Development of novel amide–derivatized 2,4-bispyridyl thiophenes as highly potent and selective Dyrk1A inhibitors. Part II: Identification of the cyclopropylamide moiety as a key modification

Article abstract of DOI:10.1016/j.ejmech.2018.08.097

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) is a potential target in Alzheimer's disease (AD) because of the established correlation between its over-expression and generation of neurofibrillary tangles (NFT) as well as the accumulation of amyloid plaques. However, the use of Dyrk1A inhibitors requires a high degree of selectivity over closely related kinases. In addition, the physicochemical properties of the Dyrk1A inhibitors need to be controlled to enable CNS permeability. In the present study, we optimized our previously published 2,4-bispyridyl thiophene class of Dyrk1A inhibitors by the synthesis of a small library of amide derivatives, carrying alkyl, cycloalkyl, as well as acidic and basic residues. Among this library, the cyclopropylamido modification (compound 4b) was identified as being highly beneficial for several crucial properties. 4b displayed high potency and selectivity against Dyrk1A over closely related kinases in cell-free assays (IC₅₀: Dyrk1A = 3.2 nM; Dyrk1B = 72.9 nM and Clk1 = 270 nM) and inhibited the Dyrk1A activity in HeLa cells with high efficacy (IC₅₀: 43 nM), while no significant cytotoxicity was observed. In addition, the cyclopropylamido group conferred high metabolic stability and maintained the calculated physicochemical properties in a range compatible with a potential CNS activity. Thus, based on its favourable properties, 4b can be considered as a candidate for further in vivo testing in animal models of AD.

Full text of DOI:10.1016/j.ejmech.2018.08.097

Accepted Manuscript
Development of novel amide–derivatized 2,4-bispyridyl thiophenes as highly potent
and selective Dyrk1A inhibitors. Part II: Identification of the cyclopropylamide moiety
as a key modification
Sarah S. Darwish, Mohammad Abdel-Halim, Ahmed K. ElHady, Mohamed Salah,
Ashraf H. Abadi, Walter Becker, Matthias Engel
PII:
S0223-5234(18)30769-4
10.1016/j.ejmech.2018.08.097
EJMECH 10708
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 June 2018
Revised Date: 29 August 2018
Accepted Date: 31 August 2018
Please cite this article as: S.S. Darwish, M. Abdel-Halim, A.K. ElHady, M. Salah, A.H. Abadi, W. Becker,
M. Engel, Development of novel amide–derivatized 2,4-bispyridyl thiophenes as highly potent and
selective Dyrk1A inhibitors. Part II: Identification of the cyclopropylamide moiety as a key modification,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.097.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Development of novel amide–derivatized 2,4-
bispyridyl thiophenes as highly potent and
selective Dyrk1A inhibitors. Part II:
Identification of the cyclopropylamide moiety as
a key modification
AUTHOR NAMES
1
1
1
2
Sarah S. Darwish , Mohammad Abdel-Halim , Ahmed K. ElHady , Mohamed Salah , Ashraf H.
1
3
2
Abadi , Walter Becker and Matthias Engel
AUTHOR ADDRESS
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German
University in Cairo, Cairo 11835, Egypt
2
Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123
Saarbrücken, Germany
3
Institute of Pharmacology and Toxicology, Medical Faculty of the RWTH Aachen University,
Wendlingweg 2, 52074 Aachen, Germany
1

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Abstract
Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) is a potential target in
Alzheimer’s disease (AD) because of the established correlation between its over-expression and
generation of neurofibrillary tangles (NFT) as well as the accumulation of amyloid plaques.
However, the use of Dyrk1A inhibitors requires a high degree of selectivity over closely related
kinases. In addition, the physicochemical properties of the Dyrk1A inhibitors need to be
controlled to enable CNS permeability. In the present study, we optimized our previously
published 2,4-bispyridyl thiophene class of Dyrk1A inhibitors by the synthesis of a small library
of amide derivatives, carrying alkyl, cycloalkyl, as well as acidic and basic residues. Among this
library, the cyclopropylamido modification (compound 4b) was identified as being highly
beneficial for several crucial properties. 4b displayed high potency and selectivity against
Dyrk1A over closely related kinases in cell-free assays (IC : Dyrk1A = 3.2 nM; Dyrk1B = 72.9
5
0
nM and Clk1 = 270 nM) and inhibited the Dyrk1A activity in HeLa cells with high efficacy
IC : 43 nM), while no significant cytotoxicity was observed. In addition, the cyclopropylamido
(
5
0
group conferred high metabolic stability and maintained the calculated physicochemical
properties in a range compatible with a potential CNS activity. Thus, based on its favourable
properties, 4b can be considered as a candidate for further in vivo testing in animal models of
AD.
Keywords: Dyrk1A, neurodegenerative diseases, cyclopropyl amide, SF3b1 phosphorylation,
CNS penetration
2

