Synthesis and in Vitro Evaluation of 8-Pyridinyl-Substituted Benzo[e]imidazo[2,1-c][1,2,4]triazines as Phosphodiesterase 2A Inhibitors

Article abstract of DOI:10.3390/molecules24152791

Phosphodiesterase 2A (PDE2A) is highly expressed in distinct areas of the brain, which are known to be related to neuropsychiatric diseases. The development of suitable PDE2A tracers for Positron Emission Tomography (PET) would permit the in vivo imaging of the PDE2A and evaluation of disease-mediated alterations of its expression. A series of novel fluorinated PDE2A inhibitors on the basis of a Benzoimidazotriazine (BIT) scaffold was prepared leading to a prospective inhibitor for further development of a PDE2A PET imaging agent. BIT derivatives (BIT1–9) were obtained by a seven-step synthesis route, and their inhibitory potency towards PDE2A and selectivity over other PDEs were evaluated. BIT1 demonstrated much higher inhibition than other BIT derivatives (82.9% inhibition of PDE2A at 10 nM). BIT1 displayed an IC50 for PDE2A of 3.33 nM with 16-fold selectivity over PDE10A. This finding revealed that a derivative bearing both a 2-fluoro-pyridin-4-yl and 2-chloro-5-methoxy-phenyl unit at the 8- and 1-position, respectively, appeared to be the most potent inhibitor. In vitro studies of BIT1 using mouse liver microsomes (MLM) disclosed BIT1 as a suitable ligand for ¹⁸F-labeling. Nevertheless, future in vivo metabolism studies are required.

Full text of DOI:10.3390/molecules24152791

molecules
Article
Synthesis and In Vitro Evaluation of
8
-Pyridinyl-Substituted
Benzo[e]imidazo[2,1-c][1,2,4]triazines as
Phosphodiesterase 2A Inhibitors
Rien Ritawidya 1,2,*, Friedrich-Alexander Ludwig , Detlef Briel , Peter Brust
Matthias Scheunemann *
1
3
1
and
1
,
1
Department of Neuroradiopharmaceuticals, Institute of Radiopharmaceuticals Cancer Research,
Helmholtz-Zentrum Dresden-Rossendorf, 04318 Leipzig, Germany
Center for Radioisotope and Radiopharmaceutical Technology, National Nuclear and Energy
Agency (BATAN), Puspiptek Area, Serpong, South Tangerang 15310, Indonesia
Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Faculty of Medicine, Leipzig University,
Brüderstraße 34, 04103 Leipzig, Germany
2
3
*
Correspondence: r.ritawidya@hzdr.de or rienrita@batan.go.id (R.R.); m.scheunemann@hzdr.de (M.S.);
Tel.: +49-341-234-179-4611 (R.R.); +49-341-234-179-4618 (M.S.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Diego Muñoz-Torrero
Received: 24 May 2019; Accepted: 26 July 2019; Published: 31 July 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Phosphodiesterase 2A (PDE2A) is highly expressed in distinct areas of the brain, which are
known to be related to neuropsychiatric diseases. The development of suitable PDE2A tracers for
Positron Emission Tomography (PET) would permit the in vivo imaging of the PDE2A and evaluation
of disease-mediated alterations of its expression. A series of novel fluorinated PDE2A inhibitors on
the basis of a Benzoimidazotriazine (BIT) scaffold was prepared leading to a prospective inhibitor
for further development of a PDE2A PET imaging agent. BIT derivatives (BIT1–9) were obtained
by a seven-step synthesis route, and their inhibitory potency towards PDE2A and selectivity over
other PDEs were evaluated. BIT1 demonstrated much higher inhibition than other BIT derivatives
(82.9% inhibition of PDE2A at 10 nM). BIT1 displayed an IC50 for PDE2A of 3.33 nM with 16-fold
selectivity over PDE10A. This finding revealed that a derivative bearing both a 2-fluoro-pyridin-4-yl
and 2-chloro-5-methoxy-phenyl unit at the 8- and 1-position, respectively, appeared to be the most
potent inhibitor. In vitro studies of BIT1 using mouse liver microsomes (MLM) disclosed BIT1 as
a suitable ligand for 18F-labeling. Nevertheless, future in vivo metabolism studies are required.
Keywords:Phosphodiesterase2A(PDE2A);PositronEmissionTomography(PET);Benzoimidazotriazine
(BIT); fluorinated; Mouse Liver Microsomes (MLM)
1
. Introduction
Cyclic nucleotide Phosphodiesterases (PDEs) represent a class of enzymes catalyzing the hydrolysis
of the intracellular second messengers, cyclic Adenosine Monophosphate (cAMP) and cyclic Guanosine
Monophosphate (cGMP) [ ]. cAMP and cGMP are involved in a great variety of cellular functions
1
related to physiological and pathophysiological processes in brain and periphery [2–6].
The 11 family members of PDEs are encoded by 21 genes and classified by their substrate
specificity [
PDEs 1, 2, 3, 10, and 11) hydrolyze both substrates [7,9,11].
The dual substrate enzyme PDE2A is highly expressed in some brain areas, such as nucleus
accumbens, cortex, hippocampus [ ], striatum, amygdala [12], substantia nigra, and olfactory
7–10]. PDEs 4, 7, and 8 are cAMP-specific and PDEs 5, 6, and 9 cGMP-specific, and others
(
7
Molecules 2019, 24, 2791; doi:10.3390/molecules24152791
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2791
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neurons [13
memory, emotion, and related diseases [8,9
–
15], thus being involved in complex neuronal processes like learning, concentration,
]. The inhibition of PDE2A will increase the intracellular
levels of cGMP and cAMP in PDE2A-abundant tissues and may result in improvement of neuroplasticity
and memory function [8].
The activity of PDE2A inhibitors related to cognitive improvement has been evaluated in animal
models [1,7,16,17]. These results suggest the use of PDE2A inhibitors for treatment of neuropsychiatric
diseases such as Alzheimer’s disease and schizophrenia [18].
Up to now, there have been several PDE2A inhibitors reported. BAY 60-7550 (Figure 1), the first
highly-selective PDE2A inhibitor, has been widely used to evaluate PDE2A activity. However,
this compound shows a poor pharmacokinetic profile, as well as poor ability to cross the blood-brain
barrier [11,16]. The finding of BAY 60-7550 triggered many pharmaceutical companies to discover
potent PDE2A inhibitors for potential use in treating a variety of brain diseases [11].
Figure 1. Different PDE inhibitors [15,19–27].
In 2010, Biotie Therapies and Wyeth claimed a series of 1,2,4-triazine- and pyrazine-containing
tricyclic compounds exhibiting dual inhibition against both PDE2A and PDE10A as therapeutic
targets [25
derivatives [27], some of which are depicted in Figure 1 (Compounds
their binding affinities. In 2015, our group reported on the first PDE2A PET tracers on the basis
of a Pyridoimidazotriazine (PIT) scaffold starting from TA1, Figure 1), as the lead compound.
Two fluoroalkyl derivatives, TA3 and TA4 (Figure 1), demonstrated high affinity towards PDE2A
with 28-fold and 125-fold selectivity over PDE10A, respectively [15 21]. More recently, we gained
a further improvement in terms of in vitro PDE2A binding via replacement of the 1-methoxy in TA1
by 1-(2-fluoroethoxy), resulting in the compound TA5, (Figure 1) [15 21 22]. However, after successful
F-labeling, the obtained tracers, [ F]TA3 and [ F]TA4, did not entirely succeed and were proven
–28]. The triazine series comprises benzo- and pyridine-fused imidazo[5,1-c][1,2,4]triazine
I
(
TA1), II, and III), along with
I
(
,
,
,
18
18
18
18
to be suitable only for in vitro autoradiographic PDE2A imaging. Beyond that, [ F]TA5 failed to
demonstrate specific binding in vitro 22]. The formation of brain-penetrating radiometabolites due
[
to the O-defluoroalkylation limited their application for in vivo PDE2A imaging. Therefore, further
structural modifications of tricyclic lead are inevitably required to allow appropriate in vivo PDE2A
18
imaging [21
,22]. A further tracer, [ F]AQ28A, obtained as a developmental compound from our
PDE10 program (AQ28A, Figure 1), was proven to be sufficiently metabolically stable and to enable

Molecules 2019, 24, 2791
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in vivo imaging of PDE10A in rodents by PET [24]. As one feature, the fluorinated aromatic ring of
this compound was ascribed to its performance as a promising tracer.
As part of our ongoing commitment in 18F-PET tracer development devoted to PDE imaging
(
2A, 5A, and 10A) [21,22,24,29], we focused on Benzoimidazotriazine (BIT) as the scaffold, which differs
in a benzo ring formally replacing the pyrido ring of our hitherto investigated TA compounds.
Because of our findings from AQ28A, we felt encouraged to combine a 2-fluorpyridine moiety as
a fluorine-bearing group with structural features required for PDE2A binding (Figure 2). Herein,
we report the synthesis and in vitro evaluation of fluorinated derivatives containing a BIT scaffold and
the selection of a promising ligand for the development of a 18F-labeled ligand for PDE2A imaging
with PET. The use of a benzo fused imidazo[2,1-c][1,2,4]triazine allows us to readily perform structural
modification in the six-membered benzo ring (8-position) while retaining the structural modifiability of
the imidazole portion (1-position) of the tricycle (Figure 2). An example of this BIT series, compound
III (Figures 1 and 2), bearing no substituent at the 6-position, was more potent to PDE2A in comparison
to II and served as the lead in our studies [26].
Figure 2. Approach for structural modification of the lead structure III.
Therefore, our strategy was to modify this structure by changing the substituents at 1-(phenyl) and
the 8-position (pyridinyl instead of F) in order to obtain potent and selective PDE2A inhibitors
(Figure 2). These can be established via a standard palladium (Pd)-catalyzed Suzuki coupling
reaction [30]. Our main interest was to obtain fluorinated compounds for the development of PDE2A
PET tracers. Therefore, the introduction of fluorine-containing groups particularly at the 8-position
(pyridinyl) is required. According to our previous work, several fluorinated pyridinyl substituents
may enhance the potency and selectivity to PDE2A, and the incorporation of the pyridine moiety is
tolerable. Besides, pyridine, as an electron-deficient aromatic ring, offers a convenient way to perform
radiofluorination via nucleophilic aromatic substitution [31]. Furthermore, fluorinated pyridinyl
substituents are relatively more stable towards metabolic degradation compared with fluoroalkyl
groups, which were found to be more prone to defluoroalkylation [15,21,22,24].
2
. Results and Discussion
2
.1. Chemistry
The first of our synthetic approaches focused on bromo-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine
(
(
4
Scheme 1, Compound
5
) as the key intermediate for further conversions into the desired final products
BIT derivatives). The 8-bromo-substituted tricycle was synthesized in four steps starting from
-bromo-2-fluoro-aniline (1) as depicted in Scheme 1.
5

