Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22

Article abstract of DOI:10.1016/j.ejmech.2017.06.011

Herein we describe the synthesis and evaluation of antidepressant properties of seven analogues (1–7) of the low affinity/high capacity transporter blocker decynium-22 (D-22). All analogues (1–7) were synthesized via base promoted coupling reactions between N-alkylated-2-methylquinolinium iodides or N-alkylated-4-methylquinolinium iodides and electrophilic N-alkylated-2-iodoquinolinium iodides. All final compounds were purified by re-crystallization or preparative HPLC and initial evaluation studies included; 1) screening for in vitro α1-adrenoceptor activity (a property that can lead to unwanted side-effects), 2) measuring antidepressant-like activity in a mouse tail suspension test (TST), and 3) measuring effects upon mouse locomotion. The results showed some analogues have lower affinities at α1-adrenoceptors compared to D-22 and showed antidepressant-like activity without the need for co-administration of SSRIs. Additionally, many analogues did not affect mouse locomotion to the same extent as D-22. Plans for additional evaluations of these promising analogues, including measurement of antidepressant-like activity with co-administration of selective serotonin re-uptake inhibitors (SSRIs), are outlined.

Full text of DOI:10.1016/j.ejmech.2017.06.011

Accepted Manuscript
Evaluation of the antidepressant therapeutic potential of isocyanine and
pseudoisocyanine analogues of the organic cation decynium-22
Anwen M. Krause-Heuer, Rheaclare Fraser-Spears, Jeremy C. Dobrowolski, Mark
E. Ashford, Naomi A. Wyatt, Maxine P. Roberts, Georgianna G. Gould, Wai-Ching
Cheah, Clarissa K.L. Ng, Mohan Bhadbhade, Bo Zhang, Ivan Greguric, Nial J.
Wheate, Naresh Kumar, Wouter Koek, Paul D. Callaghan, Lynette C. Daws, Benjamin
H. Fraser
PII:
S0223-5234(17)30457-9
10.1016/j.ejmech.2017.06.011
EJMECH 9506
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 February 2017
Revised Date: 2 June 2017
Accepted Date: 6 June 2017
Please cite this article as: A.M. Krause-Heuer, R. Fraser-Spears, J.C. Dobrowolski, M.E. Ashford, N.A.
Wyatt, M.P. Roberts, G.G. Gould, W.-C. Cheah, C.K.L. Ng, M. Bhadbhade, B. Zhang, I. Greguric, N.J.
Wheate, N. Kumar, W. Koek, P.D. Callaghan, L.C. Daws, B.H. Fraser, Evaluation of the antidepressant
therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.011.
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ACCEPTED MANUSCRIPT
Evaluation of the Antidepressant Therapeutic Potential of
Isocyanine and Pseudoisocyanine Analogues of the
Organic Cation Decynium-22
Anwen M. Krause-Heuer,1‡ Rheaclare Fraser-Spears,2‡ Jeremy C. Dobrowolski,5 Mark E. Ashford,1
Naomi A. Wyatt,1 Maxine P. Roberts,1 Georgianna G. Gould,2 Wai-Ching Cheah,1 Clarissa K. L. Ng,1
Mohan Bhadbhade,5 Bo Zhang,6 Ivan Greguric,1 Nial J. Wheate,6 Naresh Kumar,5 Wouter Koek,3,4
Paul D. Callaghan1, Lynette C. Daws,2,4* and Benjamin H. Fraser1*
1The Australian Nuclear Science and Technology Organisation, Locked Bag 2001 Kirrawee DC NSW
2232, Australia.
2Departments of Cellular and Integrative Physiology, 3Psychiatry and 4Pharmacology, University of
Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900,
USA.
5School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia.
6Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia.
‡These authors contributed equally.
*Co-senior authors.
Corresponding Author
*Email: benjamin.fraser@ansto.gov.au. Phone: +61-02-97173887 Fax: +61-02-97179262
*Email: daws@uthscsa.edu. Phone: +1-210-5674174 Fax: +1-210-5674410
Graphical abstract:

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Abstract
Herein we describe the synthesis and evaluation of antidepressant properties of seven
analogues (1-7) of the low affinity/high capacity transporter blocker decynium-22 (D-22). All
analogues (1-7) were synthesized via base promoted coupling reactions between N-alkylated-
2-methylquinolinium iodides or N-alkylated-4-methylquinolinium iodides and electrophilic
N-alkylated-2-iodoquinolinium iodides. All final compounds were purified by re-
crystallization or preparative HPLC and initial evaluation studies included; 1) screening for in
vitro α1-adrenoceptor activity (a property that can lead to unwanted side-effects), 2)
measuring antidepressant-like activity in a mouse tail suspension test (TST), and 3)
measuring effects upon mouse locomotion. The results showed some analogues have lower
affinities at α1-adrenoceptors compared to D-22 and showed antidepressant-like activity
without the need for co-administration of SSRIs. Additionally, many analogues did not affect
mouse locomotion to the same extent as D-22. Plans for additional evaluations of these
promising analogues, including measurement of antidepressant-like activity with
co-administration of selective serotonin re-uptake inhibitors (SSRIs), are outlined.
Keywords: depression, antidepressants, decynium-22, adrenoceptor, SSRIs, antidepressant-
like activity
1. Introduction
Depression and its related mood disorders cause much suffering and lost productivity
worldwide.[1] This problem is compounded by the fact that the most commonly prescribed
antidepressants – the selective serotonin re-uptake inhibitors (SSRIs) – are not effective for a
significant proportion of patients.[2] SSRIs act by blocking the high-affinity serotonin
transporter (SERT) and the subsequent increase in extracellular serotonin[3] is thought to be
critical for promoting downstream therapeutic effects.[4] A potential explanation for the

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limited therapeutic efficacy of SSRIs is the presence of low-affinity/high capacity
transporters for serotonin in the brain [i.e. organic cation transporters (OCTs) and plasma
monoamine transporter (PMAT)], which may limit the ability of SSRIs to increase the
amount of extracellular serotonin.[5] Previously the Daws group have demonstrated that
decynium-22 (D-22) (Figure 1) – a potent inhibitor of OCTs and/or PMAT – enhances the
ability of the SSRI fluvoxamine to inhibit clearance of serotonin from extracellular fluid and
to produce antidepressant-like activity in wild type mice.[6] Moreover, they showed that
when SERT is genetically compromised, D-22 inhibited serotonin clearance and produced
antidepressant-like effects when given alone.[7] This is of special interest due to the existence
of common polymorphisms in the human SERT gene, which are linked to psychiatric
disorders and their treatment.[8, 9] Since OCTs and PMAT can also take up other biogenic
amines, including norepinephrine and dopamine, it is possible that their blockade may also
potentiate the therapeutic efficacy of antidepressant drugs that act by blocking the
norepinephrine and dopamine transporters (NET and DAT, respectively).
Figure 1. Decynium-22 and related analogues 1-7

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Given the significant potential of D-22 as an adjunct treatment for depression we have
synthesized a small library of D-22 analogues (isocyanines 1-2 and pseudoisocyanines 3-7)
and evaluated their potential for further development as therapeutics for depression and other
psychiatric disorders (Figure 1). Although D-22 produces antidepressant-like effects when
SERT is genetically or pharmacologically compromised, at higher doses it markedly reduces
locomotor activity, an undesirable side-effect. In addition, D-22 has appreciable activity at
α1-adrenoceptors which could lead to unwanted side-effects such as orthostatic hypertension,
syncope and nasal congestion.[10] Therefore, our aims were to synthesize and evaluate
several D-22 analogues (1-7) for, firstly, their potential for less off-target effects via
decreased activity at α1-adrenoceptors (compared with D-22) and, secondly, for their ability
to retain or have increased antidepressant-like effects without impairing locomotor activity.
Isocyanines 1 and 2 and pseudoisocyanines 3 and 7 have been synthesized previously but
with only limited chemical characterization data.[11, 12] Pseudoisocyanines 4, 5 and 6 are
novel analogues and their synthesis and full chemical characterization is reported herein.
2. Results and discussion
2.1 Synthetic chemistry
Synthesis of N-alkylated intermediates for base promoted pseudoisocyanine and isocyanine
coupling reactions were completed using adaptations of previously reported procedures
(Scheme 1).[12-16] 4-Methylquinoline (8) and 2-chloroquinoline (9) were both alkylated
using an excess of methyl iodide and/or 2-iodopropane to the give 1,4-dimethylquinolinium
iodide (10), 1-isopropylquinolinium iodide (11) and 2-iodo-1-methylquinolinium iodide (12)
in good yields. For the subsequent base promoted coupling reactions, the appropriate
N-alkylated precursor (10-11) and 2-iodo-1-methylquinolinium iodide (12) were suspended

