Synthesis and antitrypanosomal activities of novel pyridylchalcones

Article abstract of DOI:10.1016/j.ejmech.2017.01.027

A library of novel pyridylchalcones were synthesised and screened against Trypanosoma brucei rhodesiense. Eight were shown to have good activity with the most potent 8 having an IC₅₀value of 0.29?μM. Cytotoxicity testing with human KB cells showed a good selectivity profile for this compound with a selectivity index of 47. Little activity was seen when the library was tested against Leishmania donovani. In conclusion, pyridylchalcones are promising leads in the development of novel compounds for the treatment of human African trypanosomiasis (HAT).

Full text of DOI:10.1016/j.ejmech.2017.01.027

Accepted Manuscript
Synthesis and antitrypanosomal activities of novel pyridyl-chalcones
Avninder S. Bhambra, Ketan C. Ruparelia, Hoon L. Tan, Deniz Tasdemir, Hollie
Burrell-Saward, Vanessa Yardley, Kenneth J.M. Beresford, Randolph R.J. Arroo
PII:
S0223-5234(17)30037-5
10.1016/j.ejmech.2017.01.027
EJMECH 9178
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 November 2016
Revised Date: 21 December 2016
Accepted Date: 21 January 2017
Please cite this article as: A.S. Bhambra, K.C. Ruparelia, H.L. Tan, D. Tasdemir, H. Burrell-Saward,
V. Yardley, K.J.M. Beresford, R.R.J. Arroo, Synthesis and antitrypanosomal activities of novel pyridyl-
chalcones, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.01.027.
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ACCEPTED MANUSCRIPT
Synthesis and antitrypanosomal activities of novel pyridyl-chalcones
Avninder S Bhambra,a Ketan C Ruparelia,b Hoon L Tan,b Deniz Tasdemir,c Hollie Burrell-Saward,d
Vanessa Yardley,d Kenneth J M Beresford,b* Randolph R J Arroo b
a School of Allied Health Sciences, Faculty of Health and Life Sciences, De Montfort University, The Gateway, Leicester, LE1
9BH, UK.
b Leicester School of Pharmacy, Faculty of Health and Life Sciences, De Montfort University, The Gateway, Leicester, LE1
9BH, UK.
c GEOMAR Centre for Marine Biotechnology, Research Unit Marine Natural Products Chemistry, GEOMAR Helmholtz Centre
for Ocean Research Kiel, Am Kiel-Kanal 44, D-24106 Kiel, Germany.
d Faculty of Infectious and Tropical Disease, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.
N
O
O
NaOH(aq), MeOH,
OoC then RT. (25%)
Br
Br
O
CHO
Br
+
O
N
Br
N
LiOH.H2O, 1,4-dioxane,
RT. (80%)
T. b.rho de sei nse IC = 0.29
50
µM

1
ACCEPTED MANUSCRIPT
Synthesis and antitrypanosomal activities of novel pyridylchalcones
Avninder S Bhambra,a Ketan C Ruparelia,b Hoon L Tan,b Deniz Tasdemir,c Hollie
Burrell-Saward,d Vanessa Yardley,d Kenneth J M Beresford,b* Randolph R J Arroo.b
a School of Allied Health Sciences, Faculty of Health and Life Sciences, De Montfort
University, The Gateway, Leicester, LE1 9BH, UK.
b Leicester School of Pharmacy, Faculty of Health and Life Sciences, De Montfort
University, The Gateway, Leicester, LE1 9BH, UK.
c GEOMAR Centre for Marine Biotechnology, Research Unit Marine Natural Products
Chemistry, GEOMAR Helmholtz Centre for Ocean Research Kiel, Am Kiel-Kanal 44, D-
24106 Kiel, Germany.
d Faculty of Infectious and Tropical Disease, London School of Hygiene and Tropical
Medicine, London WC1E 7HT, UK.
* Corresponding author: Tel: +44 (0)116 250 6356; email: kberesford@dmu.ac.uk
ABSTRACT
A library of novel pyridylchalcones were synthesised and screened against Trypanosoma
brucei rhodesiense. Eight were shown to have good activity with the most potent 8 having
an IC50 value of 0.29 µM. Cytotoxicity testing with human KB cells showed a good
selectivity profile for this compound with a selectivity index of 47. Little activity was seen
when the library was tested against Leishmania donovani. In conclusion, pyridylchalcones
are promising leads in the development of novel compounds for the treatment of human
African trypanosomiasis (HAT).
Keywords: Neglected tropical disease; Trypanosoma brucei rhodesiense; Leishmania
donovani; pyridylchalcone; Claisen-Schmidt.

