Bis(arylvinyl)pyrazines, -pyrimidines, and -pyridazines As imaging agents for tau fibrils and β-Amyloid plaques in alzheimers disease models

Article abstract of DOI:10.1021/jm300653b

The in vivo diagnosis of Alzheimers disease (AD) is of high socioeconomic interest and remains a demanding field of research. The biopathological hallmarks of the disease are extracellular plaques consisting of aggregated β-amyloid peptides (Aβ) and tau protein derived intracellular tangles. Here we report the synthesis and evaluation of fluorescent pyrazine, pyrimidine,and pyridazine derivatives in vitro and in vivo aiming at a tau-based diagnosis of AD. The probes were pre-evaluated on human brain tissue by fluorescence microscopy and were found to label all known disease-related alterations at high contrast and specificity. To quantify the binding affinity, a new thiazine red displacement assay was developed and selected candidates were toxicologically profiled. The application in transgenic mouse models demonstrated bioavailability and brain permeability for one compound. In the course of histological testing, we discovered an AD-related deposition of tau aggregates in the Bowmans glands of the olfactory epithelium, which holds potential for an endoscopic diagnosis of AD in the olfactory system.

Full text of DOI:10.1021/jm300653b

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Article
Bis(arylvinyl)pyrazines, -pyrimidines and -pyridazines as Imaging Agents
for Tau Fibrils and #-Amyloid Plaques in Alzheimer’s Disease Models
Alexander Boländer, Daniel Kieser, Constantin Voss, Silvia Bauer, Christian Schön, Steffen
Burgold, Tobias Bittner, Jana Hölzer, Roland Heyny-von Haußen, Gerhard Mall, Valérie Goetschy,
Christian Czech, Henner Knust, Robert Berger, Jochen Herms, Ingrid Hilger, and Boris Schmidt
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm300653b • Publication Date (Web): 22 Aug 2012
Downloaded from http://pubs.acs.org on August 27, 2012
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Journal of Medicinal Chemistry is published by the American Chemical Society.
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Bis(arylvinyl)pyrazines, -pyrimidines and -
pyridazines as Imaging Agents for Tau Fibrils and β-
Amyloid Plaques in Alzheimer’s Disease Models
Alexander Boländer,† Daniel Kieser,† Constantin Voss,† Silvia Bauer,† Christian Schön,‡ Steffen
Burgold,‡ Tobias Bittner,‡ Jana Hölzer,§ Roland Heyny-von Haußen,≠ Gerhard Mall,≠ Valérie
Goetschy,∞ Christian Czech,∞ Henner Knust,∞ Robert Berger,† Jochen Herms,‡ Ingrid Hilger,§
and Boris Schmidt*,†
†Clemens Schoepf-Institute of Organic Chemistry and Biochemistry,Technische Universität
Darmstadt, Petersenstrasse 22, 64287 Darmstadt, Germany, ‡DZNE site Munich and Center of
Neuropathology Ludwig- Maximilians- Universität, 81377 Munich, Germany, §Institute of
Diagnostic and Interventional Radiology I, Experimental Radiology, Universitätsklinikum Jena,
07747 Jena, Germany, ≠Department of Pathology, Klinikum Darmstadt, 64287 Darmstadt,
Germany, ∞F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland
KEYWORDS: Alzheimer’s disease, fluorescent probes, imaging agents, medicinal chemistry,
polarizability.
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ABSTRACT: The in vivo diagnosis of Alzheimer’s disease (AD) is of high socioeconomic
interest and remains a demanding field of research. The biopathological hallmarks of the disease
are extracellular plaques consisting of aggregated β-amyloid peptides (Aβ) and tau protein
derived intracellular tangles. Here we report the synthesis and evaluation of fluorescent pyrazine,
pyrimidine and pyridazine derivatives in vitro and in vivo aiming at a tau-based diagnosis of AD.
The probes were pre-evaluated on human brain tissue by fluorescence microscopy and were
found to label all known disease related alterations at high contrast and specificity. To quantify
the binding affinity, a new thiazine red displacement assay was developed and selected
candidates were toxicologically profiled. The application in transgenic mouse models
demonstrated bioavailability and brain permeability for one compound. In the course of
histological testing, we discovered an AD related deposition of tau aggregates in the Bowman’s
glands of the olfactory epithelium, which hold potential for an endoscopic diagnosis of AD in the
olfactory system.
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Introduction
With approximately 36 million people worldwide suffering from dementia and with that number
expected to grow to 114 million by 2050, AD is the most common type of dementia among older
adults.1 There is no cure for the disease and a distinct diagnosis can be carried out by postmortem
examination only, when the biopathological hallmarks of AD are detectable in human brain
tissue. These are two notorious protein aggregates: extracellular plaques mainly consisting of
two β-amyloid peptides Aβ40 and Aβ42, and intracellular tangles resulting from the aggregation
of abnormally hyperphosphorylated tau protein to paired helical filaments. Both deposits are
considered to impair intra- and inter-neuronal reaction cascades causing progressive
neurodegeneration almost decades before first signs of cognitive impairment emerge.2 It has
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been shown that the load of tau fibrils in brain tissue correlates far more accurately to the
severity of dementia, whereas there are several reports for amyloid plaque-burden at autopsy, yet
no signs of cognitive impairment during lifetime.3 Nevertheless, both deposits remain the most
important targets for in vivo imaging of disease progression. Especially positron emission
tomography (PET) and single photon emission computed tomography (SPECT) are under
constant further development with respect to new radiolabeled markers to visualize the
pathology. [11C]PIB is the most prominent and most frequently applied PET tracer to visualize
Aβ in vivo.4
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The insufficient ability of the markers to discriminate between intracellular tau and extracellular
Aβ and the relative amounts of these deposits limit them to Αβ detection. In the course of ligand
design tau targeting probes have emerged more slowly than Aβ probes. This may be due to the
still overwhelming interest in Aβ. Therefore tau-directed probes are needed. On the basis of
histological staining of tau fibrils and Aβ plaques, we sought non-toxic fluorescent dyes of high
photostability and a high affinity to tau over Aβ, easy to handle and applicable in biological and
histological staining.
Results and Discussion
We reported on curcumin-derived isoxazoles 2 and pyrazoles 3 as high affinity ligands for
fibrillar Aβ42 aggregates and as potent inhibitors of tau aggregation recently (Scheme 1).5
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Scheme 1. Reported synthesis of curcumin-derived isoxazoles 2 and pyrazoles 3
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9
O
O
O
O
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HO
OH
1
a
b
R
O
O
O
O
N O
N N
OH
OH
HO
HO
2
3
a) NH2OH·HCl, pyridine, EtOH, reflux. b) RNHNH2, MeOH, NEt3, rt / RNHNH2, TFA,
toluene, reflux.5
These results suggest a fundamental affinity of these compounds to tau deposits in vitro. The
same rationale was exploited to arrive at the tau ligand TH-2 (4) (Figure 1).6
Figure 1. Structure of the tau ligand TH-2 (4)6
Curcumin (1) itself is currently used in optical imaging studies and led to the identification of Aβ
plaques in the mouse retina, yet requires high intravenous dosage of 1 which is deemed toxic for
humans.7 Unfortunately the fluorescence of isoxazoles 2 and pyrazoles 3 is low and their
derivation from 1 imposes limits on the substitution pattern of their phenylenevinylene flanks.
