2), 3.38
(t, J = 6.7 Hz, 2H, CH2NHAc), 2.12–2.14 (m, 9H, 3 ꢃ AdCH2 + Ac),
1.9–2.0 (m, 11H, 3 ꢃ AdCH + ArCH3 + Ac + OCH2CH2CH2N), 1.77
(br, 6H, 3 ꢃ AdCH2), 1.24 (t, J = 7.1 Hz, 3H, COOCH2CH3) ppm. 13C
NMR (100.62 MHz, CD3OD): d 173.5 (s), 168.8 (s), 160.0 (s), 145.3
(d), 137.9 (s), 135.5 (s), 135.3 (s), 134.1 (s), 133.3 (d), 131.2 (s),
130.0 (d), 119.3 (d), 118.8 (d), 117.9 (d), 112.2 (d), 66.8 (t, ArOCH2),
61.6 (t, COOCH2CH3), 41.8 (t, 3 ꢃ AdCH2), 38.3 (t, CH2NHAc),
37.6 (t, 3 ꢃ AdCH2), 37.7 (s, AdC), 30.7 (q, 2x Ac +NAc), 30.3
(t, OCH2CH2CH2NHAc), 22.6 (d, 3 ꢃ AdCH), 19.8 (q, ArCH3), 14.7
To a mixture of 17a (0.11 g, 0.19 mmol) in EtOH (3.2 mL) was
added concentrated HCl (0.32 mL). This mixture was heated at
85 °C for 8 h and then concentrated. The residue containing the pri-
mary amine 18a was sequentially treated with CH2Cl2 (4.6 mL),
pyridine (0.24 mL, 3.01 mmol), and Ac2O (0.08 mL, 0.82 mmol)
and stirred overnight at room temperature before addiction of
H2O and extraction with EtOAc (3ꢃ). The organic layer was washed
with H2O, brine, dried (Na2SO4), the solvent was removed in vacuo
and the residue was purified by column chromatography on silica
gel (98:2 CH2Cl2/MeOH) to afford 19a (0.04 g, 58%) as a white solid
(mp 215 °C, CH2Cl2/MeOH/hexane). 1H NMR (400.13 MHz, CD3OD):
d 7.81 (d, J = 15.5 Hz, 1H, H3), 7.43 (d, J = 8.4 Hz, 1H, ArH), 7.38 (s,
1H, ArH), 7.32 (s, 1H, ArH), 7.25 (d, J = 8.4 Hz, 1H, ArH), 7.15 (d,
J = 8.3 Hz, 1H, ArH), 7.08 (d, J = 8.3 Hz, 1H, ArH), 6.46 (d,
J = 15.5 Hz, 1H, H2), 6.18 (br, 1H, NH), 4.32 (q, J = 7.1 Hz, 2H,
COOCH2CH3), 4.20 (t, J = 5.8 Hz, 2H, ArOCH2), 3.58 (q, J = 5.8 Hz,
2H, CH2NHAc), 2.49 (s, 3H, Ac), 2.13 (br, 6H, 3 ꢃ AdCH2), 2.11 (s,
6H, 3 ꢃ AdCH or COCH3), 1.57 (m, 8H, 3 ꢃ AdCH2 + OCH2CH2CH2N),
1.40 (t, J = 7.1 Hz, 3H, COOCH2CH3) ppm. 13C NMR (100.62 MHz,
CDCl3): d 170.4 (s), 169.5 (s), 166.6 (s), 158.0 (s), 148.5 (s), 143.8
(d), 140.5 (s), 136.7 (s), 135.5 (s), 133.7 (s), 131.6 (d), 129.4 (d),
127.7 (d), 124.0 (d), 119.5 (d), 116.5 (d), 111.9 (d), 66.2 (t, OCH2),
60.4 (t, OCH2), 41.1 (t, 3 ꢃ AdCH2), 37.2 (t, CH2N), 36.8 (t + s,

1294
J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
(q, COOCH2CH3) ppm. IR (NaCl):
m
3392 (m, N–H), 2975 (m, C–H),
506 ([M+H]+ 81Br], 25), 504 ([M+H]+ 79Br], 26), 375 (34), 349
[ [
2900 (s, C–H), 1746 (m, C@O), 1701 (s, C@O), 1650 (s, C@O),
1556 (s), 1540 (s), 1521 (s), 1493 (s), 1399 (s), 1234 (s), 1178 (s),
730 (s) cmꢁ1. MS (EI): m/z (%) 531 (18), 100 (100). HRMS (EI): calcd
for C35H43NO6, 573.3090; found, 573.3106.
(30), 322 (25), 321 (100), 313 (19), 253 (35), 237 (28), 195 (63).
HRMS (ESI+): calcd for C25H3581BrNOSSi [M+H]+ 506.1368 and C25-
H3579BrNOSSi [M+H]+ 504.1386; found: 506.1392 and 504.1397.
Elem. Anal. calcd for C25H34BrNOSSi: C, 59.91; H, 6.79; N, 2.78;
found: C, 58.87; H, 6.28; N, 2.78. Data for thiazole 21a. 1H NMR
(400.13 MHz, CDCl3): d 7.86 (s, 1H, ArH), 7.84 (s, 1H, ArH), 7.65
(d, J = 8.1 Hz, 1H, ArH), 7.43 (s, 1H, H4), 7.31 (d, J = 8.1 Hz, 1H,
ArH), 6.84 (app. t, J = 7.1, 2H, H50 + H500), 2.18 (s, 6H, 3 ꢃ AdCH2),
2.16 (s, 6H, 3 ꢃ AdCH2), 2.11 (s, 6H, 6xAdCH), 1.80 (s, 12H,
6xAdCH2), 1.07 (s, 18H, 2xSiC(CH3)3), 0.38 (s, 12H, 4 ꢃ Si(CH3)2)
ppm. 13C NMR (100.62 MHz, CDCl3): d 166.8 (s), 156.5 (s), 154.9
(s), 140.2 (s), 140.1 (s), 138.7 (s), 137.6 (d), 126.4 (s), 126.0 (d),
125.6 (d), 124.6 (d), 124.5 (d), 123.9 (s), 119.4 (d), 119.3 (d),
40.3 (t, 3ꢃ), 40.2 (t, 3ꢃ), 37.0 (t, 3ꢃ), 36.9 (s, 2ꢃ), 29.0 (d, 6ꢃ),
5.7.6. (E)-3-[2-(3-Acetamidopropoxy)-3-(adamant-1-yl)-4-
hydroxybiphenyl-4-yl]acrylic acid (2c)11
To a stirred suspension of compound 19a (0.013 g, 0.023 mmol)
in MeOH was treated with Na2CO3 (0.12 mL, 2 M in H2O,
0.24 mmol). The mixture was stirred at 70 °C for 3 h, cooled down
to room temperature, acidified (10% HCl), and extracted with
EtOAc. The combined organic extracts were washed (H2O, brine),
dried (Na2SO4), and evaporated. The residue was purified by crys-
tallization (CH2Cl2/hexane) to afford 0.006 g (53%) of a white solid
(mp: 215 °C) identified as 2c.11
26.4 (q, 6ꢃ), 19.0 (s, 2ꢃ), ꢁ3.3 (q, 2ꢃ), ꢁ3.4 (q, 2ꢃ) ppm. IR:
m
2902 (m, C–H), 2850 (w, C–H), 1601 (w), 1486 (m), 1447 (w),
1391 (w), 1264 (s), 1122 (w), 920 (s), 866 (s), 837 (s), 815 (s),
788 (s), 742 (m) cmꢁ1. MS (ESI+): m/z (%) 766 ([M+H]+, 30), 566
(12), 484 (100), 433 (26), 396 (26), 391 (34), 379 (27), 365 (29),
363 (18), 359 (23), 358 (16), 343 (89), 341 (29), 317 (18), 247
(14). HRMS (ESI+): calcd for C47H68NO2SSi2 [M+H]+ 766.4504;
found: 766.4500.
5.7.7. (E)-3-[2-(3-Acetamidopropoxy)-5-(adamant-1-yl)-4-
hydroxy-2-methylbiphenyl-4-yl]acrylic acid (5c)
To a stirred suspension of compound 19b (0.03 g, 0.05 mmol) in
MeOH was treated with Na2CO3 (0.52 mL, 2 M in H2O, 1.05 mmol).
The mixture was stirred at 70 °C for 3 h, cooled down to room tem-
perature, acidified (10% HCl), and extracted with EtOAc. The com-
bined organic extracts were washed (H2O, brine), dried (Na2SO4),
and evaporated. The residue was purified by column chromatogra-
phy on silica gel (90:10 CH2Cl2/MeOH), to afford 0.015 g (57%) of a
white solid identified as 5c. 1H NMR (400.13 MHz, CD3OD): d 7.36
(d, J = 16.0 Hz, 1H, H3), 7.26 (d, J = 2.5 Hz, 1H, H3), 7.06 (d,
J = 8.5 Hz, 1H, H6), 6.97 (dd, J = 8.5, 2.5 Hz, 1H, H5), 6.75 (s, 1H,
H600), 6.60 (s, 1H, H300), 6.34 (d, J = 16.0 Hz, 1H, H2), 4.08 (t,
J = 6.1 Hz, 2H, ArOCH2), 3.38 (t, J = 6.8 Hz, 2H, CH2NHAc), 2.11 (s,
6H, 3 ꢃ AdCH2), 1.8–2.0 (m, 5H, Ac or 3 ꢃ AdCH or ArCH3 + -
ArOCH2CH2), 1.94 (s, 3H, ArCH3 or Ac or 3 ꢃ AdCH), 1.89 (s, 3H,
3 ꢃ AdCH or ArCH3 or Ac), 1.76 (br, 6H, 3 ꢃ AdCH2) ppm. 13C
NMR (100.62 MHz, CD3OD): d 172.0 (s, 2ꢃ), 158.0 (s), 155.4 (s),
143.2 (d), 136.3 (s), 134.2 (s), 134.0 (s), 133.4 (s), 131.8 (d), 129.9
(d), 128.3 (d, 2ꢃ), 117.3 (d), 116.1 (d), 110.7 (d), 65.4 (t, OCH2),
40.3 (t, 3 ꢃ AdCH2), 36.9 (t, 3 ꢃ AdCH2), 36.2 (t, CH2N), 36.1 (s,
AdC), 29.2 (d, 3 ꢃ AdCH), 28.8 (t, OCH2CH2), 21.2 (q, COCH3), 18.3
5.7.9. 5-Bromo-2-[3-adamantan-1-yl-6-methyl-4-(tert-butyldi
methylsilyloxy)phenyl]thiazole (22b)
Following the general procedure for the microwave-assisted Su-
zuki reaction, 2,5-dibromothiazole 20 (0.22 g, 0.90 mmol) and
boronic acid 16b32 (0.40 g, 1.0 mmol) were heated at 90 °C for
10 min. The residue was purified by column chromatography
(SiO2, 99:1 hexane/EtOAc), to give 0.27 g of a yellow oil (a mixture
of mono- and disubstituted thiazole derivatives), which was used
in the next step without further purification.
5.7.10. 2-(Adamantan-1-yl)-4-(5-bromothiazol-2-yl)phenol
(23a)
In accordance with the general procedure for the cleavage of si-
lyl ethers, ether 22a (0.35 g, 0.69 mmol) gave, after purification by
column chromatography (SiO2, from 80:20 hexane/EtOAc to 100%
EtOAc), 0.25 g (91%) of phenol 23a as a white solid, mp 280–
281 °C (hexane/CH2Cl3). 1H NMR (400.13 MHz, DMSO-d6): d 10.02
(s, 1H, OH), 7.85 (s, 1H, H40), 7.63 (d, J = 2.1 Hz, 1H, H3), 7.53 (dd,
J = 2.1, 8.3 Hz, 1H, H5), 6.87 (d, J = 8.3 Hz, 1H, H6), 2.09 (s, 6H,
3 ꢃ AdCH2), 2.05 (s, 3H, 3 ꢃ AdCH), 1.73 (s, 6H, 3 ꢃ AdCH2) ppm.
13C NMR (100.62 MHz, DMSO-d6): d 169.6 (s), 158.7 (s), 144.6
(d), 136.2 (s), 125.0 (d), 124.2 (d), 123.5 (s), 116.9 (d), 106.1 (s),
(q, ArCH3). IR (NaCl):
m 3388 (m, NH), 3100-2950 (br, O–H), 2972
(s, C–H), 2902 (s, C–H), 1699 (s, C@O), 1650 (s, C@O), 1555 (s),
1539 (s), 1521 (s), 1512 (s), 1493 (s), 1455 (m), 1393 (m), 1051
(m) cmꢁ1. MS (EI): m/z (%) 503 ([M+], 13), 135 (8), 101 (9), 100
([M-C5H10ON]+, 100). HRMS (EI): calcd for C31H37NO5, 503.2672;
found, 503.664.
5.7.8. 5-Bromo-2-[3-adamantan-1-yl-4-(tert-butyldimeth
ylsilyloxy)phenyl]thiazole (22a)
39.5 (t, 3ꢃ), 36.4 (t, 3ꢃ), 36.2 (s), 28.2 (d, 3ꢃ) ppm. IR:
m 3500-
3300 (br, O–H), 2898 (m, C–H), 2847 (m, C–H), 1595 (m), 1489
Following the general procedure for the Suzuki reaction, 2,5-
dibromothiazole 20 (0.70 g, 2.88 mmol) and boronic acid 16a11
(1.44 g, 3.74 mmol) were heated in benzene at 90 °C for 12 h. The
residue was purified by column chromatography (SiO2, 99:1 hex-
ane/EtOAc), to give 22a (0.72 g, 50%) as a white powder, mp
150–155 °C (hexane/CHCl3), 0.11 g (5%) of disubstituted thiazole
21a as a yellow solid, mp 237–238 °C (hexane/CHCl3) and 0.05 g
(8%) of starting material. Data for thiazole 22a. 1H NMR
(400.13 MHz, CDCl3): d 7.77 (d, J = 2.4 Hz, 1H, H20), 7.66 (s, 1H,
H4), 7.53 (dd, J = 8.4, 2.4 Hz, 1H, H60), 6.83 (d, J = 8.4 Hz, 1H, H50),
2.14 (s, 6H, 3 ꢃ AdCH2), 2.09 (s, 3H, 3 ꢃ AdCH), 1.78 (s, 6H,
3 ꢃ AdCH2), 1.05 (s, 9H, SiC(CH3)3), 0.37 (s, 6H, Si(CH3)2) ppm.
