Design, synthesis, and structure-activity relationship of novel thiophene derivatives for β-amyloid plaque imaging

Article abstract of DOI:10.1016/j.bmcl.2005.11.055

Novel 2,5-diphenylthiophene derivatives were synthesized and structure activity relationship with regard to Aβ plaque binding was studied. Binding affinities of these compounds were found to range from 3.9 to >1000 nM, depending on the substitution patter

Full text of DOI:10.1016/j.bmcl.2005.11.055

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1350–1352
Design, synthesis, and structure–activity relationship
of novel thiophene derivatives for b-amyloid plaque imaging
Rajesh Chandra,a Mei-Ping Kunga and Hank F. Kunga,b,
*
aDepartment of Radiology, University of Pennsylvania, Room 305, 3700 Market Street, Philadelphia, PA 19104, USA
bDepartment of Pharmacology, University of Pennsylvania, Room 305, 3700 Market Street, Philadelphia, PA 19104, USA
Received 11 October 2005; revised 14 November 2005; accepted 14 November 2005
Available online 1 December 2005
Abstract—Novel 2,5-diphenylthiophene derivatives were synthesized and structure activity relationship with regard to Ab plaque
binding was studied. Binding affinities of these compounds were found to range from 3.9 to >1000 nM, depending on the substitu-
tion patterns on the phenyl ring. The fluoroethyl-substituted thiophene derivatives showed excellent binding affinities. These com-
pounds may be useful for the development of novel PET tracers for the imaging of b-amyloid plaques in the brain of patients with
Alzheimerꢀs disease.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Alzheimerꢀs disease (AD) is a neurodegenerative disor-
der that impairs the normal functions of brain, ultimate-
ly leading to death. It is estimated that nearly 2% of the
population in industrialized countries is affected and the
risk is highest among older individuals. A central event
in the pathology of Alzheimerꢀs disease is the production
of b-amyloid (Ab) peptides and subsequent aggregation
to form Ab plaques.1–3 Imaging study of b-amyloid pla-
que present in the brain offers one of the most potential
diagnostic tools for the detection as well as evaluation of
the advancement of AD. Thus, it transpires that the
development of small molecular probes for labeling Ab
plaques in vivo is of major significance in Alzheimerꢀs re-
search.4 It may also assist in the development of drugs
targeting Ab plaques for the treatment of AD and in
ascertaining the effectiveness of the treatment.
X-34,7 ISB, BSB, and IMSB,8,9 are equally potent in this
regard. Positron emission tomography (PET) is a non-
invasive imaging technique available for examining the
brain functions, physiology, and metabolism. Previous-
ly, a stilbene derivative, [11C]SB-13,10 showing promise
in detecting senile plaques in AD patients has been
reported from our group. The short half-life (20 min)
of C-11, however, may limit the usefulness of [11C]SB-
13 or even [11C]PIB11,12 for a widespread application.
Comparable 18F labeled agents may supplant the clinical
need due to the longer half-life of the isotope
(109.7 min). Recently, we have successfully reported
two series of stilbene derivatives,13 both of which can
be labeled with F-18, as potential PET imaging agents
for AD patients. In an attempt to test novel highly con-
jugated biphenyl derivatives, we have selected to investi-
gate biphenyl thiophene derivatives. Our studies with
the stilbene-based probes have established the minimum
structural feature to be the presence of two phenyl rings,
at least one of which should be electron rich, in order for
these molecules to exhibit desirable Ab plaque binding
properties.14 These two phenyl rings should be in conju-
gation with each other and the molecule as a whole
should be neutral and planar. We replaced the double
bond in stilbene-based probes with a cyclic moiety, at
the same time without altering the above-mentioned
structural features. Thiophene ring was an obvious
choice, as it can be considered as a diene in s-cis confor-
mation.9 As for the attachment of phenyl rings to the
thiophene core, 2 and 5 positions seemed to be the
Several fluorescent probes, making use of the optical
properties of highly conjugated dye molecules, have
been reported to bind to amyloid plaques with high
specificity. For instance, dye molecules such as Congo
Red (CR) and Thioflavin-T and S have been used in
the fluorescent staining of plaques and tangles in post-
mortem AD brain sections.5,6 Further studies showed
that more abbreviated forms of chrysamine G, such as
Keywords: In vitro binding; PET imaging; Alzheimerꢀs disease.
