Synthesis and in vitro evaluation of diverse heterocyclic diphenolic compounds as inhibitors of DYRK1A

Article abstract of DOI:10.1016/j.bmc.2018.10.034

Dual-specificity tyrosine phosphorylation-related kinase 1A (DYRK1A) is a dual-specificity protein kinase that catalyses phosphorylation and autophosphorylation. Higher DYRK1A expression correlates with cancer, in particular glioblastoma present within the brain. We report here the synthesis and biological evaluation of new heterocyclic diphenolic derivatives designed as novel DYRK1A inhibitors. The generation of these heterocycles such as benzimidazole, imidazole, naphthyridine, pyrazole-pyridines, bipyridine, and triazolopyrazines was made based on the structural modification of the lead DANDY and tested for their ability to inhibit DYRK1A. None of these derivatives showed significant DYRK1A inhibition but provide valuable knowledge around the importance of the 7-azaindole moiety. These data will be of use for developing further structure-activity relationship studies to improve the selective inhibition of DYRK1A.

Full text of DOI:10.1016/j.bmc.2018.10.034

Accepted Manuscript
Synthesis and in vitro evaluation of diverse heterocyclic diphenolic compounds
as inhibitors of DYRK1A
Qingqing Zhou, Tristan A. Reekie, Ramzi H. Abbassi, Dinesh Indurthi Venkata,
Josep S. Font, Renae M. Ryan, Lenka Munoz, Michael Kassiou
PII:
S0968-0896(18)31456-1
https://doi.org/10.1016/j.bmc.2018.10.034
BMC 14593
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
17 August 2018
19 October 2018
27 October 2018
Please cite this article as: Zhou, Q., Reekie, T.A., Abbassi, R.H., Indurthi Venkata, D., Font, J.S., Ryan, R.M.,
Munoz, L., Kassiou, M., Synthesis and in vitro evaluation of diverse heterocyclic diphenolic compounds as inhibitors
of DYRK1A, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.034
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Synthesis and in vitro evaluation of diverse
heterocyclic diphenolic compounds as inhibitors of
DYRK1A
1
1
2
2
Qingqing Zhou , Tristan A. Reekie , Ramzi H. Abbassi , Dinesh Indurthi Venkata ,
3
3
2
1*
Josep S. Font , Renae M. Ryan , Lenka Munoz and Michael Kassiou
1School of Chemistry, The University of Sydney, New South Wales 2006, Australia
2School of Medical Sciences, Discipline of Pathology and Charles Perkins Centre,
The University of Sydney, New South Wales 2006, Australia
3School of Medical Sciences, Discipline of Pharmacology, The University of Sydney,
New South Wales 2006, Australia
*
Author to whom correspondence should be addressed.
e-mail: michael.kassiou@sydney.edu.au
1

ABSTRACT
Dual-specificity tyrosine phosphorylation-related kinase 1A (DYRK1A) is a dual-
specificity protein kinase that catalyses phosphorylation and autophosphorylation.
Higher DYRK1A expression correlates with cancer, in particular glioblastoma
present within the brain. We report here the synthesis and biological evaluation
of new heterocyclic diphenolic derivatives designed as novel DYRK1A inhibitors.
The generation of these heterocycles such as benzimidazole, imidazole,
naphthyridine, pyrazole-pyridines, bipyridine, and triazolopyrazines was made based
on the structural modification of the lead DANDY and tested for their ability to
inhibit DYRK1A. None of these derivatives showed significant DYRK1A inhibition
but provide valuable knowledge around the importance of the 7-azaindole moiety.
These data will be of use for developing further structure-activity relationship studies
to improve the selective inhibition of DYRK1A.
2

INTRODUCTION
Protein kinases catalyse protein phosphorylation and are therefore linked to the
occurrence of multiple diseases including neurodegenerative disorders and cancer.1, 2
Inhibitors targeting protein kinases are regarded as potential new therapeutic agents
for researchers.3, 4 DYRK1A (dual-specificity tyrosine phosphorylation-related kinase
1
A) is a dual-specificity protein kinase that catalyses not only autophosphorylation on
its own tyrosine residue, but the phosphorylation of serine and threonine residues of
5
its substrates. The DYRK1A gene is the only DYRK member (others including
DYRK1B, DYRK2, DYRK3 and DYRK4) located on 21q22.2 of human
6
chromosome 21, which encodes more than 300 annotated genes, one third of which
7
are overexpressed in Down syndrome (DS). Recent studies indicated that the
DYRK1A expression level is correlated with cancer. For example, an upregulation of
over 30% of DYRK1A levels has been found in glioblastoma cells, a cancer that
8
represents approximately 15% of brain tumours. It has been suggested that DYRK1A
9
inhibition by DYRK1A inhibitors leads to cancer cell apoptosis. The development of
novel DYRK1A inhibitors has consequently attracted considerable interest as new
chemotherapeutic agents.
There are several compounds with inhibitory activity for DYRK1A. That have either
1
0
11
been isolated from natural sources, with examples including harmine, variolins ,
meriolins12 and staurosporine.13,
14
All of these natural leads possess an
indolocarbazole ring system. Purely synthetic inhibitors also exist, though they
possess very similar structural features namely with the heterocyclic cores, which
include
thiazoloquinazoline,18 pyrrolopyrimidines.9, 19 Recently discovered lead compounds
imidazopyridine,15
imidazopyridazine,16
pyridoquinazoline,17
1
9
DANDY (1) represent one chemotype of the most potent DYRK1A inhibitors. That
study showed that variable hydroxy substitution gave potent compounds, including
mono substitution (1a, IC = 23.1 nM) and dihydroxylation (1b, IC = 3.0 nM)
5
0
50
9
among others. In our previous studies, we were able to improve potency without
increasing phenyl substitution and thus generate a molecule that was more likely to
cross the blood-brain barrier. Our previous studies also suggested that the N-C-N
moiety of DANDY plays an important role in maintaining DYRK1A inhibition.9, 20 It
is still unclear if other heterocyclic cores with a similar arrangement are tolerated, or
potentially provide a more potent scaffold. Herein, we present the synthesis of a new
3

and diverse group of heterocyclic compounds. They correspond to the 7-azaindole
DANDY by maintaining the characteristic diphenol moieties and dinitrogen
functionality (shown in pink in Figure 1). Different spatial arrangements of two
nitrogen atoms were studied by designing heterocycles such as benzimidazole-2-
amine 2, 2-acetamide 3, imidazole 4, naphthyridine 5, pyrazole-pyridines 6 and 7,
bipyridine 8, and triazolopyrazines 9 and 10. Additionally, the 7-membered analogue
1
1 with an intrinsic diphenol has also been investigated. We decided to maintain
single hydroxy substitution on the phenyl groups to improve drug-like properties and
reduce toxicity associated with oxidation to the quinone. With these novel analogues
in hand, we then explored their inhibitory activity against DYRK1A.
Figure 1. Generalised heterocyclic cores based on 7-azaindole core of DANDY.
RESULTS & DISCUSSION
We synthesised benzimidazole-2-amine 2 and benzimidazole-2-acetamide 3 starting
from commercially available 2-fluoroaniline (12) (Scheme 1). The selectively
brominated intermediate 13 was achieved by treatment with N-bromosuccinimide
(
NBS) in chloroform in 90% yield. The aniline 13 was then oxidised to nitro
compound 14 in the presence of hydrogen peroxide in triflouroacetic acid (TFA) at 75
C for 2 h. It was planned that the subsequent nucleophilic substitution could be

achieved by treatment with tert-butyldimethylsilyl ether (OTBS)-aniline 15a under
basic conditions. Unexpectedly, the desired product 16a was not obtained, and the by-
product 16b was produced instead. We proposed that the silicon atom in the TBS
protecting group favoured bond formation with the fluorine atom of 14, resulting in
4

the formation of a four-membered transition state (Scheme 1), and eventually
producing ether 16b.
Scheme 1. Reagents and conditions: (a) NBS (1.0 equiv.), CHCl , RT, 2 h, 90%; (b)
3
aq. H O (30%, 5.0 equiv.), TFA, 75 C, 2 h, 75%; (c) 15a (1.1 equiv.), K CO (1.5
2
2
2
3
equiv.), DMF, 90 C, 3 h, 80%.
To avoid the formation of 16b, the compound 14 was treated with 4-aminophenol and
potassium carbonate in anhydrous dimethylformamide at 90 C (Scheme 2), and the
desired amine 16c was successfully obtained in moderate yield. Nitro reduction to 17
by iron powder in acetic acid was realised at room temperature in excellent yield.
Cyclisation to intermediate 18 was achieved in the presence of cyanogen bromide to
2
1
proceed in 78% yield at room temperature. The 2-aminobenzimidazole analogue 2
was achieved using a standard Suzuki coupling reaction, followed by acetylation to
afford the 2-acetamide benzimidazole analogue 3 in moderate 65% yield (Scheme 2).
5

