Synthesis and in?vitro evaluation of new fluorinated quinoline derivatives with high affinity for PDE5: Towards the development of new PET neuroimaging probes

Article abstract of DOI:10.1016/j.ejmech.2017.03.091

The increasing incidence of Alzheimer's disease (AD) worldwide is a major public health problem. Current treatments provide only palliative solutions with significant side effects. Therefore, new efficient treatment options and novel early diagnosis tools are urgently needed. Recently, strong pre-clinical evidences suggested that phosphodiesterase 5 (PDE5) may be clinically relevant both as biomarker and drug-target in AD. In this study, we intended to develop a new radiofluorinated tracer for the visualisation of PDE5 in brain using PET imaging. Based on currently known PDE5 inhibitors, a series of novel fluorinated compounds bearing a quinoline core have been synthesised via multi-steps reaction pathways. Their affinity for PDE5 and selectivity over other PDE families have been investigated. According to the data collected from this in vitro screening, fluorinated derivatives 24a, b bearing a fluoroethoxy group at the C-3 position of the quinoline core appeared to be the most promising structures and will be further radiolabelled with fluorine-18 for in vitro and in vivo evaluations as PET radiotracer for neuroimaging of PDE5.

Full text of DOI:10.1016/j.ejmech.2017.03.091

Accepted Manuscript
Synthesis and in vitro evaluation of new fluorinated quinoline derivatives with high
affinity for PDE5: Towards the development of new PET neuroimaging probes
Jianrong Liu, Aurélie Maisonial-Besset, Barbara Wenzel, Damien Canitrot, Ariane
Baufond, Jean-Michel Chezal, Peter Brust, Emmanuel Moreau
PII:
S0223-5234(17)30236-2
10.1016/j.ejmech.2017.03.091
EJMECH 9388
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 January 2017
Revised Date: 3 March 2017
Accepted Date: 26 March 2017
Please cite this article as: J. Liu, Auré. Maisonial-Besset, B. Wenzel, D. Canitrot, A. Baufond, J.-M.
Chezal, P. Brust, E. Moreau, Synthesis and in vitro evaluation of new fluorinated quinoline derivatives
with high affinity for PDE5: Towards the development of new PET neuroimaging probes, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.091.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and in vitro evaluation of new fluorinated quinoline
derivatives with high affinity for PDE5: towards the development of
new PET neuroimaging probes.
Jianrong Liu,a Aurélie Maisonial-Besset,*a Barbara Wenzel,b Damien Canitrot,a Ariane
Baufond,a Jean-Michel Chezal,a Peter Brust,b and Emmanuel Moreaua
aUniv. Clermont Auvergne, INSERM, UMR 1240, IMOST, F-63005 Clermont-Ferrand,
France;
damien.canitrot@ucamail.fr;
liujianrong2005@163.com;
aurelie.maisonial@ucamail.fr;
j-michel.chezal@ucamail.fr;
ariane.baufond@ucamail.fr;
emmanuel.moreau@ucamail.fr.
bHelmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer
Research, Research Site Leipzig, Dept. of Neuroradiopharmaceuticals, Permoserstrasse 15,
04318 Leipzig, Germany; b.wenzel@hzdr.de; p.brust@hzdr.de.
* Corresponding author: Aurélie Maisonial-Besset, aurelie.maisonial@ucamail.fr; UMR
1240, IMOST, Univ. Clermont Auvergne, INSERM, BP 184, 58 rue Montalembert, F-63005
Clermont-Ferrand, France.
Keywords:
Quinoline ; fluorination ; Phosphodiesterase 5 ; radiotracer ; positron emission tomography.
Abbreviations used: AD: alzheimer’s disease; AllocCl: allyl chloroformate; ATR: attenuated
total reflectance; cAMP: cyclic adenosine 3’,5’-monophosphate; cGMP: cyclic guanosine
3’,5’-monophosphate; CNS: central nervous system; CREB: responsive element binding
protein; DAST: (diethylamino)sulfur trifluoride; DIBALH: diisobutylaluminium hydride; DIPEA:
N,N-diisopropylethylamine;
DMAP:
N,N-dimethylaminopyridine;
DMF:
N,N-
dimethylformamide; DMSO: dimethylsulfoxide; EMME: ethoxy methylene malonic diethyl
ester; ESI-MS: electrospray ionization mass spectra; HMBC: heteronuclear multiple bond
correlation; HRMS: high resolution mass spectroscopy; HSQC: heteronuclear single quantum
correlation; IC50: inhibition concentration 50; IR: infrared spectroscopy; Mp: melting point; n-
BuLi: n-butyl lithium; NDs: neurodegenerative disorders; NMR: nuclear magnetic resonance;
PDE: phosphodiesterase; PDE5Is: phosphodiesterase 5 inhibitors; PET: positron emission
tomography; Rf: retention factor; rt: room temperature; SAR: structure-activity relationship;
SPR: structure-property relationship; THF: tetrahydrofuran; TLC: thin layer chromatography;
UK: United Kingdom; UV: ultra-violet.

