Synthesis and antileishmanial activity of fluorinated rhodacyanine analogues: The ‘fluorine-walk’ analysis

Article abstract of DOI:10.1016/j.bmc.2019.115187

In a search for potent antileishmanial drug candidates, eighteen rhodacyanine analogues bearing fluorine or perfluoroalkyl substituents at various positions were synthesized. These compounds were tested for their inhibitory activities against Leishmania martiniquensis and L. orientalis. This ‘fluorine-walk’ analysis revealed that the introduction of fluorine atom at C-5, 6, 5′, or 6′ on the benzothiazole units led to significant enhancement of the activity, correlating with the less negative reduction potentials of the fluorinated analogues confirmed by the electrochemical study. On the other hand, [sbnd]CF₃ and [sbnd]OCF₃ groups were found to have detrimental effects, which agreed with the poor aqueous solubility predicted by the in silico ADMET analysis. In addition, some of the analogues including the difluorinated species showed exceptional potency against the promastigote and axenic amastigote stages (IC₅₀ = 40–85 nM), with the activities surpassing both amphotericin B and miltefosine.

Full text of DOI:10.1016/j.bmc.2019.115187

Journal Pre-proofs
Synthesis and antileishmanial activity of fluorinated rhodacyanine analogues:
the ‘fluorine-walk’ analysis
Thitiya Lasing, Atchara Phumee, Padet Siriyasatien, Kantima Chitchak,
Parichatr Vanalabhpatana, Kit-Kay Mak, Chew Hee Ng, Tirayut Vilaivan,
Tanatorn Khotavivattana
PII:
S0968-0896(19)31342-2
https://doi.org/10.1016/j.bmc.2019.115187
BMC 115187
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
11 August 2019
16 October 2019
25 October 2019
Please cite this article as: T. Lasing, A. Phumee, P. Siriyasatien, K. Chitchak, P. Vanalabhpatana, K-K. Mak, C.
Hee Ng, T. Vilaivan, T. Khotavivattana, Synthesis and antileishmanial activity of fluorinated rhodacyanine
analogues: the ‘fluorine-walk’ analysis, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.bmc.2019.115187
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Synthesis and antileishmanial activity of
fluorinated rhodacyanine analogues: the
‘fluorine-walk’ analysis
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Thitiya Lasing, Atchara Phumee, Padet Siriyasatien, Kantima Chitchak, Parichatr Vanalabhpatana, Kit-Kay
Mak, Chew Hee Ng, Tirayut Vilaivan and Tanatorn Khotavivattana

Bioorganic & Medicinal Chemistry
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Synthesis and antileishmanial activity of fluorinated rhodacyanine analogues: the
‘fluorine-walk’ analysis
Thitiya Lasinga , Atchara Phumeeb, c , Padet Siriyasatienc , Kantima Chitchakd , Parichatr Vanalabhpatanae ,
Kit-Kay Makf , Chew Hee Ngf , Tirayut Vilaivang and Tanatorn Khotavivattanaa, h,

aCenter of Excellence in Natural Products Chemistry, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
bThai Red Cross Emerging Infectious Diseases-Health Science Centre, World Health Organization Collaborating Centre for Research and Training on Viral
Zoonoses, Chulalongkorn Hospital, Bangkok 10330, Thailand
cVector Biology and Vector Borne Disease Research Unit, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330,
Thailand
dProgram of Petrochemistry and Polymer Science, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
eElectrochemistry and Optical Spectroscopy Center of Excellence, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330,
Thailand
fDepartment of Pharmaceutical Chemistry, School of Pharmacy, International Medical University, Kuala Lumpur 57000, Malaysia
gOrganic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
hResearch Unit for Metabolic Bone Disease in CKD Patients, Chulalongkorn University, Bangkok 10330, Thailand
———
ARTICLE INFO
ABSTRACT
 Corresponding author. Tel.: +66-2-218-7621; fax: +66-2-218-7598; e-mail: tanatorn.k@chula.ac.th
Article history:
Received
Received in revised form
Accepted
In a search for potent antileishmanial drug candidates, eighteen rhodacyanine analogues bearing
fluorine or perfluoroalkyl substituents at various positions were synthesized. These compounds
were tested for their inhibitory activities against Leishmania martiniquensis and L. orientalis.
This ‘fluorine-walk’ analysis revealed that the introduction of fluorine atom at C-5, 6, 5’, or 6’
on the benzothiazole units led to significant enhancement of the activity, correlating with the
less negative reduction potentials of the fluorinated analogues confirmed by the electrochemical
study. On the other hand, -CF3 and -OCF3 groups were found to have detrimental effects, which
agreed with the poor aqueous solubility predicted by the in silico ADMET analysis. In addition,
some of the analogues including the difluorinated species showed exceptional potency against
the promastigote and axenic amastigote stages (IC50 = 40-85 nM), with the activities surpassing
both amphotericin B and miltefosine.
Available online
Keywords:
Antileishmanial activity
Drug discovery
Fluorine
Rhodacyanines
Structure-activity relationships
2009 Elsevier Ltd. All rights reserved.
1. Introduction
medications suffer from various limitations such as high cost,
lack of efficacy, serious side effects, low bioavailability, and
Leishmaniasis is a vector-borne disease caused by over 20
species of the protozoan parasites belonging to the genus
Leishmania which are transmitted between mammalian hosts by
sand flies.1 According to the World Health Organization (WHO),
leishmaniasis was classified as one of the major neglected
tropical diseases (NTDs), ranking second only to malaria.
Currently, there are more than 12 million people across 98
countries affected by leishmaniasis and it is estimated that
approximately 2 million new cases will occur each year with
more than 350 million people who are at risk of contracting the
disease.2 In Thailand, an increasing number of autochronous
leishmaniasis infections have been reported; L. martiniquensis, L.
donovani, L. infantum, and L. orientalis, the indigenous species
of Thailand, were responsible for these infections.3 Depending on
the species of the parasite, there are various symptoms associated
with the leishmaniases ranging from skin ulcers to life-
threatening internal organ failure;4 hence, a potent medication is
highly in demand. However, only a handful of drugs are effective
for the treatment of leishmaniasis and the currently available
drug resistance.5 Different Leishmania species may also have
varied susceptibility against each drug; for example, miltefosine,
(a) previous work
(b) this work
X
CH3
N
CH3
N
Rf
S
S
Rf'
S
S
N
CH3
Cl
N
CH3
Cl
S
S
N
N
O
C2H5
: IC50a = 0.106 M; selectivityb = 75
(SJL-01); IC50a = 0.011 M; selectivityb = >15000
O
C2H5
11a 11q
X
X
H
F
-
=
=
Rf Rf'
or
F CF OCF
, ,
3 3
=
Figure 1. (a) The enhancement of antileishmanial activity and selectivity by
an introduction of a fluorine atom in rhodacyanines;6 (b) The ‘fluorine-walk’
analysis of the fluorinated rhodacyanine analogues (this work); a in vitro IC50
b
against promastigotes of L. donovani; Calculated as IC50 against L-6
myocytes / IC50 against promastigotes of L. donovani.
the only approved oral antileishmanial drug, has been shown to
be highly effective against L. donovani (IC50 = 0.430 µM).6
However, our preliminary result revealed that the potency was

drastically reduced in the case of L. martiniquensis (IC50 = 15.8
µM).
could lead to various beneficial effects, for instance, the increase
in the potency, membrane permeability, and metabolic stability.
Herein, we report the ‘fluorine-walk’ analysis of the
antileishmanial rhodacyanines to gain further understanding of
the effect of fluorine in this class of compounds. A series of
rhodacyanines containing fluorine and/or perfluoroalkyl
substituents at different positions on one or both benzothiazole
units were synthesized (Figure 1b). These compounds were then
tested for their antileishmanial properties against L.
martiniquensis and L. orientalis to study the structure-activity
relationship, which will provide crucial knowledge for further
development of rhodacyanine-based antileishmanial drugs.
To date, several research groups have introduced a variety of
synthetic compounds as potential drug candidates for treating
leishmaniasis,7 one of which is a series of compounds belonging
to the class of rhodacyanine dyes. It was reported that these
compounds have a diverse range of bioactivities, including
anticancer,8 antimalarial,9 and antileishmanial properties.6,10 One
of the most potent compounds among the series was reported by
Ihara and co-workers in 2010 (Figure 1a).6 The fluorinated
rhodacyanine analogue, SJL-01, exhibited exceptionally high
efficacy and excellent selectivity index (>15000) in the in vivo
testing against L. donovani. Comparing with the non-fluorinated
analogue, a single fluorine atom at the benzothiazolium unit
enhanced the activity by almost 10 times and the selectivity was
improved by over 200 times. Nevertheless, the exact role of the
fluorine substituent in SJL-01 has not yet been clarified and their
activities against L. martiniquensis and L. orientalis are
unknown. Fluorine has found widespread applications in drug
discovery and development owing to its unique properties such
as high electronegativity, low polarizability and the extremely
strong C–F bond strength.11 Installation of fluorine or
perfluoroalkyl groups at a suitable position in drug molecule
2. Results and discussion
The fluorinated rhodacyanines were synthesized starting by
preparing a range of novel fluorinated or perfluoroalkylated
benzothiazolium salts 6a-6h to be used as the fluorine-containing
building blocks. This can be achieved using one of the two
possible pathways (Scheme 1). In pathway I, o-bromoanilines
with various fluorine-containing substituents were acetylated to
give 1a-1e,12 then converted into o-bromothioacetamide 2a-2e in
good yields by the use of Lawesson’s reagent.13
S
Pathway I
O
AcOH
EtOAc, 110 o
NH2
HN
CH3
Br
HN
CH3 Lawesson's
Pd2(dba)3, JohnPhos
Br
KOtBu, dioxane, 80 o
C
C
reagent
Br
Rf
Rf
or
Rf
THF, r.t.
(42-72%)
Ac2O (1M in DCM), r.t.
(54-95%)
TsO
1a 1e
-
2a 2e
-
CH3
(96-100%)
N
N
a
b
c
d
e
Rf = :3-F, :5-F, :6-F, :4-CF3 or :4,5-diF
MeOTs
130 o
CH3
Rf
Rf
CH3
C
S
S
(39-89%)
6a 6h
-
5a 5h
-
Pathway II
O
S
5a 6a
, : Rf = H
5b 6b
,
: Rf = 4-F
: Rf = 5-F
: Rf = 6-F
NH2
K3Fe(CN)6, NaOH
AcOH
EtOAc, 110 o
or
HN
CH3
Lawesson's
reagent
HN
CH3
5c 6c
,
EtOH/H2O, 80 o
C
C
5d 6d
,
Rf
5e,6e: Rf = 7-F
5f 6f
Rf
Rf
THF, r.t.
(50-59%)
,
: Rf = 6-CF3
Ac2O (1M in DCM), r.t.
(71-97%)
(71-81%)
5g 6g
5h 6h
,
,
: Rf = 6-OCF3
: Rf = 5,6-diF
3a 3b
-
4a 4b
-
a
b
Rf = :4-F or :4-OCF3
Scheme 1. The synthesis procedures of fluorinated or perfluoroalkylated benzothiazolium tosylates 6a-6h.
CH3
OTs
CH3
N
O
Rf'
Ph
Ph
C
CH3
N
S
6a 6f
N
N
Ph
N
-
H
1)
S
S
S
CH3CN, 70 o
1) Ac2O, CH3CN, 50 o
C
Rf'
S
S
S
S
N
N
2) NEt3, 60 o
C
2) Propionic anhydride
N
O
C2H5
O
C2H5
NEt3, 110 o
C
O
C2H5
8a 8f
-
7
(56-77%)
(97%)
MeOTs, DMF
115 oC, 3 h
(40-80%)
CH3
N
CH3
N
OTs
CH3
OTs
CH3
N
Rf
Rf
Rf'
S
N
S
6a 6h
-
S
SCH3
S
Rf'
S
N
CH3
X
S
NEt3, CH3CN, 75 o
C
9a 9f
-
O
C2H5
N
8a 9a
,
: Rf' = H
O
C2H5
(66-84%)
8b 9b
,
: Rf' = 4-F
: Rf' = 5-F
: Rf' = 6-F
: Rf' = 7-F
: Rf' = 6-CF3
8c 9c
,
10a 10q
-
X = OTs
X = Cl
HCl, MeOH, 80 o
(43-90%)
C
8d 9d
,
8e 9e
,
11a 11q
-
8f 9f
,
Scheme 2. The construction of fluorine-containing rhodacyanines from 3-ethylrhodanine and the synthesized benzothiazolium tosylates.