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ACCEPTED MANUSCRIPT
1. Introduction
Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic
disease transmitted by the bite of tsetse flies. It occurs in 36 countries in sub-Saharan
Africa, where millions of people are at risk of infection [1-3]. HAT is caused by 2 sub-
species of Trypanosoma brucei and exists as a chronic infection with T. b. gambiense or as
an acute infection with T. b. rhodesiense. Untreated, both forms are usually fatal [1-3].
There are two clinical stages in the progression of HAT. The first corresponds to the
multiplication of the parasites in the blood and lymphatic system. The second occurs when
these cross the blood-brain barrier to infect the central nervous system causing
neurological symptoms to appear. Stage 1 can be treated with pentamidine and suramin
whereas melarsoprol, eflornithine, and their combinations with nifurtimox are the only
treatments for stage 2. All of these drugs suffer from serious drawbacks including low
efficacy, severe toxic side effects and the need for parenteral administration [1-3]. With no
vaccine available and limited therapeutic alternatives the continuing emergence of drug
resistance is seen as a major public health problem [4]. An urgent need exists for the
development of more effective, less toxic and orally available treatments for this neglected
tropical disease.
Chalcones (1,3-diphenyl-2-propen-1-ones) are a major class of natural products often found
in edible plants. They have received considerable attention due to their wide range of
biological actions and have been used as a scaffold in the development of anticancer,
antioxidant, antiangiogenic, and anti-inflammatory agents [5]. A number of natural and
synthetic chalcones have been shown to possess anti-parasitic activities including
licochalcone A 1, a natural prenylated chalcone isolated from liquorice root (Glycyrrhiza
glabra), and a potent anti-leishmanial agent [6]. However, the therapeutic use of chalcones
has been limited due to their poor bioavailability and rapid metabolic clearance from
biological systems [7]. Nevertheless, hybrid molecules incorporating the chalcone moiety
are emerging as promising leads for targeting Leishmania and Trypanosoma species [8-
11]. For example, the chalcone-benzoxaborole hybrid 2 gave 100% mouse survival rate
and complete elimination of parasites in a T. b. brucei murine model [9].

3
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O CH O
O
3
B
O CH
3
O
HO
O H
N2H
1
2
O
O
O
O
O CH
3
S
Cl
O CH
N
N
H
3
N
Cl
O CH
3
3
4
Fig. 1. Anti-parasitic chalcones
Pyridylchalcones, such as 3 and 4, have been found to exhibit both antimalarial and anti-
leishmanial activity [6, 11, 12, 13]. However, there are no examples of them being tested
against Trypanosoma brucei. With the antitrypanosomal activity of chalcone hybrids in mind
[8-11], we were intrigued to determine whether pyridylchalcones would also be active
against this target. It was anticipated that pyridyl analogues would exhibit the same low
toxicity shown by compounds containing the chalcone nucleus but with improved
bioavailability [14]. The introduction of a pyridine group will reduce the lipophilicity of the
molecule and increase water solubility, both of which should ease formulation. In addition,
lowering the lipophilicty of a chalcone and the inclusion of a heterocyclic A-ring has been
associated with an increase in anti-parasitic activity [15].
We report here the synthesis and in vitro evaluation of a series of novel pyridylchalcones
against T. b. rhodesiense and L. donovani. Their cytotoxicity against human KB cells was
also determined to establish their selectivity profile.
2. Chemistry
Attempts to prepare the 3-(3-pyridyl)chalcone 8 using standard Claisen-Schmidt conditions
with 2 equivalents of aqueous sodium hydroxide in methanol yielded an unexpected result
[16]. Instead of the desired chalcone, recrystallization from ethanol afforded the diketone 7
in 25% yield (Scheme 1). This product presumably results from conjugate addition of the
enolate of 3-bromoacetophenone to the previously formed chalcone. Similar results were
obtained when using 3-chloroacetophenone and 4-chloroacetophenone. To our knowledge
this unusual reactivity has not previously been reported for 3-pyridinecarboxaldehyde 5,

4
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N
O
O
Br
Br
(i)
7
O
CHO
Br
+
N
(ii)
O
5
6
Br
N
8
Scheme 1. Reagents and conditions: (i) NaOH(aq), MeOH, 0°C then RT (25%). (ii)
LiOH.H2O, 1,4-dioxane, RT (80%).
although it has been observed with both 2-pyridinecarboxaldehyde and 2-
quinolinecarboxaldehyde when reacted with acetophenone under similar conditions [17].
The addition of one equivalent of pyridine to these reactions was found to prevent this
conjugate addition. This was thought to be due to the pyridine competing with the aldolate
nitrogen for chelation of the metal ion in the intermediate. Unfortunately, this result was not
repeated when one equivalent of pyridine was added to our reaction.
Bhagat et al report good yields of chalcone with all three isomers of pyridinecarboxaldehyde
when reacted with 4-methoxyacetophenone and 10% lithium hydroxide monohydrate in
ethanol [18]. Using these conditions, we obtained 8 in a disappointing yield of 39%. It was
clear from TLC that 7 was still being formed during this reaction. This proved difficult to
remove by recrystallization from ethanol, and column chromatography was required to
obtain pure 8. In a bid to improve yields and simplify purification we investigated the use of
potassium hydroxide as a catalyst in 1,4-dioxane. This couple has been found to give good
yields of quinoline-2-one based chalcones which had proved difficult to prepare using other
conditions [19]. However, with 1.6 equivalents of potassium hydroxide we again obtained a
mixture of 8 and 7. Maintaining 1, 4-dioxane as the solvent but changing the base to lithium
hydroxide monohydarate proved the solution to our problems with 8 being obtained in 80%
yield after recrystallization from ethanol. TLC showed little or no diketone formation. This
method proved suitable for the synthesis of a range of pyridylchalcones (Table 1) in high
yield.
3. Results and discussion