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Highlights
New fluorinated inhibitors of the PDE5 enzyme were synthesised and evaluated in vitro
Small fluoro-containing alkyl side chains did not alter the inhibitory potency
Compounds 24a, b appeared to be most promising regarding affinity and selectivity
Compounds 24a, b were selected for radiolabelling with fluorine-18 and PET imaging

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Abstract
The increasing incidence of Alzheimer’s disease (AD) worlwide is a major public health
problem. Current treatments provide only palliative solutions with significant side effects.
Therefore, new efficient treatment options and novel early diagnosis tools are urgently
needed. Recently, strong pre-clinical evidences suggested that phosphodiesterase 5 (PDE5)
may be clinically relevant both as biomarker and drug-target in AD. In this study, we intended
to develop a new radiofluorinated tracer for the visualisation of PDE5 in brain using PET
imaging. Based on currently known PDE5 inhibitors, a series of novel fluorinated compounds
bearing a quinoline core have been synthesised via multi-steps reaction pathways. Their
affinity for PDE5 and selectivity over other PDE families have been investigated. According
to the data collected from this in vitro screening, fluorinated derivatives 24a, b bearing a
fluoroethoxy group at the C-3 position of the quinoline core appeared to be the most
promising structures and will be further radiolabelled with fluorine-18 for in vitro and in vivo
evaluations as PET radiotracer for neuroimaging of PDE5.

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1. Introduction
In the last decades, the neurodegenerative disorders (NDs) became a major concern
of public health in industrialized countries. The risk of developing diseases like
Alzheimer’s, Parkinson’s, Huntington’s diseases or amyotrophic lateral sclerosis, is
clearly increasing with advancing age [1]. The common feature of these pathologies is
the progressive deterioration of neuronal activity leading for example to dementia,
cognitive and mental degeneration and/or movement disorders and changes in
individual personality. Among NDs, Alzheimer’s disease (AD) accounts for around 50-
75% of diagnosed dementias in elderly people [2, 3]. Currently, 35 million patients are
identified worldwide [3] and one case of AD is revealed every 7 second [1, 4]. Early
diagnosis of AD is highly challenging [3, 5] and there is no remedial treatment for this
pathology. The medical care and treatment of AD patients at the earliest stage of the
pathology can considerably reduce the progression rate of the disease and delay the
hospitalisation necessity, but most of the time the treatment often begins far too late
[3]. Due to the inefficiency of current strategies focused on β-amyloid plaques or
neurofibrillary tangles, the identification of new biological targets to offer innovative
diagnostic and therapeutic options are urgently needed.
The phosphodiesterases (PDEs) are a large family of enzymes that regulate the
intracellular levels of two important second messengers: cyclic adenosine 3’,5’-
monophosphate (cAMP) and cyclic guanosine 3’,5’-monophosphate (cGMP), by
cleaving their phosphodiester bond. To date, 11 subtypes of these enzymes have
been identified and classified mainly according to their amino acid sequences [6].
Phosphodiesterase 5 (PDE5) is specific for cGMP and is located in some peripheral
organs such as heart, lungs and smooth muscle cells but also in the central nervous
system (CNS) and particularly in cerebellar Purkinje cells, hippocampus, caudate and
substantia nigra [7-10]. There is currently a growing interest in using inhibitors of
PDE5 such as Sildenafil (Viagra®) or Tadalafil (Cialis®), commonly used as treatment
for erectile dysfunction or chronic pulmonary hypertension, to reverse or decrease
memory damages caused by AD [7, 11]. There are strong pre-clinical evidences
suggesting that PDE5 may be both a clinically relevant biomarker and a disease-
relevant drug-target in AD. For example, the crucial nitric oxide/cGMP/CREB
(responsive element binding protein) signalling pathway is known to be altered in
several processes including neurotransmission, synaptic plasticity and memory [12-
16]. Restoring normal cGMP levels in the CNS of AD patients by action of PDE5
inhibitors (PDE5Is) could decrease neuroinflammation, improve cerebral blood flow,
long-term memory consolidation, and synaptic plasticity [12, 17, 18]. However, the