13C NMR (100.62 MHz, CDCl3): d 170.3 (s), 157.0 (s), 144.4 (d),
140.4 (s), 125.7 (s), 125.6 (d), 124.8 (d), 119.4 (d), 106.9 (s), 40.2
(t, 3ꢃ), 37.0 (s), 36.9 (t, 3ꢃ), 28.9 (q, 3ꢃ), 26.4 (d, 3ꢃ), 19.0 (s),
(m), 1412 (m), 1375 (m), 1271 (m), 1244 (s), 1125 (m), 999 (w),
816 (s) cmꢁ1. MS (ESI+): m/z (%) 392 ([M+H]+ 81Br], 100), 390
[
([M+H]+ 79Br], 94), 349 (31), 321 (89), 279 (29), 253 (58). HRMS
[
79-
(ESI+): calcd for C19H2181BrNOS [M+H]+ 392.0502 and C19H21
BrNOS [M+H]+, 390.0522; found: 392.0484 and 390.0508.
5.7.11. 2-(Adamantan-1-yl)-4-(5-bromothiazol-2-yl)-5-
methylphenol (23b)
In accordance with the general procedure for the cleavage of
silyl ethers, the mixture obtained above gave, after purification
by column chromatography (SiO2, from 80:20 hexane/EtOAc to
100% EtOAc), 0.11 g (30%, two steps) of phenol 23b as a white so-
lid, mp 237–238 °C (hexane/CH2Cl2), and the disubstituted thia-
zole 21b (0.03 g, 5% two steps) as a white solid, mp 237–238 °C.
Data for 23b: 1H NMR (400.13 MHz, DMSO-d6): d 9.86 (s, 1H,
OH), 7.91 (s, 1H, H40), 7.44 (s, 1H, H3), 6.71 (s, 1H, H6), 2.40 (s,
3H, ArCH3), 2.05 (s, 6H, 3 ꢃ AdCH2), 2.02 (s, 3H, 3 ꢃ AdCH), 1.71
(s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, DMSO-d6): d
ꢁ3.4 (q, 2ꢃ) ppm. IR:
m 2905 (m, C–H), 2882 (m, C–H), 2845 (w,
C–H), 1597 (w), 1475 (s), 1422 (w), 1272 (s), 1251 (s), 1132 (m),
921 (s), 837 (s), 818 (s), 788 (s), 727 (m) cmꢁ1. MS (ESI+): m/z (%)

J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
1295
169.3 (s), 157.7 (s), 144.1 (d), 134.5 (s), 133.9 (s), 127.8 (d), 122.7
(s), 119.2 (d), 106.9 (s), 39.8 (t, 3ꢃ), 36.5 (t, 3ꢃ), 35.9 (s), 28.3 (d,
3ꢃ), 20.7 (q) ppm. IR: 3500–3300 (br, O–H), 2900 (s, C–H), 2843
(m, C–H), 1606 (m), 1494 (m), 1444 (m), 1392 (s), 1244 (s), 1120
(s), 999 (m), 845 (s) cmꢁ1. MS (ESI+): m/z (%) 406 ([M+H]+ 81Br],
5.7.14. Methyl (E)-3-[2-(3-adamantan-1-yl-4-hydroxyphenyl)
thiazol-5-yl]acrylate (24a)
m
Following the general procedure for the Heck reaction with
methyl acrylate, bromide 23a (0.09 g, 0.24 mmol) gave, after puri-
fication by column chromatography (SiO2, 80:20 hexane/EtOAc),
0.06 g (68%) of ester 24a as a yellow solid, mp 240–241 °C (hex-
[
92), 404 ([M+H]+ [79Br], 100), 363 (16), 359 (8), 321 (9), 319 (66),
309 (9), 241 (11), 210 (13), 192 (7). HRMS (ESI+): calcd for C20-
H2381BrNOS [M+H]+ 406.0657 and C20H2379BrNOS [M+H]+,
404.06782; found: 406.0655 and 404.0675. Data for thiazole
21b. 1H NMR (400.13 MHz, DMSO-d6): d 9.81 (s, 1H, OH), 9.61
(s, 1H, OH), 7.83 (s, 1H, H4), 7.62 (s, 1H), 7.18 (s, 1H), 6.82 (s,
2H; H50 + H500), 2.60 (s, 9H, 3 ꢃ AdCH2 + ArCH3), 2.57 (s, 3H,
ArCH3), 2.37 (s, 3H, 3 ꢃ AdCH), 2.2–2.1 (m, 6H, 3 ꢃ AdCH2), 2.12
(s, 3H, 3 ꢃ AdCH), 1.81 (s, 12H, 6xAdCH2) ppm. 13C NMR
(100.62 MHz, DMSO-d6): d 166.2 (s), 157.0 (s), 156.1 (s), 140.1
(d), 137.0 (s), 134.2 (s), 133.9 (s), 133.7 (s), 133.6 (s), 128.4 (d),
127.8 (d), 123.3 (s), 120.4 (s), 119.2 (d), 118.6 (d), 39.9 (t, 3ꢃ),
39.8 (t, 3ꢃ), 36.6 (t, 3ꢃ), 35.9 (s), 35.8 (s), 28.3 (d, 6ꢃ), 20.9 (q),
1
ane/CHCl3). H NMR (400.13 MHz, CD3OD): d 7.95 (s, 1H, H40),
7.86 (d, J = 15.6 Hz, 1H, H3), 7.78 (d, J = 2.3 Hz, 1H, H200), 7.63 (dd,
J = 8.4, 2.3 Hz, 1H, H600), 6.81 (d, J = 8.4 Hz, 1H, H500), 6.26 (d,
J = 15.6 Hz, 1H, H2), 3.78 (s, 3H, OCH3), 2.20 (s, 6H, 3 ꢃ AdCH2),
2.08 (s, 3H, 3 ꢃ AdCH), 1.83 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR
(100.62 MHz, CD3OD): d 173.4 (s), 168.6 (s), 161.2 (s), 148.3 (d),
138.6 (s), 135.8 (d), 134.8 (s), 127.1 (d), 127.0 (d), 125.5 (s),
119.8 (d), 118.0 (d), 52.4 (q), 41.5 (t, 3ꢃ), 38.3 (t, 3ꢃ), 38.2 (s),
30.7 (d, 3ꢃ) ppm. IR:
m 2901 (m, C–H), 2877 (m, C–H), 2847 (m,
C–H), 1720 (s, C@O), 1624 (m), 1600 (m), 1433 (m), 1359 (s),
1331 (m), 1269 (s), 1235 (s), 1167 (s), 1151 (s), 1120 (s), 959
(m), 819 (s) cmꢁ1. MS (ESI+): m/z (%) 396 ([M+H]+, 100), 349 (6),
321 (8), 253 (6), 203 (7), 201 (8). HRMS (ESI+): calcd for C23H26NO3-
S [M+H]+ 396.1628; found: 396.1623.
20.2 (q) ppm. IR:
m 3500–3000 (br, O–H), 2900 (s, C–H), 2848
(m, C–H), 1602 (w), 1562 (w), 1494 (w), 1449 (w), 1393 (s),
1239 (s), 1120 (s) cmꢁ1. MS (ESI+): m/z (%) 566 ([M+H]+, 100),
363 (8), 186 (13). HRMS (ESI+): calcd for C37H44NO2S [M+H]+
566.3087; found: 566.3083.
5.7.15. Methyl (E)-3-[2-(5-adamantan-1-yl-4-hydroxy-2-
methylphenyl)thiazol-5-yl]acrylate (24b)
Following the general procedure for the Heck reaction with
methyl acrylate, bromide 23b (0.10 g, 0.25 mmol) gave, after puri-
fication by column chromatography (SiO2, 80:20 hexane/EtOAc),
0.06 g (56%) of ester 24b as a yellow solid, mp 200–201 °C (hex-
5.7.12. 5-Bromo-2-[3-adamantan-1-yl-4-[(2-methoxyethoxy)
methoxy]phenyl]thiazole (23c)
According to the general procedure for the protection of phe-
nols with MEMCl, 23a (0.09 g, 0.23 mmol) gave, after purification
by column chromatography (SiO2, 85:15 hexane/EtOAc), com-
pound 23c (0.10 g, 88%) as a white solid, mp 125–126 °C (hex-
ane/CHCl3). 1H NMR (400.13 MHz, CDCl3): d 7.78 (d, J = 2.3 Hz,
1H, H20), 7.67 (s, 1H, H4), 7.61 (dd, J = 8.6, 2.3 Hz, 1H, H60), 7.20
(d, J = 8.6 Hz, 1H, H50), 5.37 (s, 2H, OCH2O), 3.9–3.8 (m, 2H,
OCH2), 3.6–3.5 (m, 2H, OCH2), 3.40 (s, 3H, OCH3), 2.13 (s, 6H,
3 ꢃ AdCH2), 2.09 (s, 3H, 3 ꢃ AdCH), 1.78 (s, 6H, 3 ꢃ AdCH2).ppm.
13C NMR (100.62 MHz, CDCl3): d 170.1 (s), 158.3 (s), 144.5 (d),
139.3 (s), 126.6 (s), 125.2 (d), 125.0 (d), 114.8 (d), 107.2 (s), 93.2
(t), 71.5 (t), 68.0 (t), 59.1 (q), 40.5 (t, 3ꢃ), 37.2 (s), 37.0 (t, 3ꢃ),
1
ane/CHCl3). H NMR (400.13 MHz, CDCl3): d 7.93 (s, 1H, H40),
7.82 (d, J = 15.6 Hz, 1H, H3), 7.63 (s, 1H, H600), 6.52 (s, 1H, H300),
6.38 (s, 1H, OH), 6.22 (d, J = 15.6 Hz, 1H, H2), 3.82 (s, 3H, OCH3),
2.44 (s, 3H, ArCH3), 2.11 (s, 6H, 3 ꢃ AdCH2), 2.07 (s, 3H, 3 ꢃ AdCH),
1.76 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3): d
170.9 (s), 167.0 (s), 156.6 (s), 146.1 (d), 135.7 (s), 134.7 (s), 134.5
(d), 134.1 (s), 129.5 (d), 124.4 (s), 119.8 (d), 118.9 (d), 51.9 (q),
40.4 (t, 3ꢃ), 36.9 (t, 3ꢃ), 36.4 (s), 28.9 (d, 3ꢃ), 20.9 (q) ppm. IR:
m
3500–3200 (br, O–H), 2902 (m, C–H), 2847 (m, C–H), 1693 (s,
C@O), 1622 (s), 1606 (m), 1453 (m), 1435 (m), 1383 (s), 1331 (s),
1175 (s), 1126 (m), 953 (s), 842 (s) cmꢁ1. MS (ESI+): m/z (%) 410
([M+H]+, 100), 210 (3), 143 (7). HRMS (ESI+): calcd for C24H28NO3S
[M+H]+ 410.1784; found: 410.1779.
28.9 (d, 3ꢃ) ppm. IR:
m 2898 (m, C–H), 2849 (w, C–H), 1597 (w),
1472 (m), 1223 (m), 1096 (s), 980 (s), 846 (m), 814 (m) cmꢁ1. MS
(ESI+): m/z (%) 480 ([M+H]+
[
81Br], 100), 478 ([M+H]+
[
79Br], 99),
5.7.16. Methyl (E)-3-[2-(3-adamantan-1-yl-4-[(2-methoxy
ethoxy)methoxy]phenyl)thiazol-5-yl]acrylate (24c)
402 (7), 401 (10), 400 (41), 324 (4). HRMS (ESI+): calcd for C23H29
BrNO3S [M+H]+ 480.1025, and C23H2979BrNO3S 478.1046; found:
480.1020 and 478.1039.
81-
According to the general procedure for the protection of the
phenols with MEMCl, phenol 24a (0.05 g, 0.13 mmol) gave, after
purification by column chromatography (SiO2, 80:20 hexane/
EtOAc), compound 24c (0.05 g, 70%) as a yellow oil. 1H NMR
(400.13 MHz, CDCl3): d 7.88 (s, 2H, H40 + H200), 7.79 (d, J = 15.6 Hz,
1H, H3), 7.72 (dd, J = 8.6, 2.2 Hz, 1H, H600), 7.21 (d, J = 8.6 Hz, 1H,
H500), 6.19 (d, J = 15.6 Hz, 1H, H2), 5.38 (s, 2H, OCH2O), 3.9–3.8 (m,
2H, OCH2), 3.80 (s, 3H, OCH3), 3.6–3.5 (m, 2H, OCH2), 3.40 (s, 3H,
OCH3), 2.15 (s, 6H, 3 ꢃ AdCH2), 2.10 (s, 3H, 3 ꢃ AdCH), 1.79 (s, 6H,
3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3): d 170.6 (s), 166.8
(s), 158.8 (s), 147.0 (d), 139.3 (s), 134.4 (d), 133.7 (s), 126.5 (s),
125.9 (d), 125.7 (d), 118.9 (d), 114.9 (d), 93.2 (t), 71.5 (t), 68.1 (t),
59.1 (q), 51.8 (q), 40.5 (t, 3ꢃ), 37.2 (s), 37.0 (t, 3ꢃ), 29.0 (d, 3ꢃ)
5.7.13. 5-Bromo-2-[5-adamantan-1-yl-4-[(2-methoxy
ethoxy)methoxy]-2-methylphenyl]thiazole (23d)
According to the general procedure for the protection of phe-
nols with MEMCl, 23b (0.04 g, 0.10 mmol) gave, after purification
by column chromatography (SiO2, 85:15 hexane/EtOAc), com-
pound 23d (0.03 g, 66%) as a colorless oil. 1H NMR (400.13 MHz,
CDCl3): d 7.73 (s, 1H, H60), 7.55 (s, 1H, H4), 7.05 (s, 1H, H30),
5.36 (s, 2H, OCH2O), 3.9–3.8 (m, 2H, OCH2), 3.6–3.5 (m, 2H,
OCH2), 3.41 (s, 3H, OCH3), 2.51 (s, 3H, ArCH3), 2.10 (s, 6H,
3 ꢃ AdCH2), 2.07 (s, 3H, 3 ꢃ AdCH), 1.77 (s, 6H, 3 ꢃ AdCH2) ppm.