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Structurally, they resemble methoxy-XO4 (5) (Figure 2), the benchmark ligand for fluorescence
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microscopy of Aβ plaques in vivo, which has also been used for Aβ-PET imaging as its C-
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labeled isotopologue8 but displays very low clearance and non-ideal single-photon excitation in
tissue ruling out a clinical application.8-10
OH
O
HO
methoxy-XO4 (5)
Aryl
N
Aryl
N
N
N
N
N
Aryl
Aryl
Aryl
Aryl
R
8
6
7
Aryl: see Table 1
Figure 2. 5, bis(arylvinyl)pyrazines 6, -pyrimidines 7 and –pyridazines 8
The replacement of the anisole core of 5 by pyrazine, pyrimidine or pyridazine as aromatic cores
with a high electron-withdrawing character and significant aromaticity should lead to molecules
with high conjugation length and permits a large structural variability within the flanks of the
molecule. This allows for an improvement of physicochemical properties by introducing
functional groups affecting lipophilicity and fluorescence. Herein we describe the synthesis of
bis(arylvinyl)pyrazines 6, -pyrimidines 7 and -pyridazines 8 (Figure 2), and their histological and
toxicological evaluation with respect to AD-pathology aiming at an in vivo diagnosis of AD
associated protein deposits.
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The styryl scaffold of 6 and 7 is formed in an atom efficient reaction by the condensation of
aromatic aldehydes, and dimethylpyrazine 9 or dimethylpyrimidine 10 according to a slightly
modified published procedure (Scheme 2).11
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Scheme 2. Synthesis of bis(arylvinyl)pyrazines 6 and -pyrimidines 7
Aryl
O
KOtBu
N
N
N
2
2
N
N
N
N
Aryl
Aryl
H
H
DMF
Aryl
Aryl
9
6
7
O
KOtBu
N
DMF
Aryl
10
Aryl: see Table 1
The selectivity was sufficiently high to generate trans isomers only within the limits of NMR
1
detection by the coupling constant for the vinylic protons in the H-NMR spectra (J ca. 16 Hz).
The products obtained are listed in Table 1.
The synthesis of the bis(arylvinyl)pyridazines 8 was carried out via a ten step synthesis (Scheme
3).
Scheme 3. Synthesis of bis(arylvinyl)pyridazines 8
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COOMe
COOMe
R
)
*
O
(a)
R
(b)
N
N
N
N
N
N
HCl H2N
OEt
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COOMe
COOMe
11
12
13a-c
14a-c
PO(OEt)2
Cl
R
OH
R
R
N
N
(d)
N
N
(c)
N
N
PO(OEt)2
Cl
OH
15a-c
16a-c
17a-c
a: R = phenyl
b: R = 4-methoxyphenyl
c: R = 2-fluorophenyl
13-17
PO(OEt)2
R
N
Aryl
O
N
N
N
(e)
Aryl
H
Aryl
R
PO(OEt)2
8a-e
a-c: R = phenyl
17a-c
d: R = 4-methoxyphenyl
e: R = 2-fluorophenyl
Aryl: see Table 1
*)Five step synthesis of tetrazinedimethylcarboxylate 12 according to a published procedure12
and further five step conversion of 12 to bis(arylvinyl) pyridazines 8a-e; (a) dioxane, reflux; (b)
LiAlH4, 60 °C; (c) SOCl2, CH2Cl2, rt; (d) P(OEt)3, 110 °C; (e) NaH, DMF, rt.
Starting from glycine ethyl ester hydrochloride 11, tetrazinedimethylcarboxylate 12 was obtained
according to a strategy reported for 13a (R = phenyl).12 The diversity of the aromatic substituent
R on the pyridazines 14a-c was introduced by a subsequent Diels-Alder reaction with inverse
electron demand utilizing 12 and the aromatic alkynes 13a-c to provide novel derivatives.
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Reduction of 14a-c and conversion of the resulting dialcohols 15a-c yielded 3,6-
bis(chloromethyl)pyridazines 16a-c. From the latter resulted the corresponding diphosphonates
17a-c which underwent a Horner-Wadsworth-Emmons reaction with aromatic aldehydes to yield
the desired bis(arylvinyl)pyridazines 8a-e (Table 1).
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Table 1. 5 and the synthesized bis(arylvinyl)pyrazines 6a-h, -pyrimidines 7a-f and -pyridazines
8a-e with determined affinities, spectroscopical data and polarizability
IC50 (nM)a
cLogPb
max
λabs
(nm)
max
λabs
(nm)
max
λem
(nm)
compound
α’
(a0 )
3 d
(calculated)c
Tau
Aβ
246
140
5.35
5
Bis(arylvinyl)pyrazines 6
n.m.e
n.m.e
4.10
1.38
405
435
453
420
570
544
507
362
6a
6b
13
17
3.89
6.29
2.87
380
375
380
414
403
448
486
457
564
421
455
560
6c
inactive
6d
6
43
51
6e
6f
42
3.12
5.29
4.66
390
385
416
454
454
458
556
574
565
608
585
568
n.m.e
n.m.e
6g
6h
Bis(arylvinyl)pyrimidines 7
4
54
4.54
430
428
575
462
7a
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inactive
3.32
3.56
380
405
424
427
538
552
517
563
7b
7469
2381
7c
7d
7e
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17
2
156
52
3
5.73
5.10
5.68
414
440
450
429
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476
576
581
587
540
522
635
2
7f
Bis(arylvinyl)pyridazines 8
inactive
6.13
4.91
3.41
411
365
409
455
448
429
537
535
509
559
614
419
8a
4707
7591
8b
8c
inactive
1591
4571
5.96
6.19
410
415
453
457
534
535
580
560
8d
8e
1251 10066
aIC50 values were determined in a thiazine red displacement assay as duplicates of replicate
experiments13; calculated cLogP values14; calculated absorption maxima13; calculated static
b
c
d
electronic polarizability volume
3
α’ = α / (4πε0) in units of the Bohr radius cubed
e
(a0 ), α = calculated polarizability13, ε0 = vacuum permittivity13; n.m.: not measurable due to
spectral overlap with the reference compound thiazine red
For all compounds we calculated13 the absorption maxima and determined the absorption and
emission maxima in solution. In a preliminary in vitro study the synthesized dyes were applied to
human brain tissue with AD pathology.15 The affinity to tau fibrils and Aβ plaques was evaluated
qualitatively by fluorescence microscopy.13 All of the tested compounds showed good to
excellent staining of both tau fibrils and Aβ plaques at high contrast, low background staining
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and high photostability in accord with immunohistochemical staining applying the antibodies
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anti-PHF-Tau clone AT8 mAb for tau deposits and Amyloid A4 for Aβ plaques (Figure 3 A-D).