Next, palladium-catalyzed intramolecular cyclization using
Pd2(dba)3/JohnPhos as the catalytic system led to the formation
of substituted benzothiazoles in moderate yields.13 Alternatively,
pathway II can also be followed as a more economical option if
regioselectivity is not a problem, starting from simple para-
substituted anilines. After being converted into thioacetamides 4a
and 4b using the same protocol as pathway I, the Jacobson
cyclization using potassium ferricyanide (K3Fe(CN)6) as a
reagent led to C–H activation/cyclization to form the desired
benzothiazoles in moderate yields.14 Finally, methylation of the
fluorine-containing benzothiazoles prepared from both pathways
using methyl p-toluenesulfonate (MeOTs) provided the desired
fluorinated benzothiazolium tosylates 6a-6h.
47.9%, respectively for the latter group. Nevertheless, all the
fluorinated analogues exhibited higher inhibitory effect than the
non-fluorinated analogue 11a (36.8%) under the same conditions,
hence confirming that the introduction of the fluorine substituent
is crucial for the good activity. Moreover, the result revealed that
the novel fluorinated analogue 11d was the most effective species
against the promastigotes of L. orientalis with the IC50 value as
low as 40 nM. On the other hand, the introduction of
perfluoroalkyl substituents such as trifluoromethyl (11j) and
trifluoromethoxyl (11k) led to drastic decrease in the
antileishmanial activity, especially against the promastigote of L.
martiniquensis. This effect is also observed when an additional
trifluoromethyl group was installed at the position 6’ of the
previously described SJL-01 (11c), resulting in the analogue 11q
with almost no antileishmanial activity at the tested concentration
range. Moreover, since the installation of fluorine substituents on
either position 5, 6, 5’, or 6’ led to the analogues with highest
potency, we also attempted to investigate the difluorinated
analogues with various combinations of these positions (11l-
11p). The result showed that most of the difluorinated analogues
exhibited lower inhibition activity against promastigotes of both
Leishmania species, except for 11l with fluorine substituents at
positions 5 and 6 which exhibited comparable activity to those of
11c and 11d. On the other hand, for the axenic amastigote stage,
most difluorinated analogues (11l, 11m, 11o, and 11p) showed
similar activity to that of 11c with IC50 in the range of 77 to 85
nM, which are all slightly more potent than amphotericin B (IC50
= 108 nM). Finally, the cytotoxicity of each analogue against
normal cells was evaluated using J774A.1 macrophage as a
model. At 0.25 M, almost all compounds exhibited no
observable toxicity except for the two trifluoromethylated
analogues (11j and 11q). Even when the concentration was
increased to 4.0 M, the toxicity against normal cells of most
analogues remained low to moderate.
The construction of fluorinated rhodacyanines from the
benzothiazolium building blocks was subsequently achieved
using the established procedure with slight modifications
(Scheme 2).15 In this process, 3-ethylrhodanine was treated with
N,N-diphenylformamidine followed by propionic anhydride and
triethylamine to give 7. Reaction between 7 and the first
benzothiazolium salt to provide 2-thioxothiazolidin-4-ones 8a-8f,
which were then S-methylated with MeOTs to give the
2-(methylthio)-4-oxo-4,5-dihydrothiazol-3-ium intermediates 9a-
9f. These intermediates were then condensed with another
benzothiazolium salt to give 10a-10q, followed by anion
exchange of the resulting salts with hydrochloric acid in
methanol at 80 °C to furnish the desired rhodacyanine analogues
11a-11q with high overall yields. The structures of all analogues
are tabulated in Table 1.
The synthesized rhodacyanine analogues (10c and 11a-11q)
were screened for their antileishmanial activities against the
promastigotes of L. martiniquensis and L. orientalis (Table 2).
The non-fluorinated analogue 11a exhibited moderate
antileishmanial activities (IC50 = 0.658 µM and 0.302 µM,
respectively). The previously described SJL-01 (11c) with a
single fluorine substituent at position 5 is almost 8 times more
effective against the promastigotes of L. martiniquensis;
however, the difference is smaller in the case of L. orientalis
(approximately 3 times more effective). No significant difference
in the activities between the chloride salt 11c and the tosylate salt
10c was observed. These findings are in agreement with the
previously reported data when these compounds were tested
against L. donovani.7 Although the IC50 of 11c are slightly higher
than that of amphotericin B, it is almost 200 times more potent
than miltefosine against the promastigotes of L. martiniquensis.
Additionally, when tested against axenic amastigotes of L.
orientalis, 11c was almost 12 times more potent than the non-
fluorinated analogue, and in this case, it is also more potent than
both amphotericin B and miltefosine. The strong inhibition
ability towards the amastigote stage is highly desirable for the
further development of antileishmanial drugs, since it is the stage
found in patients who will be receiving the medication while the
promastigote stage is found in sand flies. Unfortunately, this
study cannot be tested with axenic amastigotes of L.
martiniquensis due to the limited capacity to grow outside the
host cell.16
To become useful drugs and to save overall cost of new drug
development, new compounds need early evaluation for their
drug-likeness and their absorption, distribution, metabolism,
excretion, and toxicity (ADMET) properties before moving into
clinical trial.17 The in silico ADMET properties of the
synthesized rhodacyanine analogues were predicted using
Maestro Schrödinger’s QikProp tool18 and are tabulated in Table
1. Compounds with reactive functional groups, such as aldehyde
or ketone, are known to react in a non-selective manner with
proteins, resulting in unjustified success in hits. Thus, the scores
of ‘0’ for #rtvFG across the series of rhodacyanine analogues
suggest that the activities shown by rhodacyanine analogues in
the Table 2 reflect their true biological activities, and not
chemical reactivities of the compounds.19 All the compounds
have QPlogPo/w values outside the recommended range,
indicating that they are rather hydrophobic. By comparing the
predicted octanol/water partition coefficient (QPlogPo/w) and
aqueous solubility (QPlogS) of fluorinated (11b-11q) and non-
fluorinated (11a) rhodacynine analogues, it is noted that the
fluorine substituent on either or both rings (compounds 11b-11i
or 11l-11p, respectively) have no effect on these properties. The
compounds also lie within the limit of predicted brain/blood
partition coefficient QPlogBB and compound 11a was predicted
to have no central nervous system (CNS) activity, while all of the
fluorinated analogues show good activity with score of 1 each,
suggesting their potential use for cerebral leishmaniasis.20 In
addition, they have high cell permeation (QPPCaco) values with
scores of more than 500, and have high percent human oral
absorption at 100%. These scores signify that the compounds
have good permeation.
Interestingly, the analogues with varied positions of the
fluorine substituent on both benzothiazole units (11b-11i)
showed significant difference in their antileishmanial activities.
In general, the installation of fluorine at positions 5 (11c), 6
(11d), 5’ (11g), or 6’ (11h) improved the potency more
significantly than at positions 4 (11b), 7 (11e), 4’ (11f), or 7’
(11i). For example, the inhibition against axenic amastigote of L.
orientalis at 0.25 M were 86.5%, 78.5%, 92.2%, 91.2%,
respectively for the former group, and 58.2%, 42.9%, 40.2%,