Scheme 2. Reagents and conditions: (a) 4-aminophenol (1.1 equiv.), K CO (1.5
2
3
equiv.), anhydrous DMF, 90 C, 3 h, 65%; (b) Fe (5.0 equiv.), AcOH, RT, 2 h, 95%;
c) BrCN (5 M in CH CN, 1.2 equiv.), MeCN-H O, 3:1 (v/v), RT, 16 h, 78%; (d) (4-
(
3
2
hydroxyphenyl)boronic acid (1.5 equiv.), Pd(PPh ) (2 mol%), aq. K CO (2 M, 3.0
3
4
2
3
equiv.), 1,4-dioxane, 110 C, 4 h, 30%; (e) Ac O (1.1 equiv.), AcOH, 110 C, 3 h,
2
6
5%.
Imidazole-based analogue 4 could be easily obtained from imidazole 19 first by
treatment with iodine in the presence of potassium iodide and aqueous sodium
hydroxide at room temperature. The regioselective diiodinated intermediate 20 was
2
2
acquired in excellent yield (Scheme 3). The diarylation of 20 was achieved through
a double-Suzuki coupling reaction. The conditions described previously by using
potassium carbonate as a base were initially trialled, but failed to afford the desired
product 4. We assumed that the strong basicity of imidazole combined with the
addition of potassium carbonate increased the pH to a point that was detrimental to
the reaction.23 The use of milder base dipotassium phosphate and the addition of
phase transfer catalyst tetra-n-butylammonium bromide (TBAB) afforded analogue 4
in 35% yield.
Scheme 3. Reagents and conditions: (a) NaOH (4 M), KI (5.0 equiv.), I (2.2 equiv.),
2
H O, RT, 10 h, 80%; (b) (4-hydroxyphenyl)boronic acid (3.0 equiv.), Pd(PPh ) (6
2
3 4
mol%), K HPO (3.8 equiv.), TBAB (0.1 equiv.), MeOH-H O, 4:1 (v/v), MW, 100
2
4
2

C, 60 min, 35%.
Analogue 5 was achieved through known synthetic routes.24 The regioselective
bromination of 2-aminonicotinate 21 gave the 3-bromopyridine 22 in 90% yield,25
and subsequent esterification under classical conditions furnished 23 in excellent
yield (Scheme 4). The cyclisation of 23 was achieved by treatment with N,N-
dimethylformamide dimethyl acetal (DMF-DMA) in a sealed tube at high
6

temperatures to afford the imine intermediate, followed by the treatment with n-BuLi
and MeCN at 78 C and subsequent addition of acetic acid to give 24 in 35% yield
in a one pot reaction sequence. Decyanation of 24 was performed by treatment with
concentrated hydrochloric acid at 150 C in a sealed tube to give compound 25 in
3
3% yield after neutralisation. Given that triflates act as pseudohalides in coupling
2
6
reactions, we initially attempted the triflation of the hydroxy group on compound 25
not shown here). However, the triflate intermediate appeared unstable in this
(
heterocyclic system as observed by TLC monitoring. Instead, chlorination was
achieved by treatment with phosphoryl chloride at 105 C in a sealed tube to give
chloro-substituted intermediate 26. Subsequent Suzuki coupling was achieved using
the more reactive [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride
catalyst and microwave irradiation at 130 C. Thus, analogue 5 could be obtained in
good overall yield.
Scheme 4. Reagents and conditions: (a) Br (1.3 equiv.), AcOH, RT, 20 h, 90%; (b)
2
H SO (8.0 equiv.), MeOH, 80 C, 18 h, 94%. (c) (1) DMF-DMA (10.0 equiv.), 110
2
4

C, 3 h; (2) n-BuLi (2.2 equiv.), MeCN (2.2 equiv.), THF, –78 C 1 h; (3) AcOH (3.0
equiv.), –40 C, 2 h, then RT, 20 h, 35% over three steps; (d) aq. HCl (32%), 150 C,
h, 33%; (e) POCl , 105 C, 3 h, 93%; (f) (4-hydroxyphenyl)boronic acid (2.5
3
3
equiv.), PdCl (dppf) (20 mol%), aq. K CO (2 M, 6.0 equiv.), 1,4-dioxane, MW, 130
2
2
3

C, 3 h, 85%. dppf = 1,1′-ferrocenediyl-bis(diphenylphosphine).
For the synthesis of 6, we initially elected to use benzaldehyde 28a as the reactant by
treatment with 27a in the presence of lithium diisopropylamide at 78 C to proceed
7

intermediate 29a. Unfortunately, the reaction was sluggish and yielded a complex
mixture of products after stirring for another 12 h at room temperature (Scheme 5).
We assumed that the HOMO of the nucleophile 27a and the LUMO of the
electrophile 28a might not match with each other due to the electronic effects from
the aromatic systems. Therefore, we decided to swap the functional groups of ketone
2
7a and aldehyde 28a to aldehyde 27b and ketone 28b, respectively (Scheme 5). As
predicted, the desired aldol product 29b was successfully achieved in 78% yield by
treatment with lithium diisopropylamide in a similar protocol as described above.
With the intermediate 29b available, subsequent oxidation with Dess-Martin
periodinane (DMP) was employed to furnish 1,3-dione 30 in 95% yield (Scheme 5).
The formation of pyrazole intermediate 31 was achieved in 75% yield by treatment
2
7
with hydrazine monohydrate in methanol at room temperature. No reaction occurred
when this substrate was treated with (4-hydroxyphenyl)boronic acid under standard
Suzuki coupling conditions. A proposed explanation for this lies in the formation of a
complex between the paladium catalyst and pyridine-N and pyrazole moieties (a
typical bidentate coordination site), thus reducing the catalytic reactivity of the
palladium catalyst in the reaction. Therefore, the pyrazole-N was protected with the
tosyl group and the resulting 32 was treated with (4-hydroxyphenyl)boronic acid
under the Suzuki coupling conditions, which finally afforded aryl-substituted
compound 33 in 85% yield. Removal of the tosyl and benzyl protecting groups could
be easily achieved in two sequential steps, and analogue 6 was eventually furnished in
reasonable yield.
8

Scheme 5. Reagents and conditions: aldehyde (1.0 equiv.), ketone (1.05 equiv.), LDA
1.1 equiv.), THF, 78 C to RT, 1 h, 78%; (b) DMP (1.5 equiv.), CH Cl , RT, 30
(
2
2
min, 95%; (c) N H H O (2.0 equiv.), MeOH, RT, 48 h, 75%; (d) TsCl (1.1 equiv.),
2
4
2
NaH (60% in mineral oil, 2.0 equiv.), CH Cl , 0 CRT, 30 min, 95%; (e) (4-
2
2
hydroxyphenyl)boronic acid (1.5 equiv.), Pd(PPh ) (2 mol%), aq. K CO (2 M, 3.0
3
4
2
3
equiv.), 1,4-dioxane, 100 C, 4 h, 85%; (f) 1) KOH (5.0 equiv.), MeOH, 75 C, 1 h; 2)
Pd/C (10 wt. %), H , MeOH, RT, 5 h, 45% over two steps.
2
The pyrazole-pyridine based analogue 7, with the phenol substitution at the C-4
position was also investigated (Scheme 6). In a similar manner to that of analogue 6,
compound 7 could be derived from the cyclisation of a 1,3-dione, which could be
originally achieved from the aldol condensation between aldehyde and ketone with
the optimised conditions described above. 2-Amino-4-bromopyridine (34) was then
chosen as the starting material which was first coupled with boronic acid 35 to afford
4
-aryl intermediate 36. Iodination at the C-2 position of 36 could be furnished by a
Sandmeyer reaction using diiodomethane as the solvent and halogen source with
2
8
catalytic hydrogen iodide to give the desired compound 37 in 25% yield. The 2-
aldehyde containing compound 38 was furnished by the lithium-halogen exchange
9

and quenching with dimethylformamide in a moderate 50% yield. The subsequent
aldol condensation with 28b gave the aldol product 39 in 60% yield. Oxidation by
DMP and subsequent pyrazole formation yielded intermediate 41 and the two
sequential deprotection steps eventually afforded analogue 7.
Scheme 6. Reagents and conditions: (a) 35 (1.5 equiv.), Pd(PPh ) (2 mol%),
3
4
aq. K CO (2 M, 3.0 equiv.), 1,4-dioxane, 100 C, 4 h, 87%; (b) isopentyl nitrite (20.0
2
3
equiv.), aq. HI (57%, 2 mol%), CH I , RT, 24 h, 25%; (c) n-BuLi (1.1 equiv.), DMF
2
2
(1.5 equiv.), toluene, 78 C, 1 h, 50%; (d) 28b (1.05 equiv.), LDA (1.1 equiv.), THF,