Molecules 2019, 24, 2791
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◦
. (i) Oxidation, NaBO3 4H O, acetic acid, 65 C, 2–3 d;
2
Scheme 1. Synthesis of Compound
5
·
(
ii) 4-methyl-1H-imidazole, K CO , DMF, room temperature (rt), 1–2 d; (iii) Fe (5 eq), HOAc/EtOH,
2
3
reflux, 2.5 h; (iv) NaNO , acetic acid, H O, rt, 1 h.
2
2
The oxidation of the aromatic amino group with sodium perborate tetrahydrate (NaBO3
in acetic acid afforded nitro compound in a 51% yield. To avoid significant by-product formation,
the reaction temperature was kept below 65 C, and the aniline
agent [23]. The obtained 4-bromo-2-fluoro-nitrobenzene ( ) was reacted with 4-methyl-1H-imidazole
to provide the corresponding N-aryl-4- and N-aryl-5-methylimidazoles and 3b. This nucleophilic
·
4H O)
2
2
◦
1
was slowly added to the oxidizing
2
3
aromatic substitution (S Ar) employed 1.2 eq of 4-Me-imidazole and 2.0 eq of K CO in DMF and
N
2
3
formed a mixture of
3
and 3b in a ratio of ~4:1 [32
,
33]. However, the main regioisomer
using iron
in high yield (92%). The iron powder represents a preferred reducing agent for nitro
compounds bearing sensitive functional groups such as halides and/or other reducible groups [34].
A subsequent one-pot diazotation-intramolecular azo coupling of compound by use of aqueous
in a satisfactory yield of 93%. The overall yield
3 could
be purified via repeated recrystallization from methanol (50% yield). The reduction of
afforded aniline
3
4
4
sodium nitrite in acetic acid gave the intermediate
was 22%.
5
The synthesis of diaryl-substituted compounds (BIT derivatives) starting from 5 is depicted in
Scheme 2. Positions 1 and 8 were substituted with different aryl moieties by two independent Suzuki
couplings, possible through an intermediate bromination on position 1. The first Suzuki coupling
was used to introduce a substituted pyridine ring to the 8-position. Six different Boronic Acids (BAs),
including fluoropyridinyl boronic acids (8a
–8d) and pyridinyl boronic acids (8e and 8f) as well,
were coupled in the presence of K CO as a base and Pd(Ph ) as a catalyst under standard conditions
2
3
3 4
(Scheme 1, Table 1).
Cross-coupling products 6a–6f were obtained after flash column chromatography purification in
yields from 28%–98%, as depicted in Table 1. Afterwards, the bromination of the imidazo fused ring in
the 1-position was carried out using N-bromosuccinimide (NBS) in acetonitrile at room temperature
and provided Compounds 7a–7f in moderate to high yields (Table 1).

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Scheme 2. Approach to achieve a series of nine BIT derivatives. (i) R B(OH) (8a–8f), K CO , Pd(PPh ) ,
2 3 3 4
1
2
1
,4-dioxane:H O (4:1), reflux, 1–2 d; (ii) NBS, MeCN, rt 4–6 h; (iii) R B(OH) (9a [phe-1-B(OH)2],
2
2
2
9
b [phe-2-B(OH) ]), or R B(pin) (9c, [phe-3-B(pin)]), K CO , Pd(PPh ) , dioxane:H O (4:1), reflux,
2 2 2 3 3 4 2
1
–2 d.
Table 1. Yields of Suzuki coupling with compound
5
(products: 6a
–6f) and of bromination (products:
7
a–7f).
Product of First
Suzuki Coupling
Product of
Bromination
Entry
Starting Boronic Acid (BA)
Yield
Yield
1
2
3
4
5
6
2-fluoro-pyridine-4-yl-BA (8a)
6a
6b
6c
6d
6e
6f
76%
28%
98%
56%
77%
87%
7a
7b
7c
7d
7e
7f
79%
63%
88%
98%
87%
92%
6-fluoro-pyridine-3-yl-BA (8b
)
2-fluoro-pyridine-3-yl-BA (8c)
3-fluoro-pyridine-5-yl-BA (8d
pyridine-3-yl-BA (8e)
)
pyridine-4-yl-BA (8f)
By a second Suzuki coupling, the five-membered imidazole portion was functionalized (Scheme 2).
Initially, two ortho-chlorophenyl boronic acid (BA) derivatives 9a and 9c were used for coupling
0
0
with 7a and 7b, as well. In addition, to include the 5 -methoxy substituted BA 9a, we investigated
a derivative with a modified propoxy substitution by using 9c with a (3-methyloxetan-3-yl)methoxy
0
0
moiety at the C-5 -position (Scheme 2, 9c [phe-3-B(pin)]). It has been reported that an oxetane residue
may modulate and positively influence physicochemical, as well as biological properties of a drug
candidate [35,36]. Besides, an oxetane moiety is regarded to induce stability towards metabolic attack
similar to a corresponding geminal dimethyl unit (gem-Me ) [37], but in contrast will not increase the
2
lipophilicity of the compound [35,36]. To make this oxetane derivative available, we prepared boronic
ester 9c in three steps, starting from 3-bromo-phenol (10) (Scheme 3).

Molecules 2019, 24, 2791
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Scheme 3. Preparation of 9c. (i) 3-methyloxetan-3-yl-methyl-mesylate, Cs CO , K CO , MeCN,
2
3
2
3
◦
5–79 C, 18 h; (ii) B pin , PdCl (dppf), KOAc, 2-Me-THF, Ar; (iii) NCS, DMF, rt.
2 2 2
7
First, phenol 10 was reacted with 3-methyloxetan-3-yl-methyl-mesylate to provide aryl ether 11
85% yield). Afterwards, Miyaura-borylation was performed to react 11 with bis(pinacolato)diboron
(
(
B pin ) in the presence of PdCl (dppf) and KOAc as a catalyst and base, respectively, providing
2
2
2
boronate ester 12 in a good yield of 81%. Finally, the chlorination of 12 ortho to the boronate ester by
means of N-chloro-succinimide (NCS) in DMF at room temperature afforded 9c in a yield of 56% [38].
Boronic acid derivatives 9a and 9c were reacted with 7a and 7b via cross-coupling under conditions
similar to those described for the reactions of BAs 8a–8f with compound 5. Reaction products BIT1,
BIT2, and BIT3 were obtained in satisfactory yields, as depicted in Table 2 (Entries 1–3).
Table 2. Series of BIT derivatives according to Scheme 2.
Boronic Acid
Derivative
Entry
Bromo Compound
R /R
1
Product
Yield
2
1
2
3
4
5
6
7
8
9
7a
7b
7a
7b
7c
7a
7d
7e
7f
9a
9c
9c
9a
9a
9b
9a
9b
9b
2-F-pyr-4/phe-1
6-F-pyr-3/phe-3
2-F-pyr-4/phe-3
6-F-pyr-3/phe-1
2-F-pyr-3/phe-1
2-F-pyr-4/phe-2
5-F-pyr-3/phe-1
pyr-3/phe-2
BIT1
BIT2
BIT3
BIT4
BIT5
BIT6
BIT7
BIT8
BIT9
80%
79%
68%
64%
69%
72%
56%
82%
39%
pyr-4/phe-2
In vitro evaluation data of the first series of final compounds revealed BIT1 to have a higher
inhibitory potency towards PDE2A in contrast to BIT2 and BIT3 (see the In Vitro Evaluation section).
Therefore, on the basis of this first finding, we prepared further BIT derivatives and investigated the
effect of an alteration of the pyridinyl substitution in 8-position on the PDE2A inhibition. For that
00
–BIT9 with the methoxy group in the 5 -position, since this substitution
purpose, we synthesized BIT4
appeared to be the most promising for the inhibition of PDE2A. Of the newly-synthesized compounds,
00
BIT6, BIT8, and BIT9 possessed the fluoro substitution in position 2 of the phenyl residue. BIT4–BIT9
were obtained in moderate to good yields as shown in Table 2 (Entries 4–9). After characterization by
one- and two-dimensional NMR spectroscopy and HR-MS (see Supplementary Materials), the new
derivatives were evaluated in vitro (see the In Vitro Evaluation Section).
2
.2. In Vitro Evaluation: Structure-Activity Relationships of BIT Derivatives
Final products (BIT derivatives) were evaluated in vitro towards PDEs by means of radioligand
binding assays. Inhibition of PDE2A was measured at an inhibitor concentration of 10 nM and those
of other PDEs at a concentration of 1 µM for each BIT derivative. The inhibitory potencies of all BIT
compounds are summarized in Table 3.

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Table 3. Percentage inhibition of PDE sub-types from synthesized compounds; for PDE2A3,
the compounds were measured at a concentration of 10 nM and for PDE4A1, PDE5A1, PDE9A1,
and PDE10A1 at 1 µM.
Inhibition of PDE Sub-Types (%)
2
A3
4A1
5A1
9A1
10A1
BIT1
BIT2
BIT3
BIT4
BIT5
BIT6
BIT7
BIT8
BIT9
TA1
82.9
8.52
13.2
2.63
NI
56.6
50.6
NI
58.6
64.1
72.7
NI
29.3
NI
NI
NI
NI
13.6
25.1
36.2
57.7
3.08
NI
NI
NI
NI
NI
28.4
11.5
NI
17.5
NI
32.4
2.46
28.2
10.8
22.1
74.0
87.0
55.5
96.6
41
NI
54.2
65.7
13.9
86.8
24.9
80.5
93.5
NI: No Inhibition
We first focused our attention on modifications at the 1-position while keeping the
2
-fluoropyridin-4-yl (2-F-pyr-4) fixed at the 8-position as for compounds BIT1 and BIT3. According
to our previous work [21], a longer alkyl side chain length (3-fluoropropoxy to 4-fluorobutoxy,
as TA3 TA4) contributes to improved potency, as well as selectivity towards PDE2A, as was also
shown for TA1 (Figure 1) [15
–
00
21]. However, the substitution of the 5 -methoxy group (BIT1) by
,
a (3-methyloxetan-3-yl)methoxy moiety (BIT3) in the 1-phenyl moiety resulted in a strong reduction of
inhibitory potency towards PDE2A3 from 82.9% to 13.2% (at 10 nM). One possible explanation for
that result might be the increased polarity of the chosen oxetanyl-methoxy residue compared with
alkoxy groups, which possibly is unfavorable for key interactions towards PDE2A. It was reported
that the phenylpropyl portion in the imidazole ring of BAY 60-7550 interacts with Leu770, one of
the amino acids that is known to be responsible for hydrophobic pocket (H-pocket) formation in the
PDE2A active site [39]. Moreover, BAY 60-7550 binding to the PDE2A active site suggested that the
selectivity for PDE2A was generated from the ability of the ligand to induce a conformational change
of Leu770 [1,39]. Therefore, it is hypothesized that BIT3 may not induce the conformational change of
the Leu770 amino acid that generates the selectivity pocket formation, thus leading to potency losses
towards PDE2A [1,39].
In the case of a pair of 6-F-pyr-3-substituted products, BIT2 and BIT4, a weak inverse effect of
0
0
PDE2A binding was observed [40], depending on the nature of the 5 substituent and in comparison
to BIT3 BIT1. Strikingly, the much lower inhibition displayed by BIT4 (2.63%) in contrast to BIT1
82.9%), bearing the same phe-1 substituent, points to the impact of the pyridine substitution pattern in
/
(
exerting inhibition on PDE2A.
In this connection, another factor that contributes to the selectivity of BAY 60-7550 is
a glutamine-switch mechanism [39], which is generally proposed for dual substrate-specific PDEs,
such as PDE2A [
Gln859 in a similar mode as described for the pyrazine portion in bioisosteric triazoloquinoxaline-based
PDE2A inhibitors [16 20]. We further investigated the influence of different pyridinyl substituents at
1,39]. We assume that triazine N-5 in our scaffold might accept a hydrogen bond from
,
the 8-position on PDE2A inhibition while keeping the phe-1 fixed at the 1-position. In addition to
BIT4, containing a 6-F-pyr-3, the PDE2A inhibition also dropped significantly with BIT5, containing
a 2-F-pyr-3 at the 8-position. Interestingly, a potency of at least 50% remained with the introduction of
the 5-F-pyr-3 residue (BIT7) at the 8-position. It is not unambiguously clear whether this was caused
only by the position of the pyridine nitrogen, which may affect rotational freedom of the ring via
active site H-bonding to the pyridine N [16], or in the case of BIT1 and BIT7, additionally by X-H···F-C
interactions with fluorine [41,42]. Considering this, we found an influence of substituent variation at
the 8-position while keeping phe-1 at the 1-position in decreasing order of activity towards PDE2A