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in dry ethanol and treated with ethanolic potassium hydroxide following an adaptation of
procedure of Russ et al. (Scheme 1).[12]
Scheme 1. Synthesis of N-alkylated quinoline precursors (10-12) and isocyanines (1-2)
Upon addition of ethanolic potassium hydroxide, an immediate color change to purple was
observed which was expected due to formation of the isocyanine product – a known class of
dye compound.[17] The purple solids were collected by filtration and analysis by HPLC,
however, showed both samples contained the desired isocyanines 1 and 2 but in purity levels
less than 95%. Attempts to purify 1 and 2 by crystallization were unreliable and gave
products with purity levels ranging from 79-95% by microanalysis. This was attributed to
varying amounts of ethanol and salt (NaI or NaOH) by-product being co-crystallised with the
isocyanine product. The procedure of Russ and co-workers does not report HPLC or micro-
analytical data for compounds 1 and 2 but only 1H and 13C NMR data which would not detect
by-product salt content.[12] Given the concerns regarding the purity of isocyanines 1 and 2,
conditions for purification by preparative HPLC were developed. The solids were first
dissolved in excess trifluoroacetic acid (TFA) (~5 mL) and heated to 80 °C in order to
exchange the iodide counter-ion for trifluoroacetate. It was necessary to convert the iodides
into trifluoroacetate salts before preparative HPLC as the trifluoroacetate salts purified more

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readily under reverse phase HPLC conditions. This also ensured the isocyanine products
contained a single counter-ion for chemical and biological characterization. For HPLC
purification, the crude solids were dissolved in the minimum amount of ACN/DMSO (90/10)
and were eluted using a water/ACN mobile phase with a 10% TFA modifier. This gave the
isocyanines 1 and 2 in moderate yields (54% and 48%) and purity greater than 98% (HPLC).
Elemental analysis confirmed the presence of the trifluoroacetate counter ion with no
detectable amount (<0.3%) of iodide counter ion. Characterization data including 1H and 13C
NMR, LRMS, HRMS, IR and elemental analysis were all consistent with the structures
1
shown for isocyanines 1 and 2 (Scheme 1). H NMR data for both isocyanines 1 and 2 were
near identical to the previously published data for their corresponding iodide salts.[12] The
1H signal assigned to the methine hydrogen (C=CH-C) appears at 6.48 ppm for both the
iodide and trifluoroacetate salt compounds[12] (See supplementary information for details).
For synthesis of pseudoisocyanine analogues (3-7), the N-alkylated coupling precursors
(19-24) were prepared by reaction of 6-substituted-2-methylquinolines (13-17) or
2-chloroquinoline (18) with an excess of iodoethane at 90 °C for 2-7 days in a sealed tube
(Scheme 2).

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Scheme 2. Synthesis of N-alkylated quinoline precursors (19-24) and
pseudoisocyanines (3-7)
Although all previous methylation reactions were complete in 24 h the ethylation reactions
took considerably longer (2-7 days) owing to the increased steric hindrance of the
6-substituted-2-methylquinoline starting materials and the ethyl iodide electrophile.[18, 19] It
was generally observed that alkylation reactions performed at temperatures above 90 °C gave
diminished yields and significant amounts of unreacted starting material. This observation is
consistent with the known phenomenon for N-alkylation reactions, where equilibrium lies
back in favour of starting materials at higher temperatures.[19] Alkylation reactions
performed with excess alkyl iodide (3 equiv.) in a sealed tube at 85-90 °C minimized this
effect and consistently gave the highest yields of N-alkylated products (see supplementary
information for details). All characterization data for the electrophilic coupling partner
1-ethyl-2-iodoquinolinium iodide (24) were consistent with that previously reported,[12]
however additionally, single crystals of 24 were obtained from ethanol and were suitable for
x-ray diffraction crystallography. The resulting crystal structure confirmed the presence of
the 2-iodo leaving group and an iodide counter ion. This is the first report of an x-ray crystal

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structure for 1-ethyl-2-iodoquinolinium iodide (24) which is used extensively in coupling
reactions for the synthesis of cationic organic compounds and dyes (Figure 2)
(see supplementary data for details).
Figure 2. Crystal structure ORTEP diagram of coupling reaction intermediate 1-ethyl-2-
iodoquinolinium iodide (24)
Base promoted coupling reactions for synthesis of pseudoisocyanines 3-7 were also
performed following an adaptation of the procedure of Russ et al. (Scheme 2).[12] A
suspension of the appropriate 6-substituted-2-methylquinolinium iodide (19-23) and 2-iodo-
1-ethylquinolinium iodide (24) in dry ethanol were treated with potassium hydroxide in
ethanol and heated to 35 °C for 2 days. An immediate color change to purple/red was
observed and after concentration by rotary evaporation and filtration of the solid product,
analysis by HPLC indicated a purity of less than 95%. The final compounds were then either
treated with TFA (~5 mL) and purified by preparative HPLC (pseudoisocyanine 4) or re-
crystallized from ethanol (pseudoisocyanines 3 and 5-7). Microanalysis confirmed the
presence of a trifluoroacetate counter ion for pseudoisocyanine 4 with no detectable (<0.3%)
amount of iodide present. For pseudoisocyanines 3 and 5-7 elemental analysis were
consistent with that expected for the products with an iodide counter ion. After re-
crystallization of pseudoisocyanine 6, single crystals suitable for x-ray diffraction
crystallography were obtained from ethanol (Figure 3).

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Figure 3. Crystal structure ORTEP diagram of (E)-6-chloro-N-ethyl-2-((N
2(1H)-ylidene)methyl)quinolinium iodide (6)
′-ethylquinolin-
The x-ray crystal structure of pseudoisocyanine 6 confirmed the presence of an iodide
counter-ion, two molecules of co-crystallized water (supported by elemental analysis) and
(E)-geometry of the central methine (C-CH=C) group; the carbon-carbon bond lengths for the
methine unit (C6A-C5BA (1.387 Å) and C5BA-C6B (1.394 Å)) are intermediate between
single and double bonds which is consistent with π-electron delocalization over the two
heterocyclic ring systems (see supplementary information for details).

