Elongation of the molecular probe DDNP with phenylethynylidene or phenyldiazenylidene spacers

Article abstract of DOI:10.1016/j.tetlet.2014.01.002

In this work, we report the synthesis, X-ray structures, and optical properties of two DDNP analogs containing an unsaturated spacer inserted between the donor N,N-dimethylamino group and the naphthalene ring. The new compounds have longer distances betwe

Full text of DOI:10.1016/j.tetlet.2014.01.002

Tetrahedron Letters 55 (2014) 1218–1221
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Elongation of the molecular probe DDNP with phenylethynylidene
or phenyldiazenylidene spacers
a
a
a
´
ˇ a
ˇ a,b,
⇑
Luka Rejc , Jan Fabris , Armin Adrovic , Marta Kasunic , Andrej Petric
a
ˇ
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerceva 5, 1000 Ljubljana, Slovenia
b EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, we report the synthesis, X-ray structures, and optical properties of two DDNP analogs con-
taining an unsaturated spacer inserted between the donor N,N-dimethylamino group and the naphtha-
lene ring. The new compounds have longer distances between the donor and acceptor groups and are
intended to serve as molecular probes for the assessment of spatial and polarity requirements for binding
to protein aggregates found in the central nervous system (CNS) of patients with neurodegenerative
diseases.
Received 4 September 2013
Revised 27 November 2013
Accepted 2 January 2014
Available online 9 January 2014
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
Molecular probes
Sonogashira coupling
Knoevenagel reaction
Diazo coupling
Optical properties
With increasing lifespan, the number of elderly people with Alz-
heimer’s (AD) and other neurodegenerative diseases is growing.
Extracellular protein aggregate formation and accumulation is
common to a number of such diseases.1 Detection of these aggre-
gates in the brains of living patients allows early diagnosis and
assessment of treatment. Traditionally, fluorescent dyes such as
thioflavin T and Congo red are used to stain brain slices for post
mortem diagnosis of AD by fluorescence microscopy. These dyes
are not suitable for in vivo detection of protein aggregates in the
central nervous system (CNS) because they are charged at physio-
logical pH and thus do not cross the blood–brain barrier (BBB). In
contrast to these dyes, 1,1-dicyano-2-[6-(dimethylamino)naphtha-
len-2-yl]propene (DDNP; Fig. 1)2 is uncharged under these condi-
tions and is easily able to pass the BBB. DDNP is fluorescent and
can be used to label protein aggregates in vitro. By using a side-
chain modification, DDNP has been transformed into its fluorinated
analog FDDNP (Fig. 1), and has also been synthesized containing
the positron emitter 18F, thus allowing the development of the first
non-destructive method for the diagnosis of AD with positron
emission tomography (PET).3
labels protein aggregates, which characterize a number of other
neurodegenerative diseases.5,6 In parallel to successful in vivo
diagnostic applications of [18F]FDDNP PET imaging and clarifica-
tion of molecular processes responsible for neurodegenerative dis-
eases,7 the parent molecule has been systematically modified in
order to obtain an insight into the molecular mechanisms of bind-
ing and to enhance the labeling specificity of the probe.8,9 Until re-
cently, attempts to obtain high resolution crystal structures of
complexes of FDDNP/DDNP with amyloid-beta or tau proteins have
failed, due to the inability to grow suitable single crystals. Studies
of the atomic structures of complexes of fiber-forming segments of
proteins involved in AD with small molecules, including DDNP,
have revealed that these molecules reside in the cylindrical cavity,
roughly parallel to the fiber axis.10
A set of DDNP/FDDNP analogs in which the donor (amine) and
acceptor (dicyanopropene) sides of the parent molecule are
11.28 Å
Semi-automated and automated procedures for the synthesis of
large quantities (110–170 mCi) of [18F]FDDNP have been devel-
oped to provide the tracer for multiple PET experiments on the
same day, and for distribution of the tracer to surrounding PET
imaging centers.4 It has been proven that FDDNP also efficiently
N
N
F
N
N
N
N
FDDNP
DDNP
⇑
Corresponding author. Tel.: +386 1 241 9232; fax: +386 1 241 9220.
ˇ
E-mail address: andrej.petric@fkkt.uni-lj.si (A. Petric).
Figure 1. Structures of DDNP (with acceptor–donor distance indicated) and FDDNP.
0040-4039/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2014.01.002

