N-Alkylated 2,3,3-trimethylindolenines and 2-methylbenzothiazoles. Potential lead compounds in the fight against Saccharomyces cerevisiae infections Dedication in memory of the late Derek Bennett 17-07-1931 to 05-04-2013.

Article abstract of DOI:10.1016/j.ejmech.2013.03.031

The synthesis of a variety of N-alkylated 2,3,3-trimethylindolenines and 2-methylbenzothiazoles is reported herein. Their potential as antifungal agents is evaluated by preliminary screening against Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe), and Candida albicans (C. albicans). Statistical analyses illustrate a strong relationship between chain length and growth inhibition for S. cerevisiae and S. pombe (p < 0.0001 in every case). Of particular interest is the activity of both sets of compounds against S. cerevisiae, as this is emerging as an opportunistic pathogen, especially in immunosuppressed and immunocompromised patients. Bioassays were set up to compare the efficacy of our range of N-alkylated compounds against classic antifungal agents; Amphotericin B and Thiabendazole.

Full text of DOI:10.1016/j.ejmech.2013.03.031

European Journal of Medicinal Chemistry 64 (2013) 222e227
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
N-Alkylated 2,3,3-trimethylindolenines and 2-methylbenzothiazoles.
Potential lead compounds in the fight against Saccharomyces cerevisiae
infections
Andrew R. Tyler a, Adeyi Okoh Okoh b, Clare L. Lawrence c, Vicky C. Jones c, Colin Moffatt b,
,
Robert B. Smith b
*
a Chemistry, School of Forensic and Investigative Sciences, University of Central Lancashire, Preston PR1 2HE, UK
b Centre for Material Sciences, School of Forensic and Investigative Sciences, University of Central Lancashire, Preston PR1 2HE, UK
c School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston PR1 2HE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a variety of N-alkylated 2,3,3-trimethylindolenines and 2-methylbenzothiazoles is re-
ported herein. Their potential as antifungal agents is evaluated by preliminary screening against
Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe), and Candida albicans
(C. albicans). Statistical analyses illustrate a strong relationship between chain length and growth inhi-
bition for S. cerevisiae and S. pombe (p < 0.0001 in every case).
Of particular interest is the activity of both sets of compounds against S. cerevisiae, as this is emerging
as an opportunistic pathogen, especially in immunosuppressed and immunocompromised patients.
Bioassays were set up to compare the efficacy of our range of N-alkylated compounds against classic
antifungal agents; Amphotericin B and Thiabendazole.
Received 23 January 2013
Received in revised form
15 March 2013
Accepted 19 March 2013
Available online 6 April 2013
Dedication In memory of the late Derek
Bennett 17-07-1931 to 05-04-2013.
Keywords:
Antifungal
Ó 2013 Elsevier Masson SAS. All rights reserved.
Drug design
2-Methylbenzothiazole
Structure activity relationship
2,3,3-Trimethylindolenine
Yeast
1. Introduction
(1,3)-b-D-glucan; their structure being based on natural lipopeptide
products [3]. The shift towards natural products is not particularly
surprising; these appear to be more acceptable to both the phar-
maceutical industry and patients from a toxicity viewpoint. Indeed
many natural products which are being derived from plants, are
beingidentified as antifungalagents[4]. Benzothiazoles are one such
type of compound, whose core molecular structure can be found in a
large number of natural products [5]. The benzathiazoles exhibit
great pharmaceutical importance due to their potent biological ac-
tivities, these include anti-bacterial [6], anti-ulcer [7], anti-cancer
[8], anti-inflammatory [9] and anti-viral [10] to mention a few.
Inspired by the aforementioned medicinal activity of the ben-
zothiazoles, we have set out to create two small libraries of struc-
turally similar N-alkylated molecules: 2,3,3-trimethylindolenine
(1aem) and 2-methylbenzothiazole (2aem). Each of these sub-
classes has a step-wise increase in linear alkyl chain length (C1e
C10). The resulting derivatives show varying degrees of lip-
opihilicity, as judged by their log P (base 10 logarithm of the
partition coefficient) values (shown in Tables 1 and 2, respectively).