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Figure 3. AD related deposits on human brain tissue, top: immunohistologically stained tau
fibrils with antibody AT8 mAb (A) and Aβ-Plaques with antibody Amyloid A4 (B), middle: Tau
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fibrils (C) and Aβ-Plaques (D) stained with 6a, bottom: Intracellular tau tangle and extracellular
tau tangles (arrow-marked) (E) and Aβ-angiopathy (F) stained with 6a15
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Though staining with antibodies is still the generally accepted benchmark to evaluate new
imaging markers, antibodies often display a lack of sensitivity. AT8-staining fails to detect up to
38 % of neurofibrillary tangles.16 Especially so-called ghost tangles, which are released from
affected neurons after cell-degeneration and are typical of late stages of AD do not show any
positive immune-tau-staining.17 Indeed the detection of ghost tangles is of high interest if it
comes to a post-mortem classification of the stage of AD at hand. They may be identified by
immunostaining of ubiquitin, a polypeptide, which is also a part of neurofibrillary tangles. Since
ubiquitin is expressed in all eukaryotic cells, it is not a specific indicator though.18 Our ligands
stain both intracellular fibrillar threads of tau and apparent extracellular aggregates of tau (Figure
3 E). In addition to extracellular Aβ plaques, brain matter affected by AD pathology harbors
additional amyloid deposits within the vascular walls of blood vessels called Aβ-angiopathies,
which are also considerably stained by 6a (Figure 3 F).19 The primary screen on human AD brain
preparations filtered test compounds for tau-staining properties. Eventually all tau positives also
stained amyloid plaques yet at variable degree. Thus all AD related pathological protein deposits
are stained by our dyes in a one-time procedure and can be detected by fluorescence microscopy.
One example is provided in Figure 3.13
Histological in vitro staining experiments provide little information on absolute affinities and are
not directly comparable with respect to prospective in vivo imaging. Thus we developed a new in
vitro affinity assay to determine the IC50 values versus thiazine red displacement from
aggregated tau and Aβ40.13 Thiazine red was selected as reference marker because it was
previously shown to be an accurate marker for β-pleated sheet structures and to reliably stain Aβ
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plaques, tau fibrils and diffuse neurofibrillary tangles (Kd for aggregated tau = 18 nM, Kd for
aggregated Aβ = 49 nM).13, 16, 20, 21 We evaluated the established Aβ marker 5 in this assay. 11C-
labeled 5 was reported to display an affinity of Ki = 26.8 nM to aggregated Aβ40 in a
displacement assay versus unlabeled 5.8 In our assay it turned out to be a much weaker ligand in
competition to thiazine red, displaying an IC50 of 140 nM on Aβ40. All IC50 values are listed in
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Table 1. Almost all of our compounds show higher affinity to aggregated tau than to Aβ40,
except for 7c. Even though 5 is considered a selective amyloid marker, we could only determine
a 1.8-fold higher affinity to Aβ over tau. It is reported that the relative amounts of amyloid and
tau deposits require a 20- to 50-fold higher selectivity towards tau to allow in vivo detection of
tau. Compounds that do not reach this threshold are limited to the detection of tau in the
enthorhinal cortex.22 If it comes to tau selective markers, 7e meets this criterion. 7a has a
comparable tau affinity, however it is just 13.5-fold higher than its Aβ affinity. Caveat: This
selectivity was observed in in vitro assays which may not reflect the physiological situation in
vivo.
A distinct structure-activity-relationship cannot be deduced from the affinity results. The variable
substituents of the tested molecules do not solely account for a probe’s affinity, though a trend
can be observed indicating that N- or O-containing substituents in para position on the aromatic
flanks drastically increase affinity. Nevertheless they do not guarantee high affinity; both shape
and size of the entire molecule seem to provide an equipotent influence: 6e shows a very high
affinity to aggregated tau (IC50 = 6 nM) and a moderate to Aβ40 (IC50 = 43 nM). Its angular
isomer 7b shows no notable affinity to tau or Aβ40 at all. 8b has a nearly linear equilibrium
structure in its electronic ground state, the additional phenyl ring on the pyridazine core buckles
the angle between the 4-morpholinostyryl-branches marginally, but significantly enlarges the
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size of the molecule, which seems to strongly diminish its target affinity (aggr. tau IC50= 4707
nM, aggr. Aβ40 IC50= 7591 nM). This observation is valid for all tested
bis(arylvinyl)phenylpyridazines 8. They all show an extremely low target binding. Where a
direct comparison is possible, bis(arylvinyl)pyrazines 6 show higher affinities than their
bis(arylvinyl)pyrimidine isomers 7. On the other hand angular bis(arylvinyl)pyrimidines 7 can
show high to excellent affinity, especially 7a, 7e and 7f. As a general correlation between
substitution pattern and affinity does not seem to exist, we tried to correlate affinities to
physicochemical parameters, starting with the calculated cLogP values (Table 1). We clearly
emphasize that these values are obtained from calculations based on specific algorithms for
calculating cLogP from fragment-based methods and can differ from experimental
determinations by a value of 2-3.14 (For 5 the calculated cLogP value is 5.35 whereas the
experimentally determined is reported as 2.6).8 The plotting of the IC50 values against the cLogP
values indicates a lack of exploitable correlation.13
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Besides, polarizability has been proposed as a descriptor for tau binding affinity within
individual scaffold families.23 Inspired by these results we calculated the static electronic
polarizabilities
α ’ of our compounds (Table 1) and plotted the corresponding IC50 values against
them.13 Again, there is no significant correlation between polarizability and affinity of this ligand
series to tau and Aβ.13 Due to the poor correlation of both cLogP and static electronic
polarizability
α ’ to both tau and Aβ affinity, neither parameter seems useful for further
improvement of our markers. Since 7a and 7e show the highest affinity to aggregated tau (7a:
IC50 = 4 nM, 7e: IC50 = 2 nM) over a moderate Aβ affinity (7a: IC50 = 54 nM, 7e: IC50 = 52 nM),
we decided to test both compounds in vivo. After the toxicological evaluation in human liver
carcinoma cell lines Hep G2 did not show any significant decline in cell vitality, we evaluated
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selected candidates in vivo in a transgenic mouse model.13, 24 However recent in vivo studies and
histological staining experiments indicate severe discrepancies between labelling experiments of
human and murine tau, which refers to significant conformational differences between the two
and suggests the absence of the appropriate ligand binding sites on transgenic murine tau versus
human tau aggregates.25 Thus we relied on the Aβ affinity of both ligands to determine the brain
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permeability and bioavailability in a transgenic mouse model overexpressing Aβ (APPswePS1∆E9
)
and equipped with a cranial window. While 7e did not show any positive results, Aβ plaques
were very well detectable with 7a in the mouse brain by in vivo fluorescence microscopy 4 hours
after injection of 2 mg/kg (Figure 4A). Counterstaining with 5 was performed in the same mouse
2 days later by the additional injection of 2 mg/kg and confirmed the results as the areas stained
by 7a were overlayed by the areas stained by 5 (Figure 4B).
