Insights into the mechanism of the site-selective sequential palladium-catalyzed cross-coupling reactions of dibromothiophenes/ dibromothiazoles and arylboronic acids. Synthesis of PPARβ/δ agonists

Article abstract of DOI:10.1039/b612235c

A reactivity study, aided by NMR spectroscopy, allowed a mechanistic rationale to be postulated for the palladium-catalyzed regioselective coupling of arylboronic acid (and arylstannane where feasible) at the position next to the sulfur atom in functional

Full text of DOI:10.1039/b612235c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Insights into the mechanism of the site-selective sequential palladium-
catalyzed cross-coupling reactions of dibromothiophenes/dibromothiazoles
and arylboronic acids. Synthesis of PPARb/d agonists†
Raquel Pereira,a Audrey Furst,b Beatriz Iglesias,a Pierre Germain,b Hinrich Gronemeyerb and
Angel R. de Lera*a
Received 24th August 2006, Accepted 26th October 2006
First published as an Advance Article on the web 13th November 2006
DOI: 10.1039/b612235c
A reactivity study, aided by NMR spectroscopy, allowed a mechanistic rationale to be postulated for
the palladium-catalyzed regioselective coupling of arylboronic acid (and arylstannane where feasible) at
the position next to the sulfur atom in functionalized dibromothiophenes and dibromothiazoles. The
analysis of the NMR spectra (using 19F from the boronic acid CF3 group and 31P from the phosphine of
the catalyst as probes) of the entire reaction starting from the dibromoheterocycles allowed the
qualitative proposal that the transmetalation is the rate-limiting step for both sequential substitution
processes. The extremely facile oxidative addition at the C–Br bond next to the sulfur atom of the
heterocycle instead determines the positional selectivity. An additional Stille reaction then replaced the
second halogen, providing the trisubstituted heterocyclic scaffolds of PPAR ligands, which displayed
PPARb/d agonist activity, as revealed by reporter assays in living cells.
of a functionalized dibromothiazole and organometallics.5 This
Introduction
approach serves as an attractive alternative to the classical
In recent years, there has been a growing interest in the study
Hantzsch reaction commonly used for thiazole formation, and
of site-selective palladium-catalyzed cross-coupling reactions of
facilitates the preparation of a group of derivatives with increased
dihaloheteroarenes.1–4 These halogenated heterocycles have proved
structural diversity by incorporating aryl and heteroaryl substi-
to be suitable precursors for the synthesis of a plethora of carbon-
tuents at the thiazole C4-position (Scheme 1).
substituted heterocycles with interesting biological activities.
We envisioned that the synthetic methodology thus developed
We have recently described a new total synthesis of thiazole
derivative GW501516 1a, a potent PPARb/d selective agonist,
based on the palladium-catalyzed site-selective cross-coupling
could be successfully applied to the preparation of some other
substituted heterocyclic compounds with promising biological
activity. For instance, the case of highly substituted thiophenes,
which find wide application in materials science and medicinal
chemistry. In fact, the thiophene analogues of GW501516, namely
aDepartamento de Qu´ımica Orga´nica, Universidade de Vigo, 36310, Vigo,
3a, 3b,6 4a and 4b are interesting synthetic targets as potential
Spain
bDepartment of Cell Biology and Signal Transduction, Institut de
Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (IGBMC)/CNRS/
INSERM/ULP, BP 163, 67404, Illkirch Cedex, C. U. de Strasbourg, France
† Electronic supplementary information (ESI) available: NMR monitoring
experiments, experimental procedures, spectroscopic characterization data
for selected compounds, 1H and 13C NMR spectra of synthesized products.
See DOI: 10.1039/b612235c
PPARb/d selective agonists, an activity that has been proven
for 3b6 (Fig. 1). Analogues 3–4 can be efficiently prepared using
the same methodology, namely site-selective palladium-catalyzed
cross-coupling reactions of the appropriate starting materials
(5a, 5b) with organometallics. We report a new total synthesis
of compounds 3–4 based on site-selective palladium-catalyzed
Scheme 1
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Results and discussion
Regioselectivity on the palladium catalyzed cross-coupling of 5a
and 5b
With the focus on the potential PPARb/d targets, we selected
methyl 4,5-dibromothiophene-2-carboxylate (5a7) and ethyl 3,5-
dibromothiophene-2-carboxylate (5b,8 Scheme 2) as substrates.
These are positional isomers of dibromothiophene carboxylate
which keep the ester and the bromine vicinal to the sulfur atom,
but differ on the location of the second halogen. They are readily
prepared from commercially available 4,5-dibromothiophene-2-
carboxylic acid and 2,3,5-tribromothiophene,9 respectively. Aimed
at establishing the optimal experimental conditions for the regiose-
lective cross-coupling reactions of substrates 5a and 5b, a thorough
investigation of their reactions with different organometallics
was carried out. Previous reports on the reactivity of dibromo-
thiophenes have shown regioselective reactions occurring on the
carbon atom close to the sulfur. The disubstituted derivative was
also produced as the result of a competitive process.2d,f ,g,h,j Whereas
these precedents might anticipate position-selective replacement
of the Br at C5 in 5a by the aryl group, the presence of an
electron-withdrawing substituent vicinal to the C3–Br bond in
5b was expected to alter the reactivity at this position, making the
prediction on selectivity less reliable.
In keeping with the anticipated enhanced reactivity at C5, the
first organometallic component of the sequence was chosen to be
the 4-trifluoromethylphenyl metal derivative 6 en route to targets 3
and 4. We surveyed the common metal derivatives 6 prepared
from commercially available 4-bromotrifluoromethylbenzene.10
The results of the palladium-catalyzed cross-coupling reactions
of 5a and 5b with 6 are listed in Table 1. The electron-withdrawing
nature of the trifluoromethyl substituent reduces the reactivity of
the organometal partner 6 relative to the unsubstituted analogue,
and temperatures in excess of 70 ◦C were required for efficient
coupling.
Fig. 1
cross-coupling reactions with organometallic compounds, and the
study of their biological activity as PPAR ligands.
From a mechanistic point of view, palladium-catalyzed cross-
coupling reactions have been thoroughly studied. The complex
reaction mechanism can be simplified to include three key steps:
oxidative addition, transmetalation and reductive elimination
(Fig. 2). In close dependence with the substrate and the reaction
conditions employed for the cross-coupling process either the
oxidative addition, the transmetalation or the reductive elimi-
nation have been proposed as the rate-determining step of the
catalytic cycle. Site-selectivity, i.e. the differentiation in favour
of a certain position in di- or polyhalogenated electrophiles can
conceivably occur at any of these three steps. A detailed study
on the regioselectivity of the palladium-catalyzed cross-coupling
reactions of substituted dibromothiophenes and dibromothiazoles
with organometallics and a mechanistic investigation of the site-
selectivity observed in the reaction with arylboronic acids will
also be presented. Our findings support that transmetalation is
the rate-determining step in these coupling reactions.
The organotin derivative 6 (M = SnBu3) coupled at 80 ◦C
with dibromothiophene 5a using the conditions developed by
Farina11 [Pd2(dba)3–AsPh3 as palladium–ligand combination in
NMP] to provide a 96 : 4 mixture of the monocoupled and
bis-coupled derivatives 7a and 8a, respectively, in 78% overall
yield (entry 1). However, use of the same reaction conditions
for 5b at 60 ◦C provided a disappointing 57 : 43 mixture of the
mono-coupled 7b and bis-coupled 8b derivatives in unacceptable
yield (40%), thus proving the anticipated greater reactivity of
the second bromine of 5b relative to 5a (entry 2). Attempts at
reducing the reaction temperature by employing the modification
described by◦Fu, with the bulky PtBu3 [Pd2(dba)3–PtBu3, CsF,
dioxane, 25 C],12 proved unrewarding (entries 3 and 4). The
Fig. 2
Scheme 2
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Table 1 Position-selective Pd-catalyzed cross-coupling of dibromothiophenes 5a and 5b
Yield (%)
Entry
M in 6 (mol equiv.)