Table 1. Structures and in silico ADMET analysis of 10c and 11a-11q.a
6
7
5
Rf
CH3
4'
S
N
5'
4
Rf'
S
N
CH3
X
6'
S
7'
N
O
C2H5
QPlog
Po/w
QPPCaco
(nm·s-1)
QPlog
HERG
Percentage
of HOA
Compounds
Rf
Rf’
-
X
#rtvFG
0
QPlogS
-9.410
QPlogBB CNS
#Metab
5
vLROF
1
10c
5-F
OTs
7.746
0.059
1
3901.428
-6.852
100
11a
11b
11c
11d
-
-
-
-
-
Cl
Cl
Cl
Cl
0
0
0
0
7.479
7.637
7.746
7.706
-8.941
-9.157
-9.410
-9.286
-0.050
0.022
0.059
0.061
0
1
1
1
4015.842
4065.763
3901.428
4048.873
-6.925
-6.838
-6.852
-6.784
5
5
5
5
100
100
100
100
1
1
1
1
4-F
5-F
6-F
11e
11f
7-F
-
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
0
0
0
0
0
0
0
0
0
0
0
0
0
7.688
7.585
7.741
7.748
7.659
8.509
8.643
7.946
7.988
7.993
7.987
7.994
8.710
-9.262
-8.993
-9.375
-9.416
-9.152
-10.498
-10.479
-9.680
-9.777
-9.786
-9.775
-9.794
-10.766
0.042
0.026
0.062
0.059
0.049
0.224
0.173
0.159
0.176
0.179
0.176
0.175
0.332
1
1
1
1
1
1
1
1
1
1
1
1
1
4030.470
4041.975
3992.543
3897.276
4034.065
3977.860
4020.452
4040.863
3972.619
4000.565
3989.158
3941.058
4037.635
-6.810
-6.764
-6.824
-6.852
-6.767
-6.865
-6.89
5
5
5
5
5
5
6
5
5
5
5
5
5
100
100
100
100
100
100
100
100
100
100
100
100
100
1
1
1
1
1
2
2
2
2
2
2
2
2
-
-
4’-F
5’-F
6’-F
7’-F
-
11g
11h
11i
-
-
11j
6-CF3
6-OCF3
5,6-diF
5-F
6-F
6-F
5-F
11k
11l
-
-
-6.688
-6.700
-6.707
-6.704
-6.702
-6.681
11m
11n
11o
11p
11q
5’-F
6’-F
5’-F
6’-F
5-F 6’-CF3
a #rtvFG, number of reactive functional groups: 0 = no reactive functional groups, 1 = mild presence of reactive functional groups, 2 = high presence of reactive
functional groups; QPlogPo/w, predicted octanol/water partition coefficient: recommended range -2.0 to 6.5; QPlogS, prediction of aqueous solubility level:
recommended range -6.5 to 0.5; QPlogBB, predicted brain/blood partition coefficient: recommended range -3.0 to 1.2; CNS, central nervous system activity: -2 =
completely inactive, -1 = very low activity, 0 = low activity, 1 = medium activity, 2 = completely active; QPPCaco, predicted apparent gut-blood barrier
permeability: <25 = poor, >500 = great; QPlogHERG, predicted IC50 value for blockage of HERG K+ channels: <-5 = concern; #Metab, number of likely
metabolic reactions (1 - 8); Percentage of HOA, percentage of human oral absorption level: >80% = high absorption, <25% = poor absorption; vLROF,
violations to Lipinski’s rule of five.
Table 2. Biological activities of the synthesized rhodacyanine analogues (10c and 11a-11q).
L. martiniquensis
L. orientalis
J774A.1 macrophage
%cytotoxicitya
Promastigote
Promastigote
%inhibitiona
0.1 M 0.25 M
78.8 ± 1.8 91.7 ± 1.2
Axenic amastigote
a
a
a
%inhibitiona
%inhibitiona
IC50
IC50
IC50
(M)
(M)
(M)
0.1 M
0.25 M
0.25 M
0.25 M
4.0 M
10c 85.4 ± 0.5
11a 21.0 ± 1.5
11b 37.7 ± 2.6
11c 82.8 ± 0.7
11d 57.1 ± 0.9
11e 41.1 ± 2.6
11f 30.0 ± 2.1
11g 76.9 ± 0.5
11h 40.3 ± 10.4
89.5 ± 0.7
70.8 ± 3.5
64.1 ± 4.3
89.3 ± 1.1
71.1 ± 1.1
85.2 ± 2.8
73.0 ± 0.4
89.2 ± 0.4
87.8 ± 0.8
92.7 ± 1.0
24.9 ± 3.3
27.1 ± 1.8
91.2 ± 0.5
79.8 ± 0.4
62.9 ± 1.6
75.9 ± 2.7
93.4 ± 0.2
39.2 ± 2.9
inactiveb
0.076 ± 0.008
0.658 ± 0.028
n.d.b
0.098 ± 0.001
0.302 ± 0.030
0.065 ± 0.004
0.104 ± 0.001
0.040 ± 0.002
n.d.b
91.0 ± 0.6
36.8 ± 5.0
58.2 ± 1.5
86.5 ± 2.9
78.5 ± 1.5
42.9 ± 4.7
40.2 ± 1.6
92.2 ± 1.3
91.2 ± 0.6
47.9 ± 0.8
71.1 ± 5.4
59.2 ± 2.8
92.8 ± 0.4
87.9 ± 0.7
62.1 ± 3.2
88.9 ± 0.5
93.0 ± 0.4
68.4 ± 8.0
24.9 ± 3.3
91.1 ± 1.0
0.110 ± 0.012
0.909 ± 0.004
n.d.b
n.t.b
n.t. b
n.t.b
31.1 ± 5.6
19.2 ± 0.7
24.9 ± 0.9
26.9 ± 1.5
41.3 ± 3.8
88.5 ± 7.5
13.4 ± 4.1
37.0 ± 1.1
61.8 ± 10.4
72.8 ± 8.8
33.3 ± 0.8
48.6 ± 9.5
18.6 ± 3.8
13.8 ± 4.9
46.0 ± 4.4
21.0 ± 1.9
20.7 ± 3.8
11.5 ± 3.5
67.5 ± 3.0
66.2 ± 7.4
85.7 ± 1.3
13.9 ± 7.1
33.0 ± 7.4
50.1 ± 2.9
60.0 ± 7.8
inactiveb
39.0 ± 8.1
12.5 ± 9.8
61.6 ± 4.8
57.5 ± 6.4
1.9 ± 1.9
81.5 ± 5.4
92.9 ± 1.2
91.7 ± 0.9
95.4 ± 0.6
88.9 ± 2.1
84.2 ± 2.1
90.4 ± 0.6
96.0 ± 1.0
75.4 ± 4.0
87.6 ± 0.9
60.9 ± 5.3
89.5 ± 0.8
81.5 ± 0.3
76.0 ± 1.9
79.3 ± 0.7
93.0 ± 0.3
33.3 ± 2.8
4.0 ± 4.5
0.085 ± 0.015
0.080 ± 0.002
0.200 ± 0.007
n.d.b
n.t.b
n.d.b
n.t.b
n.d.b
n.t.b
n.d.b
n.d.b
n.d.b
n.t.b
0.139 ± 0.005
0.133 ± 0.003
0.088 ± 0.001
n.d.b
0.134 ± 0.026
0.223 ± 0.014
n.d.b
n.t.b
n.d.b
n.t.b
11i
56.1 ± 2.4
11j 10.5 ± 0.5
0.272 ± 0.006
n.d.b
n.t.b
n.d.b
n.d.b
0.5 ± 1.1
11k
11l
8.7 ± 0.9
80.6 ± 2.2
11m 56.4 ± 4.0
11n 17.6 ± 1.3
11o 43.9 ± 8.0
11p 54.3 ± 8.1
n.d.b
n.d.b
n.d.b
n.t.b
0.106 ± 0.009
0.114 ± 0.002
0.161 ± 0.008
n.d.b
0.077 ± 0.001
0.080 ± 0.003
n.d.b
n.t.b
n.d.b
n.t.b
n.d.b
n.t.b
n.d.b
20.4 ± 4.4
32.3 ± 7.8
inactiveb
n.d.b
0.081 ± 0.018
0.085 ± 0.005
n.d.b
n.t.b
0.142 ± 0.022
n.d.b
n.t.b
11q
5.9 ± 3.2
n.d.b
n.d.b
13.3 ± 1.2 52.2 ± 3.6
MFb
inactiveb
15.790 ± 3.564
0.031 ± 0.016
inactiveb
n.d.b
n.d.b
7.3 ± 0.2
12.4 ± 4.3
16.7 ± 1.1
AMb 94.2 ± 0.2
97.5 ± 0.4
96.2 ± 0.2
96.7 ± 0.1
0.023 ± 0.003
0.108 ± 0.003
n.t.b
a IC50 (the half maximal inhibitory concentration, M), %inhibition at specified concentration of compounds (%), and %cytotoxicity at specified concentration of
b
compounds (%) were expressed as the mean values of three replicates  standard deviations (SD). inactive = no significant difference to the negative control
(the absence of the test compound); n.d. = not determined; n.t. = nontoxic to murine macrophages cell line, no significant difference to the negative control (the
absence of the test compound). c MF: miltefosine; AM: amphotericin B.

