Synthesis and antileishmanial evaluation of thiazole orange analogs

Article abstract of DOI:10.1016/j.bmcl.2019.126725

Cyanine compounds have previously shown excellent in vitro and promising in vivo antileishmanial efficacy, but the potential toxicity of these agents is a concern. A series of 22 analogs of thiazole orange ((Z)-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium salt), a commercial cyanine dye with antileishmanial activity, were synthesized in an effort to increase the selectivity of such compounds while maintaining efficacy. Cyanines possessing substitutions on the quinolinium ring system displayed potency against Leishmania donovani axenic amastigotes that differed little from the parent compound (IC₅₀ 12–42 nM), while ring disjunction analogs were both less potent and less toxic. Changes in DNA melting temperature were modest when synthetic oligonucleotides were incubated with selected analogs (ΔTm ≤ 5 °C), with ring disjunction analogs showing the least effect on this parameter. Despite the high antileishmanial potency of the target compounds, their toxicity and relatively flat SAR suggests that further information regarding the target(s) of these molecules is needed to aid their development as antileishmanials.

Full text of DOI:10.1016/j.bmcl.2019.126725

Journal Pre-proofs
Synthesis and Antileishmanial Evaluation of Thiazole Orange Analogs
Ahmed Abdelhameed, Xiaoping Liao, Craig A. McElroy, April C. Joice, Liva
Rakotondraibe, Junan Li, Carla Slebodnick, Pu Guo, W. David Wilson, Karl A.
Werbovetz
PII:
S0960-894X(19)30683-3
DOI:
https://doi.org/10.1016/j.bmcl.2019.126725
BMCL 126725
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 July 2019
26 September 2019
28 September 2019
Please cite this article as: Abdelhameed, A., Liao, X., McElroy, C.A., Joice, A.C., Rakotondraibe, L., Li, J.,
Slebodnick, C., Guo, P., David Wilson, W., Werbovetz, K.A., Synthesis and Antileishmanial Evaluation of Thiazole
Orange Analogs, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.126725
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Synthesis and Antileishmanial Evaluation of Thiazole Orange Analogs
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Ahmed Abdelhameeda,b, Xiaoping Liaoa, Craig A. McElroya, April C. Joicea, Liva Rakotondraibea,
Junan Lic, Carla Slebodnickd, Pu Guoe, W. David Wilsone, and Karl A. Werbovetza,*
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aDivision of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus, Ohio, USA
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bPharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University, Cairo, Egypt
cCollege of Pharmacy, The Ohio State University, Columbus, Ohio, USA
dDepartment of Chemistry, Virginia Tech, Blacksburg, VA, USA
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eDepartment of Chemistry, Georgia State University, Atlanta, Georgia, USA
*Corresponding author
Phone: 1-614-292-5499
Email: werbovetz.1@osu.edu
Keywords:
Leishmaniasis
Drug discovery
Cyanine
Declarations of interest: none
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Abstract – Cyanine compounds have previously shown excellent in vitro and promising in vivo
antileishmanial efficacy, but the potential toxicity of these agents is a concern. A series of 22
analogs
of
thiazole
orange
((Z)-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-
ylidene)methyl)quinolin-1-ium salt), a commercial cyanine dye with antileishmanial activity, were
synthesized in an effort to increase the selectivity of such compounds while maintaining efficacy.
Cyanines possessing substitutions on the quinolinium ring system displayed potency against
Leishmania donovani axenic amastigotes that differed little from the parent compound (IC50 12-
42 nM), while ring disjunction analogs were both less potent and less toxic. Changes in DNA
melting temperature were modest when synthetic oligonucleotides were incubated with selected
analogs (∆Tm ≤ 5 °C), with ring disjunction analogs showing the least effect on this parameter.
Despite the high antileishmanial potency of the target compounds, their toxicity and relatively flat
SAR suggests that further information regarding the target(s) of these molecules is needed to aid
their development as antileishmanials.
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Leishmaniasis is a neglected tropical disease prominent in developing regions. Due to the
increase in international travel a surge in leishmaniasis has been observed in non-endemic
countries [1]. There are two major forms of the disease: cutaneous and visceral leishmaniasis.