Substrate
Reaction conditions
T/◦C
t/h
Selectivity 7 : 8
7
8
5
1
2
3
4
5
6
7
8
9
10
11
12
13i
14i
15i
16i
17i
18i
19i,k
SnBu3 (1.5)
SnBu3 (1.3)
SnBu3 (1.3)
5a
5b
5a
5b
5a
5b
5a
5b
5a
5b
5a
5b
5a
5b
5a
5b
5a
5b
5b
Pd2(dba)3, AsPh3, NMP
Pd2(dba)3, AsPh3, NMP
Pd2(dba)3, PtBu3, CsF, dioxane
80
60
25 to 60
5
4
25
96 : 4a
57 : 43b
92 : 8a
50 : 50b
—
—
—
—
—
75
3
4
71
100
100
100
—
—
52
40c
44
—
28c
—
—
—
—
—
—
d
ZnCl (1.6)
Pd(PPh3)4, THF
70
100
100
100
21
2
d
d
B(OMe)2 (2)
B(OiPr)2 (2)
B(OMe)2 (2)
B(OMe)2 (2)
B(OMe)2 (1.6)
B(OMe)2 (1.2)
B(OMe)2 (2)
Pd(PPh3)4, Tl2CO3, THF
Pd(PPh3)4, Tl2CO3, THF
Pd(PPh3)4, K2CO3, PhMe
Pd(PPh3)4, K2CO3, PhMe
Pd(PPh3)4, K2CO3, PhMe
Pd2(dba)3, PtBu3, Cs2CO3, dioxane
Pd(PPh3)4, K2CO3, PhMe
e
f
2
—
—
f
—
18
95 : 5a
75 : 25b
36 : 64a
70 : 30b
100 : 0a
78 : 22b
89 : 11a
71 : 29b
∼100 : 0b
60 : 40b
100 : 0b
100 : 0b
82
4g
—
18
—
50c,h
31
70
100
70
24
19
25
54
—
<1
—
7
59
—
70c,j
90
—
36
89c
57
25 to 60
50
25
41c
90c
72
96
96
24
20i
21i
B(OMe)2 (1.2)
B(OMe)2 (1.2)
5b
5b
Pd(PPh3)4, K2CO3, PhMe
Pd(PPh3)4, K2CO3, PhMe
50
70
40
90
—
—
44
—
a Selectivity was determined after isolation of the substituted thiophenes by chromatography. b Selectivity was determined by 1H NMR integration of the
crude mixture. c Yield refers to the inseparable mixture of the substituted thiophenes. d The homocoupling product 4,4ꢀ-bistrifluoromethylbiphenyl 9 was
isolated in 100% yield. e Mixture of the esters (Me and Et) of the starting dibromide and the substituted thiophene. f Unidentifiable mixture of products.
g The homocoupling product 4,4ꢀ-bistrifluoromethylbiphenyl 9 was isolated in 20% yield. h The homocoupling product 4,4ꢀ-bistrifluoromethylbiphenyl 9
was isolated in 17% yield. i The reaction mixture was thoroughly degassed with freeze–thaw cycles (3×). j Trace amounts of the homocoupling product 9
were detected. k Reaction progress was monitored by 1H NMR.
reaction appears to stop at about 30–50% conversion and seems
unaffected by higher reaction temperatures (up to 60◦C). Likewise,
the reaction of the organozinc derivative [Pd(PPh3)4, THF]13
or the dimethyl organoboronate under thallium carbonate rate-
acceleration conditions [Pd(PPh3)4, Tl2CO3, THF]14 led to recov-
ery of the starting dibromide accompanied by the homocoupling
product, 4,4ꢀ-bis-trifluoromethylbiphenyl 9 (entries 5, 6 and 7),
or scrambling of ester groups both in the recovered starting
material and in the product (entry 8). The use of the di-isopropyl
organoboronate under the same reaction conditions (entries 9
and 10) led to an intractable mixture of products. Changing
the base of the Suzuki reaction to K2CO3 and using toluene
as solvent15 provided a better ratio (5a 95 : 5, entry 11; 5b
75 : 25, entry 12) of the above reaction mixture, in particular
for 5a, but the homocoupling product 9 (entries 11 and 12)
and recovered starting material (entry 12) were also isolated.
Mindful of precedents showing a direct influence of oxygen in the
formation of homocoupling products in Pd-catalyzed reactions,16
the reaction flasks were thoroughly degassed. Only trace amounts
of the secondary product 9 were now detected under these
conditions. The disubstituted thiophene 8a was the main product
(36 : 64 ratio) when two equiv. of the boronate were employed with
substrate 5a (entry 13). Furthermore, lowering the quantities of
boronate to 1.6 equivalents, and heating the degassed mixture for
25 h at 70 ◦C proved optimal, and a 90% yield of compound 7a,
the bromothiophene-2-carboxylate selectively substituted at the
C5 position, was obtained (entry 15). However, for the positional
isomer 5b, the ratio of 7b to 8b did not improve even when
the reaction temperature or the amounts of organoborane were
lowered (entries 14 and 16). Attempts at accelerating the cross-
coupling with PtBu3–Cs2CO3 faced incomplete conversion at room
temperature, with no further evolution observed upon heating up
to 60 ◦C (entries 17 and 18).17 Interestingly, at the lowest reaction
temperatures required for detectable coupling of 5b with excess
organoborane (50 ◦C) we observed complete selectivity albeit slow
conversion (72 h). Upon extended reaction time (96 h) selectivity
was lost (entry 19). This result, however, was encouraging and
suggested that selectivity could be achieved through a precise
control of the reaction variables. Lowering the amount of boronate
to 1.2 equivalents and heating for the same time period (96 h)
returned 44% of starting dibromide, although position-selectivity
was maintained (entry 20). Upon further optimization, conditions
were developed (entry 21) that afforded the desired product 7b in
high yield and complete position-selectivity using a slight excess
of organoborane (1.2 equivalent) and heating to 70 ◦C for 24 h.
The positional selectivity of the cross-coupling was determined
by NOE experiments on advanced intermediate 11 of the planned
synthetic sequence (Scheme 3). A second Stille cross-coupling
of compounds 7a and 7b with tetramethyltin in DMA at 90 ◦C
afforded the trisubstituted thiophene derivatives 10a and 10b in
92% and 83% yield, respectively. Reduction of the esters to the
alcohols using LAH furnished the thiophene carbinols 11a and
11b in 93% and 79% yield, respectively. The latter compound is
an intermediate in the previously reported synthesis of analog
3b.6 NOE experiments on 11a revealed cross-peaks at the C3-H
and ArC-H signals upon irradiation of the methyl substituent,
whereas 11b showed cross-peaks at the -CH2OH and C4-H signals
on irradiating its methyl group. These experiments confirm the
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Scheme 3 Reagents and conditions: (a) Me4Sn, PdCl2(PPh3)2, DMA, 90 ◦C, 7 h, 92% (10a); 15 h, 83% (10b); (b) LiAlH4, THF, 0 ◦C, 2.5 h, 93% (11a);
2 h, 79% (11b).
substitution pattern depicted in structures 11a and 11b (and
hence, that of precursors 7a and 7b) and discard the alternative
constitutional isomer, the product of sequential cross-coupling at
C4 or C3 and then at C5.
ambient temperature to afford exclusively the palladium complex
16 resulting from oxidative addition on the carbon atom at position
C5 (Scheme 4), which was isolated and fully characterized.24
None of the conditions listed in Table 1 led to the monosub-
stituted thiophene isomer by coupling at C4 of 5a or C3 of 5b.