78 C to RT, 1 h, 60%. (e) DMP (1.5 equiv.), CH Cl , RT, 30 min, 75%; (f)
2
2
N H H O (2.0 equiv.), MeOH, RT, 48 h, 70%; (g) 1) Pd/C (10 wt. %), H , MeOH,
2
4
2
2
RT, 5 h; 2) TBAF (2.0 equiv.), THF, RT, 30 min, 55% over two steps.
2
9
Analogue 8 is a known compound reported by Constable and co-workers. Using
,2'-bipyridine 42 as the starting material, it was first treated with hydrogen peroxide
2
in acetic acid to achieve N-oxide 43 in high yield. The dinitro-substitution at the para-
positions of 43 was achieved by treatment with a mixture of sulfuric acid and nitric
acid at 100 C (Scheme 7). The low yield of 20% after 20 h of heating presumably
resulted from the use of the normal nitric acid instead of fuming nitric acid as
previously reported.30 The following chloro-substitution could be achieved by
treatment with acetyl chloride in acetic acid at 100 C to afford 45 in good yield.30
Reduction of N-oxide 45 was performed in the presence of phosphorus trichloride to
1
0

give 46 in excellent yield, which was further coupled with (4-hydroxyphenyl)boronic
acid to furnish the desired compound 8 in moderate overall yield.30
Scheme 7. Reagents and conditions: (a) aq. H O (30%, 5.0 equiv.), AcOH, RT to
2
2
7
0 C, 15 h; (b) H SO (21.0 equiv.), HNO (8.4 equiv.), 100 C, 20 h, 20%; (c)
2
4
3
acetyl chloride (5.0 equiv.), AcOH, 100 C, 12 h, 85%. (d) PCl (20.0 equiv.), 65 C,
3
2
0 h, 95%; (e) (4-hydroxyphenyl)boronic acid (2.5 equiv.), PdCl (dppf) (5 mol%), aq.
2
K CO (2 M, 5.0 equiv.), 1,4-dioxane, 110 C, 4 h, 40%.
2
3
The next core under investigation was triazolopyrazine. Considering the spatial
arrangements of diphenol substitutions on lead compound 1, we designed
triazolopyrazine analogues with linkers between the phenols and triazolopyrazine core
to give the optimal structural arrangements. Therefore, analogues 9a and 10a were
proposed, in addition to meta-hydroxy-substituted 9b and 10b, in order to investigate
more diversified analogues (see as Schemes 8 and 9, respectively).
The synthesis of triazolopyrazine analogues 9ab could be achieved from the
commercially available 2,6-dichloropyrazine 47 shown in Scheme 8. The formation
of hydrazone 48 was achieved in two sequential steps. Firstly, treatment of 47 with
hydrazine monohydrate in ethanol gave the hydrazine intermediate, which was then
reacted with 4-(benzyloxy)benzaldehyde 28a to give hydrazone 48 in 70% yield over
two steps.31 The oxidative heterocyclisation of hydrazone 48 to triazolopyrazine
intermediate 49 was easily achieved using (diacetoxyiodo)benzene in
3
2
dichloromethane at room temperature in excellent yield. The following nucleophilic
substitution reactions with alcohols 50ab, using 18-crown-6 and potassium
1
1

hydroxide, could be achieved to give ethers 51ab in moderate yields (69–73%).
Hydrogenolysis of 51ab then gave the desired compounds 9ab in reasonable yields
(Scheme 8).
Scheme 8. Reagents and conditions: (a) N H H O (1.1 equiv.), ethanol, 80 C, 8 h;
2
4
2
(b) 4-(benzyloxy)benzaldehyde 28a (1.1 equiv.), ethanol, 80 C, 10 h, 70% over two
steps. (c) PhI(OAc) (1.0 equiv.), CH Cl , RT, 12 h, 85%; (d) 50a or 50b (1.1 equiv.),
2
2
2
KOH (3.0 equiv.), 18-crown-6 (0.07 equiv.), toluene, 40 C, 4 h, 6973%; (e) Pd/C
10 wt. %), H , MeOH, RT, 2 h, 6770%.
(
2
The synthesis of triazolopyrazine analogues 10ab could be achieved in a similar
manner as that described above (Scheme 9). Pyrazine-2-carboxylic acid 52 was first
oxidised to give N-oxide intermediate as reported previously.33 The subsequent
chlorination34 was achieved by treatment with phosphoryl chloride at high
temperature to give the chloro-intermediate 53 in 70% yield over two steps. Amide
formation was performed via the acetyl chloride intermediate, followed by the
treatment with para- or meta-OTBS aniline 15ab and triethylamine in anhydrous
dichloromethane to give the amides 54ab in good yields (80–85%). The following
reactions were performed in a similar manner to those described in Scheme 8,
hydrazones 56ab were obtained in 78–85% yields over two steps and further
1
2

converted to the cyclised compounds 57ab in good yields. The deprotection of the
TBS groups gave the desired compounds 10ab in excellent yields.35
Scheme 9. Reagents and conditions: (a) (1) Na WO 2H O (0.025 equiv.), aq. H O
2
4
2
2
2
(
30%, 1.1 equiv.), H SO (1 M), pH = 2, RT to 80 C, 2 h; (2) POCl , 120 C, 2 h,
2
4
3
7
0
0% over two steps; (b) (1) oxalyl chloride (1.5 equiv.), DMF (0.1 equiv.), CH Cl ,
2 2
C to RT, 2 h; (2) 15a or 15b (1.0 equiv.), triethylamine (1.1 equiv.), CH Cl , RT, 2
2
2
h, 8085% over two steps; (c) N H H O (1.1 equiv.), ethanol, 80 C, 8 h; (d) 55 (1.1
2
4
2
equiv.), ethanol, 80 C, 10 h, 7885% over two steps. (e) PhI(OAc) (1.0 equiv.),
2
CH Cl , RT, 12 h, 8087%; (f) TBAF (2.0 equiv.), THF, RT, 30 min, 9597%.
2
2
The synthesis of 7-membered analogue 11 could be achieved from commercially
available starting material 58. Nucleophilic substitution with 4-methoxy-2-
nitroaniline was unsuccessful under basic conditions at high temperatures (Scheme
1
0). However, a Buchwald-Hartwig cross coupling reaction could be utilised by first
protecting the aldehyde 58 as the dioxolane 59, then coupling with 4-methoxy-2-
nitroaniline to obtain the desired secondary amine 60. The following acetal
deprotection was easily carried out by treatment with trifluoroacetic acid in
chloroform to give the desired intermediate 61 in 90% yield. Compound 62 could be
obtained from 61 in one pot via a cascade process. Initial reduction of the nitro moiety
to the aniline was followed by intramolecular condensation with the aldehyde.
1
3

Subsequent imine reduction in the presence of hydrogen gave 62.36, 37 Finally, the 7-
membered analogue 11 was successfully obtained after demethylation under classical
conditions in reasonable yield.
Scheme 10. Reagents and conditions: (a) NaH (60% in mineral oil, 1.5 equiv.), 4-
methoxy-2-nitroaniline (1.0 equiv.), THF, 0 to 70 C, 24 h; (b) TsOH (0.01 equiv.),
ethane-1,2-diol (5.0 equiv.), toluene, 125 C, 24 h, 95%; (c) 4-methoxy-2-nitroaniline
(
1.0 equiv.), Pd(OAc) (10 mol%), Cs CO (1.5 equiv.), toluene, 120 C, 3 h, 89%;
2
2
3
(
d) TFA (6.0 equiv.), CHCl , RT, 30 min, 90%; (e) Pd/C (10 wt. %), H , MeOH, RT,
3
2
4
h, 75%; (f) BBr (6.0 equiv.), CH Cl , RT, 10 h, 50%; TsOH = p-toluenesulfonic
3 2 2
acid.
With the completed synthesis of these derivatives, their potency for DYRK1A
inhibition was determined in a kinase inhibition assay using recombinant DYRK1A,
Woodtide as a substrate and an ATP concentration of 100 μM. The lead compound 1a
at 1 M was confirmed to have almost complete inhibitory potency against
9
DYRK1A. Next, we tested the analogues described above at two concentrations (1
M vs 10 M) (Table 1).
1
4