Cutaneous leishmaniasis leads to lifelong scars while the visceral form affects internal organs
and is lethal if left untreated. Visceral leishmaniasis is the second most deadly parasitic infection
after malaria [2]. Currently the drugs used for treating leishmaniasis are limited to pentavalent
antimonial drugs, amphotericin B formulations, the aminoglycoside paromomycin, and the first
orally active drug, miltefosine. However, these drugs suffer from significant weaknesses, which
include the need for a long treatment course, the development of resistance, and the occurrence
of toxic side effects [3-5]. Antimonial drug treatment requires 20-30 days; the long misuse of
these agents due to their availability led to resistance in some regions [6]. Liposomal amphotericin
B possesses excellent clinical efficacy, but is the most expensive antileishmanial drug [7] and is
only administered by injection. Hospitalization of the patient during therapy increases the cost of
treatment for both antimonials and amphotericin B [8, 9]. Paromomycin is useful in treating
visceral leishmaniasis on the Indian subcontinent and is the least expensive antileishmanial drug
[7], but a higher dose of paromomycin is required for efficacy against visceral leishmaniasis in
Sudan [10, 11]. Also, paromomycin must be given parenterally over at least a 21 day treatment
course and carries the threat of developing resistance in the future. Miltefosine is the only oral
drug approved for leishmaniasis treatment, but it is teratogenic, shows gastrointestinal side
effects, and its efficacy has decreased in India since its initial approval [12], most likely due to
inadequate exposure [13]. Efforts toward the development of new antileishmanial drugs that are
effective and orally available should be a high priority.
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Cyanine dyes contain two nitrogen atoms, one being positively charged, that are separated by a
conjugated chain. This chain is typically part of a heterocyclic system and consists of an odd
number of carbon atoms [14]. Cyanines are known to display potent biological activity. The
rhodacyanine MKT-077 (Figure 1) shows activity against several cancer cell lines, is effective in
murine cancer models, and advanced to clinical trials against solid tumors. In Phase I human
clinical trials, MKT-077 was administered i.v. and displayed a half-life of 37 hours and a large
volume of distribution [15]. More recently, other rhodacyanines have shown excellent anticancer
activity [16-18], with JG-231 displaying efficacy in a xenograft model of triple negative breast
cancer [18]. Rhodacyanines also exhibit potent activity against various parasites including those
responsible for Chagas disease [19], malaria [19-24], African sleeping sickness [19], and
leishmaniasis [19, 25]. SJL-01 displayed ≥95% inhibition of liver parasitemia in L. donovani-
infected BALB/c mice when given at i.v. doses of 1.3, 4.1, and 12.5 mg/kg/day for five consecutive
days [25]. While modest antiparasitic activity was observed with high oral doses of some
rhodacyanines [20, 25], these compounds were administered by injection in the vast majority of
in vivo studies reported. Benzothiazole cyanines lacking a rhodanine moiety also exhibit potent
antileishmanial activity [26]. Thiazole orange displayed an IC50 value of 21 nM against L. donovani
intracellular amastigotes with more than 25-fold selectivity towards the parasite compared to
murine peritoneal macrophages [26]. In vivo testing of thiazole orange in a murine visceral
leishmaniasis model at a dose of 1.2 mg/kg/day i.p. for 5 days led to 48% reduction in liver
parasitemia. However, i.p. doses of thiazole orange at 2 mg/kg/day or greater were poorly
tolerated in mice [26].
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3

Br
S
N
N
S
Cl-
Cl-
N
S
S
N
S
S
N
N
Cl
O
O
MKT-077
JG-231
F
N
N
S
S
O
N
S
Cl-
S
O
S
-O
N
Thiazole orange
N
O
SJL-01
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Figure 1. Cyanines previously shown to possess in vitro and in vivo anticancer or antileishmanial
activity.
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While oral activity is desirable for any new antileishmanial drug candidate, the reduction of host
toxicity is also required for the further development of cyanines as antileishmanial agents. Since
the thiazole orange scaffold has been underexplored compared to the rhodacyanines in terms of
antiparasitic evaluation, a series of 22 thiazole orange analogs were synthesized and evaluated
for their antileishmanial activity and toxicity to J774 murine macrophages. Twelve analogs were
prepared where systematic changes were made at positions 5-8 of the quinolinium moiety aiming
to determine the effect of substitution with groups bearing different electronic, hydrophobic, and
steric properties on antileishmanial potency and cytotoxicity to J774 cells [27, 28]. Four other
analogs were synthesized by placing a phenyl ring at these positions to evaluate the impact of an
additional aromatic ring on biological activity. The N-methyl group of the quinolinium ring was
also replaced with other groups based on negative results obtained in genotoxicity tests with the
N-propyl derivative of thiazole orange [29]. One phenyl ring was also removed from either the
benzothiazole or quinolinium moieties to examine the effects of structural simplification on
antileishmanial potency and selectivity.