Figure 4. A Detection of Aβ plaques stained with 7a in APPswePS1∆E9 transgenic mouse model
(2 mg 7a/kg intraperitoneal injection (ip.) after 4 hours) through cranial window in vivo. B
Counterstaining by additional injection of 5 in the same mouse on day 2 (2 mg 4/kg ip. after 4
hours)13, 24
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The fluorescence properties of 7a in solution are characterized by a broad excitation range and a
remarkable Stokes shift of about 140 nm (Figure 5A). The emission of 7a after target binding
shows a maximum at 510 nm, which enables for an easy distinction from stained lipofuscin
deposits at 570 nm, whose accumulation is also implicated in several neurodegenerative diseases
(Figure 5 B-C).26
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Figure 5. A: 2D-absorption/emission spectra of 7a (measured in methanol). B-D: In vitro on
target emission spectra of 7a on human hippocampal tissue (B: tau fibril, C: Aβ plaque, D:
lipofuscin)13
Conclusion and Outlook
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In summary, we present a series of fluorescent probes for in vitro detection of AD associated
protein deposits in human brain tissue by fluorescence microscopy offering a broad excitation
and emission range by the choice of suitable candidates. Two of these compounds (7a, 7e) even
show a remarkably higher selectivity for aggregated tau, which qualifies for further tau selective
imaging techniques with respect to an early onset diagnosis of AD. The ability of 7a to pass the
blood-brain barrier was demonstrated in a transgenic mouse model by the Aβ binding of that
compound in vivo. In the course of our histological studies of ligand profiling on human AD-
altered brain material, we discovered that certain areas of the olfactory epithelium, which can be
considered an extension of the brain actually, harbor tau deposits13, 15 These are the Bowman’s
glands, which are involved in the production of nasal discharge but also contain nerve tissue
(Figure 6 B-D). Our staining results correlated with TAU Ab-3 immunostaining (Figure 6 A) and
thioflavin-S-staining (data not shown), whereas no Aβ deposits could be observed in these
regions.
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Figure 6. Bowman’s glands of the olfactory epithelium containing tau aggregates extracted from
a deceased AD patient (88y, Braak V, CERAD C). A: immunohistological staining with antibody
TAU Ab-3 B: stained with 7a C-D: stained with 6a13, 15
These findings have been confirmed within the investigation of olfactory epithelium tissue of 20
confirmed AD patients (classification analysis: NIA Reagan Institute criteria27) in comparison to
5 healthy controls. The severity of tau load in the Bowman’s glands and its correlation to the
progress of AD is subject to ongoing investigation. These results hold potential to detect AD
related tau deposits endoscopically and therefore noninvasively in the olfactory epithelium at
very high resolution. The advantages of such a method compared to imaging techniques based on
radiotracers, which require elaborate infrastructure and on-site radiochemical synthesis of
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suitable tracers is apparent. Moreover, the diameter of a Bowman’s gland is approximately 50
µm, which is far below the resolution of PET or SPECT probes. These are thus limited to the
quantification of an averaged protein deposition, which in turn is rather low prior to the onset of
symptoms, resulting in a signal to noise limitation of these techniques.
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The applied fluorescent markers for Bowman’s gland-imaging should display cell permeability
whereas a brain penetration would no longer be necessary, since the olfactory bulb is not
confined by the blood-brain barrier. A contamination of the brain by brain-penetrating markers
and their metabolites may be avoided, since tracers as 5 clear from the brain extremely slowly
and can still be detected 100 days post injection (pi) at barely diminished intensity compared to
day 1 pi.10
Experimental Section
General Information
All chemicals were purchased as reagent grade from commercial suppliers and used without
further purification, unless otherwise noted. Proton (1H-NMR) and carbon (13C-NMR) nuclear
magnetic resonance spectra were recorded on Bruker DRX 500 at 500 MHz and 125.75 MHz
respectively at 293 K. The chemical shifts are given in parts per million (ppm) on the delta scale
(δ) and are referenced to tetramethylsilane (δ = 0 ppm), 1H-NMR. Abbreviations for NMR data:
s = singlet; d = doublet; t = triplet; q = quartet; qi = quintet; dd = doublet of doublets; dt =
doublet of triplets; dq = doublet of quartets; tt = triplet of triplets; m = multiplet. Coupling
constants (J values) are given in hertz (Hz). Mass spectra were recorded on a Finnigan MAT 95
magnetic sector spectrometer. Thin layer chromatography (TLC) was performed using Merck
TLC silica gel 60 F 254 (0.2 mm) on Aluminium sheets. Silica gel columns for chromatography
were prepared with E. Merck silica gel 60 (0.063-0.20 mesh ASTM). UV/vis-spectra in
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methanolic solution were recorded using a Shimadzu UV-2401 PC UV-VIS Recording
Spectrometer, fluorescence spectra in methanolic solution were recorded using a Shimadzu RF-
1501 Spectrofluorophotometer. 2D fluorescence spectra were recorded on a Tecan Infifite®
M1000.
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High performance liquid chromatographies (HPLC) were carried out on an Agilent 1100
(column: reversed phase, Zorbax Eclipse XDB-C18, 4.6 mm x 150 mm; detector: Agilent 1100
Series Refractive Index Detector, 254 nm). Solvent gradient = 90% A at 0 min, 30% A at 2 min,
10% A at 5 min; solvent A = 0.1% trifluoroacetic acid in water; solvent B = acetonitrile; flow
rate 1.0 mL/min; temperature 35 °C.
All compounds that were evaluated in biological assays had >95% purity, which has been
determined using this method.
Synthesis of bis(arylvinyl)pyrazines (6a-h) and bis(arylvinyl)pyrimidines (7a-e).
2,5-Dimethylpyrazine (0.125 g, 1.16 mmol) or 4,6-dimethylpyrimidine (0.125 g, 1.16 mmol)
was dissolved in 10 mL of dry dimethylformamide, potassium-tert-butoxide (0.26 g, 2.31 mmol)
was added and the mixture was heated to 80 °C. After 15 minutes, the corresponding aromatic
aldehyde (2.31 mmol) was added and the reaction mixture was stirred for 4 hours at 80 °C. After
cooling to ambient temperature, the resulting solid was filtered off and washed with ethyl
acetate. If necessary, the obtained product was purified by column chromatography.
4,4'-(1E,1'E)-2,2'-(pyrazine-2,5-diyl)bis(ethene-2,1-diyl)bis(N,N-dimethylaniline) (6a). Yield
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0.370g, 86%, red solid. H-NMR (CDCl3, 500 MHz): δ [ppm] = 3.02 (12H, s), 6.72 (4H, d, J =
8.9 Hz), 6.96 (2H, d, J = 16 Hz), 7.49 (4H, d, J = 8.9 Hz), 7.62 (2H, d, J = 16 Hz), 8.49 (2H, s).
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13C-NMR (CDCl3, 125 MHz): δ [ppm] = 39.3, 111.2, 118.7, 123.7, 127.5, 132.8, 141.7, 148.0,
8
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149.7. HPLC: 98%; tR 3.20 min. HRMS: calcd. for C24H26N4: 370.2158, found: 370.21641.