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2.2 Biological evaluation studies
2.2.1 In vitro α1-adrenoceptor activity
Analogues 1-7 were first screened for in vitro α1-adrenoceptor activity (decreased activity
being a predictor of less undesirable side effects in vivo), and then in vivo for
antidepressant-like activity in the TST and for effect upon mouse locomotion. Affinity of
analogues 1-7 for α1-adrenoceptors was measured by displacement of [3H]prazosin (Perkin
Elmer, 81.7-84.2 Ci/mmol), a selective α-1 receptor antagonist. D-22 has appreciable affinity
for α1-adrenoceptors[10] and since this is an off-target site of action, with potential unwanted
side-effects, including orthostatic hypertension, syncope and nasal congestion, it was
important to compare the affinities of D-22, and lead analogues (those not lethal at a high
dose), for α1-adrenoceptors. Although analogues 1, 3, and 4 showed similar α-1 adrenoceptor
activity to that of decynium-22 at a single concentration (IC50 not determined), promising
analogues with lower affinity were identified as analogues 2, 5, 6 and 7. [3H]Prazosin binding
and competition binding assays were based on prior studies[10, 20, 21] (See supplementary
information for details). Figure 4 shows the concentration response analysis for displacement
of [3H]prazosin by each competing ligand. Prazosin displacement by D-22 and 2, 5, 6 and 7
was compared with the displacement by the high affinity adrenoceptor blocker, tamsulosin.
All competing ligands were less potent than tamsulosin, with D-22 being at least 10-fold less
potent and 2, 5, 6 and 7 at least 100-fold less potent. Corticosterone, a known inhibitor of
OCT3,[22] was found here to be a weak inhibitor at adrenoceptors. Summarized IC50 values
obtained from displacement experiments are provided in Table 1. Comparison of non-linear
regression curves for each ligand with either tamsulosin or D-22 was performed to determine
the statistical significance of the observed differences. The near 2 nM IC50 for the high-
affinity adrenoceptor ligand tamsulosin was 30-fold lower than the IC50 for D-22 (IC50 = 60
nM, p=0.0003), and was 150-500-fold lower than the D-22 analogues (p<0.0001): analogue 2

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(IC50 ≈ 1 ꢀM), analogue 5 (IC50 = 0.3 ꢀM), analogue 6 (IC50 = 0.5 ꢀM), and analogue 7 (IC50
= 0.7 ꢀM). In terms of structure/activity relationship, moving the methine link to the 4-
position (analogue 2) or the presence of 6-substituents on pseudoisocyanines (analogues 5-7)
markedly reduces affinity for α1-adrenoceptors expressed in the hippocampus. This warrants
their potential application as therapeutic analogues essentially devoid of α1-adrenergic
receptor-mediated behavioral effects.
Figure 4. Concentration effect curve for [3H]prazosin binding displacement by competing
ligands, including corticosterone, D-22, tamsulosin, and analogues 2, 5, 6 and 7. Curve fit
determined by non-linear regression analysis with GraphPad Prism 6 software.
IC50 (nM)
[95% CI], (n)
Ligand
60
D22
[18.7-190], (5)
1066†*
2
[526-2160], (3)
348†*
5
[229-528], (3)
577†*
6
[286-1166], (3)
711†*
7
[326-1549], (5)
Corticosterone
Ambiguous fit
1.8
Tamsulosin
[0.7-5.0], (3)
Table 1. Summary of IC50 values (nM) for ligand displacement of 1.5 nM [3H]prazosin
binding in mouse hippocampal homogenate. Estimations and statistical comparisons
determined by non-linear regression analysis with GraphPad Prism. Ligand curve fit

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comparison to tamsulosin †P values < 0.0001 or D22 *P values as given (analogue 2, p =
0.0007; analogue 5, p = 0.015; analogue 6, p = 0.025; analogue 7, p = 0.0005). CI, confidence
intervals.
2.2.2 Antidepressant-like activity assay (tail suspension test)
Given the significant potential for further development of analogues 2, 5, 6 and 7 (having
decreased in vitro α1-adrenoceptor activity) and that analogues 1, 3 and 4 retained similar
α1-adrenoceptor activity compared to D-22, all analogues 1-7 were then evaluated for
antidepressant-like activity in a mouse tail suspension test (TST) assay. The TST has been
used to investigate antidepressant-like effects of drugs in mice, and shows predictive validity
for drugs with antidepressant-like properties[23] (see supplementary information for details).
Figure 5 shows the effect of D-22 and analogues 1-7 on immobility time in the TST. With the
exception of 5 and 6, none of the analogues, including D-22, displayed antidepressant-like
activity (i.e. a reduction in immobility time). This is not surprising since in our previously
published studies, we found D-22 to be without effect on immobility time unless paired with
an SSRI[6] or given to mice with a constitutive reduction in SERT expression,[7] where
OCT3 expression is increased as a compensatory result. In contrast, a 3.2 mg/kg dose of
analogues 5 and 6 caused mice to spend significantly less time immobile than saline treated
controls (Figure 5). Given that this dose, if it affects locomotion, likely suppresses locomotor
activity (see dose-response relationships in Figure 5F-G), this increase in time spent mobile
in the TST is indicative of these analogues having independent antidepressant-like activity. In
terms of SAR, the electron withdrawing substituents for 5 (fluoro) and 6 (chloro) impart
independent antidepressant-like activity, while the larger and slightly weaker electron
withdrawing substituents for 3 (methoxy), 4 (bromo) and 7 (iodo) and moving the methine
linker to the 4-position (1 and 2) do not impart independent antidepressant-like activity.

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Decynium-22 (D22)
250
200
150
100
50
0
Compound 7
250
200
150
100
50
0
Figure 5. Effects of D-22 and analogues 1-7 on immobility time in the tail suspension test. *
P < 0.05, one-way ANOVA with Dunnett’s post-hoc comparisons versus saline.

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2.2.3 Locomotor activity assay
Given analogues 5 and 6 show significant potential due to independent antidepressant like
activity (not requiring co-administration of an SSRI) and that analogues 1-4 and 7 will be
evaluated in future work with co-administration of an SSRI, all analogues 1-7 were then
evaluated for their effect upon locomotor activity.
Locomotor activity was measured by placing the mouse in a chamber containing infrared
emitters and receivers (see supplementary information for details). Mice received an
intraperitoneal (ip) injection of D-22 (0.01 – 10.0 mg/kg), or equivalent dose of new analogue
(1-7) immediately before being placed in the chamber. Locomotion was measured as infrared
beam breaks per 5 min period during a four-hour session. Data are shown for cumulative
beam breaks occurring 60 to 120 minutes following injection (Figure 6) (see supplementary
information for details).

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Figure 6. Dose-effect of D-22 and analogues 1-7 on locomotor activity. * P < 0.05
compared with the same dose of D22. *The number adjacent to symbols indicates the number of animals
tested in each condition.
Figure 6A shows the dose-response relationship for D-22 to influence locomotor activity.
This relationship is replotted in Figure 6B-H to facilitate comparison with analogues 1-7. At
1.0 mg/kg, D-22 completely suppressed locomotor activity, whereas none of the analogues
caused complete suppression of activity at this dose. These data suggest that all analogues

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(1-7) may have a more favorable profile for use in vivo than D-22. For analogues 1-6
(Figure 6B-G) maximal or near maximal suppression of locomotor activity was observed at a
dose of 10 mg/kg. For analogue 1, 3 and 4 (Figure 6B, D & E), one out of six mice in each
test group died at this 10 mg/kg dose. Of great interest, 10 mg/kg of analogue 7 reduced
locomotor activity by only half that of saline-injected control mice (Figure 6H), indicating
analogue 7 is an excellent candidate for further development for use in vivo. One-way
ANOVA followed by Dunnett’s test was used 1) to examine the effects of D-22 on
locomotion, 2) to compare the effects of 0.1 and 1 mg/kg of each analogue (1-7) with the
effects of 0.1 and 1 mg/kg of D-22, and 3) to compare the maximal effect of each analogue
with the maximal effect of D-22. D-22 decreased locomotion at 1 mg/kg (p<0.0001) but not
at 0.1 mg/kg (p=0.44). At 0.1 mg/kg, none of the analogues had effects on locomotion that
differed significantly from those of D-22 (F[7,53]=1.82, p=0.10). At 1 mg/kg, 6 and 7
affected locomotion significantly less than D-22 (p=0.046 and 0.013, respectively). At
10 mg/kg, only analogue 7 had significantly less effect on locomotion than the maximally
effective dose (1 mg/kg) of D-22 (p=0.0096). In terms of overall SAR, moving the methine
linker to the 4-position (analogues 1-2) affords less decrease in locomotor activity,
particularly at higher doses of 1 mg/kg and 10 mg/kg compared to D-22. For the
pseudoisocyanines, the electron donating methoxy substituent (3) does not significantly affect
locomotor activity compared to D22, but electron withdrawing substituents, especially bromo
(6) and iodo (7) give significantly less decreases in locomotor activity.
3. Conclusions
We have evaluated two isocyanine (1-2) and five pseudoisocyanine (3-7) analogues based
upon the non-selective, parent analogue D-22, for their potential as novel antidepressants.
The majority of analogues, and in particular analogues 6 and 7, were less potent than D-22 in