It’s widely accepted that an increase in the length of the carbon
The rise in number of immunocompromised patients (either
through infections, nutritional irregularities or medical treatment)
has led to an increase in opportunistic fungal infections [1]. This in
turn, is forcing health care professionals to seek more aggressive
forms of chemotherapeutic treatments, which inevitably leads to
fungal infections with greater resistance to current therapies.
Although systemic antifungal agents have been available since the
1950s, the end of the last century saw the development of a new
generation of antifungal agent; the triazoles; which revolutionised
clinical mycology. Itraconazole 1 and Fluconazole 2 (Fig.1) were two
such triazoles, licensed by the Food and Drug Administration (FDA)
for topical use [2]. The dawn of the new millennium brought the new
echinocandin antifungals, such as Micafungin 3, as shown in Fig. 2.
These drugs inhibit cell wall synthesis, in particular the synthesis of
* Corresponding author. Tel.: þ44 (0)1772 894384; fax: þ44 (0)1772 894981.
E-mail address: rbsmith@uclan.ac.uk (R.B. Smith).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.031

A.R. Tyler et al. / European Journal of Medicinal Chemistry 64 (2013) 222e227
223
1
2
Fig. 1. Two licensed triazole antifungals, Itraconazole 1 and Fluconazole 2.
chain, goes hand in hand with an increase in pharmacological ac-
tivity (this is usual from C1eC10), due to an increase in lipid
membrane penetration. However, above C10 a decrease in activity
is usually seen and this is attributed to poor transport through
aqueous media [10]. Our intention was to examine the growth in-
hibition of three yeast strains up to a linear chain length of C10 and
assess any potential antifungal activity observed.
Scheme 1. The salts of 2,3,3-trimethylindolenine and 2-
methylbenzothiazole were prepared by alkylation with the corre-
sponding alkyl/benzyl halides or sultones to afford the compounds
in excellent to poor yield as shown in Tables 1 and 2. Compounds
1aed, kem and 2aem precipitated from the reaction mixture and
were isolated by filtration under reduced pressure. What is
noticeable is the increased hygroscopic nature of the indolenium
bromide salts with increased chain length (C5eC10). To counteract
this problem, a counter ion exchange based on the Finkelstein re-
action was performed to yield the iodide salts which are not as
hygroscopic as the bromides; thus easier to handle and work with
The compounds were tested against three different yeast spe-
cies; Schizosaccharomyces pombe (S. pombe),
a fission yeast;
Saccharomyces cerevisiae (S. cerevisiae), budding yeast and
a
Candida albicans (C. albicans) a diploid fungus known for oppor-
tunistic oral and genital infections in humans [11]. Both S. pombe
and S. cerevisiae yeast species are used extensively in eukaryotic
microbiological research and demonstrate close homology to a
number of pathogenic fungi. For example, S. cerevisiae is closely
related to C. albicans, which, as mentioned above, is a widespread
commensal and important pathogen of humans. Similarly, S. pombe
is closely related to Pneumocystis jiroveci, a yeast-like fungus which
commonly causes pneumonia in immunocompromised patients
[12]. S. cerevisiae is of particular importance, emerging as an
opportunistic pathogen, especially in immunosuppressed and
immunocompromised patients, and has been associated with
fungemia, endocarditis, peritonitis, meningitis, ventriculitis, and
with polymicrobial fatal pneumonia in HIV/AIDS patients [13e19].
These yeast species can serve as excellent models to learn more
about pathogenic fungi, in particular with regard to regulatory
features and drug therapy, because they share many characteristics
with their pathogenic relatives [20e22].
from
a biological viewpoint. The synthesis of four 2,3,3-
trimethylindolenine salts (1a, e, k and l) are shown in the experi-
mental section below for clarity. The synthesis of the full set of
compounds (1aem and 2aem) is also shown in the Supporting
information.
2.2. Antifungal activity
The synthesised compounds were tested in vitro to determine
growth inhibitory activity. Minimum inhibitory concentration
(MIC) values were determined in sets, by comparison with 2,3,3-
trimethylindolenine (1n) and 2-methylbenzathiazole (2n) (pur-
chased from Alfa Aesar), under the same conditions noted in
Table 1
The antifungal data for compounds 1aen.