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Scheme 1 highlights the synthesis of 1,4-dimethyl quinolinium salts bearing electron donating
groups following Knorr’s quinoline synthesis [30]. Briefly, o-, m- and p-substituted anilines 1a-g
reacted with ethyl acetoacetate (2) under neat conditions followed by acid catalyzed cyclization
using sulfuric acid or the milder polyphosphoric acid to yield compounds 3a-g in low yields [31,
32]. Wlodarczyk et al. [33] mentioned that the synthesis of anilides by Knorr’s method was
achieved in low yield due to side products. Such impurities were separated by column
chromatography or simply by trituration from ether [34]. Compounds 4a-g were synthesized in
excellent yields firstly by chlorination using phosphorous oxychloride [35, 36] followed by transfer
hydrogenation employing palladium on carbon using hydrazine as the hydrogen source [36].
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Finally the dimethyl quinolinium salts 5a-j were synthesized by reaction with different alkyl iodides
under sealed tube conditions.
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O
e
c, d
a, b
R
R
R
R
N
H
O
NH2
N
N
R
EtO
O
1 I
1a-g
2
3a-g
4a-g
5a-j
1
1a
1b-1d
1e-1g
3a, 4a
3b-3d, 4b-4d
3e-3g, 4e-4g
: R = H
: R = H
5a
: R = H; R = CH3
1
: R = o-, m- or p-CH3
: R = o-, m- or p-OCH3
: R = 6-, 7- or 8-CH3
: R = 6-, 7- or 8-OCH3
5b
5c
5d
5e
: R = H; R = CH2H3
1
: R = H; R = CH2CH2CH3
1
: R = H; R = Bn
1
: R = 6-CH3; R = CH3
1
5f
: R = 7-CH3; R = CH3
1
5g
5h
: R = 8-CH3; R = CH3
1
: R = 6-OCH3; R = CH3
1
5i
5j
: R = 7-OCH3; R = CH3
1
: R = 8-OCH3; R = CH3
Scheme 1
. Reagents and conditions: (a) neat, 180 °C, 24-48 h; (b) H2SO4 or PPA, 95 °C, 3h; (c) POCl3, reflux, 2-3 h;
(d) hydrazine hydrate 80%, Pd/C 10%, EtOH, reflux, 4-8 h; (e) RI, MeOH, sealed tube, 80 °C, 2-8 h.
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Scheme 2 shows the Skraup-Doebner-von Miller quinoline synthesis [37] used to prepare 1,4-
dimethyl quinolinium salts bearing electron withdrawing substituents. Knorr’s quinoline synthesis
is a longer synthetic procedure relative to the one step Skraup quinoline synthesis but provided
electron rich quinolines with good yields and purity. However, the Knorr approach cannot be used
for the synthesis of halogenated quinolines as it would lead to unwanted dehalogenation during
step d of Scheme 1. Halogenated anilines 1h-m were reacted with methyl vinyl ketone (6) under
acidic conditions to yield quinolines 7a-f [38]. Formation of the corresponding 1,4-dimethyl
quinolinium salts 8a-b was achieved through N-methylation using methyl iodide except for the 8-
chloro analog 7c, as the reaction of this compound with methyl iodide was sluggish. The stronger
alkylating agent methyl triflate was critical for the synthesis of the quinolinium salt 8c [39] and was
used with caution as methyl triflate is a strong alkylating agent.
5

O
a
R
R
+
N
NH2
7a-f
1h-m
6
7a-c
: R = 6-, 7- or 8-Cl
7d-f
: R = 6-, 7- or 8-Br
b or c
-
-
8a
8b
8c
: 6-Cl; X = I
Cl
: 7-Cl; X = I
N
X
-
: 8-Cl; X = CF3SO3
8a-c
Scheme 2
. Reagents and conditions: (a) dioxane, H2SO4, reflux, 48 h; (b)
CH3I, MeOH, sealed tube, 80 °C, 4-8 h; (c) CF3SO3CH3, toluene, rt, 24 h.