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1
Introduction
Dual-specificity tyrosine-regulated kinases (Dyrks) belong to the larger human kinome family
known as the CMGC group[1] which includes cyclin-dependent kinases (CDKs), mitogen-
activated protein kinase (MAPKs), glycogen synthase kinases (GSKs), and cdc-like kinases
(Clks).[2] The Dyrk family comprises the mammalian subtypes 1A, 1B, 2, 3, and 4.[3] As their
name implies, dual specificity kinases are capable of catalyzing their self-activation by
autophosphorylation at a conserved tyrosine residue, whereas the resulting mature forms of the
kinases can only phosphorylate exogenous protein substrates at serine or threonine residues.[1, 2]
Due to its location in the Down Syndrome (DS) critical region on human chromosome 21,
Dyrk1A is the most studied isoform in its family.[4-6] During embryonic development, Dyrk1A
plays an important role in neurogenesis as well as neuronal differentiation,[7] hence its about 1.5
fold overexpression in DS-affected individuals (trisomy 21) is believed to contribute to the
neurodevelopmental alterations associated with DS. Moreover, dysregulated Dyrk1A activity
was shown to play a pathogenic role in the development of Alzheimer’s disease (AD) in DS
individuals.[1, 8] The main hallmarks of AD include neurofibrillary tangles (NFT) as well as the
accumulation of amyloid plaques.[9] In vitro studies showed that Dyrk1A directly
phosphorylates tau protein, leading to the loss of tau biological function as well as sequestration
of normal tau and neurofibrillary degeneration.[10-12] Additionally, Dyrk1A was found
associated with NFTs in individuals with DS in the age group of 38–51 years, suggesting that
Dyrk1A contributes to the early onset of neurofibrillary degeneration.[13] Furthermore, over-
expressed Dyrk1A hyperphosphorylates amyloid precursor protein (APP) which enhances the
production of amyloid-β, the main component of amyloid plaques.[9, 13, 14]
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More recently, evidence has accumulated that Dyrk1A can also contribute to α-synuclein
aggregation and fibrillization in Lewy bodies in Parkinson’s disease (PD) and Lewy body
dementia. [15-19] Dyrk1A binds to and phosphorylates α-synuclein at Ser87.[15] In addition,
Dyrk1A phosphorylates the neurodegeneration –related septin 4,[16] and complexes of these
proteins may contribute to the cytoplasmic aggregation/fibrillization observed in PD, Lewy
body dementia and multiple-system atrophy.[17, 18] Moreover, parkin was reported to co-
localize and bind to Dyrk1A.[19] Dyrk1A directly phosphorylates parkin at Ser-131, causing
the inhibition of its E3 ubiquitin ligase activity, which may also contribute to the
pathogenesis of PD.
Furthermore, both AD and PD are also driven by aberrant splicing events which may be co-
regulated by Dyrk1A, too.[20-23] Dyrk1A was reported to phosphorylate SR proteins which
are considered among the main determinants of splice site recognition in pre-mRNA. [24]
Dyrk1A is additionally known to catalyze the phosphorylation of the non-SR protein SF3b1
which is an important modulator of splicing reactions.[25] In AD, a misbalance of the two
splicing–dependent 3R and 4R tau isoforms was shown to be caused by Dyrk1A in a mouse
model.[26] This imbalance can be linked to the ability of Dyrk1A to phosphorylate relevant
SR proteins such as SRp55, SC35 and ASF. [20-22] Besides the proven influence on the
3
R/4R tau protein ratio, Dyrk1A over-dosage models also showed changes in the neuroligin
mRNAs transcript composition as well as changes in the splicing pattern of
acetylcholinesterase (AChE), leading to different AChE mRNA variants.[27]
Thus, pharmacological inhibition of Dyrk1A might not only suppress the pathogenic
hyperphosphorylation of neuroproteins, it may also restore the normal pattern of splicing
products of the respective proteins. In light of these pleiotropic functions of Dyrk1A,
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potential pharmacological inhibitors of Dyrk1A should be highly selective to avoid the
accumulation of adverse side effects due to additional off target inhibitions. Although many
published Dyrk1A inhibitors, including harmine[28], INDY[29], leucettine L41[30], EHT
5
372 [31], and 7-chloro-2-phenyl-1H-indole-3-carbonitrile[32] exhibited a good overall
selectivity in a larger kinase screening panel, they still showed co-inhibition of at least one of
the off-targets Dyrk1B, Clk1 and/or haspin, all of which show a high degree of similarity
with Dyrk1A in their ATP binding site. The catalytic domain of Dyrk1A and Dyrk1B possess
a sequence identity of 85%, and their ATP binding sites differ by only one amino acid
residue: Met240 of Dyrk1A is replaced by Leu192 in Dyrk1B.[32] Dyrk1B was proposed as
an anticancer target,[33-35] however, in the CNS, it was shown to be implicated in the
regulation of astrocyte activation[36] which can have pro-inflammatory and neurotoxic but
also neuroprotective roles.[37-39] In addition, prolonged inhibition of Dyrk1B may trigger or
aggravate metabolic syndrome as a side effect, since the loss of Dyrk1B activity due to a
point mutation that decreased the protein stability was shown to cause an autosomal-
dominant form of metabolic syndrome.[40] Clk1 is involved in the regulation of alternative
splicing,[41] too, hence co-inhibition of Dyrk1A and Clk1 might potentiate the effects on
alternative splicing of numerous pre-mRNAs, which could be poorly tolerated during
prolonged treatments. Finally, haspin is indispensable for cell division,[42] and its inhibition
is expected to interfere with normal cell proliferation.
In a previous study, we introduced the 2,4-bispyridyl thiophene core as a favourable scaffold
for Dyrk1A inhibition,[43] The selectivity of this scaffold over some of the common off
target kinases was increased in a subsequent study using an amide–linked structural
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extension.[44] In the current work, we present new derivatives of the 2,4-bispyridyl
thiophene amides with further improved selectivity profiles and physicochemical properties.
2
2
Results and Discussion
.1 Design strategy.
In our previous attempt to improve the selectivity towards Dyrk1A, we extended the bispyridyl
thiophene scaffold by introducing an additional amino function on the pyridine at the thiophene-
2
position, yielding compound I (compound 2 in ref.[44], Fig. 1). Its increased potency prompted
us to attach a variety of side chains through amide linkage. In the library thus generated, we had
identified Compound II (compound 31b in ref.[44]) as the most potent inhibitor of that series. It
also showed a great improvement in selectivity towards Dyrk1B and Clk1 (IC : Dyrk1B = 383
5
0
nM, Clk1 > 2µM, [ATP]=15 µM), however, we noticed that II still showed significant cross
reactivity with haspin (IC = 36 nM). In addition, the clogP value of the benzylamide derivative
50
II (clogP = 4.11) was above the optimal average range reported for CNS active compounds,
which is about 2.8.[45]
Therefore, it seemed straightforward to extend our diversification strategy by including mainly
non aromatic and polar amide moieties, in order to enable alternative interactions with pocket
side chains and decrease the lipophilicity. We selected different alkyl, cycloalkyl, as well as
acidic and basic amide side chain extensions, which could potentially address pocket residues
located at the hinge region of the ATP binding site (e.g., Tyr243 and Ile165) but also at the
opposite side (e.g. Asp307, Phe170). We used the previously reported compound I as our
extendable hit (Fig. 1).
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2
.2 Chemistry.
The main synthetic scheme for attaining the planned amide functionalized 2,4-bispyridyl
thiophenes amide derivatives constitutes four steps (Scheme 1). Primarily, a Miyaura reaction
was carried out on 3-amino-5-bromopyridine and bis(pinacolato)diboron in the presence of
potassium acetate and Pd(dppf)Cl as a catalyst to yield the corresponding boronic acid pinacol
2
ester (Compound A). In the second step, Suzuki cross coupling reaction with 2,4-
dibromothiophene in the presence of Cs
2 3
CO and palladium-tetrakis(triphenylphosphine) to give
4
-bromothiophen-2-yl pyridine amines takes place to yield compound B. Afterwards, the amino
group in compound B undergoes a coupling reaction with diverse carboxylic acids and
methylsulfonyl chloride to yield a series of amides and sulfonamide, respectively. Finally, the
free bromo in the previous amides/sulfonamides make the compounds accessible for a second
Suzuki reaction with 3-pyridine boronic acid to synthesize the bispyridyl thiophene
functionalized amides (1b-8b). Alkylation of the secondary amide of compound 4a was done by
deprotonation of the amide with KH followed by the reaction with the respective alkyl iodide
then the product was coupled with 3-pyridine boronic acid to yield compounds 9-10 (Scheme 1).
To have an amide with basic side chains, either an additional BOC deprotection was performed
on compound 7b using TFA to release compound 11 (Scheme 2) or the chloroacetamide
derivative (compound E) was reacted with the appropriate alicyclic amines followed by
subsequent Suzuki coupling to yield compounds 12-13 (Scheme 3). In order to omit the terminal
pyridyl of compound 4b, compound B was coupled with 2-bromothiophene, followed by amide
coupling with cyclopropyl carboxylic acid to give compound 14 (Scheme 4). In addition to the
compounds mentioned above, a second unique cluster of compounds was synthesized by linking
the pyrazol-4-yl moiety to position 4 of the thiophene core of compound 4a. In compounds17-19,
7

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the 4-pyrazoleboronic acid pinacol ester was initially alkylated by its reaction with the
corresponding alkyl iodides in the presence of Cs CO as a base (Scheme5).
2
3
2
2
.3 Biological evaluation.
.3.1 Investigation of the amide linker.
Firstly, we investigated which type of amide was preferred as linking functionality with small
alkyl groups. Comparing the methyl amide and the methyl sulfonamide derivatives (compounds
1
b and 2b, respectively) of precursor compound I (Fig. 1), we found that the amide linkage was
better tolerated (Table 1), although the activity of 1b fell below that of I. However, an initial loss
of potency was not unexpected for the simple amide derivative, because the mesomeric effect of
the former amine was strongly diminished, thus lowering the electron density and H-bond
acceptor strength at the pyridine nitrogen.
2
.3.2 The effect of alkylamido extensions.
Since the Dyrk1A inhibitory activity was totally abolished with the methyl sulfonamide (2b,
Table 1), further alkyl extensions were subsequently introduced via the carboxamide linker,
hoping to over-compensate the drop of potency brought about this functional group. While a
simple homologation did not seem promising, as indicated by the lack of potency gain with the
ethyl amide derivative 3b (Table 1), the cyclopropyl substituent in compound 4b caused a
dramatic and unexpected increase in activity, lowering the IC to 3.2 nM. Interestingly, a one-
5
0
atom ring expansion through the use of cyclobutyl in compound 5b reduced the inhibitory
activity by more than 18-fold (Table 1). This trend was confirmed with larger cycloalkyl ring
expansions, e. g., with the cyclohexyl derivative 6b (40.3% inhibition at 250 nM), indicating that
the cyclopropyl amide moiety in 4b had the optimal size. It is likely that larger cycloalkyl rings
8