The activating nature of the 4-trifluoromethyl group in 7 does
accelerate a second coupling reaction at C4 or C3 but the bis-
coupled product 8 only competes when excess organometallic
reagent is present in the reaction media. The presence of an
electron-withdrawing group at the neighboring position to the
C3–Br bond in 5b increases the reactivity at this position, calling
for a strict control of the reaction conditions in order to achieve
selectivity.
In accordance to our published results on the synthesis of
the thiazole derivatives GW501516 and analogues,5 appropriate
experimental conditions for the selective synthesis of the C-
5 monosubstituted thiophene derivatives 7a and 7b have been
designed, completely avoiding the other possible monosubstituted
products (at C-4 or C-3, respectively), which are not isolated or
even detected.
Mechanistic studies
Scheme 4
Mechanistic studies of cross-coupling reactions using different
techniques (31P NMR, 19F NMR, UV, cyclic voltammetry, etc.)
have been applied to all key steps of the catalytic cycle (oxidative
addition,18 transmetalation or reductive elimination19) as well as
to the entire process.20
More recently, the tools of computational chemistry have
also added a new view on the metal-catalyzed reaction through
the characterization of reaction profiles of the catalytic cycles,
including transition structures.21
In order to gain insights into both the origin of the disubstituted
product and the nature of the rate-determining step of the cross-
coupling processes, the reactions of 5a and 5b were carefully
monitored by NMR using the 31P and 19F signals for the
catalyst and the organometallic reagent, respectively, as probes. In
addition, since similar regioselectivity had been previously noticed
for the dibromothiazole analogues,5 the study was extended to
include both types of dibromoheteroarenes with two functional
groups of different electronic properties.
A great number of studies have focused on the oxidative
addition of aromatic halides to palladium complexes.18,22 Some
lines of evidence suggest that, in many cases, the oxidative
addition is the rate-limiting step of the cycle.23 Strikingly, the
reaction of methyl 4,5-dibromothiophene-2-carboxylate 5a with
stoichiometric quantities of Pd(PPh3)4 in toluene proceeded at
Dibromoheteroarenes 5b, 12, 13,25 14 and 1526 were converted
in a similar fashion into the C-5 (C-2 for the thiazole derivatives)
palladium insertion complexes in good yields (Scheme 4).
Complexes 16, 17, 20 and 21 have been characterized by X-ray
analysis and drawings of their molecular structures are given in
Fig. 3. They all show a square-planar geometry with the heteroaryl
group and the bromine atom in trans disposition, and two
triphenylphosphine ligands completing the coordination sites. The
finding that only the complexes resulting from oxidative addition
on the carbon–halogen bond close to the sulfur were isolated not
only corroborates the reactivity pattern experimentally observed
throughout this study, but also confirms previous reports by
different authors.2,5
In turn, preparation of the oxidative addition palladium com-
plex 22 derived from coupling product 7b required the reaction
temperature to be raised to 90 ◦C. The molecular structure of 22
was also elucidated by single-crystal X-ray analysis and a plot
with thermal ellipsoids is displayed in Fig. 4. The more drastic
conditions employed for oxidative addition of monosubstituted
thiophene 7b further confirm the significant difference in reactivity
between positions C-3 and C-5 of the dibromoheteroarene. This
in turn suggests that the selectivity in the palladium catalyzed
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Fig. 3 Representation of molecular structures of 16, 17, 20 and 21. Ellipsoids are shown at the 30% probability level. Cocrystallized solvent molecules
in 16 and 20, hydrogen atoms of the PPh3 ligands and one set of disordered ligands in 17 and 21 have been omitted for clarity.
the experiment was performed at the temperatures required for the
C–C bond forming process. The reaction of 5b and boronic acid
6 was monitored by 31P NMR spectroscopy using increasingly
higher reaction temperatures. A similar study (not shown: Sup-
porting Information†) was carried out for the remaining analogues
depicted in Scheme 4 with comparable findings.
The oxidative addition of palladium to the C–Br bond of
5b is complete at room temperature after just a few minutes
(spectra acquisition time). The 31P NMR spectrum of the mixture
reveals the presence of three signals: a singlet for complex 18 at
d 23.9 ppm, a second singlet at d − 4.2 ppm, characteristic of
free triphenylphosphine, and a third peak at d 24.2 ppm, tenta-
tively assigned (vide infra) to some palladium–triphenylphospine
species (Supporting Information†). Signals for other substrate–
phosphorus-containing species were conspicuously absent, which
appears to discard alternative oxidative additions on the C–Br
bond more distant from the sulfur atom either alternatively or in
addition to (double oxidative addition) that at C5.
The reaction between monobromothiophene derivative 7b and
Pd(PPh3)4 was also monitored by 31P NMR. The signals can be
attributed to complex 22 (singlet at d 23.8 ppm) and the unidenti-
fied Pd(PPh3)n species at d 24.2 ppm (Supporting Information†).
The presence of this Pd(0) species in solution starting also from a
monobrominated substrate confirms that these signals resonating
at d 24.2 ppm are due to some species comprising only palladium
and triphenylphosphine. On the other hand, heating to 90 ◦C
for 3 h afforded a spectrum exhibiting a peak at d 23.8 ppm,
Fig. 4 Representation of molecular structure of 22. Ellipsoids are shown
at the 30% probability level. Hydrogen atoms of the PPh3 ligands and
the minor component of the disordered CF3 group have been omitted for
clarity.
cross-coupling reactions of dibromothiophene derivatives27 is
primarily due to this rate difference. To confirm this assumption,
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which corresponds to 22, the intensity of which is notably smaller
than that attributed to the palladium species, and this leads to
the conclusion that the oxidative addition process is considerably
slower at C-3 than at C-5.
The reaction of the product substituted at C5 7b with Pd(PPh3)4
◦
at 90 C produces, as seen before by 31P NMR, complex 22 and
the Pd(0)–(PPh3)n species. After addition of 1 mol equivalent of
boronic acid (and base), the latter peak at d 24.2 ppm disappears
in favour of a broad signal at lower field, confirming the nature
of these substances as Pd(0) species in equilibrium with free
triphenylphosphine in the reaction medium. On the other hand,
the peak corresponding to complex 22 progressively diminishes its
intensity until the reaction is complete, which occurs after 15 h
(Supporting Information†). The failure to detect any additional
signal that could be assigned to the proposed transmetalation
intermediate 25 (Scheme 5) seems to confirm that transmetalation
(TM-2) is the rate-limiting step of the process.