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The 6-, 7- and 8-substituted lepidines were made by either the Knorr or Skraup-Doebner-von
Miller quinoline synthesis, leading to the preparation of the corresponding 1,4-dimethyl
quinolinium salts. Neither of these procedures could be used directly for the synthesis of the 5-
substituted lepidines, however, as the m-substituted anilines favor formation of the 7-substituted
lepidine rather than the 5-substituted isomer. Thus, a blocking strategy was employed as outlined
in Scheme 3 starting with the reduction of the commercially available substituted
chloronitrobenzenes 9a and 9b to give the corresponding anilines 10a and 10b [40]. The Skraup-
Doebner-von Miller quinoline synthesis was then used for the preparation of 5-substituted-8-
chloro lepidines followed by dehalogenation to yield 4h and 4i. The quinolinium salts 5k and 5l
were synthesized by the reaction of 4h and 4i with methyl iodide.
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R
R
R
R
R
a
b
c
d
N
N
NO2
9a, b
N
NH2
I
Cl
Cl
Cl
10a, b
11a, b
4h, i
5k, l
4h, 5k
9, 10, 11a
: R = CH3
: R = CH3
4i, 5l
9, 10, 11b
: R = OCH3
: R = OCH3
Scheme 3
6
. Reagents and conditions: (a) Fe, acetic acid, rt to 90 C, 30 min; (b) , dioxane, H2SO4, reflux, 24 h;
(c) NH2NH2.H2O 80%, Pd/C 10%, EtOH, reflux, 4-8 h; (d) CH3I, MeOH, sealed tube, 80 °C, 2-4 h.
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Although demethylation of 4i according to a literature procedure for the demethylation of closely
related compounds [41] yielded the corresponding 5-hydroxylepidine, chlorination of this
derivative was unsuccessful. An alternative route to prepare 5-halo quinolines was thus required
as shown in Scheme 4. Nitration of 8-bromolepidine (7f) employing potassium nitrate and sulfuric
acid gave compound 12 followed by reduction and dehalogenation to prepare 5-aminolepidine
(13) whose structure was confirmed by 2D COSY and NOE NMR experiments. The 5-halo
derivatives 7g and 7h were synthesized by the Sandmeyer reaction using copper(I) chloride and
copper(I) bromide, respectively [42, 43]. Compound 8d was prepared in excellent yield by
reaction of 7g with methyl iodide.
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R
NH2
NO2
Br
a
b,c
d or e
7g
7h
: R = Cl
: R = Br
N
N
N
N
N
Br
7f
12
13
7g, h
f
Cl
I
8d
Scheme 4
. Reagents and conditions: (a) H2SO4, KNO3, 0 °C to rt, 20 h; (b) Fe, acetic acid, rt to 90
C, 30 min; (c) NH2NH2.H2O 80%, Pd/C 10%, EtOH, reflux, 8 h; (d) NaNO2, HCl, CuCl, -10 C to rt,
10 h; (e) NaNO2, 48% HBr, CuBr, -10 C to rt, 10 h; (f) CH3I, MeOH, sealed tube, 80 °C, 2-4 h.
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Scheme 5 outlines the synthesis of the quinolinium salts bearing a phenyl group at positions 5-8
(15a-d) via Suzuki coupling [44] between phenylboronic acid and the bromo analogs 7d-f and 7h,
followed by N-methylation as discussed previously.
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a
Ph
Br
N
14a-d
N
7d-f, 7h
b
14, 15a
: 5-Ph
: 6-Ph
: 7-Ph
: 8-Ph
Ph
14, 15b
14, 15c
14, 15d
N
I
15a-d
Scheme 5
. Reagents and conditions:(a) PhB(OH)2, Pd[P(Ph)3]4, EtOH,
Cs2CO3, reflux, 12 h; (b) CH3I, MeOH, sealed tube, 80 °C, 2-4 h .
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204
Scheme 6 displays the synthesis of cyanines following previously published procedures with slight
modifications [45, 46]. Methylation of the commercially available 2-methylthio benzothiazole (16)
with methyl iodide or methyl triflate provided 17a and 17b. Triethylamine-catalyzed condensation
of 17a and 17b with the quinolinium salts 5a-l, 8a-d and 15a-d yielded the corresponding cyanine
dyes 18a-t (Compound 18a corresponds to thiazole orange prepared as the iodide salt for
comparison with its analogs in this study). The synthesis of thiazolyl cyanine 18u (Scheme 7)
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210
and pyridyl cyanines 18v and 18w (Scheme 8) followed previously published procedures [47, 48].