According to these results, the improved antileishmanial
activity observed for the fluorinated analogues compared to that
of the non-fluorinated compound (11a) cannot be attributed to
the change in solubility or permeability since their predicted
values are very similar. On the other hand, the poorer
antileishmanial activity of analogues containing -CF3 (11j, 11q)
or -OCF3 (11k) substituents, may be influenced by their lower
aqueous solubility. The QPlogHERG scores of all compounds are
slightly outside the accepted range, suggesting potential toxic
effect on the heart. Out of eight metabolic reactions, it is
predicted that the rhodacyanine analogues may undergo up to 5-6
metabolic reactions in the human body. These may affect
bioavailability or drug clearance and potential metabolism-based
drug-drug interactions.21
Figure 2. Cyclic voltammograms recorded with a glassy carbon electrode
(area = 0.071 cm2) at 100 mVs-1 for DMF containing 0.10 M TBAP in the
presence of 1.0 mM 11a (solid lines) and DMF containing only 0.10 M
TBAP (dashed lines). Potential scans go from -0.70 to -1.80 to -0.70 V and
-0.70 to +0.90 to -0.70 V.
The mechanism of action for the antimalarial activity of
rhodacyanines has recently been investigated by incubating a
fluorescent rhodacyanine analogue with Malaria parasites,
revealing a selective accumulation of these species within the
plasmodium mitochondria.22 However, the mechanism for the
antileishmanial activity of these rhodacyanine analogues has not
yet been revealed. Nevertheless, it has been reported that the
bioactivity of various antileishmanial agents involves free radical
mechanisms, including ones that lead to the generation of
reactive oxygen or nitrogen species. These processes are usually
facilitated by the concomitant reduction of the antileishmanial
agent itself, hence the pronounced antileishmanial activity is
typically associated to less negative reduction potential of the
compounds as observed in several SAR studies on the
nitro(hetero)aromatic compounds.23 It is well known that the
nitro group can undergo facile reduction into amine or
hydroxylamine intermediate.24 In this regard, the rhodacyanine
analogues contain a benzothiazolium moiety, which has also
been reported to undergo reduction in many electrochemical
studies.25 Therefore, the introduction of an electron-withdrawing
fluorine substituent could potentially affect the reduction
potential, hence the antileishmanial activity. As a result, the
electrochemical properties of the non-fluorinated (11a) and the
fluorinated rhodacyanine analogues (11c and 11g) were
examined. Figure 2 illustrates cyclic voltammograms recorded at
100 mVs-1 in the potential range of -1.80 to +0.90 V for the
reduction and the oxidation of 1.0 mM 11a (solid lines) at a
glassy carbon electrode in DMF containing 0.10 M TBAP. The
dashed line voltammograms, collected as backgrounds, show no
significant peaks of TBAP–DMF electrolyte solution. When the
potential was negatively scanned from -0.70 to -1.80 V, the
reduction of 11a gave rise to a single irreversible combined wave
with a peak potential of approximately -1.35 V and a broad
shoulder beginning at -1.53 V. In the positive potential region,
the oxidation of this compound reveals three successive
irreversible anodic waves with peak potentials of +0.30, +0.49,
and +0.72 V as well as small cathodic peak at -0.34 V on the
reverse scan. Similar cyclic voltammograms have been observed
from the cationic cyanine dye solutions at platinum electrode.26
In cases of the fluorinated rhodacyanine analogues (11c and 11g),
their electrochemical behaviors are comparable to that of non-
fluorinated one (11a) (as displayed in Figure S1, Supporting
Information, Section 3). Table 3 summarizes electrochemical
information obtained from the cyclic voltammograms of these
three compounds. It is interesting to point out that the presence of
fluorine substituent on the benzothiazole units induces a slight
positive shift of the cathodic peak potential (40-60 mV),
implying thermodynamically more favorable characteristics
towards electro-reduction. Other publications27 have previously
reported similar observations with halogen-containing
compounds.
Table 3. Peak potential values for cyclic voltammograms of 11a,
11c, and 11g.
Reduction
(forward)
Epc1 (V)
Oxidation
(forward)
Epa1 (V) Epa2 (V)
(reverse)
Compound
Epa3 (V)
E
pc1 (V)
-0.34
-0.25
-0.27
11a
11c
11g
-1.35
-1.29
-1.31
+0.30
+0.32
+0.33
+0.49
+0.48
+0.49
+0.72
+0.65
+0.65
Epc = cathodic peak potential; and Epa = anodic peak potential.
The potential is quoted with respect to Ag/Ag+ reference electrode having a
potential of 0.542 V versus standard hydrogen electrode (SHE).28
In addition, scan rate studies (i.e., plots of cathodic peak
currents versus square root of scan rates) of 11a, 11c, and 11g
(Figure S2, Supporting Information, Section 3) demonstrate
linear behavior which indicates the diffusion-controlled reduction
processes of these compounds. Based on earlier results in this
work, the electrochemical data suggest the correlation between
the antileishmanial activity and the electron-accepting ability of
the rhodacyanine analogues.
3. Conclusions
In conclusion, we have successfully synthesized a series of
fluorine-containing rhodacyanine analogues including fifteen
novel and three known derivatives. The antileishmanial activities
were screened against promastigote and axenic amastigote stages
of L. martiniquensis and L. orientalis. Comparing with the non-
fluorinated rhodacyanine 11a, most of the fluorinated analogues
showed strong inhibition activities, even surpassing the currently
available medications for some analogues such as 11c, 11l, 11m,
11o, and 11p. The apparent correlation of less negative reduction
potentials of the tested fluorinated analogues (11c and 11g)
compared to 11a in the electrochemical study with their enhanced
antileishmanial activities suggests further investigation into
potential involvement of free radical mechanism of action. The
SAR revealed that the position of the fluorine substituent
strongly affects the antileishmanial activity. Moreover, it was
found that the -CF3 and the -OCF3 substituents significantly
decreased the antileishmanial activity, which could be explained
by the decrease in aqueous solubility predicted by the in silico
ADMET analysis of these analogues. The ADMET prediction
also suggested that this class of compounds might be suitable to

be administered orally and their suitability for treating cerebral
leishmaniasis. Whether the potential cardiac toxicity is reversible
or not, and the predicted metabolism by several enzymes need
further investigation. The knowledge obtained from this
‘fluorine-walk’ study could become a valuable information for
the development of a new cyanine-based antileishmanial drug in
the future.
triethylamine (3.0 equiv.). The mixture was stirred at 75 ºC