The entire coupling process starting from ethyl 3,5-
dibromothiophene-2-carboxylate 5b and an equimolar amount
of Pd(PPh3)4 at 90 ◦C in toluene was then monitored by NMR
(31P and 19F). Signals corresponding to complex 18 (d 23.9 ppm,
31P NMR, Scheme 5) arose rapidly. After addition of 1 mol
equivalent of p-trifluoromethylphenylboronic acid 6 and base (t2),
the peak at d 24.2 ppm shifted to a downfield position, varying
its location throughout the analysis in accordance to its proposed
nature as equilibrating Pd(0) species. The new peak that resonates
at d 23.8 ppm increases its intensity with time (the reaction
requires 13 h at 90 ◦C to reach completion), and corresponds
to complex 22, resulting from the oxidative addition at C-3 of
the monosubstituted product 7b. Signals for additional species
corresponding to other reaction intermediates that could be
The transmetalation step of the catalytic cycle has received
considerable attention, especially in the context of the Stille
reaction.28 In many cases, this has proven to be the rate-limiting
step,29 a consideration that also extends to the Suzuki reaction.30
The characterization of the intermediate palladium complexes 18
and 22 resulting from the oxidative addition of 5b and 7b allowed
the remaining steps of the catalytic cycle to be investigated in
order to elucidate which of them is responsible for the observed
selectivity and which, if different, is the rate-determining step.
Scheme 5 shows possible reaction pathways leading to the
disubstituted product 8b, with indication of chemical shifts (31P
NMR and 19F NMR resonances) for the selected nuclei determined
previously on known isolated intermediates.
Judging from the spectroscopic characterization of the products
resulting from the oxidative addition step just discussed, and the
absence of the alternative oxidative addition complexes 23 (OA-
ꢀ
1, k1 negligible) in solution, we concluded that the disubstituted
product 8b must arise from two coupling reactions occurring in
a sequential manner: first at position C-5 and then at position
C-3. Monitoring the reaction by 31P and 19F NMR confirmed this
assumption and further clarified which of the remaining steps of
the cycle, transmetalation and reductive elimination, must be rate-
limiting.
Scheme 5
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accumulating were absent, thus confirming that transmetalation
is likely the rate-determining step. Consequently, for the first
coupling process, OA-1 and RE-1 are the rapid steps leading to
monosubstituted product 7b, and TM-1 is the slow, limiting event.
Addition of the second molar equivalent of boronic acid and base
(t3) induces slow disappearance (the second coupling requires more
than 15 h) of the signal corresponding to complex 22, pointing to
the transmetalation TM-2 as the rate-limiting step of the second
cross-coupling as well (Scheme 5).
The entire experiment starting from dibromide 5b was likewise
monitored by 19F NMR spectroscopy (Supporting Information†).
19F NMR analysis of the cross-coupling process shows evidence
for formation of the monosubstituted product 7b (d −62.95 ppm),
since the peak corresponding to the boronic acid 6 (d −63.0 ppm)
diminishes in intensity, concomitantly with the oxidative addi-
tion of Pd(PPh3)4 to thiophene 7b to provide complex 22 (d
−62.76 ppm). A second equivalent of boronic acid 6 leads to a
complete transformation of 7b into complex 22 and the latter
into disubstituted product 8b (signals at d −62.85 ppm and d
−62.69 ppm are already present at early stages of the process, t1 =
5 h, albeit their intensity is low). Like the experiment followed by
31P NMR, monitoring the reaction by 19F NMR reveals solely the
oxidative addition species and corroborates that transmetalation
is the limiting process for both cross-coupling reactions, with a
qualitative estimate of k2 (TM-1) slightly higher than k5 (TM-2)
judging from the reaction times required for completion.
In consequence, the control of the reaction temperature and the
quantities of the organometal counterpart are crucial for the
selective synthesis of the monosubstituted coupling products at
the position next to the sulfur atom in both (functionalized)
dibromothiophenes and dibromothiazoles.
Synthesis of PPARb/d agonists
Compound 11b is an intermediate in the previously reported
synthesis of these analogues.6 With the patent synthesis intercepted
at this stage, the remaining steps followed the sequence depicted
in Scheme 6. Alcohols 11a and 11b were converted into the
corresponding chlorides 26a and 26b upon treatment with mesyl
chloride in Et3N. Aryl thiols 276,31 and 2831 displaced the chlorides
at room temperature when treated with Cs2CO3 in CH3CN, and
the esters were then saponified to the desired targets, 3b and its yet
undescribed analogs 3a, 4a and 4b.
Biological studies
The transcriptional activity of the newly synthesized compounds
has been studied using a PPARb/d ‘reporter’ cell line and
compared to the activity of the previously identified potent agonist
GW501516 1a. At 1 lM all investigated compounds (3a, 3b, 4a,
4b) are quite similar in their ability to activate reporter gene
transcription through PPARb/dand work as full agonists (Fig. 5).
Dose–response curves with increasing ligand concentrations were
performed to assess the cellular potency of the synthesized
compounds to PPARb/d (Figs. 6 and 7). Similar curves were
obtained with all four analogues (3a, 3b, 4a, 4b), albeit all of
them exhibited higher EC50 values than the reference compound
GW501516 1a (5 × 10−10, 3 × 10−7, 9 × 10−6, 4 × 10−7, and 1 ×
10−7 M, respectively for 1a, 3a, 3b, 4a and 4b).
With regard to the clear preference for monosubstitution
observed in the first cross-coupling reaction of all the dibromo-
heteroarenes under study, we interpret this as a consequence of
the pronounced difference in reaction rates for the two sequen-
tial oxidative additions: k1 (OA-1) ꢁ k4 (OA-2). Nevertheless,
although the transmetalation is proposed to be the limiting step,
the high temperature required for promoting the coupling turns
the second oxidative addition (OA-2) into a competitive process.
Scheme 6 Reagents and conditions: (a) ClSO2Me, Et3N, CH2Cl2, 0 ◦C, 2 h, then 25 ◦C, 16 h, 97% (26a); 90% (26b); (b) 27 or 28, Cs2CO3, CH3CN, 25 ◦C,
2 h, 87% (29a); 67% (30a); 77% (29b); 80% (30b); (c) K2CO3, MeOH, 80 ◦C, 1 h, 92% (3a); 89% (4a); 88% (3b); 91% (4b).
4520 | Org. Biomol. Chem., 2006, 4, 4514–4525
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Conclusion
Control of the reaction variables, at a particular temperature
and equivalents of arylstannane or arylboronic acid, allowed
the regioselective (or position-selective) coupling at the position
next to the sulfur atom in functionalized dibromothiophenes
and dibromothiazoles. An additional Stille reaction under more
drastic conditions then replaced the second halogen, providing
trisubstituted heterocyclic scaffolds of interest for the diversity-
oriented preparation of PPAR ligands, as shown by the synthesis
of agonists 3 and 4. Mechanistic insights were gleaned from
the analysis of the NMR spectra (19F and 31P as probes) of the
entire reaction starting from the dibromoheterocycles once the
signals for the putative intermediates were previously assigned
and the oxidative addition products properly characterized by
X-ray diffraction. We conclude that the transmetalation is likely
to be the rate-limiting event for both steps of the cycle, whereas
the positional selectivity is determined by the extremely favorable
oxidative addition at the C–Br bond next to the sulfur atom of
the heterocycle, which then enters the transmetalation step at a
rate comparable to that of the oxidative addition at the alternative
C–Br bond. Using the isolated oxidative addition intermediate as
starting material, it was noticed that the coupling to the boronic
acid releases Pd(0) which then inserts into the alternative Pd–Br
bond to complete the second palladium-catalyzed cross-coupling
leading to the doubly substituted derivative.