13C NMR (100.62 MHz, CDCl3): d 169.7 (s), 157.3 (s), 143.8 (d),
136.5 (s), 135.3 (s), 128.5 (d), 125.7 (s), 117.2 (d), 108.0 (s), 93.1
(t), 71.5 (t), 67.9 (t), 59.0 (q), 40.6 (t, 3ꢃ), 37.0 (t, 3ꢃ), 36.8 (s),
ppm. IR:
m 2903 (s, C–H), 2849 (m, C–H), 1716 (s, C@O), 1623 (s),
1493 (w), 1424 (m), 1327 (m), 1224 (s), 1164 (s), 1102 (m), 982 (s)
cmꢁ1. MS (ESI+): m/z (%) 484 ([M+H]+, 100), 321 (5). HRMS (ESI+):
calcd for C27H34NO5S [M+H]+ 484.2152; found: 484.2136.
28.9 (d, 3ꢃ), 21.0 (q) ppm. IR:
m 2900 (s, C–H), 2847 (m, C–H),
1604 (w), 1490 (m), 1450 (m), 1360 (m), 1242 (m), 1220 (m),
1097 (s), 1015 (s), 990 (s), 923 (m), 847 (m) cmꢁ1. MS (ESI+): m/
5.7.17. Methyl (E)-3-[2-(5-adamantan-1-yl-4-[(2-methoxy
ethoxy)methoxy]-2-methylphenyl)thiazol-5-yl]acrylate (24d)
According to the general procedure for the protection of the
phenols with MEMCl, phenol 24b (0.03 g, 0.08 mmol) gave,
after purification by column chromatography (SiO2, 80:20
z (%) 494 ([M+H]+ 81Br], 100), 492 ([M+H]+ 79Br], 94), 418 (5),
[ [
416 (5). HRMS (ESI+): calcd for C24H3181BrNO3S [M+H]+
494.1182, and C24H3179BrNO3S [M+H]+ 492.1189; found:
494.1178 and 492.1199.

1296
J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
hexane/EtOAc), compound 24d (0.03 g, 78%) as a yellow oil. 1H
NMR (400.13 MHz, CDCl3) d 7.93 (s, 1H, H40), 7.81 (d, J = 15.6 Hz,
1H, H3), 7.69 (s, 1H, H600), 7.06 (s, 1H, H300), 6.21 (d, J = 15.6 Hz,
1H, H2), 5.37 (s, 2H, OCH2O), 3.9–3.8 (m, 2H, OCH2), 3.80 (s, 3H,
OCH3), 3.6–3.5 (m, 2H, OCH2), 3.41 (s, 3H, OCH3), 2.58 (s, 3H,
ArCH3), 2.11 (s, 6H, 3 ꢃ AdCH2), 2.08 (s, 3H, 3 ꢃ Ad), 1.77 (s, 6H,
3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3): d 170.3 (s),
166.8 (s), 157.6 (s), 146.3 (d), 136.6 (s), 135.9 (s), 134.4 (d), 134.2
(s), 128.9 (d), 125.6 (s), 118.9 (d), 117.3 (d), 93.0 (t), 71.5 (t), 68.0
(t), 59.0 (q), 51.8 (q), 40.6 (t, 3ꢃ), 37.0 (t, 3ꢃ), 36.8 (s), 28.9 (d,
3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3): d 171.3 (s),
170.3 (s), 158.9 (s), 147.7 (d), 139.3 (s), 136.4 (d), 133.4 (s), 126.3
(s), 126.0 (d), 125.8 (d), 118.0 (d), 114.9 (d), 93.1 (t), 71.5 (t),
68.1 (t), 59.1 (q), 40.5 (t, 3ꢃ), 37.2 (s), 37.0 (t, 3ꢃ), 28.9 (d, 3ꢃ)
ppm. IR:
m 2901 (s, C–H), 2848 (m, C–H), 1699 (s, C@O), 1616
(m), 1497 (m), 1271 (m), 1222 (s), 1147 (s), 1101 (s), 987 (s), 855
(m) cmꢁ1. MS (ESI+): m/z (%) 470 ([M+H]+, 100). HRMS (ESI+): calcd
for C26H32NO5S [M+H]+ 470.1996; found: 470.1991. Elem. Anal.
Calcd for C26H31NO5S ½H2O C, 65.25; H, 6.74; N, 2.93; found: C,
65.55; H, 6.29; N, 2.69.
3ꢃ), 21.4 (q) ppm. IR:
m 2902 (s, C–H), 2848 (m, C–H), 1718 (s,
C@O), 1624 (s), 1503 (m), 1447 (m), 1432 (m), 1327 (m), 1217
5.7.21. (E)-3-[2-(5-Adamantan-1-yl-4-[(2-methoxyethoxy)
methoxy]-2-methylphenyl)thiazol-5-yl]acrylic acid (7d)
In accordance with the general procedure for the hydrolysis of
esters, ester 24d (0.03 g, 0.06 mmol) gave, after crystallization,
0.02 g (92%) of acid 7d as a yellow solid, mp 169–170 °C (hex-
(s), 1160 (s), 1122 (m), 1098 (m), 1016 (s), 989 (s), 847 (m), 752
(s) cmꢁ1
.
5.7.18. (E)-3-[2-(3-Adamantan-1yl-4-hydroxyphenyl)thiazol-5-
yl]acrylic acid (7a)
1
ane/CH2Cl2). H NMR (400.13 MHz, CDCl3): d 7.99 (s, 1H, H40),
In accordance with the general procedure for the hydrolysis of
esters, ester 24a (0.01 g, 0.04 mmol) gave, after crystallization,
0.01 g (95%) of acid 7a as a yellow solid, mp 284 °C (dec) (hex-
ane/CH2Cl2). 1H NMR (400.13 MHz, DMSO-d6): d 10.16 (s, 1H,
OH), 8.11 (s, 1H, H40), 7.78 (d, J = 15.6 Hz, 1H, H3), 7.72 (s, 1H,
H200), 7.63 (d, J = 7.6 Hz, 1H, H600), 6.90 (d, J = 7.6 Hz, 1H, H500),
6.16 (d, J = 15.6 Hz, 1H, H2), 3.4–3.2 (br, OH) 2.10 (s, 6H,
3 ꢃ AdCH2), 2.06 (s, 3H, 3 ꢃ AdCH), 1.74 (s, 6H, 3 ꢃ AdCH2) ppm.
13C NMR (100.62 MHz, DMSO-d6): d 169.8 (s), 167.0 (s), 159.2 (s),
147.5 (d), 136.3 (s), 133.8 (d), 133.0 (s), 125.6 (d), 125.0 (d),
123.7 (s), 120.1 (d), 117.0 (d), 39.6 (t, 3ꢃ), 36.5 (t, 3ꢃ), 36.3 (s),
7.90 (d, J = 15.6 Hz, 1H, H3), 7.70 (s, 1H, H600), 7.07 (s, 1H, H300),
6.22 (d, J = 15.6 Hz, 1H, H2), 5.38 (s, 2H, OCH2O), 3.9–3.8 (m, 2H,
OCH2), 3.6–3.5 (m, 2H, OCH2), 3.42 (s, 3H, OCH3), 2.57 (s, 3H,
ArCH3), 2.12 (s, 6H, 3 ꢃ AdCH2), 2.08 (s, 3H, 3 ꢃ AdCH), 1.78 (s,
6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3): d 171.1 (s),
171.0 (s), 157.7 (s), 146.8 (d), 136.7 (s), 136.3 (d), 136.0 (s), 134.0
(s), 129.0 (d), 125.4 (s), 118.5 (d), 117.4 (d), 93.1 (t), 71.5 (t), 68.0
(t), 59.0 (q), 40.6 (t, 3ꢃ), 37.0 (t, 3ꢃ), 36.8 (s), 29.0 (d, 3ꢃ), 21.5
(q) ppm. IR:
m 2900 (s, C–H), 2850 (m, C–H), 1689 (s, C@O), 1618
(s), 1323 (m), 1268 (m), 1220 (s), 1100 (s), 1017 (s), 993 (s), 963
(m), 847 (m) cmꢁ1. MS (ESI+): m/z (%) 484 ([M+H]+, 100), 408 (4).
HRMS (ESI+): calcd for C27H34NO5S [M+H]+ 484.2152; found:
484.2143. Elem. Anal. calcd for C27H33NO5S: C, 67.05; H, 6.88; N,
2.90; found: C, 66.91; H, 6.48; N, 2.84.
28.3 (d, 3ꢃ) ppm. IR:
m 3500–3000 (s, O–H), 2900 (m, C–H), 2848
(w, C–H), 1682 (m, C@O), 1619 (s), 1599 (s), 1385 (s), 1322 (m),
1251 (s), 1179 (s), 1149 (s), 1120 (s), 963 (s), 823 (s) cmꢁ1. MS
(ESI+): m/z (%) 382 ([M+H]+, 100), 375 (20), 321 (27), 253 (53).
HRMS (ESI+): calcd for C22H24NO3S [M+H]+ 382.1471; found:
382.1464. Elem. Anal. Calcd for C22H23NO3S ½[CH3)2SO] C, 65.69;
H, 6.32; N, 3.33; found: C, 65.73; H, 5.78; N, 2.94.
5.7.22. Methyl 4-[2-(3-adamantan-1-yl-4-hydroxyphenyl)
thiazol-5-yl]benzoate (25a)
Following the general procedure for the microwave-assisted Su-
zuki reaction, bromothiazole 23a (0.02 g, 0.05 mmol) and 4-
(methoxycarbonyl)phenylboronic acid (0.01 g, 0.08 mmol) were
heated at 120 °C for 10 min. The residue was purified by column
chromatography (SiO2, 70:30 hexane/EtOAc), to give 25a (0.02 g,
78%) as a yellow solid, mp >300 °C (hexane/CHCl3). 1H NMR
(400.13 MHz, DMSO-d6): d 10.06 (s, 1H, OH), 8.36 (s, 1H, H40),
8.00 (s, 2H, H2 + H6), 7.84 (s, 2H, H3 + H5), 7.73 (s, 1H, H200),
7.64 (d, J = 7.1 Hz, 1H, H600), 6.90 (d, J = 7.1, 1H, H500), 3.87 (s, 3H,
OCH3), 2.12 (s, 6H, 3 ꢃ AdCH2), 2.06 (s, 3H, 3 ꢃ AdCH), 1.74 (s,
6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, DMSO-d6): d 168.4
(s), 165.7 (s), 158.8 (s), 141.2 (d), 136.3 (s), 135.6 (s), 135.5 (s),
130.1 (d, 2ꢃ), 128.6 (s), 126.1 (d, 2ꢃ), 125.2 (d), 124.7 (d), 123.9
(s), 117.0 (d), 52.2 (q), 36.5 (t, 3ꢃ), 36.3 (t, 3ꢃ), 28.3 (d, 3ꢃ) ppm.
5.7.19. (E)-3-[2-(5-Adamantan-1-yl-4-hydroxy-2-methylphenyl)
thiazol-5-yl]acrylic acid (7b)
In accordance with the general procedure for the hydrolysis of
esters, ester 24b (0.03 g, 0.07 mmol) gave, after crystallization,
0.02 g (76%) of acid 7b as a yellow solid, mp 164–165 °C (hex-
1
ane/CH2Cl2). H NMR (400.13 MHz, CD3OD): d 7.96 (s, 1H, H40),
7.82 (d, J = 15.6 Hz, 1H, H3), 7.53 (s, 1H, H600), 6.65 (s, 1H, H300),
6.21 (d, J = 15.6 Hz, 1H, H2), 2.46 (s, 3H, ArCH3), 2.14 (s, 6H,
3 ꢃ AdCH2), 2.04 (s, 3H, 3 ꢃ AdCH), 1.79 (s, 6H, 3 ꢃ AdCH2) ppm.
13C NMR (100.62 MHz, CD3OD): d 172.9 (s), 169.7 (s), 159.8 (s),
147.3 (d), 136.9 (s), 136.0 (s), 135.8 (d), 135.5 (s), 130.2 (d),
124.6 (s), 120.9 (d), 120.4 (d), 41.6 (t, 3ꢃ), 38.3 (t, 3ꢃ), 37.8 (s),
30.7 (d, 3ꢃ), 21.4 (q) ppm. IR:
m
3500–3000 (s, O–H), 2901 (s, C–
IR: m 3600–3000 (br, O–H), 2900 (m, C–H), 2878 (m, C–H), 1701
H), 2848 (w, C–H), 1684 (s, C@O), 1606 (s), 1449 (m), 1382 (s),
1361 (m), 1222 (s), 1159 (s), 1125 (s), 964 (m), 851 (m), 754 (s)
cmꢁ1. MS (ESI+): m/z (%) 396 ([M+H]+, 100), 326 (4), 148 (5), 143
(4). HRMS (ESI+): calcd for C23H26NO3S [M+H]+ 369.1628; found:
396.1624. Elem. Anal. Calcd for C23H25NO3S C, 69.84; H, 6.37; N,
3.54; found: C, 69.52; H, 6.15; N, 3.37.