overnight. After being cooled to room temperature, the
precipitate formed was collected and washed with acetonitrile to
give the tosylate salts of the corresponding rhodacyanines 10a-
10q. Finally, 10a-10q (1.0 equiv.) were dissolved in methanol.
The resulting solution was stirred at 80 ºC for 30 min before
slowly adding conc. HCl. The mixture was stirred for an
additional 30 min. After cooling to room temperature, the
precipitate formed was filtered and washed with methanol to give
the corresponding products 11a-11q.
4. Experimental Section
4.1. Materials and instrumentation for chemical synthesis
Data for 10c: dark-green solid (97 mg, 0.14 mmol); Mp: 293-295
°C; H NMR (400 MHz, DMSO-d6) δ 8.22 (d, J = 5.6 Hz, 1H,
All reagents and solvents were obtained from Sigma-Aldrich
(St. Louis, MO, USA), TCI chemicals (Tokyo, Japan),
Fluorochem (Hadfield, Derbyshire, UK) and Merck (Darmstadt,
Germany). All solvents for column chromatography from RCI
Labscan (Samutsakorn, Thailand) were distilled before use.
Reactions were monitored by thin-layer chromatography (TLC)
1
ArH), 7.94 – 7.69 (m, 2H, ArH), 7.63 (d, J = 12.8 Hz, 1H,
methine CH), 7.48 (d, J = 7.8 Hz, 2H, ArH), 7.45 – 7.33 (m, 3H,
ArH), 7.28 (s, 1H, ArH), 7.11 (d, J = 7.9 Hz, 2H, ArH), 6.70 (s,
1H, methine CH), 5.92 (d, J = 13.1 Hz, 1H, methine CH), 4.16
(q, J = 6.9 Hz, 2H, CH2CH3), 3.97 (s, 3H, CH3), 3.69 (s, 3H,
using aluminium Merck TLC plates coated with silica gel 60 F254
.
CH3), 2.28 (s, 3H, CH3), 1.28 (t, J = 6.9 Hz, 3H, CH2CH3); 13C
Normal phase column chromatography was performed using
silica gel 60 (0.063-0.200 mm, 70-230 mesh ASTM, Merck,
Darmstadt, Germany). Proton, carbon, fluorine and two-
dimensional nuclear magnetic resonance (1H, 13C, 19F and 2D
NMR) spectra were recorded on a Bruker Advance (III) 400WB
spectrometer. Chemical shifts were expressed in parts per million
(ppm), J values were in Hertz (Hz). High-resolution mass spectra
1
NMR (126 MHz, DMSO-d6) δ 164.14, 163.22, 162.73 (d, JCF
=
250.2 Hz), 157.69, 145.38, 141.74, 138.33, 128.46, 127.81,
3
126.34, 125.77, 125.09, 124.54, 123.99, 123.30 (d, JCF = 9.7
Hz), 122.77, 122.19 (d, 3JCF = 9.5 Hz), 121.64, 114.64 (d, 2JCF
=
2
24.7 Hz), 112.53, 108.59 (d, JCF = 27.2 Hz), 102.18, 91.35,
86.66, 39.19, 34.88, 33.03, 21.04, 12.60; 19F NMR (376 MHz,
DMSO-d6) δ -111.16; IR (neat): 1679 (C=N), 1560 (C=N), 1493
(C=C), 1463 (C=O), 1341 (SO3), 1190 (C-N), 1124 (C-S) 1030
(SO3), 1058 (C-S), 915 (C-F), 816 (C-S), 680 (C-S) cm-1; HRMS
(HRMS) were obtained with
a
micrOTOF-Q II mass
spectrometer (Bruker Daltonics) with electrospray ionization. IR
spectra were recorded using the Thermo Scientific™ Nicolet™
iS50 FTIR spectrometer with ATR module. Melting points (Mp)
were determined using a Stuart SMP20 melting point apparatus.
+
(ESI+): m/z calcd for C24H21FN3OS3 [M]+ 482.0825, found
482.0808; Data consistent with the literature values.6
Data for 11a: dark-green solid (31 mg, 0.062 mmol); Mp: 254-
256 °C; H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 8.0 Hz,
4.2. Chemical synthesis
1
1H, ArH), 7.90 (d, J = 8.1 Hz, 1H, ArH), 7.84 (d, J = 7.8 Hz, 1H,
ArH), 7.74 – 7.62 (m, 2H, ArH, methine CH), 7.60 – 7.51 (m,
1H, ArH), 7.50 – 7.38 (m, 2H, ArH), 7.32 – 7.24 (m, 1H, ArH),
6.75 (s, 1H, methine CH), 5.94 (d, J = 13.3 Hz, 1H, methine CH),
4.17 (q, J = 6.8 Hz, 2H, CH2CH3), 4.06 (s, 3H, CH3), 3.72 (s, 3H,
CH3), 1.27 (t, J = 7.0 Hz, 3H, CH2CH3); 13C NMR (101 MHz,
DMSO-d6) δ 163.62, 162.95, 162.88, 156.94, 141.89, 140.37,
134.55, 128.83, 127.57, 126.05, 125.80, 124.15, 123.95, 123.42,
122.58, 114.66, 112.40, 101.83, 90.70, 86.32, 39.52, 34.69,
32.89, 12.38 (s); IR (neat): 1673 (C=N), 1562 (C=N), 1512
(C=C), 1471 (C=O), 1342 (C-N), 1181 (C-N), 1019 (C-S), 812
The synthesis of the benzothiazolium tosylate building blocks
6a-6h and the full experimental detail for the synthesis of the
rhodacyanine analogues 11a-11q are described in the Supporting
Information, Section 1.
N-((3-Ethyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)-N-
phenylpropionamide (7): the title compound was synthesized
following literature reported procedure.15 yellow solid (11.41 g,
36 mmol, 97% yield); Rf: 0.33 (1:4 EtOAc:hexanes); Mp: 172-
1
173 °C; H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H, methine
CH ), 7.70 – 7.52 (m, 3H, ArH), 7.28 (d, J = 4.8 Hz, 2H, ArH),
4.09 (q, J = 7.1 Hz, 2H, CH2CH3), 2.25 (q, J = 7.2 Hz, 2H,
CH2CH3), 1.21 (t, J = 7.1 Hz, 3H, CH2CH3), 1.13 (t, J = 7.3 Hz,
3H, CH2CH3); 13C NMR (101 MHz, DMSO-d6) δ 194.12, 173.48,
168.40, 136.46, 131.32, 131.20, 130.75, 129.70, 106.07, 39.61,
28.65, 12.22, 8.94; IR (neat): 1718 (C=O of amide), 1604 (cyclic
C=O), 1443 (C-N), 1330 (C-N), 1298 (C-N), 1102 (C=S), 883
(C-S) cm-1.
+
(C-S) cm-1; HRMS (ESI+): m/z calcd for C24H22N3OS3 [M]+
464.0920, found 464.0909; Data consistent with the literature
values.6
Data for 11b: dark-green solid (68 mg, 0.13 mmol); Mp: 231-233
1
°C; H NMR (400 MHz, DMSO-d6) δ 8.04 (d, J = 8.9 Hz, 1H,
ArH), 7.88 (d, J = 7.2 Hz, 1H, ArH), 7.72 (d, J = 13.0 Hz, 1H,
methine CH), 7.58 – 7.43 (m, 4H, ArH), 7.32 (t, J = 6.9 Hz, 1H,
ArH), 6.72 (s, 1H, methine CH), 6.03 (d, J = 13.1 Hz, 1H,
methine CH), 4.20 – 4.12 (m, 5H, CH2CH3, CH3), 3.75 (s, 3H,
CH3), 1.26 (t, J = 7.1 Hz, 3H, CH2CH3); 13C NMR (101 MHz,
DMSO-d6) δ 163.58, 163.17, 161.40, 161.35 (d, 1JCF = 235.1 Hz),
157.98, 150.76, 145.54, 141.89, 135.11, 127.69, 124.39, 123.31
General procedure for the synthesis of 11a-11q: A mixture of
7 (1 equiv.) and the benzothiazolium tosylates 6a-6f (1 equiv.) in
acetonitrile was added acetic anhydride (1.4 equiv.). After
stirring at 50 °C for 1 h, triethylamine (3.7 equiv.) was added and
the resulting mixture was stirred at 60 °C for an additional 4 h.
Then, the solution was cooled to room temperature. The
precipitate was filtered and washed with acetonitrile. The solid
was dried in vacuo to obtain the corresponding 2-
thioxothiazolidin-4-ones 8a-8f.15 A mixture of 8a-8f (1 equiv.)
and methyl p-toluenesulfonate (3 equiv.) in DMF was stirred at
115 °C for 3 h. After being cooled to room temperature, the
mixture was stirred with acetone for further 30 min to allow
precipitation. The solid formed was collected and washed with
acetone to give the corresponding 2-(methylthio)-4-oxo-4,5-
dihydrothiazol-3-ium tosylates 9a-9f. To a mixture of 9a-9f (1.0
equiv.) and 6a-6h (1.0 equiv.) in acetonitrile was added
2
2
(d, JCF = 25.1 Hz), 122.65, 119.70, 116.04 (d, JCF = 20.5 Hz),
115.99, 112.59, 110.01, 91.17, 86.02, 39.02, 37.29, 33.00, 12.36;
19F NMR (376 MHz, DMSO-d6) δ -126.65; IR (neat): 1688
(C=N), 1556 (C=N), 1516 (C=C), 1469 (C=O), 1349 (C-N), 1224
(C-F), 1185 (C-N), 1021 (C-S), 921 (C-F), 808 (C-S) cm-1;
+
HRMS (ESI+): m/z calcd for C24H21FN3OS3 [M]+ 482.0825,
found 482.0834.
Data for 11c: dark-green solid (40 mg, 0.070 mmol); Mp: 275-
1
277 °C; H NMR (400 MHz, DMSO-d6) δ 8.25 (dd, J = 8.8, 5.1
Hz, 1H, ArH), 7.82 (d, J = 7.7 Hz, 1H, ArH), 7.74 (d, J = 8.3 Hz,