*
Corresponding author. Tel.: +1 215 662 3096; e-mail: kunghf@
sunmac.spect.upenn.edu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.055

R. Chandra et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1350–1352
1351
obvious choices as it will avoid any steric interactions
between the phenyl rings. Reported herein are the
synthesis and the structure–activity relationship of a
novel series of potential Ab plaque imaging agents based
on the biphenyl thiophene core.
materials in one step, often in good yields.17 The subse-
quent functional group transformations of the resulting
primary products (3b–d) afforded compounds (3e–g)
(Scheme 1). Compound 3c has been prepared according
to de Boer et al.18
The 2,5-disubstituted thiophene derivatives (3a–q) em-
ployed in the study were prepared according to Schemes
1 and 2. The key step in the synthesis of N,N-, O,O-, and
S,S-disubstituted derivatives is the Suzuki coupling reac-
tion of 2,5-dibromothiophene with the corresponding
phenylboronic acids. It is worthmentioning that the
compound 3d had been prepared by the multi-step se-
quence involving the Pd(0) catalyzed coupling of thei-
nylzinc chloride with iodoanisole,15 while compound
3b had been synthesized starting from the acyclic precur-
sor 4-nitroacetophenone.16 However, we opted the Su-
zuki coupling procedure, as the target compounds are
readily obtained from commercially available starting
The synthesis of N,O-disubstituted derivatives started
from the acyclic precursor 4-methoxy acetophenone 4,
which upon treatment with POCl3 and DMF afforded
b-chloroacrolein 5. Treatment of 5 sequentially with
Na2SÆ9H2O and 4-nitrobenzyl bromide followed by
cyclization with NaOMe yielded 2-(4-nitrophenyl)-5-
(4-methoxyphenyl)-thiophene 3h.19 Further manipula-
tions with the nitro and methoxy groups in 3h as
depicted in Scheme 2 resulted in compounds (3i–q).
For the preparation of N-methyl derivative 3q, the rou-
tinely used method of reductive alkylation20 was found
to be inefficient. In order to achieve it, the aniline
nitrogen in 3i was initially protected with Boc, subse-
quently deprotonated using NaH and methylated using
MeI. The deprotection of the N-Boc group and O-de-
methylation were achieved in one step using microwave
heating to afford 3q.21
B(OH)
2
i)
+
S
R
Br
Br
S
1
2
R
R
The in vitro binding studies of the thiophene derivatives
(3a–q) have clearly established the functional group tol-
erance of these molecules (Table 1).
2
1
3(a-g)
2
1
3a R = R = 2-OMe
3b R = R = 4-NO
3c R = R = 4-SMe
3d R = R = 4-OMe
3e R = R = 4-OH
3f R = 4-OH; R = OMe
3g R = R = 4-NH
1
2
2
1
2
1
2
Table 1. Inhibition constants (Ki, nM) of compounds (3a–q) on ligand
binding to AD brain homogenatesa
ii)
iv)
1
2
iii)
1
2
2,5-Diphenylthiophene derivative
Inhibition constants
(Ki, nM)
1
2
2
3a R1 = R2 = 2-OMe
625 ( 20)
185 ( 30)
>1000
Scheme 1. Reagents and conditions: (i) 2 M Na2CO3/Pd(PPh3)4,
DMF, 100 ꢁC, 24 h; (ii) BBr3 (2 equiv)/CH2Cl2, 50 ꢁC, 6 h; (iii) BBr3
(1 equiv)/CH2Cl2, 50 ꢁC, 6 h; (iv) SnCl2/ethanol, 85 ꢁC, 12 h.