Fig. 5 Transactivation studies were performed to assess PPARb/dactivity
of synthetic compounds using stably transfected HeLa cells expressing
chimeric proteins containing the GAL4 DNA-binding domain fused to
the ligand binding domain of PPARb/d and a luciferase gene driven by a
pentamer of the Gal4 recognition sequence (‘17m’) in front of the b-globin
promoter, as illustrated at the top. This reporter system is unaffected by
the presence of endogeneous receptors as they cannot recognize the Gal4
binding. Cells were incubated with various synthetic compounds at 1 lM.
Experimental
General
Solvents were dried according to published methods and were
distilled before use. All other reagents were commercial com-
pounds of the highest purity available. Analytical thin-layer
chromatography (TLC) was performed using Merck silica gel (60
F-254) plates (0.25 mm) precoated with a fluorescent indicator.
Column chromatography was performed using Merck silica gel 60
(particle size 0.040–0.063 lm). Melting points (mp) were measured
on a Gallenkamp apparatus and a Stuart SMP10 apparatus
and are uncorrected. Magnetic resonance spectra (NMR) were
Fig. 6 Transactivation studies to assess PPARb/d activity of compounds
3a and 3b. Gal-PPARb/d reporter cells were incubated with increasing
concentrations of 1a (ꢀ), 3a (ꢁ), or 3b (᭺) for 16 hours.
1
recorded on a Bruker AMX-400 [400 MHz for H, 100 MHz
for 13C, 162 MHz for 31P and 376 MHz for 19F] Fourier transform
spectrometer, and chemical shifts are expressed in parts per million
(d) relative to tetramethylsilane (TMS, 0 ppm) or chloroform
(CHCl3, 7.26 ppm for 1H and 77.00 ppm for 13C) as internal
reference, or 85% H3PO4 (31P NMR) or CFCl3 (19F NMR) as
external standard. 13C multiplicities (s, singlet; d, doublet; t, triplet;
q, quartet) were assigned with the aid of the DEPT pulse sequence.
Infrared spectra (IR) were obtained on a MIDAC Prospect
Model FT-IR spectrophotometer. Absorptions are recorded in
wavenumbers (cm−1). Low-resolution mass spectra were taken on
an HP59970 instrument operating at 70 eV. High-resolution mass
spectra were taken on a VG Autospec M instrument. Elemental
analyses were obtained on a Carlo Erba Elemental Analyzer
EA1108.
X-Ray diffraction
Fig. 7 Transactivation studies for compounds 4a and 4b. Gal-PPARb/d
reporter cells were incubated with increasing concentrations of 1a (ꢀ), 4a
(ꢂ), or 4b (᭹) for 16 hours.
Crystallographic data were collected on a Bruker Smart 1000 CCD
diffractometer at CACTI (Universidade de Vigo) at 20 C using
◦
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3
ꢀꢀ
˚
125.7 (d, 2C, JC–F = 3.7 Hz), 129.1 (d, 2C), 129.4 (d, 2C), 129.7
graphite monochromated Mo Ka radiation (k = 0.71073 A),
and were corrected for Lorentz and polarisation effects. The
frames were integrated with the Bruker SAINT32 software package
and the data were corrected for absorption using the program
SADABS.33 The structures were solved by direct methods using
the program SHELXS97.34 All non-hydrogen atoms were refined
with anisotropic thermal parameters by full-matrix least-squares
calculations on F2 using the program SHELXL97.35 Hydrogen
atoms were inserted at calculated positions and constrained
with isotropic thermal parameters. Drawings were produced with
PLATON.36 Crystal data and structure refinement parameters are
summarized in Table 2 (see Electronic Supplementary Informa-
tion). Selected bond distances and angles are listed in Table 3 (see
Electronic Supplementary Information).‡
2
2
(s, JC–F = 33.8 Hz), 130.5 (s, JC–F = 32.6 Hz), 132.8 (s), 135.7
(d, C3), 136.3 (s), 138.1 (s), 138.4 (s), 144.2 (s), 162.1 (s, CO2Me)
=
ppm. IR (NaCl) m 2956 (w, C–H), 1718 (s, C O), 1617 (w), 1451
(w), 1326 (s), 1294 (m), 1252 (m), 1168 (m), 1124 (s), 1069 (s), 839
(m) cm−1. MS m/z (%) 432 ([M + 2]+, 7), 430 (M+, 100), 411 (12),
400 (15), 399 ([M − OCH3]+, 71), 327 (25), 302 (15). HRMS (EI+)
calcd for C20H12F6O2S 430.0462, found 430.0461. Anal. Calcd for
C20H12F6O2S: C, 55.82; H, 2.81; S, 7.45. Found: C, 55.77; H, 2.78;
S, 7.40%.
Preparation of 7a. General procedure for Suzuki reactions
To a cold (−78 ◦C) solution of 4-bromotrifluoromethylbenzene
(0.05 g, 0.22 mmol) in THF (1 mL) was added dropwise n-BuLi
(0.16 mL, 1.44 M in hexane, 0.23 mmol), and the mixture was
stirred for 30 min at −78 ◦C. Then B(OMe)3 (0.03 mL, 0.23 mmol)
was added dropwise. After stirring for an additional 30 min, the
mixture was allowed to warm to 25 ◦C. The solvent was removed
and toluene (1 mL) was added. The solution was transferred
to a Schlenk tube charged with methyl 4,5-dibromothiophene-
2-carboxylate 5a (0.05 g, 0.17 mmol) and Pd(PPh3)4 (0.01 g,
0.008 mmol) in toluene (0.5 mL). After addition of K2CO3
(0.11 mL, 3 M in H2O, 0.33 mmol), the reaction mixture was
thoroughly degassed with three freeze–thaw cycles, the Schlenk
tube was sealed, placed in a 70 ◦C oil bath and stirred for 25 h. After
cooling to room temperature, water was added and the mixture
was extracted with TBME (3×). The combined organic extracts
were dried (Na2SO4), and the solvent was evaporated. Purification
of the residue by chromatography (SiO2, 98 : 2 hexane–AcOEt)
yielded 0.05 g (90%) of thiophene 7a as a white solid (mp 62 ◦C,
hexane).
Preparation of methyl 4-bromo-5-(4-trifluoromethylphenyl)-
thiophene-2-carboxylate (7a). General procedure for Stille
reactions
To a solution of Pd2(dba)3 (0.05 g, 0.05 mmol) in NMP (0.5 mL)
was added, in one portion, AsPh3 (0.1 g, 0.33 mmol), followed
by a solution of methyl 4,5-dibromothiophene-2-carboxylate 5a
(0.5 g, 1.67 mmol) in NMP (2 mL). After stirring at 25 ◦C for
10 min, a solution of tributyl(4-trifluoromethylphenyl)stannane 6
(1.1 g, 2.5 mmol) in NMP (2.5 mL) was added and the resulting
mixture was heated to 80 ◦C for 5 h. After cooling down to
◦
25 C, a KF saturated solution was added and the mixture was
stirred for 30 min. It was then extracted with TBME (3×), the
combined organic extracts were washed with H2O (2×) and KF
saturated solution, dried (Na2SO4) and evaporated. Purification
of the residue by chromatography (SiO2, 98 : 2 hexane–AcOEt)
afforded 0.46 g (75%) of a white solid (mp 62 ◦C, hexane) identified
as 7a and 0.02 g (3%) of 8a as a white solid (mp 102 ◦C, hexane–
AcOEt). Spectroscopic data for 7a: 1H NMR (400 MHz, CDCl3)
d 3.92 (s, 3H, CO2CH3), 7.72 (d, J = 8.3 Hz, 2H, H3ꢀ + H5ꢀ), 7.76
(s, 1H, H3), 7.79 (d, J = 8.3 Hz, 2H, H2ꢀ + H6ꢀ) ppm. 13C NMR
(100 MHz, CDCl3) d 52.6 (q, CO2CH3), 109.0 (s, C4), 123.8 (s,
CF3, 1JC–F = 272.3 Hz), 125.7 (d, 2C, C3ꢀ + C5ꢀ, 3JC–F = 3.7 Hz),
Preparation of methyl 4-methyl-5-(4-trifluoromethylphenyl)-
thiophene-2-carboxylate (10a). General procedure for the second
Stille coupling
A Schlenk tube was charged with methyl 4-bromo-5-(4-trifluoro-
methylphenyl)thiophene-2-carboxylate (7a) (0.42 g, 1.16 mmol),
PdCl2(PPh3)2 (0.04 g, 0.06 mmol), SnMe4 (0.◦41 g, 2.32 mmol)
and DMA (5 mL), sealed and placed in a 90 C oil bath. After
stirring for 7 h, the reaction mixture was cooled to 25 ◦C. A
NH4Cl saturated aqueous solution was then added and the
mixture was extracted with TBME (3×). The combined organic
extracts were dried (Na2SO4), and the solvent was evaporated.
