Synthesis of fluorescent probes based on stilbenes and diphenylacetylenes targeting β-amyloid plaques

Article abstract of DOI:10.1016/j.tetlet.2008.03.130

Three fluorescent probes were synthesized aiming for optical imaging to detect amyloid plaques present in the patients with Alzheimer's disease (AD). These compounds were prepared via Sonogashira coupling of a well-defined fluorophore (4-bora-3a,4a-diaza-

Full text of DOI:10.1016/j.tetlet.2008.03.130

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3395–3399
Synthesis of fluorescent probes based on stilbenes
and diphenylacetylenes targeting b-amyloid plaques
Ajit K. Parhi, Mei-Ping Kung, Karl Ploessl, Hank F. Kung *
Department of Radiology, University of Pennsylvania, 3700 Market Street, Room 305, Philadelphia, PA 19104, United States
Received 10 October 2007; revised 20 March 2008; accepted 25 March 2008
Available online 29 March 2008
Abstract
Three fluorescent probes were synthesized aiming for optical imaging to detect amyloid plaques present in the patients with Alzhe-
imer’s disease (AD). These compounds were prepared via Sonogashira coupling of a well-defined fluorophore (4-bora-3a,4a-diaza-s-inda-
cene, BODIPY) with the pharmacophore possessing either a stilbene or a diphenylacetylene moiety. Different polyethylene glycol chain
lengths were used as linkers between the fluorophore and the pharmacophore to adjust the lipophilicity of these probes. These
compounds exhibit strong fluorescence emission between 665 and 680 nm and have very high extinction coefficients comparable to
the parent fluorophore, BODIPY dye.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Fluorescent probes; Alzheimer’s disease; BODIPY; Near-IR imaging
1. Introduction
ies. To meet the requirements of such fluoroscent probes,
we have employed a tether approach for designing the
probes. All these fluorescent probes have three common
structural units, that is, a fluorophore, a linker, and a phar-
macophore, where the fluorophore is tethered to the
pharmacophore through the polypegylated linker (Fig. 1).
The polypegylated linker is added to improve the water-
solubility. This series of fluorescent probes is not designed
to penetrate intact blood-brain barrier; instead, due to their
higher molecular weight, rather these will only bind to Ab-
plaques accumulated on the cerebral blood vessels, at the
extravascular space, commonly observed in the patients
with cerebral angiopathy.9–11
The formation of b-amyloid (Ab) plaques, consisting of
b-sheets of Ab protein aggregates, in the brain is a pivotal
event in the pathogenesis of Alzheimer’s disease (AD).1,2
Thus, the development of specific agents targeting Ab
aggregates is important for the diagnosis, and it may be
possible for the evaluation of the therapeutic course of
the disease progression.3,4 Recently, significant advance-
ment for the detection of Ab plaques in AD patients has
been achieved in using positron emission tomography
(PET) and single photon emission tomography (SPECT)
imaging ligands.5–8
For our effort to develop complementary techniques, we
envisioned to make new water-soluble neutral fluorescent
compounds that would emit in the near-infrared region
and be able to bind to Ab plaques as tools for in vitro stud-
For all our probes prepared for this Letter, we opted for
the same neutral fluorophore, a 4-bora-3a,4a-diaza-s-inda-
cene (BODIPY) dye, 1, previously synthesized and charac-
terized by Kim et al.12 The reason for choosing 1 as the
fluorophore owes to its strong red shifted, sharp fluores-
cence emission at 654 nm and its neutral nature. The choice
of pharmacophores is based on our previous findings that a
series of 4-amino-40-hydroxy substituted stilbene13 and
diphenylacetylene derivatives14 showed high binding affini-
ties (Ki = 1–10 nM range) to Ab aggregates. Similarly,
Abbreviations: BODIPY, 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-
diaza-s-indacene; PEG, poly ethylene glycol
*
Corresponding author. Tel.: +1 215 662 3096; fax: +1 215 349 5035.
E-mail address: kunghf@sunmac.spect.upenn.edu (H. F. Kung).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.03.130