(s, C@O), 1601 (s), 1392 (s), 1287 (s), 1271 (s), 1183 (s), 1111
(m), 1005 (s), 824 (s), 767 (s) cmꢁ1. MS (ESI+): m/z (%) 446
([M+H]+, 100), 210 (9), 192 (9). HRMS (ESI+): calcd for C27H28NO3S
[M+H]+ 446.1784; found: 446.1782. Elem. Anal. Calcd for C27H27-
NO3S C, 67.05; H, 6.88; N, 2.90; found: C, 66.91; H, 6.48; N, 2.84.
5.7.23. Methyl 4-[2-(5-adamantan-1-yl-4-hydroxy-2-meth
ylphenyl)thiazol-5-yl]benzoate (25b)
5.7.20. (E)-3-[2-(3-Adamantan-1-yl-4-[(2-methoxyethoxy)
methoxy]phenyl)thiazol-5-yl]acrylic acid (7c)
Following the general procedure for the microwave-assisted Su-
zuki reaction, bromothiazole 23b (0.04 g, 0.10 mmol) and 4-
(methoxycarbonyl)phenylboronic acid (0.03 g, 0.15 mmol) were
heated at 120 °C for 10 min. The residue was purified by column
chromatography (SiO2, 70:30 hexane/EtOAc), to give 25b (0.02 g,
53%) as a yellow solid, mp 210–215 °C (hexane/CHCl3). 1H NMR
(400.13 MHz, DMSO-d6): d 9.88 (s, 1H, OH), 8.39 (s, 1H, H40), 7.99
(d, J = 8.4 Hz, 2H, H2 + H6), 7.83 (d, J = 8.4 Hz, 2H, H3 + H5), 7.60
(s, 1H, H600), 6.75 (s, 1H, H300), 3.87 (s, 3H, OCH3), 2.51 (s, 3H,
In accordance with the general procedure for the hydrolysis of
esters, ester 24c (0.05 g, 0.10 mmol) gave, after crystallization,
0.04 g (91%) of acid 7c as a yellow solid, mp 232–233 °C (hexane/
1
CH2Cl2). H NMR (400.13 MHz, CDCl3): d 7.93 (s, 1H, H40), 7.9–7.8
(m, 2H, H3 + H200), 7.74 (d, J = 8.4 Hz, 1H, H600), 7.22 (d, J = 8.4 Hz,
1H, H500), 6.19 (d, J = 15.5 Hz, 1H, H2), 5.39 (s, 2H, OCH2O), 3.9–
3.8 (m, 2H, OCH2), 3.6–3.5 (m, 2H, OCH2), 3.40 (s, 3H, OCH3),
2.15 (s, 6H, 3 ꢃ AdCH2), 2.10 (s, 3H, 3 ꢃ AdCH), 1.79 (s, 6H,

J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
1297
ArCH3), 2.09 (s, 6H, 3 ꢃ AdCH2), 2.04 (s, 3H, 3 ꢃ AdCH), 1.73 (s, 6H,
3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, DMSO-d6): d 168.3 (s),
165.7 (s), 157.7 (s), 140.6 (d), 136.0 (s), 135.6 (s), 134.7 (s), 133.9
(s), 130.0 (d, 2ꢃ), 128.6 (s), 128.1 (d), 126.2 (d, 2ꢃ), 122.9 (s),
119.4 (d), 52.2 (q), 39.9 (t, 3ꢃ), 36.6 (t, 3ꢃ), 35.9 (s), 28.4 (d, 3ꢃ),
7.99 (d, J = 8.2 Hz, 2H, H2 + H6), 7.82 (d, J = 8.2 Hz, 2H, H3 + H5),
7.74 (d, J = 1.4 Hz, 1H, H200), 7.64 (dd, J = 8.3, 1.4 Hz, 1H, H600),
6.90 (d, J = 8.3 Hz, 1H, H500), 2.11 (s, 6H, 3 ꢃ AdCH2), 2.06 (s, 3H,
3 ꢃ AdCH), 1.74 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz,
DMSO-d6): d 168.2 (s), 166.8 (s), 158.8 (s), 141.0 (d), 136.3 (s),
135.7 (s), 135.2 (s), 130.2 (d, 2ꢃ), 129.8 (s), 126.1 (d, 2ꢃ), 125.2
(d), 124.7 (d), 123.9 (s), 117.0 (d), 39.7 (t, 3ꢃ), 36.5 (t, 3ꢃ), 36.3
21.1 (q) ppm. IR:
m 3300–3000 (br, O–H), 2902 (m, C–H), 2881
(m, C–H), 1708 (s, C@O), 1603 (m), 1435 (m), 1402 (m), 1278 (s),
1248 (m), 1183 (m), 1107 (m), 1021 (s), 1001 (s), 765 (s) cmꢁ1
.
(s), 28.3 (d, 3ꢃ) ppm. IR:
m 3582 (w, O–H), 3400–2800 (br, O–H),
MS (ESI+): m/z (%) 460 ([M+H]+, 100), 397 (14), 379 (18), 359
(16), 327 (16), 326 (55), 319 (33). HRMS (ESI+): calcd for C28H30-
NO3S [M+H]+ 460.1941; found: 460.1937.
2898 (m, C–H), 2877 (m, C–H), 2849 (m, C–H), 1681 (s, C@O),
1604 (s), 1425 (m), 1391 (s), 1297 (s), 1250 (m), 1187 (m), 1100
(m), 817 (m), 766 (m) cmꢁ1. MS (ESI+): m/z (%) 432 ([M+H]+,
100). HRMS (ESI+): calcd for C26H26NO3S [M+H]+ 432.1628; found:
432.1624. Elem. Anal. calcd for C26H25NO3Sꢀ1/2H2O C, 70.88; H,
5.95; N, 3.18; found: C, 71.19; H, 5.44; N, 3.29.
5.7.24. Methyl 4-[2-(3-adamantan-1-yl-4-[(2-methoxyethoxy)
methoxy]phenyl)thiazol-5-yl]benzoate (25c)
Following the general procedure for the microwave-assisted Su-
zuki reaction, bromothiazole 23c (0.03 g, 0.06 mmol) and 4-
(methoxycarbonyl)phenylboronic acid (0.02 g, 0.09 mmol) were
heated at 120 °C for 10 min. The residue was purified by column
chromatography (SiO2, 75:25 hexane/EtOAc), to give 25c (0.02 g,
61%) as a yellow solid, mp 157–158 °C (hexane/CHCl3). 1H NMR
(400.13 MHz, CDCl3): d 8.07 (s, 1H, H40), 8.06 (d, J = 8.2 Hz, 2H,
H2 + H6), 7.89 (d, J = 1.8 Hz, 1H, H200), 7.74 (dd, J = 8.5, 1.8 Hz, 1H,
H600), 7.66 (d, J = 8.2 Hz, 2H, H3 + H5), 7.22 (d, J = 8.5 Hz, 1H,
H500), 5.38 (s, 2H, OCH2O), 3.94 (s, 3H, OCH3), 3.9–3.8 (m, 2H,
OCH2), 3.6–3.5 (m, 2H, OCH2), 3.40 (s, 3H, OCH3), 2.16 (s, 6H,
3 ꢃ AdCH2), 2.10 (s, 3H, 3 ꢃ AdCH), 1.79 (s, 6H, 3 ꢃ AdCH2) ppm.
13C NMR (100.62 MHz, CDCl3): d 169.0 (s), 166.5 (s), 158.4 (s),
140.2 (d), 139.2 (s), 136.9 (s), 136.0 (s), 130.4 (d, 2ꢃ), 129.3 (s),
126.8 (s), 126.1 (d, 2ꢃ), 125.4 (d), 125.3 (d), 114.9 (d), 93.2 (t),
71.5 (t), 68.0 (t), 59.1 (q), 52.2 (q), 40.5 (t, 3ꢃ), 37.2 (s), 37.0 (t,
5.7.27. 4-[2-(5-Adamantan-1-yl-4-hydroxy-2-methylphenyl)
thiazol-5-yl]benzoic acid (8b)
According to the general procedure for the hydrolysis of esters,
25b (0.02 g, 0.05 mmol) gave, after crystallization, acid 8b (0.02 g,
77%) as a yellow solid, mp >300 °C (hexane/CHCl3). 1H NMR
(400.13 MHz, DMSO-d6): d 9.87 (s, 1H, OH), 8.39 (s, 1H, H40), 7.99
(d, J = 8.3 Hz, 2H, H2 + H6), 7.83 (d, J = 8.2 Hz, 2H, H3 + H5), 7.59
(s, 1H, H600), 6.75 (s, 1H, H300), 2.50 (s, 3H, ArCH3), 2.09 (s, 6H,
3 ꢃ AdCH2), 2.04 (s, 3H, 3 ꢃ AdCH), 1.73 (s, 6H, 3 ꢃ AdCH2) ppm.
13C NMR (100.62 MHz, DMSO-d6): d 168.1 (s), 166.8 (s), 157.6 (s),
140.4 (d), 136.2 (s), 135.2 (s), 134.7 (s), 133.9 (s), 130.2 (d, 2ꢃ),
129.8 (s), 128.1 (d), 126.1 (d, 2ꢃ), 122.9 (s), 119.4 (d), 39.9 (t,
3ꢃ), 36.6 (t, 3ꢃ), 35.9 (s), 28.3 (d, 3ꢃ), 21.1 (q) ppm. IR:
m 3400–
3000 (br, O–H), 2902 (m, C–H), 2874 (m, C–H), 2849 (m, C–H),
1697 (s, C@O), 1605 (s), 1388 (s), 1259 (s), 1234 (s), 1180 (m),
1124 (m), 1020 (m), 976 (s), 850 (s), 773 (s) cmꢁ1. MS (ESI+): m/z
(%) 446 ([M+H]+, 100), 373 (11), 331 (29), 327 (19), 326 (91). HRMS
(ESI+): calcd for C27H28NO3S [M+H]+ 446.1784; found: 446.1785.
Calcd for C27H27NO3Sꢀ1/5H2O C, 64.91; H, 6.66; N, 2.80; found: C,
65.35; H, 6.29; N, 2.69.
3ꢃ), 29.0 (d, 3ꢃ) ppm. IR:
m 2874 (m, C–H), 2846 (w, C–H), 1717
(s, C@O), 1603 (m), 1434 (m), 1385 (m), 1275 (s), 1220 (m), 1184
(m), 1106 (s), 1071 (m), 979 (s), 836 (s) cmꢁ1. MS (ESI+): m/z (%)
534 ([M+H]+, 100). HRMS (ESI+): calcd for C31H36NO5S [M+H]+
534.2309; found: 534.2296.
5.7.25. Methyl 4-[2-(5-adamantan-1-yl-4-[(2-methoxyethoxy)
methoxy]-1-methylphenyl)thiazol-5-yl]benzoate (25d)
5.7.28. 4-[2-(3-Adamantan-1-yl-4-[(2-methoxyethoxy)methoxy]
phenyl)thiazol-5-yl]benzoic acid (8c)
Following the general procedure for the microwave-assisted Su-
zuki reaction, bromothiazole 23d (0.03 g, 0.06 mmol) and 4-
(methoxycarbonyl)phenylboronic acid (0.02 g, 0.09 mmol) were
heated at 120 °C for 10 min. The residue was purified by column
chromatography (SiO2, 80:20 hexane/EtOAc and SiO2-C18, 5:95
CH2Cl2/CH3CN), to give 25d (0.02 g, 61%) as a yellow solid, mp
116–117 °C (hexane/CHCl3). 1H NMR (400.13 MHz, CDCl3): d 8.12
(s, 1H, H40), 8.07 (d, J = 8.6 Hz, 2H, H2 + H6), 7.69 (s, 1H, H600),
7.67 (d, J = 8.6 Hz, 2H, H3 + H5), 7.08 (s, 1H, H300), 5.38 (s, 2H, OCH2-
O), 3.94 (s, 3H, OCH3), 3.9–3.8 (m, 2H, OCH2), 3.6–3.5 (m, 2H,
OCH2), 3.42 (s, 3H, OCH3), 2.60 (s, 3H, ArCH3), 2.13 (s, 6H,
3 ꢃ AdCH2), 2.08 (s, 3H, 3 ꢃ AdCH), 1.78 (s, 6H, 3 ꢃ AdCH2) ppm.