3b R1 = R2 = 4-NO2
3c R1 = R2 = 4-SMe
3d R1 = R2 = 4-OMe
108 ( 22)
4.0 ( 0.6)
6.1 ( 0.5)
6.1 ( 0.8)
18.5 ( 5.0)
5.6 ( 0.4)
9.6 ( 0.8)
500 ( 20)
7.5 ( 0.4)
21.5 ( 0.5)
3.9 ( .05)
31.2 ( 3.0)
3e R1 = R2 = 4-OH
3f R1 = 4-OH; R2 = 4-OMe
3g R1 = R2 = 4-NH2
CHO
3h R1 = 4-NO2; R2 = 4-OMe
3i R1 = 4-NH2; R2 = 4-OMe
3j R1 = 4-NH2; R2 = 4-OH
3k R1 = 4-NMe2; R2 = 4-OMe
3m R1 = 4-NMe2; R2 = 4-OH
3n R1 = 4-NH(CH2CH2F); R2 = 4-OMe
3p R1 = 4-NH(CH2CH2F); R2 = 4-OH
3q R1 = 4-NHMe; R2 = 4-OH
Cl
O
i)
ii)
S
1
2
R
R
OMe
4
OMe
5
3(h-q)
1
2
3h R = OMe; R = NO
2
iii)
iv)
a Values are means SEM of three independent experiments, each in
duplicate. Binding assays were carried out in 12 · 75 mm borosilicate
glass tubes. For the competition binding, the reaction mixture con-
tained 50 lL tissue homogenates (20–50 lg), 50 lL of [125I]IMPY
(diluted in PBS, 0.02–0.04 nM) and 50 lL of inhibitors (10À5 to
10À10 M diluted serially in PBS containing 0.1% bovine serum albu-
min) in a final volume of 1 mL. Non-specific binding was defined in
the presence of 600 nM IMPY in the same assay tubes. The mixture
was incubated at 37 ꢁC for 2 h, and the bound and the free radio-
activity were separated by vacuum filtration through Whatman GF/B
filters using a Brandel M-24R cell harvester followed by 2 · 3 mL
washes of PBS at room temperature. Filters containing the bound I-
125 ligand were counted in a gamma counter (Packard 5000) with
70% counting efficiency. Protein determinations were performed with
Lowyꢀs method using bovine serum albumin as a standard. The
results of inhibition experiments were subjected to non-linear
regression analysis using EBDA by which Ki values were calculated.
1
2
3i R = OMe; R = NH
iv)
2
1
2
3j R = OH; R = NH
2
v)
1
2
3k R = OMe; R = NMe
2
1
2
3mR = OH; R = NMe
2
vii)
vi)
1
2
3n R = OMe; R = NH(CH CH F)
2
2
viii)
1
2
3p R = OH; R = NH(CH CH F)
2
2
1
2
3q R = OH; R = NHMe
Scheme 2. Reagents and conditions: (i) POCl3/DMF, 60 ꢁC, 3 h; (ii)
a—Na2S/DMF, rt, 2 h; b—4-nitrobenzylbromide, 50 ꢁC, 3 h; c—
NaOMe; 10 min; (iii) SnCl2/ethanol, 85 ꢁC, 12 h; (iv) BBr3 (1 equiv)/
CH2Cl2, 50 ꢁC, 6 h; (v) (CH2O)n, AcOH, NaCNBH3, 18 h; (vi) K2CO3/
DMF, 90 ꢁC, 1 h then BrCH2CH2F, 90 ꢁC, 12 h; (vii) a—(Boc)2O,
THF, reflux, 18 h; b—NaH, DMF, 50 ꢁC, 30 min then MeI, 50 ꢁC, 3 h;
c—BBr3/CH2Cl2, microwave, 140 ꢁC, 5 min; (viii) BBr3 (1 equiv)/
CH2Cl2, À78 ꢁC to rt, 18 h.

1352
R. Chandra et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1350–1352
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16. Ling, C.; Lahti, P. M. J. Am. Chem. Soc. 1994, 116, 8784.
17. General procedure for the Suzuki reaction: to a mixture of
2,5-dibromothiophene (1 mmol) and phenylboronic acid
(1.66 mmol) in 10 mL of anhydrous DMF was added 2 M
Na2CO3 (5 mL). After degassing the mixture for 15 min,
Pd(PPh3)4 (5 mol %) was added and the mixture was
heated at 100 ꢁC for 24 h and cooled to room temperature.