Number
MIC
g/mL)
(
m
2. Results and discussion
2.1. Chemistry
R
% Yield Log P
Sc
500
Sp
500
500
250
250
125
125
62.5 1000
62.5 1000
31.3 1000
15.6
1000
Ca
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
CH3
CH2CH3
58
86
85
67
38 (99)
22 (99)
19 (86)
11 (40)
20 (27)
53 (99)
77
ꢀ0.691
1000
1000
1000
1000
1000
1000
The synthesis of both sets of salts was straightforward and
required no harsh or unusual synthetic methodologies, as shown in
ꢀ0.315
0.188
0.747
1.252
1.758
2.263
2.768
3.273
3.778
500
250
250
250
125
125
62.5
31.3
15.6
CH2CH2CH3
CH2(CH2)2CH3
CH2(CH2)3CH3
CH2(CH2)4CH3
CH2(CH2)5CH3
CH2(CH2)6CH3
CH2(CH2)7CH3
CH2(CH2)8CH3
CH2C6H5
500
1000
1000
1000
500
0.904 1000
CH2CH2CH2SOꢀ3
58
ꢀ4.33 1000
ꢀ4.114 1000
3.286 1000
1000
1000
250
CH2CH2CH2CH2SOꢀ3 50
NA
e
Minimal Inhibitory Growth Concentration (MIC) of synthesised compounds tested
in S. cerevisiae, S. pombe, and C. albicans. Cells were inoculated at a concentration of
3 ꢁ 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 h incubation at 30 ꢂC. The MIC of the compounds was
determined to be the well before yeast growth was first seen. The experiment was
repeated twice. Sc e (S. cerevisiae), Sp e (S. pombe) and Ca e (C. albicans). Yielded
brackets highlight yield for iodo counter ion exchange. Xꢀ Indicates either a bromide
or iodide counter ion.
3
Fig. 2. Micafungin 3, an echinocandin natural product antifungal.

224
A.R. Tyler et al. / European Journal of Medicinal Chemistry 64 (2013) 222e227
Table 2
to be the least potent with the highest MIC values, yet compounds
2iej with N-alkylated chain length > C9 show the lowest MIC
The antifungal data for compounds 2aen.
values, again with compound 2j displaying most potency at 7.8 mg/
mL.
Number
MIC (
m
g/mL)
Sp
It is also noted that the sulfonic acid salts of both the 2,3,3-
trimethylindolenine and the 2-methylbenzothiazole (1lem and
2lem) showed no antifungal activity, it is assumed that the pres-
ence of the sulfonic acid groups tends to increase their solubility
and reduces their growth inhibitory characteristics, possibly
through suppressing membrane permeability and cellular uptake.
It is also noted that the benzyl substituent showed no activity
against any of the three fungi types and again this could possibly be
due to the molecule having little membrane permeability due to
the sp2 hybridised ring system. It is interesting to note that the
starting material 2,3,3-trimethylindolenine (1n) showed slight
R
% Yield Log P
Sc
Ca
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
CH3
CH2CH3
87
41
44
51
11
7
10
15
9
ꢀ1.077
ꢀ0.701
ꢀ0.199
0.36
1.252
1.371
1.876
2.381
2.887
3.392
500
500
250
250
62.5
62.5
31.3
15.6
7.8
500
1000
1000
1000
1000
1000
1000
1000
250
250
250
250
250
125
62.5 1000
31.3
7.8
CH2CH2CH3
CH2(CH2)2CH3
CH2(CH2)3CH3
CH2(CH2)4CH3
CH2(CH2)5CH3
CH2(CH2)6CH3
CH2(CH2)7CH3
CH2(CH2)8CH3
CH2C6H5
500
250
1000
1000
1000
1000
6
3.9
90
50
7
0.517 1000
ꢀ4.576 1000
ꢀ4.411 1000
2.085 1000
1000
growth inhibition of S. pombe and C. albicans with MIC’s of 250
mL and 500 g/mL respectively. The 2-methylbenzathiazole (2n)
also showed growth inhibition of S. pombe again at 250 g/mL but
mg/
CH2CH2CH2SO-3
CH2CH2CH2CH2SO-3
N/A
1000
1000
250
m
m
e
showed no growth inhibition against S. cerevisiae or C. albicans. This
indicates that varying the alkyl chain length has a very positive
impact on growth inhibition.