3396
A. K. Parhi et al. / Tetrahedron Letters 49 (2008) 3395–3399
by p-chloroanil also proceeded rapidly.23 These two steps
Fluorophore
Pharmacophore
Linker
can be accomplished within a day in good overall yield
(more than 50%). Previously, the target compound, 1,
was prepared in a one-pot two-step reaction beginning with
dipyrromethene 4. Boron complexation of dipyrromethene
4 followed by the demethylation using BBr3 furnished com-
pound 1 in 93% yield.16 In a slightly modified way, we iso-
lated the borane complex 5 and upon treatment with 1.0 M
BBr3 in DCM afforded the target dye 1 as a green solid,
which was identical with the reported compound in all
aspects. This slight modification enabled us to use 1.0 M
BBr3 in DCM rather than neat BBr3.
I
HO
HO
NH
O
O
9
n
N
N
PEG, n = 5 or 12
B
O
O
N
1
14
Fig. 1. Examples of fluorophore, linkers, and pharmacophores.
With the BODIPY dye 1 in hand, the next issue was to
connect the fluorophore to the rest of molecule. Alkynyl
connectors are thought to be the most appropriate because
it is known that these connectors are particularly useful by
favoring electronic communication and extending the con-
jugation length.24 This is especially true when the connec-
tion is through the para position of the central benzene
ring of the BODIPY dye. Moreover, the iodo-substituted
BODIPY dye can be suitably utilized in the cross-coupling
reaction with the terminal alkynes.25 The terminal alkynes
10a and 10b were prepared by the reactions shown in
Scheme 2.
Firstly the PEGs (n = 3 or 10) were monoalkylated with
propargyl bromide to afford the terminal alkynes 7a–b in
45% and 39% yield, respectively. The free OH groups of
7a–b were converted into mesylates 8a–b by reacting with
MsCl in the presence of triethyl amine. To prepare com-
pounds with five or 12 ethoxy group as the PEG linkage,
mesylates 8a–b were coupled with the OH-group of 4-
methyl amino-40-hydroxy stilbene 926 to obtain 10a–b in
82% and 78% yield, respectively.
various units of polyethylene glycol (PEG, n = 5 or 12) as
linkers on the 40-hydroxyl group of stilbene and diphenyl-
acetylene through which the fluorophore is attached at
the end of PEG chain provides a flexible tool to adjust
the lipophilicity of the dye.15,13 Reported herein are
the synthesis and spectroscopic properties of the three
compounds as potential fluorescent probes targeting Ab
plaques.
The synthesis began with the preparation of the BOD-
IPY dye 1 by slightly modifying the procedure reported
by Kim et al.16 Two common methods exist to build a bor-
adiazoindacene core: (1) starting either from an aldehyde17
or (ii) from an acyl chloride.18,19 In the previously reported
synthesis, dipyrromethene 4 was prepared by the condensa-
tion of 4-iodobenzoyl chloride with 2-aryl pyrrole 220,21 in
a moderate yield. Although this approach is attractive
owing to the acid sensitive nature of the 2-aryl pyrrole 2,
the method is hampered by its very long reaction time (usu-
ally required 2–4 days of refluxing). Therefore we decided
to explore the first approach as illustrated in Scheme 1.
The condensation of p-iodobenzaldehyde with 2-aryl
pyrrole 2 at 0 °C was found to be extremely fast in the pres-
ence of catalytic amounts of TFA. The corresponding
dipyrromethane 3 could be isolated in 52% yield when
the reaction was quenched within 5 min after the addition
of TFA.22 The subsequent oxidation to dipyrromethene 4
In order to introduce the PEG functionalized stilbene
pharmacophore into the BODIPY core, compound 1 was
coupled with the propargyl-PEG-stilbene 10a–b (Scheme 3).
Thus 4-iodo substituted dye 1 was treated with 10a–b
under catalyzed condition using Pd(PPh3)2Cl2 and CuI,
I
I
OMe
a
c
b
N
H
HN
NH
N
NH
OMe
MeO
OMe
MeO
I
2
4
3
I
d
N
N
F
N
N
OMe
B
B
MeO
F
O
O
5
1
Scheme 1. Synthesis of 4-bora-3a,4a-diaza-s-indacene (BODIPY) dye 1. Reagents and conditions: (a) p-iodo benzaldehyde, TFA, DCM, 0 °C, 52%; (b) p-
chloroanil, DCM, 1 h, 85%; (c) Et3N, BF3–Et2O, MePH, 80 °C, 88%; (d) 1.0 M BBr3 in DCM, 0 °C to rt, 4 h, 82%.