5
ACCEPTED MANUSCRIPT
The activity of our chalcones against T. b. rhodesiense (STIB900) was determined using
the Alamar blue growth inhibition assay [20]. Their cytotoxicity against human KB cells was
also obtained in vitro (Table 1) allowing the calculation of a selectivity index (SI) for each
compound where: SI = IC50 (KB cells)/ IC50 (T. b. r). Of the 17 compounds synthesised, 8
showed good activity against T. b. rhodesiense (IC50 <0.2 mg/ mL), 8 moderate activity
(IC50 0.2-3 mg/ mL) and 1 was inactive (IC50 > 3 mg/ mL). The most active compound, with
an IC50 value of 0.29 µM, was 8 which contained a 3-pyridyl A-ring and a 3-bromophenyl B-
N
O
O
Br
Br
O
A r
A r'
CH COA r'
A rCHO
(i)
3
+
7
Scheme 2. Reagents and conditions: (i) LiOH.H2O, 1,4-dioxane, RT.
Table 1. Antitrypanosomal activity and cytotoxicity of synthesized chalcones
Ar (A-ring)
Ar' (B-ring)
SIb
N°
IC50 /
µM
T. b.
Cytotoxicity
KB Cells
rhodesiense
STIB900a
12.2
7
8
9
15
47
19
11
6
181
13.5
18.4
7.7
−
−
3-Py-
Ph-
3-Br-Ph-
3-Br-Ph-
Ph-
0.29
0.96
0.72
10 3-Py-
11 3-Py-
12 3-Py-
13 3-Py-
14 3-Py-
15 3-Py-
16 3-Py-
17 4-Py-
18 2-Py-
19 3-Br-Ph-
20 3-Py-
21 3-Py-
22 3-Py-
23 3-Py-
24 3-Py-
3-MeO-Ph-
4-Br-Ph-
2-Br-Ph-
2-MeO-Ph-
4-MeO-Ph-
3,4-(MeO)2-Ph-
3-Br-Ph-
3-Br-Ph-
3-Py-
3-Cl-Ph-
3,4-(OCH2O)-Ph-
3,4-F2-Ph-
3-HO-Ph-
3-Py-
0.41
0.34
37.8
1.14
0.94
0.98
0.65
0.40
0.69
0.62
4.83
0.72
1.15
1.02
0.011
0.005
−
2.3
7.9
23
>8
20
23
24
36
37
63
21
12
29
19
17
−
>300
22.7
21.7
22.8
23.7
14.7
43.7
12.7
59.4
20.7
21.7
17.0
−
Pentamidine
Melarsoprol
Podophyllotoxin
−
−
−
0.058
a Values are the mean of two independent experiments in triplicate.
b Selectivity index (SI) = IC50 KB cells/ IC50 T. b. rhodesiense STIB900.

6
ACCEPTED MANUSCRIPT
ring. This compound also showed good selectivity with an SI of 47. Although it was difficult
to establish a clear structure activity relationship, certain trends were apparent. The
presence of a pyridyl A-ring appears to be important for high activity. When this was
replaced by a phenyl, as in 9, the activity dropped to 0.96 µM. The position of the nitrogen
in the pyridine ring does not seem to be as crucial with the 2-pyridyl derivative 18 still
having reasonable activity (IC50 = 0.40 µM). The 4-pyridyl derivative 17 had a lower IC50
value of 0.65 µM but was still considered active. Modifying the B-ring while keeping a 3-
pyridyl A-ring can have a significant effect on activity. This was particularly apparent in 13
and 14 which both have substituents at the 2-position in the B-ring. The latter with a
methoxy group shows only moderate activity with an IC50 value of 1.14 µM, while the former
with a bromo is our only inactive compound. Moving the bromo group in 8 to the para
position produced a slight reduction in activity with 12 having an IC50 of 0.34 µM. A more
significant change was evident in 20 where the bromo group in 8 was replaced by a chloro
resulting in a doubling of the IC50 value to 0.62 µM. With a methoxy group at the meta
position in ring B, 11 showed reasonable activity (IC50 0.41 µM) but had a relatively low SI
of 6. Moving the methoxy group to the para position significantly reduced activity with 15
having an IC50 of 0.94 µM. The only other derivatives with an electron releasing group at
this position, 16 (3,4-dimethoxy) and 21 (3,4-methylenedioxy), both exhibited only moderate
activity suggesting that electron releasing groups in the B-ring are not conducive to high
activity. The phenyl derivative 10 and its 3, 4-difluoro analogue 22 were both active with the
same IC50 value of 0.72 µM. The phenolic chalcone 23 had moderate activity with an IC50
value of 1.15 µM. Taken in conjunction with our other results this could suggest that high
activity is favoured by a hydrophobic group at the 3-position in the B-ring. A 3-pyridyl group
in the B-ring is not optimum for activity with 19 and 24 giving only moderate IC50 values of
0.69 and 1.02 µM respectively. The diketone 7 was found to be inactive.
In view of the literature precedent [11, 12] and our above results with T. b. rhodesiense we
decided to screen our compound library against Leishmania donovani [21]. Using HU3
intracellular amastigotes at 30 µM only 8, 9, 12 and 20 showed any activity, with 39.7, 13.8,
11.6 and 2.7% inhibition respectively. At 10 µM no inhibition was observed. Thus, although
a number of our derivatives showed promise as potential antitrypanosomal agents they
appear to have little activity against Leishmania.