L. Rejc et al. / Tetrahedron Letters 55 (2014) 1218–1221
1219
N
N
N
intention to utilize diazonium salt coupling to an aniline deriva-
tive. The amino derivative 6 was prepared from the naphthol 1
via a Bucherer reaction with ammonia. However, our attempt to
transform 6 into the corresponding diazonium salt followed by
the addition of N,N-dimethylaniline under several tested condi-
tions led to mixtures of products. Based on the missing H5 signals
in the 1H NMR spectrum of the crude products, we presumed that
the problems were caused by the high susceptibility of position 5
in the naphthalene ring to electrophilic reactions. To test our
assumption, we subjected 6 to a solution of bromine in CH2Cl2 at
0 °C. After one hour, the bromo compound 7 was isolated as a sin-
gle product. The bromine at position 5 protected effectively com-
C
C
C
C
C
C
C
N
C
N
N
pound
7 in the reaction with nitrous acid to give the
corresponding diazonium salt. Without isolating the intermediate,
N,N-dimethylaniline was added to the reaction mixture, leading to
the azo-dye 8. In the presence of the hydrogenation-prone azo
group, debromination was achieved with triphenyl phosphine/
Pd(OAc)2, leading to the elongated 1-[6-(dimethylamino)naphtha-
len-2-yl]ethanone (ADMAN) analog 9. In the last step, a Knoevena-
gel reaction with malononitrile was performed to produce the
desired DDNP analog 10.
Figure 2. Valence bond (VB) representation of electron density delocalization from
the donor to acceptor group in 5.
modified to produce defined conformational changes, has already
been prepared.11,12 It was found that even drastic changes at the
amine position, for example, replacing dimethylamino- with a 4-
methylpiperidino group, have little effect on the binding affinity.
The binding properties are much more influenced by the ability
of the acceptor group to attain a co-planar arrangement with the
naphthalene ring. Formal replacement of the methyl group at the
acceptor side by the tert-butyl group forces the side-chain out of
plane and the binding affinity decreases; Ki increases from 10 to
520 nM.12 On the other hand, if the acceptor side-chain is incorpo-
rated into an annealed six-membered ring, and thus forced into a
co-planar conformation, the compound binds to protein aggregates
roughly three orders of magnitude tighter (Ki 0.01 nM).12 Polar
contacts between the bound probe, tyrosine, and valine residues
in the VQIVYK pseudofibril model suggest that the dipole moment
of the probe molecule could be a significant factor in binding.
In this work, we report the preparation, structure, and optical
properties of two DDNP analogs in which a spacer (consisting of
a phenyl ring and an ethynyl or azo group) is inserted between
the donor group and the naphthalene ring. The modifications aim
to alter the distance between the donor and the acceptor groups,
but to retain the conformational freedom and the total conjugation
between them (Fig. 2), as much as possible. We expected that the
larger dipole and increased number of delocalized electrons would
result in a bathochromic shift of absorption and emission maxima.
Most importantly, however, we expected that in the future the
determination of the binding affinity of these longer molecules to
protein aggregates would test the validity of the hypothesized
influence of a larger dipole moment on the binding properties.
Single crystal X-ray analyses
An unexpected number of signals and coupling patterns for pro-
tons 1, 3, and 4 were observed in the NMR spectrum of 7. Instead of
three signals of equal intensity and a typical coupling pattern (d for
H-1 with meta coupling, dd for H-3 with meta and ortho coupling,
and d for H-4 with ortho coupling), two signals at 8.04 (d, 2H,
J = 1.3 Hz) and 8.29 (t, 1H, J = 1.3 Hz) were observed. Single crystal
X-ray analysis was used to clarify the structural ambiguity of 7.
Using the results of the X-ray analysis (for details see the Supple-
mentary material), we attributed the unexpected appearance of
the proton spectrum of 7 to the close proximity of the bromine
at position 5 and proton H-4. This effect made protons 3 and 4
magnetically equivalent, resulting in an AX2 coupling pattern in
the NMR spectrum. Crystals of compounds 5 and 10, suitable for
X-ray analysis (Fig. 3), were grown from a benzene solution under
a hexane atmosphere. In the crystal unit cell of 5, two conformers
were present (only one is shown in Fig. 3). The major difference be-
tween the two conformers was the orientation of the propanedinit-
rile side-chain. The measured dihedral angles (x) were 148.2°
(C24–C25–C32–C34) for the isomer shown in Figure 3, and
À41.7° (C1–C2–C9–C11) for the other isomer. In both isomers,
the side-chain was rotated out of coplanarity with the naphthalene
ring by 30–40°. Similar out-of-coplanarity of the side-chain (139°)
was measured in the only conformer in the unit cell of 10. The rel-
ative orientations of the acceptor side-chain (propanedinitrile) and
naphthalene ring in the new analogs 5 and 10 were found to be
similar to DDNP/FDDNP. However, the distances between the do-
nor (N,N-dimethylamino) and acceptor groups in DDNP, 5, and
10 were markedly different [11.28, 18.04 (18.12 in the second con-
former), and 17.42 Å, respectively].16
Syntheses
Elongated DDNP analogs were prepared by the synthetic ap-
proach depicted in Scheme 1. The hydroxyl group in naphthol 113
was first activated for substitution by transformation into the tri-
flate 2. The latter was subjected to a Sonogashira-type coupling
with 4-ethynyl-N,N-dimethylaniline (3), which was prepared from
4-iodo-N,N-dimethylaniline14 and 2-methylbut-3-yn-2-ol (meby-
nol) by a modified literature procedure,15 to give the acetyl com-
pound 4. The Knoevenagel condensation of 4 with malononitrile
yielded the DDNP analog 5, containing a phenylethynylidene
spacer between the dimethylamino group and the naphthalene
ring.
Optical properties
In comparison to ADMAN and DDNP,2,12 compounds 4, 5, 9, and
10 show bathochromic shifts of the respective absorption maxima
(Table 1). This can be attributed to the increase in the molecular
size and, in the cases of 9 and 10, also to the presence of the azo
chromophore. To explore the effect of solvent polarity on the
absorption and fluorescence, UV/vis, emission and excitation spec-
tra of the new compounds were recorded in dichloromethane, N,N-
N
For the preparation of the phenyldiazenylidene-containing ana-
log, a classical synthetic approach was envisaged. It was our
dimethylformamide (DMF), and methanol (respective ET values
0.309, 0.386, and 0.762).17 The low solubility of all the new