Molecules 2019, 24, 2791
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as 2-F-pyr-4 > 5-F-pyr-3 > 6-F-pyr-3 > 2-F-pyr-3. It was supposed that 2-F-pyr-4 and 5-F-pyr-3 likely
maintained the conformational locking by the H-bond, resulting in higher PDE2A potency of BIT1 and
BIT7. Moreover, compared to BIT7, both BIT4 and BIT5 demonstrated eight- and four-fold selectivity
over PDE10A, respectively.
We then directed our attention to investigate the influence of ortho-fluorophenyl (phe-2) at the
1
-position. In the case of BIT6, having a 2-F-pyr-4 at the 8-position, a weakly lower potency towards
PDE2A of 56% was observed, when compared to BIT1, which is in accordance with the known positive
0
0
00
PDE2A inhibitory effect of 2 -Cl in comparison to 2 -F [20,28]. A non-fluorinated pyridine (pyr-4
in BIT9) also maintained the inhibitory activity towards PDE2A of 80.5%, close to BIT1 (82.9%),
however at the expense of increasing inhibition towards PDE10A and PDE4A (96.6% and 86.8% at
a 1
µM inhibitor concentration) in contrast to BIT1 (32.4% and 58.6% at 1
µM). Incorporation of pyr-3
resulted in a significant loss of PDE2A inhibition, which is consistent with the result of BIT2
,
BIT4
,
and BIT5, having also a substituted pyridin-3-yl at the 8-position, but not with BIT7, which may be
due to reasons discussed above. Again, the strong effect of the N-atom position in the pyridine ring
on PDE2A inhibition may be explained by differing conformational preferences between pyr-4 and
pyr-3 [11]. It was assumed that pyr-3 allows more rotational freedom, resulting in energy loss of
binding [16]. The selectivity towards certain PDEs was decreased when exchanging phe-1 with phe-2.
Thus, in addition to lower potency towards PDE2A, BIT6 displayed higher inhibition of PDE10A when
compared to BIT1 (74% vs. 32%, at 1
phe-1) at 1-position was useful for PDE2A potency and selectivity.
Three compounds, BIT1 BIT6, and BIT9, were selected for estimation of IC50 values of PDE2A
µM). Therefore, these results suggest that the ortho-chlorophenyl
(
,
and PDE10A inhibition, to determine the potency in more detail and also the PDE10A/PDE2A selectivity
ratio. The related IC50 values are shown in Table 4.
Table 4. Affinity and selectivity of three new fluorinated compounds towards PDE2A and PDE10A.
IC50 PDE2A
nM)
IC50 PDE10A
(nM)
Compounds
Selectivity Ratio
(
BIT1
BIT6
BIT9
3.33
65.06
17.7
53.23
168.4
20.24
16
2.5
1.1
Finally, the potencies of BIT1 towards a range of other PDEs were evaluated as depicted in Table 5.
BIT1 showed a good selectivity over other PDEs (12.6%–23.3% for other PDEs, measured at 1 M).
µ
Summarizing, BIT1 had a good selectivity over PDE10A (16-fold), while BIT6 and BIT9 had 2.5-fold
and 1.1-fold selectivity over PDE10A, respectively. Applying similar assay conditions, TA1 and BAY
6
0-7550 displayed an IC50 value towards PDE2A of 10.8 nM and 0.66 nM, respectively. Thus, BIT1,
possessing phe-1 and 2-F-pyr-4 at the 1- and 8-positions, proved to be a prospective ligand for labeling
with 18F for PDE2A PET imaging.
Table 5. Percentage inhibition of PDE sub-types by BIT1 (measured at 1 µM).
PDE Subtypes
%Inhibition
PDE1A3
PDE3A
12.6
20
PDE6AB
PDE7A
PDE8A1
PDE11A
16.8
16.4
23.3
14.5

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2
.3. Incubations with Mouse Liver Microsomes
To gain first information about whether BIT1 is prone to metabolic degradation, the compound
was investigated in an in vitro test system. Briefly, BIT1 was incubated with Mouse Liver Microsomes
◦
(
MLM), in the presence of NADPH, at 37 C for 90 min [43]. After protein precipitation by addition of
acetonitrile and subsequent centrifugation, the supernatant was examined by HPLC-UV-MS.
After incubation with MLM for 90 min, unchanged BIT1 could still be detected in high amounts
(Figure 3). The main fractions of in vitro metabolites were products of a mono-oxygenation of BIT1,
namely Metabolites M3, M5, and M6 (see Table 6).
Figure 3. HPLC-UV chromatogram after incubation of BIT1 with MLM in the presence of NADPH
(
for conditions, see the experimental part): (a) UV chromatogram (partial) recorded during gradient
elution (Method A); (b) UV chromatogram recorded during isocratic elution (Method B).
Table 6. Overview of HPLC and MS data of in vitro metabolites of BIT1 formed by MLM in the
presence of NADPH.
tR (min)
Method A
Gradient
tR (min)
Method B
Isocratic
m/z
Found
m/z
Theoret.
Metabolite
Identification
BIT1
M1
M2
M3
M4
M5
M6
11.28
8.47
9.05
9.33
9.86
10.67
16.10
420.7
422.7
452.8
436.7
406.6
436.7
436.7
420.1
422.1
452.1
436.1
406.1
436.1
436.1
parent (M + H)+
reduction
2.85
(
M + 2H + H)+
di-oxygenation
M + 2O + H)+
mono-oxygenation
M + O + H)+
demethylation
4.24
(
5.02
(
7.03
+
(
M-CH + H)
3
mono-oxygenation
12.04
17.89
(
M + O + H)+
not
detected
mono-oxygenation
(M + O + H)+
The metabolite M6 was detectable only by an HPLC method with isocratic elution as shown
in Figure 3b. Furthermore, one metabolite resulting from a two-fold oxygenation (M2) was found.
Due to the limitations of a single quadrupole MS, it was not possible to obtain information regarding

Molecules 2019, 24, 2791
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the sites of functionalization in the molecule, neither to distinguish products from N-oxidation or
C-hydroxylation [44]. In contrast, for the formation of M1 and M4 by reduction and demethylation,
respectively, the chloro(methoxy)-phenyl moiety is expected to undergo these reactions under the
conditions applied.
The considerable metabolic stability of BIT1 in vitro supports the potential suitability of the
corresponding 18F-labelled compounds as a radioligand for PET. However, due to the retention
properties of the metabolite M6 in HPLC this metabolite may have a similar or higher lipophilicity in
comparison to BIT1. Therefore, of the metabolites detected, M6 most likely bears the risk of passing the
blood-brain barrier and influencing brain PET results. Hence, future in vivo metabolism studies should
pay attention to the occurrence of this metabolite, as well as to distinguish it chromatographically from
the parent compound.
3
. Materials and Methods
3
.1. General Information
Chemicals were purchased from following suppliers: Manchester Organics, abcr, VWR, Fluka,
Acros, Roth, ChemPure, Merck, Sigma Aldrich, Apollo scientific, and Fluorochem. Solvents were
dried before use, if required. Air- and moisture-sensitive reactions were carried out under argon
◦
atmosphere. Room temperature (rt) refers to 20–25 C. The progress of a reaction was monitored
by thin layer chromatography using pre-coated TLC sheets POLYGRAM® SIL G/UV254 purchased
from Macherey-Nagel. Detected spots were observed under UV light at
λ 254 nm and 365 nm.
Flash chromatography was performed with silica gel 40–63 µm from VWR Chemicals.
1
13
19
NMR spectra ( H, C, F) were recorded on Mercury 300/Mercury 400 (Varian, Palo Alto,
CA, USA) or Fourier 300/Avance DRX 400 Bruker (Billerica, MA, USA) instruments. Signals of solvents
1
13
were used as internal standards for H-NMR (CDCl ,
δ = 7.26; DMSO-d6, δ = 2.50) and C-NMR
H H
3
(
CDCl3, δ = 77.16; DMSO-d6, δ = 39.52). The chemical shifts (δ) are reported in ppm as follows: s,
C C
singlet; d, doublet; t, triplet; m, multiplet; and the coupling constants (J) are reported in Hz. Mass
spectra were recorded on an ESQUIRE 3000 Plus (ESI, low resolution) and a 7 T APEX II (ESI, high
resolution) from Bruker Daltonics.
3
4
.2. Syntheses
-Bromo-2-fluoro-nitrobenzene (
2
): A suspension of sodium perborate tetrahydrate (20.24 g, 0.13 mol)
◦
in glacial acetic acid (80 mL) was stirred at 65 C. 4-Bromo-2-fluoro-aniline (5 g, 26.31 mmol, 1 eq) in
35 mL acetic acid was dropwise added over 5 h. The reaction was heated overnight, and subsequently,
another portion of NaBO3 4H O (12.2 g, 78.9 mmol) was added. After full consumption of the
·
2
starting material, the reaction mixture was cooled to room temperature, the solid filtered off, and the
filtrate quenched with ice-cold water (600 mL). Then, the precipitate was filtrated and purified with
column chromatography (hexane/chloroform, 2:1) to give the product as yellow solid 2 (2.96 g, 51%).
1
TLC (hexane/CHCl (2:1)): R = 0.32. H-NMR (400 MHz, CDCl )
δ = 8.01–7.93 (m, 1H), 7.50 (dd,
H
3
f
3
13
J = 10.1, 1.9 Hz, 1H), 7.48–7.44 (m, 1H). C-NMR (101 MHz, CDCl3)
δ = 155.48 (d, J = 269.9 Hz),
C
1
(
36.60 (d, J = 7.2 Hz), 129.56 (d, J = 8.9 Hz), 128.24 (d, J = 4.3 Hz), 127.28 (d, J = 2.5 Hz), 122.21
d, J = 23.6 Hz). LR-MS (EI): m/z = 219 (calcd. 219 for C H BrFNO2+ [M] )
7
9
+
6 3
4
-Bromo-2-(4-methyl-1H-imidazol-1-yl) nitrobenzene (
3
): A mixture of compound
2
(9.46 g, 43 mmol) and
◦
K CO (11.9 g, 86 mmol) in DMF (40 mL) was stirred at 4 C, while a solution of 4-methyl imidazole
2
3
(3.78 g, 46 mmol) in DMF (10 mL) was slowly added in the course of 2 h. Afterwards, stirring was
continued for 10 h at room temperature. The reaction mixture was poured into water (250 mL), and the
formed precipitate was filtered off, washed, and dried to give a brown yellow solid (10.47 g), which was
found to be an impure mixture of regioisomeric Products
3 and 3b in a ratio of ~4:1, according to
1
H-NMR. The solid was dissolved in CHCl (80 mL) and filtered through a plug of silica gel (2 g).
3
The solvent was evaporated, and the remaining solid was recrystallized twice from aqueous EtOH