7
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4. Conclusion
Initial attempts to prepare 3-pyridylchalcones using standard Claisen-Schmidt conditions
with sodium hydroxide were unsuccessful resulting in the formation of a diketone. This
problem was overcome by the use of lithium hydroxide monohydrate as base in 1, 4-
dioxane. Of the 17 derivatives synthesised eight were shown to have good activity against
T. b. rhodesiense with the most active 8 having an IC50 value of 0.29 µM. This compound
showed good selectivity with an SI of 47. Little activity was observed when the library was
evaluated against L. donovani. In conclusion, pyridylchalcones are promising leads in the
development of novel compounds for the treatment of HAT.
5. Experimental section
5.1 Synthetic chemistry
5.1.1 General
All solvents and chemicals were used as purchased without further purification. The 1H and
13C-NMR spectra were recorded on a Bruker Avance AV400 NMR spectrometer at 30°C.
Chemical shifts are reported in δ units (ppm) relative to either TMS or the residual solvent
signal. IR spectra were recorded as KBr discs on a Perkin-Elmer 298 spectrophotometer.
HRMS was performed using a Thermo Scientific LTQ Orbitrap XL at the EPSRC UK
National Mass Spectrometry Facility at Swansea University. Melting points (uncorrected)
were determined on a Gallenkamp melting point apparatus in open glass capillary tubes.
TLC was performed on Merck Silica Gel 60F254 coated plates. Plates were visualised under
UV light (254/366nm) and stained with either 2, 4-dinitrophenylhydrazine, iodine or
phosphomolybdic acid. Fluka silica gel 60 (30-45µ) was used for flash chromatography.
Elemental analyses (C, H, N) was carried out by Warwick Analytical Services using a
CE440 elemental analyser. Results were within ±0.4% of the theoretical values.
5.1.2 1, 5-Di(3-bromophenyl)-3-(3-pyridyl)pentane-1,5-dione (7)
Sodium hydroxide solution (0.8 mL, 50% w/v, 10.00 mmol) was added to a stirred and
cooled (ice bath) solution of 3-bromoacetophenone (0.40 mL, 5.00 mmol) in ethanol (2.5
mL). 3-Pyridinecarboxylate (0.47 mL, 5.00 mmol) was added and stirring continued for 2 h

8
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before allowing the reaction to warm to RT. On the formation of a cream precipitate the
reaction was quenched with water (30 mL) and the solid removed by filtration. After
washing with water the solid was recrystallized from ethanol affording white crystals (0.305
g, 25%). mp 152-154oC; 1H NMR (CDCl3): δ 3.32 (2H, dd, J = 6.6, 16.7 Hz), 3.54 (2H, dd, J
= 6.6, 16.7 Hz), 4.08 (1H, quin, J = 6.6 Hz), 7.15-7.26 (2H, m), 7.36 (2H, t, J = 8.3, Hz),
7.60-7.70 (2H, m), 7.86 (2H, d, J = 8.3 Hz), 8.07 (2H, s), 8.48 (1H, d, J = 1.2 Hz), 8.59 (1H,
s); 13C NMR (CDCl3) δ 34.2, 44.2, 123.1, 123.5, 126.6, 130.3, 131.2, 135.4 136.3, 138.3,
138.9, 148.4, 149.2, 196.4; IR (KBr) /cm-1 1678 (C=O); HRMS found [M+1]+ 485.9686,
C22H17Br2NO2 requires [M+1]+ 485.9699; Anal. Calcd C22H17Br2NO2: C, 54.24; H, 3.52, N,
2.88; Found C, 54.08; H, 3.54; N, 2.91.
5.1.3 General method for chalcone synthesis
Lithium hydroxide monohydrate (0.63 g, 14.95 mmol) was added to a stirred solution of the
appropriate aldehyde (9.35 mmol) and ketone (9.35 mmol) in 1,4-dioxane (2.5 mL) at RT.
Upon completion, as indicated by the formation of a precipitate and confirmed by TLC the
reaction was quenched with water (30 mL). The resulting mixture was extracted with ethyl
acetate (3x30 mL). The combined organic extracts were washed with brine (50 mL), dried
(MgSO4) and the solvent removed under vacuum. The crude product was purified by either
recrystallization from ethanol or flash chromatography on silica gel.
5.1.4 (E)-1-(3-Bromophenyl)-3-(3-pyridinyl)prop-2-en-1-one (8)
Recrystallization from ethanol. Pale yellow crystals (80%). mp 126-128oC; 1H NMR (DMSO-
d6): δ 7.51 (1H, dd, J = 7.4, 3.9 Hz), 7.56 (1H, t, J = 7.7 Hz), 7.81 (1H, d, J = 15.8 Hz), 7.87-
7.92 (1H, m), 8.11 (1H, d, J = 15.8 Hz), 8.16-8.19 (1H, m), 8.35-8.42 (2H, m), 8.64 (1H, dd,
J = 2.1, 7.4 Hz), 9.06 (1H, d, J = 2.8 Hz); 13C NMR (DMSO-d6) δ 122.4, 123.4, 123.9, 127.6,
130.37, 131.04, 135.31, 135.96, 139.27, 141.4, 150.5, 151.2, 187.7; IR (KBr) /cm-1 1665
(C=O); HRMS found [M]+ 288.0025, C14H10BrNO requires [M]+ 288.0019; Anal. Calcd
C14H10BrNO: C, 58.36; H, 3.50, N, 4.86; Found C, 58.26; H, 3.44; N, 4.83.
5.1.5 (E)-1-(3-Bromophenyl)-3-(phenyl)prop-2-en-1-one (9)
1
Recrystallization from ethanol. Pale yellow crystals (67%). mp 82-84oC; H NMR (DMSO-
d6): δ 7.44-7.47 (3H, m), 7.53 (1H, t, J = 9.2 Hz), 7.76 (1H, d, J = 15.3 Hz), 7.84-7.90 (3H,
13
m), 7.93 (1H, d, J = 15.3 Hz), 8.14 (1H, dd, J = 1.2, 7.5 Hz), 8.30-8.34 (1H, m); C NMR