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2,5-bis((E)-2-(1-methyl-1H-pyrrol-2-yl)vinyl)pyrazine (6b). Yield 0.301 g, 89%, orange solid.
1H-NMR (CDCl3, 500 MHz): δ [ppm] = 3.76 (6H, s), 6.19 (2H, m), 6.65 (2H, m), 6.70 (2H, m),
6.88 (2H, d, J = 15.6 Hz), 7.62 (2H, d, J = 15.6 Hz), 8.40 (2H, s). 13C-NMR (CDCl3, 125 MHz):
δ [ppm] = 33.3, 107.8, 107.9, 119.3, 120.6, 124.1, 130.5, 142.1, 147.8. HPLC: 96%; tR 7.53 min.
HRMS: calcd. for C18H18N4: 290.1532, found: 290.15118.
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2,5-bis(4-methoxystyryl)pyrazine (6c). Yield 0.322 g, 80%, yellow solid. H-NMR (CDCl3, 500
MHz): δ [ppm] = 3.85 (6H, s), 6.93 (4H, d, J = 9 Hz), 7.04 (2H, d, J = 16 Hz), 7.55 (4H, d, J = 9
Hz), 7.67 (2H, d, J = 16 Hz), 8.54 (2H, s). 13C-NMR (CDCl3, 125 MHz): δ [ppm] = 53,4, 112.3,
120.0, 126.7, 131.7, 141.0, 146.9, 158.3. HPLC: 99%; tR 4.63 min. HRMS: calcd. for
C22H20N2O2: 344.1525, found: 344.15173.
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2,5-bis(4-isopropylstyryl)pyrazine (6d). Yield 0.373 g, 86%, light yellow solid. H-NMR
(CDCl3, 500 MHz): δ [ppm] = 1.28 (12H, d, J = 6.9 Hz), 2.94 (2H, d, J = 6.9 Hz), 7.14 (2H, d, J
= 16.1 Hz), 7.26 (4H, d, J = 8.1 Hz), 7.54 (4H, d, J = 8.1 Hz), 7.71 (2H, d, J = 16.1 Hz), 8.58
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(2H, s). C-NMR (CDCl3, 125 MHz): δ [ppm] = 23.9, 34.0, 123.3, 127.0, 127.3, 134.0, 134.2,
143.1, 149.1, 150.0. HPLC: 97%; tR 5.25 min. HRMS: calcd. for C26H28N2: 368.2253, found:
368.22192.
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2,5-bis(4-morpholinostyryl)pyrazine (6e). Yield 0.417 g, 79%, orange solid. H-NMR (CDCl3,
500 MHz): δ [ppm] = 3.25 (8H, t, J = 4.8 Hz), 3.90 (8H, t, J = 4.8 Hz), 6.95 (4H, d, J = 8.8 Hz),
7.03 (2H, d, J = 16.2 Hz), 7.53 (4H, d, J = 8.8 Hz), 7.66 (2H, d, J = 16.2 Hz), 8.53 (2H, s).
HPLC: 99%; tR 7.35 min. HRMS: calcd. for C28H30N4O2: 454.2369, found: 454.2354.
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2,5-bis(4-(4-methylpiperazin-1-yl)styryl)pyrazine (6f). Yield 0.463 g, 83%, yellow solid. H-
NMR (500 MHz, CDCl3, 300 K): δ [ppm] = 2.36 (s, 6H), 2.58 (8H, t, J = 5.0 Hz), 3.30 (8H, t, J
= 5.0 Hz), 6.92 (4H, d, J = 8.8 Hz), 7.00 (2H, d, J = 16.1 Hz), 7.50 (4H, d, J = 8.8 Hz), 7.63 (2H,
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d, J = 16.1 Hz), 8.51 (2H, s). C-NMR (125 MHz, CDCl3, 300 K): δ [ppm] = 45.2, 47.3, 54.0,
114.4, 120.1, 126.4, 127.4, 132.6, 141.9, 148.0, 150.4. HPLC: 98%; tR 1.80 min. HRMS: calcd.
for C30H36N6: 480.3003, found: 480.30034.
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2,5-bis(4-(piperidin-1-yl)styryl)pyrazine (6g). Yield 0.413 g, 79%, orange solid. H-NMR (500
MHz, CDCl3, 300 K): δ [ppm] = 1.61 (4H, m), 1.70 (8H, m), 3.26 (8H, t, J = 5.3 Hz), 6.91 (4H,
d, J = 8.5 Hz), 6.99 (2H, d, J = 15.9 Hz), 7.49 (4H, d, J = 8.5 Hz), 7.62 (2H, d, J = 15.9 Hz), 8.50
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(2H, s). C-NMR (125 MHz, CDCl3, 300 K): δ [ppm] = 24.4, 25.6, 49.7, 115.6, 120.6, 126.7,
128.4, 133.7, 142.8, 149.0, 152.1. HPLC: 96%; tR 6.51 min. HRMS: calcd. for C30H34N4:
450.2784, found: 450.27531.
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2,5-bis(4-(pyrrolidin-1-yl)styryl)pyrazine (6h). Yield 0.402 g, 82%, brown solid. H-NMR (500
MHz, CDCl3, 300 K): δ [ppm] = 2.03 (8H, m), 3.34 (8H, t, J = 6.6 Hz), 6.56 (4H, d, J = 8.7 Hz),
6.93 (2H, d, J = 16.0 Hz), 7.48 (4H, d, J = 8.7 Hz), 7.61 (2H, d, J = 16.0 Hz), 8.48 (2H, s). 13C-
NMR (125 MHz, CDCl3, 300 K): δ [ppm] = 25.5, 47.6, 111.8, 119.0, 123.9, 128.7, 134.0, 142.7,
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149.0, 151.3. HPLC: 99%; tR 6.28 min. HRMS: calcd. for C28H30N4: 422.2471, found:
422.24976.
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4,4'-(1E,1'E)-2,2'-(pyrimidine-4,6-diyl)bis(ethene-2,1-diyl)bis(N,N-dimethylaniline) (7a). Yield
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0.383 g, 89%, yellow solid, H-NMR (500 MHz, CDCl3, 300 K): δ [ppm] = 2.93 (12H, s), 6.62
(4H, d, J = 8.6 Hz), 6.76 (2H, d, J = 15.8 Hz), 7.06 (1H, s), 7.42 (4H, d, J = 8.6 Hz), 7.73 (2H, d,
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J = 15.8 Hz), 8.90 (1H, s). C-NMR (125 MHz, CDCl3, 300 K): δ [ppm] = 40.6, 112.4, 115.4,
121.2, 124.2, 129.5, 137.6, 151.5, 158.5, 163.4. HPLC: 98%; tR 6.35 min. HRMS: calcd. for
C24H26N4: 370.2158, found: 370.21534.