13C NMR (100.62 MHz, CDCl3): d 168.6 (s), 166.6 (s), 157.3 (s),
139.6 (d), 137.5 (s), 136.5 (s), 136.0 (s), 135.5 (s), 130.4 (d, 2ꢃ),
129.3 (s), 128.7 (d), 126.2 (d, 2ꢃ), 125.9 (s), 117.3 (d), 93.1 (t),
71.5 (t), 68.0 (t), 59.1 (q), 52.2 (q), 40.7 (t, 3ꢃ), 37.0 (t, 3ꢃ), 36.8
According to the general procedure for the hydrolysis of esters,
25c (0.01 g, 0.02 mmol) gave, after crystallization, acid 8c (0.09 g,
99%) as a white solid, mp 275 ꢁ276 °C (hexane/CHCl3). 1H NMR
(400.13 MHz, DMSO-d6): d 8.40 (s, 1H, H40), 8.00 (d, J = 8.5 Hz,
2H, H2 + H6), 7.84 (d, J = 8.5 Hz, 2H, H3 + H5), 7.83 (d, J = 2.3 Hz,
1H, H200), 7.78 (dd, J = 8.6, 2.3 Hz, 1H, H600), 7.19 (d, J = 8.6 Hz, 1H,
H500), 5.39 (s, 2H, OCH2O), 3.8–3.7 (m, 2H, OCH2), 3.5–3.4 (m, 2H,
OCH2), 3.24 (s, 3H, OCH3), 2.12 (s, 6H, 3 ꢃ AdCH2), 2.08 (s, 3H,
3 ꢃ AdCH), 1.76 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz,
DMSO-d6): 167.5 (s), 166.8 (s), 157.8 (s), 141.2 (d), 138.4 (s),
136.5 (s), 135.0 (s), 130.2 (d, 2ꢃ), 130.0 (s), 126.2 (d, 2ꢃ), 125.9
(s), 125.3 (d), 124.5 (d), 114.9 (d), 92.9 (t), 71.0 (t), 68.0 (t), 58.1
(q), 39.9 (t, 3ꢃ), 36.7 (s), 36.4 (t, 3ꢃ), 28.3 (d, 3ꢃ) ppm. IR:
m
2902 (m, C–H), 2848 (w, C–H), 1690 (w, C@O), 1585 (m), 1546
(s), 1415 (s), 1223 (s), 1101 (s), 983 (s), 849 (m), 785 (s) cmꢁ1
.
MS (ESI+): m/z (%) 559 ([M+K+H]+, 82), 520 ([M+H]+, 100), 459
(16), 455 (9), 445 (9), 415 (17), 401 (9), 371 (8), 210 (21). HRMS
(ESI+): calcd for C30H34NO5S [M+H]+ 520.2152; found: 520.2145.
Calcd for C30H33NO5Sꢀ1/2 [CH3)2SO] C, 66.64; H, 5.51; N, 2.51;
found: C, 66.12; H, 5.51; N, 2.62.
(s), 29.0 (d, 3ꢃ), 21.3 (q) ppm. IR:
m 2899 (m, C–H), 2848 (w, C–
H), 1713 (s, C@O), 1603 (m), 1444 (m), 1271 (s), 1239 (m), 1161
(s), 1110 (s), 1018 (m), 979 (s), 848 (s), 770 (s) cmꢁ1. MS (ESI+):
m/z (%) 548 ([M+H]+, 100). HRMS (ESI+): calcd for C32H38NO5S
[M+H]+ 548.2465; found: 548.2452.
5.7.29. 4-[2-(5-Adamantan-1-yl-4-[(2-methoxyethoxy)
5.7.26. 4-[2-(3-Adamantan-1-yl-4-hydroxyphenyl)thiazol-5-yl]
benzoic acid (8a)
According to the general procedure for the hydrolysis of esters,
25a (0.01 g, 0.02 mmol) gave, after crystallization, acid 8a (0.09 g,
99%) as a yellow solid, mp >310 °C (hexane/CHCl3). 1H NMR
(400.12 MHz, DMSO-d6): d 10.06 (s, 1H, OH), 8.35 (s, 1H, H40),
methoxy]-2-methylphenyl)thiazol-5-yl]benzoic acid (8d)
According to the general procedure for the hydrolysis of esters,
25d (0.02 g, 0.03 mmol) gave, after crystallization, acid 8d (0.01 g,
94%) as a yellow solid, mp 235–236 °C (hexane/CHCl3). 1H NMR
(400.13 MHz, DMSO-d6): d 8.43 (s, 1H, H40), 7.99 (d, J = 8.4 Hz,
2H, H2 + H6), 7.84 (d, J = 8.4 Hz, 2H, H3 + H5), 7.66 (s, 1H, H600),

1298
J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
7.04 (s, 1H, H300), 5.37 (s, 2H, OCH2O), 3.8–3.7 (m, 2H, OCH2), 3.5–
3.4 (m, 2H, OCH2), 3.25 (s, 3H, OCH3), 2.56 (s, 3H, ArCH3), 2.08 (s,
6H, 3 ꢃ AdCH2), 2.05 (s, 3H, 3 ꢃ AdCH), 1.74 (s, 6H, 3 ꢃ AdCH2)
ppm. 13C NMR (100.62 MHz, DMSO-d6): d 167.4 (s), 166.8 (s),
156.7 (s), 140.5 (d), 136.9 (s), 135.8 (s), 135.0 (s, 2ꢃ), 130.2 (d,
2ꢃ), 130.0 (s), 127.9 (d), 126.2 (d, 2ꢃ), 124.9 (s), 117.3 (d), 92.8
(t), 71.0 (t), 68.0 (t), 58.1 (q), 38.1 (t, 3ꢃ), 36.5 (t, 3ꢃ), 36.3 (s),
MeOH/THF) and 75 mg (19%) of deprotected starting pyrazine
29a as a yellowish solid, mp 203–204 °C (hexane/MeOH/THF).
Data for methyl (E)-3-[5-(3-adamantan-1-yl-4-hydroxyphenyl)-6-
methoxypyrazin-2-yl]acrylate (28a). 1H NMR (400.13 MHz,
DMSO-d6): d 9.88 (br, 1H), 8.48 (s, 1H, H30), 7.98 (d, J = 2.1 Hz,
1H, H200), 7.86 (dd, J = 8.5, 2.1 Hz, 1H, H600), 7.68 (d, J = 15.5 Hz,
1H, H3), 6.89 (d, J = 15.5 Hz, 1H, H2), 6.87 (d, J = 8.5 Hz, 1H, H500),
4.02 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 2.10 (s, 6H, 3 ꢃ AdCH2),
2.04 (s, 3H, 3 ꢃ AdCH), 1.73 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR
(100.62 MHz, DMSO-d6): d 166.1 (s), 158.1 (s), 156.2 (s), 144.0
(s), 140.8 (s), 139.9 (d), 137.3 (d), 135.2 (s), 128.0 (d), 127.7 (d),
125.4 (s), 121.0 (d), 115.9 (d), 53.4 (q), 51.6 (q), 39.8 (t, 3ꢃ), 36.5
28.4 (d, 3ꢃ), 21.3 (q) ppm. IR:
m 3500–3200 (br, OH), 2900 (m, C–
H), 2879 (m, C–H), 2849 (m, C–H), 1683 (s, C@O), 1604 (s), 1441
(m), 1426 (m), 1299 (m), 1225 (m), 1099 (s), 1016 (s), 984 (s),
845 (m), 767 (s) cmꢁ1. MS (ESI+): m/z (%) 534 ([M+H]+, 100), 319
(6), 210 (4). HRMS (ESI+) calcd for C31H36NO5S [M+H]+ 534.2309;
found: 534.2303. Elem. Anal. calcd for C31H35NO5S: C, 69.77; H,
6.61; N, 2.62; found: C, 69.52; H, 6.26; N, 2.59.
(t, 3ꢃ), 36.3 (s), 28.3 (d, 3ꢃ) ppm. IR:
m 3300–3100 (br, O–H),
2900 (m, C–H), 2847 (w, C–H), 1712 (s, C@O), 1645 (w), 1532
(w), 1602 (w), 1449 (m), 1370 (s), 1162 (s), 1122 (m), 972 (m)
cmꢁ1. MS (FAB+): m/z (%) 421 ([M+H]+, 100), 420 ([M]+, 70), 419
(15), 307 (25), 289 (14), 155 (26), 154 (83). HRMS (FAB+): calcd
for C25H28N2O4 [M]+, 420.2049; found: 420.2050. Data for 5-bro-
mo-2-[3-adamantan-1-yl-4-hydroxyphenyl]-3-methoxypyrazine
29a. 1H NMR (400.13 MHz, CDCl3): d 8.28 (s, 1H, H6), 7.93 (d,
J = 2.0 Hz, 1H, H20), 7.76 (dd, J = 8.3, 2.1 Hz, 1H, H60), 6.68 (d,
J = 8.3 Hz, 1H, H50), 5.36 (br. s, OH), 4.06 (s, 3H, OCH3), 2.16 (s,
6H, 3 ꢃ AdCH2), 2.09 (s, 3H, 3 ꢃ AdCH), 1.78 (s, 6H, 3 ꢃ AdCH2)
ppm. 13C NMR (100.62 MHz, CDCl3): d 156.8 (s), 156.0 (s), 141.7
(s), 137.5 (d), 136.4 (s), 132.8 (s), 128.4 (d), 127.8 (d), 127.0 (s),
116.6 (d), 54.7 (q), 40.4 (t, 3ꢃ), 37.0 (t, 3ꢃ), 36.9 (s), 29.0 (d, 3ꢃ)
5.7.30. 5-Bromo-2-[3-adamantan-1-yl-4-(tert-butyldimeth
ylsilyloxy)phenyl]-3-methoxypyrazine (27a)
Following the general procedure for the Suzuki reaction, pyra-
zine 26 (0.50 g, 1.59 mmol) and boronic acid 16a11 (0.80 g,
2.07 mmol) were heated in benzene at 50 °C for 12 h. The residue
was purified by column chromatography (C18-SiO2, 100% CH3CN)
and crystallized, to give 27a (0.64 g, 76%) as a white powder, mp
143–144 °C (hexane/CHCl3). 1H NMR (400.13 MHz, CDCl3): d 8.28
(s, 1H, H6), 7.94 (d, J = 2.3 Hz, 1H, H20), 7.78 (dd, J = 8.5, 2.3 Hz,
1H, H60), 6.86 (d, J = 8.5 Hz, 1H, H50), 4.06 (s, 3H, OCH3), 2.15 (s,
6H, 3 ꢃ AdCH2), 2.08 (s, 3H, 3 ꢃ AdCH), 1.78 (s, 6H, 3 ꢃ AdCH2),
1.05 (s, 9H, SiC(CH3)3), 0.37 (s, 6H, Si(CH3)2) ppm. 13C NMR
(100.62 MHz, CDCl3): d 156.6 (s), 156.1 (s), 141.7 (s), 139.4 (s),
137.6 (d), 132.6 (s), 128.3 (d), 127.3 (d), 126.7 (s), 118.7 (d), 54.5
(q), 40.2 (t, 3ꢃ), 37.0 (t, 3ꢃ), 36.9 (s), 28.9 (q, 3ꢃ), 26.3 (d, 3ꢃ),
ppm. IR:
m 3400–3100 (br, O–H), 2902 (w, C–H), 2882 (w, C–H),
2847 (w), 1599 (m), 1461 (m), 1409 (s), 1357 (s), 1284 (m), 1225
(s), 1177 (s), 1146 (s), 912 (s), 875 (m), 823 (m), 753 (s), 732 (s)
cmꢁ1
.
MS (ESI+): m/z (%) 417 ([M+H]+[81Br], 100), 415
18.9 (s), –3.5 (q, 2ꢃ) ppm. IR:
m
2904 (s, C–H), 2853 (m, C–H),
([M+H]+[79Br], 95), 359 (10), 279 (4), 201 (24). HRMS (ESI+): calcd
for C21H2481BrN2O2 [M+H]+ 417.0998 and C21H2479BrN2O2 [M+H]+
415.1016; found: 417.1000 and 415.1030. Elem. Anal. calcd for
1600 (m), 1522 (w), 1492 (m), 1419 (m), 1357 (s), 1248 (s), 1144
(s), 900 (s) cmꢁ1. MS (ESI+): m/z (%) 531 ([M+H]+
[
81Br], 35), 530
([M]+
[
81Br], 97), 529 ([M+H]+
[
79Br], 34), 528 ([M]+
[
79Br], 94),
C21H24 BrN2O2: C, 60.73; H, 5.58; N, 6.71; found: C, 60.84; H,
5.60; N, 6.71.
t
473 ([Mꢁ Bu]+, 100), 353 (16), 351 (34), 349 (20), 135 (93), 73
(20). HRMS (ESI+): calcd for C27H3781BrN2O2Si [M]+ 530.1787 and
C
27H3779BrN2O2Si [M]+ 528.1808; found: 530.1786 and 528.1791.
5.7.33. Methyl (E)-3-[5-(5-adamantan-1-yl-4-hydroxy-2-
methylphenyl)-6-methoxypyrazin-2-yl]acrylate (28b)
5.7.31. 5-Bromo-2-[5-adamantan-1-yl-2-methyl-4-(tert-
butyldimethylsilyloxy)phenyl]-3-methoxypyrazine (27b)
Following the general procedure for the Heck reaction, bromide
27b (0.09 g, 0.17 mmol) gave, after purification by column chroma-
tography (SiO2, 70:30 hexane/EtOAc), 0.04 g (57%) of ester 28b as a
yellow oil. 1H NMR (400.13 MHz, CDCl3): d 8.25 (s, 1H, H30), 7.68 (d,
J = 15.4 Hz, 1H, H3), 7.22 (s, 1H, H600), 7.04 (d, J = 15.4 Hz, 1H, H2),
6.49 (s, 1H, H300), 4.00 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 2.11 (s, 9H,
ArCH3 + 3 ꢃ AdCH2), 2.05 (s, 3H, 3 ꢃ AdCH), 1.75 (s, 6H, 3 ꢃ AdCH2)
ppm. 13C NMR (100.62 MHz, CDCl3): d 167.1 (s), 157.6 (s), 155.5 (s),
147.5 (s), 142.9 (s), 139.8 (d), 136.5 (d), 135.6 (s), 133.9 (s), 129.0
(d), 127.1 (s), 122.6 (d), 118.6 (d), 53.7 (q), 51.9 (q), 40.5 (t, 3ꢃ),
Following the general procedure for the Suzuki reaction, pyra-
zine 26 (0.36 g, 1.15 mmol) and boronic acid 16b32 (0.51 g,
1.26 mmol) were heated in benzene at 50 °C for 12 h. The residue
was purified by column chromatography (SiO2, 97:3 hexane/EtOAc
and C18-SiO2, 95:5 CH3CN/CH2Cl2), to give 27b (0.18 g, 30%) as a
foam. 1H NMR (400.13 MHz, CDCl3): d 8.32 (s, 1H, H6), 7.20 (s,
1H, H60), 6.70 (s, 1H, H30), 4.00 (s, 3H, OCH3), 2.13 (s, 3H, ArCH3),
2.11 (s, 6H, 3 ꢃ AdCH2), 2.05 (s, 3H, 3 ꢃ AdCH), 1.76 (s, 6H,
3 ꢃ AdCH2), 1.07 (s, 9H, OSiC(CH3)3), 0.38 (s, 6H, OSi(CH3)2) ppm.