9
ACCEPTED MANUSCRIPT
(DMSO-d6) δ 121.6, 122.1, 127.6, 128.1, 128.9, 130.5, 130.9, 139.5, 187.8; IR (KBr) /cm-1
1661 (C=O); HRMS found [M+1]+ 288.0048, C15H11BrO requires [M+1]+ 288.0046; Anal.
Calcd C15H11BrO: C, 62.74; H, 3.86; Found C, 62.38; H, 3.87.
5.1.6 (E)-1-(Phenyl)-3-(3-pyridinyl)prop-2-en-1-one (10)
Flash chromatography, 40% ethyl acetate: hexane. Lemon yellow solid (67%). mp 93-95oC;
1H NMR (CDCl3): δ 7.36 (1H, dd, J = 4.9, 9.9 Hz), 7.49-7.56 (2H, m), 7.58-7.64 (2H, m),
7.79 (1H, d, J = 14.8 Hz), 7.91-7.97 (1H, m), 8.00-8.07 (2H, m), 8.63 (1H, dd, J = 1.0, 4.9
Hz), 8.62 (1H, d, J = 1.0 Hz); 13C NMR (CDCl3) δ 123.8, 128.6, 130.7, 132.9, 133.2, 134.6,
137.7, 140.9, 150.0, 151.1, 189.8; IR (KBr) /cm-1 1660 (C=O); HRMS found [M+1]+
210.0913, C14H11NO requires [M+1]+ 210.0913; Anal. Calcd C14H11NO: C, 80.36; H, 5.30,
N, 6.69; Found C, 80.36; H, 5.26; N, 6.60.
5.1.7 (E)-1-(3-Methoxyphenyl)-3-(3-pyridinyl) prop-2-en-1-one (11)
Flash chromatography, 50% ethyl acetate: hexane. Light yellow solid (70%). mp 83-84°C;
1H NMR (CDCl3): δ 3.90 (3H, s), 7.15 (1H, dd, J = 3.7, 8.8 Hz), 7.36 (1H, dd, J = 4.7, 8.8
Hz), 7.43 (1H, t, J = 8.4 Hz), 7.54-7.63 (3H, m), 7.79 (1H, d, J = 15.8 Hz), 7.93-7.98 (1H,
m), 8.64 (1H, dd, J = 1.9, 4.7 Hz), 8.87 (1H, d, J = 2.7 Hz). 13C NMR (CDCl3): δ 55.5, 112.9,
119.6, 120.8, 121.3, 123.8, 129.7, 130.7, 139.1, 140.9, 150.3, 151.1, 160.0, 189.5; IR (KBr)
/cm-1 1663 (C=O); HRMS found [M+1]+ 240.1019, C15H13NO2 requires [M+1]+ 240.1019;
Anal. Calcd C15H13NO2: C, 75.30; H, 5.48, N, 5.85; Found C, 75.53; H, 5.42; N, 5.67.
5.1.8 (E)-1-(4-Bromophenyl)-3-(3-pyridinyl)prop-2-en-1-one (12)
1
Recrystallization from ethanol. Fine off white crystals (80%). mp 126-128oC; H NMR
(CDCl3): δ 7.36 (1H, dd, J = 5.3, 9.5 Hz), 7.54 (1H, d, J = 15.9 Hz), 7.65 (2H, d, J = 8.4 Hz),
7.80 (1H, d, J = 15.8 Hz), 7.89 (2H, d, J = 8.4 Hz), 7.93-7.98 (1H, m), 8.65 (1H, d, J = 3.1
Hz), 8.86 (1H, s); 13C NMR (CDCl3): δ 123.2, 123.8, 128.3, 130.1, 130.5, 131.8, 134.6,
141.5, 150.1, 151.3, 188.7; IR (KBr) /cm-1 1663 (C=O); HRMS found [M+1]+ 289.9997,
C14H10BrNO requires [M+1]+ 289.9998; Anal. Calcd C14H10BrNO: C, 58.36; H, 3.50, N, 4.86;
Found C, 58.34; H, 3.46; N, 4.81.
5.1.9 (E)-1-(2-Bromophenyl)-3-(3-pyridinyl)prop-2-en-1-one (13)