A. K. Parhi et al. / Tetrahedron Letters 49 (2008) 3395–3399
3397
b
O
O
a
O
OH
HO
HN
O
HO
O
n = 3 or 10
n = 3 or 10
6 a-b
7 a-b
HN
OH
O
O
O
O
MsO
O
O
9
c
O
n = 3 or 10
n = 3 or 10
8 a-b
10 a-b
Scheme 2. Preparation of terminal alkynes 10a and 10b. Reagents and conditions: (a) NaH, propargyl bromide, THF, 45% (n = 3), 39% (n = 10); (b)
MsCl, Et3N, DCM, 91% (n = 2), 88% (n = 10); (c) K2CO3, DMF, 100 °C, 12 h, 82% (n = 3), 78% (n = 10).
N
O
I
B
HN
O
O
O
N
O
O
n = 3 or 10
10a n = 3
1
Pd(PPh3)2Cl2
CuI, Triethyl amine
10b n = 10
THF, 3-4 h, 66%(n=3), 61%(n=10)
N
O
O
O
B
O
O
O
NH
N
n=3 or 10
11a n = 3
11b n = 10
Scheme 3. Sonogashira coupling to synthesize compounds 11a–b.
Table 1
and yielded the target compounds 11a–b in 66% and 61%
yield, respectively, as green solids. These compounds were
isolated via chromatography on deactivated alumina col-
umn. Extensive decomposition occurred when silica gel
was used for chromatography. Similarly, the synthesis of
the fluorescent compound 16 via Sonogashira coupling is
shown in Scheme 4.
Spectroscopic data for 11a, 11b, and 16
Compound
Solvent
k
max/nm (abs)
e/Mꢀ1 cmꢀ1
kmax/nm (em)
11a
11b
16
Water
Water
Water
338
364
345
38,000
34,000
35,000
680
665
678
Palladium-catalyzed coupling of alkyne 12 with 4-iodo-
benzaldehyde 13 produced the internal alkyne 15 in 51%
yield. The alkylation of the OH group of 4,4-dimethyl
amino 40-hydroxy diphenylacetylene 15 with mesylate 8a
furnished the terminal alkyne 15 in 62% yield. Sonogashira
coupling of 15 with 1 gave the target compound 16 in 67%
yield, again as a green solid.
Spectroscopic data relevant to the present discussion are
shown in Table 1. A typical fluorescence emission spectrum
as shown by compound 16 is given in Figure 2.
The fluorescence properties were examined under ambi-
ent conditions either in water or in ethanol. It is important
to point out that all dyes are soluble in water and exhibit
the expected absorption and emission patterns typical of
BODIPY dye 1.
While compound 1 exhibits fluorescence emission at
654 nm in chloroform, all newly synthesized compounds
11a, 11b, and 16 are red-shifted by 10–20 nm relative to
the parent compound 1. This is probably due to the
a
b
O
O
N
N
O
I
OH
N
OH
O
O
n=3
13
14
12
15
N
N
O
c
O
N
B
O
O
O
n=3
16
Scheme 4. Synthesis of Fluorescent probe 16. Reagents and conditions: (a) Pd(PPh3)2Cl2, Cul, Et3N, THF, 3 h, 51%; (b) 8a, K2CO3, DMF, 62%; (c) 1,
Pd(PPh3)2Cl2, Cul, Et3N, THF, 4 h, 67%.