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caused steric clashes with the predicted binding site at the ATP binding pocket (cf. below and
Fig. 2).
2
.3.3 The effect of polar and ionizable amide extensions.
Despite the large boost of potency observed with the cyclopropyl moiety, our further attempts
focused on establishing polar interactions with the hydrophilic amino acid residues at the pocket
border. Especially the introduction of basic moieties seemed worthwhile with respect to a
potential enhancement of the CNS availability of our inhibitors – besides a general improvement
of drug-like characteristics. The pyrrolidine-3-carboxamide derivative 11, in which a secondary
amine was integrated in the cycloalkylamide moiety, indicated some degree of tolerance toward
basic functions, but did not reach by far the potency of the best compound, 4b (Table 1). Shifting
the position of the protonable nitrogen and introducing a methylene spacer, as in the
piperidinylacetamide 12, decreased the biological activity against Dyrk1A to the level of the
cyclohexane carboxamide 6b. The morpholino analogue 13 exhibited a further drop of activity,
indicating that multiple polar atoms are not tolerated. A tetrazolyl acetamide derivative with
reversed, partially anionic charge was also synthesized and tested (8b); its low inhibitory activity
(24% at 250 nM) suggested that an anionic charge cannot compensate for the unfavourable
influence of increased polarity (Table 1). Compound 7b, the protected synthetic precursor of 11,
was also tested to probe whether a further structural extension by bulky side chains could be
promising; however, this was not the case, as indicated by the total loss of activity with 7b.
2
.3.4 The effect of N-alkylation of the cyclopropylamide moiety.
Two more derivatives were synthesized by the N-alkylation of our most potent compound 4b to
afford the N-methyl and N-ethyl derivatives (compounds 9 and 10, respectively). Both tertiary
amide derivatives almost lost the Dyrk1A inhibitory activity (Table 1). N-alkylation of an amide
9

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function not only removes the H-bond acceptor hydrogen, but also has a considerable impact on
the preferred conformation, hydrophobicity and rotational flexibility of the respective molecule
part. Most likely, the N-alkyl caused the carboxamide function to rotate out of the pyridine ring
plane, due to steric hindrance between the alkyl and the ortho H atoms, thus stabilizing a
biologically less active conformation. In our binding model, compound 4b bound with a coplanar
carboxamide–pyridine conformation (cf. Fig. 2). On the other hand, the loss of the HBD function
did not play a role, at least according to our predicted binding model. Nevertheless, to exclude
experimentally that an alternative binding mode could be formed, involving tandem H-bonding
of the pyridine N and the amide carbonyl or NH to the hinge region, we synthesized the
truncated derivative 14, which should adopt this hypothetical binding mode more easily than the
full length parent compound 4b. As indicated by the complete loss of activity of 14, the
compounds did not switch to a new binding mode driven by tandem H-bonding to the hinge
region, rather the 4-pyridyl ring was still essential, probably interacting with Lys188 as
illustrated in Fig. 2.
2
.3.5 Replacement of the 4-pyridyl in 4b by pyrazole.
With the last set of compounds, we investigated whether the nitrogen in a heteroaromatic five-
membered ring could further increase the potency. In our previously reported series, the methyl
pyrazole moiety proved to be a good surrogate of ring B as shown in compound C30 in Fig. 1
(compound 30 in Ref.[43]), since it mediated a higher potency towards Dyrk1A than the 4-
pyridyl in C4 (compound 4 in Ref. [43], Fig. 1) (IC ´s: 130 nM vs. 300 nM). Hence, it was
5
0
straightforward to combine the favourable cyclopropylamide feature (this work) with the methyl
pyrazolyl ring (previous work). The resulting compound 16 was also potent against Dyrk1A
(
IC =42.6 nM, Table 2), still exhibiting a 3-fold increase in potency compared to compound
50
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C30 due to the cyclopropyl amide extension. However, when compared to 4b, compound 16 was
almost 13-fold less potent, indicating that in the presence of the cyclopropylamide extension,
pyridine was preferred as ring B (cf. Fig. 1) over N-methyl pyrazole. This was partially
attributable to a steric hindrance of the N-methyl group in 16, because the demethylated 1H-
pyrazolyl derivative 15 was clearly more potent (IC = 18 nM, Table 2).
5
0
Nevertheless, we aimed at exploring the possibility of utilizing the pyrazole nitrogen as a
convenient attachment point for further molecule extensions. Therefore, we probed the effect of
installing ethyl, propyl and isopropyl groups at the pyrazole N1 (compounds 17, 18 and 19,
respectively). Interestingly, placing an ethyl group instead of the methyl in compound 16 slightly
improved the inhibitory potency (compound 17, IC50 = 29.7 nM), suggesting that an
enhancement of the hydrophobic effect partially compensated for the steric hindrance. However,
further elongation of the alkyl chain to n-propyl (18) reduced the inhibitory activity again
(
IC =76 nM). Moreover, the branched isopropyl derivative (19) was almost inactive against
50
Dyrk1A. These data suggested that pyrazole N1 in 15 was not suitable for molecule extensions
because of the unfavourable vector, probably causing steric clashes of the substituents inside the
binding pocket. In addition, a further disadvantage of the pyrazole containing scaffold was noted
with respect to the selectivity. In general, the scaffold was better tolerated by Dyrk1B, thus
lowering the selectivity factor for Dyrk1A over Dyrk1B to 8 for 15 (compared with 24 for 4b),
while the selectivity was even lost with the N-alkylated derivatives 16–18. Fig. 3 summarizes the
structure activity relationships of the current series toward Dyrk1A.
2
4
.3.6 Predicted binding mode of 4b to Dyrk1A.
b showed a remarkable boost of potency, which was attributable to the rather small
cyclopropylamide moiety. To identify the binding mode of 4b and the particular interactions of
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the cyclopropylamide extension, we performed docking simulations to the ATP binding pocket
using the coordinates from PDB entry 3ANR (human Dyrk1A/harmine complex). While in
theory, binding in two different orientations was conceivable, each with the pyridine nitrogens
forming H-bonds with Lys188 and the Leu241 NH, only one of the binding orientations placed
the key cyclopropylamide group close to a pocket side chain with which it could interact (Fig.
2
A). In this binding model, a CH-π interaction between the hinge region residueTyr243 and the
cyclopropyl carbon in 4b was observed. Furthermore, the cyclopropyl moiety showed packing
against the Ile165 side chain, leading to additional hydrophobic interactions (Fig. 2B). Thus, the
notable impact of the cyclopropyl may arise from a combination of CH–π and hydrophobic
interactions with different side chains, leading to a synergistic enhancement of binding affinity.
Such a cooperativity between different simultaneous interactions, also including hydrophobic
interactions, is common in protein–ligand binding and has been experimentally confirmed.[46]
Hence, if one of the specific interactions is weakened, the total binding affinity is over-
proportionally affected. This might explain the low activity of 1b; according to our docking
model, the methyl at the amide function could also engage in CH–π interaction with Tyr243 but
cannot reach Ile165 (see Fig. S1, Supplementary Information).
2
.3.7 Selectivity against Dyrk1B and other selected kinases.
The selectivity of all new compounds was initially evaluated against Dyrk1B, the isoform most
closely related to Dyrk1A, by screening at 250 nM, as shown in Tables 1 and 2. All of the new
potent compounds were clearly more active against Dyrk1A, similar to what we observed with
the benzamide and benzylamide derivatives.[44] The most potent cycloalkylamide derivatives of
bispyridyl thiophene, 4b and 5b, showed selectivity factors (Dyrk1A over -1B) of approximately
2
4- and 4-fold, respectively. While the pyrazole analogue 15 also exhibited a significant 8-fold
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selectivity toward Dyrk1A, this was lost with the N1-alkylated congeners (see above).
Altogether, 4b possessed the greatest selectivity for Dyrk1A in the present series, nearly
approximating the 27 fold selectivity observed with the previous 4-fluorobenzyl amide derivative
II (depicted in Fig. 1). To further examine the selectivity of 4b, it was additionally tested against
a panel of kinases (Table 3) which are supposed to have structurally similar ATP binding pockets
and were frequently reported to show cross reactivity with diverse Dyrk1A inhibitors. As can be
seen in Table 3, 4b displayed a remarkable selectivity in this crucial selection of kinases, ranking
4
b among the most selective Dyrk1A inhibitors published so far. Even haspin, a kinase which
was still slightly co-inhibited by the previous compound II, was 24 times less strongly inhibited
by 4b than the target Dyrk1A.
How was the selectivity over the most closely related isoform Dyrk1B achieved?
Despite the high sequence similarity between Dyrk1A and Dyrk1B, our most potent compound
4
b showed a remarkable 24 fold selectivity towards Dyrk1A. Indeed, the ATP binding sites of
two closely related homologues differ only by a single amino acid (Met240 in Dyrk1A
corresponds to Leu192 in Dyrk1B). Our docking model gave a hint on how this difference might
translate into reduced affinity to Dyrk1B. Although the Met240 side chain in Dyrk1A is not
directly facing the ATP binding pocket, it could be attracted by a hydrophobic patch created or
enhanced upon binding of the inhibitor, thereby engaging in van der Waals and hydrophobic
interactions with the pyridine ring in 4b which is H-bonded to Leu241. The latter interactions
could possibly contribute to anchoring the pyridine ring at this position in Dyrk1A, and might
explain, at least partially, the lower potency of 4b against Dyrk1B, where the van der Waals
interaction surface to the corresponding Leu192 is considerably smaller (see Fig. 2B).
Concomitantly, the essential H-bond of the pyridine N to the hinge region NH might be less
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effectively shielded from water molecules in Dyrk1B. Furthermore, attractive interactions
between aliphatic sulfur and pyridine nitrogen are well documented[47] and might play a role
with Dyrk1A, but would be absent with Dyrk1B.
2
.3.8 Inhibition of Dyrk1A in HeLa cells.
To evaluate the potency of 4b against Dyrk1A in intact cells, we analyzed the effects on the
intracellular phosphorylation of the splicing factor 3b1 (SF3b1) using western blot analysis (Fig.
4
). The phosphorylation of Thr434 on SF3b1 was previously shown to be solely dependent on
Dyrk1A activity in HeLa cells.[25] The inactive analogue 9 served as a negative control. In this
assay, treatment of HeLa cells with 4b reduced the pT343-signal in a concentration dependent
manner with an IC value of 43 nM (Fig. 4). These results were in good agreement with the
–
5
0
strong Dyrk1A inhibition observed in the cell free assay.
In parallel, we assessed the potential cytotoxicity of our most potent compound 4b with the same
cell line. As can be seen in Table 4, our test compounds showed minimal cytotoxicity on HeLa
cells up to a concentration of 3 µM, which ensures the safety of our inhibitor in concentrations
that fully inhibit Dyrk1A in cells.
2
.4 Evaluation of CNS drug like properties.
Since the major indication for Dyrk1A inhibitors is the treatment of neurodegenerative diseases,
it was straightforward to assess the ability of our novel analogues to cross the BBB. A set of
physicochemical properties has been previously reported in literature to be associated with high
probability of CNS penetration. [45] CNS active drug candidates are assumed to have definite
–
attributes exemplified in molecular weight: 181-427, logP: 0.4-5.1 (median: 2.8) ,[45] HBA: 2-3,
HBD: 0-1, and TPSA (topological polar surface area) < 76 Å.[48] Accordingly, we calculated
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different physicochemical parameters using ACD/Labs software for our most potent inhibitors
(4b and 15b) to estimate the CNS drug-likeness of the current series (Table 5). Most of the
calculated parameters for both inhibitors were in good agreement with the ideal ranges for brain
penetration as reported in literature. In comparison to the analogue from the previous series, II,
Compounds 4b and 15 showed more favourable characteristics, such as reduced logP and
molecular weight, thus increasing the likeliness to penetrate the BBB. With respect to the TPSA
2
values which exceeded the limit of 76 Å , it should be mentioned that this depends on whether
the thiophene sulphur is included in the calculation or not. Sulphur actually shows little polarity
in the aromatic ring system, and thiophene is therefore often employed as bioisosteric
replacement for benzene. Without counting the thiophene sulphur, the TPSA values for 4b and
2
1
5 are 54.9 and 70.7 Å , respectively.
Interestingly, searching the literature for structurally similar compounds that had shown high
brain penetration in vivo, we found a series of pyridine–containing glutamate receptor
antagonists, e. g., 2-{2-[3-(pyridin-3-yloxy)phenyl]-2H-tetrazol-5-yl}pyridine,[49] that even
showed a larger polar surface due to the central tetrazole core. Altogether, our assessment
suggested a high probability of CNS penetration for our most potent compounds 4b and 15.
2
.5 Evaluation of metabolic stability.
To further assess whether 4b and 15 are suitable for in vivo studies, the phase I and phase II
metabolic stability were measured using human hepatic liver S9 fractions. A definite set of
samples were taken at defined time points, and the remaining percentage of parent compound
was determined by LC-MS/MS. The calculated half-life times almost reached 2.5 h for 4b and
1
5 (Table 6), indicating a high metabolic stability, which exceeded that of the previous
15