10
ACCEPTED MANUSCRIPT
Recrystallization from ethanol. Pale yellow crystals (37%). mp 248-250oC (decomposed);
1H NMR (CDCl3): δ 7.38-7.42 (2H, m), 7.52 (1H, d, J = 15.6 Hz), 7.67 (1H, dd, J = 2.2, 5.4
Hz), 7.79 (1H, d, J = 15.6 Hz), 7.93-8.14 (2H, m), 8.15 (1H, t, J = 7.2 Hz), 8.65 (1H, d, J =
4.7 Hz), 8.87 (1H, s); 13C NMR (CDCl3): δ 123.1, 127.0, 130.3, 131.6, 134.6, 136.0, 139.5,
141.8, 150.2, 151.4, 186.1; IR (KBr) /cm-1 1666 (C=O); HRMS found [M+1]+ 289.9999,
C14H10BrNO requires [M+1]+ 289.9998; Anal. Calcd C14H10BrNO: C, 58.36; H, 3.50, N, 4.86;
Found C, 58.30; H, 3.42; N, 4.88.
5.1.10 (E)-1-(2-Methoxyphenyl)-3-(3-pyridinyl)prop-2-en-1-one (14)
1
Recrystallization from ethanol. Pale yellow crystals (44%). mp 91-93oC; H NMR (DMSO-
d6): δ 3.91 (3H, s), 7.02 (1H, d, J = 8.3 Hz), 7.07 (1H, t, J = 7.5 Hz), 7.35 (1H, dd, J = 4.2,
7.9 Hz), 7.48-7.54 (1H, m), 7.50 (1H, d, J = 15.8 Hz), 7.64 (1H, d, J = 15.8 Hz), 7.67 (1H,
dd, J = 1.7, 7.5 Hz), 7.89 (1H, d, J = 7.9 Hz), 8.61 (1H, d, J = 4.2 Hz), 8.82 (1H, s); 13C
NMR (CDCl3): δ 55.5, 11.4, 120.7, 123.5, 128.7 130.4, 130.6, 133.5, 134.7, 138.9, 149.9,
150.8, 158.3, 192.1; IR (KBr) /cm-1 1656 (C=O); HRMS Found [M+1]+ 240.1017, C15H13NO2
requires [M+1]+ 240.1019; Anal. Calcd C15H13NO2: C, 75.30; H, 5.48, N, 5.85; Found C,
75.14; H, 5.58; N, 5.64.
5.1.11 (E)-1-(4-Methoxyphenyl)-3-(3-pyridinyl)prop-2-en-1-one (15) [12]
Recrystallization from ethanol. Yellow solid (60%). mp 105-106°C; 1H NMR (CDCl3): δ 3.90
(3H, s), 7.00 (2H, d, J = 8.8 Hz), 7.35 (1H, dd, J = 4.6, 8.3 Hz), 7.61 (1H, d, J = 16.2 Hz),
7.78 (1H, d, J = 16.2 Hz), 7.92-7.96 (1H, m), 8.04 (2H, d, J = 8.8 Hz), 8.62 (1H, dd, J = 4.2,
1.3 Hz), 8.87 (1H, d, J = 2.5 Hz); 13C NMR (CDCl3): δ 55.6, 114.0, 123.8, 130.7, 130.9,
134.6, 137.4, 140.1, 149.9, 150.9, 163.7, 188.0; IR (KBr) /cm-1 1662 (C=O); HRMS found
[M+1]+ 240.1017, C15H13NO2 requires [M+1]+ 240.1019; Anal. Calcd C15H13NO2.0.15H2O:
C, 74.46; H, 5.54, N, 5.79; Found C, 74.41; H, 5.43; N, 5.77.
5.1.12 (E)-1-(3, 4-Dimethoxyphenyl)-3-(3-pyridyl)prop-2-en-l-one (16)
Flash chromatography, 60-80% ethyl acetate: hexane. Light yellow solid (67%). mp 91-
1
93°C; H NMR (CDCl3): δ 3.99 (6H, s), 6.94 (1H, d, J = 7.5 Hz), 7.36 (1H, dd, J = 5.0, 8.0
Hz), 7.63 (1H, d, J = 15.9 Hz), 7.64 (1H, d, J = 2.0 Hz), 7.68 (1H, dd, J = 2.0, 7.5 Hz), 7.78
(1H, d, J = 15.9 Hz), 7.93-7.96 (1H, m), 8.63 (1H, dd, J = 1.5, 5.0 Hz), 8.87 (1H, d, J = 1.5
Hz); 13C NMR (CDCl3) δ 56.2, 110.0, 110.8, 122.7, 123.7, 124.5, 130.9, 134.6, 140.1,