An analysis of the results gave some important insights into the structure-activity
relationship about these molecules. Interestingly, naphthyridine 5, the most
structurally related compound to the lead compound 1a with the NH moiety replaced
with N, showed the best DYRK1A inhibitory potency among these new derivatives
(reducing activity to 67% and 30% at 1 M and 10 M, respectively), though much
lower potency than that of compound 1a, highlighting the importance of the NH
moiety in maintaining DYRK1A inhibition. Furthermore, pyrazole-pyridine-based
analogue 6 or 11 failed to show any inhibitory potency against DYRK1A at both 1
M and 10 M. Compound 11 with its fused ring structure showed no inhibition of
DYRK1A, which may indicate that aromaticity between the two nitrogen atoms is
essential, as is the phenol group on each side chain. Analogue 7, with a closer
distance between the two phenol substituents in comparison to 6, exhibited reduced
DYRK1A activity at both 1 M (64%) and 10 M (9%). This may suggest that the
two phenols at a specific distance between each other may play an important role in
maintaining DYRK1A inhibition. However, this is not replicated with para-hydroxy
analogues 9a and 10a and meta-hydroxy analogues 9b and 10b, all of which were
mostly ineffectual against DYRK1a with activity staying above 90%. The slight
change between 9a and 9b (99% and 91% at 10 M, respectively) suggests that the
hydroxyl group plays an important role in DYRK1A inhibition, as even subtle
modification from para to meta substitution changes inhibitory activity.
Table 1. DYRK1A inhibition assay at different concentrations (1 M vs 10 M).
DYRK1A
remaining remaining
activity at activity at
μM (%) 10 μM (%)
DYRK1A
DYRK1A
remaining remaining
activity at activity at
1 μM (%) 10 μM (%)
DYRK1A
Compound
Structures
Compound Structures
1
1
a
3.3 ± 0.3
3.3 ± 0.3
43 ± 21.5
8
90 ± 15.3
94 ± 11.2
83 ± 9.5
99 ± 5.5
2
74 ± 5.5
9a
3
106 ± 10.3
107 ± 14.7
9b
94 ± 10.5
91 ± 9.0
1
5

4
100 ± 13.0
79 ± 11.0
10a
99 ± 12.1
99 ± 10.7
5
6
67 ± 15.8
110 ± 4.1
30 ± 9.5
10b
11
104 ± 8.4
102 ± 8.7
98 ± 10.6
104 ± 16.7
113 ± 14.7
7
64 ± 5.1
9 ± 1.2
CONCLUSIONS
We have presented a series of synthetic methods in this paper that have generated 12
novel compounds containing a variety of heterocyclic cores. All these novel
compounds were assessed in a DYRK1A inhibition assay. However, none of these
derivatives showed significant DYRK1A inhibition. Regardless, biological studies
have provided us with the valuable knowledge that the 7-azaindole heterocyclic core
shows more potent activity against DYRK1A than other heterocycles, even when
maintaining the diphenolic characteristics. Analogue 5 is the most potentially
bioactive molecule among these new series of compounds though, comparing
analogue 5 to lead compound 1a has hinted that the NH moiety is important, probably
to act as a hydrogen bond donor in the binding modes. By comparing analogues 6
with 7, we have shown that the distance between the two phenols has strong effects
on the DYRK1A inhibition. These data will be of use for designing further structure-
activity relationship studies to further improve the selective inhibition of DYRK1A.
ACKNOWLEDGEMENTS
This work was supported by the National Health & Medical Research Council of
Australia (APP1106145).
EXPERIMENTAL
1
6

General chemical synthesis details
Unless noted otherwise, commercially obtained reagents were used as purchased
without further purification. Solvents for flash chromatography were distilled prior to
use, or used as purchased for HPLC grade, with the eluent mixture reported as the
volume/volume ratio (v/v). Unless otherwise stated, reactions were performed under
an atmosphere of nitrogen. Flash chromatography was performed using Merck
Kieselgel 60 (230–400 mesh) silica gel. Analytical thin-layer chromatography (TLC)
was performed using Merck aluminum-backed silica gel 60 F254 (0.2 mm) plates
(Merck, Darmstadt, Germany), which were visualized using shortwave (254 nm)
ultraviolet fluorescence. Melting points were measured with open capillaries using a
Stanford Research Systems (SRS) MPA160 melting point apparatus with a with a rate
of 5 C/min and are uncorrected. Infrared absorption spectra were reported as
–
1
vibrational frequency (cm ). Nuclear magnetic resonance spectra were recorded at
00 K on Bruker Advance DRX200, DRX300, DRX400 or DRX500
3
spectrophotometers. The data are reported as chemical shift (δ ppm) relative to the
residual protonated solvent resonance (CDCl : δ 7.26, d -DMSO: δ 2.50, d -MeOD: δ
3
6
3
3
.31, D O: δ 4.79 and d -acetone: δ 2.05), the carbon of the solvent resonance
2 6
(
CDCl : δ 77.16, d -DMSO: δ 39.52, d -MeOD: δ 49.00 and d -acetone: δ
3 6 3 6
3
8
2
9.84/206.26), relative integral, multiplicity (s = singlet, br.s = broad singlet, d =
doublet, dd = doublet of doublets, t = triplet, m = multiplet, etc.) and coupling
constants (J Hz). Low-resolution mass spectra (LRMS) was obtained from a
ThermoQuest Finnigan LCQ Deca ion trap mass spectrometer with electro-spray
ionisation in either positive (+ESI) or negative (-ESI) mode. Data is expressed as
observed mass (m/z), assignment (M = molecular ion), and relative intensity (%).
High-resolution mass spectra (HRMS) was performed on a Bruker Apex Qe 7T
Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometer equipped
with an Apollo  ESI dual source. Samples were run with syringe infusion at 150
L/hr on a Cole Palmer syringe pump into electrospray ionization (ESI). Atmospheric
pressure chemical ionization (APCI) was performed by untilising MeOH. High
performance liquid chromatography (HPLC) analysis of organic purity was conducted
on a Waters Alliance 2695 instrument using a SunFireTM C18 column (5 m, 2.1 x
1
2
50 mm) and detected using a Waters 2996 photodiode array (PDA) detector set at
54 nm. Separation was achieved using water (solvent A) and acetonitrile (solvent B)
1
7

at flow rate of 0.2 mL/min and a gradient of 0% B to 100% (HPLC Method A) or 0%
B to 40% (HPLC Method B) over 30 min. HPLC data is reported as percentage purity
and retention time (RT) in minutes.
4
-Bromo-2-fluoroaniline (13). This compound was prepared following a literature
procedure.39 To a suspension of 2-fluoroaniline (12) (2.22 g, 20 mmol) in CHCl3
50 mL) was added NBS (3.56 g, 20 mmol), and the resulting mixture was stirred at
(
RT for 2 h. After completion monitored by TLC, the mixture was quenched with sat.
Na S O (aq.) and extracted with CH Cl (3 x 20 mL). The combined organic layers
2
2
3
2
2
were dried over MgSO and concentrated in vacuo. The crude product was purified by
4
flash chromatography (hexane/ethyl acetate 10:1) to give the product 13 as an orange
1
oil (3.42 g, 90%); R (hexane/ethyl acetate 6:1): 0.35; H NMR (300 MHz, CDCl ): δ
f
3
7
.14 (1H, dd, J = 2.1, 10.5 Hz), 7.05 (1H, d, J = 8.4 Hz), 6.65 (1H, t, J = 9.0 Hz), 3.72
1
3
(
2H, br.s); C NMR (75 MHz, CDCl ): δ 151.5 (d, J = 241.5 Hz), 133.9 (d, JCF =
3 CF
1
1
2.8 Hz), 127.5 (d, JCF = 3.0 Hz), 118.8 (d, JCF = 21.8 Hz), 117.9 (d, JCF = 3.8 Hz),
09.0 (d, JCF = 9.0 Hz). The spectroscopic data matched that reported in the
literature.39
4
-Bromo-2-fluoro-1-nitrobenzene (14). This compound was prepared following a
3
9
literature procedure. To a solution of 13 (1.26 g, 6.6 mmol) in TFA (13 mL) was
added aq. H O (30%, 3.4 mL, 33 mmol) dropwise over 30 min, and the resulting
2
2
mixture was stirred at 75 C for 1 h. After completion monitored by TLC, the mixture
was poured into ice (20 mL), the pale yellow precipitate was obtained and filtered,
washed by cold water and dried under reduced pressure to get the desired compound
1
1
4 without further purification (1.09 g, 75%). R (hexane/ethyl acetate 10:1): 0.45; H
f
NMR (400 MHz, CDCl ): δ 7.97 (1H, t, J = 8.0Hz), 7.50 (1H, dd, J = 2.0, 10.0 Hz),
3
1
3
7
1
.47-7.44 (1H, m); C NMR (100 MHz, CDCl ): δ 155.4 (d, J = 268 Hz), 136.4,
3 CF
29.4 (d, JCF = 9.0 Hz), 128.1 (d, JCF = 4.0 Hz), 127.1 (d, JCF = 2.0 Hz), 122.1 (d, JCF
24.0 Hz). The spectroscopic data matched that reported in the literature.39
=
General procedure A for the TBS protection
The compounds could be prepared according to the literature.40 To a solution of
hydroxy substrate (1.0 equiv.) in anhydrous DMF (0.05 M) was added imidazole (2.5
1
8