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4,6-bis(4-morpholinostyryl)pyrimidine (7b). Yield 0.464 g, 88%, yellow solid. H-NMR (500
MHz, CDCl3, 300 K): δ [ppm] = 3.24 (8H, t, J = 5.0 Hz), 3.87 (8H, t, 5.0 Hz), 6.91 (4H, d, J =
8.9 Hz), 6.91 (2H, d, J = 15.9 Hz), 7.20 (1H, s), 7.53 (4H, d, J = 8.9 Hz), 7.82 (2H, d, J = 15.9
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Hz), 9.03 (1H, s). C-NMR (125 MHz, CDCl3, 300 K): δ [ppm] = 48.4, 66.7, 115.0, 115.7,
122.8, 127.0, 129.0, 136.6, 151.9, 158.6, 163.0. HPLC: 96%; tR 6.48 min. HRMS: calcd. for
C28H30N4O2: 454.2369, found: 454.23502.
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4,6-bis(4-(4-methylpiperazin-1-yl)styryl)pyrimidine (7c). Yield 0.468 g, 84%, yellow solid. H-
NMR (500 MHz, CDCl3, 300 K): δ [ppm] = 2.35 (6H, s), 2.57 (8H, t, J = 5.1 Hz), 3.30 (8H, t, J
= 5.1 Hz), 6.89 (2H, d, J = 15.9 Hz), 6.91 (4H, d, J = 8.8 Hz), 7.19 (1H, s), 7.51 (4H, d, J = 8.8
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Hz), 7.81 (2H, d, J = 15.9 Hz), 9.02 (1H, s). C-NMR (125 MHz, CDCl3, 300 K): δ [ppm] =
45.2, 47.1, 48.0, 53.9, 114.2, 114.5, 121.2, 121.6, 125.6, 128.0, 135.6, 150.8, 157.6, 162.0.
HPLC: 99%; tR 1.80 min. HRMS: calcd. for C30H36N6: 480.3003, found: 480.29930.
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4,6-bis(4-(piperidin-1-yl)styryl)pyrimidine (7d). Yield 0.481 g, 92%, yellow solid. H-NMR
(500 MHz, CDCl3, 300 K): δ [ppm] = 1.60 (8H, t, J = 4.1 Hz), 1.68 (8H, m), 3.27 (8H, t, J = 5.8
Hz), 6.88 (2H, d, J = 15.9 Hz), 6.90 (4H, d, J = 8.8 Hz), 7.18 (1H, s), 7.50 (4H, d, J = 8.8 Hz),
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7.80 (2H, d, J = 15.9 Hz), 9.00 (1H, s). C-NMR (125 MHz, CDCl3, 300 K): δ [ppm] = 24.3,
25.6, 49.5, 115.3, 115.4, 122.0, 125.8, 129.0, 136.8, 152.5, 158.5, 163.1, 170.6. HPLC: 98%; tR
5.20 min. HRMS: calcd. for C30H34N4: 450.2784, found: 450.27606.
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4,6-bis(4-(pyrrolidin-1-yl)styryl)pyrimidine (7e). Yield 0.422 g, 86%, brown solid. H-NMR
(500 MHz, CDCl3, 300 K): δ [ppm] = 2.02 (8H, t, J = 6.5 Hz), 3.34 (8H, t, J = 6.5 Hz), 6.56 (4H,
d, J = 8.8 Hz), 6.82 (2H, d, J = 15.9 Hz), 7.14 (1H, s), 7.50 (4H, d, J = 8.8 Hz), 7.80 (2H, d, J =
15.9 Hz), 8.97 (1H, s). 13C-NMR (125 MHz, CDCl3, 300 K): δ [ppm] = 25.5, 47.6, 111.7, 115.0,
120.4, 123.1, 129.3, 137.2, 148.6, 158.5, 163.2. HPLC: 96%; tR 5.07 min. HRMS: calcd. for
C30H34N4: 422.2471, found: 422.24958.
4,4'-(1E,1'E,3E,3'E)-4,4'-(pyrimidine-4,6-diyl)bis(buta-1,3-diene-4,1-diyl)bis(N,N-
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dimethylaniline) (7f). Yield 0.358 g, 73%, red solid. H-NMR (500 MHz, CDCl3, 300 K): δ
[ppm] = 2.93 (12H, s), 6.41 (2H, d, J = 15.1 Hz), 6.61 (4H, d, J = 8.7 Hz), 6.75 (4H, d, J = 5.1
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Hz), 6.98 (1H, s), 7.31 (4H, d, J = 8.7 Hz), 7.57 (2H, m), 8.90 (1H, s). C-NMR (125 MHz,
CDCl3, 300 K): δ [ppm] = 39.3, 111.2, 114.3, 122.5, 123.9, 126.0, 127.3, 137.2, 137.7, 149.7,
157.5, 161.8. HPLC: 99%; tR 5.88 min. HRMS: calcd. for C28H30N4: 422.2471, found:
422.24540.
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Synthesis of dimethyl 4-arylpyridazine-3,6-dicarboxylates (14a-c)
Dimethyl 4-arylpyridazine-3,6-dicarboxylates (14a-c) were obtained according to a published
procedure.12
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dimethyl 4-phenylpyridazine-3,6-dicarboxylate (14a). Yield 3.302 g, 96%, brown solid. H-
NMR (500 MHz, CDCl3, 300 K): δ [ppm] = 3.87 (3H, s), 4.10 (3H, s), 7.56 (5H, m), 8.27 (1H,
s). 13C-NMR (125 MHz, CDCl3, 300 K): δ [ppm] = 53.3, 53.6, 127.9, 128.2, 129.2, 130.2, 133.9,
140.0, 151.7, 154.3, 164.1, 165.5. EI-MS: m/z = 272 [M]+.
dimethyl 4-(4-methoxyphenyl)pyridazine-3,6-dicarboxylate (14b). Yield 0.751 g, 74%, yellow
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solid. H-NMR (500 MHz, CDCl3, 300 K): δ [ppm] = 3.86 (3H, s), 3.90 (3H, s), 4.09 (3H, s),
8.23 (1H, s), 7.39 (2H, d, J = 8.9 Hz), 7.01 (2H, d, J = 8.9 Hz). 13C-NMR (125 MHz, CDCl3, 300
K): δ [ppm] = 53.3, 53.6, 55.5, 114.8, 125.9, 127.8, 129.6, 139.5, 151.6, 154.3, 161.4, 164.2,
165.9. EI-MS: m/z = 302 [M]+.
dimethyl 4-(2-fluorophenyl)pyridazine-3,6-dicarboxylate (14c). Yield 1.07 g, 73%, brown solid.
1H-NMR (500 MHz, CDCl3, 300 K): δ [ppm] = 3.93 (3H, s), 4.10 (3H, s), 7.25 (4H, m), 8.26
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(1H, s). C-NMR (125 MHz, CDCl3, 300 K): δ [ppm] = 53.3, 53.6, 115.9, 122.4, 125.0, 129.4,
130.1, 132.2, 135.6, 151.9, 153.4, 159.0, 163.6, 164.7. EI-MS: m/z = 290 [M]+.