13C NMR (100.62 MHz, CDCl3): d 157.3 (s), 155.3 (s), 143.9 (s),
137.4 (d), 137.0 (s), 134.9 (s), 134.0 (s), 128.9 (d), 126.5 (s), 120.9
(d), 54.6 (q), 40.4 (t, 3ꢃ), 37.0 (t, 3ꢃ), 36.6 (s), 29.0 (d, 3ꢃ), 26.4
37.0 (t, 3ꢃ), 36.4 (s), 29.0 (d, 3ꢃ), 19.1 (q) ppm. IR:
(br, O–H), 2908 (s, C–H), 2852 (m, C–H), 1720 (s, C@O), 1448 (s),
1365 (s), 1328 (s), 1227 (m), 1185 (m), 1142 (m), 756 (m) cmꢁ1
MS (ESI+): m/z (%) 435 ([M+H]+, 100). HRMS (ESI+): calcd for
26H31N2O4 [M+H]+ 435.2278; found: 435.2268.
m 3600–3100
.
(q, 3ꢃ), 19.3 (q), 18.9 (s), –3.4 (q, 2ꢃ) ppm. IR:
2850 (m, C–H), 1605 (w), 1525 (w), 1497 (w), 1456 (m), 1420
(m), 1357 (s), 1251 (s), 1155 (s), 900 (s), 837 (s), 782 (s) cmꢁ1
m
2900 (m, C–H),
C
.
5.7.34. Methyl (E)-3-[5-(3-adamantan-1-yl-4-[(2-methoxy
ethoxy)methoxy]phenyl)-6-methoxypyrazin-2-yl]acrylate (28c)
According to the general procedure for the protection of the
phenols with MEMCl, phenol 28a (0.29 g, 0.70 mmol) gave, after
purification by column chromatography (SiO2, 70:30 hexane/
EtOAc), compound 28c (0.32 g, 92%) as a white solid, mp 145–
146 °C (hexane/MeOH/THF). 1H NMR (400.13 MHz, DMSO-d6): d
8.54 (s, 1H, H30), 8.02 (s, 1H, H200), 7.96 (d, J = 8.6 Hz, 1H, ArH),
7.72 (d, J = 15.6 Hz, 1H, H3), 7.14 (d, J = 8.6 Hz, 1H, ArH), 6.94 (d,
J = 15.6 Hz, 1H, H2), 5.38 (s, 2H, OCH2O), 4.04 (s, 3H, OCH3), 3.79
(t, J = 3.9 Hz, 2H, O(CH2)2O), 3.76 (s, 3H, OCH3), 3.51 (t, J = 3.9 Hz,
2H, O(CH2)2O), 3.24 (s, 3H, OCH3), 2.10 (s, 6H, 3 ꢃ AdCH2), 2.06
MS (ESI+): m/z (%) 545 ([M+H]+ 81Br], 100), 543 ([M+H]+ 79Br],
[ [
97), 279 (7). HRMS (ESI+): calcd for C28H4081BrN2O2Si [M+H]+
545.2016 and C28H4079BrN2O2Si [M+H]+ 543.2037; found:
545.2010 and 543.2030.
5.7.32. Methyl (E)-3-[5-(3-adamantan-1-yl-4-hydroxyphenyl)-6-
methoxypyrazin-2-yl]acrylate (28a)
Following the general procedure for the Heck reaction, bromide
27a (0.50 g, 0.95 mmol) gave, after purification by column chroma-
tography (SiO2, 70:30 hexane/EtOAc) and crystallization, 0.27 g
(67%) of ester 28a as a yellow solid, mp 245–246 °C (hexane/

J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
1299
(s, 3H, 3 ꢃ AdCH), 1.75 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR
41.7 (t, 3ꢃ), 38.4 (t, 3ꢃ), 37.8 (s), 30.7 (d, 3ꢃ), 19.4 (q) ppm. IR:
m
3500–2800 (br, O–H), 2904 (s, C–H), 2851 (m, C–H), 1692 (s,
C@O), 1449 (s), 1364 (s), 1235 (m), 1184 (m), 1142 (m) cmꢁ1. MS
(ESI+): m/z (%) 421 ([M+H]+, 100), 394 (4), 359 (6), 201 (26). HRMS
(ESI+): calcd for C25H29N2O4 [M+H]+ 421.2122; found: 421.2114.
Purity: 98% (RP HPLC, gradient from 50:50 to 0:100 H2O/CH3CN,
1 mL/min).
(100.62 MHz, DMSO-d6): d 166.1 (s), 157.1 (s), 156.4 (s), 143.6
(s), 141.5 (s), 139.9 (d), 137.4 (d), 137.3 (s), 128.2 (d), 127.6 (s),
127.5 (d), 121.6 (d), 113.7 (d), 92.8 (t), 70.9 (t), 67.9 (t), 58.0 (q),
53.6 (q), 51.7 (q), 40.1 (t, 3ꢃ), 36.6 (t, 3ꢃ), 36.4 (s), 28.3 (d, 3ꢃ)
ppm. IR:
m 2899 (w, C–H), 2850 (w, C–H), 1721 (w, C@O), 1641
(m), 1600 (w), 1522 (w), 1443 (w), 1361 (m), 1224 (m), 1158
(m), 1112 (s), 979 (s) cmꢁ1. MS (FAB+): m/z (%) 509 ([M+H]+,
100), 508 ([M]+, 46), 433 (12), 307 (11), 155 (15), 154 (46). HRMS
(FAB+): calcd for C29H37N2O6 [M+H]+, 509.2652; found: 509.2660.
5.7.38. (E)-3-(5-(3-Adamantan-1-yl-4-[(2-methoxy
ethoxy)methoxy]phenyl)-6-methoxypyrazin-2-yl)acrylic acid
(9c)
5.7.35. Methyl (E)-3-[5-(5-adamantan-1-yl-4-[(2-methoxy
ethoxy)methoxy]-2-methylphenyl)-6-methoxypyrazin-2-yl]
acrylate (28d)
In accordance with the general procedure for the hydrolysis of
esters, ester 28c (0.18 g, 0.36 mmol) gave, after crystallization, ac-
rylic acid 9c (0.11 g, 62%) as a yellow solid, mp 215–216 °C (hex-
1
According to the general procedure for the protection of the
phenols with MEMCl, phenol 28b (0.02 g, 0.05 mmol) gave, after
purification by column chromatography (SiO2, 70:30 hexane/
EtOAc), compound 28d (0.02 g, 87%) as a yellow oil. 1H NMR
(400.13 MHz, CDCl3): d 8.26 (s, 1H, H30), 7.67 (d, J = 15.3 Hz, 1H,
H3), 7.23 (s, 1H, H600), 7.06 (s, 1H, H300), 7.04 (d, J = 15.3 Hz, 1H,
H2), 5.35 (s, 2H, OCH2O), 4.00 (s, 3H, OCH3), 3.9–3.8 (m, 2H,
OCH2), 3.85 (s, 3H, OCH3), 3.6–3.5 (m, 2H, OCH2), 3.41 (s, 3H,
OCH3), 2.18 (s, 3H, ArCH3), 2.10 (s, 6H, 3 ꢃ AdCH2), 2.04 (s, 3H,
3 ꢃ AdCH), 1.75 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz,
CDCl3): d 167.0 (s), 157.5 (s), 157.1 (s), 147.5 (s), 142.9 (s), 139.8
(d), 136.7 (d), 135.9 (s), 135.8 (s), 128.5 (d), 128.3 (s), 122.6 (d),
116.3 (d), 93.2 (t), 71.6 (t), 67.8 (t), 59.0 (q), 53.7 (q), 51.9 (q),
ane/THF). H NMR (400.13 MHz, DMSO-d6): d 8.51 (s, 1H, H30),
8.02 (s, 1H, H200), 7.96 (d, J = 8.6 Hz, 1H, ArH), 7.63 (d, J = 15.5 Hz,
1H, H3), 7.13 (d, J = 8.6 Hz, 1H, ArH), 6.87 (d, J = 15.5 Hz, 1H, H2),
5.38 (s, 2H, OCH2O), 4.03 (s, 3H, OCH3), 3.79 (t, J = 4.4 Hz, 2H,
O(CH2)2O), 3.51 (t, J = 4.4 Hz, 2H, O(CH2)2O), 3.24 (s, 3H, O(CH2)2-
OCH3), 2.10 (s, 6H, 3 ꢃ AdCH2), 2.06 (s, 3H, 3 ꢃ AdCH), 1.75 (s,
6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, DMSO-d6): d 167.0
(s), 157.1 (s), 156.4 (s), 143.3 (s), 141.8 (s), 139.2 (d), 137.3 (s),
137.2 (d), 128.1 (d), 127.6 (s), 127.5 (d), 123.2 (d), 113.7 (d), 92.7
(t), 70.9 (t), 67.9 (t), 58.0 (q), 53.5 (q), 40.1 (t, 3ꢃ), 36.6 (t, 3ꢃ),
36.4 (s), 28.3 (d, 3ꢃ) ppm. IR:
m 3100–2800 (br, O–H), 1682 (s,
C@O), 1630 (w), 1598 (w), 1523 (w), 1443 (w), 1361 (m), 1300
(m), 1223 (m), 1115 (s), 979 (s) cmꢁ1. MS (FAB+): m/z (%) 495
([M+H]+, 100), 494 ([M]+, 43), 419 ([M – OCH2CH2OCH3]+, 14),
176 (12). HRMS (FAB+): calcd for C28H35N2O6 [M+H]+, 495.2495;
found: 495.2502. Purity: 98% (RP HPLC, gradient from 50:50 to
0:100 H2O/CH3CN, 1 mL/min).
40.7 (t, 3ꢃ), 37.1 (t, 3ꢃ), 36.8 (s), 29.0 (d, 3ꢃ), 19.5 (q) ppm. IR:
m
2921 (s, C–H), 2853 (m, C–H), 1722 (s, C@O), 1449 (s), 1362 (s),
1323 (m), 1229 (m), 1162 (m), 1015 (m), 981 (m) cmꢁ1. MS
(ESI+): m/z (%) 523 ([M+H]+, 100), 435 (3). HRMS (ESI+): calcd for
C
30H39N2O6 [M+H]+ 523.2803; found: 523.2787.
5.7.39. (E)-3-[5-(5-Adamantan-1-yl-4-[(2-methoxyethoxy)
methoxy]-2-methylphenyl)-6-methoxypyrazin-2-yl]acrylic acid
(9d)
5.7.36. (E)-3-[5-(3-Adamantan-1-yl-4-hydroxyphenyl)-6-meth
oxypyrazin-2-yl]acrylic acid (9a)
In accordance with the general procedure for the hydrolysis of
esters, ester 28a (0.18 g, 0.42 mmol) gave, after crystallization, ac-
rylic acid 9a (0.12 g, 73%) as a yellow solid, mp 256–257 °C (hex-
In accordance with the general procedure for the hydrolysis of
esters, ester 28d (0.02 g, 0.04 mmol) gave, after crystallization, ac-
rylic acid 9d (0.015 g, 65%) as a yellow solid, mp 107–108 °C (hex-
1
1
ane/THF). H NMR (400.13 MHz, CD3OD): d 8.23 (s, 1H, H30), 7.97
ane/CH2Cl2). H NMR (400.13 MHz, CD3OD): d 8.28 (s, 1H, H30),
(d, J = 1.8 Hz, 1H, H200), 7.80 (dd, J = 8.4, 1.8 Hz, 1H, H600), 7.64 (d,
J = 15.4 Hz, 1H, H3), 6.93 (d, J = 15.4 Hz, 1H, H2), 6.78 (d,
J = 8.4 Hz, 1H, H500), 4.08 (s, 3H, OCH3), 2.20 (s, 6H, 3 ꢃ AdCH2),
2.06 (s, 3H, 3 ꢃ AdCH), 1.82 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR
(100.62 MHz, CD3OD): d 169.9 (s), 159.7 (s), 158.4 (s), 146.5 (s),
143.3 (s), 141.1 (d), 137.8 (d), 137.2 (s), 129.5 (d), 129.4 (d),
127.3 (s), 123.7 (d), 116.9 (d), 54.1 (q), 41.5 (t, 3ꢃ), 38.3 (t, 3ꢃ),
7.62 (d, J = 16.0 Hz, 1H, H3), 7.15 (s, 1H, H600), 7.05 (s, 1H, H300),
7.05 (d, J = 16.0 Hz, 1H, H2), 5.36 (s, 2H, OCH2O), 4.02 (s, 3H,
OCH3), 3.9–3.8 (m, 2H, OCH2), 3.6–3.5 (m, 2H, OCH2), 3.37 (s, 3H,
OCH3), 2.14 (s, 6H, 3 ꢃ AdCH2), 2.13 (s, 3H, ArCH3), 2.05 (s, 3H,
3 ꢃ AdCH), 1.81 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz,
CD3OD): d 170.9 (s), 159.3 (s), 158.4 (s), 148.3 (s), 145.9 (s),
139.6 (d, 2ꢃ), 137.1 (d+2s, 3ꢃ), 129.7 (s), 129.5 (d), 117.4 (d),
94.6 (t), 73.0 (t), 69.3 (t), 59.3 (q), 54.3 (q), 42.1 (t, 3ꢃ), 38.3 (t,
38.1 (s), 30.6 (d, 3ꢃ) ppm. IR:
m 3500–3100 (br, O–H), 2900 (m,
C–H), 2848 (w, C–H), 1683 (s, C@O), 1632 (w), 1601 (w), 1524
(w), 1444 (m), 1359 (s), 1298 (m), 1227 (s), 1177 (m), 1125 (m),
973 (w) cmꢁ1. MS (FAB+): m/z (%) 407 ([M+H]+, 100), 406 ([M]+,
69). HRMS (FAB+): calcd for C24H27N2O4 [M+H]+, 407.1971; found:
407.1966. Purity: 96% (RP HPLC, gradient from 50:50 to 0:100 H2O/
CH3CN, 1 mL/min).