or 5.0 equiv.) at 0 C, followed by the addition of TBSCl (1.5 or 3.5 equiv.). The
reaction mixture was allowed to warm to RT for 12 h. After completion monitored by
TLC, the mixture was extracted with ethyl acetate (3 x 30 mL) and H O (50 mL), the
2
organic layers were dried over MgSO and concentrated in vacuo.
4
4
-((tert-Butyldimethylsilyl)oxy)aniline (15a). This compound was prepared
according to general procedure A by treating 4-aminophenol (2.18 g, 0.02 mol),
imidazole (3.2 g, 0.05 mol) and TBSCl (4.53 g, 0.03 mol). The residue was purified
by flash chromatography (hexane/ethyl acetate 10:1 → 5:1) to give the product 15a as
1
pale yellow oil (4.0 g, 87%). R (hexane/ethyl acetate 2:1): 0.45; H NMR (300 MHz,
f
d6-DMSO): δ 6.53 (2H, d, J = 8.7 Hz), 8.46 (2H, d, J = 8.7 Hz), 4.59 (2H, s), 0.93
1
3
(
9H, s), 0.11 (6H, s); C NMR (75 MHz, d -DMSO): δ 145.5, 142.8, 119.9, 114.9,
6
2
5.6, 17.8, –4.6. The spectroscopic data matched that reported in the literature.41
3
-((tert-Butyldimethylsilyl)oxy)aniline (15b). This compound was prepared
according to general procedure A by treating 3-aminophenol (1.09 g, 0.01 mol),
imidazole (1.60 g, 0.025 mol) and TBSCl (2.27 g, 0.015 mol). The crude product was
purified by flash chromatography (hexane/ethyl acetate 10:1 → 5:1) to give the
1
product 15b as pale yellow oil (2.07 g, 93%). R (hexane/ethyl acetate 2:1): 0.45; H
f
NMR (500 MHz, CDCl ): δ 6.99 (1H, t, J = 8.0 Hz), 6.31-6.26 (2H, m), 6.21-6.20
3
1
3
(
1H, m), 3.46 (2H, br.s), 0.99 (9H, s), 0.20 (6H, s); C NMR (125 MHz, CDCl ): δ
3
1
56.9, 147.8,130.1, 110.7, 108.7, 107.3, 25.9, 18.3, –4.2. The spectroscopic data
matched that reported in the literature.42
4
-(5-Bromo-2-nitrophenoxy)aniline (16b). To a solution of 14 (220 mg, 1 mmol) in
DMF (10 mL) was added K CO (207 mg, 1.5 mmol), followed by the addition of 4-
2
3
((tert-butyldimethylsilyl)oxy)aniline 15a (245 mg, 1.1 mmol). The resulting mixture
was heated to 90 C and stirred for 3 h. After completion monitored by TLC, the
mixture was extracted with ethyl acetate (3 x 10 mL) and H O. The combined organic
2
layers were dried over MgSO and concentrated in vacuo. The crude product was
4
purified by flash chromatography (hexane/ethyl acetate 5:1  2:1) to give the product
1
1
6b as a pale yellow semisolid (247 mg, 80%); R (hexane/ethyl acetate 4:1): 0.25; H
f
NMR (400 MHz, d -DMSO): δ 7.96 (1H, d, J = 8.8 Hz), 7.42 (1H, dd, J = 2.0, 8.8
6
1
9

Hz), 6.96 (1H, d, J = 2.0 Hz), 6.88 (2H, dd, J = 2.0, 10.8 Hz), 6.65 (2H, dd, J = 2.0,
1
3
1
1
0.8 Hz), 5.26 (2H, br. s); C NMR (100 MHz, d -DMSO): δ 152.6, 146.7, 143.6,
6
38.8, 127.4, 127.2, 125.1, 121.1, 120.3, 115.0; HRMS (ESI+) Calc. for
+
-
C H N O Br [M+Na] 330.9689/332.9668, found 330.9691/332.9670. IR (neat, cm
1
2
9
2
3
1)
:

3475, 3385, 3098, 3085, 1615, 1505, 1466, 1231, 824, 528.
4
-((5-Bromo-2-nitrophenyl)amino)phenol (16c). To a solution of 14 (220 mg, 1
mmol) in anhydrous DMF (10 mL) was added K CO (207 mg, 1.5 mmol), followed
2
3
by the addition of 4-aminophenol (120 mg, 1.1 mmol). The resulting mixture was
heated to 90 C and stirred for 3 h. After completion monitored by TLC, the mixture
was extracted with ethyl acetate (3 x 10 mL) and H O. The combined organic layer
2
was dried over MgSO and concentrated in vacuo. The crude product was purified by
4
flash chromatography (hexane/ethyl acetate 10:1  4:1) to give the product 16c as a
pale orange solid (201 mg, 65%); m.p. 151-152 C; R (hexane/ethyl acetate 4:1):
f
1
0
.30; H NMR (300 MHz, d -DMSO): δ 9.65 (1H, br.s), 9.40 (1H, br.s), 8.02 (1H, d,
6
1
3
J = 9.0 Hz), 7.14 (2H, d, J = 8.1 Hz), 6.92-6.84 (4H, m); C NMR (75 MHz, d -
6
DMSO): δ 156.1, 145.0, 131.0, 130.2, 129.0, 128.2, 127.7, 119.4, 117.7, 116.3;
+
HRMS (ESI+) Calc. for C H N O Br [M+Na] 330.9689/332.9668, found
1
2
9
2
3
-1

3
7
30.9692/332.9672. IR (neat, cm ):
3349, 3089, 1602, 1556, 1475, 1241, 1198,
47, 531.
4
-((2-Amino-5-bromophenyl)amino)phenol (17). To a solution of 16c (308 mg, 1
mmol) in acetic acid (15 mL) was added iron powder (280 mg, 5 mmol), and the
resulting mixture was stirred at RT for 12 h. After completion monitored by TLC, the
volatile was removed under reduced pressure and the residue was washed with sat.
NaHCO (aq.) and extracted with ethyl acetate (3 x 15 mL). The combined organic
3
layers were dried over MgSO and concentrated in vacuo. The crude product was
4
purified by flash chromatography (CH Cl /MeOH 100:1) to give the product 17 as a
2
2
1
brown solid (265 mg, 95%); m.p. 145-147 C; R (hexane/ethyl acetate 1:1): 0.25; H
f
NMR (400 MHz, d -DMSO): δ 6.91 (1H, dd, J = 2.4, 8.4 Hz), 6.75 (2H, dd, J = 2.4,
6
6
.8 Hz), 6.68 (1H, d, J = 8.4 Hz), 6.59 (2H, dd, J = 2.4, 6.8 Hz), 6.55 (1H, d, J = 2.4
1
3
Hz), 5.08 (2H, br.s), 4.97 (1H, br.s); C NMR (100 MHz, d -DMSO): δ 146.1, 145.7,
6
1
45.3, 138.4, 125.1, 120.2, 118.4, 116.2, 114.8, 105.5; HRMS (ESI+) Calc. for
2
0

+
-
C H BrN O [M+H] 279.0128/281.0107, found 279.0125/281.0104. IR (neat, cm
1
2
11
2
1)
:

3500, 3400, 2896, 1610, 1598, 1510, 1220, 756, 520.
4
-(2-Amino-6-bromo-1H-benzo[d]imidazol-1-yl)phenol (18). This compound was
2
1
prepared following a literature procedure. To a solution of 17 (200 mg, 0.72 mmol)
in the mixture solvent of MeCN-H O (4:1 (v/v), 10 mL) was added cyanogen bromide
2
(158 L, 0.79 mmol, 5 M in MeCN) at 0 C. and the resulting mixture was allowed to
warm to RT for 16 h. After completion monitored by TLC, the mixture was treated
with sat. NaHCO (aq.) (10 mL) and extracted with ethyl acetate (3 x 15 mL). The
3
combined organic layers were dried over MgSO and concentrated in vacuo. The
4
residue was purified by flash chromatography (CH Cl /MeOH 100:1  20:1) to give
2
2
the product 18 as a pale pink solid (171 mg, 78%); m.p. 273-274 C; Rf
1
(
CH Cl /MeOH 15:1): 0.20; H NMR (400 MHz, d -DMSO): δ 9.98 (1H, s), 7.24
2
2
6
(2H, dd, J = 2.0, 6.4 Hz), 7.14 (1H, dd, J = 0.4, 8.4 Hz), 7.11 (1H, dd, J = 1.6, 8.4
1
3
Hz), 6.96 (2H, dd, J = 2.0, 6.4 Hz), 6.81 (1H, dd, J = 0.4, 1.6 Hz), 6.35 (2H, s); C
NMR (100 MHz, d -DMSO): δ 157.8, 155.3, 141.6, 136.8, 128.5, 125.0, 123.8,
6
+
1
16.8, 116.4, 110.4, 110.3; HRMS (ESI+) Calc. for C H BrN O [M+H]
1
3
10
3
-1