2-(Methylthio)thiazole (19) was converted to the corresponding thiazolium salt 20, which was then
stirred with 1,4-dimethylquinolinium iodide 5a in dichloromethane at room temperature with 5
equivalents of triethylamine to yield compound 18u (Scheme 7). In similar fashion, 22a and 22b
[obtained by methylation of 4-picoline (21a) or 2-picoline (21b)] reacted with 17a to obtain the
cyanines 18v and 18w, respectively (Scheme 8).
211
X
N
S
N
S
N
X
a or b
SMe
SMe
S
16
-
17a, b
c
17a
17b
: X = I
-
: X = CF3SO3
R
N
R1
R
18a-t
N
R1
X
5a-l
8a-d
15a-d
1
-
1
-
18a
18k
: R = 7-OCH3; R = CH3; X = I
: R = H; R = CH3; X = I
1
-
1
-
18b
18c
18d
18e
18l
: R = H; R = CH2CH3; X = I
: R = 8-OCH3; R = CH3; X = I
1
-
1
-
18m
: R = H; R = CH2CH2CH3; X = I
: R = 5-Cl; R = CH3; X = I
1
-
1
-
18n
18o
18p
18q
: R = H; R = Bn; X = I
: R = 6-Cl; R = CH3; X = I
1
-
1
-
: R = 5-CH3; R = CH3; X = I
: R = 7-Cl; R = CH3; X = I
1
-
1
-
18f
: R = 6-CH3; R =CH3; X = I
: R = 8-Cl; R = CH3; X = CF3SO3
1
-
1
-
18g
18h
: R = 7-CH3; R = CH3; X = I
: R = 5-Ph; R = CH3; X = I
1
-
1
-
18r
: R = 8-CH3; R = CH3; X = I
: R = 6-Ph; R = CH3; X = I
1
-
-
1
-
18i
18j
18s
: R = 5-OCH3; R = CH3; X = I
: R = 7-Ph; R = CH3; X = I
1
1
-
18t
: R = 6-OCH3; R = CH3; X = I
: R = 8-Ph; R = CH3; X = I
Scheme 6
. Reagents and conditions: (a) CH3I, MeOH, sealed tube, 80 °C, 8 h; (b) CF3SO3CH3, rt, 24 h; (c) NEt3, EtOH:
DMF (1:1), reflux, 12 h.
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213
N
S
I
N
N
b
a
MeS
MeS
S
S
N
I
19
20
18u
Scheme 7
. Reagents and conditions: (a) CH3I, MeOH, sealed
5a
tube, 80 °C, 8 h; (b) , NEt3, DCM, rt, 48 h.
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a
CH3
CH3
N
N
I
22a, b
21a, b
b
N
N
S
S
N
I
18v
18w
N
`
I
Scheme 8
. Reagents and conditions: (a) CH3I, MeOH, sealed
17a
tube, 80 °C, 2-4 h; (b)
, NEt3, DCM, rt, 48 h.
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218
219
220
221
222
223
224
225
To examine the geometry of the cyanine target compounds, an X-ray crystal structure was
obtained for 18q (Figure 2). This crystal structure (deposition number CCDC 1920794) shows
the quinoline and benzothiazole ring systems to be nearly coplanar (angle between calculated
mean planes = 23.49), as expected from the delocalized π-electron system present in the
compound and in agreement with a previous report [49]. The conformation of the quinoline and
thiazole ring systems in 18q are also similar to the conformation observed based on Nuclear
Overhauser data and the calculated low energy conformation for thiazole orange reported earlier
[50]. Distortion from co-planarity is primarily due to steric interactions between the phenyl ring at
position 5 and the methyl group on the benzothiazole. The mean plane of the phenyl ring at
position 5 is at an angle of 52.28 relative to the mean plane of the quinoline ring system.
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228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
Figure 2. X-ray crystal structure of 18q.
Compounds 18a, 18b, 18d, 18e, 18k, 18n, 18u, 18v, and 18w were submitted to Badapple [51],
a publically available web application designed to identify promiscuous compounds. The analysis
of substructures generated by these searches for promiscuity is shown in Figure 3 below. The
(Z)-4-(benzo[d]thiazol-2(3H)-ylidenemethyl)quinolin-1-ium scaffold present in all compounds
except for 18u-w exhibited a low probability of promiscuity as assessed by this web application,
while most other substructures identified by Badapple that are present in these compounds
showed high probabilities of promiscuous activity. Although similar molecules were identified as
Pan Assay Interference Compounds (PAINS) in the original paper by Baell and Holloway that
coined this term, the same paper also suggested that such compounds (often dyes) may
nonetheless serve as useful anti-infective leads due to their high antimicrobial potency [52].