Purification of the residue by chromatography (SiO2, 95 : 5
hexane–AcOEt) afforded 0.32 g (92%) of methyl 4-methyl-5-(4-
trifluoromethylphenyl)thiophene-2-carboxylate (10a) as a white
solid (mp 65 ◦C, hexane–TBME). 1H NMR (400 MHz, CDCl3) d
2.32 (s, 3H, C4-CH3), 3.89 (s, 3H, CO2CH3), 7.58 (d, J = 8.2 Hz,
2H, H2ꢀ + H6ꢀ), 7.64 (s, 1H, H3), 7.69 (d, J = 8.2 Hz, 2H, H3ꢀ
+ H5ꢀ) ppm. 13C NMR (100 MHz, CDCl3) d 14.9 (q, C4-CH3)
52.2 (q, CO2CH3), 124.0 (s, CF3, 1JC–F = 272.3 Hz), 125.7 (d, 2C,
C3ꢀ + C5ꢀ, 3JC–F = 3.7 Hz), 129.2 (d, 2C, C2ꢀ + C6ꢀ), 130.1 (s, C4ꢀ,
2JC–F = 32.5 Hz), 131.5 (s), 135.1 (s), 137.0 (d, C3), 137.3 (s), 143.3
(s), 162.5 (s, CO2Me) ppm. IR (NaCl) m 2955 (m, C–H), 2927 (m,
129.4 (d, 2C, C2ꢀ + C6ꢀ), 131.1 (s, C4ꢀ, JC–F = 32.7 Hz), 133.0
2
(s), 135.5 (s), 137.2 (d, C3), 143.1 (s), 161.4 (s, CO2Me) ppm. IR
=
(NaCl) m 2956 (w, C–H), 1724 (s, C O), 1616 (m), 1532 (w), 1513
(m), 1448 (m), 1326 (s), 1289 (s), 1249 (s), 1169 (s), 1129 (s), 1073
(s), 1017 (m), 830 (s), 750 (m) cm−1. MS m/z (%) 368 ([M + 2]+
[81Br], 5), 366 (M+ [81Br], 97), 365 ([M + 1]+ [79Br], 15), 364 (M+
[79Br], 91), 335 ([M − OMe]+ [81Br], 100), 333 ([M − OMe]+ [79Br],
95), 226 (54), 69 (22). HRMS (EI+) calcd for C13H881BrF3O2S
365.9360 and C13H879BrF3O2S 363.9380, found 365.9358 and
363.9379. Anal. Calcd for C13H8BrF3O2S: C, 42.76; H, 2.21; S,
8.78. Found: C, 43.04; H, 2.27; S, 8.70%. Spectroscopic data
for methyl 4,5-bis(4-trifluoromethylphenyl)thiophene-2-carboxylate
(8a): 1H NMR (400 MHz, CDCl3) d 3.94 (s, 3H, CO2CH3), 7.35 (d,
J = 8.1 Hz, 2H, ArH), 7.40 (d, J = 8.1 Hz, 2H, ArH), 7.58 (d, J =
8.2 Hz, 4H, ArH), 7.85 (s, 1H, H3) ppm. 13C NMR (100 MHz,
1
CDCl3) d 52.4 (q, CO2CH3), 123.7 (s, CF3, JC–F = 272.4 Hz),
1
3
123.8 (s, CF3, JC–F = 272.4 Hz), 125.6 (d, 2C, JC–F = 3.7 Hz),
=
C–H), 2852 (w, C–H), 1717 (s, C O), 1615 (m), 1551 (w), 1447
(s), 1325 (s), 1292 (s), 1251 (s), 1193 (s), 1168 (s), 1127 (s), 1073
‡ CCDC reference numbers 612416–612420 for compounds 16, 17, 20, 21
and 22, respectively. For crystallographic data in CIF or other electronic
format see DOI: 10.1039/b612235c
(s), 1016 (m), 842 (s), 753 (m) cm−1. MS m/z (%) 302 ([M + 2]+, 5),
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301 ([M + 1]+, 15), 300 (M+, 100), 197 (39), 171 (20), 128 (28),
115 (21), 69 (17). HRMS (EI+) calcd for C14H11F3O2S 300.0432,
found 300.0430. Anal. Calcd for C14H11F3O2S: C, 55.99; H, 3.70;
S, 10.67. Found: C, 55.56; H, 3.64; S, 11.03%.