Molecules 2019, 24, 2791
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◦
(
(
7
80%) to give pure 3, as bright yellow crystals (7.09 g, 58%). Mp. 151–152.5 C; TLC (CHCl /MeCN
3
1
10:1)]: R = 0.33. H-NMR (400 MHz, CDCl ) δ = 7.85 (d, J = 8.7 Hz, 1H), 7.70 (dd, J = 8.7, 2.0 Hz, 1H),
H
.61 (d, J = 2.0 Hz, 1H), 7.52 (d, J = 0.9 Hz, 1H), 6.75 (s, 1H), 2.27 (d, J = 0.9 Hz, 3H). C-NMR (101 MHz,
= 143.91, 140.24, 136.31, 132.37, 131.95, 131.47, 127.95, 126.68, 116.35, 13.71. LR-MS (ESI+):
f
3
13
CDCl3)
δ
C
79
+
+
m/z = 282, (calcd. 282 for C H BrN O [M + H] ).
10
9
3
2
The minor regioisomer was isolated from a sample (0.36 g) of the filtrate obtained from the first
recrystallization. It was purified via flash purification applying a gradient from CHCl (100%) to
3
◦
CHCl /MeCN (30:1) to give pure 3b, as a yellow solid (0.11 g). Mp. 107.5–109 C; TLC [CHCl /MeCN
10:1)]: R = 0.26. H-NMR (400 MHz, CDCl ) δ = 7.97 (d, J = 8.7 Hz, 1H), 7.81 (dd, J = 8.7, 2.1 Hz, 1H),
H
.59 (d, J = 2.1 Hz, 1H), 7.46 (s, 1H), 6.91 (s, 1H), 2.04 (s, 3H). C-NMR (101 MHz, CDCl3) δ = 145.38,
C
3
3
1
(
7
f
3
13
1
36.78, 133.60, 133.54, 131.16, 128.99, 128.22, 127.82, 126.78, 9.24.
-Bromo-2-(4-methyl-1 H-imidazol-1-yl) aniline ( ): Nitro compound
dissolved under argon in a 1:1 mixture of ethanol (50 mL) and acetic acid (50 mL). Iron powder (5.25 g,
4
4
3 (4.42 g, 15.7 mmol, 1 eq) was
9
4 mmol) was added, and the reaction mixture was stirred under reflux for 2.5 h (bath temperature
◦
1
15–120 C). The mixture was filtered through celite, and the filtrate was adjusted to pH 8 with Na CO
2
3
until a greenish solid formed. The mixture was extracted with Ethyl Acetate (EE, 3
×
100 mL), and the
organic phase was washed with water (2 100 mL) and saturated NaCl solution (100 mL), and dried
×
(
Na SO )
.
The solvent was evaporated to give a beige solid (3.64 g, 92%). TLC (CHCl /MeOH/30%
2
4
3
1
NH (10:1:0.1)): R = 0.41. H-NMR (400 MHz, CDCl )
δ = 7.52 (d, J = 1.0 Hz, 1H), 7.30 (dd, J = 8.6,
H
3
f
3
2
2
.3 Hz, 1H), 7.23 (d, J = 2.2 Hz, 1H), 6.80 (s, 1H), 6.71 (t, J = 8.0 Hz, 1H), 3.68 (d, J = 48.0 Hz, 2H),
13
.30 (d, J = 0.6 Hz, 3H). C-NMR (101 MHz, CDCl )
δ = 141.22, 139.48, 136.72, 132.47, 129.50, 124.52,
C
3
79
+
+
1
17.94, 116.32, 109.38, 13.77. LR-MS (ESI+): m/z = 274, (calcd. 274 for C H
BrN Na [M + Na] ).
10 3
10
8
-Bromo-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (5): A solution of NaNO (1.28 g, 18.6 mmol) in
2
H O (10 mL) was added to a stirred solution of 4 (3.13 g, 12.4 mmol) in acetic acid (60 mL) at room
2
temperature. A yellow precipitate formed immediately. After 5 h of stirring, the reaction mixture
was evaporated to half of its original volume and diluted with H O (50 mL). The pH was carefully
2
adjusted to pH 8 by the addition of solid NaHCO . The yellow solid was filtered off and dried to give
5
3
(
3.24 g, 98% sufficiently pure for the next step). The solid was dissolved in CHCl (150 mL) and filtered
3
through a plug of silica gel (2 g). The filtrate was concentrated and triturated with CHCl /heptane
3
1
mixtures to afford a yellow powder (2.9 g, 89%). TLC (hexane/ethyl acetate (1:3)): R = 0.45. H-NMR
f
(
2
1
400 MHz, DMSO-d6) δ = 9.16 (s, 1H), 8.70 (d, J = 2.0 Hz, 1H), 8.29 (d, J = 8.6 Hz, 1H), 7.88 (dd, J = 8.6,
H
13
.0 Hz, 1H), 2.74 (s, 3H). C-NMR (101 MHz, DMSO-d6) δ = 137.71, 135.87, 134.37, 131.40, 130.55,
C
27.23, 126.01, 123.04, 118.35, 12.39. LR-MS (ESI+): m/z = 263 (calcd. 263 for C H BrN4+ [M + H] ).
7
9
+
10
8
3.2.1. General Procedure A for the Suzuki Coupling of Bromo Derivatives
5,
7a
,
7b
, 7c, 7d, 7e, and 7f
Brominated compound (1 eq), boronic acid derivative (BA, 8a
were combined in a mixture of 1,4-dioxane and water (4:1). The suspension was degassed with argon
for 5–10 min, and Pd(PPh ) (5–10 mol%) was added. The mixture was refluxed (bath temperature
–8f, 9a–9c, 1.1–1.3 eq), and K CO (2–3 eq)
2 3
3
4
◦
1
02–108 C) for 1–2 days until completion of the reaction (as indicated by TLC). The solvent was
removed, and the residue was partitioned between CHCl and water. The aqueous layer was extracted
3
twice with CHCl , and the combined organic layers were dried (Na SO ) and the solvent evaporated.
3
2
4
Unless stated otherwise, the obtained residue was purified by flash chromatography on silica gel using
CHCl /MeOH (10:1) as the eluent. The following products were isolated:
3
8
-(2-Fluoropyridin-4-yl)-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (6a): Based on General Procedure
A of Suzuki coupling, a mixture of compound (101 mg, 0.38 mmol,1 eq), BA 8a (65 mg,0.46 mmol,
.2 eq), and K CO (131 mg, 0.95 mmol, 2.5 eq), in dioxane/water (5 mL) were reacted in the presence
5
1
2
3
of Pd(PPh ) (44 mg, 10 mol%) to give after purification compound 6a (81 mg, 76%) as a yellow solid.
3
4
1
TLC (hexane/ethyl acetate (1:1)): R = 0.25. H-NMR (400 MHz, DMSO-d )
δ = 9.28 (s, 1H), 8.92
H
f
6

Molecules 2019, 24, 2791
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(
(
[
d, J = 1.9 Hz, 1H), 8.52 (d, J = 8.5 Hz, 1H), 8.46 (d, J = 5.3 Hz, 1H), 8.25 (dd, J = 8.5, 1.9 Hz, 1H), 7.97
+
dt, J = 5.3, 1.8 Hz, 1H), 7.82 (s, 1H), 2.79 (s, 3H). LR-MS (ESI+): m/z = 280, (calcd. 280 for C H FN
5
1
5
11
+
M + H] ).
-(6-Fluoropyridin-3-yl)-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (6b): According to General Procedure
A, compound (500 mg, 1.9 mmol,1 eq), BA 8b (321 mg, 2.28 mmol, 1.2 eq), K CO (788 mg, 5.7 mmol,
eq), and Pd(PPh ) (220 mg, 10 mol%) were reacted in dioxane/water (24 mL) to yield after purification
8
5
2
3
3
3
4
1
compound 6b (150 mg, 28%), as a yellow powder. TLC (CH Cl /MeOH (10:1)): R = 0.38. H-NMR
3
3
f
(
400 MHz, CDCl3) δ = 8.62–8.52 (m, 3H), 8.12 (ddd, J = 8.4, 7.4, 2.7 Hz, 1H), 7.98 (d, J = 1.8 Hz, 1H),
H
7
2
.82 (dd, J = 8.4, 1.8 Hz, 1H), 7.13 (dd, J = 8.5, 3.1 Hz, 1H), 2.91 (s, 3H). LR-MS (ESI+): m/z = 280, (calcd.
+
+
80 for C H FN [M + H] ).
15
11
5
8
-(2-Fluoropyridin-3-yl)-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (6c): According to General Procedure
(501 mg, 1.9 mmol, 1 eq), BA 8c (325 mg, 2.28 mmol, 1.2 eq), K CO (661 mg, 4.75 mmol,
A, compound
5
2
3
2
.5 eq), and Pd(PPh ) (219 mg, 10 mol%) were reacted in dioxane/water (15 mL) to afford after
3
4
purification compound 6c (0.52 g, 98%), as a yellow solid. TLC (CHCl /MeOH (10:1)): R = 0.38.
3
f
1
H-NMR (400 MHz, CDCl3)
δ = 8.58–8.52 (m, 1H), 8.34 (dt, J = 5.0, 1.5 Hz, 1H), 8.10–7.99 (m, 1H),
H
7
.85 (dd, J = 8.4, 1.6 Hz, 1H), 7.71–7.62 (m, 1H), 7.58–7.37 (m, 2H), 2.92 (s, 3H). LR-MS (ESI+): m/z = 280
+
+
(calcd. 280 for C H FN [M + H] ).
15 11 5
8
-(5-Fuoropyridin-3-yl)-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (6d): According to General Procedure
(200 mg, 0.76 mmol, 1 eq), BA 8d (118 mg, 0.84 mmol, 1.1 eq), K CO (315 mg,
A, compound
5
2
3
2
.28 mmol, 3.0 eq), and Pd(PPh ) (44 mg, 5 mol%) were reacted in dioxane/water (10 mL) to give after
3
4
purification Compound 6d (0.12 g, 56%), as a yellow powder. TLC (CHCl /MeOH (10:1)): R = 0.29.
3
f
1
H-NMR (400 MHz, DMSO-d ) δ = 9.26 (s, 1H), 9.09 (d, J = 1.9 Hz, 1H), 8.88 (d, J = 1.8 Hz, 1H), 8.71
6
H
(d, J = 2.7 Hz, 1H), 8.49 (d, J = 8.5 Hz, 1H), 8.36 (dt, J = 10.3, 2.3 Hz, 1H), 8.19 (dd, J = 8.5, 1.9 Hz, 1H),
2
.77 (s, 3H).
3-Methyl-8-(pyridin-3-yl)benzo[e]imidazo[5,1-c][1,2,4]triazine (6e): According to General Procedure A,
compound
5 (1.0 g, 3.8 mmol, 1 eq), BA 8e (0.61 mg, 4.94 mmol, 1.3 eq), K CO (1.58 g, 11.4 mmol,
2 3
3
eq), and Pd(PPh ) (220 mg, 5 mol%) were reacted in dioxane/water (50 mL) to give after purification
3
4
by flash chromatography compound 6e (0.76 g, 77%), as a yellow solid. TLC (CHCl /MeOH (10:1)):
3
1
R = 0.20. H-NMR (400 MHz, DMSO-d )
δ = 9.23 (s, 1H), 9.16 (d, J = 2.1 Hz, 1H), 8.77 (d, J = 1.8 Hz,
H
f
6
1
1
H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 8.45 (d, J = 8.4 Hz, 1H), 8.35–8.30 (m, 1H), 8.11 (dd, J = 8.5, 1.8 Hz,
+
H), 7.60 (ddd, J = 8.0, 4.8, 0.9 Hz, 1H), 2.75 (s, 3H). LR-MS (ESI+): m/z = 262, (calcd. 262 for C H N
5
15
12
+
[M+H] ).
3-Methyl-8-(pyridin-4yl)benzo[e]imidazo[5,1-c][1,2,4]triazine (6f): According to General Procedure A,
compound
5
(200 mg, 0.76 mmol, 1 eq), K CO (265 mg, 1.92 mmol, 2.52 eq), BA 8f (119 mg, 0.968
2
3
mmol, 1.27 eq), and Pd(PPh ) (44 mg, 0.038 mmol, 5 mol%) were reacted in dioxane/water (5 mL) to
3
4
afford after purification compound 6f (173 mg, 87%), as a yellow solid. TLC (CHCl /MeOH (10:1)):
3
1
R = 0.23. H-NMR (300 MHz, DMSO-d6)
δ = 9.26 (s, 1H), 8.82 (d, J = 1.8 Hz, 1H), 8.78–8.75 (m, 2H),
H
f
8
.47 (d, J = 8.5 Hz, 1H), 8.15 (dd, J = 8.5, 1.9 Hz, 1H), 7.96–7.92 (m, 2H), 2.75 (s, 3H).
3
.2.2. General Procedure B for Bromination of Compounds 6a, 6b, 6c, 6d, 6e, and 6f
N-Bromosuccinimide (NBS, ~1.5 eq) was added to a suspension of compounds 6a
The reaction mixture was protected from light and stirred at room temperature for 1–2 d. After the
full conversion of the starting material, the mixture was partitioned between CHCl and H O.
–6f in acetonitrile.
3
2
The organic layer was washed with aqueous NaHCO (5%), water, and saturated NaCl solution.
3
After drying (Na SO ) and evaporation, the residue was purified by flash chromatography, eluting
2
4
with CHCl /MeOH (10:1).
3