1H, ArH), 7.59 (d, J = 13.1 Hz, 1H, methine CH), 7.42 – 7.32
(m, 3H, ArH), 7.30 – 7.22 (m, 1H, ArH), 6.71 (s, 1H, methine
CH), 5.89 (d, J = 13.2 Hz, 1H, methine CH), 4.16 (q, J = 7.1 Hz,
2H, CH2CH3), 3.98 (s, 3H, CH3), 3.68 (s, 3H, CH3), 1.29 (t, J =
7.1 Hz, 3H, CH2CH3); 13C NMR (101 MHz, DMSO-d6) δ 163.68,
Data for 11g: dark-green solid (31 mg, 0.06 mmol); Mp: 265-267
°C; H NMR (400 MHz, DMSO-d6) δ 8.25 (d, J = 9.2 Hz, 1H,
1
ArH), 7.92 (dd, J = 17.3, 8.4 Hz, 1H, ArH), 7.83 (dd, J = 8.8, 5.6
Hz, 1H, ArH), 7.78 – 7.64 (m, 1H, ArH), 7.65 – 7.50 (m, 2H,
ArH, methine CH), 7.50 – 7.36 (m, 1H, ArH), 7.12 (t, J = 8.1 Hz,
1H, ArH), 6.76 (s, 1H, methine CH), 5.92 (d, J = 12.6 Hz, 1H,
methine CH), 4.17 (q, J = 7.1 Hz, 2H, CH2CH3), 4.07 (s, 3H,
1
163.44, 163.06, 162.66 (d, JCF = 285.4 Hz), 157.31, 141.76,
3
141.68, 134.63, 127.52, 125.01 (d, JCF = 10.1 Hz), 124.21,
2
123.85, 122.56, 121.54, 113.53 (d, JCF = 24.7 Hz), 112.37,
CH3), 3.67 (s, 3H, CH3), 1.28 (t, J = 7.1 Hz, 3H, CH2CH3); 13C
2
1
102.18 (d, JCF = 28.9 Hz), 101.72, 90.91, 86.49, 39.00, 34.81,
NMR (101 MHz, DMSO-d6) δ 163.74, 163.63, 162.02 (d, JCF
=
32.87, 12.35; 19F NMR (376 MHz, DMSO-d6) δ -111.13; IR
(neat): 1668 (C=N), 1560 (C=N), 1513 (C=C), 1466 (C=O), 1191
(C-N), 1127 (C-S), 1055 (C-S), 944 (C-F), 824 (C-S) cm-1;
231.5 Hz), 156.92, 140.39, 134.19, 128.87, 126.15, 125.94,
3
123.80 (d, JCF = 10.5 Hz), 123.45, 119.38, 114.79, 111.11 (d,
2
2JCF = 24.5 Hz), 105.08, 104.92, 103.01, 100.38 (d, JCF = 29.0
+
HRMS (ESI+): m/z calcd for C24H21FN3OS3 [M]+ 482.0825,
Hz), 90.97, 86.61, 39.53, 34.77, 33.06, 12.34; 19F NMR (376
MHz, DMSO-d6) δ -113.59; IR (neat): 1668 (C=N), 1513 (C=C),
1468 (C=O), 1332 (C-N), 1183 (C-N), 1055 (C-S), 927 (C-F),
found 482.0841; Data consistent with the literature values.6
Data for 11d: dark-green solid (32 mg, 0.062 mmol); Mp: 264-
+
830 (C-S) cm-1; HRMS (ESI+): m/z calcd for C24H21FN3OS3
1
266 °C; H NMR (400 MHz, DMSO-d6) δ 8.16 (dd, J = 7.9, 1.9
[M]+ 482.0825, found 482.0836.
Hz, 1H, ArH), 7.80 (d, J = 8.1 Hz, 2H, ArH), 7.64 – 7.46 (m, 2H,
ArH, methine CH), 7.37 (s, 2H, ArH), 7.24 (s, 1H, ArH), 6.68 (s,
1H, methine CH), 5.87 (d, J = 12.9 Hz, 1H, methine CH), 4.15
(q, J = 6.9 Hz, 2H, CH2CH3), 4.01 (s, 3H, CH3), 3.69 (s, 3H,
CH3), 1.28 (t, J = 7.0 Hz, 3H, CH2CH3); 13C NMR (101 MHz,
DMSO-d6) δ 163.53, 163.09, 162.96, 159.30 (d, 1JCF = 244.8 Hz),
Data for 11h: dark-green solid (40 mg, 0.077 mmol); Mp: 231-
1
234 °C; H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 8.0 Hz,
1H, ArH), 7.87 (d, J = 8.8 Hz, 1H, ArH), 7.77 (d, J = 8.4 Hz, 1H,
ArH), 7.66 (t, J = 7.7 Hz, 1H, ArH), 7.62 – 7.45 (m, 2H, ArH,
methine CH), 7.39 (s, 1H, ArH), 7.27 (dd, J = 22.5, 13.7 Hz, 1H,
ArH), 6.72 (s, 1H, methine CH), 5.89 (d, J = 12.8 Hz, 1H,
methine CH), 4.15 (q, J = 7.0 Hz, 2H, CH2CH3), 4.05 (s, 3H,
2
157.02, 141.80, 137.18, 134.51, 127.58, 124.05 (d, JCF = 27.6
2
Hz), 122.57, 116.83 (d, JCF = 27.2 Hz), 116.70, 116.17, 112.34,
110.39, 110.10, 101.69, 90.76, 86.43, 38.96, 34.95, 32.88, 12.36;
19F NMR (376 MHz, DMSO-d6) δ -114.81; IR (neat): 1685
(C=N), 1574 (C=N), 1524 (C=C), 1477 (C=O), 1308 (C-N), 1202
(C-F), 1191 (C-N), 1124 (C-S), 1052 (C-S), 810 (C-S) cm-1;
CH3), 3.68 (s, 3H, CH3), 1.27 (t, J = 7.0 Hz, 3H, CH2CH3); 13C
1
NMR (101 MHz, DMSO-d6) δ 163.60, 162.87, 158.68 (d, JCF
=
242.0 Hz), 156.94, 140.29, 138.65, 134.32, 128.83, 128.00,
2
126.02, 125.81, 125.47, 123.37, 114.79 (d, JCF = 24.7 Hz),
+
3
2
HRMS (ESI+): m/z calcd for C24H21FN3OS3 [M]+ 482.0825,
114.60, 113.32 (d, JCF = 8.8 Hz), 109.85 (d, JCF = 28.8 Hz),
102.17, 90.84, 86.34, 38.91, 34.67, 33.12, 12.34; 19F NMR (376
MHz, DMSO-d6) δ -117.86; IR (neat): 1657 (C=N), 1562 (C=N),
1505 (C=C), 1474 (C=O), 1335 (C-N), 1185 (C-S), 1063 (C-S),
905 (C-F), 805 (C-S) cm-1; HRMS (ESI+): m/z calcd for
C24H21FN3OS3+ [M]+ 482.0825, found 482.0825.
found 482.0831.
Data for 11e: dark-green solid (73 mg, 0.14 mmol); Mp: 268-271
1
°C; H NMR (400 MHz, DMSO-d6) δ 7.86 (d, J = 7.3 Hz, 1H,
ArH), 7.81 – 7.66 (m, 3H, ArH, methine CH), 7.54 (d, J = 8.3
Hz, 1H, ArH), 7.45 (dd, J = 16.7, 8.6 Hz, 2H, ArH), 7.29 (t, J =
7.2 Hz, 1H, ArH), 6.76 (s, 1H, methine CH), 6.25 (d, J = 13.7
Hz, 1H, methine CH), 4.18 (q, J = 6.4 Hz, 2H, CH2CH3), 4.07 (s,
3H, CH3), 3.80 (s, 3H, CH3), 1.27 (t, J = 6.4 Hz, 3H, CH2CH3);
13C NMR (101 MHz, DMSO-d6) δ 164.04, 163.49, 162.01,
159.39, 158.38, 156.01 (d, 1JCF = 247.2 Hz), 143.04 (d, 3JCF = 5.3
Data for 11i: dark-green solid (31 mg, 0.06 mmol); Mp: 277-279
1
°C; H NMR (400 MHz, DMSO-d6) δ 8.25 (d, J = 8.1 Hz, 1H,
ArH), 7.96 (d, J = 8.2 Hz, 1H, ArH), 7.71 (t, J = 7.7 Hz, 1H,
ArH), 7.68 – 7.53 (m, 2H, ArH, methine CH), 7.45 (dd, J = 14.1,
8.3 Hz, 2H, ArH), 7.31 (d, J = 8.2 Hz, 1H, ArH), 7.27 – 7.01 (m,
1H, ArH), 6.79 (s, 1H, methine CH), 5.96 (d, J = 13.0 Hz, 1H,
methine CH), 4.16 (q, J = 7.1 Hz, 2H, CH2CH3), 4.10 (s, 3H,
CH3), 3.71 (s, 3H, CH3), 1.26 (t, J = 7.1 Hz, 3H, CH2CH3); 13C
NMR (101 MHz, DMSO-d6) δ 163.72, 163.36, 161.10, 156.85,
3
Hz), 141.94, 139.63, 135.52, 130.80 (d, JCF = 7.6 Hz), 127.63,
124.30 (d, 2JCF = 22.0 Hz), 122.62, 112.71, 111.54 (d, 2JCF = 18.0
Hz), 110.97, 101.24, 91.80, 86.25, 39.53, 35.25, 33.25, 12.47; 19
F
NMR (376 MHz, DMSO-d6) δ -114.99; IR (neat): 1663 (C=N),
1556 (C=N), 1511 (C=C), 1474 (C=O), 1323 (C-N), 1245 (C-F),
1188 (C-N), 1062 (C-S), 939 (C-F), 810 (C-S) cm-1; HRMS
1
3
155.76 (d, JCF = 244.9 Hz), 144.56 (d, JCF = 6.0 Hz), 140.36,
133.93, 130.92, 129.56 (d, 3JCF = 7.9 Hz), 128.92, 126.13 (d, 2JCF
= 20.3 Hz), 123.45, 114.85, 110.16 (d, 2JCF = 22.5 Hz), 108.59 (d,
4JCF = 2.9 Hz), 104.24, 90.92, 89.03, 86.84, 39.13, 34.87, 33.36,
12.35; 19F NMR (376 MHz, DMSO-d6) δ -115.98; IR (neat):
1658 (C=N), 1507 (C=C), 1476 (C=O), 1344 (C-N), 1193 (C-S),
1054 (C-S), 927 (C-F) cm-1; HRMS (ESI+): m/z calcd for
C24H21FN3OS3+ [M]+ 482.0825, found 482.0825.
+
(ESI+): m/z calcd for C24H21FN3OS3 [M]+ 482.0825, found
482.0825.
Data for 11f: dark-green solid (39 mg, 0.07 mmol); Mp: 270-273
1
°C; H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 7.8 Hz, 1H,
ArH), 7.81 (d, J = 8.0 Hz, 1H, ArH), 7.64 (dd, J = 15.5, 7.7 Hz,
2H, ArH), 7.60 – 7.50 (m, 2H, ArH, methine CH), 7.31 – 7.13
(m, 2H, ArH), 6.75 (s, 1H, methine CH), 5.85 (d, J = 13.1 Hz,
1H, methine CH), 4.17 (q, J = 6.8 Hz, 2H, CH2CH3), 4.04 (s, 3H,
CH3), 3.78 (s, 3H, CH3), 1.29 (t, J = 6.8 Hz, 3H, CH2CH3); 13C
NMR (101 MHz, DMSO-d6) 13C NMR (101 MHz, DMSO) δ
Data for 11j: dark-green solid (43 mg, 0.076 mmol); Mp: 244-
1
247 °C; H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H, ArH),
8.08 – 7.92 (m, 2H, ArH), 7.85 (d, J = 7.8 Hz, 1H, ArH), 7.69 (d,
J = 12.9 Hz, 1H, methine CH), 7.52 – 7.34 (m, 2H, ArH), 7.29 (t,
J = 7.6 Hz, 1H, ArH), 6.77 (s, 1H, methine CH), 6.00 (d, J = 13.1
Hz, 1H, methine CH), 4.20 (q, J = 7.0 Hz, 2H, CH2CH3), 4.07 (s,
3H, CH3), 3.76 (s, 3H, CH3), 1.28 (t, J = 7.1 Hz, 3H, CH2CH3);
13C NMR (101 MHz, DMSO-d6) δ 164.22, 163.95, 163.66,
158.52, 143.35, 142.01, 135.44, 127.88, 126.92, 124.71, 124.65,
124.56 (q, 1JCF = 259.5 Hz), 124.30, 122.88, 121.37 (q, 3JCF = 4.2
1
163.62, 163.11, 162.19, 156.90, 148.37 (d, JCF = 246.6 Hz),
140.21, 134.22, 129.53 (d, 3JCF = 9.1 Hz), 128.81, 127.90, 126.23
2
3
(d, JCF = 26.8 Hz), 125.88, 124.69 (d, JCF = 7.2 Hz), 123.35,
118.80, 115.04 (d, 2JCF = 20.5 Hz), 114.66, 103.52, 90.40, 86.66,
39.52, 35.49, 34.72, 12.26; 19F NMR (376 MHz, DMSO-d6) δ -
130.05; IR (neat): 1688 (C=N), 1504 (C=C), 1479 (C=O), 1337
(C-N), 1190 (C-N), 1122 (C-F), 1055 (C-S), 918 (C-F), 810 (C-
2
Hz), 115.39, 114.64 (q, JCF = 27.0 Hz), 112.83, 101.45, 91.58,
+
S) cm-1; HRMS (ESI+): m/z calcd for C24H21FN3OS3 [M]+
86.88, 39.10, 35.07, 33.25, 12.67; 19F NMR (376 MHz, DMSO-
482.0825, found 482.0800.
d6) δ -59.96; IR (neat): 1691 (C=N), 1516 (C=C), 1469 (C=O),

