N-alkylated linear heptamethine polyenes as potent non-azole leads against Candida albicans fungal infections

Article abstract of DOI:10.1016/j.bioorg.2020.104070

In this study, eighteen heptamethine dyes were synthesised and their antifungal activities were evaluated against three clinically relevant yeast species. The eighteen dyes were placed within classes based on their core subunit i.e. 2,3,3-trimethylindolenine (5a-f), 1,1,2-trimethyl-1H-benzo[e]indole (6a-f), or 2-methylbenzothiazole (7a-f). The results presented herein imply that the three families of cyanine dyes, in particular compounds 5a-f, show high potential as selective scaffolds to treat C. albicans infections. This opens up the opportunity for further optimisation and investigation of this class compounds for potential antifungal treatment.

Full text of DOI:10.1016/j.bioorg.2020.104070

Journal Pre-proofs
Short communication
N-Alkylated Linear Heptamethine Polyenes as Potent Non-Azole Leads
against Candida Albicans Fungal Infections
Clare L. Lawrence, Adeyi Okoh Okoh, Vinod Vishwapathi, Sean T.
McKenna, Megan E. Critchley, Robert B. Smith
PII:
S0045-2068(20)31367-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.104070
YBIOO 104070
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
27 April 2020
30 June 2020
2 July 2020
Please cite this article as: C.L. Lawrence, A. Okoh Okoh, V. Vishwapathi, S.T. McKenna, M.E. Critchley, R.B.
Smith, N-Alkylated Linear Heptamethine Polyenes as Potent Non-Azole Leads against Candida Albicans Fungal
Infections, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104070
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N-Alkylated Linear Heptamethine Polyenes as Potent Non-
Azole Leads against Candida Albicans Fungal Infections
Clare L. Lawrencea*, Adeyi Okoh Okohb, Vinod Vishwapathib, Sean T. McKennab,
Megan E. Critchleyb and Robert B. Smithb*
a UCLan Research Centre for Drug Design and Development, School of Pharmacy
and Biomedical Sciences, University of Central Lancashire, Preston, PR1 2HE, UK
b UCLan Research Centre for Smart Materials, University of Central Lancashire,
Preston PR1 2HE, UK
Corresponding Authors: cllawrence@uclan.ac.uk & rbsmith@uclan.ac.uk
Abstract
In this study, eighteen heptamethine dyes were synthesised and their antifungal
activities were evaluated against three clinically relevant yeast species.. The eighteen
dyes were placed within classes based on their core subunit
i.e. 2,3,3-
trimethylindolenine (5a-f), 1,1,2-trimethyl-1H-benzo[e]indole (6a-f), or 2-
methylbenzothiazole (7a-f). The results presented herein imply that the three families
of cyanine dyes, in particular compounds 5a-f, show high potential as selective
scaffolds to treat C. albicans infections. This opens up the opportunity for further
optimisation and investigation of this class compounds for potential antifungal
treatment.
Keywords
Antifungal, Candida Albicans, Cyanine, Hepthamethine, Polyene.
Submitted to
Bioorganic Chemistry
Short Communication

Immunocompromised patients, such as those with cancer, HIV/AIDs, or recent
organ/tissue transplants, are susceptible to infection by opportunistic fungal strains
[1]. The morbid effect of these fungal pathogens is having a significant impact on
human health worldwide. Estimates of mortality are comparable to that of malaria or
tuberculosis [2]. While a limited number of systemic antifungal agents are available to
clinicians, resistance to these widely used compounds is increasing globally [3]. This
resistance not only has significant direct consequences to human health but also to
agriculture, where resistant fungi can have a severe negative impact on food
production [4]. Despite this looming threat, the worldwide increase in antifungal
resistance is not widely appreciated, particularly when compared with the attention
given to the dangers presented by antibiotic resistance [5].
Azoles and their variants (such as imidazoles and triazoles) are particularly important
in antifungal chemotherapy. Upon the discovery of their activity in the 1940s, azole
compounds revolutionised the treatment of systemic fungal infections, with promising
activity against a range of fungal pathogens [6]. Azoles target the cytochrome P450
dependant enzyme lanosterol 14-demthylase CYP51. This interrupts the biosynthesis
of ergosterol, resulting in disruption to the fungal cell membrane and the build-up of
toxic ergosterol precursors that inhibit cell growth [7]. Despite their effectiveness,
azoles are not without problems. Resistance to azoles has been reported for various
fungi of clinical significance including Candida and Aspergillus species [8, 9].
Moreover, azole resistance has also been observed in infections caused by the normally
benign and well-known human commensal organism, Saccharomyces cerevisiae (S.
cerevisiae) [10], a budding yeast prevalent in the brewing industry [11]. Additionally,
azoles have associated toxicity due to their coordination with haem-groups in
mammalian cytochrome P450 enzymes. Azoles create a considerable clinical challenge
in the treatment of immunocompromised patients that may be more vulnerable to the
hepatotoxic effects of azole therapy. Ketoconazole, shown in figure 1, was first available
as a broad-spectrum antifungal but was later replaced due to adverse gastrointestinal
side effects, highlighting another issue associated with this class of compound [12].
N
N
O
O
O
N
N
H3C
Cl
Cl
Figure 1.
The broad-spectrum antifungal Ketoconazole.
Due to increasing resistance to the limited range of available frontline antifungal
drugs, there is a clear and urgent need for novel drug candidates which focus on other
classes of antifungal, such as polyenes. Polyene compounds, such as amphotericin B
(a systemic antifungal) and nystatin (used orally or topically), are a well-established
class of antifungal drug, shown in figure 2. Amphotericin B, in particular, has been in
use for over 60 years and resistance remains relatively rare (although reports of