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suppressing spontaneous locomotor activity in mice, indicating a much improved safety
margin for in vivo applications of these analogues in comparison to D-22. Tail suspension
tests revealed that 5 and 6 reduced immobility time suggesting that these analogues have
potential independent action as antidepressants at the highest tested dose. Our data also
showed 2, 5, 6 and 7 to have significantly reduced activity at adrenergic receptors.
Antagonism at α1-adrenergic receptors by prazosin has been reported to decrease the
behavioral effects of antidepressants.[24] Despite its activity at adrenoceptors shown here and
previously,[10] D-22 did enhance the antidepressant-like effects of the SSRI fluvoxamine,[6]
suggesting that its activity at α1-adrenoceptors is insufficient to obscure its ability to enhance
the antidepressant-like effects of fluvoxamine. As our current results show 2, 5, 6 and 7 to
have less affinity at α1-adrenoceptors, the behavioral effects of these analogues in
combination with selective serotonin reuptake inhibitors should be evaluated. Given their
lower affinity for α1-adrenoceptors, one prediction is that their ability to enhance the
antidepressant-like effect of SSRIs might be considerably greater than that of D-22. Indeed,
their reduced affinity at α1-adrenoceptors might contribute to the ability of 5 and 6 to produce
antidepressant-like effects by themselves. At this stage the mode of action of these analogues
is most likely due to inhibition of OCTs and/or PMAT but this has yet to be proven. In order
to determine this we are currently developing cell based assays and studies are planned that
will evaluate the relative potency and selectivity of the D-22 derived analogues at specific
low-affinity/high-capacity transporters, OCT2, OCT3 and PMAT that are widely expressed in
the brain. Although D-22 does not have any appreciable affinity for the high-affinity/low
capacity biogenic amine neurotransmitter transporters (SERT, NET and DAT) it will also be
important to assess affinity values for D-22 derived analogues at these transporters in order to
understand their true potential as novel antidepressants.

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4. Experimental section
4.1 Commercial compounds
6-fluoro-2-methylquinoline,
6-methoxy-2-methylquinoline,
6-chloro-2-methylquinoline,
6-bromo-2-methylquinoline,
4-methylquinoline,
6-iodo-2-methylquinoline,
2-chloroquinoline, methyl iodide, ethyl iodide, 2-iodopropane, potassium hydroxide,
trifluoroacetic acid, copper(I) iodide, sodium iodide, N,N'-dimethyl-cyclohexane-1,2-
diamine, dichloromethane, ethanol and dioxane were of reagent grade quality and purchased
from Sigma-Aldrich. Acetonitrile, water and methanol (HPLC grade) were purchased from
Merck and were dried using the Braun MB SPS-800 solvent purification system. Deuterated
solvents were purchased from Cambridge Isotope Laboratories (Novachem, Collingwood,
Australia).
4.2 General chemistry methods
Nuclear magnetic resonance (13C and 1H NMR) spectra were recorded using a Bruker Avance
DPX-400, Bruker Avance III 300 or a Bruker Avance III 600 Cryo spectrometer. Low-
resolution mass spectrometry (LRMS) were performed on a Micromass ZQ quadrupole mass
spectrometer, and high-resolution mass spectrometry (HRMS) was performed at the
University of Wollongong, Australia using a Bruker Daltonics BioApex-II 7T FTICR
spectrometer equipped with an off-axis analytical electron spray ionization source. Melting
points were measured using an Optimelt melting point apparatus and are uncorrected. Infra-
red (IR) spectra were collected on an Agilent Cary 630 FTIR spectrometer fitted with a
diamond attenuated total reflectance (ATR) sample interface. Flash chromatography was
performed on a Reveleris® Flash Chromatography System fitted with an ELSD (isopropanol
support) and dual-wavelength UV (254 nm and 280 nm) detectors; solvent systems, column
sizes and flow-rates are described where appropriate. Elemental or microanalysis was
performed by the Campbell Microanalytical Laboratory, Chemistry Department, University

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of Otago, Dunedin, New Zealand. HPLC purity of all final analogues was greater than 95%
and was performed using a Waters Empower 2 system with a Waters 600 pump, Waters in-
line degasser AF, Waters temperature control module II, Waters 717 autosampler and Water
2996 PDA. An Alltech (C18 (150 × 4.6 mm, 5 ꢀm pore size) analytical column was used,
with absorbance measured using a photo-diode array (PDA) detector. Samples were prepared
as 1 mg/mL, with a 10 ꢀL injection. Percentage purity was calculated from the peak area
under using Empower software (Waters). Solvent system: A = ACN, B = H2O, C = H2O (1%
TFA). Gradient profile 5%A 85%B (with constant 10%C) to 90%A (with constant 10%C)
over 30 mins. UV detection wavelength = 254 nm. Preparative HPLC was performed using a
Waters Empower 2 system with a Waters 600 pump, Waters 600 controller, Waters in-line
degasser AF, Waters 717 autosampler and Waters 486 tunable absorbance detector. Column
and eluent specifications are provided below for each compound.
4.3 Synthetic chemistry procedures
4.3.1 1,4-Dimethylquinolinium iodide (10). An adaptation of the procedure of Russ et al was
used.[12] 4-methylquinoline (8) (1 equiv, 5.27 g, 36.8 mmol) and methyl iodide (1.5 equiv,
7.85 g, 55.2 mmol) were dissolved in DCM (20 mL) and added to a 50 mL RBF. The solution
was stirred at room temperature (rt) for 16 h and filtration of the resulting yellow solid and
washing with DCM (3 x 5 mL) afforded the title compound (10) as a yellow crystalline solid
(8.50 g, 29.8 mmol, 81%). Mp: decomposed 176-177 °C. 1H NMR (300 MHz, d6-DMSO): δ
9.35 (1H, d, J = 6.0 Hz), 8.54 (1H, dd, J = 8.5 Hz, 1.0 Hz), 8.48 (1H, d, J = 8.9 Hz),
13
8.26-8.28 (1H, m), 8.04-8.08 (2H, m), 4.58 (3H, s), 3.00 (3H, s). C NMR (100 MHz, d6-
DMSO): δ 158.12, 148.97, 137.68, 134.92, 129.64, 128.45, 126.79, 122.45, 119.52, 45.01,
19.60. IR (neat); υ = 3057 (s), 3027 (s), 3010 (s), 1525 (s), 783 (s) cm-1. ESI-MS (LRMS,
ES+, 30 V) m/z [M]+ for C11H12N predicted 158.10; found 158.06. ESI-MS (HRMS, TOF MS