Synthesis of (4-arylpyridazine-3,6-diyl)dimethanols (15a-c)
(4-Arylpyridazine-3,6-diyl)dimethanols (15a-c) were obtained according to a published
procedure:12
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(4-phenylpyridazine-3,6-diyl)dimethanol (15a). Yield 0.551 g, 51%, brown oil. H-NMR
(CDCl3, 300 MHz, 300 K): δ [ppm] = 3.22 (2H, s), 4.88 (2H, s), 5.04 (2H, s), 7.47 (1H, s), 7.41
(5H, m). 13C-NMR (CDCl3, 125 MHz, 300 K): δ [ppm] = 61.8, 63.5, 125.1, 128.3, 129.1, 129.6,
134.5, 140.2, 157.5, 161.0. EI-MS: m/z = 216 [M]+.
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(4-(4-methoxyphenyl)pyridazine-3,6-diyl)dimethanol (15b). Yield 0.948 g, 77%, brown oil. H-
NMR (CDCl3, 300 MHz, 300 K): δ [ppm] = 3.22 (2H, s), 3.92 (3H, s), 4.88 (2H, s), 5.04 (2H, s),
7.02 (2H, d, J = 9.0 Hz), 7.51 (1H, s), 7.55 (2H, d, J = 9.0 Hz). 13C-NMR (CDCl3, 125 MHz, 300
K): δ [ppm] = 53.4, 62.9, 66.4, 114.6, 126.2, 132.8, 135.2, 141.3, 156.9, 158.4, 161.2. EI-MS:
m/z = 246 [M]+.
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(4-(2-fluorophenyl)pyridazine-3,6-diyl)dimethanol (15c). Yield 0.761 g, 65%, brown oil. H-
NMR (CDCl3, 300 MHz, 300 K): δ [ppm] = 4.78 (2H, s), 4.99 (2H, s), 7.52 (4H, m), 7.58 (1H,
s). 13C-NMR (CDCl3, 125 MHz, 300 K): δ [ppm] = 62.1, 64.0, 127.5, 127.9, 128.2, 128.3, 131.8,
132.9, 138.1, 141.9, 158.5, 161.2. EI-MS: m/z = 234 [M]+.
Synthesis of 3,6-bis(chloromethyl)-4-arylpyridazines (16a-c)
To a solution of the corresponding (4-arylpyridazine-3,6-diyl)dimethanol (15a-c) (1.5 mmol) in
dichloromethane, SOCl2 (0.450 g, 3.75 mmol) was added and the solution was stirred at ambient
temperature for 20 minutes, during which its colour changed from brown to dark red. After the
addition of water and extraction with dichloromethane the united organic layers were dried over
sodium sulfate. The resulting product could be used for the next step without further purification.
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3,6-bis(chloromethyl)-4-phenylpyridazine (16a). Yield 0.114 g, 30%, red oil. H-NMR (CDCl3,
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300 MHz, 300 K): δ [ppm] = 4.84 (2H, s), 4.94 (2H, s), 7.41 (5H, m), 7.63 (1H, s). C-NMR
(CDCl3, 125 MHz, 300 K): δ [ppm] = 42.7, 44.2, 127.1, 128.4, 129.0, 129.6, 134.9, 141.6, 156.8,
158.9. EI-MS: m/z = 253 [M]+.
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3,6-bis(chloromethyl)-4-(4-methoxyphenyl)pyridazine (16b). Yield 0.149 g, 35%, red oil. H-
NMR (CDCl3, 300 MHz, 300 K): δ [ppm] = 3.89 (3H, s), 4.86 (2H, s), 4.94 (2H, s), 7.06 (2H, d,
J = 8.9 Hz), 7.47 (2H, d, J = 8.9 Hz), 7.60 (1H, s). 13C-NMR (CDCl3, 125 MHz, 300 K): δ [ppm]
= 42.8, 44.1, 53.4, 127.4, 127.9, 129.2, 131.7, 135.0, 142.5, 153.8, 158.3. EI-MS: m/z = 283
[M]+.
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3,6-bis(chloromethyl)-4-(2-fluorophenyl)pyridazine (16c). Yield 0.317 g, 78%, brown oil. H-
NMR (CDCl3, 300 MHz, 300 K): δ [ppm] = 4.83 (2H, s), 4.92 (2H, s), 7.56 (4H, m), 7.61 (1H,
s). 13C-NMR (CDCl3, 125 MHz, 300 K): δ [ppm] = 42.7, 44.2, 127.1, 128.4, 129.0, 129.2, 130.6,
131.1, 136.8, 142.3, 155.8, 160.9. EI-MS: m/z = 271 [M]+.
Synthesis of tetraethyl (4-arylpyridazine-3,6-diyl)bis(methylene)diphosphonates (17a-c)
The corresponding dichloride (16a-c) (0.5 mmol) was dissolved in P(OEt)3 (8.310 g, 50 mmol)
and the mixture was stirred for 18 hours at 110 °C whereupon the colour of the solution changed
from dark red to black. After cooling to room temperature, residual P(OEt)3 was distilled off
(high vacuum, 100 °C) and the diphosphonate was obtained as a highly viscous dark oil, which
was used for the next step without further purification.
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tetraethyl (4-phenylpyridazine-3,6-diyl)bis(methylene)diphosphonate (17a). Yield 0.21 g, 92%,
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dark brown oil. H-NMR (CDCl3, 500 MHz, 300 K): δ [ppm] = 1.24 (6H, t, J = 7.1 Hz), 1.27
(6H, t, J = 7.1 Hz), 3.60, (2H, d, J = 20.97), 3.61 (2H, d, J = 21.9), 4.06 (4H, m), 4.10 (4H, m),
7.47 (6H, m). 13C-NMR (CDCl3, 125 MHz, 300 K): δ [ppm] = 16.2, 16.4, 31.5, 33.9, 62.3, 62.5,
128.0, 128.8, 128.9, 129.0, 135.9, 141.0, 152.9, 154.0. EI-MS: m/z = 456 [M]+.
tetraethyl (4-(4-methoxyphenyl)pyridazine-3,6-diyl)bis(methylene)diphosphonate (17b). Yield
0.163 g, 69%, dark brown oil. EI-MS: m/z = 486 [M]+.
tetraethyl (4-(2-fluorophenyl)pyridazine-3,6-diyl)bis(methylene)diphosphonate (17c). Yield
0.209 g, 88%, dark brown oil. EI-MS: m/z = 472 [M]+.