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149.40 149.9, 150.9, 153.7, 187.9; IR (KBr) /cm-1 1655 (C=O); HRMS found [M+1]+
270.1125, C16H15NO3 requires [M+1]+ 270.1125; Anal. Calcd C16H15NO3: C, 71.36; H, 5.61,
N, 5.20; Found C, 71.03; H, 5.65; N, 5.14.
5.1.13 (E)-1-(3-Bromophenyl)-3-(4-pyridinyl)prop-2-en-1-one (17)
1
Recrystallization from ethanol. Pale yellow crystals (34%). mp 104-106oC; H NMR
(CDCl3): δ 7.34 (1H, t, J = 7.1 Hz), 7.41 (2H, d, J = 6.1 Hz), 7.53 (1H, d, J = 15.6 Hz), 7.64
(1H, d, J = 15.6 Hz), 7.66-7.69 (1H, m), 7.87 (1H, dt, J = 1.0, 7.1 Hz), 8.07 (1H, t, J = 1.0
Hz), 8.63 (2H, d, J = 6.1 Hz); 13C NMR (CDCl3): δ 122.1, 123.2, 125.4, 127.1, 130.4, 131.6,
136.2, 139.2, 141.8, 142.4, 150.7, 188.4; IR (KBr) /cm-1 1666 (C=O); HRMS found [M+1]+
289.9998, C14H10BrNO requires [M+1]+ 289.9998; Anal. Calcd C14H10BrNO: C, 58.36; H,
3.50, N, 4.86; Found C, 58.20; H, 3.46; N, 4.81.
5.1.14 (E)-1-(3-Bromophenyl)-3-(2-pyridinyl)prop-2-en-1-one (18)
1
Recrystallization from ethanol. Pale yellow crystals (47%). mp 110-112oC; H NMR
(CDCl3): δ 7.40-7.49 (2H, m), 7.55 (1H, t, J = 8.8 Hz), 7.72 (1H, d, J = 16.0 Hz), 7.85-8.00
(2H, m), 8.10-8.15 (2H, m), 8.20 (1H, s), 8.70 (1H, d, J = 4.5 Hz); 13C NMR (CDCl3): δ
125.0, 126.9, 127.5, 130.9, 135.7, 137.2, 143.8, 150.1, 152.6, 188.3; IR (KBr) /cm-1 1664
(C=O); HRMS found [M+1]+ 289.0056, C14H10BrNO requires [M+1]+ 289.0052; Anal. Calcd
C14H10BrNO: C, 58.36; H, 3.50, N, 4.86; Found C, 58.30; H, 3.45; N, 4.86.
5.1.15 (E)-3-(3-Bromophenyl)-1-(3-pyridinyl)prop-2-en-1-one (19)
1
Recrystallization from ethanol. Pale yellow crystals (40%). mp 114-116oC; H NMR
(CDCl3): δ 7.32-7.44 (2H, m), 7.52 (1H, d, J = 16.5 Hz), 7.72 (1H, d, J = 6.5 Hz), 7.81 (1H,
d, J = 16.5 Hz), 7.93-7.98 (2H, m), 8.14 (1H, t, J = 1.5Hz), 8.64 (1H, d, J = 2.7 Hz), 8.87
(1H, s); 13C NMR (CDCl3): δ 123.1, 123.2, 126.6, 130.4, 131.6, 134.6, 136.0, 142.3, 150.2,
151.4, 188.4; IR (KBr) /cm-1 1658 (C=O); HRMS found [M+1]+ 289.0055, C14H10BrNO
requires [M+1]+ 289.0052; Anal. Calcd C14H10BrNO: C, 58.36; H, 3.50, N, 4.86; Found C,
58.30; H, 3.45; N, 4.81.
5.1.16 (E)-1-(3-Chlorophenyl)-3-(3-pyridinyl)prop-2-en-1-one (20)
Recrystallization from ethanol. Off white crystals (65%). mp 128-130oC; 1H NMR (CDCl3): δ
7.37 (1H, dd, J = 5.2, 7.7 Hz), 7.45 (1H, t, J = 6.6 Hz), 7.51-7.60 (2H, m), 7.80 (1H, d, J =

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16.0 Hz), 7.89 (1H, d, J = 7.6 Hz), 7.94-8.02 (2H, m), 8.65 (1H, d, J = 5.2 Hz), 8.86 (1H, s);
13C NMR (CDCl3): δ 123.1, 123.8, 126.6, 128.6, 130.1, 130.4, 133.0, 135.1, 139.3, 141.8,
150.2, 151.4, 188.4; IR (KBr) /cm-1 1665 (C=O); HRMS found [M+1]+ 244.0524, C14H10NOCl
requires [M+1]+ 244.0524; Anal. Calcd C14H10NOCl: C, 69.00; H, 4.14, N, 5.75; Found C,
68.86; H, 4.12; N, 5.66.
5.1.17 (E)-1-(3,4-Methylenedioxyphenyl)-3-(3-pyridinyl)prop-2-en-1-one (21)
Recrystallization from ethanol. Off white crystals (55%). mp 138-140oC; 1H NMR (CDCl3): δ
6.10 (2H, s), 6.92 (1H, d, J = 7.8 Hz), 7.37 (1H, dd, J = 4.3, 7.1 Hz) 7.54 (1H, d, J = 2.1 Hz),
7.57 (1H, d, J = 14.9 Hz) 7.67 (1H, dd, J = 2.1, 7.8 Hz), 7.78 (1H, d, J = 14.9 Hz), 7.93-7.98
(1H, m), 8.63 (1H, dd, J = 1.1, 4.3 Hz), 8.86 (1H, d, J = 1.2 Hz); 13C NMR (CDCl3) δ 102.0,
108.0, 109.4, 123.5, 123.8, 124.9, 130.8, 132.5, 134.6, 140.4, 148.5, 149.9, 151.0, 152.0,
187.6; IR (KBr) /cm-1 1666 (C=O); HRMS found [M+1]+ 254.0811, C15H11NO3 requires
[M+1]+ 254.0812; Anal. Calcd C15H11NO3: C, 71.14; H, 4.38, N, 5.53; Found C, 70.91; H,
4.36; N, 5.48.
5.1.18 (E)-1-(3,4-Difluorophenyl)-3-(3-pyridinyl)prop-2-en-1-one (22)
Recrystallization from ethanol. Pale green crystals (47%). mp 120-121oC; 1H NMR (DMSO-
d6): δ 7.50-7.53 (1H, m), 7.63-7.70 (1H, m), 7.78 (1H, d, J = 15.0 Hz), 8.10 (2H, d, J = 15.0
Hz), 8.24-8.29 (1H, m), 8.37-8.39 (1H, m), 8.64 (1H, dd, J = 1.5, 4.3 Hz), 9.05 (1H, d, J =
1.2, 4.3 Hz); 13C NMR (DMSO-d6) δ 117.9, 118.1, 123.1, 126.5, 130.3, 134.7, 141.4, 150.5,
150.9, 186.5; IR (KBr) /cm-1 1661 (C=O); HRMS found [M]+ 246.0724, C14H9NOF2 requires
[M]+ 246.0725; Anal. Calcd C14H9F2NO: C, 68.57; H, 3.70, N, 5.71; Found C, 68.48; H,
3.70; N, 5.66.
5.1.19 (E)-1-(3-Hydroxyphenyl)-3-(3-pyridinyl)prop-2-en-1-one (23)
After quenching with water the mixture was acidified with 1 M HCl before extraction with
ethyl acetate. Recrystallization from ethanol. Light yellow crystals (55%). mp 182-184oC;
1H NMR (DMSO-d6): δ 7.09 (1H, dd, J = 2.1, 8.5 Hz), 7.39 (1H, t, J = 7.7 Hz)), 7.48-7.51
(2H, m), 7.65 (1H, d, J = 6.8 Hz), 7.75 (1H, d, J = 14.9 Hz), 8.01 (1H, d, J = 14.9 Hz), 8.33
13
(1H, d, J = 7.7 Hz)), 8.62 (1H, s), 9.02 (1H, s), 9.85 (1H, brs, OH); C NMR (DMSO-d6) δ
114.7, 119.7, 120.5, 123.9, 124.1, 129.9, 135.1, 138.7, 140.4, 150.3, 157.8, 188.9; IR (KBr)