ACCEPTED MANUSCRIPT
AP+) m/z [M]+ for C11H12N predicted 158.0964; found 158.0961. HPLC purity = 99.5%,
retention time = 7.30 mins, solvent system: A = ACN, B = H2O, C = H2O (1% TFA).
Gradient profile 5%A 85%B (with constant 10%C) to 90%A (with constant 10%C) over 30
mins.
4.3.2 1-Isopropyl-4-methylquinolinium iodide (11). An adaptation of the procedure of Russ et
al was used.[12] 4-methylquinoline (8) (1 equiv, 5.01 g, 35.0 mmol) and isopropyl iodide
(2 equiv, 11.9 g, 70.0 mmol) were added to a sealed tube reaction vessel. The solution was
stirred and heated to 90 °C for 48 h. Filtration of the resulting yellow solid and trituration
with diethyl ether (3
x 20 mL) afforded the title compound (11) as a yellow crystalline solid
(8.22 g, 26.2 mmol, 75%). Mp 122-123 °C. 1H NMR (400 MHz, DMSO): δ 9.48 (1H, d, J =
6.2 Hz), 8.72 (1H, d, J = 9.0 Hz), 8.55 (1H, dd, J = 1.2, 8.5 Hz), 8.22-8.30 (1H, m), 8.03-8.12
13
(2H, m), 5.87 (1H, septuplet, J = 6.6 Hz,), 3.02 (3H, s), 1.71 (6H, d, J = 6.6 Hz). C NMR
(100 MHz, d6-DMSO): δ 158.02, 144.82, 136.90, 135.08, 129.43, 128.96, 127.27, 122.87,
118.83, 56.62, 22.14, 19.69. IR (neat); υ = 3020 (s), 2983 (s), 2929 (s), 1615 (s), 770 (s), 707
(s) cm-1. ESI-MS (LRMS, ES+, 30 V) m/z [M]+ for C13H16N predicted 186.13; found 186.24.
ESI-MS (HRMS, TOF MS AP+) m/z [M]+ for C13H16N predicted 186.1277; found 186.1279.
HPLC purity = 98.9%, retention time = 7.31 mins, solvent system: A = ACN, B = H2O, C =
H2O (1% TFA). Gradient profile 5%A 85%B (with constant 10%C) to 90%A (with constant
10%C) over 30 mins.
4.3.3 2-Iodo-1-methylquinolinium iodide (12). 2-chloroquinoline (9) (1 equiv, 5.00 g, 30.6
mmol) and methyl iodide (2 equiv, 8.68 g, 61.1 mmol) were dissolved up in DCM (30 mL)
and the solution was heated to reflux for 48 h. Filtration of the resulting yellow solid and
washing with DCM (3
x
10 mL) afforded the title compound (12) as a yellow crystalline solid
1
(11.22 g, 92%). Mp: Decomposed 220-221 °C. H NMR (300 MHz, d6-DMSO): δ 7.90 (1H,
d, J = 9.0 Hz), 7.71 (1H, dd, J = 7.7, 1.6 Hz), 7.62 (1H, ddd, J = 8.6, 7.1, 1.6 Hz, 1H),

ACCEPTED MANUSCRIPT
7.47-7.57 (1H, m), 7.26 (1H, ddd, J = 7.7, 7.1, 1.1 Hz), 6.61 (1H, d, J = 9.5 Hz), 3.62 (3H, s).
13C NMR (100 MHz, d6-DMSO): δ 161.06, 139.71, 139.19, 130.78, 128.71, 121.92, 121.06,
120.05, 114.59, 29.00. IR (neat); υ = 3038 (s), 3003 (s), 1617 (s), 830 (s), 758 (s) cm-1. ESI-
MS (LRMS) (ES+, 40 V) m/z [M]+ for C10H9IN predicted 270.0; found 269.94. ESI-MS
(HRMS, TOF MS AP+) m/z [M]+ for C10H9IN predicted 269.9774; found 269.9774. HPLC
purity = 98.6%, retention time = 7.31 mins, solvent system: A = ACN, B = H2O, C = H2O
(1% TFA). Gradient profile 5%A 85%B (with constant 10%C) to 90%A (with constant
10%C) over 30 mins.
4.3.4 6-Methoxy-1-ethyl-2-methylquinolinium iodide (19). 6-methoxy-2-methylquinoline
(13) (1 equiv, 2.00 g, 11.6 mmol) and ethyl iodide (5 equiv, 57.8 mmol, 9.01 g) were heated
to 90 °C in a sealed tube for 3 days. Filtration of the resulting yellow/brown solid and
washing with DCM (3
x 10 mL) afforded the title compound (19) as a brown/yellow solid
(3.57 g, 94%). Mp: decomposition 200-201 °C. 1H NMR (300 MHz, d6-DMSO): δ 8.94 (1H,
dd, J = 8.7, 0.7 Hz), 8.53 (1H, d, J = 10.6 Hz), 8.06 (1H, d, J = 8.6 Hz), 7.78-7.89 (2H, m),
13
4.96 (2H, q, J = 7.2 Hz), 3.99 (3H, s), 3.05 (3H, s), 1.51 (3H, t, J = 7.2 Hz). C NMR (75
MHz, DMSO): δ 158.49, 157.19, 144.01, 133.60, 130.20, 126.73, 125.85, 120.56, 108.57,
56.24, 47.25, 21.84, 13.57. IR (neat); υ = 3196 (s), 2895 (s), 854 (s), 674 (s) cm-1. ESI-MS
(LRMS, ES+, 15 V) m/z [M]+ for C13H16NO predicted 202.12; found 202.10. ESI-MS
(HRMS, TOF MS AP+) m/z [M]+ for C13H16NO predicted 202.1232; found 202.1227. HPLC
purity = 99.2%, retention time = 9.87 mins, solvent system: A = ACN, B = H2O, C = H2O
(1% TFA). Gradient profile 5%A 85%B (with constant 10%C) to 90%A (with constant
10%C) over 30 mins.
4.3.5 6-Bromo-1-ethyl-2-methylquinolinium iodide (20). 6-bromo-2-methylquinoline (14)
(1 equiv, 4.00 g, 18.0 mmol) and ethyl iodide (5 equiv, 90.1 mmol, 14.05 g) were added to a
sealed tube reaction vessel. The solution was stirred and heated to 90 °C for 7 days. Filtration

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of the resulting yellow/green solid and washing with DCM (3 x 10 mL) afforded the title
compound (20) as a yellow/green solid (5.31 g, 78%). Mp: 208-209 °C. 1H NMR (300 MHz,
d6-DMSO): δ 9.02 (1H, d, J = 8.6 Hz), 8.74 (1H, d, J = 2.3 Hz), 8.57 (1H, d, J = 9.5 Hz), 8.36
(1H, dd, J = 9.5, 2.3 Hz), 8.17 (1H, d, J = 8.6 Hz), 4.99 (2H, q, J = 7.2 Hz), 3.10 (3H, s), 1.52
(3H, t, J = 7.2 Hz). 13C NMR (75 MHz, d6-DMSO): δ 161.71, 144.97, 138.08, 137.57,
132.79, 130.08, 127.26, 122.55, 121.72, 47.99, 22.90, 13.89. IR (neat); υ = 3012 (s), 2977 (s),
1510 (s), 894 (s), 813 (s) cm-1. ESI-MS (LRMS, ES+, 30 V) m/z [M]+ for C12H13BrN
predicted 250.0; found 249.8. ESI-MS (HRMS, TOF MS AP+) m/z [M]+ for C12H13BrN
predicted 250.0226; found 250.0220. HPLC purity = 98.6%, retention time = 10.02 mins,
solvent system: A = ACN, B = H2O, C = H2O (1% TFA). Gradient profile 5%A 85%B (with
constant 10%C) to 90%A (with constant 10%C) over 30 mins.
4.3.6 6-Fluoro-1-ethyl-2-methylquinolinium iodide (21). 6-fluoro-2-methylquinoline (15)
(1 equiv, 0.52 g, 3.24 mmol) and ethyl iodide (5 equiv, 16.2 mmol, 2.53 g) were added to a
sealed tube reaction vessel. The solution was stirred and heated to 90 °C for 5 days. Filtration
of the resulting yellow solid and washing with DCM (3
x
10 mL) afforded the title compound
1
(21) as a yellow solid (0.75 g, 73%). Mp: decomposed 150-170 °C. H NMR (300 MHz, d6-
DMSO): 9.05 (1H, dd, J = 8.8, 0.7 Hz), 8.73 (1H, dd, J = 9.8, 4.4 Hz), 8.29 (1H, dd, J = 8.4,
3.0 Hz), 8.23-8.10 (2H, m), 5.01 (2H, q, J = 7.3 Hz), 3.12 (3H, s), 1.53 (3H, t, J = 7.3 Hz).
13C NMR (100 MHz, d6-DMSO): δ 160.43, 160.86, 160.84, 159.11, 145.29, 145.23, 135.72,
130.33, 130.19, 127.18, 125.17, 124.82, 122.95, 122.83, 114.67, 114.37, 48.25, 22.89, 13.99.
IR (neat); υ = 3449 (s), 3071 (s), 2971 (s), 817 (s) cm-1. ESI-MS (LRMS, ES+, 25 V) m/z
[M]+ for C12H13NF predicted 190.02; found 190.10. ESI-MS (HRMS, TOF MS AP+) m/z
[M]+ for C12H13NF predicted 190.1032; found 190.1098. HPLC purity = 98.7%, retention
time = 9.49 mins, solvent system: A = ACN, B = H2O, C = H2O (1% TFA), gradient profile
5%A 85%B (with constant 10%C) to 90%A (with constant 10%C) over 30 mins.