3
2
1352 (C-N), 1316 (C-CF3), 1191 (C-N), 1074 (C-CF3), 819 (C-
Hz), 124.78 (d, JCF = 7.5 Hz), 118.88, 117.31 (d, JCF = 20.7
+
2
3
S), 799 (C-F) cm-1; HRMS (ESI+): m/z calcd for C25H21F3N3OS3
Hz), 116.89 (d, JCF = 24.8 Hz), 116.24 (d, JCF = 10.0 Hz),
[M]+ 532.0793, found 532.0790.
115.09 (d, JCF = 20.3 Hz), 110.22 (d, JCF = 28.6 Hz), 103.34,
90.48, 86.81, 38.96, 35.53, 35.04, 12.29; 19F NMR (376 MHz,
DMSO-d6) δ -114.66 (1F), -130.18 (1F); IR (neat): 1662 (C=N),
1557 (C=N), 1499 C=C), 1471 (C=O), 1313 (C-N), 1204 (C-N),
1021 (C-S), 910 (C-F) cm-1; HRMS (ESI+): m/z calcd for
C24H20F2N3OS3+ [M]+ 500.0731, found 500.0689.
2
2
Data for 11k: dark-green solid (46 mg, 0.08 mmol); Mp: 243-246
1
°C; H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 1H, ArH), 7.99 –
7.74 (m, 2H, ArH), 7.73 – 7.53 (m, 2H, ArH, methine CH), 7.44
– 7.19 (m, 3H, ArH), 6.72 (s, 1H, methine CH), 5.90 (d, J = 13.4
Hz, 1H, methine CH), 4.17 (q, J = 6.8 Hz, 2H, CH2CH3), 4.02 (s,
3H, CH3), 3.70 (s, 3H, CH3), 1.29 (t, J = 6.9 Hz, 3H, CH2CH3);
13C NMR (101 MHz, DMSO-d6) δ 163.67, 163.48, 163.11,
157.59, 145.04, 141.79, 141.68, 139.44, 139.33, 134.78, 127.60,
Data for 11o: dark-green solid (47 mg, 0.085 mmol); Mp: 271-
274 °C; H NMR (400 MHz, DMSO-d6) δ 8.18 (dd, J = 7.9, 2.0
1
Hz, 1H, ArH), 7.94 (dd, J = 9.1, 4.0 Hz, 1H, ArH), 7.82 (dd, J =
8.6, 5.2 Hz, 1H, ArH), 7.64 – 7.56 (m, 2H, ArH, methine CH),
7.44 (dd, J = 9.4, 0.8 Hz, 1H, ArH), 7.12 (td, J = 8.4, 1.4 Hz, 1H,
ArH), 6.73 (s, 1H, methine CH), 5.91 (d, J = 13.1 Hz, 1H,
methine CH), 4.17 (q, J = 6.9 Hz, 2H, CH2CH3), 4.06 (s, 3H,
1
125.41, 124.83 (q, JCF = 249.4 Hz), 124.29, 122.61, 122.19 (q,
3JCF = 6.8 Hz), 116.45, 112.40, 101.48, 90.96, 86.55, 39.11,
34.91, 32.91, 12.38; 19F NMR (376 MHz, DMSO-d6) δ -57.10; IR
(neat): 1690 (C=N), 1515 (C=C), 1468 (C=O), 1352 (C-N), 1243
(C-F), 1193 (C-O-CF3), 1021 (C-S), 808 (C-S) cm-1; HRMS
CH3), 3.68 (s, 3H, CH3), 1.28 (t, J = 7.1 Hz, 3H, CH2CH3); 13C
+
1
(ESI+): m/z calcd for C25H21F3N3O2S3 [M]+ 548.0743, found
NMR (101 MHz, DMSO-d6) δ 163.62, 163.26, 162.09 (d, JCF
=
=
548.0730.
250.4 Hz), 159.35 (d, 1JCF = 245.0 Hz), 156.95, 143.30 (d, 3JCF
3
12.1 Hz), 137.20, 134.13, 127.94, 127.51, 123.79 (d, JCF = 9.6
Data for 11l: dark-green solid (65 mg, 0.097 mmol); Mp: 266-
Hz), 119.33 (d, 4JCF = 2.2 Hz), 116.85 (d, 2JCF = 25.2 Hz), 116.27
1
268 °C; H NMR (400 MHz, DMSO-d6) δ 8.41 (dd, J = 9.2, 8.0
3
2
2
(d, JCF = 9.3 Hz), 111.12 (d, JCF = 23.0 Hz), 110.26 (d, JCF
=
Hz, 1H, ArH), 8.19 (dd, J = 10.8, 6.5 Hz, 1H, ArH), 7.87 (d, J =
8.1 Hz, 1H, ArH), 7.70 (d, J = 13.3 Hz, 1H, methine CH), 7.56 –
7.43 (m, 2H, ArH), 7.31 (t, J = 7.7 Hz, 1H, ArH), 6.74 (s, 1H,
methine CH), 5.97 (d, J = 13.4 Hz, 1H, methine CH), 4.18 (q, J =
6.9 Hz, 2H, CH2CH3), 4.03 (s, 3H, CH3), 3.75 (s, 3H, CH3), 1.27
(t, J = 6.9 Hz, 3H, CH2CH3); 13C NMR (101 MHz, DMSO-d6) δ
28.6 Hz), 102.80, 100.31 (d, 2JCF = 29.3 Hz), 90.99, 86.70, 39.10,
35.03, 33.03, 12.31; 19F NMR (376 MHz, DMSO-d6) δ -113.56
(1F), -114.64 (1F); IR (neat): 1690 (C=N), 1518 (C=C), 1476
(C=O), 1343 (C-N), 1202 (C-F), 1185 (C-N), 1060 (C-S), 932
(C-F) cm-1; MS (MALDI-TOF): m/z calcd for C24H20F2N3OS3
+
[M]+ 500.0731, found 500.0729.
1
2
163.95, 163.51, 163.35, 157.33, 151.32 (dd, JCF = 271.2, JCF
=
1
2
29.8 Hz), 150.46 (dd, JCF = 249.5, JCF = 28.3 Hz), 142.52,
Data for 11p: dark-green solid (48 mg, 0.087 mmol); Mp: 274-
276 °C; H NMR (400 MHz, DMSO-d6) δ 8.29 (dd, J = 8.4, 5.4
1
141.88, 137.36, 134.89, 127.62, 124.32, 124.03, 122.68, 112.54,
2
112.14 (dd, JCF = 23.6, 1.9 Hz), 104.16, 101.41, 91.00, 86.65,
Hz, 1H, ArH), 7.82 (dd, J = 9.7, 1.3 Hz, 1H, ArH), 7.65 (dd, J =
5.4, 3.2 Hz, 1H, ArH), 7.57 (d, J = 13.1 Hz, 1H, methine CH),
7.44 (td, J = 9.2, 1.8 Hz, 1H, ArH), 7.24 (dd, J = 7.0, 6.3 Hz, 2H,
ArH), 6.75 (s, 1H, methine CH), 5.88 (d, J = 12.9 Hz, 1H,
methine CH), 4.16 (q, J = 6.9 Hz, 2H, CH2CH3), 4.01 (s, 3H,
39.01, 35.20, 32.96, 12.38; 19F NMR (376 MHz, DMSO-d6) δ -
134.60 (d, J = 22.2 Hz, 1F), -138.72 (d, J = 22.1 Hz, 1F); IR
(neat): 1665 (C=N), 1554 (C=N), 1518 (C=C), 1468 (C=O), 1340
(C-N), 1224 (C-F), 1182 (C-N), 1026 (C-S), 819 (C-S) cm-1;
HRMS (ESI+): m/z calcd for C24H20F2N3OS3 [M]+ 500.0731,
CH3), 3.81 (s, 3H, CH3), 1.29 (t, J = 6.9 Hz, 3H, CH2CH3); 13C
NMR (101 MHz, DMSO-d6) δ 164.57, 163.60, 162.48 (d, JCF
245.3 Hz), 157.32, 148.46 (d, JCF = 246.8 Hz), 134.61, 129.57
+
1
found 500.0721.
=
1
Data for 11m: dark-green solid (61 mg, 0.11 mmol); Mp: 279-
3
4
(d, JCF = 8.5 Hz), 126.47 (d, JCF = 2.7 Hz), 125.47, 125.02 (d,
1
282 °C; H NMR (400 MHz, DMSO-d6) δ 8.27 (dd, J = 8.8, 5.1
2JCF = 24.1 Hz), 124.93 (d, JCF = 21.2 Hz), 121.75, 118.91,
2
Hz, 1H, ArH), 7.79 (dd, J = 8.6, 5.2 Hz, 1H, ArH), 7.68 (dd, J =
9.8, 1.9 Hz, 1H, ArH), 7.49 (d, J = 13.1 Hz, 1H, methine CH),
7.42 – 7.34 (m, 1H, ArH), 7.19 (dd, J = 10.0, 1.6 Hz, 1H, ArH),
7.08 (td, J = 8.9, 2.1 Hz, 1H, ArH), 6.69 (s, 1H, methine CH),
5.83 (d, J = 13.1 Hz, 1H, methine CH), 4.15 (q, J = 7.1 Hz, 2H,
CH2CH3), 3.95 (s, 3H, CH3), 3.60 (s, 3H, CH3), 1.30 (t, J = 7.1
Hz, 3H, CH2CH3); 13C NMR (101 MHz, DMSO-d6) δ 164.26,
2
2
115.08 (d, JCF = 21.1 Hz), 113.74 (d, JCF = 24.4 Hz), 103.30,
102.49, 102.20, 90.63, 86.85, 39.10, 35.47, 34.95, 12.31; 19F
NMR (376 MHz, DMSO-d6) δ -110.98 (1F), -129.91 (1F); IR
(neat): 1659 (C=N), 1556 (C=N), 1504 (C=C), 1455 (C=O), 1321
(C-N), 1200 (C-F), 1026 (C-S), 918 (C-F) cm-1; HRMS (ESI+):
m/z calcd for C24H20F2N3OS3+ [M]+ 500.0731, found 500.0712.
1
1
163.60, 163.50, 162.49 (d, JCF = 245.5 Hz), 162.03 (d, JCF
=
=
=
Data for 11q: dark-green solid (77 mg, 0.13 mmol); Mp: 255-258
°C; 1H NMR (400 MHz, DMSO-d6) δ 8.28 (s, 2H, ArH), 7.94 (d,
J = 8.3 Hz, 1H, ArH), 7.76 (d, J = 8.6 Hz, 1H, ArH), 7.65 (d, J =
12.9 Hz, 1H, methine CH), 7.60 (d, J = 8.5 Hz, 1H, ArH), 7.45 (t,
J = 9.0 Hz, 1H, ArH), 6.79 (s, 1H, methine CH), 6.01 (d, J = 13.0
Hz, 1H, methine CH), 4.18 (q, J = 6.8 Hz, 2H, CH2CH3), 4.07 (s,
3H, CH3), 3.74 (s, 3H, CH3), 1.27 (t, J = 6.9 Hz, 3H, CH2CH3);
13C NMR (101 MHz, DMSO-d6) δ 172.24, 165.15, 163.03,
3
3
242.9 Hz), 157.33, 143.06 (d, JCF = 12.2 Hz), 141.60 (d, JCF
12.3 Hz), 134.13, 125.09 (d, JCF = 10.4 Hz), 123.78 (d, JCF
3
3
2
10.2 Hz), 121.56, 119.17, 113.65 (d, JCF = 24.5 Hz), 111.18 (d,
2JCF = 23.7 Hz), 102.97, 102.20 (d, JCF = 29.0 Hz), 100.24 (d,
2
2JCF = 28.7 Hz), 91.19, 86.76, 39.10, 34.84, 33.00, 12.30; 19F
NMR (376 MHz, DMSO-d6) δ δ -111.17 (1F), -113.57 (1F); IR
(neat): 1671 (C=N), 1518 (C=C), 1460 (C=O), 1327 (C-N), 1268,
1199 (C-N), 1060 (C-S), 932 (C-F) cm-1; HRMS (ESI+): m/z
calcd for C24H20F2N3OS3+ [M]+ 500.0731, found 500.0724.
1
162.79 (d, JCF = 252.5 Hz), 157.53, 145.13, 142.18, 134.64,
133.68, 130.85, 128.33, 125.29, 123.56 (q, JCF = 253.4 Hz),
120.40, 120.27, 114.14 (d, JCF = 24.7 Hz), 112.67, 104.50,
1
2
Data for 11n: dark-green solid (54 mg, 0.09 mmol); Mp: 254-256
2
102.73 (d, JCF = 27.4 Hz), 91.64, 87.31, 39.18, 35.32, 33.28,
1
°C; H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 7.6 Hz, 1H,
12.56; 19F NMR (376 MHz, DMSO-d6) δ -59.96 (3F), -111.03
(1F); IR (neat): 1670 (C=N), 1509 (C=C), 1468 (C=O), 1373 (C-
CF3), 1311 (C-N), 1181 (C-N), 1065 (C-S), 941 (C-F) cm-1;
ArH), 7.89 (dd, J = 8.2, 4.3 Hz, 1H, ArH), 7.68 – 7.51 (m, 3H,
ArH, methine CH), 7.37–7.11 (m, 2H, ArH), 6.74 (s, 1H, methine
CH), 5.86 (d, J = 13.1 Hz, 1H, methine CH), 4.16 (q, J = 6.4 Hz,
2H, CH2CH3), 4.05 (s, 3H, CH3), 3.82 (s, 3H, CH3), 1.28 (t, J =
6.4 Hz, 3H, CH2CH3); 13C NMR (101 MHz, DMSO-d6) δ 163.58,
HRMS (ESI+): m/z calcd for C25H20F4N3OS3 [M]+ 550.0699,
+
found 550.0699.
1
1
162.28, 159.36 (d, JCF = 245.3 Hz), 156.98, 148.39 (d, JCF
=
4
246.9 Hz), 137.11, 134.27, 129.56, 127.38, 126.39 (d, JCF = 2.4