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/cm-1 1660 (C=O); HRMS found [M+1]+ 226.0861, C14H11NO2 requires [M+1]+ 226.0863;
Anal. Calcd C14H11NO2: C, 74.65; H, 4.92, N, 6.22; Found C, 74.51; H, 4.92; N, 6.03.
5.1.20 (E)-1-(3-pyridinyl)-3-(3-pyridinyl)prop-2-en-1-one (24)
1
Recrystallization from ethanol. Pale yellow crystals (41%). mp 148-150oC; H NMR
(DMSO-d6): δ 7.51 (1H, dd, J = 4.8, 9.0 Hz), 7.62 (1H, dd, J = 4.8, 9.0 Hz), 7.83 (1H, d, J =
13.7 Hz), 8.12 (1H, d, J = 13.7 Hz), 8.38 (1H, d, J = 7.2 Hz), 8.48 (1H, d, J = 7.2 Hz), 8.63
(1H, d, J = 4.8 Hz), 8.85 (1H, d, J = 4.8 Hz), 9.06 (1H, s), 9.37 (1H, s); 13C NMR (DMSO-d6)
δ 123.6, 123.9, 130.3, 132.5, 135.3, 141.3, 149.8, 151.3, 153.5, 188.3; IR (KBr) /cm-1 1666
(C=O); HRMS found [M+1]+ 211.0864, C13H10N2O requires [M+1]+ 211.0866; Anal. Calcd
C13H10N2O: C, 74.27; H, 4.79, N, 13.33; Found C, 73.90; H, 4.79; N, 13.36.
5.2 Biological assays
5.2.1 Trypanocidal activity
Trypanosoma brucei rhodesiense STIB 900, a clone of a population isolated in 1982 from a
patient in Tanzania. Stock drug solutions were prepared in DMSO at 20 mM and further
diluted to the appropriate concentration using medium. Assays were performed in 96-well
microtiter plates with each well containing 100 ml of parasite culture (1 x 103 bloodstream
forms) with serial drug dilutions at 37°C for 72 h in 5% CO2. Each compound was tested in
triplicate with 30 mg/ml the highest concentration of compound used and a 3-fold serial
dilution was performed down to a suitable concentration to obtain an IC50 value. Control
wells were without drug and blanks were medium only. After 72 h of incubation the plates
were inspected to assure growth in control wells and to determine the minimum inhibitory
concentration (MIC). Subsequently, 20 µL of Alamar Blue™ was added to each well and the
plates incubated for another 2-4 h. Plates were read on a Gemini Plate Reader (Molecular
Devices) using an excitation wave length of 530 nm and an emission wave length of 580
nm (cut off 550 nm). IC50 values were calculated using Prism © software. Pentamidine and
melarsoprol were used as positive controls.
5.2.2 Toxicity on KB cells
KB cells, derived from a human carcinoma of the nasopharynx and typically used in assays
for antineoplastic agents, were maintained in RPMI 1640 medium (Sigma, UK), 10% heat-

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inactivated foetal calf serum, 37°C, 5% CO2. KB cell monolayers, prepared in 96-well
plates, were exposed to the test compounds for 72 h. Podophyllotoxin was used as a
positive control. 20 µL of Alamar Blue™ was added to each well. After a further 2–4 h
incubation the plates were read (Molecular Devices Gemini™) at EX/EX 530/580, cut-off
550 nm. The IC50 values were calculated by sigmoidal regression analysis (Prism ©).
Acknowledgements
The Higher Education Funding Council for England (HEIF programme). The EPSRC UK
National Mass Spectrometry Facility at Swansea University.
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ACCEPTED MANUSCRIPT
Highlights
•
Improved Claisen-Schmidt condensation using lithium hydroxide monohydrate
in 1,4-dioxane.
•
Pyridylchalcones show good activity and selectivity against Trypanosoma
brucei.
•
•
Pyridylchalcones show little activity against Leishmania donovani.
Promising leads in the development of novel compounds for the treatment of
sleeping sickness.