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4.3.7 6-Chloro-1-ethyl-2-methylquinolinium iodide (22). 6-chloro-2-methylquinoline (16)
(1 equiv, 1.00 g, 5.63 mmol) and ethyl iodide (5 equiv, 28.2 mmol, 4.39 g) were added to a
sealed tube reaction vessel. The solution was stirred and heated to 90 °C for 4 days. Filtration
of the resulting yellow solid and washing with DCM (3
x
10 mL) afforded the title compound
1
(22) as a yellow solid (1.84 g, 98%). Mp: decomposed 207-208 °C. H NMR (300 MHz,
d6-DMSO): δ 9.02 (1H, d, J = 8.4 Hz), 8.65 (1H, d, J = 9.6 Hz), 8.59 (1H, d, J = 2.4 Hz), 8.23
(1H, dd, J = 2.8, 9.6 Hz), 8.17 (1H, d, J = 8.4 Hz), 4.99 (2H, q, J = 7.2 Hz), 3.11 (3H, s, 3H),
13
1.52 (t, J = 7.2 Hz, 3H). C NMR (75 MHz, d6-DMSO): δ 161.19, 144.56, 136.81, 135.01,
133.50, 129.25, 129.03, 126.84, 121.33, 47.60, 22.42, 13.42. IR (neat); υ = 3062 (s), 2983 (s),
904 (s), 684 (s) cm-1. LRMS (ES+, 30 V) m/z [M]+ for C12H13ClN predicted 206.1; found
206.1. HRMS (TOF MS AP+) m/z [M]+ for C12H13ClN predicted 206.0731; found 206.0741.
HPLC purity = 99.0%, retention time = 9.48 mins.
4.3.8 6-Iodo-1-ethyl-2-methylquinolinium iodide (23). 6-iodo-2-methylquinoline (17)
(1 equiv, 0.55 g, 2.04 mmol) and ethyl iodide (5 equiv, 10.2 mmol, 1.60 g) were added to a
sealed tube reaction vessel. The solution was stirred and heated to 90 °C for 5 days. Filtration
of the resulting yellow solid and washing with DCM (3
x
10 mL) afforded the title compound
1
(23) as a yellow solid (0.56 g, 64%). Mp: decomposed 251-253 °C. H NMR (300 MHz,
DMSO-d6): δ 8.97 (1H, d, J = 9.4 Hz), 8.88 (1H, d, J = 2.0 Hz), 8.46 (1H, dd, J = 9.3, 2.0
Hz), 8.37 (1H, d, J = 9.4 Hz), 8.13 (1H, d, J = 8.6 Hz), 4.96 (2H, q, J = 7.3 Hz), 3.07 (3H, s),
13
1.50 (3H, t, J = 7.2 Hz). C NMR (75 MHz, d6-DMSO): δ 161.47, 144.75, 143.39, 139.05,
137.92, 130.26, 126.94, 121.09, 96.41, 47.79, 22.88, 13.87. IR (neat); υ = 3013 (s), 2972 (s),
1510 (s), 820 (s) cm-1. ESI-MS (LRMS, ES+, 25 V) m/z [M]+ for C12H13NI predicted 298.01;
found 297.96. ESI-MS (HRMS, TOF MS AP+) m/z [M]+ for C12H13NI predicted 298.0093;
found 298.0080. HPLC purity = 98.5%, retention time = 11.04 mins, solvent system: A =

ACCEPTED MANUSCRIPT
ACN, B = H2O, C = H2O (1% TFA). Gradient profile 5%A 85%B (with constant 10%C) to
90%A (with constant 10%C) over 30 mins.
4.3.9 2-Iodo-1-ethylquinolinium iodide (24). 2-chloroquinoline (9) (1 equiv, 5.00 g,
30.6 mmol) and ethyl iodide (5 equiv, 152.8 mmol, 23.8 g) were heated to 90 °C in a sealed
tube for 5 days. Filtration of the resulting yellow solid and washing with DCM (3
x 10 mL)
afforded the title compound (24) as a yellow solid (7.29 g, 58%). Mp: Decomposed
212-213 °C. 1H NMR (300 MHz, d6-DMSO): δ 7.90 (1H, dt, J = 9.5, 0.5 Hz), 7.72 (1H, dd, J
= 7.7, 1.5 Hz), 1.69-7.52 (2H, m), 7.26 (1H, ddd, J = 7.7, 6.6, 1.5 Hz), 6.60 (1H, d, J = 9.5
Hz), 4.27 (2H, q, J = 7.1 Hz), 1.21 (3H, t, J = 7.1 Hz). 13C NMR (75 MHz, d6-DMSO):
δ 161.65, 139.29, 138.59, 130.85, 129.03, 121.77, 121.08, 120.34, 114.32, 36.44, 12.67. IR
(neat); υ = 3041 (s), 2988 (s), 1565 (s), 823 (s), 770 (s) cm-1. ESI-MS (LRMS, ES+, 30 V) m/z
[M]+ for C11H11IN predicted 284.0; found 284.3. ESI-MS (HRMS, TOF MS AP+) m/z [M]+
for C11H11IN predicted 283.9931; found 283.9930. HPLC purity = 99.4%, retention time =
9.92 mins, solvent system: A = ACN, B = H2O, C = H2O (1% TFA). Gradient profile 5%A
85%B (with constant 10%C) to 90%A (with constant 10%C) over 30 mins.
4.3.10 1,1′-Dimethyl-2,4′-cyanine trifluoroacetate (1). An adaptation of the procedure of Russ
et al. was used.[12] 1,4-dimethylquinolinium iodide (10) (1 equiv, 0.20 g, 0.70 mmol) and
2-iodo-1-methylquinolinium iodide (12) (1 equiv, 0.28 g, 0.70 mmol) were dispersed in dry
EtOH (10 mL) at rt. To this suspension was added a solution of KOH (1.5 equiv, 0.06 g, 1.40
mmol) in dry EtOH (5 mL). The solution turned immediately dark red/purple and was stirred
for 18 h at rt. Filtration of the solution gave a purple solid which was dried under vacuum and
then dissolved up in TFA (5 mL). The solution was then heated to 80 °C for 18 h and then
cooled to rt. The excess TFA was removed by distillation under reduced pressure to give
purple solid which was then purified by prep. HPLC (column: Atlantis T3 (10 ꢀm 30
x
150mm); isocratic (10% MeOH, 80% H2O, 10% H2O (1% TFA)); flow rate: 25 mL/min; inj.