ACCEPTED MANUSCRIPT
benzylamide
–
extended compound II (half-life against human S9 fraction: 118 min,[44] tested in
parallel).
3
Conclusions
We developed a novel Dyrk1A inhibitor series of amide–functionalized bispyridyl thiophenes
carrying different alkyl, cycloalkyl and polar side chains. In this series, the cyclopropyl amide in
compound 4b was identified as a key modification, which led to an enhancement of several
essential properties. Firstly, 4b exhibited the most potent Dyrk1A inhibition with an IC of 3.2
5
0
nM. In addition, the cyclopropyl amide modification effected a significant increase in selectivity
over closely related kinases including haspin (selectivity factor: 24), which was often found to be
co-inhibited even by some of the most selective Dyrk1A inhibitors, such as TG003 and
harmine.[50, 51] Of note, the benzylamide–modified previous inhibitor II had only achieved a
moderate 2.5 fold selectivity over haspin.[44] The cyclopropylamide modification did not only
increase potency and selectivity, but moreover, it also enhanced the metabolic stability against
human liver S9 fractions, leading to a half-life of almost 2.5 h.
As anticipated by the high cell free potency, 4b inhibited Dyrk1A in HeLa cells with an IC of
5
0
only 43 nM. This cellular potency, in combination with the high selectivity, low cytotoxicity and
high metabolic stability render 4b a promising candidate for in vivo studies using models of
neurodegenerative diseases.
4
Experimental Section
16

ACCEPTED MANUSCRIPT
4
.1 Chemistry.
Solvents and reagents were obtained from commercial suppliers and used as received. Melting
points were determined on a Stuart SMP3 melting point apparatus. All final compounds had a
percentage purity of at least 95%, and this could be verified by means of HPLC coupled with
mass spectrometry. Mass spectra (HPLC−ESIMS) were obtained using a TSQ quantum (Thermo
Electron Corp.) instrument prepared with a triple quadrupole mass detector (Thermo Finnigan)
and an ESI source. All samples were injected using an autosampler (Surveyor, Thermo Finnigan)
by an injection volume of 10 µL. The MS detection was determined using a source CID of 10 V
5
and carried out at a spray voltage of 4.2 kV, a nitrogen sheath gas pressure of 4.0 × 10 Pa, a
capillary temperature of 400 °C, a capillary voltage of 35 V, and an auxiliary gas pressure of 1.0
5
×
10 Pa. The stationary phase used was an RP C18 NUCLEODUR 100-3 (125 mm × 3 mm)
column (Macherey & Nagel). The solvent system consisted of water containing 0.1% TFA (A)
and 0.1% TFA in acetonitrile (B). The HPLC method used a flow rate of 400 µL/min. The
percentage of B started at 5%, was increased up to 100% during 7 min, was kept at 100% for 2
min, and was flushed back to 5% in 2 min and was kept at 5% for 2 min. A Bruker DRX 500
1
13
spectrometer was used to obtain the H NMR and C NMR spectra. The chemical shifts are
referenced to the residual protonated solvent signals.
4
4
.1.1 General synthetic procedures and experimental details.
.1.1.1 Procedure A, Procedure for synthesis of Compounds A-B, H.
A mixture of 5 mmol of the 3-amino-5-bromo pyridine and 1.96 gm (20 mmol) of potassium
acetate and 0.18 gm (0.25 mmol) of Pd(dppf)Cl2 and 5.08 gm (20 mmol) of
bis(pinacolato)diboron in dioxane was heated to reflux under argon for 2 hours to yield
17