Preparation of 5-chloromethyl-3-methyl-2-(4-trifluoromethyl-
phenyl)thiophene (26a). General procedure for the preparation of
chlorides from alcohols
Methanesulfonyl chloride (0.05 mL, 0.88 mmol) was added drop-
wise to the solution of [4-methyl-5-(4-trifluoromethylphenyl)thien-
2-yl]methanol 11a (0.06 g, 0.22 mmol) and Et3N (0.12 mL,
0.88 mmol) in CH2Cl2 (2 mL) at 0 ◦C. After 2 h at 0 ◦C, the
mixture was stirred at 25 ◦C for 16 h. Then CH2Cl2 (3 mL)
was added and the mixture was washed with saturated NaHCO3
aqueous solution (2×) and water (2×). Drying (Na2SO4) and
evaporation of the solvent afforded 0.62 g (97%) of chloride 26a
as a yellow oil, which was used in the next step without further
purification, due to its instability. A small sample was purified by
column chromatography (SiO2–C18, CH3CN) for characterization
Preparation of [4-methyl-5-(4-trifluoromethylphenyl)thien-
2-yl]methanol (11a). General procedure for the reduction of esters
to alcohols
A solution of methyl ester 10a (0.2 g, 0.67 mmol) in diethyl ether
(1 mL) was added to a cold (0 ◦C) suspension of LiAlH4 (0.03 g,
0.80 mmol) in Et2O (1 mL). The reaction mixture was stirred at
◦
0 C for 2.5 h, after which time a mixture of MeOH–H2O (90 :
10) was added, followed by a 10% NH4Cl aqueous solution. After
extraction with TBME (3×), the combined organic extracts were
washed with H2O, dried (Na2SO4) and evaporated. Purification
of the residue by column chromatography (SiO2, 80 : 20 hexane–
AcOEt) afforded 0.17 g (93%) of a colourless oil identified as [4-
1
purposes. H NMR (400 MHz, CDCl3) d 2.29 (s, 3H, C3-CH3),
4.77 (s, 2H, CH2Cl), 6.95 (s, 1H, H4). 7.55 (d, J = 8.2 Hz, 2H,
H2ꢀ + H6ꢀ), 7.66 (d, J = 8.2 Hz, 2H, H3ꢀ + H5ꢀ) ppm. 13C NMR
(100 MHz, CDCl3) d 14.9 (q, C3-CH3), 40.4 (t, CH2Cl), 124.1 (s,
CF3, 1JC–F = 271.7 Hz), 125.5 (d, 2C, C3ꢀ + C5ꢀ, 3JC–F = 3.7 Hz),
1
methyl-5-(4-trifluoromethylphenyl)thien-2-yl]methanol (11a). H
NMR (400 MHz, CDCl3) d 1.83 (t, J = 6.0 Hz, 1H, OH), 2.30 (s,
3H, C4ꢀ-CH3), 4.80 (d, J = 6.0 Hz, 2H, 2H1), 6.87 (s, 1H, H3ꢀ),
7.55 (d, J = 8.6 Hz, 2H, H2ꢀꢀ + H6ꢀꢀ), 7.66 (d, J = 8.6 Hz, 2H, H3ꢀꢀ
+ H5ꢀꢀ) ppm. 13C NMR (100 MHz, CDCl3) d 14.9 (q, C4ꢀ-CH3),
2
129.0 (d, 2C, C2ꢀ + C6ꢀ), 129.4 (s, C4ꢀ, JC–F = 32.2 Hz), 131.9
(d, C4), 134.2 (s), 137.6 (s), 137.9 (s), 138.9 (s) ppm. IR (NaCl) m
2927 (w, C–H), 1615 (m), 1407 (w), 1325 (s), 1259 (m), 1167 (s),
1126 (s), 1069 (s), 1016 (m), 840 (s), 678 (w) cm−1. MS m/z (%)
292 (M+ [37Cl], 7), 291 ([M + 1]+ [35Cl], 3), 290 (M+ [35Cl], 19), 257
(5), 256 (16), 255 ([M − Cl]+, 100), 185 (3). HRMS (EI+) calcd
for C13H1035ClF3S 290.0144 and C13H1037ClF3S 292.0114, found
290.0149 and 292.0118.
1
59.9 (t, C1), 124.1 (s, CF3, JC–F = 272.0 Hz), 125.4 (d, 2C, C3ꢀꢀ
3
+ C5ꢀꢀ, JC–F = 3.8 Hz), 128.9 (d, 2C, C2ꢀꢀ + C6ꢀꢀ), 129.0 (s, C4ꢀꢀ,
2JC–F = 32.5 Hz), 129.7 (d, C3ꢀ), 134.1 (s), 136.1 (s), 138.3 (s), 142.9
(s) ppm. IR (NaCl) m 3600–3000 (br, O–H), 2927 (w, C–H), 1614
(m), 1408 (w), 1326 (s), 1166 (m), 1115 (s), 1070 (m), 1015 (m),
840 (m) cm−1. MS m/z (%) 274 ([M + 2]+, 6), 273 ([M + 1]+, 16),
272 (M+, 100), 271 ([M − 1]+, 15), 255 (36), 243 (51), 239 (17), 86
(12), 84 (18). HRMS (EI+) calcd for C13H11F3OS 272.0483, found
272.0481.
Preparation of methyl (2-methyl-4-[4-methyl-5-(4-trifluoromethyl-
phenyl)thien-2-ylmethylthio]phenoxy)acetate (29a). General
procedure for the nucleophilic displacement of chlorides by thiols
Cs2CO3 (0.23 g, 0.70 mmol) was added to a solution of
chloride 26a (0.09 g, 0.32 mmol) and methyl (4-mercapto-2-
methylphenoxy)acetate 27 (0.09 g, 0.42 mmol) in CH3CN (2 mL).
After stirring the mixture at 25 ◦C for 2 h, water was added
and the solution was extracted with TBME (3×). The combined
organic extracts were washed with brine (2×), dried (Na2SO4)
and evaporated. Purification of the residue by chromatography
(SiO2, 87 : 10 : 3 hexane–AcOEt–Et3N) afforded 0.13 g (87%) of
General procedure for the synthesis of palladium complexes 16, 17,
18, 19, 20, 21 and 22
A mixture of Pd(PPh3)4 (1 mmol) and bromo heterocycle (1 mmol)
in toluene (0.1 M) was deoxygenated and stirred overnight in
a Schlenk flask under argon at room temperature for 16, 17,
18, 19, 20, 21 and 90 ◦C for 22. The resultant yellow solution
or suspension was evaporated to dryness in vacuo. The solid
residue was triturated with Et2O and the ether solution discarded.
Washing was repeated twice and the residual product purified
by recrystallization from CHCl3-hexane or CH2Cl2–hexane. Data
collection and refinement parameters for X-ray structures of
complexes 16, 17, 20, 21 and 22 are given in Table 2 (See
Electronic Supplementary Information). For the spectroscopic
characterization of complexes 16–22 see also ESI.†
1
thiophene 29a as a yellow solid. H NMR (400 MHz, CDCl3) d
2.24 (s, 3H, Ar-CH3) 2.26 (s, 3H, Ar-CH3), 3.79 (s, 3H, CO2CH3),
4.16 (s, 2H, SCH2), 4.64 (s, 2H, 2H2), 6.62 (d, J = 8.4 Hz, H6ꢀ),
6.66 (s, 1H, H3ꢀꢀ), 7.19 (dd, J = 8.4, 2.1, 1H, H5ꢀ), 7.23 (br s, 1H,
H3ꢀ), 7.52 (d, J = 8.1 Hz, 2H, H2ꢀꢀꢀ + H6ꢀꢀꢀ), 7.64 (d, J = 8.1 Hz,
2H, H3ꢀꢀꢀ + H5ꢀꢀꢀ) ppm. 13C NMR (100 MHz, CDCl3) d 14.9 (q,
Ar-CH3), 16.0 (q, Ar-CH3), 35.4 (t, CH2S), 52.1 (q, CO2CH3), 65.4
(t, C2), 111.4 (d, C6ꢀ), 124.0 (s, CF3, 1JC–F = 271.7 Hz), 125.3 (d,
2C, C3ꢀꢀꢀ + C5ꢀꢀꢀ, 3JC–F = 3.3 Hz), 126.5 (s), 128.1 (s), 128.6 (d, 2C,
C2ꢀꢀꢀ + C6ꢀꢀꢀ), 128.7 (s, C4ꢀꢀꢀ, 2JC–F = 32.5 Hz), 130.5 (d, C3ꢀꢀ), 130.8
(d, C5ꢀ), 133.9 (s), 135.0 (d, C3ꢀ), 135.3 (s), 138.2 (s), 140.2 (s),
155.7 (s), 169.2 (s, C1) ppm.
31P NMR monitoring of the heteroarenes cross-coupling reaction
The heteroarene (1 mmol) and Pd(PPh3)4 (0.1 mmol) were added to
d8-toluene (0.75 mL) in an NMR tube under an argon atmosphere.
The tube was warmed to 90 ◦C (0.5 h or 1 h). 31P NMR data were
obtained at this temperature. Then the organometallic (1 mmol)
and base (2 mmol) were added, and the reaction was monitored by
31P NMR at 90 ◦C until the organometallic reagent was consumed.
New portions of organometallic (1 mmol) and base (2 mmol)
were subsequently added and 31P NMR spectroscopic data were
collected at various reaction times.