Indeed, MKT-077 reached clinical anticancer trials and structurally related compounds continue
to be explored as candidate anticancer agents as mentioned earlier, providing the motivation for
the further exploration of cyanines related to thiazole orange for their antileishmanial potential.
10

HN
HN
S
S
HN
S
N
N
N
H
H
H
low
high
moderate
HN
HN
S
S
N
N
H
H
high
moderate
high
high
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245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
Figure 3. Probability of promiscuous activity for substructures occurring in target compounds as
assessed by Badapple [51].
To provide a clear picture of the inherent selectivity of the target compounds for Leishmania
compared to a mammalian cell line, the effect of these compounds on L. donovani axenic
amastigotes [53] and J774 murine macrophages [54] was measured (Table 1). Cyanine target
compounds displayed exceptional potency against L. donovani axenic amastigotes, with IC50
values ranging from 0.012 µM to 0.29 µM. Among compounds substituted on the quinoline ring
system of the cyanine scaffold, structural modifications had little effect on antileishmanial potency.
For example, the placement of different substituents at the quinoline nitrogen atom ranging in size
from a methyl group to a benzyl group (18a-d) resulted in antileishmanial potencies varying only
by 2.2-fold. Similarly, within the series of compounds substituted at positions 5-8 on the quinoline
ring system with either a methyl (18e-h), methoxy (18i-l), chlorine (18m-p), or phenyl group (18q-
t), antileishmanial potencies varied by only 1.5-, 1.4-, 2.1-, and 3.2-fold, respectively. While in
general the substitution on the quinoline ring system had little effect on antiparasitic activity,
substitution with phenyl rings at positions 5 and 6 of the quinoline core caused a slight decrease
in antiparasitic activity, which for 5-phenyl derivative 18q may be due to the small deviation from
planarity described above. Despite retaining significant activity against L. donovani, substitution
of a thiazole ring (18u) for the benzothiazole system led to an 11-fold decrease in activity against
L. donovani axenic amastigotes compared to parent compound 18a. Likewise, compounds where
a 4-pyridyl substituent (18v) or a 2-pyridyl substituent (18w) replaced the quinoline system were
less potent than the corresponding 18a by 10- and 21-fold, respectively. Truncated derivatives
18u-w were also less toxic to J774 macrophages than 18a-t. Cyanine derivatives 18a-w were in
all cases less toxic to J774 macrophages than to L. donovani axenic amastigotes with selectivity
indexes ranging from 2 to 20, although they nonetheless displayed low IC50 values against this
mammalian cell line (J774 IC50 values ranging from 0.059 µM to 2.8 µM).
272
273
274
275
11

276
277
Table 1. In Vitro Potency of Target Cyanines against L. donovani Axenic Amastigotes and J774
Macrophages
N
N
N
S
S
S
N
R
S
N
N
R1
N
N
X
I
I
I
18a-t
18u
18v
18w
278
Comp.
R1
R
X
Axenic Ld
J774
Selectivity
amastigotes
macrophages
Index
IC50 in µMa
18a
18b
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
18m
18n
18o
18p
18q
18r
Me
Et
Pr
Bn
Me
Me
Me
Me
H
H
H
H
5-Me
6-Me
7-Me
8-Me
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
0.014 ± 0.000
0.012 ± 0.002
0.013 ± 0.001
0.026 ± 0.004
0.024 ± 0.002
0.031 ± 0.003
0.037 ± 0.003
0.030 ± 0.006
0.025 ± 0.003
0.028 ± 0.002
0.035 ± 0.002
0.030 ± 0.002
0.016 ± 0.003
0.031 ± 0.002
0.034 ± 0.003
0.022 ± 0.005
0.042 ± 0.010
0.037 ± 0.011
0.013 ± 0.003
0.024 ± 0.003
0.16 ± 0.01
0.19 ± 0.03
0.17 ± 0.03
0.14 ± 0.02
0.19 ± 0.03
0.12 ± 0.01
0.20 ± 0.03