ACCEPTED MANUSCRIPT
compounds A. The mixture was left to attain room temperature and then filtered under vacuum.
Without further purification, a reaction flask containing the filterate was charged with 6.5 gm (20
mmol) of Cs CO , 0.29 gm (0.25 mmol) of palladium-tetrakis(triphenylphosphine) and 6 mmol
2
3
of the appropriate bromothiophene together with 30% water in a Suzuki coupling reaction. The
reaction was left to reflux under argon for 3.5 hours. The mixture was concentrated in vacuo.
The residue was partitioned between 150 mls ethyl acetate and 50 mls brine solution and then the
aqueous layer was re-extracted using 3 portions of 100 mls ethyl acetate. The organic layers were
collected and the volume was reduced under reduced pressure. Afterwards the product was
purified by CC to yield compounds B, H.
4
.1.1.2 Procedure B, General procedure for the amide synthesis of compounds 1a, 3a - 8a,
E, 14.
0
.18 gm (0.7 mmole) of compound D was added to a mixture of 0.4 gm (1.05 mmol) of HBTU
(in compounds 1a, 3a-7a, E, 14) or 0.4 gm (1.05 mmol) HATU (in compounds 8a) and 0.36 gm
(1.05 mmol) of DIPEA together with 2.1 mmol of the appropriate acid in DCM. The mixture was
left to stir at room temperature overnight, and afterwards, the solvent was evaporated in vacuo
and the product was purified by CC.
4
.1.1.3 Procedure C, General procedure for amide alkylation (C– D)
0
.14 gm (3.5 mmol) KH was added gradually to stirred solution of 0.23 gm (0.35 mmol) of
compound 4a in 2 mls DMF under ice cooling. The reaction mixture was left to stir for one hour
and then 1 equiv of the appropriate alkyl iodide was added to the reaction vessel and the mixture
was left to stir for 2 days at room temperature. The mixture was partitioned between aqueous
brine solution and ethyl acetate layers and the aqueous layer was extracted by three 50-ml
18

ACCEPTED MANUSCRIPT
portions of ethyl acetate. The organic layers were collected and the solvent was removed under
vacuum, this was followed by purification of the desired products using CC.
4
.1.1.4 Procedure D, General procedure for synthesis of compounds F – G.
0
.27 gm (0.7 mmol) of compound E was added to a solution of 7 mmol of the appropriate
alicyclic amine dissolved in methanol. The reaction mixture was heated to refux for 1 hour.
Afterwards, the solvent was removed in vacuo and the produced residue was purified using CC.
4
.1.1.5 Procedure E, General procedure for synthesis of compounds 1b–8b, 9-10, 12-13,
5-19.
The bromo derivative was added to a suspension of 4 equiv of Na CO and 5 mmol% of
1
2
3
Pd(dppf)Cl2 in dioxane/water mixture. This was followed by the addition of the
pyridine/pyrazole boronic acid derivative. The reaction was heated to reflux for 2 hours under
argon atmosphere. The solvent was removed in vacuo. Small amount of brine solution was added
and extraction was done using ethyl acetate (3 x 50 mls). The ethyl acetate portions were
collected and the volume was reduced in vacuo. Afterwards the product was purified by CC
4
.1.1.6 Procedure F, General Procedure for pyrazole alkylation (compounds I, J, K)
To a solution of 4-pyrazoleboronic acid pinacol ester dissolved in acetone, 2 equiv of Cs CO
2
3
and 2 equiv of the appropriate alkyl iodide were added. The reaction mixture was left to reflux
overnight. The solvent was evaporated in vacuo and the product entered the following reaction
without further purification
4
.1.1.7 5-(4-Bromothiophen-2-yl)pyridin-3-amine (B). The compound was synthesized
1
according to Procedure A: yield 80%. The product was purified by CC (ethyl acetate); H NMR
(500 MHz, DMSO) δ 8.08 (d, J = 2.0 Hz, 1H), 7.91 (d, J = 2.5 Hz, 1H), 7.70 (d, J = 1.4 Hz, 1H),
19

ACCEPTED MANUSCRIPT
1
3
7
1
.51 (d, J = 1.5 Hz, 1H), 7.16 – 7.05 (m, 1H), 5.50 (s, 2H); C NMR (126 MHz, DMSO) δ
44.98, 142.31, 136.28, 133.61, 128.34, 126.16, 123.47, 115.68, 109.89; MS (ESI) m/z = 254.82
+
(M+H)
4
.1.1.8 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)-N-methylcyclopropanecarboxamide (C).
The compound was synthesized according to Procedure C using methyl iodide: yield 83%. The
1
product was purified by CC (DCM/MeOH 100:1.5); H NMR (500 MHz, DMSO) δ 8.86 (s, 1H),
8
3
.57 (d, J = 1.9 Hz, 1H), 8.19 (s, 1H), 7.83 (d, J = 1.4 Hz, 1H), 7.81 (d, J = 1.3 Hz, 1H), 3.34 (s,
1
3
H), 1.54 – 1.37 (m, 1H), 0.86 – 0.82 (m, 2H), 0.69 (t, J = 4.3 Hz, 2H); C NMR (126 MHz,
DMSO) δ 172.32, 147.61, 144.17, 140.69, 140.12, 130.89, 129.22, 127.99, 124.84, 110.20,
+
3
6.93, 12.39, 8.24; MS (ESI) m/z =336.91 (M+H)
4
.1.1.9 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)-N-ethylcyclopropanecarboxamide (D).
The compound was synthesized according to Procedure C using ethyl iodide: yield 79%. The
1
product was purified by CC (DCM/MeOH 100:1); H NMR (500 MHz, DMSO) δ 8.88 (s, 1H),
8
3
1
.51 (s, 1H), 8.15 (s, 1H), 7.83 (t, J = 1.8 Hz, 2H), 3.75 (s, 2H), 1.25 – 1.13 (m, 1H), 1.04 (t,
1
3
H), 0.88 – 0.77 (m, 2H), 0.66 (s, 2H); C NMR (126 MHz, DMSO) δ 171.69, 148.59, 144.64,
40.00, 139.00, 131.89, 129.40, 128.11, 124.86, 110.22, 43.53, 12.98, 12.62, 8.17; MS (ESI) m/z
+
=
350.89 (M+H)
4
.1.1.10 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)acetamide (1a). The compound was
synthesized according to procedure B using acetic acid: yield 98%.The product was purified by
1
CC (ethyl acetate); H NMR (500 MHz, DMSO) δ 10.30 (s, 1H), 8.64 (dd, J = 3.1, 2.4 Hz, 2H),
1
3
8
.29 (t, J = 2.2 Hz, 1H), 7.79 (d, J = 1.4 Hz, 1H), 7.66 (d, J = 1.4 Hz, 1H), 2.10 (s, 3H); C
20