resistance are increasing) [13]. The antifungal action of polyenes involves the binding
of the drug to ergosterol in the fungal cell membrane [6]. This disrupts cell membrane
structural stability, leading to porin channel formation and subsequent cellular
leakage. This allows the free flow of molecules across the membrane, leading to
impaired cellular functions and ultimately cell death. The effective nature of polyene
compounds, combined with the relatively limited rate of resistance in medically
relevant fungi, suggests that polyenes are an interesting target for novel antifungal
development.
Figure 2.
The structures of nystatin (A) and amphotericin B (B)
This work presents a series of N-alkylated linear heptamethine polyenes as potential
non-azole leads. This series of synthetic compounds have been tested in vitro against
clinically relevant yeast species to establish their effectiveness as antifungal structural
leads. Information relating to the synthesis of the compounds within this study can be
found within the supporting information.
The synthesis of the linear heptamethine cyanine dyes (5a-f – 7a-f) was
straightforward and required no harsh or unusual synthetic methodology. The salts of
2,3,3-trimethylindolenine, 1,1,2-trimethylbenz[e]indole, and 2-methylbenzothiazole
were prepared as per literature methods via alkylation with the corresponding alkyl/
benzyl halides or 1,4-butanesultone [14]. The linear cyanine dyes (5a-f – 7a-f) were
produced through an in-situ cascade reaction as shown in scheme 1, via the ring-
opening of N-(2,4-dinitrophenyl)-pyridinium chloride with aniline to produce 5-
anilino-N-phenyl-2,4-pentadienylideniminium chloride (4). This was immediately
followed by the direct substitution of the aniline subunit with the N-alkylated
substituted indolene salt. The reaction takes place under basic conditions at room
temperature over a period of 12 hours [15, 16]. The formation of cyanines was
monitored by TLC, but an indication of the dye-forming was seen through an intense
colour change (blue/green) after ten minutes of stirring. The crude dyes were purified
by column chromatography using silica gel to obtain the reported compounds. For
each cyanine dye produced both 1D and 2D NMR experiments were used to confirm
thier structure, with other forms of analytical techniques giving further data such mass
spectrometry, infrared, UV and melting point. The synthesised compounds (5a-f – 7a-
f) were tested in vitro to determine the minimum inhibitory concentration (MIC)
using the micro-dilution method previously reported [17].

Scheme 1.
The synthetic strategy to make the linear cyanine dyes (5a-f – 7a-f).
These were screened against three yeast species, which either demonstrate close
homology to several pathogenic fungi or are directly associated with infection.
Schizosaccharomyces pombe (S. pombe), a fission yeast, is closely related to
Pneumocystis jiroveci which is a fungal infection of the lungs [18]. As noted above, S.
cerevisiae is becoming an emerging opportunistic pathogen [19, 20] and is associated
with several conditions such as fungemia [21, 22], peritonitis [23], and meningitis
[24]. Finally, Candida albicans (C. albicans) is a diploid fungus well known for
opportunistic yeast infections [25], especially oral and genital infections in humans
[26, 27]. These fungal species serve as excellent models to learn more about pathogenic
fungi as they share many characteristics with their pathogenic relatives, in particular
concerning regulatory features and drug therapy [28-30]. To validate the results, two
well-characterised antifungal agents were investigated. Amphotericin B and
thiabendazole were employed to compare the efficacy of the compounds presented in
this manuscript. Amphotericin B is an extremely potent polyene antifungal and targets
sterol in fungal cell membranes. As a positive control, amphotericin B showed high
potency against all three species of fungi at 0.061 g/mL respectively, which is
consistent with previously published values [31-32].
Thiabendazole is a known agricultural fungicide and has been used as a positive
control when investigating antifungal activity [33]. It has a similar core structure to
the molecules presented in this paper. However, thiabendazole has previously shown
poor activity against C. albicans and S. cerevisiae, which may be explained by poor
aqueous solubility [34, 35]. This is reinforced by results which showed no growth
inhibition when the aforementioned yeast strains were treated with thiabendazole.
However, inhibition of S. pombe was seen at 62.5 g/mL.
The compounds described have been separated into classes for ease of comparison and
are found in Table 1. The compounds have been separated into three classes: class 1
(5a–1f), class 2 (6a–2f) and class 3 (7a–3f).