Synthesis of bis(arylvinyl)pyridazines (8a-e)
The corresponding tetraethyl (4-arylpyridazine-3,6-diyl)bis(methylene)diphosphonate (17a-c)
(0.25 mmol) was dissolved in dry DMF and NaH (0.024 g, 1 mmol) was added. After stirring for
30 minutes, a solution of the corresponding aldehyde (0.5 mmol) in dry DMF was added. After
stirring for 18 hours, 1 N HCl was added and the mixture was extracted with DCM. The united
organic layers were washed with saturated brine, dried over sodium sulfate and the solvent was
evaporated. After column chromatography the products were obtained as colored solids.
4,4'-(1E,1'E)-2,2'-(4-phenylpyridazine-3,6-diyl)bis(ethene-2,1-diyl)bis(N,N-dimethylaniline)
(8a). Yield 0.035 g, 31%, red solid. 1H-NMR (CDCl3, 500 MHz, 300 K): δ [ppm] = 2.98 (6H, s),
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3.02 (6H, s), 6.66 (2H, d, J = 8.8 Hz), 6.73 (2H, d, J = 8.8 Hz), 6.94 (1H, d, J = 15.6 Hz), 7.26
(1H, d, J = 16.4 Hz), 7.37 (2H, d, J = 8.8 Hz), 7.42 (s, 1H), 7.49 (5H, m), 7.51 (2H, d, J = 8.8
Hz), 7.59 (1H, d, J = 16.4 Hz), 8.05 (1H, d, J = 15.6 Hz). 13C-NMR (CDCl3, 125 MHz, 300 K): δ
[ppm] = 40.2, 40.3, 112.1, 112.3, 117.4, 120.5, 123.4, 124.5, 125.1, 128.6, 128.8, 128.9, 129.1,
129.2, 135.2, 136.9, 138.4, 138.7, 150.7, 150.9, 153.8, 156.1. HPLC: 98%; tR 6.88 min. HRMS:
calcd. for C30H30N4: 446.2471, found: 446.24664.
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4,4'-(4,4'-(1E,1'E)-2,2'-(4-phenylpyridazine-3,6-diyl)bis(ethene-2,1-diyl)bis(4,1-
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phenylene))dimorpholine (8b). Yield 0.050 g, 37%, red solid. H-NMR (CDCl3, 500 MHz, 300
K): δ [ppm] = 3.19 (4H, t, J = 5.0 Hz), 3.23 (4H, t, J = 5.0 Hz), 3.86 (8H, m), 6.85 (2H, d, J = 9.0
Hz), 6.91 (2H, d, J = 9.0 Hz), 7.01 (1H, d, J = 15.8 Hz), 7.30 (1H, d, J = 16.3 Hz), 7.40 (2H, d, J
= 9.0 Hz), 7.43 (1H, s), 7.45 (5H, m), 7.53 (2H, d, J = 9.0 Hz), 7.61 (1H, d, J = 16.3 Hz), 8.06
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(1H, d, J = 15.8 Hz). C-NMR (CDCl3, 125 MHz, 300 K): δ [ppm] = 48.6, 48.6, 66.7, 115.1,
115.2, 119.1, 122.1, 123.6, 127.7, 128.2, 128.5, 128.6, 128.7, 128.9, 129.0, 129.3, 133.9, 134.8,
136.7, 138.5, 151.4, 151.5, 153.7, 156.1. HPLC: 99%; tR 7.18 min. HRMS: calcd. for
C34H34N4O2: 530.2682, found: 530.26589.
3,6-bis(4-(1-methyl-1H-pyrrol-2-yl)styryl)-4-phenylpyridazine (8c). Yield 0.036 g, 39%, orange
solid. 1H-NMR (CDCl3, 500 MHz, 300 K): δ [ppm] = 8.07 (d, J = 15.5 Hz, 1H), 7.84 (d, J = 15.9
Hz, 1H), 7.53-7.44 (m, 5H), 7.23 (s, 1H), 7.00 (d, J = 15.9 Hz, 1H), 6.89 (d, J = 15.5 Hz, 1H),
6.72 (m, 1H), 6.69-6.65 (m, 2H), 6.43 (m, 1H), 6.20 (m, 1H), 6.12 (m, 1H), 3.76 (s, 3H), 3.72 (s,
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3H). C-NMR (CDCl3, 125 MHz, 300 K): δ [ppm] = 155.6, 153.4, 142.7, 136.6, 131.8, 131.3,
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129.0, 128.8, 128.1, 125.2, 124.9, 124.4, 122.5, 121.4, 120.4, 118.3, 108.9, 108.8, 108.6, 108.5,
34.3, 34.2. HPLC: 97%; tR 6.63 min. HRMS: calcd. for C24H22N4: 366.1845, found: 366.18023.
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4,4'-(1E,1'E)-2,2'-(4-(4-methoxyphenyl)pyridazine-3,6-diyl)bis(ethene-2,1-diyl)bis(N,N-
dimethylaniline) (8d). Yield 0.043 g, 36%, light red solid. 1H-NMR (CDCl3, 500 MHz, 300 K): δ
[ppm] = 2.99 (6H, s), 3.02 (6H, s), 3.91 (3H, s), 6.67 (2H, d, J = 8.7 Hz), 6.73 (2H, d, J = 8.7
Hz), 6.98 (1H, d, J = 15.8 Hz), 7.04 (2H, d, J = 8.7 Hz), 7.22 (1H, d, J = 16.2 Hz), 7.38 (5H, m),
7.51 (2H, d, J = 8.7 Hz), 7.58 (1H, d, J = 16.2 Hz), 8.04 (1H, d, J = 15.8 Hz). 13C-NMR (CDCl3,
125 MHz, 300 K): δ [ppm] = 40.2, 40.3, 55.4, 112.2, 112.2, 114.2, 117.9, 120.9, 123.1, 124.6,
125.2, 128.5, 128.7, 129.2, 130.5, 133.9, 134.8, 137.8, 150.7, 150.8, 153.9, 156.2, 160.0. HPLC:
98%; tR 7.27 min. HRMS: calcd. for C31H32N4O1: 476.2577, found: 476.25247.
4,4'-(1E,1'E)-2,2'-(4-(2-fluorophenyl)pyridazine-3,6-diyl)bis(ethene-2,1-diyl)bis(N,N-
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dimethylaniline) (8e). Yield 0.041 g, 35%, red solid. H-NMR (CDCl3, 500 MHz, 300 K): δ
[ppm] = 2.98 (6H, s), 3.02 (6H, s), 6.66 (2H, d, J = 8.8 Hz), 6.73 (2H, d, J = 8.8 Hz), 6.77 (1H, d,
J = 15.7 Hz), 7.22 (1H, d, J = 16.4 Hz), 7.25 (6H, m), 7.42 (1H, s), 7.51 (2H, d, J = 8.8 Hz), 7.58
(1H, d, J = 16.4 Hz), 8.05 (1H, d, J = 15.7 Hz). HPLC: 96%; tR 6.74 min. HRMS: calcd. for
C30H28N4F1: 463.2299, found: 463.22797.
Tissue Probe Preparation
Human hippocampal tissue fixed in 10% buffered formalin solution and embedded in paraffin
was cut to 4 µm sections in a microtome and mounted on standard microscope slides. Subsequent
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