0.12 ± 0.02
0.12 ± 0.02
0.086 ± 0.012
0.20 ± 0.02
0.14 ± 0.01
0.059 ± 0.028b
0.13 ± 0.02
0.31 ± 0.04
0.18 ± 0.02
0.11 ± 0.01
0.16 ± 0.03
0.18 ± 0.03
0.069 ± 0.009
0.20 ± 0.04
1.5 ± 0.1
14
14
11
7.0
5.0
6.5
3.2
4.0
3.4
7.1
4.0
2.0
8.1
10
5.3
5.0
3.8
4.9
5.3
8.3
9.4
20
Me 5-OMe
Me 6-OMe
Me 7-OMe
Me 8-OMe
Me
Me
Me
Me
Me
Me
Me
Me
-
5-Cl
6-Cl
7-Cl
8-Cl CF3SO3
5-Ph
6-Ph
7-Ph
8-Ph
-
-
-
-
-
I
I
I
I
I
I
I
-
-
18s
18t
18u
18v
18w
-
-
-
-
0.14 ± 0.03
0.29 ± 0.02
3.1 ± 0.2
2.8 ± 0.2
2.5 ± 0.3
8.6
-
-
Pentamidine
Podophyllotoxin
-
-
0.023 ± 0.002
279
280
281
aMean ± standard error of at least three independent determinations
bMean ± range of two independent determinations
12

282
283
284
285
286
287
288
289
290
291
The activities of the most potent quinoline-containing cyanine (18b) and the two pyridyl cyanines
(18v and 18w) were determined against intracellular L. donovani (Table 2) using a high content
assay that was recently described [55]. Consistent with the work of Yang et al. concerning the
antileishmanial activity of rhodacyanines [25], 18b, 18v, and 18w were less potent in the
intracellular assay compared to the axenic amastigote assay. Nonetheless, each of these
compounds showed good activity in the intracellular assay, with the quinoline-containing cyanine
18b again displaying higher potency than the two pyridyl cyanines 18v and 18w, approaching the
potency of amphotericin B in this assay.
Table 2. In Vitro Potency of Cyanines 18b, 18v, and 18w against Intracellular L. donovani
Compound
18b
IC50 (µM) against intracellular L. donovania
0.072 ± 0.009
0.96 ± 0.18
1.7 ± 0.4
0.045 ± 0.009b
18v
18w
Amphotericin B
aMean ± standard error of three independent determinations
bMean ± standard error of five independent determinations
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
The ability of selected cyanine target compounds to bind double-stranded DNA (Table 3) was
assessed by measuring the increase in melting temperature of two mixed sequence synthetic
hairpin DNA oligomers (5'-dCCAAAATTTTG CTCT CAAAATTTTGG-3' [AAAATTTT]; 5'-
dCCAAAAGTTTTG CTCT CAAAACTTTTGG [AAAAGTTTT] with the hairpin CTCT loop
underlined) that have been used in previous studies with synthetic compounds [56]. Circular
dichroism studies were also conducted with a representative cyanine, 18f, binding to an AT rich
DNA (Supplementary Figure 1) and giving a negative induced CD signal which is characteristic
of an intercalation binding mode [57]. For Tm determinations, the synthetic compounds were
incubated with the DNA samples prior to Tm measurements as previously described [56, 58]. The
compounds chosen for this analysis examined the effect on DNA binding of a methyl group at
positions 5-8 of the quinoline system (18e-h) in addition to a phenyl group at position 5 (18q).
The truncated derivatives 18u-w were also tested in this assay. For the oligomer containing the
double-stranded AAAATTTT sequence, compounds 18a, 18e-h, and 18q increased the DNA Tm
by only 3-5 degrees. Truncated derivatives 18u-v were poorer binders to this oligomer, increasing
Tm by 2 degrees or less. The other DNA gave similar, relatively low Tm increases with melting
temperature increases below 5 °C in the presence of these compounds. Again, the truncated
derivatives 18u-w gave the lowest increase in melting temperature of all of the derivatives. The
relatively low Tm values of the DNA complexes of these compounds and the lack of any clear
correlation with biological activity suggests that DNA binding is not a major component of their
antileishmanial potency, although it may be one aspect of a complex mechanism of action for
these compounds.