1220
L. Rejc et al. / Tetrahedron Letters 55 (2014) 1218–1221
18.1 Å
N
N
O
O
(c)
(b)
TfO
4
5
2
(a)
N
N
N
3
O
HO
(d)
O
1
O
O
O
8
1
7
(e)
(f)
(g)
2
3
X
N2
N
H2N 6
N
5
4
7
H2N
Br
N
8
Br
Br
6
17.42 Å
(h)
N
N
O
(i)
N
N
N
N
10
9
N
N
Scheme 1. Reagents and conditions: (a) Tf2O/pyridine, À15 °C, 2 h, 95%; (b) THF, DIPA, CuI, PPh3, Pd(OAc)2, 70 °C, 3 h, 76%; (c) CH2(CN)2, pyridine, 80 °C, 23 h, 93%; (d) steel
bomb, NH3/H2O, NaHSO3, 140 °C, 96 h; (e) Br2 in CH2Cl2, 0 °C, 1 h, 82%; (f) NaNO2/HCl, 0 °C, 15 min; (g) N,N-dimethylaniline in CH2Cl2, 0 °C, 15 min, 51%; (h) NaHCO3, PPh3,
Pd(OAc)2, BuOH, 100 °C, 6 h, 87%; (i) CH2(CN)2, pyridine, 90 °C, 6 h, 26%.
Figure 3. ORTEP representations of the structures determined by single crystal X-ray diffraction: (A) one of the two conformers present in the unit cell of 5; (B) compound 10.
Table 1
Optical absorption and emission properties in different solvents, selected dihedral angles (for definition see the text), and donor–acceptor distances in the solid-state
kab (nm)
kem (nm)
e
 10À4 (L molÀ1 cmÀ1
)
xd (°)
de (Å)
a
a
a
CH2Cl2
DMFb
MeOHc
CH2Cl2
DMFb
MeOHc
CH2Cl2
DMFb
MeOHc
ADMANf
4
358g
382
445
438
441
470
—
366
376
440
426
432
455
436g
539
—
560
558
—
—
468
—
—
612
—
506
508
—
610
595
—
1.9g
3.2
3.5
2.0
4.0
3.8
—
1.8
3.2
3.4
2.0
3.7
3.6
—
—
—
152
148h
139
—
—
—
11.28
18.04i
17.42
384
460
—
446
435
3.0
2.0
—
2.5
2.5
9
DDNPf
5
10
a
b
c
d
e
f
1–10
1–10
1–10
l
l
l
M in CH2Cl2 containing 1% EtOH.
M in DMF containing 1% EtOH.
M in MeOH containing 1% EtOH.
x
is defined as the dihedral angle C1–C2–C9–C11.
The distance between the electron donor and the electron acceptor group.
From Ref. 2.
Chloroform was used instead of CH2Cl2.
g
h
i
The dihedral angle in the second conformer in the unit cell was À41.7°.
The distance between the electron donor and the electron acceptor group in the other conformer was 18.12 Å.