Molecules 2019, 24, 2791
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1-Bromo-8-(2-fluoropyridin-4-yl)methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (7a): According to General
Procedure B, for bromination, compound 6a (300 mg, 1.07 mmol) and NBS (287 mg, 1.61 mmol) were
reacted in acetonitrile (20 mL) to give after purification compound 7a, as a yellow solid (306 mg, 79%).
1
TLC (CHCl /MeOH (10:1)): R = 0.74. H-NMR (400 MHz, CDCl )
δ = 9.25 (t, J = 10.2 Hz, 1H), 8.59
H
3
f
3
(
2
d, J = 8.4 Hz, 1H), 8.42 (t, J = 8.7 Hz, 1H), 7.94 (dd, J = 8.4, 1.8 Hz, 1H), 7.55–7.48 (m, 1H), 7.25 (s, 1H),
79
BrFN5+ [M + H] )
+
.89 (d, J = 3.6 Hz, 3H). LR-MS (ESI+): m/z = 358 (calcd. 358 for C H
15
10
1-Bromo-8-(6-fluoropyridin-3-yl)-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (7b): According to General
Procedure B, compound 6b (354 mg, 1.27 mmol) and NBS (338 mg, 1.9 mmol) were reacted in acetonitrile
(
16 mL) to give after purification Compound 7b, as a yellow solid (290 mg, 63%). TLC (CHCl /MeOH
3
1
(
10:1)): R = 0.71. H-NMR (400 MHz, CDCl )
δ
= 9.15 (d, J = 1.8 Hz, 1H), 8.59 (d, J = 2.6 Hz, 1H), 8.56
f
3
H
(
d, J = 8.4 Hz, 1H), 8.12 (ddd, J = 8.5, 7.4, 2.7 Hz, 1H), 7.87 (dd, J = 8.4, 1.8 Hz, 1H), 7.13 (dd, J = 8.5, 3.0
79
BrFN5+, [M + H] ).
+
Hz, 1H), 2.88 (s, 3H). LR-MS (ESI+): m/z = 358 (calcd. 358 for C H
15
10
1-Bromo-8-(2-fluoropyridin-3-yl)-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (7c): According To General
Procedure B, compound 6c (169 mg, 0.61 mmol) and NBS (162 mg, 0.91 mmol) were reacted in acetonitrile
(
6 mL) to give after purification compound 7c, as a yellow solid (192 mg, 88%). TLC (CHCl /MeOH
3
1
(
10:1)), R = 0.66. H-NMR (400 MHz, CDCl ) δH = 9.23 (t, J = 1.5 Hz, 1H), 8.54 (d, J = 8.4 Hz, 1H), 8.34
f
3
(
dt, J = 4.9, 1.5 Hz, 1H), 8.08–8.00 (m, 1H), 7.89 (dt, J = 8.4, 1.6 Hz, 1H), 7.46–7.37 (m, 1H), 2.88 (s, 3H).
79
BrFN5+ [M + H] ).
+
LR-MS (ESI+): m/z = 358, (calcd. 358 for C H
15
10
1-Bromo-8-(5-fluoropyridin-3-yl)-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (7d): According to General
Procedure B, compound 6d (120 mg, 0.43 mmol) and NBS (115 mg, 0.65 mmol) were reacted in
acetonitrile (4 mL) to afford after purification Compound 7d, (151 mg, 98%), as a yellow solid.
1
TLC (CHCl /MeOH (10:1)), R = 0.71. H-NMR (300 MHz, CDCl )
δ = 9.20–9.19 (m, 1H), 8.83–8.81
H
3
f
3
(
1
m, 1H), 8.61–8.59 (m, 1H), 8.56 (d, J = 0.4 Hz, 1H), 7.90 (dd, J = 8.4, 1.8 Hz, 1H), 7.73 (ddd, J = 9.1, 2.7,
79
BrFN5+ [M + H] ).
+
.9 Hz, 1H), 2.88 (s, 3H). LR-MS (ESI+): m/z = 358, (calcd. 358 for C H
15
10
1-Bromo-3-methyl-8-(pyridin-3-yl)benzo[e]imidazo[5,1-c][1,2,4]triazine (7e): According to General
Procedure B, compound 6e (300 mg, 1.15 mmol, 1 eq) and NBS (306 mg, 1.72 mmol) were reacted
in acetonitrile (8 mL) to afford after purification compound 7e (343 mg, 87%), as a yellow solid.
1
TLC (CHCl /MeOH (10:1)): R = 0.47. H-NMR (400 MHz, CDCl )
δ = 9.19 (d, J = 1.7 Hz, 1H), 9.00
H
3
f
3
(dd, J = 2.5, 0.9 Hz, 1H), 8.73 (dd, J = 4.8, 1.6 Hz, 1H), 8.55 (d, J = 8.4 Hz, 1H), 8.03 (ddd, J = 8.0, 2.5,
1
.6 Hz, 1H), 7.91 (dd, J = 8.4, 1.8 Hz, 1H), 7.50 (ddd, J = 8.0, 4.8, 0.9 Hz, 1H), 2.87 (s, 3H). LR-MS (ESI+):
79
BrN5+ [M + H] ).
+
m/z = 340, (calcd. 340 for C H
15
11
1
-Bromo-3-methyl-8-(pyridin-4-yl)benzo[e]imidazo[5,1-c][1,2,4]triazine (7f): According to General Procedure
B, compound 6f (100 mg, 0.383 mmol, 1 eq) and NBS (102 mg, 0.573 mmol) were reacted in MeCN
(
(
(
(
4 mL) to give after purification compound 7f (120 mg, 92%), as a yellow powder. TLC (CHCl /MeOH
3
1
10:1)): R = 0.54. H-NMR (400 MHz, DMSO-d )
δ = 9.27 (d, J = 1.8 Hz, 1H), 8.85–8.78 (m, 2H), 8.58
H
f
6
d, J = 8.4 Hz, 1H), 8.24 (dd, J = 8.4, 1.8 Hz, 1H), 7.97–7.91 (m, 1H), 2.76 (s, 3H). LR-MS (ESI+): m/z = 340,
79
BrN5+ [M + H] ).
+
calcd. 340 for C H
1
5
11
3
.2.3. Synthesis of Oxetanyl Building Block 9c
3-((3-Bromophenoxy)methyl)-3-methyloxetane (11): A mixture of (3-methyloxetan-3-yl) methanol (2.09 g,
2
0.4 mmol) and triethylamine (TEA, 3.2 mL, 23 mmol) in MeCN (8 mL) was stirred and cooled at
◦
15–25 C, while a solution of methanesulfonyl chloride (1.55 mL, 20 mmol) in MeCN (3 mL) was
◦
◦
dropwise added in the course of 20 min. The mixture was stirred at 22 C for 4 h and at 0–2 C for
0 min. The precipitated TEA hydrochloride was filtered off and washed with MeCN (2 × 2.5 mL).
To the filtrate was successively added 3-bromophenol (10, 3.46 g, 20 mmol) and Cs CO (3.26 g,
3
2
3
1
7
0 mmol) along with K CO (2.07 g, 15 mmol). The resulting suspension was stirred and heated at
2 3
5–79 C for 18 h (progress monitored by TLC). Upon completion, the suspension was stirred with
◦