Minimal Inhibitory Growth Concentration (MIC) of synthesised compounds tested
in S. cerevisiae, S. pombe, and C. albicans. Cells were inoculated at a concentration of
3 ꢁ 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 h incubation at 30 ꢂC. The MIC of the compounds was
determined to be the well before yeast growth was first seen. The experiment was
repeated twice. Sc e (S. cerevisiae), Sp e (S. pombe) and Ca e (C. albicans). Xꢀ In-
dicates either a bromide or iodide counter ion.
2.2.1. Control bioassays
Two bioassays with Amphotericin B 4 and Thiabendazole 5
(Fig. 4) were set up to compare the efficacy of our range of N-alky-
lated compounds against classic antifungal agents. The rationale
behind the choice of these antifungals was as follows. Amphotericin
B is an extremely potent antifungal and targets the membrane sterol
of fungal cell membranes, this choice of antifungal agent is crucial as
it provides a similar mode of action in comparison to our com-
pounds. Thiabendazole inhibits nucleic acid metabolism and protein
synthesis and has a similar core structure when compared against
our compounds. However, the Thiabendazole has previously shown
poor activity against C. albicans [25] and S. cerevisiae, which may be
explained by poor aqueous solubility [26]. Amphotericin B showed
Tables 1 and 2. It is important to note that the yeast cell surfaces
carry a negative charge [23] and thus a good interaction between
the yeast cell surface should be observed with the quaternary N-
alkylated compounds (1aem and 2aem).
We chose to vary one physiochemical property (log P) to
examine how this affected growth inhibition (log 1/C), due to the
medicinal target i.e. yeast cell wall. Using a virtual method, the log P
(the base 10 logarithm was used throughout) values were obtained
[24]. Neither sets of compounds inhibited the growth of C. albicans,
so this species was removed from subsequent analyses.
From analysing the MIC values in Tables 1 and 2, the assumption
can be made that the 2,3,3-trimethylindolenine salts (1aeh)
showed a poor growth inhibition relationship against both the
S. cerevisiae and S. pombe yeast strains. However, compounds 1iej
with an N-alkylated chain length > C9, are shown to be the most
potent of the set. The lowest MIC value was recorded against both
high potencyagainst all three species of yeast at 0.49
in line with published data [25]. Thiabendazole, showed poor results
against S. cerevisiae and C. albicans species at 1000 g/mL, however
against S. pombe species the MIC was 31.25 g/mL. We thus conclude
that the longer chain N-alkylated 2,3,3-trimethylindolenines and 2-
methylbenzothiazoles (1iej and 2gej) at concentrations <35 g/mL
mg/mL, which is
m
m
m
show a comparable efficacy profile with the known antifungals used
in the treatment of fungal infections. This suggests further investi-
gation into these compounds would be beneficial for the treatment
of fungal infections. The bioassays were set-up in line with experi-
mental procedure highlighted in Section 4.2.
the aforementioned stains at 15.6
mg/mL for compound 1j. In
comparison the 2-methylbenzathiazole salts (2aej) showed vary-
ing degrees of growth inhibition. Against S. cerevisiae, compound
2aef showed poor growth inhibition. Conversely, compounds 2gej
with N-alkylated chain length > C7, gave the lowest MIC values for
growth inhibition, with the compound 2j displaying most potency
2.3. Statistical analysis
at 3.9 mg/mL. However, upon comparison with the S. pombe yeast
strain, a different result is observed. Compounds 2aeh are deemed
The intentionwas to analyse the relationship between increasing
chain length and growth inhibition by ordinary least squares
Alkyl Halide
or Sultone
X = S or C(CH3)2
Reflux
a =Me
h = n-Oct
b = Et
i = n-Non
c = n-Pr
d = n-Bu
e = n-Pent
f = n-Hex
g = n-Hept
j = n-Dec
k = Bn
l = CH2(CH2)2SO3-
m = CH2(CH2)3SO3-
Scheme 1. The synthetic strategy to making the 2,3,3-trimethylindolenine (1aem) and 2-methylbenzothiazole (2aem) salts.

A.R. Tyler et al. / European Journal of Medicinal Chemistry 64 (2013) 222e227
225
distributed. For these reasons linear regression was deemed inap-
propriate, and a correlationwas used. This is unfortunate as it means
the relationship between the variables could not be quantified as a
straight line equation. As the log (1/C) variable was not normally
distributed in three out of four cases, a non-parametric method was
required. Spearman’s rank correlation coefficient is simply Pearson’s
ProducteMoment correlation on ranks rather than original values.