ACCEPTED MANUSCRIPT
NMR (126 MHz, DMSO) δ 169.23, 141.17, 140.57, 140.02, 136.16, 128.35, 127.07, 124.41,
+
1
21.90, 110.23, 23.95; MS (ESI) m/z = 297.95 (M+H)
4
.1.1.11 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)methanesulfonamide (2a). The title
compound was synthesized through adding 1.4 mmol of the methanesulfonyl chloride to a stirred
solution of 0.18 gm (0.7 mmol) of compound B dissolved in pyridine. The reaction was heated to
6
0°C and left overnight. This was followed by the removal of solvent in vacuo: yield 69.1%. The
1
product was purified by CC (ethyl acetate); H NMR (500 MHz, DMSO) δ 10.16 (s, 1H), 8.68
(d, J = 2.0 Hz, 1H), 8.39 (d, J = 2.4 Hz, 1H), 7.81 (d, J = 1.4 Hz, 1H), 7.77 (t, J = 2.2 Hz, 1H),
1
3
7
1
.71 (d, J = 1.5 Hz, 1H), 3.13 (s, 3H); C NMR (126 MHz, DMSO) δ 141.43, 140.71, 135.35,
+
33.06, 128.82, 127.53, 124.65, 122.78, 110.19, 40.11; MS (ESI) m/z = 331.69 (M)
4
.1.1.12 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)propionamide (3a). The compound was
synthesized according to procedure B using propionic acid: yield 64%. The product was purified
1
by CC (ethyl acetate/petroleum ether 8:2); H NMR (500 MHz, DMSO) δ 10.36 (s, 1H), 8.69 (s,
1
H), 8.63 (s, 1H), 8.34 (t, J = 2.2 Hz, 1H), 7.79 (d, J = 1.4 Hz, 1H), 7.66 (d, J = 1.4 Hz, 1H), 2.39
+
(q, J = 7.5 Hz, 2H), 1.10 (t, J = 7.5 Hz, 3H).MS (ESI) m/z = 310.99 (M+H)
4
.1.1.13 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)cyclopropanecarboxamide (4a). The
compound was synthesized according to procedure B using cyclopropanecarboxylic acid: yield
1
9
4%. The product was purified by CC (ethyl acetate/petroleum ether 6:4); H NMR (500 MHz,
DMSO) δ 10.57 (d, J = 1.2 Hz, 1H), 8.66 (d, J = 2.3 Hz, 1H), 8.63 (d, J = 2.1 Hz, 1H), 8.32 (t, J
2.2 Hz, 1H), 7.79 (d, J = 1.4 Hz, 1H), 7.66 (d, J = 1.4 Hz, 1H), 1.80 (dd, J = 12.4, 6.3 Hz, 1H),
=
1
3
1
.46 (m, J = 21.2, 15.9, 10.8, 6.4 Hz, 2H), 1.30 – 1.11 (m, 2H); C NMR (126 MHz, DMSO) δ
21

ACCEPTED MANUSCRIPT
1
75.51, 141.14, 140.41, 139.93, 136.13, 128.31, 127.02, 124.33, 121.86, 110.16, 14.56, 7.65; MS
+
(ESI) m/z = 322.93 (M+H)
4
.1.1.14 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)cyclobutanecarboxamide (5a). The
compound was synthesized according to procedure B using cyclobutanecarboxylic acid: yield
1
9
4.6%. The product was purified by CC (ethyl acetate/petroleum ether 7:3); H NMR (400 MHz,
DMSO) δ 10.15 (s, 1H), 8.69 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.35 (s, 1H), 7.79 (d,
J = 1.1 Hz, 1H), 7.66 (d, J = 1.2 Hz, 1H), 3.09 – 2.99 (m, 1H), 1.10 – 1.00 (m, 2H), 0.97 (td, J =
1
3
7
1
.0, 1.7 Hz, 2H), 0.82 (dd, J = 15.2, 7.5 Hz, 2H); C NMR (101 MHz, DMSO) δ 173.82,
41.21, 140.44, 140.13, 136.26, 128.30, 127.04, 121.99, 116.94, 110.22, 24.59, 17.73; MS (ESI)
+
m/z = 337 (M+H)
4
.1.1.15 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)cyclohexanecarboxamide (6a). The
compound was synthesized according to procedure B using cyclohexanecarboxylic acid: yield
1
5
1%. The product was purified by CC (ethyl acetate/petroleum ether 6:4); H NMR (500 MHz,
DMSO) δ 10.17 (s, 1H), 8.67 (d, J = 2.3 Hz, 1H), 8.62 (d, J = 2.1 Hz, 1H), 8.35 (t, J = 2.2 Hz,
H), 7.78 (d, J = 1.4 Hz, 1H), 7.65 (d, J = 1.5 Hz, 1H), 2.36 (ddd, J = 11.6, 8.1, 3.5 Hz, 1H), 1.83
1
(d, J = 11.8 Hz, 2H), 1.80 – 1.73 (m, 2H), 1.41 (m, J = 12.4, 2.9 Hz, 2H), 1.34 – 1.11 (m, 4H);
1
3
C NMR (126 MHz, DMSO) δ 175.14, 141.21, 140.40, 140.07, 136.33, 128.28, 127.01, 124.34,
+
1
21.88, 110.19, 44.82, 29.01, 25.36, 25.16; MS (ESI) m/z = 364.97 (M+H)
4
.1.1.16 tert-Butyl 3-((5-(4-bromothiophen-2-yl)pyridin-3-yl)carbamoyl)pyrrolidine-1-
carboxylate (7a). The compound was synthesized according to procedure B using N-Boc-
pyrrolidine-3-carboxylic acid: yield 88%. The product was purified by CC (DCM/MeOH 100:3);
1
H NMR (500 MHz, DMSO) δ 10.40 (s, 1H), 8.66 (dd, J = 6.6, 2.2 Hz, 2H), 8.32 (t, J = 2.2 Hz,
22

ACCEPTED MANUSCRIPT
1
H), 7.79 (d, J = 1.4 Hz, 1H), 7.67 (d, J = 1.4 Hz, 1H), 3.03 (dd, J = 16.4, 8.6 Hz, 2H), 2.13 (d, J
1
3
=
2.9 Hz, 1H), 2.03 (s, 2H), 2.00 – 1.90 (m, 2H), 1.41 (s, 9H); C NMR (126 MHz, DMSO) δ
1
5
53.35, 141.06, 140.77, 140.20, 135.94, 132.94, 128.34, 127.11, 124.41, 122.19, 110.21, 78.34,
+
4.89, 53.55, 41.81, 28.16, 28.14; MS (ESI) m/z =453.84 (M+H)
4
.1.1.17 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)-2-(1H-tetrazol-5-yl)acetamide (8a). The
compound was synthesized according to procedure B using 1H-Tetrazole-5-acetic acid: yield
9
7%. The product was purified by extraction with 2 M NaOH and ethyl acetate (3 X 50 mls).
1
The aqueous layer was normalized using 1 M HCl and dried in vacuo; H NMR (500 MHz,
DMSO) δ 10.78 (s, 1H), 8.68 (d, J = 2.3 Hz, 1H), 8.61 (d, J = 2.1 Hz, 1H), 8.33 (t, J = 2.2 Hz,
1
3
1
H), 7.77 (d, J = 1.4 Hz, 1H), 7.64 (d, J = 1.4 Hz, 1H), 3.77 (s, 2H), 3.42 (s, 1H); C NMR (126
MHz, DMSO) δ 169.31, 155.55, 141.19, 140.54, 140.13, 136.22, 128.34, 127.08, 124.39, 121.97,
+
1
10.19, 34.83; MS (ESI) m/z = 364.90 (M+H)
4
.1.1.18 2-(Piperidin-1-yl)-N-(5-(4-(pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)acetamide (F).
The compound was synthesized according to procedure D using piperidine: yield 83.8%. The
1
product was purified by CC (ethyl acetate/MeOH 100:1); H NMR (500 MHz, DMSO) δ 9.81 (s,
1
H), 8.61 (d, J = 2.3 Hz, 1H), 8.46 (d, J = 2.1 Hz, 1H), 8.16 (t, J = 2.2 Hz, 1H), 7.61 (d, J = 1.4
Hz, 1H), 7.49 (d, J = 1.4 Hz, 1H), 2.94 (s, 2H), 2.35 – 2.30 (m, 4H), 1.39 (m, J = 11.1, 5.6 Hz,
1
3
4
1
H), 1.22 (d, J = 4.2 Hz, 2H); C NMR (126 MHz, DMSO) δ 164.59, 141.07, 140.84, 140.67,
35.52, 128.31, 127.14, 124.40, 122.64, 110.17, 62.55, 54.11, 25.32, 23.51; MS (ESI) = 379.96
+
(M+H)
4
.1.1.19 N-(5-(4-Bromothiophen-2-yl)pyridin-3-yl)-2-morpholinoacetamide (G). The
compound was synthesized according to procedure D using morpholine: yield 83.3%. The
23