Molecules 2019, 24, 2791
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methyl tert-butyl ether (MTBE, 50 mL) for 2 h. The solid was filtered off and the filtrate washed with
sodium hydroxide solution (0.8 M, 1 20, 2 10 mL) and dried (Na CO ). The solvent was evaporated
×
×
2
3
◦
1
and the remaining oil bulb-to-bulb distilled (4 mbar, air bath 150–190 C) to afford compound 11
,
as a colorless oil (4.35 g, 85%). TLC (heptane/MTBE (3:2)): R = 0.4. H-NMR (400 MHz, CDCl3)
f
δ
= 7.18–7.12 (m, 1H), 7.12-7.08 (m, 2H), 6.87 (ddd, J = 8.1, 2.5, 1.3 Hz, 1H), 4.61 (d, J = 6.0 Hz, 2H),
H
13
4
.46 (d, J = 6.0 Hz, 2H), 4.00 (s, 2H), 1.43 (s, 3H). C-NMR (101 MHz, CDCl3) δ = 159.86, 130.70,
C
1
24.24, 122.94, 117.97, 113.62, 79.81 (2C), 73.06, 39.74, 21.33.
4,4,5,5-Tetramethyl-2-(3-((3-methyloxetan-3-yl) methoxy) phenyl)-1,3,2-dioxaborolane (12): A mixture of
3
-bromophenylether 11 (1.75 g, 6.8 mmol), KOAc (1.5 g, 15.3 mmol), and bis(pinacolato)diboron (1.77 g,
15 mmol, in 2-methyltetrahydrofuran (24 mL)) was degassed with argon for 10 min. After addition
of Pd(dppf)Cl (0.07 g, 0.096 mmol), the mixture was refluxed for 7 h. Upon completion (monitored
2
by TLC), CH Cl (25 mL) was added, the resulting suspension stirred for 1 h, and the solid filtered
2
2
off. The filtrate was evaporated, and the viscous residue (3.2 g) was dissolved in MTBE (70 mL)
and subsequently filtered through a short silica gel column (H 2 cm D 2 cm). The filtrate was
×
extracted with aqueous NaOH (0.75 M, 4
addition of aqueous HCl (4 M) at 0–2 C (
×
12 mL). The alkaline extract was neutralized by slow
pH 6–7). The separated oil was extracted with MTBE
◦
→
(3
×
15 mL), and after drying (MgSO ), the solvent was evaporated to leave a viscous residue (2.15 g).
Crystallization was achieved by trituration with MTBE/heptane to yield 12, as a colorless solid (1.67 g,
4
1
8
1%). TLC (heptane/MTBE (2:1)): R = 0.38. H-NMR (300 MHz, CDCl )
δ
= 7.42 (dt, J = 7.3, 1.1 Hz,
f
3
H
1
2
H), 7.38–7.35 (m, 1H), 7.31 (t, J = 7.7 Hz, 1H), 7.04 (ddd, J = 8.2, 2.8, 1.2, 30 Hz, 1H), 4.63 (d, J = 5.9 Hz
,
H), 4.45 (d, J = 5.9 Hz, 2H), 4.06 (s, 2H), 1.44 (s, 3H), 1.35 (s, 12H). 13C-NMR (75 MHz, CDCl3)
δC = 158.65, 129.15, 127.59, 119.86, 118.27, 84.01, 80.01, 72.87, 39.86, 25.01, 21.47 (the boron-substituted
carbon atom was not detectable).
2
-(2-Chloro-5-((3-methyloxetan-5 3-yl) methoxy) phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (9c):
N-Chlorosuccinimide (NCS, 0.4 g, 3.0 mmol) was added to a solution of compound 12 (0.73 g,
.4 mmol) in DMF (7 mL). The mixture was stirred at room temperature for 20 h (monitored by TLC).
The solvent was removed in vacuo, and the viscous residue was dissolved in MTBE (20 mL). The solution
was successively washed with aqueous Na S O (10%, 9 mL) and water (6 mL) and extracted with
2
2
2
3
aqueous NaOH (0.75 M, 3
×
6 mL). The alkaline extract was neutralized (
→
pH 7–8) by slow addition
◦
of aqueous HCl (4 M) at 0–2 C. The separated oil was extracted with MTBE (3 × 10 mL), and after
drying (MgSO ), the solvent was evaporated to leave a viscous residue. Crystallization was achieved
4
by trituration with MTBE/heptane to yield 9c, as a colorless solid (0.45 g, 56%). TLC (heptane/MTBE
1
(
(
(
2:1)): R = 0.30. H-NMR (300 MHz, CDCl )
δ
= 7.25 (d, J = 8.8 Hz, 1H), 7.23 (d, J = 3.2 Hz, 1H), 6.90
f
3
H
dd, J = 8.8, 3.2 20 Hz, 1H), 4.61 (d, J = 5.9 Hz, 2H), 4.44 (d, J = 5.9 Hz, 2H), 4.01 (s, 2H), 1.42 (s, 3H), 1.37
s, 12H). 13C-NMR (75 MHz, CDCl3)
δ
= 157.11, 131.36, 130.49, 121.90, 121.88, 118.33, 84.39, 79.90,
C
7
9.88, 73.15, 39.82, 24.95, 21.40.
3
.2.4. Synthesis of Compounds BIT1–BIT9 via Suzuki Coupling According to General Procedure A
1
-(2-Chloro-5-methoxyphenyl)-8-(2-fluoropyridin-4-yl)-3methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (BIT1):
Based on General Procedure A for Suzuki coupling, a mixture of compound 7a (100 mg, 0.28 mmol,
eq), BA 9a (63.4 mg,0.34 mmol, 1.2 eq), and K CO (97 mg, 0.7 mmol, 2.5 eq) in dioxane/water
1
2
3
(
5 mL) was reacted in the presence of Pd(PPh ) (33 mg, 10 mol%) to give after purification by flash
3 4
chromatography (gradient: hexane/ethyl acetate, 9:1
→
ethyl acetate, 100%) compound BIT1 (92 mg,
1
80%) as a yellow powder. TLC (hexane/ethyl acetate (1:1)): R = 0.33. H-NMR (400 MHz, CDCl )
f
3
δ
= 8.55 (d, J = 8.4 Hz, 1H), 8.27 (d, J = 5.2 Hz, 1H), 7.85 (dd, J = 8.4, 1.7 Hz, 1H), 7.56 (d, J = 8.8 Hz,
H
1
6
H), 7.48 (d, J = 1.6 Hz, 1H), 7.21 (d, J = 3.0 Hz, 1H), 7.19 (d, J = 3.1 Hz, 1H), 7.16 (d, J = 3.0 Hz, 1H),
.88 (s, 1H), 3.87 (s, 3H), 2.99 (s, 3H). 13C-NMR (75 MHz, CDCl3)
δ
C
= 164.59 (d, J = 239.3 Hz), 159.09,
1
1
51.71 (d, J = 8.1 Hz), 148.68 (d, J = 15.4 Hz), 140.20 (d, J = 3.3 Hz), 139.84, 137.41, 136.98, 136.18, 131.51,
31.44, 130.81, 126.00, 124.05, 119.38 (d, J = 4.1 Hz), 118.56, 117.68, 113.90, 107.44 (d, J = 38.7 Hz), 56.01,

Molecules 2019, 24, 2791
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1
2.89. 19F-NMR (282 MHz, CDCl3)
δ
+
=
−
66.98 (s, br). HR-MS (ESI+): m/z = 420.1025 and 442.0848
F
+
+
+
(calcd. 420.1022 for C H ClFN O [M+H] and 442.0841 for C H ClFN NaO [M + Na] ).
22 16 5 22 15 5
1-(2-Chloro-5-((3-methyloxetan-3-yl)methoxy)phenyl)-8-(6-fluoropyridin-3-yl)-3-methylbenzo[e]imidazo[5,1-c]
[1,2,4]triazine (BIT2): According to General Procedure A, compound 7b (144 mg, 0.40 mmol,1 eq), BA
9
c
(163 mg, 0.48 mmol, 1.2 eq), K CO (157 mg, 1.14 mmol, 2.84 eq), and Pd(PPh ) (23 mg, 5 mol%)
2
3
3 4
were reacted in dioxane/water (5 mL) to give a raw product, which was purified by two-times column
chromatography (CHCl /MeOH (10:1) to yield compound BIT2 (155 mg, 79%), as a yellow solid.
3
1
TLC (hexane/ethyl acetate (1:3)): R = 0.29. H-NMR (400 MHz, CDCl3)
δ
H
= 8.52 (d, J = 8.3 Hz,
f
1
1
H), 8.21 (d, J = 2.5 Hz, 1H), 7.90–7.81 (m, 1H), 7.77 (dd, J = 8.3, 1.8 Hz, 1H), 7.55 (d, J = 8.8 Hz,
H), 7.45 (d, J = 1.8 Hz, 1H), 7.27 (s, 1H), 7.17 (dd, J = 8.9, 2.9 Hz, 1H), 7.02 (dd, J = 8.5, 3.0 Hz, 1H),
.57 (dd, J = 6.0, 2.0 Hz, 2H), 4.44 (d, J = 5.9 Hz, 2H), 4.17–3.99 (m, 2H), 2.96 (s, 3H), 1.41 (s, 3H).
4
13
C-NMR (101 MHz, CDCl3)
δ = 163.85 (d, J = 242.3 Hz), 158.37, 146.27 (d, J = 15.2 Hz), 140.19,
C
1
1
1
39.79 (d, J = 8.1 Hz), 139.45, 137.37, 136.39, 135.92, 133.01 (d, J = 4.7 Hz), 131.74, 131.46, 130.85, 126.38,
25.94, 124.10, 118.73 (d, J = 41.2 Hz), 113.32, 110.38, 110.00, 79.66 (d, J = 3.4 Hz), 73.58, 39.77, 21.28,
2.87. 19F-NMR (282 MHz, CDCl3)
δ
= 67.82 (dd, J = 7.4, 3.2 Hz). HR-MS (ESI+): m/z = 490.1442
−
F
+
+
+
and 512.1260 (calcd. 490.1441 for C H ClFN O [M+H] and 512.1260 for C H ClFN NaO
2
2
6
22
5
2
26 21
5
+
[M + Na] ).
1-(2-Chloro-5-((3-methyloxetan-3-yl)methoxy)phenyl)-8-(2-fluoropyridin-4-yl)-3-methylbenzo[e]imidazo[5,1-c]
[
9
1,2,4]triazine (BIT3): According to General Procedure A, compound 7a (100 mg, 0.279 mmol, 1 eq), BA
(104 mg, 0.307 mmol, 1.1 eq), K CO (93 mg, 0.642 mmol, 2.3 eq), and Pd(PPh ) (16 mg, 5 mol%)
c
2
3
3 4
were reacted in dioxane/water (5 mL) to give after purification by flash chromatography (gradient:
hexane/ethyl acetate, 1:2
→
ethyl acetate, 100%) compound BIT3 (93 mg, 68%), as a yellow solid. TLC
1
(
hexane/ethyl acetate (1:3)): R = 0.29. H-NMR (400 MHz, CDCl )
δ
H
= 8.55 (d, J = 8.4 Hz, 1H), 8.27
f
3
(d, J = 5.2 Hz, 1H), 7.86 (dd, J = 8.4, 1.8 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 7.52 (d, J = 1.8 Hz, 1H), 7.28
(
4
d, J = 3.0 Hz, 1H), 7.23–7.18 (m, 2H), 6.89 (d, J = 1.6 Hz, 1H), 4.60–4.55 (m, 2H), 4.44 (d, J = 6.0 Hz, 2H),
.15–4.03 (m, 2H), 2.97 (s, 3H), 1.42 (s, 3H). C-NMR (101 MHz, CDCl ) δ = 164.60 (d, J = 239.3 Hz),
3 C
13
1
58.47, 151.79 (d, J = 8.2 Hz), 148.72 (d, J = 15.5 Hz), 140.25 (d, J = 3.4 Hz), 139.92, 137.45, 137.02,
36.10, 131.68, 131.49, 130.91, 126.37, 125.90, 124.06, 119.44 (d, J = 4.1 Hz), 118.91, 118.56, 114.00, 107.49
1
(
d, J = 38.7 Hz), 79.63 (d, J = 4.1 Hz), 73.61, 39.77, 21.26, 12.90. 19F-NMR (282 MHz, CDCl )
δ
F
= 66.93
−
3
+
+
(s, br). HR-MS (ESI+) m/z = 490.1443 and 512.1261 (calcd. 490.1441 for C26H22ClFN O2 [M + H] and
5
+
+
5
12.1260 for C26H21ClFN5NaO2 [M + Na] ).
1
-(2-Chloro-5-methoxyphenyl)-8-(6-fluoropyridin-3-yl)-3 methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (BIT4):
According to General Procedure A, compound 7b (111 mg, 0.308 mmol, 1 eq), BA 9a (69 mg,
.370 mmol, 1.2 eq), K CO (107 mg, 0.77 mmol, 2.5 eq), and Pd(PPh ) (36 mg, 10 mol%) were reacted
0
2
3
3 4
in dioxane/water (5 mL) to give after purification by flash chromatography (gradient: CHCl /MeOH,
3
5
0:1
→
CHCl /MeOH, 30:1) compound BIT4 (83 mg, 64%), as a yellow solid. TLC (hexane/ethyl acetate
3
1
(1:1)): R = 0.37. H-NMR (400 MHz, CDCl )
δ
= 8.51 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 2.3 Hz, 1H), 7.82
f
3
H
(
1
ddd, J = 8.6, 7.3, 2.7 Hz, 1H), 7.76 (dd, J = 8.4, 1.8 Hz, 1H), 7.52 (d, J = 8.9 Hz, 1H), 7.39 (d, J = 1.8 Hz,
H), 7.19 (d, J = 3.1 Hz, 1H), 7.13 (dd, J = 8.9, 3.0 Hz, 1H), 7.01 (dd, J = 8.5, 3.1 Hz, 1H), 3.84 (s, 3H), 2.96
13
(
s, 3H). C-NMR (75 MHz, CDCl ) δ = 163.82 (d, J = 242.0 Hz), 159.00, 146.24 (d, J = 15.0 Hz), 140.19,
3 C
1
1
39.76 (d, J = 8.1 Hz), 139.38, 137.33, 136.36, 136.00, 132.98 (d, J = 4.7 Hz), 131.58, 131.43, 130.77, 126.04,
25.87, 124.10, 118.64, 117.62, 113.26, 110.16 (d, J = 37.5 Hz), 55.99, 12.86. 19F-NMR (282 MHz, CDCl )
3
+
δ
= 67.90 (dd, J = 7.4, 3.1 Hz). HR-MS (ESI+): m/z = 420.1010 (calcd. 420.1022 for C H ClFN O
−
F
22 16 5
+
[M + H] ).
1
-(2-Chloro-5-methoxyphenyl)-8-(2-fluoropyridin-3-yl)-3 methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (BIT5):
According to General Procedure A, compound 7c (115 mg, 0.322 mmol, 1 eq), BA 9a (72 mg, 0.386 mmol,
.2 eq), K CO (115 mg, 0.832 mmol, 2.58 eq), and Pd(PPh ) (37 mg, 10 mol%) were reacted in
1
2
3
3 4
dioxane/water (5 mL) to give after purification by flash chromatography (gradient: light petroleum