3
1
04.0080/304.0059, found 304.0083/306.0062. IR (neat, cm ):
3330, 3205, 1647,
514, 1449, 1272, 1247, 1122, 800, 727, 586, 432.
General procedure B for Suzuki Coupling Reaction
To a solution of aryl-halide (1.0 equiv.) in 1,4-dixoane (0.02 M) was added
arylboronic acid (1.2 equiv.), aq. K CO (2 M, 2.0 equiv.), and Pd(PPh ) (2 mol%),
2
3
3 4
and the reaction was heated to 100-110 C for 4 h under argon. After completion
monitored by TLC, the reaction mixture was cooled to RT, solvent was removed
under reduce pressure, and then partitioned between H O and ethyl acetate. The
2
aqueous layer was extracted with ethyl acetate, and the combined organic layers were
dried over MgSO and concentrated in vacuo.
4
4
,4'-(2-Amino-1H-benzo[d]imidazole-1,6-diyl)diphenol (2). This compound was
prepared according to general procedure B by treating 18 (61 mg, 0.2 mmol) with
4-hydroxyphenyl)boronic acid (33 mg, 0.24 mmol) at 110 C. The residue was
purified by flash column chromatography (CH Cl : MeOH = 50:1  10:1) to afford
(
2
2
2
1

the product 2 as a pale yellow solid (10 mg, 30%). m.p. 249-250 C; Rf
1
(
CH Cl /MeOH 10:1): 0.35; H NMR (400 MHz, d -DMSO): δ 9.85 (1H, br.s), 9.36
2
2
6
(1H, s), 7.42 (2H, dd, J = 2.0, 6.4 Hz), 7.35 (1H, d, J = 1.6 Hz), 7.26 (2H, dd, J = 2.0,
6
.8 Hz), 7.06 (1H, dd, J = 1.6, 8.0 Hz), 6.97 (2H, dd, J = 2.0, 6.8 Hz), 6.82 (2H, dd, J
1
3
=
2.0, 6.4 Hz), 6.79 (1H, d, J = 8.0 Hz), 6.14 (2H, br.s); C NMR (100 MHz, d -
6
DMSO): δ 157.3, 156.2, 154.9, 143.4, 134.5, 133.8, 132.6, 128.1, 127.5, 125.8, 117.1,
+
1
16.5, 115.5, 112.6, 107.6; HRMS (ESI+) Calc. for C H N O [M+H] 318.1237,
1
9
15
3
2
-1

found 318.1241. IR (neat, cm ): 3337, 3135, 2954, 2921, 2852, 1633, 1608, 1540,
1
1
513, 1459, 1218, 1170, 1101, 835, 801, 513. HPLC: 96.0% (HPLC Method A), RT:
6.8 min.
N-(1,6-bis(4-hydroxyphenyl)-1H-benzo[d]imidazol-2-yl)acetamide (3). To
solution of 2 (30 mg, 0.10 mmol) in acetic acid (1 mL) was added acetic anhydride
10 L, 0.11 mmol) at RT. And the resulting mixture was stirred at 110 C for 3 h.
a
(
After completion monitored by TLC, the solvent was removed under reduced pressure
and the residue was neutralised with sat. NaHCO (aq.) and extracted with ethyl
3
acetate (3 x 15 mL). The combined organic layers were dried over MgSO and
4
concentrated in vacuo. The crude product was purified by flash chromatography
(
CH Cl : MeOH = 50:1  10:1) to afford the product 3 as a pale yellow solid (25 mg,
2
2
1
3
8%). m.p. 207-209 C; R (CH Cl /MeOH 10:1): 0.25; H NMR (400 MHz, d -
f
2
2
6
DMSO): δ 10.28 (1H, br.s), 9.86 (1H, s), 9.45 (1H, s), 7.77 (1H, s), 7.50 (2H, d, J =
8
6
.4 Hz), 7.43 (1H, d, J = 8.0 Hz), 7.24 (2H, d, J = 8.0 Hz), 7.16 (1H, d, J = 8.4 Hz),
1
3
.93 (2H, d, J = 8.4 Hz), 6.85 (2H, d, J = 8.4 Hz), 1.95 (3H, s); C NMR (100 MHz,
d6-DMSO): δ 170.3, 157.9, 157.1, 141.7, 141.6, 135.6, 134.6, 132.2, 128.3, 128.0,
26.5, 122.0, 116.5, 116.4, 116.2, 110.7, 23.0; HRMS (ESI+) Calc. for C H N O
1
2
1
17
3
3
+
-1 
[
M+H] 360.1343, found 360.1346. IR (neat, cm ): 3274, 2953, 2921, 2852, 1587,
550, 1513, 1366, 1203, 834, 808, 560. HPLC: 96.6% (HPLC Method A), RT: 16.6
min.
1
4
,5-Diiodo-1H-imidazole (20). This compound was prepared following a literature
2
2
procedure. To a solution of imidazole 19 (1.11 g, 16 mmol) in NaOH (4 M, 60 mL)
was added a solution of KI (13.3 g, 80 mmol) and I (8.88 g, 35 mmol) in H O (50
2
2
mL) dropwise. The resulting mixture stirred at RT for 10 h. After completion
2
2

monitored by TLC, the mixture was reduced to pH = 8 with acetic acid, and the
resulting white precipitate was filtered and washed with cold water. The remaining
solid was air dried to afford the product 20 as a white creamy solid (4.1 g, 80%). m.p.
1
1
7
88-190 C; R (hexane/ethyl acetate 1:1): 0.15; H NMR (300 MHz, d -DMSO): δ
f
6
1
3
.77 (1H, s); C NMR (75 MHz, d -DMSO): δ 141.8, 86.9. The spectroscopic data
6
matched that reported in the literature.43
4
,4'-(1H-Imidazole-4,5-diyl)diphenol (4). This compound was prepared following a
literature procedure.23 The mixture of 4,5-diiodo-1H-imidazole 20 (0.11 g, 0.34
mmol), (4-hydroxyphenyl)boronic acid (141 mg, 1.02 mmol), TBAB (11 mg, 0.034
mmol), K HPO (226 mg, 1.30 mmol) and Pd(PPh ) (25 mg, 6 mol%) were
2
4
3 4
transferred to a microwave reactor tube equipped with a magnetic stirring bar,
followed by the addition of a mixture solvent of MeOH/H O (5 mL, 4:1), and the
2
resulting mixture was degassed and back-filled with nitrogen and then stirred at 100
°
C (100 W) with microwave irradiation for 60 min. After completion monitored by
TLC, the reaction mixture was dissolved in aq. HCl (10%, 10 mL) and stirred
vigorously. The mixture was washed with ethyl acetate (2 x 10 mL), the combined
aqueous layers were neutralised with sat. NaHCO (aq.) and extracted with ethyl
3
acetate (3 x 10 mL). The combined organic layers were washed with H O, dried over
2
MgSO and concentrated in vacuo. The crude product was purified by flash
4
chromatography (CH Cl : MeOH = 50:1  10:1) to afford the product 4 as a white
2
2
1
solid (30 mg, 35%). R (CH Cl /MeOH 10:1): 0.25; H NMR (400 MHz, d -DMSO):
f
2
2
6
1
3
δ 9.44 (2H, Br.s), 7.67 (1H, s), 7.25 (4H, d, J = 8.4 Hz), 6.72 (4H, d, J = 8.4 Hz); C
NMR (100 MHz, d -DMSO): δ 156.3, 134.4, 128.6, 125.6, 124.3, 115.2. HRMS
6
+
(
ESI+) Calc. for C H N O [M+Na] 275.0791, found 275.0794. HPLC: 97.7%
1
5
12
2
2
(HPLC Method A), RT: 13.3 min. The spectroscopic data matched that reported in the
literature.44
2
-Amino-5-bromonicotinic acid (22). This compound was prepared following a
2
5
literature procedure. To a suspension of 2-aminonicotinic acid 21 (3.75 g, 27 mmol)
in glacial acetic acid (15 mL) was added a solution of bromine (1.8 mL, 35 mmol) in
glacial acetic acid (3 mL) and the mixture was stirred at RT for 20 h. After
completion monitored by TLC, the resulting precipitate was filtered and washed with
2
3

glacial acetic acid 3 times. The remaining solid was air dried and then recrystallised
from boiling methanol to afford the product 22 as white crystalline needles (5.2 g,
1
9
2
1
0%). H NMR (400 MHz, d -DMSO): δ 8.36 (1H, d, J = 2.6 Hz), 8.25 (1H, d, J =
6
1
3
.6 Hz), 7.95-7.79 (2H, br.s); C NMR (100 MHz, d -DMSO): δ 166.6, 156.5, 149.9,
6
43.8, 109.3, 103.7. The spectroscopic data matched that reported in the literature.25
Methyl 2-amino-5-bromonicotinate (23). To a solution of 2-amino-5-
bromonicotinic acid 22 (1.0 g, 4.6 mmol) in MeOH (10 mL) was added sulphuric acid
(2.0 mL, 36.8 mmol) dropwise and the mixture was stirred at 80 C for 18 h. After
completion monitored by TLC, the solvent was removed under reduced pressure. The
residue was neutralised with sat. NaHCO (aq.) and extracted with ethyl acetate (3 x
3
2
0 mL). The combined organic layers were dried over MgSO and concentrated in
4
vacuo to afford the product 23 as a white powder (1.0 g, 94%) without further
1
purification. m.p. 148-149 C; R (CH Cl /MeOH 20:1): 0.55; H NMR (400 MHz,
f
2
2
d6-DMSO): δ 8.24 (1H, d, J = 2.4 Hz), 8.21 (1H, d, J = 2.4 Hz), 6.68-6.19 (2H, br.s),
1
3
3
1
.89 (3H, s); C NMR (100 MHz, d -DMSO): δ 166.6, 158.0, 154.4, 142.0, 107.5,
6
+
06.1, 52.4. HRMS (ESI+) Calc. for C H N O Br [M+H] 230.9764/232.9743, found
7
7
2
2
-1