Table 1.
Number
The antifungal data for compounds 5a-f, 6a-f and 7a-f.
MIC (g/mL)
+
+
N
N
–
R
R
X
R
CH3
CH2CH3
CH2CH2CH3
CH2(CH2)2CH3
CH2C6H5
X
I
I
I
I
Yield LogP
28% 3.810
49% 4.562 1.95
70% 5.567
33% 6.686
Sp
1.95
Sc
62.5
500
125
125
1000
Ca
7.8
15.6
7.8
5.7
5a
5b
5c
5d
5e
5f
1.95
1.21
Br
-
22% 6.804 1.95
3.9
-
CH2(CH2)3SO3
74%
-
10.0 703.5
10.9
1.995
MIC (g/mL)
+
+
N
N
–
R
X
R
R
CH3
CH2CH3
X
I
I
I
I
Yield LogP
Sp
Sc
Ca
6
10.9
19
55.6
6a
6b
6c
6d
6e
38%
19%
21%
6.129 0.007 1250
6.881 0.03 703.5
7.881 0.03
CH2CH2CH3
CH2(CH2)2CH3
CH2C6H5
625
4750
29% 8.696 0.03
17% 8.753 6.74 5000* 1250
29% 0.323 16.6 5000* 5000*
Br
-
-
6f/ICG
CH2(CH2)3SO3
MIC (g/mL)
S
S
+
+
N
N
–
R
X
R
R
CH3
CH2CH3
X
I
I
I
I
Yield LogP
79% 2.805 7.81
47% 3.557 7.81
69% 4.562 3.91
Sp
Sc
Ca
3.9
31.3
31.3
7a
7b
7c
7d
7e
7f
1000*
1000*
1000*
CH2CH2CH3
CH2(CH2)2CH3
CH2C6H5
47% 5.681 2.93 1000* 15.63
61% 5.994 250 1000* 1000*
15.85 5000* 5000*
Br
-
-
CH2(CH2)3SO3
47%
-
3.001
MIC’s of synthesised compounds tested in S. cerevisiae, S.pombe, and C. albicans. Cells were inoculated
at a concentration of 3 x 104/ml. Culture media tested were in yeast extract broth (YE) for S.pombe and
complex growth media (YPD) for S. cerevisiae and C. albicans. Growth of yeast was determined visually
°
after 24 hours incubation at 30 C. The MIC of the compounds were determined to be the well before
yeast growth was first seen. The experiment was repeated twice. Sc - (S. cerevisiae), SP – (S.pombe) and
Ca– (C. albicans). * indicates the maximum concentration tested. LogP calculated using the
molinspiration (https://www.molinspiration.com). Compound 6f is Indocyanine Green but was made
in house for this study.