Molecules 2019, 24, 2791
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ether/ethyl acetate, 1:1
→
light petroleum ether/ethyl acetate, 1:2) compound BIT5 (93 mg, 69%),
1
as a yellow solid. TLC (light petroleum ether/ethyl acetate (1:2)): R = 0.43. H-NMR (300 MHz, CDCl )
f
3
δ
= 8.51 (d, J = 8.4 Hz, 1H), 8.22 (dd, J = 4.8, 0.7 Hz, 1H), 7.83–7.73 (m, 2H), 7.52–7.45 (m, 2H), 7.32–7.27
H
13
(
(
m, 1H), 7.17 (d, J = 3.0 Hz, 1H), 7.10 (dd, J = 8.9, 3.0 Hz, 1H), 3.83 (s, 3H), 2.97 (s, 3H). C-NMR
75 MHz, CDCl3) = 160.19 (d, J = 242.0 Hz), 158.95, 147.84 (d, J = 15.0 Hz), 140.67 (d, J = 3.5 Hz),
δ
C
1
39.44, 137.51, 137.42 (d, J = 2.1 Hz), 136.37, 136.21, 131.33, 131.01, 130.93, 127.58 (d, J = 2.8 Hz), 126.05,
23.76, 122.28 (d, J = 4.6 Hz), 121.95 (d, J = 27.2 Hz), 118.72, 117.03, 115.51 (d, J = 5.2 Hz), 55.95, 12.88.
1
19
F-NMR (282 MHz, CDCl )
δ
+
= −70.11 (d, J = 9.8 Hz). HR-MS (ESI+): m/z = 420.1014 (calcd. 420.1022
3
F
+
for C H ClFN O [M + H] ).
22
16
5
1
-(2-Fluoro-5-methoxyphenyl)-8-(2-fluoropyridin-4-yl)-3-methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (BIT6):
According to General Procedure A, compound 7a (100 mg, 0.279 mmol, 1 eq), BA 9b (62 mg, 0.365
mmol, 1.3 eq), K CO (97 mg, 0.698 mmol, 2.58 eq), and Pd(PPh ) (32 mg, 10 mol%) were reacted in
2
3
3 4
dioxane/water (5 mL) to give after purification by flash chromatography (gradient: light petroleum
ether/ethyl acetate, 1:1
→
light petroleum ether/ethyl acetate, 1:2) compound BIT6 (81 mg, 72%),
1
as an orange solid. TLC (petroleum ether/ethyl acetate (1:2)): R = 0.54. H-NMR (300 MHz, CDCl )
f
3
δ
= 8.55 (d, J = 8.4 Hz, 1H), 8.27 (dd, J = 5.3, 0.7 Hz, 1H), 7.86 (dd, J = 8.4, 1.9 Hz, 1H), 7.73 (dd, J = 3.5,
H
13
1
.8 Hz, 1H), 7.30–7.27 (m, 1H), 7.25–7.14 (m, 3H), 6.94–6.91 (m, 1H), 3.88 (s, 3H), 2.97 (s, 3H). C-NMR
(
75 MHz, CDCl3) = 164.61 (d, J = 239.5 Hz), 156.62 (d, J = 7.1 Hz), 153.86 (d, J = 246.3 Hz), 153.58,
δ
C
1
1
1
51.80, 148.73 (d, J = 15.4 Hz), 140.32, 140.24 (d, J = 2.9 Hz), 137.50 (d, J = 62.8 Hz), 133.85, 131.56, 125.94,
24.28, 120.00 (d, J = 16.3 Hz), 119.46 (d, J = 4.1 Hz), 118.76 (d, J = 7.9 Hz), 117.03 (d, J = 22.7 Hz), 116.44,
19
13.90 (d, J = 2.2 Hz), 107.56 (d, J = 38.8 Hz), 56.21, 12.88. F-NMR (282 MHz, CDCl )
δ
F
= 66.90—-66.97
−
3
+
+
(
m), 121.68–121.78 (m). HR-MS (ESI+): m/z = 404.1325 (calcd. 404.1317 for C H F N O [M + H] ).
−
22 16 2 5
1
-(2-Chloro-5-methoxyphenyl)-8-(3-fluoropyridin-5-yl)-3 methylbenzo[e]imidazo[5,1-c][1,2,4]triazine (BIT7):
According to General Procedure A, compound 7d (100 mg, 0.279 mmol, 1 eq), BA 9a (62 mg,
.335 mmol, 1.2 eq), K CO (116 mg, 0.839 mmol, 3.0 eq), and Pd(PPh ) (17 mg, 5 mol%) were reacted
0
2
3
3 4
in dioxane/water (5 mL) to give after purification by flash chromatography (gradient: hexane/ethyl
acetate, 1:2
→
hexane/ethyl acetate, 1:3) compound BIT7 (79 mg, 67
%
), as a yellow solid. TLC
1
(
(
(
(
(
hexane/ethyl acetate (1:2)): R = 0.38. H-NMR (400 MHz, CDCl )
d, J = 2.7 Hz, 1H), 8.46–8.45 (m, 1H), 7.81 (dd, J = 8.4, 1.9 Hz, 1H), 7.55 (d, J = 8.9 Hz, 1H), 7.44–7.38
m, 2H), 7.20 (d, J = 3.0 Hz, 1H), 7.14 (dd, J = 8.9, 3.0 Hz, 1H), 3.85 (s, 3H), 2.97 (s, 3H). C-NMR
101 MHz, CDCl3) = 159.71 (d, J = 258.4 Hz), 159.05, 143.99 (d, J = 4.0 Hz), 139.77, 139.56, 138.21
δ
= 8.53 (d, J = 8.4 Hz, 1H), 8.48
f
3
H
13
δ
C
d, J = 23.1 Hz), 137.37, 136.58, 136.23 (d, J = 3.8 Hz), 136.09, 131.52, 131.48, 130.84, 126.02, 125.99, 124.10,
1
21.21 (d, J = 19.0 Hz), 118.59, 117.70, 113.68, 55.99, 12.87. 19F-NMR (377 MHz, CDCl )
δ
F
= 125.95
−
3
+
+
(d, J = 9.2 Hz). HR-MS (ESI+): m/z = 420.1024 (calcd. 420.1022 for C H ClFN O [M + H] ).
22
16
5
1-(2-Fluoro-5-methoxyphenyl)-3-methyl-8-(pyridin-3-yl)benzo[e]imidazo[5,1-c][1,2,4]triazine (BIT8): According
to General Procedure A, compound 7e (100 mg, 0.294 mmol, 1 eq), BA 9b (60 mg, 0.353 mmol, 1.2 eq),
K CO (110 mg, 0.796 mmol, 2.7 eq), and Pd(PPh ) (17 mg, 5 mol%) were reacted in dioxane/water
2
3
3 4
(
5 mL) to give after purification by flash chromatography (gradient: hexane/ethyl acetate, 1:3
→
ethyl
), as an orange solid. TLC (hexane/ethyl acetate (1:3)):
δ = 8.69–8.62 (m, 2H), 8.53 (d, J = 8.4 Hz, 1H), 7.83 (dd, J = 8.4,
H
acetate, 100%) compound BIT8 (93 mg, 82
R = 0.35. H-NMR (300 MHz, CDCl )
%
1
f
3
1
0
.8 Hz, 1H), 7.77 (ddd, J = 8.0, 2.4, 1.6 Hz, 1H), 7.70 (dd, J = 3.5, 1.8 Hz, 1H), 7.39 (ddd, J = 8.0, 4.8,
.8 Hz, 1H), 7.30–7.23 (m, 2H), 7.18–7.11 (m, 1H), 3.86 (s, 3H), 2.97 (s, 3H). 13C-NMR (75 MHz, CDCl3)
δC = 156.52 (d, J = 1.8 Hz), 154.85 (d, J = 243.0 Hz), 149.93, 148.39, 140.58 (d, J = 125.3 Hz), 137.76, 136.60,
1
34.74, 134.53, 133.64, 131.43, 128.62 (d, J = 12.4 Hz), 126.12, 124.28, 123.94, 120.12 (d, J = 16.6 Hz),
18.83 (d, J = 7.9 Hz), 117.01 (d, J = 22.9 Hz), 116.29 (d, J = 2.0 Hz), 113.35 (d, J = 1.9 Hz), 56.18, 12.86.
1
19
F-NMR (282 MHz, CDCl )
δ
F
= 121.71 (td, J = 9.0, 4.0 Hz). HR-MS (ESI+): m/z = 386.1400 (calcd.
−
3
+
+
3
86.1412 for C H FN O [M + H] ).
22
17
5