2
5
30.9765/232.9745. IR (neat, cm ):
3425, 3131, 2918, 1704, 1620, 1223, 796,
26.
6
-Bromo-4-hydroxy-1,8-naphthyridine-3-carbonitrile (24). This compound was
2
4
prepared following a literature procedure. To a sealed tube was added a mixture of
methyl 2-amino-5-bromonicotinate 23 (500 mg, 2.16 mmol) and N,N-
dimethylformamide dimethyl acetal (3 mL, 21.6 mmol), and stirred at 110 °C for 3 h.
The volatiles were concentrated under reduced pressure, and the crude intermediate
was dissolved in anhydrous THF (30 mL), which would be used immediately in the
following step.
To a solution of n-BuLi (2.5 M in hexane, 1.9 mL, 4.75 mmol) in anhydrous THF (10
mL) was added anhydrous acetonitrile (248 μL, 4.75 mmol) dropwise at –78 C under
nitrogen atmosphere. The resulting reaction mixture was stirred at –78 C for 20 min.
To the resulting white suspension was added dropwise the intermediate solution
described above, and the reaction mixture was stirred at –78 C for 30 min, then at –
4
0 C for 2 h. Acetic acid (370 μL, 6.48 mmol) was added and the resulting yellow
2
4

suspension was warmed to RT and stirred for 10 h. After completion monitored by
TLC, the mixture was diluted with H O (20 mL) and extracted with ethyl acetate (3 x
2
1
5 mL). The combined organic layers were washed with H O (2 x 15 mL) and brine,
2
dried over MgSO and concentrated in vacuo. The crude product was purified by flash
4
chromatography (hexane/ethyl acetate = 5:1  1:1) to afford the product 24 as an
1
orange solid (190 mg, 35%). H NMR (400 MHz, d -DMSO): δ 13.43-13.41 (1H,
6
1
3
br.s), 8.97 (1H, d, J = 2.4 Hz), 8.85 (1H, s), 8.61 (1H, d, J = 2.4 Hz); C NMR (100
MHz, d -DMSO): δ 174.0, 154.8, 148.6, 148.4, 136.5, 121.2, 116.5, 115.9, 95.3. The
6
spectroscopic data matched that reported in the literature.24
6
-Bromo-1,8-naphthyridin-4-ol (25). This compound was prepared following a
2
4
literature procedure. To a sealed tube was added a mixture of 6-bromo-4-hydroxy-
,8-naphthyridine-3-carbonitrile 24 (100 mg, 0.40 mmol) in aq. HCl (32%, 2 mL) and
1
stirred at 150 C for 3 h. After cooling to room temperature, the reaction mixture was
neutralised with ammonia solution, extracted with ethyl acetate (3 x 10 mL). The
combined organic layers were washed with H O (2 x 10 mL), dried over MgSO and
2
4
concentrated in vacuo. The crude product was purified by flash chromatography
CH Cl : MeOH = 50:1  20:1) to afford the product 25 as a pale yellow solid (30
(
2
2
1
mg, 33%). H NMR (300 MHz, d -DMSO): δ 12.4 (1H, br.s), 8.84 (1H, s), 8.53 (1H,
6
1
3
s), 7.98 (1H, d, J = 7.5 Hz), 6.15 (1H, d, J = 7.5 Hz); C NMR (75 MHz, d -DMSO):
6
δ 176.5, 153.6, 149.1, 140.9, 136.3, 121.3, 114.3, 110.0. The spectroscopic data
matched that reported in the literature.24
3
-Bromo-5-chloro-1,8-naphthyridine (26). This compound was prepared following
a literature procedure.24 To a sealed tube was added a mixture of 6-bromo-1,8-
naphthyridin-4-ol 25 (30 mg, 0.13 mmol) in POCl (1 mL) and stirred at 105 C for 3
3
h. After cooling to RT, the reaction mixture was poured into ice, and the resulting
mixture was neutralised with sat. NaHCO (aq.) and extracted with ethyl acetate (3 x
3
5
mL). The combined organic layers were washed with H O (2 x 5 mL), dried over
2
MgSO and concentrated in vacuo. The crude product was purified by flash
4
chromatography (hexane/ethyl acetate = 5:1  3:1) to afford the product 26 as a
1
white solid (29 mg, 93%). H NMR (400 MHz, CDCl ): δ 9.16 (1H, d, J = 2.4 Hz),
3
1
3
9
.02 (1H, d, J = 4.8 Hz), 8.75 (1H, d, J = 2.4 Hz), 7.61 (1H, d, J = 4.8 Hz); C NMR
2
5

(
100 MHz, CDCl ): δ 155.8, 155.0, 153.4, 142.1, 135.3, 123.0, 122.8, 119.5. The
3
spectroscopic data matched that reported in the literature.24
4
,4'-(1,8-Naphthyridine-3,5-diyl)diphenol (5). A mixture of 3-bromo-5-chloro-1,8-
naphthyridine 26 (15 mg, 0.06 mmol), (4-hydroxyphenyl)boronic acid (21 mg, 0.15
mmol) and aq. K CO (2 M, 180 μL, 0.36 mmol) in 1,4-dioxane (1 mL) was
2
3
degassed, and then PdCl (dppf) (9 mg, 0.012 mmol) was added. The resulting mixture
2
was degassed and back-filled with nitrogen, and then stirred at 130 C (100 W) in
microwave under nitrogen atmosphere for 2 h. After completion monitored by TLC,
the reaction mixture was concentrated under reduced pressure, and diluted with H O
2
(
5 mL), neutralised with sat. NH Cl (aq.) and extracted with ethyl acetate (3 x 5 mL).
4
The combined organic layers were washed with H O (5 mL), dried over MgSO and
2
4
concentrated in vacuo. The crude product was purified by flash chromatography
(
CH Cl : MeOH = 50:1  10:1) to afford the product 5 as a yellow solid (16 mg,
2
2
1
8
5%). m.p. 211-213 C; R (CH Cl /MeOH 15:1): 0.30; H NMR (400 MHz, d -
f
2
2
6
DMSO): δ 9.90 (1H, s), 9.78 (1H, s), 9.38 (1H, s), 9.04 (1H, br.s), 8.37 (1H, d, J = 2.4
Hz), 7.62 (2H, d, J = 8.4 Hz), 7.54 (1H, d, J = 4.4 Hz), 7.48 (2H, d, J = 8.4 Hz), 7.00
1
3
(
2H, d, J = 8.4 Hz), 6.92 (2H, d, J = 8.4 Hz); C NMR (100 MHz, d -DMSO): δ
6
1
1
58.7, 158.5, 155.4, 152.8, 152.5, 149.6, 134.2, 131.5, 130.7, 129.0, 127.6, 127.3,
+
22.7, 121.1, 116.7, 116.3. HRMS (ESI+) Calc. for C H N O [M+Na] 337.0947,
2
0
14
2
2
-1

found 337.0950. IR (neat, cm ): 3363, 2921, 1606, 1512, 1265, 831, 559, 527.
HPLC: 98.5% (HPLC Method A), RT: 15.5 min.
General procedure C for condensation reactions
The compounds ware prepared following a literature procedure.45, 46 To a solution
of halogenated pyridine substrate (1.0 equiv.) in anhydrous toluene (0.05 M) was
added n-BuLi (1.1 equiv., 2.5 M in hexane) dropwise at –78 C, and the resulting
mixture was stirred for 30 min. N, N-dimethylacetamide or DMF (1.5 equiv.) was
added dropwise and the mixture was stirred for another 30 min. After completion, the
reaction was quenched with sat. NH Cl (aq.) and extracted with ethyl acetate (3 x 15
4
mL). The organic layer was dried over MgSO and concentrated in vacuo.
4
2
6