Its statistic r
(‘Spearman’s rho‘) is thus related to r2, the coefficient of
determination, so values of r2 are given in Fig. 3 to facilitate com-
parison with studies which report this quantity.
A strong relationship is clear in all four plots of Fig. 3, with that
for S. pombe and 2-methylbenzothiazole (Fig. 3d) the weakest
(
r
¼ 0.910, t ¼ 9.34, n ¼ 20, p < 0.0001); increasing the alkyl chain
length increases growth inhibition.
3. Conclusion
The quaternary N-alkylated derivatives of both 2,3,3-
trimethylindolenine and 2-methylbenzothiazole salts show vary-
ing degrees of antifungal activity with the longer chain substituents
being more potent against S. cerevisiae and S. pombe. We propose
that the growth inhibition can be attributed to the charged N-
alkylated compounds with the increasing linear chain lengths. We
postulate that these compounds are attracted to the negatively
charged yeast membrane, with the longer lipophilic chains being
absorbed into and subsequently distorting the lipid bilayer. Com-
pounds 1iej and 2gej, all show MIC values of <35 mg/mL and are
thus deemed from this study to be the most potent towards
S. cerevisiae and S. pombe. Together with further compounds, which
are being generated with higher degrees of lipophilicity these
compounds will be taken forward for mammalian screening. This
preliminary study has also highlighted that none of the synthesised
compounds (1aem and 2aem) has shown any real activity towards
C. albicans. This response may be the result of C. albicans’ ability to
adapt to the antifungal stress and develop drug tolerance. As a
diploid fungus, C. albicans contains a large number of drug exclu-
sion mechanisms with varying substrate specificities enabling it to
obtain tolerance to many novel antifungals, a resistance not seen
with the haploid yeast species, S. cerevisiae and S. pombe [27,28].
The results highlighted above imply that longer N-alkylated (i.e.
>C7) 2,3,3-trimethylindolenines and 2-methylbenzothiazole salts
show potential for selective targeting towards S. cerevisiae in-
fections which are a major problem in health care.
4. Experimental section
4.1. General procedure
1H and 13C NMR spectra were measured on either a Bruker DPX
250 MHz, Bruker Avance-III 300 MHz or a Bruker Avance 400 MHz
spectrometer at ambient temperature with tetramethylsilane
(TMS) as internal standard for 1H NMR and deuteriochloroform
(CDCl3, dC 77.23 ppm) and deuteriodimethylsulfoxide (d6-DMSO, dC
39.51 ppm) for 13C NMR unless otherwise stated. All chemical shifts
Fig. 3. Relationship between (base 10 logarithms of) the inverse of minimum con-
centration for growth inhibition and the partition coefficient for the fungal species S.
pombe and S. cerevisiae and alkylated molecule type. Two replicates were used, and
points overlap where only one appears at each log P value. 2,3,3-Trimethylindolenine
(Indol) (1aej) and 2-methylbenzathiazole (Benzo) (2aej) values for each replicate are
marked by a plus symbol. The square of Spearman’s rho (see text for explanation) on
each plot quantifies the strength of the relationship.
are quoted in
d (ppm) and coupling constants in Hertz (Hz) using
the high frequency positive convention. The abbreviations used for
the multiplicity of the NMR signals are: s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, quin ¼ quintet, sex ¼ sextet, m ¼ multiplet,
dd ¼ doublet of doublet, td ¼ triplet of doublets, dm ¼ doublet of
multiplets, br s ¼ broad singlet, etc. Mass spectra were recorded on
a Thermo Scientific Trace LC Ultra DSQ II using Electron Ionisation
(LCMS-EI). Infrared spectra were recorded on a Specac ATR with a
He Ne ꢀ633 nm laser. Thin Layer Chromatography (TLC) was car-
ried out on MacheryeNagel polygramSil/G/UV254 pre-coated plates.