ACCEPTED MANUSCRIPT
1
product was purified by CC (ethyl acetate /MeOH 100:3); H NMR (500 MHz, DMSO) δ 10.07
(s, 1H), 8.78 (d, J = 2.2 Hz, 1H), 8.66 (d, J = 2.1 Hz, 1H), 8.34 (t, J = 2.2 Hz, 1H), 7.79 (d, J =
1
4
1
.4 Hz, 1H), 7.68 (d, J = 1.4 Hz, 1H), 3.69 – 3.62 (m, 4H), 3.20 (d, J = 15.9 Hz, 2H), 2.52 (m,
1
3
H); C NMR (126 MHz, DMSO) δ 169.13, 164.60, 141.06, 140.91, 135.51, 128.31, 127.14,
+
24.42, 122.73, 110.19, 66.02, 61.95, 53.18; MS (ESI) m/z = 383.86 (M+H)
4
.1.1.20 5-(Thiophen-2-yl)pyridin-3-amine (H). The compound was synthesized according to
Procedure A using 2-bromothiophene: yield 89%.The product was purified by CC (ethyl
1
acetate/petroleum ether 9:1); H NMR (500 MHz, DMSO) δ 8.07 (d, J = 1.7 Hz, 1H), 7.87 (d, J
=
2.3 Hz, 1H), 7.57 (d, J = 5.0 Hz, 1H), 7.46 (d, J = 3.2 Hz, 1H), 7.20 – 7.09 (m, 2H), 5.47 (s,
1
3
2
1
H); C NMR (126 MHz, DMSO) δ 144.95, 140.69, 135.61, 133.80, 129.50, 128.47, 126.03,
24.03, 115.94.
4
.1.1.21 N-(5-(4-(Pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)acetamide (1b). The compound was
synthesized according to procedure E to give a brown solid: yield 13.8%. The product was
1
purified by CC (DCM/MeOH/TEA 100:5:1); mp151.3-153.2 °C; H NMR (500 MHz, DMSO) δ
1
0.50 (s, 1H), 9.06 (d, J = 1.7 Hz, 1H), 8.74 (d, J = 1.8 Hz, 1H), 8.69 (d, J = 2.0 Hz, 1H), 8.53
(d, J = 3.6 Hz, 1H), 8.38 (s, 1H), 8.19 (dd, J = 8.3, 4.6 Hz, 2H), 8.15 (s, 1H), 7.47 (dd, J = 7.9,
1
3
4
1
.8 Hz, 1H), 2.11 (s, 3H); C NMR (126 MHz, DMSO) δ 169.25, 148.41, 147.21, 140.87,
40.64, 139.70, 139.39, 136.23, 133.31, 130.43, 129.27, 123.94, 123.68, 122.93, 122.09, 23.93;
+
MS (ESI) m/z = 296.02 (M + H)
4
.1.1.22 N-(5-(4-(Pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)methanesulfonamide (2b). The
compound was synthesized according to procedure E to give a white solid: yield 45.4%. The
product was purified by extraction with water and ethyl acetate (3X50 mls) the organic layers
24

ACCEPTED MANUSCRIPT
1
were collected and the solvent was removed in vacuo; mp >280 °C; H NMR (500 MHz, DMSO)
δ 10.37 (s, 1H), 9.06 (s, 1H), 8.76 (d, J = 1.9 Hz, 1H), 8.53 (d, J = 3.4 Hz, 1H), 8.43 (d, J = 2.1
13
Hz, 1H), 8.21 (s, 2H), 8.17 (s, 1H), 7.89 (s, 1H), 7.47 (dd, J = 7.9, 4.6 Hz, 1H), 3.11 (s, 3H); C
NMR (126 MHz, DMSO) δ 148.40, 147.19, 141.33, 140.68, 140.40, 139.53, 139.36, 135.86,
+
1
33.30, 130.37, 129.70, 124.06, 123.90, 123.17; MS (ESI) m/z = 331.79 (M + H)
4
.1.1.23 N-(5-(4-(Pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)propionamide (3b). The compound
was synthesized according to procedure E to give a beige solid: yield 9.1%. The product was
1
purified by CC (DCM/MeOH 100:5); mp 211.6-212.4 °C; H NMR (500 MHz, DMSO) δ 10.21
(
s, 1H), 9.06 (d, J = 1.7 Hz, 1H), 8.73 (d, J = 2.1 Hz, 1H), 8.67 (d, J = 2.2 Hz, 1H), 8.53 (dd, J =
.7, 1.5 Hz, 1H), 8.39 (t, J = 2.2 Hz, 1H), 8.19 (ddd, J = 8.0, 2.3, 1.6 Hz, 1H), 8.17 (d, J = 1.5
Hz, 1H), 8.13 (d, J = 1.4 Hz, 1H), 7.47 (ddd, J = 7.9, 4.8, 0.8 Hz, 1H), 2.39 (q, J = 7.5 Hz, 2H),
4
1
3
1
1
.12 (t, J = 7.5 Hz, 3H); C NMR (126 MHz, DMSO) δ 172.79, 148.34, 147.17, 140.85, 140.53,
39.68, 139.36, 136.16, 133.24, 130.39, 129.23, 123.85, 123.61, 122.82, 122.07, 29.39, 9.37; MS
+
(ESI) m/z = 310.08 (M + H)
4
.1.1.24 N-(5-(4-(Pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)cyclopropanecarboxamide (4b).
The compound was synthesized according to procedure E to give a brick red solid: yield 8.5%.
1
The product was purified by CC (DCM/MeOH 100:5); mp 194-195.8 °C; H NMR (500 MHz,
DMSO) δ 10.58 (s, 1H), 9.06 (d, J = 1.9 Hz, 1H), 8.69 (dd, J = 37.5, 2.1 Hz, 2H), 8.52 (dd, J =
4
.7, 1.4 Hz, 1H), 8.39 (t, J = 2.2 Hz, 1H), 8.23 – 8.16 (m, 2H), 8.13 (d, J = 1.4 Hz, 1H), 7.47 (dd,
1
3
J = 7.9, 4.8 Hz, 1H), 1.85 – 1.78 (m, 1H), 0.95 – 0.79 (m, 4H); C NMR (126 MHz, DMSO) δ
1
1
72.53, 148.35, 147.15, 140.82, 140.52, 139.57, 139.35, 136.13, 133.24, 130.38, 129.26, 123.87,
+
23.63, 122.85, 122.03, 14.56, 7.56; MS (ESI) m/z = 321.93 (M + H)
25

ACCEPTED MANUSCRIPT
4
.1.1.25 N-(5-(4-(Pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)cyclobutanecarboxamide (5b). The
compound was synthesized according to procedure E to give an off-white solid: yield 17%. The
1
product was purified by CC (DCM/MeOH 100:4); mp 213-214.2 °C; H NMR (500 MHz,
DMSO) δ 10.09 (s, 1H), 9.06 (s, 1H), 8.73 (s, 1H), 8.68 (s, 1H), 8.53 (d, J = 4.0 Hz, 1H), 8.41 (s,
1
2
H), 8.20 (d, J = 8.7 Hz, 2H), 8.14 (s, 1H), 7.47 (dd, J = 7.7, 4.7 Hz, 1H), 3.31 – 3.22 (m, 1H),
.32 – 2.19 (m, 2H), 2.14 (d, J = 8.6 Hz, 2H), 1.96 (dd, J = 19.0, 9.3 Hz, 1H), 1.83 (d, J = 9.8
1
3
Hz, 1H); C NMR (126 MHz, DMSO) δ173.76, 148.38, 147.20, 140.87, 140.57, 139.78, 139.38,
1
36.21, 133.27, 130.42, 129.24, 123.89, 123.65, 122.86, 122.16, 24.58, 17.71; MS (ESI) m/z
+
=
336.08 (M + H)
4
.1.1.26 N-(5-(4-(Pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)cyclohexanecarboxamide (6b). The
compound was synthesized according to procedure E to give a beige solid: yield 14%. The
1
product was purified by CC (DCM/MeOH 100:3); mp 182-184 °C; H NMR (500 MHz, DMSO)
δ 10.17 (s, 1H), 9.06 (s, 1H), 8.72 (s, 1H), 8.67 (s, 1H), 8.53 (d, J = 3.7 Hz, 1H), 8.42 (s, 1H),
8
1
1
1
1
.23 – 8.15 (m, 2H), 8.13 (s, 1H), 7.53 – 7.42 (m, 1H), 2.38 (t, J = 11.4 Hz, 1H), 1.85 (d, J =
2.2 Hz, 2H), 1.77 (d, J = 11.9 Hz, 2H), 1.66 (d, J = 12.0 Hz, 1H), 1.43 (d, J = 12.2 Hz, 2H),
1
3
.26 (dd, J = 23.2, 10.8 Hz, 2H), 1.22 – 1.16 (m, 1H) C NMR (126 MHz, DMSO) δ 175.08,
48.33, 147.16, 140.87, 140.49, 139.70, 139.35, 136.30, 133.25, 130.40, 129.20, 123.86, 123.59,
+
22.82, 122.03, 44.79, 29.00, 25.35, 25.15; MS (ESI) m/z = 364.03 (M + H)
4
1
.1.1.27 tert-Butyl 3-((5-(4-(pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)carbamoyl)pyrrolidine-
-carboxylate (7b). The compound was synthesized according to procedure E to give a yellow
1
semi-solid: yield 34%. The product was purified by CC (DCM/MeOH/ TEA 100:4:1); H NMR
(500 MHz, DMSO) δ 10.49 (s, 1H), 9.08 (s, 1H), 8.73 (dd, J = 32.1, 1.9 Hz, 2H), 8.55 (d, J = 3.5
Hz, 1H), 8.41 (t, J = 2.1 Hz, 1H), 8.27 – 8.21 (m, 1H), 8.20 (d, J = 1.4 Hz, 1H), 8.16 (d, J = 1.4
26