ACCEPTED MANUSCRIPT
volume: 300 ꢀL). The appropriate fractions were evaporated under reduced pressure to
remove ACN and then the remaining water lyophilized to give the title compound (1) as a
purple solid (156 mg, 54%). Mp: 114-115 °C. 1H NMR (400 MHz, d6-DMSO): δ 8.55 (1H, d,
J = 8.4 Hz), 8.22 (1H, d, J = 7.2 Hz), 8.07 (1H, d, J = 9.5 Hz), 7.90-8.02 (4H, m), 7.88 (1H,
d, J = 7.7 Hz), 7.77-7.83 (1H, m), 7.65-7.72 (1H, m), 7.47-7.53 (1H, m), 7.40 (1H, d, J = 7.2
Hz), 6.48 (1H, s), 4.07 (3H, s), 4.00 (3H, s). 13C NMR (100 MHz, d6-DMSO): δ 153.99,
149.37, 143.51, 140.00, 138.44, 137.15, 132.89, 132.43, 128.84, 126.27, 125.83, 124.89,
124.45, 124.30, 121.29, 117.70, 116.70, 108.54, 94.42, 41.77, 37.85. IR (neat); υ = 3036 (s),
3010 (s), 2976 (s), 895 (s), 811 (s) cm-1. ESI-MS (LRMS, ES+, 40 V) m/z [M]+ for C21H19N2
predicted 299.15; found 299.04. ESI-MS (HRMS, TOF MS AP+) m/z [M]+ for C21H19N2
predicted 299.1548; found 299.1556. HPLC purity = 99.7%, retention time = 6.81 mins,
solvent system: A = ACN, B = H2O, C = H2O (1% TFA). Gradient profile 5%A 85%B (with
constant 10%C) to 90%A (with constant 10%C) over 30 mins. Elemental analysis
(C23H19F3N2O2·1.2TFA) predicted %C: 55.55, H: 3.71, N: 5.1, I: 0.0. Found %C: 55.46 and
55.59, H: 3.82 and 3.82, N: 5.39 and 5.40, I: <0.3 and <0.3.
4.3.11 1-Methyl-1’-isopropyl-2,4′-cyanine trifluoroacetate (2). An adaptation of the
procedure of Russ et al. was used.[12] 1-isopropyl-4-methyl-quinolinium iodide (11)
(1 equiv, 0.15 g, 0.48 mmol) and 2-iodo-1-methylquinolinium iodide (12) (1 equiv, 0.20 g,
0.48 mmol) were dispersed in dry EtOH (8 mL) at rt. To this suspension was added a solution
of KOH (1.5 equiv, 0.96 mmol, 40 mg) in dry EtOH (5 mL). The solution turned immediately
dark red/purple and was stirred for 18 h at rt. Filtration of the solution gave a purple solid
which was dried under vacuum and then dissolved up in TFA (4 mL). The solution was then
heated to 80 °C for 18 h and then cooled to rt. The excess TFA was removed by distillation
under reduced pressure to give a purple solid which was dissolved up in ACN (1.0 mL) and
purified by prep. HPLC (column: Atlantis T3 (10 ꢀm 30
x 150mm); isocratic

ACCEPTED MANUSCRIPT
(20% MeOH, 70% H2O, 10% H2O (1% TFA)); flow rate: 25 mL/min; inj. volume: 300 ꢀL).
The appropriate fractions were evaporated under reduced pressure to remove ACN and then
the remaining water lyophilized to give the title compound (2) as a purple solid (126 mg,
1
48%). Mp: 240-241 °C. H NMR (400 MHz, d6-DMSO): δ 8.56 (1H, d, J = 7.7 Hz),
8.36 (1H, d, J = 7.4 Hz), 8.16 (1H, d, J = 8.8 Hz), 8.08 (1H, d, J = 9.4 Hz), 7.86-8.01 (4H,
m), 7.77-7.82 (1H, m), 7.61-7.68 (1H, m), 7.47-7.52 (1H, m), 7.43 (1H, d, J = 7.4 Hz), 6.48
(1H, s), 5.31 (1H, septuplet J = 6.5 Hz), 4.01 (3H, s), 1.55 (6H, d, J = 6.5 Hz). 13C NMR (100
MHz, d6-DMSO): δ 154.27, 149.16, 140.12, 138.31, 137.80, 137.20, 133.02, 132.47, 128.89,
126.34, 126.07, 124.95, 124.58, 121.38, 117.02, 116.74, 109.26, 94.56, 51.93, 37.81, 27.72.
IR (neat); υ = 3036, 3010, 2976, 895, 811cm-1. ESI-MS (LRMS, ES+, 40 V) m/z [M]+ for
C23H23N2 predicted 327.19; found 327.10. ESI-MS (HRMS, TOF MS AP+) m/z [M]+ for
C23H23N2 predicted 327.1861; found 327.1875. HPLC purity 99.3%, retention time = 8.15
min, solvent system: A = ACN, B = H2O, C = H2O (1% TFA). Gradient profile 5%A 85%B
(with constant 10%C) to 90%A (with constant 10%C) over 30 mins. Elemental analysis
(C25H23F3N2O2·1.8TFA) predicted %C: 53.20, H: 3.87, N: 4.34, I: 0.0. Found %C: 53.18 and
53.4, H: 3.87 and 3.98, N: 4.34 and 4.44, I: <0.3 and <0.3.
4.3.12
(E)-1-Ethyl-2-((1-ethylquinolin-2(1H)-ylidene)methyl)-6-methoxy-4a,8a-dihydro-
quinolinium iodide (3). 6-methoxy-1-ethyl-2-methylquinolinium iodide (19) (1 equiv, 0.50 g,
1.51 mmol) and 1-ethyl-2-iodoquinolinium iodide (24) (1 equiv, 0.49 g, 1.51 mmol) were
dispersed in dry EtOH (40 mL) at rt. To this suspension was added a solution of KOH (1.5
equiv, 0.18 g, 2.27 mmol) in dry EtOH (30 mL). The solution turned immediately purple and
was heated to 35 °C for 48 h. After this time the solution was filtered to give a purple solid
which was then re-crystallized from EtOH to give the title compound (3) as dark purple
crystals (0.24 g, 32%). Mp: 266-267 °C. 1H NMR (400 MHz, d6-DMSO): δ 8.18 (1H, d, J =
9.4 Hz), 8.00 (2H, t, J = 10.7 Hz), 7.86-7.91 (3H, m), 7.77 (1H, t, J = 7.2 Hz), 7.71 (1H, d, J

ACCEPTED MANUSCRIPT
= 9.4 Hz), 7.52 (2H, dd, J = 2.8, 17.8 Hz), 7.46 (1H, t, J = 7.2 Hz), 5.58 (1H, s), 4.63-4.59
(2H, m), 4.48-4.52 (2H, m), 3.90 (3H, s), 1.53-1.47 (6H, m). 13C NMR (100 MHz, d6-
DMSO): δ 156.53, 152.21, 151.83, 138.82, 137.98, 136.84, 132.53, 129.13, 124.47, 122.56,
122.52, 121.35, 118.42, 115.95, 109.84, 88.37, 55.85, 44.88, 43.77, 12.14, 11.60. IR (neat); υ
= 2986 (s), 2692 (s), 854 (s) cm-1. ESI-MS (LRMS, ES+, 20 V) m/z [M]+ for C24H25N2O
predicted 357.20; found 357.09. ESI-MS (HRMS, TOF MS AP+) m/z [M]+ for C24H25N2O
predicted 357.1961; found 357.1960. HPLC purity = 99.9%, retention time = 12.91 mins,
solvent system: A = ACN, B = H2O, C = H2O (1% TFA). Gradient profile 20%A 70%B (with
constant 10%C) to 90%A (with constant 10%C) over 30 mins. Elemental analysis
(C24H27IN2O·0.1(H2O)·0.1EtOH) predicted %C: 58.98, H: 5.69, N: 5.68, I: 25.75. Found
%C: 59.22, H: 5.30, N: 5.71, I: 25.86.
4.3.13 (E)-6-Bromo-1-ethyl-2-((1-ethylquinolin-2(1H)-ylidene)methyl)quinolinium 2,2,2-
trifluoroacetate (4). 6-bromo-1-ethyl-2-methylquinolinium iodide (20) (1 equiv, 0.20 g, 0.53
mmol) and 1-ethyl-2-iodoquinolinium iodide (24) (1 equiv, 0.22 g, 0.53 mmol) were
dispersed in dry EtOH (10 mL) at rt. To this suspension was added a solution of KOH (1.5
equiv, 45 mg, 0.80 mmol) in dry EtOH (10 mL). The solution turned immediately dark
red/purple and was stirred for 18 h at rt. After this time, filtration of the solution gave a
purple/red solid which was dried under vacuum and dissolved up in TFA (5 mL). The
solution was heated to 80 °C for 18 h and then cooled to rt. The excess TFA was removed by
distillation under reduced pressure to give a purple/red solid which was purified by prep.
HPLC
(column:
Atlantis
T3,
10
ꢀm
30
x
150 mm);
isocratic
(45% MeOH, 45% H2O, 10% H2O (1% TFA)); flow rate: 25 mL/min; inj. volume: 300 ꢀL).
The appropriate fractions were evaporated under reduced pressure to remove ACN and the
remaining water lyophilized to give the title compound (4) (79 mg, 22%). Mp: 265-266 °C.
1H NMR (400 MHz, d6-DMSO): δ 8.24 (1H, d, J = 9.2 Hz), 8.18 (1H, d, J = 2.0 Hz), 8.07