3ꢃ), 38.1 (s), 30.7 (d, 3ꢃ), 19.7 (q) ppm. IR:
m 3500–2800 (br, O–
H), 2904 (s, C–H), 2851 (m, C–H), 1699 (m, C@O), 1642 (w), 1449
(m), 1361 (s), 1234 (s), 1182 (s), 1139 (m), 1017 (s), 980 (m)
cmꢁ1. MS (ESI+): m/z (%) 509 ([M+H]+, 100), 421 (8), 201 (10).
HRMS (ESI+): calcd for C29H37N2O6 [M+H]+ 509.2646; found:
509.2659.
5.7.37. (E)-3-[5-(5-Adamantan-1-yl-4-hydroxy-2-methylphenyl)
-6-methoxypyrazin-2-yl]acrylic acid (9b)
5.7.40. 5-Bromo-2-[3-adamantan-1-yl-4-phenol]-3-methoxy
pyrazine (29a)
In accordance with the general procedure for the hydrolysis of
esters, ester 28b (0.02 g, 0.05 mmol) gave, after crystallization, ac-
rylic acid 9b (0.017 g, 84%) as a yellow solid, mp 168–169 °C (hex-
In accordance with the general procedure for the cleavage of the
silyl ethers, ether 27a (0.23 g, 0.44 mmol) gave, after purification
by column chromatography (SiO2, from 90:10 hexane/EtOAc),
0.14 g (83%) of phenol 29a as a yellow solid, mp 203–204 °C
(CHCl3/hexane). Spectroscopic data matched those showed above.
1
ane/CH2Cl2). H NMR (400.13 MHz, CD3OD): d 8.27 (s, 1H, H30),
7.69 (d, J = 15.5 Hz, 1H, H3), 7.07 (s, 1H, H600), 7.01 (d, J = 15.5 Hz,
1H, H2), 6.63 (s, 1H, H300), 4.01 (s, 3H, OCH3), 2.15 (s, 6H,
3 ꢃ AdCH2), 2.07 (s, 3H, ArCH3), 2.03 (s, 3H, 3 ꢃ AdCH), 1.79 (s,
6H, 6xAdCH2) ppm. 13C NMR (100.62 MHz, CD3OD): d 169.8 (s),
159.4 (s), 158.6 (s), 149.3 (s), 145.1 (s), 141.0 (d), 137.2 (d), 136.7
(s), 135.0 (s), 129.6 (d), 127.3 (s), 124.8 (d), 119.0 (d), 54.3 (q),
5.7.41. 5-Bromo-2-[5-adamantan-1-yl-4-hydroxy-2-meth
ylphenyl]-3-methoxypyrazine (29b)
In accordance with the general procedure for the cleavage of the
silyl ethers, ether 27b (0.08 g, 0.15 mmol) gave, after purification

1300
J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
by column chromatography (SiO2, from 90:10 hexane/EtOAc),
0.04 g (63%) of phenol 29b as a yellow oil. 1H NMR (400.13 MHz,
CDCl3): d 8.31 (s, 1H, H4), 7.16 (s, 1H, H60), 6.47 (s, 1H, H30), 3.98
(s, 3H, OCH3), 2.09 (s, 6H, 3 ꢃ AdCH2), 2.07 (s, 3H, ArCH3), 2.04 (s,
3H, 3 ꢃ AdCH), 1.75 (s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR
(100.62 MHz, CDCl3): d 157.4 (s), 155.4 (s), 143.8 (s), 137.2 (d),
135.3 (s), 134.2 (s), 134.0 (s), 128.8 (d), 126.3 (s), 118.5 (d), 54.6
(q), 40.4 (t, 3ꢃ), 37.0 (t, 3ꢃ), 36.4 (s), 29.0 (d, 3ꢃ), 19.0 (q) ppm.
chromatography (SiO2, 80:20 hexane/EtOAc), to give 30a (0.03 g,
58%) as a yellow powder, mp 269–270 °C (hexane/CHCl3). 1H
NMR (400.13 MHz, DMSO-d6): d 9.80 (s, 1H, OH), 8.92 (s, 1H,
H30), 8.28 (d, J = 8.5 Hz, 2H, H2 + H6), 8.07 (d, J = 8.5 Hz, 2H,
H3 + H5), 8.01 (d, J = 2.0 Hz, 1H, H200), 7.87 (dd, J = 8.4, 2.1 Hz, 1H,
H600), 6.87 (d, J = 8.4 Hz, 1H, H500), 4.09 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 2.12 (s, 6H, 3 ꢃ AdCH2), 2.05 (s, 3H, 3 ꢃ AdCH), 1.74 (s,
6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, DMSO-d6): d 165.9
(s), 157.8 (s), 156.1 (s), 143.4 (s), 141.8 (s), 140.1 (s), 135.3 (s),
133.2 (d), 129.9 (s), 129.7 (d, 2ꢃ), 127.8 (d), 127.6 (d), 126.4 (d,
2ꢃ), 125.6 (s), 116.0 (d), 53.5 (q), 52.2 (q), 39.9 (t, 3ꢃ), 36.6 (t,
IR:
m 3400–3100 (br, O–H), 2906 (s, C–H), 2852 (s, C–H), 1609
(m), 1526 (m), 1456 (s), 1401 (s), 1364 (s), 1225 (s), 1155 (s),
1008 (m), 894 (s), 757 (s) cmꢁ1
.
MS (ESI+): m/z (%) 431
([M+H]+[81Br], 100), 429 ([M+H]+[79Br], 100). HRMS (ESI+): calcd
for C22H2681BrN2O2 [M+H]+ 431.1153 and C22H2679BrN2O2 [M+H]+
429.1172; found: 431.1142 and 429.1168.
3ꢃ), 36.3 (s), 28.4 (d, 3ꢃ) ppm. IR:
m 3300–3000 (br, O–H), 2899
(m, C–H), 2846 (m, C–H), 1723 (s, C@O), 1599 (m), 1449 (m),
1434 (m), 1409 (s), 1368 (s), 1270 (s), 1220 (s), 1190 (s), 1110
(s), 771 (m) cmꢁ1. MS (ESI+): m/z (%) 471 ([M+H]+, 79), 379 (14),
369 (19), 355 (17), 353 (100), 317 (21), 279 (21), 275 (52), 259
(12), 210 (33). HRMS (ESI+): calcd for C29H31N2O4 [M+H]+
471.2278; found: 471.2274.
5.7.42. 5-Bromo-2-[3-adamantan-1-yl-4-[(2-methoxyethoxy)
methoxy]phenyl]-3-methoxypyrazine (29c)
According to the general procedure for the protection of the
phenols with MEMCl, phenol 29a (0.04 g, 0.10 mmol) gave, after
purification by column chromatography (SiO2, 80:20 hexane/
EtOAc), compound 29c (0.04 g, 89%) as a yellow solid, mp 93–
94 °C (hexane/EtOAc). 1H NMR (400.13 MHz, CDCl3): d 8.29 (s,
1H, H6), 7.94 (d, J = 2.2 Hz, 1H, H20), 7.86 (dd, J = 8.6, 2.2 Hz, 1H,
H60), 7.22 (d, J = 8.6 Hz, 1H, H50), 5.38 (s, 2H, OCH2O), 4.05 (s, 3H,
OCH3), 3.9–3.8 (m, 2H, OCH2), 3.6–3.5 (m, 2H, OCH2), 3.40 (s, 3H,
OCH3), 2.15 (s, 6H, 3 ꢃ AdCH2), 2.08 (s, 3H, 3 ꢃ AdCH), 1.78 (s,
6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3): d 157.6 (s),
156.7 (s), 141.6 (s), 138.4 (s), 137.6 (d), 132.9 (s), 127.9 (d), 127.8
(d), 127.7 (s), 114.0 (d), 93.1 (t), 71.5 (t), 67.9 (t), 59.0 (q), 54.6
5.7.45. Methyl 4-[5-(3-adamantan-1-yl-4-[(2-
methoxyethoxy)methoxy]phenyl)-6-methoxypyrazin-2-
yl]benzoate (30c)
Following the general procedure for the Suzuki reaction,
bromopyrazine 29c (0.05 g, 0.10 mmol) and 4-(methoxycar-
bonyl)phenylboronic acid (0.03 g, 0.14 mmol) were heated in
1,4-dioxane at 120 °C for 12 h. The residue was purified by col-
umn chromatography (SiO2, from 85:15 to 75:25 hexane/EtOAc),
to give 30c (0.03 g, 66%) as a yellow powder, mp 125–126 °C
(hexane/CHCl3). 1H NMR (400.13 MHz, CDCl3): d 8.74 (s, 1H,
H30), 8.15 (s, 4H, H2 + H3 + H5 + H6), 8.07 (d, J = 2.2 Hz,
1H, H200), 7.98 (dd, J = 8.6, 2.2 Hz, 1H, H600), 7.24 (d, J = 8.6 Hz,
1H, H500), 5.39 (s, 2H, OCH2O), 4.14 (s, 3H, OCH3), 3.95 (s, 3H,
OCH3), 3.9–3.8 (m, 2H, OCH2), 3.6–3.5 (m, 2H, OCH2), 3.41 (s,
3H, OCH3), 2.18 (s, 6H, 3 ꢃ AdCH2), 2.10 (s, 3H, 3 ꢃ AdCH), 1.79
(s, 6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3): d 166.7
(s), 157.5 (s), 156.9 (s), 144.9 (s), 142.4 (s), 140.5 (s), 138.3 (s),
133.0 (d), 130.5 (s), 130.0 (d, 2ꢃ), 128.5 (s), 128.1 (d), 128.0 (d),
126.3 (d, 2ꢃ), 114.0 (d), 93.1 (t), 71.5 (t), 67.9 (t), 59.0 (q), 53.6
(q), 52.2 (q), 40.6 (t, 3ꢃ), 37.2 (s), 37.0 (t, 3ꢃ), 29.0 (d, 3ꢃ) ppm.
(q), 40.5 (t, 3ꢃ), 37.2 (s), 37.0 (t, 3ꢃ), 29.0 (d, 3ꢃ) ppm. IR:
m
2906 (m, C–H), 2885 (m, C–H), 1599 (w), 1520 (m), 1456 (m),
1418 (m), 1356 (s), 1219 (s), 1146 (s), 1100 (s), 1004 (s), 909 (s),
830 (m) cmꢁ1. MS (ESI+): m/z (%) 505 ([M+H]+[81Br], 96), 503
([M+H]+[79Br], 100), 430 (7), 429 (24), 427 (27), 209 (8), 192 (7).
HRMS (ESI+): calcd for C25H3281BrN2O4 [M+H]+ 505.1525 and C25-
H3279BrN2O4 [M+H]+ 503.1540; found: 505.1513 and 503.1534.
Elem. Anal. calcd for C25H31BrN2O4: C, 59.64; H, 6.21; N, 5.56;
found: C, 59.10; H, 5.84; N, 5.41.
5.7.43. 5-Bromo-2-[5-adamantan-1-yl-4-[(2-methoxyethoxy)
methoxy]-2-methylphenyl]-3-methoxypyrazine (29d)
IR: m 2898 (m, C–H), 2852 (w, C–H), 1719 (s, C@O), 1603 (w),
1444 (m), 1360 (m), 1269 (s), 1114 (s), 1028 (m), 984 (s), 820
(m) cmꢁ1. MS (ESI+): m/z (%) 559 ([M+H]+, 100). HRMS (ESI+):
calcd for C33H39N2O6 [M+H]+ 559.2803; found: 559.2791. Elem.
Anal. Calcd for C33H38N2O6 C, 70.95; H, 6.86; N, 5.01; found: C,
71.08; H, 6.38; N, 4.99.
According to the general procedure for the protection of the
phenols with MEMCl, phenol 29b (0.02 g, 0.05 mmol) gave, after
purification by column chromatography (SiO2, 95:5 hexane/
EtOAc), compound 29d (0.023 g, 99%) as a colourless oil. 1H NMR
(400.13 MHz, CDCl3): d 8.32 (s, 1H, H6), 7.18 (s, 1H, H60), 7.05 (s,
1H, H30), 5.35 (s, 2H, OCH2O), 3.99 (s, 3H, OCH3), 3.9–3.8 (m, 2H,
OCH2), 3.6–3.5 (m, 2H, OCH2), 3.41 (s, 3H, OCH3), 2.15 (s, 3H,
ArCH3), 2.09 (s, 6H, 3 ꢃ AdCH2), 2.05 (s, 3H, 3 ꢃ AdCH), 1.75 (s,
6H, 3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, CDCl3): d 157.3 (s),
157.0 (s), 143.8 (s), 137.5 (d), 136.0 (s), 135.6 (s), 134.3 (s), 128.4
(d), 127.5 (s), 116.3 (d), 93.2 (t), 71.6 (t), 67.8 (t), 59.0 (q), 54.6
(q), 40.7 (t, 3ꢃ), 37.1 (t, 3ꢃ), 36.8 (s), 29.0 (d, 3ꢃ), 19.4 (q) ppm.