orientalis (1 × 107 cells·mL–1, 90 µL) was transferred into 96-
wells plate containing fluorinated rhodacyanines at 0.25 µM
(final concentration) with 0.5% or 1% DMSO in cultured
solution. Cultures treated without the synthesized compounds
were used as negative controls and medium without cells was
used as blank. After adjusting the total volume of 100 µL using
Schneider’s insect medium, the plates were incubated further 72
h at 37 ºC in 5% CO2. Then, the living cells were quantified
using the colorimetric assay.
4.3. Biological assays
Material and instrumentations: Schneider’s insect medium
(Sigma-Aldrich, USA) containing 10% fetal bovine serum (FBS)
was used for Leishmania cell culture. Dulbecco’s modified
Eafle’s medium (DMEM) (Gibco, life technologies, USA)
supplemented with 10% heat-inactivated FBS was used for
macrophages cell culture. Phosphate buffered saline (PBS) was
purchased from Apsalagen, Bangkok, Thailand. The research
grade of dimethyl sulfoxide (DMSO) (SERVA Electrophoresis
GmbH, Heidelberg, Germany) was used for the preparation of
compound solution. Resazurin sodium salt (TCI, Tokyo, Japan)
was used as an indicator for colorimetric assay. Cells were
inspected under an inverted microscope (Olympus, Japan). The
fluorescence intensities were obtained using the Thermo
Scientific Varioskan® Flash microplate reader.
The half maximal inhibitory concentration (IC50) evaluation
of axenic proliferation inhibition: The promastigotes of L.
martiniquensis or L. orientalis (1 × 107 cells·mL–1) 90 µL were
transferred into 96-wells plate containing two-fold dilution of the
selected fluorinated rhodacyanines with 0.5% or 1% DMSO in
culture solution. Cultures treated without the synthesized
compounds were used as negative controls and medium without
cells was used as blank. After adjusting the total volume to 100
µL using Schneider’s insect medium, the plates were incubated
further for 72 h at 37 ºC in 5% CO2. Then, the living cells were
quantified using the colorimetric assay.
Cell culture: The Leishmania martiniquensis strains, LSCM1,
were isolated from the bone marrow of a Thai immunocompetent
patient from Lamphun province, northern Thailand. The isolation
of this strain was assigned WHO code MHOM/MQ/92/MAR;
LEM2494. The Leishmania orientalis strains, PCM2, were
isolated from the bone marrow of a Thai immunocompetent
patient from Trang province, southern Thailand. The isolation of
this strain was assigned WHO code MHOM/TH/2010/PCM2;
Trang. 100 µL of thawing cells was loaded into 10 mL
Schneider’s insect medium + 10% FBS in a 25-cm3 flask and
maintained at 25 ± 2 ºC. As for Cytotoxicity: Murine macrophage
J774A.1 cells were purchased from American Type Culture
Collection and were cultured in DMEM + 10% FBS at 37 ºC in
5% CO2.
Colorimetric assay: After incubation for 72 h, 10 µL of
0.0125% resazurin in PBS was added to each well. The resazurin
solution was prepared by dissolving 12.5 mg of resazurin sodium
salt (TCI, Tokyo, Japan) in 100.00 mL of PBS, then stored at 4
ºC. The plates were further incubated at 37 ºC in 5% CO2 for 2-3
h. The fluorescence intensities were calculated with
a
fluorescence plate reader using an excitation wavelength of 536
nm and an emission wavelength of 588 nm. The percentage of
inhibition was calculated using the formula: %inhibition =
[{Absorbance (control) – Blank} – {Absorbance (sample) –
Blank}] / {Absorbance (control) – Blank} × 100.
Determination of the number of Leishmania parasites and
macrophage cells: To the solution of 20 µL of promastigote or
macrophage cells in 20 µL of trypan blue solution was gently
mixed at a dilution factor of 2. Then, 10 µL of the stained cell
mixtures was transferred to the hemocytometer and incubated at
room temperature for 5 min. Viable cells, unstained cells, were
counted in 5 squares under a microscope at 40x magnification.
The number of cells was calculated using the following equation:
Number of cells (cells·mL–1) = Average cells × 104 × dilution
factor.
Cytotoxicity: Murine macrophage J774A.1 cells were purchased
from American Type Culture Collection and were cultured in
DMEM + 10% FBS at 37 ºC in 5% CO2 in a humidified
atmosphere. The cultures were inspected for the parasites every
24 h under an inverted microscope (Olympus, Japan). After that,
200 µL of J774A.1 cells (2 × 105 cells·mL–1) was added into 96-
wells plate and allowed cell adhesion by incubation at 37 ºC in
5% CO2 for 24 h. Cells were washed twice with DMEM and 99
µL of fresh medium was added followed by adding 1 µL of
fluorinated rhodacyanines at the final concentration of 0.25 µM
or 4.0 µM. Then, the plates were incubated at the same
conditions for an additional 72 h. The percentages of viable cells
were obtained using the mentioned colorimetric assay.
The %inhibition of promastigote proliferation inhibition: The
cultured solution of promastigotes of L. martiniquensis or L.
orientalis (1 × 106 cells·mL–1, 90 µL) was transferred into 96-
wells plate containing fluorinated rhodacyanines at 0.1 µM or
0.25 µM (final concentration) with 1% DMSO in cultured
solution. Cultures treated without the synthesized compounds
were used as negative controls and medium without cells was
used as blank. After adjusting the total volume to 100 µL using
Schneider’s insect medium, the plates were incubated further for
72 h at 25 ± 2 ºC. Then, the living cells were quantified using the
colorimetric assay.
4.4. In silico ADMET prediction analysis
QikProp v3.9 module of Maestro v9.7 interface of
Schrödinger from Schrödinger Release 2019-2 was used to
evaluate the in silico ADMET properties of the rhodacyanine
analogues. Various physicochemical descriptors were calculated,
including number of reactive functional groups (#rtvFG),
octanol/water partition coefficient (QPlogPo/w), aqueous
solubility level (QPlogS), brain/blood partition coefficient
(QPlogBB), central nervous system activity (CNS), apparent gut-
blood barrier permeability (QPPCaco), IC50 value for blockage of
HERG K+ channels (QPlogHERG), number of likely metabolic
reactions (#Metab), and percentage of human oral absorption
level (Percentage of HOA). In addition, violation of the
Lipinski’s rule (vLROF) was assessed using obtained values for
the physicochemical descriptors.
The half maximal inhibitory concentration (IC50) evaluation
of promastigote proliferation inhibition: The cultured solution
of promastigotes of L. martiniquensis or L. orientalis (1 × 106
cells·mL–1, 90 µL) was transferred into 96-wells plate containing
two-fold dilution of the selected fluorinated rhodacyanines with
1% DMSO in cultured solution. Cultures treated without the
synthesized compounds were used as negative controls and
medium without cells was used as blank. After adjusting the total
volume to 100 µL using Schneider’s insect medium, the plates
were incubated further for 72 h at 25 ± 2 ºC. Then, the living
cells were quantified using the colorimetric assay.
4.5. Electrochemistry
Cyclic voltammetric measurements were obtained with an
Autolab PGSTAT101 potentiostat/galvanostat (Eco Chemie, The
The %inhibition of axenic proliferation inhibition: The
cultured solution of promastigotes of L. martiniquensis or L.

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Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
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This work was supported by Research Grant for New Scholar,
[MRG6280024] from the Thailand Research Fund and the Office
of the Higher Education Commission (to T.K.) and the DPST
scholarship from the Royal Thai Government (studentship to
T.L.). Partial support from Ratchadaphiseksomphot Endowment
Fund from Chulalongkorn University is gratefully acknowledged.
Special thanks to Prof. Dr. Masataka Ihara, Prof. Dr. Masayoshi
Tsubuki, Dr. Kazunori Takahashi, and Prof. Dr. Khanitha
Pudhom for their academic advice throughout this project.
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Appendix A. Supplementary data
Supplementary data (including experimental procedures,
characterization such as NMR and HRMS spectra of
intermediates and target compounds, and more electrochemical
data) to this article can be found online at …
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