Melting point (Mp) analysis was carried out using the Griffin
regression, since, with the limited range of log P values, a straight
line relationship was expected. However, the relationship was not
sufficiently linear to use this method, as a slight upward curve e
made more noticeable in a plot of residuals against fitted values (not
shown) e is evident (Fig. 3aed). Furthermore, since the dependent
variable was restricted by the small numbers of serial dilution
concentrations, consistent with experimental protocol, residual
values from the model were very unlikely to be normally

226
A.R. Tyler et al. / European Journal of Medicinal Chemistry 64 (2013) 222e227
4
5
Fig. 4. The structures of Amphotericin B 4 and Thiabendazole 5.
melting point apparatus. All chemicals were purchased from
SigmaeAldrich or Alfa Aesar and used without purification.
132.7, 130.1, 129.5, 129.3, 128.1, 124.2, 116.5, 55.1, 51.2, 15.1, 9.81. IR
(ATR, cmꢀþ1): 2969, 1603, 1454, 931, 741, 701, 567. MS (ESI) m/z:
250.33 [M ]. Melting point ¼ 226e230 ꢂC.
4.1.1. 1,2,3,3-Tetramethyl-3H-indol-1-ium iodide (1a)
2,3,3-Trimethylindolenine (63.0 mmol) was dissolved in iodo-
methane (168 mmol) and with constant stirring, the solution was
refluxed for 24 h. The precipitate produced was filtered under
suction, washed with n-hexane and dried in vacuo to yield the
product (58%) as a pink solid.
4.1.4. 2,3,3-Trimethyl-1-(3-sulfonatopropyl)-3H-indol-1-ium (1l)
To a solution of 2,3,3-trimethylindolenine (62.3 mmol) in
toluene (50.0 mL) was added 1,3-propanesultone (93.5 mmol) and
with constant stirring, the solution was refluxed for 24 h. The
precipitate produced was filtered under suction, washed with
toluene and dried in vacuo to yield the product (58%) as a white
solid.
1H NMR (d6-DMSO, 300 MHz):
d
7.90 (t, J ¼ 6.0 Hz, 1H, AreH),
7.82 (t, J ¼ 6.0 Hz, 1H, AreH), 7.66e7.61 (m, 2H, AreH), 3.96 (s, 3H,
NeCH3), 2.75 (s, 3H, CeCH3), 1.52 (s, 6H, Ce(CH3)2). 13C NMR (d6-
1H NMR (d6-DMSO, 300 MHz):
d
7.43 (d, J ¼ 9.0 Hz, 2H, AreH),
DMSO, 75.4 MHz):
d
196.45, 142.56, 142.05, 129.77, 129.28, 123.76,
7.88 (t, J ¼ 9.0 Hz, 1H, AreH), 7.78 (t, J ¼ 9.0 Hz, 2H, AreH) 4.91
(t, J ¼ 9.0 Hz, 2H, NeCH2) 3.54 (s, 6H, Ce(CH3)2), 2.65 (t, J ¼ 6.0 Hz,
2H, CH2eCH2) 2.16 (quin, J ¼ 6.0 Hz, 2H, CH2eCH2eCH2) 2.08 (s, 3H,
115.57, 100.00, 54.38, 22.14, 14.51. IR (ATR, cmꢀ1): 2968.4, 1628.9,
1455.1, 1392.7, 1357.8, 774.4. MS (ESI) m/z: 174.09 [Mþ]. Melting
point ¼ 255e257 ꢂC.
CeCH3). 13C NMR (d6-DMSO, 75.4 MHz)
d 207.08, 177.75, 177.72,
141.35, 129.61, 128.51, 125.01, 117.31, 47.80, 47.78, 31.17, 24.72, 17.21
IR (ATR, cmꢀ1): 2904.9, 1634.1, 1455.5, 1326.4, 1161.7, 1028.6, 781.9.
MS (ESI) m/z: 282.32 [Mþ] Melting point ¼ 265e268 ꢂC.
4.1.2. 1-Pentyl-2,3,3-trimethyl-3H-indol-1-ium iodide (1e)
2,3,3-Trimethylindolenine (15.0 mmol) was dissolved in aceto-
nitrile (10.0 mL), followed by the addition of 1-bromopentane
(20.0 mmol). With constant stirring, the solution was refluxed for
24 h to produce a brown solution. The solution was concentrated
under reduced pressure to yield brown oil, which was purified by
column chromatography to yield the product as a bromide salt
(38%) which was hygroscopic. The bromide salt (1.00 mmol) was
dissolved in acetone (10.0 mL) and heated under reflux with so-
dium iodide (1.00 mmol) for 24 h. The white solid produced (KBr)
was filtered and the solution was evaporated under reduced pres-
sure yielding the iodide salt (99%) as a purple solid.