The key difference of each class is a variation of the “indole type” fragment within the
core of each molecule. These changes affect their hydrophilic/hydrophobic character,
which seems to play a key role in their activity.
Each class of compound showed excellent activity against S. pombe. This is not
surprising as S. pombe is well known to be relatively sensitive to several antibiotic
agents [36] compared to other fungal species. The most hydrophilic of the compounds,
the sulphonic acids (5f, 6f and 7f), each showed similar MICs. Compound 7e showed
limited growth inhibitory activity against S. pombe with MICs of 250 µg/ml. This is
highly unexpected and does not align with the other benzyl derivatives, 5e and 6e,
suggesting the sulphur moiety is a contributing factor to poor inhibition activity. This
may be due to the increased polarity of the molecule, reducing its interaction with the
yeast cell.
Each class of compound showed poor activity against S. cerevisiae, with 5a being the
most active at 62.5 µg/mL. Class 1 compounds showed limited growth inhibitory
activity, with similar MICs seen with compounds 5a (62.5 µg/mL), 5c (125 µg/mL)
and 5d (125 µg/mL), which contain carbon chains of increasing length. However, this
was not the case for 5b, where the addition of an N-alkylated ethyl group resulted in a
5-fold increase in the MIC (500 µg/mL). This result was unexpected as the only
difference between compounds 5a–d, is the increased carbon length of the R group.
This result was consistent across experiments and similar data was observed with C.
albicans.
The compounds in class 2 follow a similar trend when compared. Against S. cerevisiae,
little growth inhibition was observed, with MICs exceeding 600 µg/mL. This was
significantly higher than the measured values for compounds 5a–c This indicates the
presence of additional phenyl groups from the 1,1,2-trimethyl-1H-benzo[e]indole core
moiety has impacted their antifungal activity. Finally, class 3 compounds, with the
benzothiazole core, showed no activity in S. cerevisiae at the highest concentration
tested (5000 µg/mL) suggesting that the sulphur moiety, as previously described, is a
contributing factor to poor activity.
Notably, each class of compound shows increased activity against C.albicans. To
expand, the MICs of class 1 compounds were, on average, 10-times lower than those
seen in S. cerevisiae. For example, compound 5a in S. cerevisiae had an MIC of 62.5
µg/mL compared to 7.8 µg/mL in C. albicans. Overall, there was little change in the
level of growth inhibition between the class 1 compounds, with MICs ranging from 3.9
– 15.6 µg/mL. The lowest MIC against C. albicans was observed with compound 5e at
3.9 µg/mL. These results are inconsistent when comparing similar compounds in
classes 2 and 3 with “e” and “f” R groups, showing a significant increase in the MIC
compared to other compounds in their class. For example, the MIC for compound 5e
in C.albicans is 3.9 µg/mL compared to 1250 µg/mL in 6e and 1000 µg/mL in 7e. This
may indicate that the increased size of the N-alkylated ‘R’ group in compound classes
2 and 3 are negatively interacting with the core moiety, resulting in a decrease in the
growth inhibitory activity of the compounds.
For class 2 compounds, a similar level of growth inhibition was observed when
compared to class 1 for smaller R groups (5a–7c) but, as described above, the MIC

increased as the R group size increased. Finally, the compounds in class 3 show a
similar trend to the other two classes. Compound 7a showed the greatest growth
inhibition of all tested compounds, with an MIC of 3.9 µg/mL. Increasing the length
of the linear carbon chain resulted in the MIC increasing 10 fold as shown by 7b-c. It
is interesting to note that compound 7d, with the further increased butyl linear chain,
showed a decrease in MIC when compared to 7c. This doesn’t reflect the trend
observed for the class 2 compounds. However, as the change is approximately 2-fold,
this is likely within experimental error and unlikely to be significant. All three classes
of compounds share a polymethine backbone, which suggests this is key to the growth
inhibitory activity against C.albicans. In the majority of cases, the addition of the N-
alkylated R group appears to interfere with this, increasing the MIC.
To determine the mechanism of action of these compounds, comparisons can be made
with key polyene antifungals based on structural similarities. Amphotericin B forms
pores within the fungal membrane via mycosamine-mediated interaction with
ergosterol. Published work proposes that membrane-bound Amphotericin B assumes
two distinct orientations [37]. One is parallel to the membrane bilayer, with the polar
head (i.e., mycosamine and the carboxyl group) located at the membrane/water
interface and the lactone ring buried within the membrane hydrocarbon core [38-42].
The other is orientated perpendicular to the lipid bilayer with the hydrophobic polyene
portion facing the membrane interior and the hydrophilic polyol chain exposed to the
aqueous phase [43-45]. Both membrane binding modes might be relevant for
amphotericin B to exert its mechanism of action.
However, natamycin, which also contains a mycosamine group, exerts its effect by
binding to ergosterol without forming membrane pores. This results in sterols being
unable to perform their normal biological function, resulting in an antifungal effect
[46]. The antifungal activity of natamycin is lower than that seen with amphotericin B
[47]. Therefore, amphotericin B may have two effects that contribute to its activity,
sterol sequestering and membrane permealisation [48].
In comparison with these compounds, the MICs for both amphotericin B (0.03 – 0.25
µg/mL) [31] and natamycin (1.13 µg/mL) [46], are much lower. The lack of the
mycosamine group within our three classes of compounds suggests they are only able
to interact with the ergosterol core through favourable hydrophobic interactions with
the core polyene moiety of the compounds. This would allow the compounds to
sequester ergosterol, similarly to natamycin. Moreover, the cLogP values for class 3
compounds indicate that these are the most hydrophilic and, apart from 7a in
C.albicans, show the least growth inhibition. The increased hydrophilic nature of these
molecules may impact these hydrophobic interactions. The class 2 compounds are the
most hydrophobic due to the fused phenyl ring and, in C.albicans, the MICs of 6a and
6b are lower than that of their corresponding group 1 compound. This suggests that
hydrophobicity is a contributing factor to the mechanism of action of these
compounds. The reduction in activity seen with 6c–6f may be the result of the
introduction of larger N-alkylated R groups which may impact their ability to interact
with sterols and, therefore, reduce their inhibitory activity. The difference in response
of different fungal species to the antifungals might be the result of ergosterol levels