L. Rejc et al. / Tetrahedron Letters 55 (2014) 1218–1221
1221
Figure 4. Absorption and fluorescence emission spectra of 4 (A) and 5 (B) in CH2Cl2 (blue), DMF (red) and MeOH (green). The emission spectrum of 4 in MeOH (A) contains a
Raman band at approximately 420 nm.
N
compounds prevented reliable measurements in water (ET value
1.000). Although solvatochromism is well documented in the liter-
ature,17 the complex nature of solute–solvent interactions and its
effect on the HOMO–LUMO energy variations prevent its unified
prediction and explanation. For phenolate betaine dyes, such an at-
tempt has been described utilizing the chemical hardnesses of the
donor and acceptor groups.18 In compounds with a non-polar
structure dominant in the ground state, charge flows internally
from the donor toward the acceptor fragments, generating a more
polar excited state, resulting in a steeper dependence of the LUMO
in comparison to the HOMO energy on solvent polarity and posi-
tive solvatochromism. If a polar structure is dominant in the
ground state and excitation reduces the molecular polarity, nega-
tive solvatochromism is observed. In some cases, the solvent polar-
ity changes do not result in unidirectional changes in the
absorption energies (positive or negative solvatochromism). In
these cases, described as inverted solvatochromism, the HOMO
and LUMO energy dependences cross within the accessible range
of solvent polarity. With increasing solvent polarity, the absorption
maxima shift to longer wavelengths (bathochromic shift), but in
the most polar (protic) solvents, the direction is reversed (hypso-
chromic shift). This kind of behavior was observed for compounds
4, 5, and 9. Hypsochromic deviation from the expected positive sol-
vatochromism in protic polar solvents can also be attributed to
hydrogen bond formation between the solvent and molecule in
the polar excited state. A non-linear solvent effect on the fluores-
cence emission has been observed for compounds 4 and 5
(Fig. 4), similar to previous results for ADMAN and DDNP.2,12
In conclusion, through the insertion of phenylethynylidene (cf.
compound 5) or phenyldiazenylidene (cf. compound 10) spacers
between the donor group and the naphthalene ring in DDNP, elon-
gation of the molecule was achieved. The distances between the
respective donor and acceptor groups in the solid state increased
from 11.28 Å (DDNP) to 18.04 (5) or 17.42 Å (10). The absorption
maxima exhibit bathochromic shifts with increased dipoles and
Acknowledgments
Financial support from the Slovenian Research Agency (Grant
P1-0230) is acknowledged. This work was partially supported
within the infrastructures of the EN-FIST Centre of Excellence,
Dunajska 156, SI-1000 Ljubljana, Slovenia, and the Centre for Re-
search Infrastructure at the Faculty of Chemistry and Chemical
Technology of the University of Ljubljana.
Supplementary data
Supplementary data (experimental procedures, characteriza-
tion data, 1H and 13C NMR spectra of all new compounds; ORTEP
drawings of crystal structures of compounds 5, 7, 8, and 10) asso-
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.tetlet.2014.01.002.
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