5.7.46. 4-(5-(3-Adamantan-4-hydroxyphenyl)pyrazin-2-
yl)benzoic Acid (10a)
In accordance with the general procedure for the hydrolysis of
esters, ester 30a (0.01 g, 0.02 mmol) gave, after crystallization,
acid 10a (0.09 g, 99%) as a white solid, mp 301–302 °C (hexane/
CHCl3). 1H NMR (400.13 MHz, DMSO-d6): d 9.82 (s, 1H, OH),
8.94 (s, 1H, H30), 8.29 (d, J = 7.0 Hz, 2H, H2+H6), 8.07 (d,
J = 7.0 Hz, 2H, H3 + H5), 8.01 (s, 1H, H200), 7.88 (d, J = 7.8 Hz, 1H,
H600), 6.88 (d, J = 7.8 Hz, 1H, H500), 4.11 (s, 3H, OCH3), 2.13 (s, 6H,
3 ꢃ AdCH2), 2.06 (s, 3H, 3 ꢃ AdCH), 1.75 (s, 6H, 3 ꢃ AdCH2) ppm.
13C NMR (100.62 MHz, DMSO-d6): d 167.0 (s), 157.7 (s), 156.2
(s), 143.7 (s, 2ꢃ), 141.7 (s), 139.7 (s), 135.3 (s), 133.1 (d), 129.9
(d, 2ꢃ), 127.8 (d), 127.5 (d), 126.3 (d, 2ꢃ), 125.6 (s), 116.0 (d),
53.6 (q), 39.9 (t, 3ꢃ), 36.6 (t, 3ꢃ), 36.4 (s), 28.4 (d, 3ꢃ) ppm. IR:
IR:
m 2904 (s, C–H), 2852 (m, C–H), 1526 (m), 1455 (m), 1421
(m), 1361 (s), 1232 (m), 1155 (s), 1100 (m), 1012 (s), 890 (m)
cmꢁ1
.
MS (ESI+): m/z (%) 519 ([M+H]+[81Br], 100), 517
([M+H]+[79Br], 97), 476 (7), 431 (4), 429 (4). HRMS (ESI+): calcd
for C26H3481BrN2O4 [M+H]+ 519.1676 and C26H3479BrN2O4 [M+H]+
517.1696; found: 519.1656 and 517.1686.
5.7.44. Methyl 4-[5-(3-adamantan-1-yl-4-hydroxyphenyl)-6-
methoxypyrazin-2-yl]benzoate (30a)
Following the general procedure for the Suzuki reaction,
bromopyrazine 29a (0.04 g, 0.10 mmol) and 4-(methoxycar-
bonyl)phenylboronic acid (0.03 g, 0.14 mmol) were heated in
1,4-dioxane at 120 °C for 12 h. The residue was purified by column
m 3531 (m, OH), 3300–2700 (br, O–H), 2902 (s, C–H), 2848 (m,
C–H), 1677 (s, C@O), 1604 (m), 1529 (m), 1426 (m), 1409 (s),
1359 (s), 1290 (s), 1215 (s), 1118 (s), 862 (s), 841 (s), 776 (s)
cmꢁ1. MS (ESI+): m/z (%) 457 ([M+H]+, 100), 359 (5), 348 (4).
HRMS (ESI+): calcd for C28H29N2O4 [M+H]+ 457.2122; found:
457.2117.

J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
1301
5.7.47. 4-[5-(3-Adamantan-1-yl-4-[(2-methoxyethoxy)
25 mM KCl, 5 mM EGTA, 1 mM DTT, 10
lM cytochalasin B, 0.5%
methoxy]phenyl)6-methoxypyrazin-2-yl]benzoic acid (10c)
In accordance with the general procedure for the hydrolysis of
esters, ester 30c (0.03 g, 0.06 mmol) gave, after crystallization, acid
10c (0.03 g, 93%) as a yellow solid, mp 252–253 °C (hexane/CHCl3).
1H NMR (400.13 MHz, DMSO-d6): d 8.95 (s, 1H, H30), 8.28 (d,
J = 8.4 Hz, 2H, H4+H6), 8.07 (d, J = 8.4 Hz, 2H, H3 + H5), 8.04 (d,
J = 2.1 Hz, 1H, H200), 7.97 (dd, J = 8.8, 2.0 Hz 1H, H600), 7.14 (d,
J = 8.8 Hz, 1H, H500), 5.37 (s, 2H, OCH2O), 4.10 (s, 3H, OCH3), 3.8–
3.7 (m, 2H, OCH2), 3.5–3.4 (m, 2H, OCH2), 3.25 (s, 3H, OCH3),
2.11 (s, 6H, 3 ꢃ AdCH2), 2.06 (s, 3H, 3 ꢃ AdCH), 1.75 (s, 6H,
3 ꢃ AdCH2) ppm. 13C NMR (100.62 MHz, DMSO-d6): d 167.0 (s),
156.9 (s), 156.3 (s), 144.3 (s), 141.2 (s), 139.6 (s), 137.4 (s), 133.2
(d), 131.4 (s), 129.9 (d, 2ꢃ), 127.9 (d), 127.8 (s), 127.4 (d), 126.4
(d, 2ꢃ), 113.8 (d), 92.8 (t), 71.0 (t), 67.9 (t), 58.1 (q), 53.6 (q),
NP-40, and a mixture of protease inhibitors consisting of 1 mM
PMSF, 1 g/mL leupeptine, and 1 g/mL aprotinin) and DEVDase
activity was measured following addition of an equal volume of
2ꢃ caspase buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM
EDTA, 5 mM DTT, 0.1% CHAPS and 10% sucrose) containing
l
l
100 lM of Ac-DEVD-AFC (Assay Biotechnology) essentially as
described.27 The emission at 510 nm was measured upon excita-
tion at 390 nm every two minutes continuously for 1 h in a Victor
2 multilabel reader set at 37 °C and the slope of the linear part of
the plot was calculated as a measure of DEVDase activity. The
activity in untreated cells was measured as basal activity.
5.8.4. RAR/RXR transactivation assay
Gal4-RARa or Gal4-RXRa were transfected into HEK-293-lucif-
40.2 (t, 3ꢃ), 36.7 (s), 36.5 (t, 3ꢃ), 28.4 (d, 3ꢃ) ppm. IR:
m
3500–
erase reporter cells that express the luciferase gene under the con-
trol of 5 copies of an UAS element. HEK-293-luc cells were seeded
in 96 well plates (30,000 cells per well) the day before transfection.
Cells were transfected following a standard calcium phosphate
3000 (br, O–H), 2900 (m, C–H), 2848 (m, C–H), 1681 (m, C@O),
1603 (w), 1442 (m), 1361 (m), 1301 (m), 1215 (m), 1116 (s),
1026 (m), 972 (s), 821 (m) cmꢁ1. MS (ESI+): m/z (%) 545 ([M+H]+,
100), 413 (30), 245 (40). HRMS (ESI+): calcd for C32H37N2O6
[M+H]+ 545.2646; found: 545.2641. Elem. Anal. calcd for
DNA precipitation protocol using 10 ng Gal4-RARa or Gal4-RXRa
vector together with 2 ng of b-galactosidase expression vector
(50 ng total amount of DNA per well). Expression vectors have
been described elsewhere.27 Sixteen hours after transfection, cells
were washed with PBS, replenished with fresh medium supple-
mented with 5% charcoal-treated FBS, and left to recover for 2 h
C32H36N2O6: C, 70.57; H, 6.66; N, 5.14; found: C, 70.46; H, 6.68;
N, 5.00.
5.8. Biology
prior to stimulation with 4
cells were stimulated with
CD3254 (Gal4-RXR ), or solvent (0.1% v/v DMSO) for basal activity.
Cells were harvested 6 h (RAR ) or 24 h (RXR ) after ligand
stimulation and luciferase and b-galactosidase activities were mea-
sured using a Dual-Light chemiluminiscence assay system (Applied
Biosystems) following the manufacturer’s instructions. Normalized
luciferase/b-galactosidase ratio was used to calculate the ligand-
dependent RAR/RXR transactivation activity as fold induction over
control non-stimulated cells. In a separate experiment, we tested
the ability of compounds to function as RAR/RXR antagonists by
l
M of the test compounds. As control,
5.8.1. Kinase assays
1
lM
atRA (Gal4-RAR ),
a
1 lM
Kinase activity was measured using a LANCE Ultra time-re-
solved fluorescence resonance energy transfer (TR-FRET) assay
and purified recombinant IKKs (Carna Biosciences). Kinases 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 concentra-
a
a
a
tion of 4 nM (IKK
and Ulight-rpS6 (Perkin Elmer) were used as peptide substrates
for IKK /b and IKK , respectively. All assays were performed with
an ATP concentration close to the apparent Km for each enzyme
(1.25 M for IKK /b and 5 M for IKK ). After 1 h (IKKb) or 2 h
incubation (IKK at room temperature, the reaction was
a), 1 nM (IKKb), or 2 nM (IKKe). 50 nM Ulight-Ij-
Ba
a
e
l
a
l
e
stimulating cells with 0.1
lM atRA or 0.1
lM CD3254 in the
a/e
)
absence or in the presence of 4 lM of the AdArs. Luciferase activity
stopped by addition of 20 mM EDTA in LANCE detection buffer,
containing 2 nM Europium-labelled phospho-specific antibody
(Perkin Elmer). Two hours later, the TR-FRET signals at 615 and
was normalized by b-galactosidase activity following background
subtraction, and all activities were represented as percentage of
control cells stimulated in the presence of atRA or CD3254.
665 nm were measured upon excitation at 340 nm with a 50 ls de-
lay in a Victor V multilabel reader. LANCE counts were normalized
following Perkin Elmer’s instructions. IC50 values for active com-
pounds were determined using a 8 point titration experiment
and GraphPrism software.
5.8.5. RXR LBD-coactivator peptide recruitment assay
We used two different TR-FRET-based homogeneous assays to
evaluate the interactions of recombinant GST-RXR
a LBD with
fluorescein labeled D22 coactivator peptide (Fl-LPYEGSLLLKLLRAP-
VEEV) (Life Technologies) and with biotinylated SRC-1-676-700
peptide (CPSSHSSLTERHKILHRLLQEGSPS-K-biotin) (AnaSpec). In
5.8.2. Cell proliferation assay
A luminescence based CellTiter-GLO assay (Promega) was used
to determine the ATP levels as a measure of cell viability. Jurkat
cells were grown in RPMI supplemented with 10% heat inactivated
FBS. Cells were seeded the day before treatment in medium con-
taining 0.5% FBS in a 384 well CulturPlate (Perkin Elmer) at a den-
sity of 10,000 cells per well (400,000 cells/mL). Cells were treated
with increasing concentrations of the compounds in triplicate
points. Control cells were treated with the same amount of solvent,
DMSO, up to 0.1% v/v. After 24 h of treatment, a CellTiter-GLO as-
say was carried out following the manufacturer’s instructions. A
parallel CellTiter-GLO assay was performed at time 0 to determine
cell growth and distinguish between inhibition of cell growth
(cytostatic effect) versus induction of cell killing (cytotoxic effect).
both assays, 5 nM GST-RXR
bated at room temperature with 150 nM coactivator peptide in a
20 l final volume of binding buffer (25 mM Hepes pH 7.4
100 mM NaCl2, 0.025% BSA, 5% glycerol, 0.005% Tween-20 and
2.5 mM DTT). Increasing concentrations of ligand or solvent con-
trol (61% DMSO v/v) were included as indicated. For detection,
2 nM Terbium labeled anti-GST antibody (Life Technologies) was
included in the Fl-D22 reaction and the TR-FRET signal was mea-
sured at 495 nm and 520 nm after 4 h of incubation at RT. The
520/495 ratio was calculated for each sample as a direct measure
a LBD (Life Technologies) was incu-
l
of RXRa-D22 interaction. Alternatively, 2 nM Eu-W1024-labeled
anti-GST antibody (Perkin Elmer) together with 37.5 nM (4:1
biotin/streptavidin ratio) streptavidin conjugated SureLight-allo-
phycocyanin (Perkin Elmer) were included in the binding reaction
with biotin-SRC-1 peptide. The TR-FRET signal at 615 nm and
665 nm were measured after 4 h of incubation and the 665/615
5.8.3. DEVDase assay
Jurkat cells (20,000 per well) were seeded in 0.5% FBS-RPMI in a
384 well black OptiPlate. Following a 4 h incubation with 5
l
M of
ratio was used as RXR
a-SRC-1 binding. To compare the results
test compounds, cells were lysed for 15 min (25 mM PIPES pH 7,
from different experiments, the percentage of change (Delta F%)

1302
J. García-Rodríguez et al. / Bioorg. Med. Chem. 22 (2014) 1285–1302
Peterson, V. J.; Zhang, X. K.; Muhammad, R.; Lu, J. S.; Willman, C.; VanBuren, E.;
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with respect to negative control samples containing no GST-RXR
was used as defined by CisBio:
a
Ratio of sample ꢁ ratio of negative control
Delta F% ¼
ꢃ 100
Ratio of negative control
18. Lopez-Hernandez, F. J.; Ortiz, M. A.; Bayon, Y.; Piedrafita, F. J. Mol. Cancer Ther.
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This work was supported by funds from the Spanish MICINN
(SAF2010-17935-FEDER), Xunta de Galicia (Grant 08CSA052383PR
from DXI+D+i; Consolidación 2006/15 from DXPCTSUG;
INBIOMED-FEDER ‘Unha maneira de facer Europa’), EU (FP7/REG-
POT-2012-2013.1] under grant agreement No. 316265, BIOCAPS),
and NIH grant (CA133475, to F.J.P.).
27. Lorenzo, P.; Ortiz, M. A.; Álvarez, R.; Piedrafita, F. J.; de Lera, Á. R.
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Supplementary data
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