4.2. Determination of antifungal activity
The growth inhibitory activity of the compounds (1aen and 2ae
n) were determined by screening S. pombe, S. cerevisiae and C.
albicans using the following method:
Yeast species were inoculated into relevant media; S. pombe (NJ2
h- ura4-D18 leu1-32 ade6-M210 his7-366) [29] into yeast extract
broth (YE) [30], and S. cerevisiae (strain BY4741a, a derivative of
S288C), (MATahis31 leu20 met150 ura30) [31] and C. albicans (strain
SC5314) [32] into complex media (YPD) [33]. The culture was then
incubated for 12 h at 30 ꢂC with shaking at 200 rpm. Stock solutions
of the compounds were prepared in 20% (v/v) DMSO and culture
media. DMSO and culture media were all used as controls for the
experiment. 3 ꢁ 104 yeast cells were transferred into the wells of a
96-well plate. A 1:2 serial dilution of the compounds was then
performed. The wells were inspected visually for growth of yeast
after 24 h of incubation at 30 ꢂC. Growth was indicated by full or
partial white appearance of yeast on the bottom of the wells. The
MIC values of the compounds were determined to be the well
before yeast growth was first seen. The experiment was repeated
two times to ensure reproducibility of the results.
1H NMR (d6-DMSO, 300 MHz):
d
7.97 (t, J ¼ 6.0 Hz,1H, AreH), 7.84
(t, J ¼ 6.0 Hz,1H, AreH), 7.64e7.61 (m, 2H, AreH), 4.44 (t, J ¼ 9.0 Hz,
2H, NeCH2), 2.83 (s, 3H, CeCH3),1.82 (sex, J ¼ 9.0 Hz, 2H, CH2eCH2e
CH3), 1.53 (s, 6H, Ce(CH3)2), 1.38e1.31 (m, 4H, CeCH2), 0.88 (t,
J ¼ 6.0 Hz, 3H, CH2eCH3). 13C NMR (d6-DMSO, 75.4 MHz):
d 196.65,
142.22, 141.34, 129.83, 129.40, 124.20, 105.20, 54.70, 28.42, 27.47,
26.85, 22.61, 22.18, 15.91, 10.33. IR (ATR, cmꢀ1): 3412.6, 3213.4,
1605.1. MS (ESI) m/z: 230.20 [Mþ]. Melting point ¼ 139e140 ꢂC.
4.1.3. 1-Benzyl-2,3,3-trimethyl-3H-indol-1-ium bromide (1k)
Benzyl bromide (6.80 mmol) dissolved in acetonitrile (20.0 mL)
was heated with constant stirring until a state of reflux was
established. 2,3,3-Trimethylindolenine (6.30 mmol) dissolved in
acetonitrile (20.0 mL) was added dropwise to the reaction mixture
from a dropping funnel. Once all the reactants had been added the
reaction was continued for 48 h. The precipitate produced was
filtered under suction, washed with n-hexane and dried in vacuo to
yield the product (77%) as a red hygroscopic solid.
Acknowledgements
We are pleased to acknowledge the financial support from the
Centre for Material Sciences, School of Forensic and Investigative
Sciences and the Undergraduate Research Intern Scheme which is
operated by the Centre for Research-informed Teaching. We would
also like to thank Kerry Ann Rostron for helping with the pre-
liminary stages of the antifungal studies and the Royal Society of
1H NMR (d6-DMSO, 250 MHz):
d
7.89 (d, J ¼ 7.0 Hz, 1H, AreH),
7.84 (d, J ¼ 7.0 Hz, 1H, AreH), 7.67e7.59 (m, 2H, AreH), 7.45e7.38
(m, 5H, AreH), 5.87 (s, 2H, NeCH2), 3.59 (s, 3H, NeCeCH3), 1.55 (s,
6H Ce(CH3)2). 13C NMR (d6-DMSO, 75.4 MHz):
d 198.7, 142.5, 141.6,

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