Molecules 2019, 24, 2791
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1
-(2-Fluoro-5-methoxyphenyl)-3-methyl-8-(pyridin-4-yl)benzo[e]imidazo[5,1-c][1,2,4]triazine (BIT9): According
to General Procedure A, compound 7f (100 mg, 0.294 mmol, 1 eq), BA 9b (61 mg, 0.366 mmol, 1.2 eq),
K CO (103 mg, 0.745 mmol, 2.53 eq), and Pd(PPh ) (19 mg, 5 mol%) were reacted in dioxane/water
2
3
3 4
(
5 mL) to give a raw product, which was purified by two-times flash chromatography: CHCl /MeOH
3
(
10:1) was used first, followed by hexane/ethyl acetate (gradient 1:3
→
ethyl acetate, 100%) to obtain
1
compound BIT9 (44 mg, 39
%), as a yellow solid. TLC (hexane/ethyl acetate (1:1)): R = 0.29. H-NMR
f
(
300 MHz, CDCl3) δ = 8.68–8.65 (m, 2H), 8.53 (d, J = 8.5 Hz, 1H), 7.87 (dd, J = 8.4, 1.8 Hz, 1H), 7.74
H
(
2
ddd, J = 3.5, 1.9, 0.5 Hz, 1H), 7.33–7.30 (m, 2H), 7.29–7.22 (m, 2H), 7.19–7.13 (m, 1H), 3.87 (s, 3H),
.96 (s, 3H). 13C-NMR (101 MHz, CDCl3)
δ
C
= 156.57 (d, J = 2.0 Hz), 154.85 (d, J = 243.4 Hz), 150.60,
1
46.38, 141.51, 140.07, 137.81, 137.04, 133.73, 131.46, 125.98, 124.28, 121.65, 120.11 (d, J = 16.6 Hz),
18.86 (d, J = 7.9 Hz), 117.01 (d, J = 22.6 Hz), 116.24 (d, J = 2.0 Hz), 113.67 (d, J = 2.3 Hz), 56.21, 12.87.
1
19
F-NMR (282 MHz, CDCl )
δ
F
= −121.63—121.71 (m). HR-MS (ESI+): m/z = 386.1404 (calcd. 386.1412
3
+
+
for C H FN O [M + H] ).
22
17
5
3
.3. Biology
3
.3.1. In Vitro Evaluation of BIT Derivatives Towards PDEs
The inhibitory potency of BIT derivatives against the human recombinant PDE subtype was
determined in enzyme assays, conducted by SB Drug Discovery (Scotland, U.K.). The phosphodiesterase
assays were performed using recombinant human PDE enzymes expressed in a baculoviral system.
This had similarity to PDE enzymes taken from human tissue using known inhibitor standards
where available. The radiometric assay was a modification of the two-step method of Thompson and
Appleman [45], which was adapted for 96-well plate format.
0
0
Firstly, tritium-labeled cyclic AMP or cyclic GMP was hydrolyzed to 5 -AMP or 5 -GMP by
0
0
phosphodiesterase. The 5 -AMP or 5 -GMP was then further hydrolyzed to adenosine or guanosine
by nucleotidase in snake venom. An anion-exchange resin bound all charged nucleotides and left
H]adenosine or [ H]guanosine as the only labeled compound to be counted by liquid scintillation.
3
3
[
3
3
Briefly, 50
and 11 L of 50% DMSO (or compound dilution) for 20 min (at 30 C). Reactions were carried out
in a Greiner 96-deep well 1-mL master-block. The enzyme was diluted in 20 mM Tris HCl pH 7.4
µL of diluted human PDE enzyme were incubated with 50 µL of [ H]-cAMP or [ H]-cGMP
◦
µ
3
3
and [ H]-cAMP or [ H]-cGMP are diluted in 10 mM MgCl , 40 mM Tris HCl pH 7.4. The reaction is
terminated by denaturing the PDE enzyme (at 70 C for 2 min), after which 25
nucleotidase were added and incubated for 10 min (at 30 C). Plates were centrifuged for 9 seconds
2
◦
µ
L of snake venom
◦
before incubation. After incubation, 200
µL of Dowex resin were added, and the plate was shaken for
20 min then centrifuged for 3 min at 2500 r.p.m. Fifty microliters of supernatant were removed and
added to 200 µL of MicroScint-20 in white plates (Greiner 96-well Optiplate) and shaken for 30 min
before reading on a Perkin Elmer TopCount Scintillation Counter.
Compounds were tested at a concentration of 10 nM against human PDE2A3 and at 1 µM for
PDE4A1, PDE5A, PDE6AB, PDE9A1, and PDE10A1. The percentage of inhibition of the compounds
and standard inhibitors was determined and compared to historical assay data to ensure that they fell
within acceptable ranges. For IC50 values’ measurements, BIT1 was tested at concentrations of 0.1,
0.5, 1.0, 5.0, 10.0, and 100 nM, and BIT6 and BIT9 were tested at concentrations of 0.5, 1.0, 5.0, 10.0,
100, and 1000 nM against PDE2A3, while the concentration against PDE10A1 at 0.25, 5.0, 10, 100, 250,
and 1000 nM (n = 2).
Data generated were analyzed using Prism software (GraphPad Inc.).
3
.3.2. Incubations with Mouse Liver Microsomes
For microsome experiments, the following instruments were used: BioShake iQ (QUANTIFOIL
Instruments, Jena, Germany), Centrifuge 5424 (Eppendorf, Hamburg, Germany), DB-3D TECHNE Sample
Concentrator (Biostep, Jahnsdorf, Germany), and UltiMate 3000 UHPLC System (Thermo Scientific,

Molecules 2019, 24, 2791
18 of 21
Germering, Germany) including a DAD detector (DAD-3000RS) coupled to an MSQ Plus Single
Quadrupole Mass Spectrometer (Thermo Scientific, Austin, Texas, USA).
NADPH (Nicotinamide Adenine Dinucleotide Phosphate) and testosterone were purchased from
Sigma-Aldrich (Steinheim, Germany). GIBCO Mouse Liver Microsomes (MLM, 20 mg/mL) were
purchased from Life Technologies (Darmstadt, Germany). Dulbecco’s Phosphate-Buffered Saline (PBS)
without Ca2 , Mg ) was purchased from Biochrom (Berlin, Germany).
+
2+
(
Incubations had a final volume of 250
µL and were performed in PBS (pH 7.4). BIT1 was freshly
dissolved in DMSO to provide a stock solution of 2 mM that was used for the experiments and resulted
in an amount of DMSO of 1% in each incubation mixture. In the following, final concentrations are
provided in brackets [43]. PBS, MLM (1 mg/mL), and BIT1 (20
µ
M) were mixed and preincubated at
◦
3
7 C for 5 min. Analogously, preincubated NADPH (2 mM) was added, and the mixtures were gently
◦
◦
shaken at 37 C. After 90 min, portions of 1.0 mL of cold acetonitrile (
−
20 C) were added, respectively,
followed by vigorous shaking (30 s), cooling on ice (4 min), and centrifugation at 14,000 rpm (10 min).
◦
Supernatants were concentrated at 50 C under a flow of nitrogen to provide residual volumes of
4
0–60
µ
L, which were reconditioned by adding acetonitrile/water 1:1 (v/v) to obtain samples of 100
µ
L,
◦
which were stored at 4 C until analyzed by HPLC-UV-MS. As a positive control, testosterone was
used as the substrate and incubated at an appropriate concentration, similar to the protocol described
above, to give complete conversion confirmed by HPLC. Furthermore, incubations without NADPH,
microsomal protein, and BIT1, respectively, were performed as negative controls.
HPLC-UV-MS analyses were performed on a ReproSil-Pur 120 C18-AQ-column, 125 mm
m (Dr. Maisch GmbH, Ammerbuch, Germany) equipped with an appropriate precolumn at 25 C
×
3 mm,
◦
3
µ
and a flow rate of 0.7 mL/min. The solvent system consisted of Eluent A: water, containing 2 mM
ammonium acetate, and Eluent B: water/acetonitrile 20/80 (v/v), containing 2 mM ammonium acetate.
Two methods were applied. Method A (gradient elution, % acetonitrile): 0–1.5 min, 10%; 1.5–10 min,
10–80%; 10-13 min, 80%; 13–16 min 10%. Method B (isocratic elution, % acetonitrile): 0–25 min, 42%.
For both methods, UV detection was performed at a wavelength of 228 nm (maximum UV absorbance
of BIT1) and a bandwidth of 20 nm. MSQ Plus single quadrupole mass spectrometer was operated in
◦
positive electrospray ionization mode: probe temperature 500 C, needle voltage 5 V, cone voltage 75 V.
4
. Conclusions
A series of novel fluorinated PDE2A inhibitors based on a BIT (benzoimidazotriazine) scaffold
was successfully prepared and evaluated in vitro. The binding results revealed that a modification
with a 2,2-oxetanyl propoxy portion on the phenyl (phe-3) at the 1-position led to a potency loss
towards PDE2A. However, a small methoxy group at the same position (phe-1) led to a significant
increase of potency towards PDE2A. It appeared that a higher inhibition towards PDE2A was
obtained by pyridine-4-yl residues in this position, either fluorinated or non-fluorinated. Furthermore,
the introduction of ortho-fluorophenyl at the 1-position, instead of o-chlorophenyl, showed a lack
of selectivity towards PDE2A. The introduction of the 2-fluoropyridin-4-yl residue at the 8-position
provided us a promising candidate for future labeling with fluorine-18.
In vitro metabolism studies of BIT1 with MLM showed that BIT1 was sufficiently stable and
18
suitable for the development of an F-labeled radioligand. This should further be proven by in vivo
investigations in the future. Taken together, BIT1 might be a prospective ligand to be developed as
a radioligand for PDE2A-PET imaging.
Supplementary Materials: Supplementary Materials including NMR spectra of final compounds BIT1
–BIT9,
and dose response curves (BIT1, BIT6, and BIT9) can be accessed at.
Author Contributions: R.R., D.B., and M.S. designed and performed the organic syntheses; R.R., P.B., and M.S.
designed the in vitro evaluation; R.R. and F.-A.L. designed and performed the microsomal incubations; R.R.,
F.-A.L, D.B., P.B., and M.S. wrote the paper. All authors read and approved the final manuscript.
Funding: This research was funded by the Deutsche Forschungsgemeinschaft (DFG), Project Number:
SCHE 1825/3-1.

Molecules 2019, 24, 2791
19 of 21
Acknowledgments: The authors would like to thank the Indonesia Ministry of Research and Technology-Program
for Research and Innovation in Science and Technology Project (RISET-Pro), World Bank Loan No. 8245-ID,
for supporting the PhD thesis of Rien Ritawidya. We also thank the staff of the Institute of Analytical Chemistry,
Department of Chemistry and Mineralogy of University of Leipzig, for recording and processing the NMR and
LR/HR-MS spectra and Tina Spalholz (HZDR) for technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: BIT2, and BIT6 are avaliable from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).