ACCEPTED MANUSCRIPT
(1H, d, J = 8.6 Hz), 7.99-8.01 (2H, m), 7.84-7.92 (4H, m), 7.82 (1H, d, J = 9.4 Hz), 7.55-7.63
(1H, m), 5.66 (1H, s), 4.48-4.56 (2H, m), 4.58-4.69 (2H, m), 1.53 (3H, t, J = 7.2 Hz), 1.48
(3H, t, J = 7.2 Hz). 13C NMR (100 MHz, d6-DMSO): δ 153.65, 152.35, 138.70, 138.63,
137.92, 136.01, 134.83, 133.10, 130.93, 129.44, 126.21, 125.55, 125.13, 122.86, 122.02,
118.48, 116.98, 116.74, 89.64, 44.75, 44.17, 12.00, 11.63. IR (neat); υ = 3423 (s), 3034 (s),
1499 (s), 1149 (s), 649 (s) cm-1. ESI-MS (LRMS, ES+, 15 V) m/z [M]+ for C23H22NBr2
predicted 405.10; found 405.02. ESI-MS (HRMS, TOF MS AP+) m/z [M]+ for C23H22NBr2
predicted 405.0961; found 405.0967. HPLC purity = 97.4%, retention time = 20.05 mins,
solvent system: A = ACN, B = H2O, C = H2O (1% TFA). Gradient profile 20%A 70%B (with
constant 10%C) to 90%A (with constant 10%C) over 30 mins. Elemental analysis
(C25H24BrF3N2O2·1.25TFA) predicted %C: 49.75, H: 3.83, N: 4.22. Found %C: 49.64 and
49.68, H: 3.59 and 3.57, N: 4.29 and 4.28, I: <0.1 and <0.1.
4.3.14
(E)-1-Ethyl-2-((1-ethylquinolin-2(1H)-ylidene)methyl)-6-fluoro-4a,8a-dihydro
quinolinium iodide (5). 6-fluoro-1-ethyl-2-methylquinolinium iodide (21) (1 equiv, 0.50 g,
1.57 mmol) and 1-ethyl-2-iodoquinolinium iodide (24) (1 equiv, 0.65 g, 1.57 mmol) were
dispersed in dry EtOH (40 mL) at rt. To this suspension was added a solution of KOH (1.5
equiv, 0.13 g, 2.35 mmol) in dry EtOH (30 mL). The solution turned immediately red and
was heated to 35 °C and stirred for 48 h after which the solution was filtered to give a purple
solid which was re-crystallized from EtOH to give the title compound (5) as purple crystals
(0.18 g, 24%). Mp: 275-276 °C. 1H NMR (400 MHz, d6-DMSO): δ 8.18 (1H, d, J = 9.2 Hz),
8.01 (2H, dd, J = 18.1, 9.4 Hz), 7.84-7.93 (3H, m), 7.70-7.81 (2H, m), 7.54 (1H, d, J = 2.9
Hz), 7.43-7.52 (2H, m), 5.58 (1H, s), 4.58-4.65 (2H, m), 4.47-4.55 (2H, m), 1.52 (3H, t, J =
7.2 Hz), 1.49 (3H, t, 7.2 Hz). 13C NMR (151 MHz, d6-DMSO): δ 159.34, 157.72, 153.20,
152.61, 138.44 (d, J = 49 Hz), 136.81, 135.63, 132.94, 129.36, 126.03 (d, J = 10.6 Hz),
125.24, 124.87, 123.10, 121.78, 120.60 (d, J = 24.2 Hz), 118.98 (d, J = 9.06 Hz), 116.52,

ACCEPTED MANUSCRIPT
113.77 (d, J = 22.7 Hz), 89.16, 44.53 (d, J = 19.6 Hz), 11.83 (d, J = 13.6 Hz). IR (neat); υ =
2983 (s), 2935 (s), 1523 (s), 845 (s), 766 (s) cm-1. ESI-MS (LRMS, ES+, 20 V) m/z [M]+ for
C23H22FN2 predicted 345.18; found 345.13. ESI-MS (HRMS, TOF MS AP+) m/z [M]+ for
C23H22FN2 predicted 345.1767; found 345.1760. HPLC purity = 97.8%, retention time =
19.40 mins, solvent system: A = ACN, B = H2O, C = H2O (1% TFA). Gradient profile 20%A
70%B (with constant 10%C) to 90%A (with constant 10%C) over 30 mins. Elemental
analysis (C23H24FIN2·0.5H2O) predicted %C: 57.15, H: 5.21, N: 5.8, I: 26.25. Found %C:
57.39, H: 4.82, N: 5.82, I: 26.36.
4.3.15
(E)-6-Chloro-1-ethyl-2-((1-ethylquinolin-2(1H)-ylidene)methyl)quinolinium
iodide (6). 6-chloro-1-ethyl-2-methylquinolinium iodide (22) (1 equiv, 0.50 g, 1.50 mmol)
and 1-ethyl-2-iodoquinolinium iodide (24) (1 equiv, 0.48 g, 1.50 mmol) were dispersed in dry
EtOH (40 mL) at rt. To this suspension was added a solution of KOH (1.5 equiv, 2.25 mmol,
0.13 g) in dry EtOH (30 mL). The solution turned immediately purple and was heated to 35
°C and stirred for 48 h. After this time, the solution was filtered to give a purple solid which
was re-crystallized from EtOH to give the title compound (6) as a purple crystals (0.27 g,
28%). MP: 273-274 °C. 1H NMR (400 MHz, d6-DMSO): δ 8.24 (1H, d, J = 8 Hz), 8.09-7.98
(2H, m), 7.98-8.03 (2H, m), 7.96 (1H, d, J = 8 Hz), 7.79-7.91 (4H, m), 7.55-7.63 (1H, m),
5.67 (1H, s), 4.49-4.56 (2H, m), 4.63-4.69 (2H, m), 1.53 (3H, t, J = 7.2 Hz), 1.49 (3H, t, J =
7.2 Hz). 13C NMR (100 MHz, d6-DMSO): δ 153.63, 152.40, 138.68, 138.64, 137.59, 136.12,
133.10, 132.14, 129.44, 128.89, 127.89, 125.82, 125.54, 125.11, 122.95, 122.00, 118.35,
116.73, 89.58, 44.73, 44.25, 12.00, 11.65. IR (neat); υ = 3364 (s), 2694 (s), 1449 (s), 760 (s)
cm-1. ESI-MS (LRMS, ES+, 15 V) m/z [M]+ for C23H22N2Cl predicted 361.15; found 361.12.
ESI-MS (HRMS, TOF MS AP+) m/z [M+H]+ C23H23N2ClI predicted 489.0594; found
489.0573. HPLC purity = 98.1%, retention time = 19.41 mins, Solvent system: A = ACN, B
= H2O, C = H2O (1% TFA). Gradient profile 20%A 70%B (with constant 10%C) to 90%A