1
-(5-Bromopyridin-2-yl)ethan-1-one (27a). This compound was prepared according
to general procedure C by treating 2,5-dibromopyridine (237 mg, 1 mmol) with N,
N-dimethylacetamide (44 L, 1.5 mmol). The crude product was purified by flash
chromatography (hexane/ethyl acetate 10:1) to give the product 27a as a white solid
1
(
160 mg, 80%); R (hexane/ethyl acetate 8:1): 0.45; H NMR (300 MHz, CDCl ): δ
f
3
1
3
8
1
.70 (1H, s), 7.95-7.88 (2H, m), 2.66 (3H, s); C NMR (75 MHz, CDCl ): δ 199.2,
3
51.9, 150.2, 139.6, 125.4, 123.0, 25.8. The spectroscopic data matched that reported
in the literature.45
5
-Bromopicolinaldehyde (27b). This compound was prepared according to general
procedure C by treating 2,5-dibromopyridine (237 mg, 1 mmol) with DMF (115 L,
.5 mmol). The crude product was purified by flash chromatography (hexane/ethyl
1
acetate 10:1) to give the product 27b as an off-brown solid (149 mg, 80%); Rf
1
(
hexane/ethyl acetate 8:1): 0.45; H NMR (400 MHz, CDCl ): δ 10.03 (1H, s), 8.85
3
(1H, dd, J = 0.8, 2.0 Hz), 8.02 (1H, ddd, J = 0.8, 2.0, 8.0 Hz), 7.85 (1H, dd, J = 0.8,
1
3
8
.0 Hz); C NMR (100 MHz, CDCl ): δ 192.4, 151.7, 151.3, 140.0, 126.3, 122.8.
3
The spectroscopic data matched that reported in the literature.47
General procedure D for benzyl protection
4
8
The compounds were prepared following a literature procedure. To a solution of
hydroxyaryl substrate (1.0 equiv., 1 mmol) in DMF (0.05 M) was added K CO (1.5
2
3
equiv., 1.5 mmol), followed by the dropwise addition of benzyl bromide (1.2 equiv.,
.2 mmol) at RT, and the mixture was stirred at RT for 12 h. After completion
1
monitored by TLC, the solvent was removed under reduced pressure, and the residue
was washed with H O (30 mL) and extracted with ethyl acetate (3 x 20 mL), the
2
organic layers were dried over MgSO and concentrated in vacuo.
4
4
-(Benzyloxy)benzaldehyde (28a). The compounds were prepared according to
general procedure D by treating 4-hydroxybenzaldehyde (1.22 g, 0.01 mol) with
K CO (2.07 g, 0.015 mol) and benzyl bromide (1.42 ml, 0.012 mol). The crude
2
3
product was purified by flash chromatography (hexane/ethyl acetate 10:1  5:1) to
give the product 28a as a white solid (1.97 g, 93%). R (hexane/ethyl acetate 4:1):
f
1
0
.35; H NMR (300 MHz, CDCl ): δ 9.89 (1H, s), 7.84 (2H, d, J = 7.8 Hz), 7.43-7.35
3
2
7

1
3
(
5H, m), 7.08 (2H, d, J = 7.8 Hz), 5.15 (2H, s); C NMR (75 MHz, CDCl ): δ 190.9,
3
1
63.8, 136.0, 132.1, 130.2, 128.8, 128.4, 127.6, 115.3, 70.4. The spectroscopic data
matched that reported in the literature.49
1
-(4-(Benzyloxy)phenyl)ethan-1-one (28b). This compound was prepared according
to general procedure D by treating 1-(4-hydroxyphenyl)ethan-1-one (1.36 g, 0.01
mol) with K CO (2.07 g, 0.015 mol) and benzyl bromide (1.42 ml, 0.012 mol). The
2
3
crude product was purified by flash chromatography (hexane/ethyl acetate 10:1 →
5
4
:1) to give the product 28b as a white solid (2.17 g, 96%); R (hexane/ethyl acetate
f
1
:1): 0.50; H NMR (400 MHz, CDCl ): δ 7.94 (2H, dd, J = 2.0, 6.8 Hz), 7.45-7.33
3
1
3
(
5H, m), 7.01 (2H, dd, J = 2.0, 6.8 Hz), 5.12 (2H, s), 2.55 (3H, s); C NMR (100
MHz, CDCl ): δ 196.8, 162.7, 136.3, 130.7, 130.6, 128.7, 128.3, 127.5, 114.6, 70.2,
3
2
6.4. The spectroscopic data matched that reported in the literature.50
General procedure E for aldol condensation
LDA formation: To a solution of N,N-diisopropylethylamine (1.3 equiv.) in
anhydrous THF was added n-BuLi (1.2 equiv.) dropwise at –78 C. After addition,
the resulting mixture was stirred at –78 C for 30 min before using.
To a solution of LDA (1.3 equiv.) in solvent (10 mL) was added ketone (1.1 equiv.) at
–
78 C, and the resulting mixture was stirred for 30 min. To the mixture above was
added a solution of aldehyde (1.0 equiv.) in the corresponding solvent (0.1 M)
dropwise, after addition, the resulting mixture was slowly warmed up to 25 C. After
completion monitored by TLC, the reaction was quenched with sat. NH Cl (aq.) and
4
extracted with ethyl acetate (3 x 15 mL). The organic layer was dried over
MgSO and concentrated in vacuo. The crude product was purified by flash
4
chromatography.
1
-(4-(Benzyloxy)phenyl)-3-(5-bromopyridin-2-yl)-3-hydroxypropan-1-one (29b).
This compound was prepared according to general procedure E by treating 27b (186
mg, 1.0 mmol) with LDA (1.2 mmol) and 28b (249 mg, 1.1 mmol). After completion
monitored by TLC, the reaction was quenched with sat. NH Cl (aq.) and extracted
4
with ethyl acetate (3 x 15 mL). The organic layer was dried over MgSO and
4
concentrated in vacuo. The crude product was purified by flash chromatography
2
8

(hexane/ethyl acetate 10:1  3:1) to give the product 29b as a white solid (322 mg,
1
7
8%); m.p. 133-134 C; R (hexane/ethyl acetate 3:1): 0.25; H NMR (400 MHz,
f
CDCl ): δ 8.58 (1H, d, J = 2.4 Hz), 7.93 (2H, d, J = 9.0 Hz), 7.82 (1H, dd, J = 2.4, 8.4
3
Hz); 7.51 (1H, d, J = 8.0 Hz), 7.43-7.32 (5H, m), 6.99 (2H, d, J = 9.0 Hz), 5.31 (1H,
p, J = 4.0 Hz), 5.12 (2H, s), 4.31 (1H, d, J = 4.8 Hz), 3.60 (1H, dd, J = 3.6, 17.6 Hz),
1
3
3
1
4
.36 (1H, dd, J = 8.0, 17.6 Hz); C NMR (100 MHz, CDCl ): δ 198.6, 163.2, 160.7,
3
49.7, 139.4, 136.1, 130.7, 129.9, 128.8, 128.4, 127.5, 122.1, 119.2, 114.8, 70.3, 70.2,
+
5.0; HRMS (ESI+) Calc. for C H BrNO [M+Na] 434.0362/436.0342, found
2
1
18
3
-1

4
1
34.0365/436.0345. IR (neat, cm ): 3257, 2890, 1672, 1601, 1571, 1253, 1170,
011, 828, 699, 543.
General procedure F for the oxidation of alcohols
To a solution of alcohol substrate (1.0 equiv.) in CH Cl (0.05 M) was added Dess-
2
2
Martin periodinane (DMP) (1.5 equiv.), and the mixture was stirred at RT for 30 min.
After completion monitored by TLC, the mixture was quenched with sat. NaHCO3
(
aq.) (5 mL) and the resulted mixture was extracted with CH Cl (3 x 10 mL), the
2 2
organic layers were dried over MgSO and concentrated in vacuo.
4
1
-(4-(Benzyloxy)phenyl)-3-(5-bromopyridin-2-yl)propane-1,3-dione (30). This
compound was prepared according to general procedure F by treating 29b (206 mg,
.5 mmol) with DMP (318 mg, 0.75 mmol). The crude product was purified by flash
0
chromatography (hexane/ethyl acetate 10:1  5:1) to give the product 30 as a yellow
1
solid (195 mg, 95%). m.p. 162-163 C; R (hexane/ethyl acetate 8:1): 0.50; H NMR
f
(
400 MHz, CDCl ): δ 16.54 (1H, br.s), 8.73 (1H, d, J = 2.0 Hz), 8.04 (2H, d, J = 8.8
3
1
3
Hz), 8.01-7.96 (2H, m), 7.46-7.35 (6H, m), 7.05 (2H, d, J = 8.8 Hz), 5.15 (2H, s); C
NMR (100 MHz, CDCl ): δ 186.9, 180.9, 162.8, 151.2, 150.5, 139.8, 136.3, 129.9,
3
1
28.8, 128.4, 128.3, 127.6, 124.0, 123.2, 115.0, 93.1, 70.3; HRMS (ESI+) Calc. for
+
-
C H BrNO [M+Na] 432.0206/434.0185, found 432.0205/434.0185. IR (neat, cm
2
1
16
3
1)
:

3031, 2944, 1588, 1510, 1492, 1451, 1242, 1170, 999, 826, 800, 624, 518.
General procedure G for the formation of pyrazoles
The compounds were prepared following a literature procedure. To a solution of
,3-dione (1.0 equiv.) in MeOH (0.05 M) was added N H •H O (64%, 2.0 equiv.),
2
7
1
2
4
2
2
9