3398
A. K. Parhi et al. / Tetrahedron Letters 49 (2008) 3395–3399
70.0, 69.6, 67.8, 59.4, 30.9. HRMS (ESI) calcd for
1008.4
C55H52BN3O6 [M+] 894.3926 obsd 894.3965.
Compound (11b): 1H NMR (200 MHz, CDCl3) d: 7.76–
7.62 (m, 6H), 7.40–7.30 (m, 6H), 7.07–6.97 (m, 4H), 6.94–
6.87 (m, 8H), 6.58 (d, 2H, J = 8.0 Hz), 4.48 (s, 2H), 4.13 (t,
2H, J = 4.0 Hz), 3.87–3.53 (m, 46H), 2.85 (s, 3H). 13C
NMR (50 MHz, CDCl3) d: 158.2, 154.5, 150.4, 148.0,
137.8, 134.3, 134.1, 132.5, 132.2, 131.3, 130.7, 130.0,
128.1, 127.7, 127.4, 127.0, 126.0, 125.4, 124.7, 120.7,
120.0, 119.9, 116.8, 115.1, 113.4, 88.1, 85.8, 71.1, 70.8,
70.7, 70.0, 69.6, 67.7, 59.4, 31.4. HRMS (ESI) calcd for
C69H80BN3O15 [M+] 1202.576 obsd 1202.577.
800
600
400
200
0.0
1
Compound (16): H NMR (200 MHz, CDCl3) d: 7.75–
7.62 (m, 6H), 7.43–7.35 (m, 6H), 7.07–7.04 (m, 5H),
6.93–6.84 (m, 5H), 6.64 (d, 2H, J = 9.0 Hz), 4.48 (s, 2H),
4.13 (t, 2H), 3.87–3.67 (m, 18H), 2.97 (s, 6H). 13C NMR
(50 MHz, CDCl3) d: 158.5, 154.5, 150.4, 150.2, 137.9,
134.3, 134.1, 132.9, 132.7, 132.5, 132.4, 132.2, 132.1,
132.0, 130.7, 130.0, 128.8, 128.6, 126.0, 125.4, 120.7,
120.0, 116.8, 114.8, 112.1, 110.7, 89.3, 88.1, 87.3, 85.8,
71.1, 70.9, 70.8, 70.7, 69.9, 69.6, 67.7, 59.4, 40.4. HRMS
(ESI) calcd for C56H52BN3O8 [M+] 906.3926 obsd 906.396.
300.0
400
500
600
700
795.5
nm
Fig. 2. Excitation and emission (250 nM in ethanol) spectra of 16.
extended conjugation with the alkyne in these compounds.
Acknowledgments
Compounds 11a, 11b, and 16 have similar molar extinc-
tion coefficients in water (emax = 38,000 Mꢀ1 cmꢀ1
,
This work was supported by the grant from the National
Institutes of Health (AG-022559 to H.F.K and AG-21868
to M.P.K).
34,000 Mꢀ1 cmꢀ1
, , respectively)
and 35,000 Mꢀ1 cmꢀ1
compared to compound 1 (emax = 46,000 Mꢀ1 cmꢀ1) in
chloroform.
In conclusion, we present here examples of water-solu-
ble fluorescent agents with emission in the near-IR, for
targeting b-amyloid plaques. These compounds were suc-
cessfully prepared and characterized. They have high
extension coefficients showing emission around 665–
680 nm. The in vitro binding affinity and other biological
properties of these compounds will be reported later.
Supplementary data
Supplementary data (experimental procedure and chara-
cterization of some intermediate compounds and copies
1
of H, 13C, and UV spectra of some intermediate com-
pounds and all fluorescent compounds) associated with this
article can be found, in the online version, at doi:10.1016/
j.tetlet.2008.03.130.
2. General procedure for Sonogashira coupling
References and notes
To a solution of bodipy dye 1 in THF and triethyl
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room temperature for 3–4 h. The solvents were evaporated
and the crude product was purified by flash column chro-
matography on basic alumina using EtOAc/hexane mix-
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