The solvent was removed under reduced pressure and the
residue was taken in ethyl acetate. The ethyl acetate layer
was washed successively with water and brine, and dried
over anhyd MgSO4. The crude, after the evaporation of
the solvent, was purified by column chromatography on
silica gel (50% DCM in hexane) to afford products (3a–d).
18. de Boer, B.; Meng, H.; Perepichk, D. F.; Zheng, J.; Frank,
M. M.; Chabal, Y. J.; Bao, Z. Langmuir 2003, 19, 4272.
19. Kirsch, G.; Prim, D.; Leising, F.; Mignani, G. J. Hetero-
cycl. Chem. 1994, 31, 1005.
In the case of 3b, where both the phenyl rings carry
strongly electron withdrawing NO2 groups
(R1 = R2 = NO2), the Ki was found to be 185 nM. How-
ever, the replacement of these NO2 groups with NH2
(compound 3g) resulted in a steep decrease in the Ki val-
ue (6 nM). It proves that the phenyl rings should be elec-
tron rich in order for these compounds to exhibit high
binding affinities. One other important factor emerges
by comparing the Ki values of compounds (3d–f) all of
which carry electron rich phenyl rings. In the case of
3d where both R1 and R2 are OMe, the compound
showed a low binding affinity (Ki = 108 nM). When
either one or both of these OMe groups is replaced by
an OH group, the resulting compounds showed very
high binding affinities (3e: R1 = R2 = OH, Ki = 4 nM
and 3f: R1 = OH, R2 = OMe, Ki = 6 nM). It can be sur-
mised that at least one ring should carry either NH or
OH group for these molecules to exhibit effective bind-
ing to Ab plaques. This is further proven by the Ki value
(500 nM) of compound 3k, in which the rings are substi-
tuted with OMe and NMe2 moieties. It is important to
note that N-fluoroethyl-substituted derivative 3p exhib-
its a very high binding affinity (3.9 nM); the correspond-
ing [18F] labeled compound can be utilized to image Ab
plaques in brain using PET.
In conclusion, we have synthesized several novel 2,5-
diphenylthiophene derivatives with various substitu-
tion patterns on the phenyl rings. The SAR studies
of these compounds clearly vindicated our assump-
tion that the phenyl rings should be electron rich
in order for these compounds to exhibit desirable
binding affinities. Further, all compounds, which
exhibited high binding affinities, carried at least
one OH or NH moiety in the phenyl ring. The N-
fluoroethyl-substituted compound showed very good
binding to Ab plaques in vitro, which is very impor-
tant for the development of PET tracers. Further
studies to develop 2,5-diphenylthiophene-based PET
markers for the imaging of Ab plaques is currently
under way.
20. Barluenga, J.; Bayon, A. M.; Asensio, G. J. Chem. Soc.,
Chem. Commun. 1984, 1334.
Acknowledgments
21. Preparation of 3q: compound 3i (325 mg, 1.16 mmol) and
di-tert-butyldicarbonate (266 mg, 1.22 mmol) were dis-
solved in anhyd THF (12 mL) and the solution was
refluxed overnight. Subsequent workup and column
chromatography afforded N-Boc-protected 3i. The N-
Boc 3i (198 mg, 0.6 mmol) was taken in 4 mL anhyd
DMF, NaH (20 mg, 0.8 mmol, 95% powder) was added,
and the mixture was warmed at 50 ꢁC for 30 min. It was
then cooled to room temperature, MeI (426 mg, 3 mmol)
was added and stirred at room temperature for 3 h to
afford N-methyl-N-Boc 3i after workup and purification.
Microwave heating (140 ꢁC, 5 min, Biotage initiator
microwave oven) of N-methyl-N-Boc 3i (39 mg, 0.1 mmol)
and BBr3 (IM solution in DCM, 0.1 mL, 0.1 mmol) in
DCM (4 mL) afforded compound 3q.
This work was supported by grants awarded from the
National Institutes of Health (R21-AG021868, M.P.K.
and R01-AG022559, H.F.K.).
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