316
317
Table 3. DNA binding of selected cyanines to synthetic double stranded oligomers
ΔTm (°C) for AAAATTTT
18a
18e
18f
18g
18h
18q
18u
18v
18w
4
3
5
4
4
3
2
1
<1
ΔTm (°C) for AAAAGTTTT
18a
18e
18f
18g
18h
18q
18u
18v
18w
4
4
4
4
3
2
1
<1
<1
318
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354
355
356
357
358
359
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361
362
363
364
365
366
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369
Our analysis of 22 cyanines related to thiazole orange confirmed the exceptional in vitro
antileishmanial potency of the class. The SAR in this series is relatively flat when substitutions
on the quinoline ring system are considered, however. Removal of one aromatic ring from either
the quinolinium or benzothiazole portion of the scaffold led to a decrease in toxicity but also a
decrease in antileishmanial potency. While not a focus of the current study, future efforts to
improve the drug-like properties of thiazole orange analogs should consider increasing the level
of saturation of these molecules. Lovering et al. demonstrated that successful transition from
drug discovery through clinical trials correlated with the saturation level of the molecule; Fsp3
(number of sp3 carbon atoms/total number of carbon atoms) was also defined in that manuscript
as a simple, intuitive measure of saturation [59]. Calculated Fsp3 values are low for the present
target molecules, ranging from 0.08 for compounds 18d and 18q-t to 0.19 for N-propyl derivative
18c. Past studies concerning the anticancer and antiprotozoal activity of rhodacyanines have
also focused on molecules with a low percentage of sp3 hybridized carbons [17-25]. Future
discovery efforts should consider balancing activity requirements for planarity/electron
delocalization in these classes of compounds with increasing their level of saturation.
MKT-077 accumulates in the mitochondrion of cancer cells to a greater extent than in normal cells
[60], in accord with the delocalized lipophilic cation hypothesis outlined by Chen [61]. Subsequent
studies with MKT-077 indicate specific binding of this molecule to mot-2, a member of the Hsp70
family, resulting in release of the tumor suppressor protein p53 from sequestration by mot-2 in
cancer cells [62]. YM-1, a close analog of MKT-077 possessing a methyl group at the pyridinium
nitrogen atom rather than an ethyl group, was also toxic to cancer cell lines but showed a greater
distribution to the cytosol than to the mitochondrion in MCF7 cells [16]. Attempts to optimize these
rhodacyanines led to the synthesis of related molecules that bound Hsp70 [17] and later to the
preparation of JG-231 [18], which blocks the Hsp70 interaction with BAG family nucleotide
exchange factors. Thus, there is evidence that rhodacyanine dyes interact with specific molecular
targets. In terms of the activity of related molecules against parasites, studies employing
fluorescent rhodacyanine antimalarial compounds showed the selective and irreversible
accumulation of these molecules within P. falciparum infected erythrocytes compared to non-
infected erythrocytes. The most potent rhodacyanine compounds accumulated in specific
organelles while less potent derivatives exhibited diffuse staining throughout the parasite
cytoplasm; double staining experiments revealed that a potent antimalarial rhodacyanine
colocalized with MitoTracker Red CMXRos but not with the nuclear diamidine stain DAPI [22]. A
subsequent report indicated the accumulation of a different rhodacyanine in the Plasmodium
apicoplast [23]. The cationic antileishmanial drugs pentamidine and WR6026 (sitamaquine)
depolarize the mitochondrial membrane potential in L. donovani promastigotes [63]; dications
pentamidine and [1,1':4',1''-terphenyl]-4,4''-bis(carboximidamide) cause dilation of this organelle
in Leishmania [64, 65]. While cyanines may disrupt the mitochondrion in Leishmania, additional
parasite targets could also be affected by the molecules reported in this manuscript. Since
antiparasitic potency was not dissociated from toxicity in the present study, and considering that
a low dose of thiazole orange was not well tolerated in mice [26], further progress toward the
identification of cyanines with true potential as antileishmanial drug candidates will require
additional information on the targets of these compounds. Such mechanistic information is
expected to lower the risk of developing new antileishmanial candidates [66] and enable the
design of potent antiparasitic cyanines with greater selectivity toward Leishmania parasites.
Acknowledgement
Ahmed Abdelhameed was supported by a graduate fellowship from the Egyptian government.
The sponsors had no role in the study design, in the collection, analysis, or interpretation of the
data, in preparing the manuscript, or in the decision to publish this work.
14

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N
N
S
S
R = H, Me, OMe, Cl, Ph
R1 = Me, Et, Pr, Bn
R
N
N
18a-t
R1
Potent vs. L. donovani axenic amastigotes (IC50: 12-42 nM)
Toxic to J774 macrophages (selectivity index (SI): 2-14)
N
S
N
S
N
S
N
N
N
IC50 vs. Ld = 160 nM
IC50 vs. Ld = 290 nM
IC50 vs. Ld = 140 nM
SI (J774) = 9.4
SI (J774) = 8.6
SI (J774) = 20
Molecular simplification approach decreased both antileishmanial potency and toxicity
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Declaration of interests
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☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
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19

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20