within the cell, with higher levels of ergosterol being related to increased sensitivity.
However, it has been previously reported that there is no significant correlation
between MICs and ergosterol content [49].
It has been noted that the macromolecular composition of the cell wall is similar
between S. cerevisiae and C. albicans [50]. It should also be noted, although mainly
in the membrane, ergosterol is also present in the cell wall [51, 52]. Therefore, it is
possible that some of these compounds are binding to the cell wall and the presence of
larger substitutions is preventing them from accessing the cell membrane directly.
Overall these compounds showed significantly less activity than seen with
amphotericin B. Although they may have a similar mode of action via the cell
membrane, it is also possible that these compounds may be working via an alternative
mechanism. For example, the polyene motif has been demonstrated to produce
reactive oxygen species, which can cause oxidative damage to cells [53].
Within this manuscript, we have reported the preparation of three different families
of linear heptamethine dyes. Each family was tested against three yeast species
including the clinically important S. cerevisiae and C albicans. Moreover, their
activities were compared against two classical antifungals; amphotericin B and
thiabendazole. The three linear heptamethine families differed only in core sub-unit
structure i.e. 2,3,3-trimethylindolenine (5a-f), 1,1,2-trimethyl-1H-benzo[e]indole
(6a-f), or 2-methylbenzothiazole (7a-f). Each of the compound classes shows greater
activity when N-alkylated with short-chain hydrocarbons.
It’s clear to see compounds 5a-f were the most potent, in terms of growth inhibition
against C albicans. Further work needs to look at new derivatives of this class of
subunit. As such, the results presented here imply that the three families of cyanine
dyes, in particular compounds 5a-f, show high potential as selective scaffolds to treat
C. albicans infections.
Notes
The authors declare no competing financial interest.
Acknowledgement
We are pleased to acknowledge the financial support from the University of Central
Lancashire.

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Declaration of interests
☒ 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.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
Title: N-Alkylated Linear Heptamethine Polyenes as Potent Non-Azole Leads against
Candida Albicans Fungal Infections
Authors:
Clare L. Lawrencea*, Adeyi Okoh Okohb, Vinod Vishwapathib, Sean T. McKennab,
Megan E. Critchleyb and Robert B. Smithb*
a
Affiliations:
UCLan Research Centre for Drug Design and Development, School of Pharmacy
and Biomedical Sciences, University of Central Lancashire, Preston, PR1 2HE, UK

Title: N-Alkylated Linear Heptamethine Polyenes as Potent Non-Azole Leads against
Candida Albicans Fungal Infections
Authors:
Clare L. Lawrencea*, Adeyi Okoh Okohb, Vinod Vishwapathib, Sean T.
McKennab, Megan E. Critchleyb and Robert B. Smithb*
a
Affiliations:
UCLan Research Centre for Drug Design and Development, School of
Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston,
PR1 2HE, UK
b
UCLan Research Centre for Smart Materials, University of Central
Lancashire, Preston PR1 2HE, UK
Contact email:
cllawrence@uclan.ac.uk & rbsmith@uclan.ac.uk
Graphical Abstract

N-Alkylated Linear Heptamethine Polyenes as Potent Non-
Azole Leads against Candida Albicans Fungal Infections
Clare L. Lawrencea*, Adeyi Okoh Okohb, Vinod Vishwapathib, Sean T. McKennab,
Megan E. Critchleyb and Robert B. Smithb*
a UCLan Research Centre for Drug Design and Development, School of Pharmacy
and Biomedical Sciences, University of Central Lancashire, Preston, PR1 2HE, UK
b UCLan Research Centre for Smart Materials, University of Central Lancashire,
Preston PR1 2HE, UK
Corresponding Authors: cllawrence@uclan.ac.uk & rbsmith@uclan.ac.uk
Highlight
Synthesis of various Hepthamethine Cyanine Dyes.
Compounds were tested for their antifungal activity.
Compounds found to be potent against C. albicans.