ACCEPTED MANUSCRIPT
Hz, 1H), 7.51 (dd, J = 7.9, 4.8 Hz, 1H), 3.54 (dd, J = 20.8, 11.7 Hz, 2H), 3.25 – 3.16 (m, 2H),
1
3
2
.10 (dd, J = 29.5, 14.7 Hz, 2H), 1.41 (s, 9H), 1.23 (d, J = 1.2 Hz, 1H); C NMR (126 MHz,
DMSO) δ 177.88, 153.34, 147.97, 146.77, 140.80, 139.80, 139.22, 133.72, 130.57, 129.47,
1
29.15, 128.07, 124.07, 123.74, 123.10, 122.35, 78.38, 61.17, 45.66, 45.59, 28.20, 28.17; MS
+
(ESI) m/z = 451.35 (M + H)
4
.1.1.28
N-(5-(4-(Pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)-2-(1H-tetrazol-5-yl)acetamide
(8b). The compound was synthesized according to procedure E to give an off-white solid: yield
1
5
5.4%. The product was purified by CC (DCM/MeOH/TEA 100:11:3); mp 119.2-122.7 °C; H
NMR (500 MHz, DMSO) δ 10.80 (s, 1H), 9.14 – 8.99 (m, 1H), 8.73 (d, J = 2.1 Hz, 1H), 8.69 (d,
J = 2.2 Hz, 1H), 8.52 (dd, J = 4.7, 1.6 Hz, 1H), 8.40 (t, J = 2.2 Hz, 1H), 8.22 – 8.16 (m, 2H),
1
3
8
.14 (d, J = 1.4 Hz, 1H), 7.50 – 7.43 (m, 1H), 5.32 (s, 1H), 3.86 (s, 2H); C NMR (126 MHz,
DMSO) δ 172.10, 168.73, 148.35, 147.19, 140.80, 140.73, 139.73, 139.37, 136.12, 133.28,
+
1
30.42, 129.27, 123.89, 123.71, 122.89, 122.10, 34.34; MS (ESI) m/z = 364.02 (M + H)
4
.1.1.29
N-Methyl-N-(5-(4-(pyridin-3-yl)thiophen-2-yl)pyridin-3-
yl)cyclopropanecarboxamide (9). The compound was synthesized according to procedure E to
give brown oil: yield 15.6%. The product was purified by CC (DCM/MeOH 100:2); mp 142-
1
1
42.7 °C; H NMR (500 MHz, DMSO) δ 9.07 (s, 1H), 8.93 (s, 1H), 8.57 (s, 1H), 8.53 (d, J = 3.7
Hz, 1H), 8.34 (s, 1H), 8.25 (s, 1H), 8.20 – 8.16 (m, 2H), 7.48 (dd, J = 7.9, 4.8 Hz, 1H), 3.31 (s,
1
3
3
1
1
H), 1.22 (m, 1H), 0.89 – 0.84 (m, 2H), 0.70 (m, 2H); C NMR (126 MHz, DMSO) δ 172.30,
49.12, 148.40, 147.76, 147.14, 140.71, 139.67, 139.28, 134.40, 133.18, 130.32, 124.67, 123.96,
+
23.89, 123.31, 36.97, 12.35, 8.20; MS (ESI) m/z = 336.03 (M + H)
27

ACCEPTED MANUSCRIPT
4
.1.1.30
N-Ethyl-N-(5-(4-(pyridin-3-yl)thiophen-2-yl)pyridin-3-
yl)cyclopropanecarboxamide (10). The compound was synthesized according to procedure E to
give a beige solid: yield 21.2%. The product was purified by CC (DCM/MeOH 100:3); mp 140-
1
1
2
3
2
1
42.2 °C; H NMR (500 MHz, DMSO) δ 9.07 (d, J = 1.9 Hz, 1H), 8.94 (s, 1H), 8.58 – 8.44 (m,
H), 8.36 (s, 1H), 8.24 – 8.19 (m, 2H), 8.18 (d, J = 1.3 Hz, 1H), 7.48 (dd, J = 7.8, 4.8 Hz, 1H),
.78 (q, J = 6.3 Hz, 2H), 1.30 (m, 1H), 1.07 (t, J = 6.6 Hz, 3H), 0.88 – 0.81 (m, 2H), 0.67 (m,
1
3
H); C NMR (126 MHz, DMSO) δ 171.81, 148.40, 148.17, 147.16, 144.66, 144.45, 139.55,
39.29, 139.02, 133.18, 131.82, 130.30, 124.79, 123.87, 123.32, 43.57, 12.99, 12.63, 8.15; MS
+
(ESI) m/z =350.05 (M + H)
4
.1.1.31 N-(5-(4-(Pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)pyrrolidine-3-carboxamide (11).
The title compound was prepared by adding compound 7b to a mixture of TFA and DCM. The
reaction mixture was stirred overnight at room temperature and then the solvent was removed in
1
vacuo and purified by crystallization from ethanol to give yellow oil: yield 23.6%. H NMR (500
MHz, MeOD)δ 8.97 (s, 1H), 8.66 (dd, J = 7.3, 1.8 Hz, 2H), 8.52 (d, J = 4.4 Hz, 1H), 8.47 (t, J =
2
5
3
.2 Hz, 1H), 8.36 – 8.26 (m, 1H), 8.05 (s, 1H), 7.97 (dd, J = 3.7, 1.5 Hz, 2H), 7.61 (dd, J = 7.9,
.1 Hz, 1H), 3.66 (dd, J = 11.7, 5.0 Hz, 1H), 3.49 (dd, J = 11.7, 7.7 Hz, 1H), 3.45 – 3.35 (m,
1
3
H), 2.50 – 2.35 (m, 1H), 2.29 (m, J = 13.2, 5.9 Hz, 1H), 1.27 (dd, J = 14.8, 7.8 Hz, 1H); C
NMR (126 MHz, MeOD) δ 173.24, 147.11, 146.14, 142.42, 142.07, 140.58, 140.04, 137.55,
1
37.47, 133.83, 132.00, 126.28, 125.31, 124.99, 124.79, 48.76, 46.56, 44.80, 30.38.; MS (ESI)
+
m/z = 350.93 (M + H)
4
.1.1.32 2-(Piperidin-1-yl)-N-(5-(4-(pyridin-3-yl)thiophen-2-yl)pyridin-3-yl)acetamide (12).
The compound was synthesized according to procedure E to give brick red oil: yield 17.5%. The
1
product was purified by CC (DCM/MeOH 100:3); H NMR (500 MHz, DMSO) δ 11.10 (s, 1H),
28