Preparation of (2-methyl-4-[4-methyl-5-(4-trifluoromethyl-
phenyl)thien-2-ylmethylthio]phenoxy)acetic acid (3a). General
procedure for the hydrolysis of esters
A solution of methyl ester 29a (0.11 g, 0.24 mmol) and 3 M K2CO3
(0.8 mL, 2.4 mmol) in MeOH (2.5 mL) was heated to 80 ◦C for 1 h.
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◦
After cooling to 25 C, the mixture was acidified with 10% HCl
References
(pH ≈ 3). It was then extracted with AcOEt (5×), and the extracts
were dried (Na2SO4) and evaporated. Purification of the residue by
recrystallization (hexane–CHCl3) afforded 0.10 g (92%) of acid 3a
as a white solid (mp 100 ◦C). 1H NMR (400 MHz, CD3COCD3)
d 2.05 (s, 3H, Ar-CH3), 2.08 (s, 3H, Ar-CH3), 4.10 (s, 2H, SCH2),
4.58 (s, 2H, 2H2), 6.59 (s, 1H, H3ꢀꢀ), 6.66 (d, J = 8.5, 1H, H6ꢀ),
7.06 (d, J = 8.5, 1H, H5ꢀ), 7.10 (s, 1H, H3ꢀ), 7.48 (d, J = 8.0 Hz,
2H, H2ꢀꢀꢀ + H6ꢀꢀꢀ), 7.58 (d, J = 8.0 Hz, 2H, H3ꢀꢀꢀ + H5ꢀꢀꢀ) ppm.
13C NMR (100 MHz, CD3COCD3) d 15.2 (q, Ar-CH3), 16.2 (q,
Ar-CH3), 35.2 (t, CH2S), 65.5 (t, C2), 112.6 (d, C6ꢀ), 125.3 (s, CF3,
1JC–F = 270.7 Hz), 126.4 (d, 2C, C3ꢀꢀꢀ + C5ꢀꢀꢀ, 3JC–F = 3.7 Hz), 127.0
1 (a) I. Kaswasaki, M. Yamashita and S. Ohta, J. Chem. Soc., Chem.
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2
(s), 128.4 (s), 129.0 (s, C4ꢀꢀꢀ, JC–F = 32.2 Hz), 129.7 (d, 2C, C2ꢀꢀꢀ
+ C6ꢀꢀꢀ), 131.3 (d, C5ꢀ), 131.8 (d, C3ꢀꢀ), 135.1 (s), 135.1 (d, C3ꢀ),
135.6 (s), 139.5 (s), 141.9 (s), 156.8 (s), 170.1 (s, C1) ppm. IR
2 (a) J. O. Karlsson, S. Gronowitz and T. Frejd, J. Org. Chem., 1982,
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=
(NaCl) m 3400 − 2700 (br, O–H), 2925 (w, C–H), 1734 (s, C O),
1614 (m), 1490 (s), 1325 (s), 1228 (m), 1124 (s), 1069 (s), 1016 (w),
840 (m) cm−1. MS m/z (%) 453 ([M + 1]+, 4), 452 (M+, 17), 257
(18), 256 (47), 255 ([M–C9H9O3S]+, 100), 139 (10). HRMS (EI+)
calcd for C22H19F3O3S2 452.0728, found 452.0732. Anal. Calcd for
C22H19F3O3S2: C, 58.39; H, 4.23; S, 14.17. Found: C, 58.06; H,
4.16; S, 14.15%.
Cell culture
3 Several other dibromothiophene derivatives (2,5-dibromothiophene,
ethyl
4,5-dibromothiophene-2-carboxylate
or
ethyl
2,5-
HeLa cells were routinely maintained in DMEM supplemented
with 5% FCS and ligand assays were performed in the same
medium.
dibromothiophene-3-carboxylate) have been selectively substituted
through halogen–metal exchange reactions: M. Abarbri, J. Thibonnet,
L. Be´rillon, F. Dehmel, M. Rottla¨nder and P. Knochel, J. Org. Chem.,
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Stable reporter cell lines
A stable Gal4-hPPARb/d cell line was generated as described
previously to facilitate assessment of agonistic activity at PPARb/d
with minor modifications.37 Briefly, HeLa cells were cotransfected
with Gal4-hPPARb/dand (17m)5-bglobin-Luc-Neo reporter gene.
At 48 h post-transfection, the cell line was selected with Geneticin
at 0.8 mg mL−1 and puromycin at 0.3 lg mL−1. Clones expressing
PPARb/d agonist-inducible luciferase activity were selected as
described.
5 R. Pereira, C. Gaudon, B. Iglesias, P. Germain, H. Gronemeyer and
A. R. de Lera, Bioorg. Med. Chem. Lett., 2006, 16, 49–54.
6 P. J. Beswick, C. C. F. Hamlett, V. Patel, M. L. Sierra and N. G.
Ramsden, European Patent No. WO 0292590, 2002; P. J. Beswick,
C. C. F. Hamlett, V. Patel, M. L. Sierra and N. G. Ramsden, Chem.
Abstr., 2002, 137, 384743.
7 H.-L. Elbe, L. Assmann, R. Tiemann, U. Schulz, H. Haenssler and
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Assmann, R. Tiemann, U. Schulz, H. Haenssler and H.-W. Dehne,
Chem. Abstr., 1995, 124, 55787.
Assessment of agonistic activity
To test the different compounds, identical aliquots of the Gal-
hPPARb/d reporter cell line were seeded and exposed in parallel
to the known agonist GW501516 1a, the test ligand, and vehicle
for 16 h. Cells were lysed in 100 lL of lysis buffer (25 mM Tris
phosphate [pH 7.8], 2 mM EDTA, 1 mM DTT, 10% glycerol, and
1% Triton X-100). A 100 lL portion of luciferin buffer (20 mM
Tris phosphate [pH 7.8], 1.07 mM MgCl2, 2.67 mM MgSO4, 1 mM
EDTA, 33.3 mM DTT, 0.53 mM ATP, 0.47 mM luciferin, and
0.27 mM coenzyme A) was added prior to direct photon counting.
8 S. Kano, Y. Yuasa, T. Yokomatsu and S. Shibuya, Heterocycles, 1983,
20, 2035–2037.
9 O. Meth-Cohn and G. van Vuuren, J. Chem. Soc., Perkin Trans. 1, 1986,
233–243.
10 The boronates (6, M = B(OMe)2 or B(OiPr)2) were prepared by
bromine–lithium exchange (n-BuLi, THF, −78 ◦C) followed by trap-
ping with B(OMe)3 or B(OiPr)3 and were used without isolation.
The organostannane (6, M = SnBu3) was prepared by trapping
with Bu3SnCl the organolithium generated as indicated above, and
purified by reverse-phase column chromatography (V. Farina, J. Org.
Chem., 1991, 56, 4985–4987). The organozinc halide (6, M = ZnCl)
was prepared by treating with ZnCl2 the organolithium generated as
indicated above and was used without isolation.
Acknowledgements
We are grateful to the Spanish MEC (SAF2004-07131-FEDER),
the European Community (ANTICANCER RETINOIDS
QLK3-2002-02029 and EPITRON Contract No. LSHC-CT2005-
518417) and Xunta de Galicia (PGIDIT05PXIC31403PN).
4524 | Org. Biomol. Chem., 2006, 4, 4514–4525
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31 The arylthiol 27 was prepared according to the procedure described
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disulfide, which was obtained by displacement of methyl bromoacetate
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