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level of expression, exact role and involvement of this enzyme in AD are still quite
unclear and controversial. Therefore we aim at developing radiotracers for clinical
positron emission tomography (PET) imaging of PDE5 in brain under normal and
pathological conditions. By use of radiotracers with optimal pharmacokinetic profiles in
combination with high accuracy, PET imaging should proof valuable in at least two
principle areas of interest for basic scientists and clinical investigators: i) the timely
availability of diagnostic information i.e. the upfront prediction of disease states, which
involve impairments and/or deregulation of the cGMP-PDE5 pathway, and ii) for
monitoring and evaluating the disease-progression and response to therapy.
Moreover, dynamic PET imaging of PDE5 in intact living subjects should strongly
foster the paths to new therapeutic strategies and to new mechanistic insights into the
role of PDE5 for biology and pathophysiology of NDs such as AD. To date, there is no
radiopharmaceutical approved for the visualisation of PDE5 in clinic [19, 20]. Most of
the radiotracers developed at the preclinical level are labelled with carbon-11 and
suffer from a lack of specificity toward others PDEs. Only one compound radiolabelled
with fluorine-18, which is the best radionuclide for PET imaging purposes, has been
reported to date and warrant further preclinical investigations [21-23]. In such a
context, we envisaged to develop and evaluate new fluorinated analogues of PDE5
inhibitors as candidates for PET imaging of PDE5 in the CNS.
An extensive study of the literature shows that, on the basis of their structural
similarities, PDE5 inhibitors can be grouped as follows: i) cGMP-based derivatives,
represented by Sildenafil and Vardenafil [24-26]; ii) β-carboline-derived molecules,
represented by Tadalafil; iii) pyrazolopyridine, phtalazine, pyrazolopyridopyridazine,
quinoline-based inhibitors; iv) isoquinoline, naphthyridinone and pyridopyrazinone
derivatives [27-33]. As the work presented herein is directed toward the development
of new radiofluorinated inhibitors for PET imaging of PDE5 in brain, we decided to
focus our attention on the heterocyclic scaffold quinoline which presents a high PDE5
potency and selectivity over mainly PDE6 and PDE11 and is described to cross
readily the blood-brain barrier (Figure 1) [12]. This lead compound is the result of
considerable structure-activity (SAR) and structure-property relationship (SPR) studies
which revealed important structural features of this family of PDE5 inhibitors [12, 31].
The quinoline scaffold bearing a hydroxymethyl group at the C-3 position (“east
region”) improves the solubility compared to the other core ring systems tested. The
binding to the protein is highly dependent on the presence of the benzylamino moiety
at the C-4 position (“north region”). Moreover the combination of a cyano or a
trifluoromethyl group at the C-6 position (“west region”) and ethyl or cyclopropyl
substituents at the C-8 position (“south region”) enhance PDE5 potency and appear to

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be the optimal parameters for a high selectivity over others PDEs (based on IC50
values).
Figure 1. Strategy of introduction of a fluorine atom on the lead heterocyclic quinoline
scaffold presenting a high PDE5 potency and selectivity.
In order to develop a radiotracer for PET imaging of PDE5 according to the SAR
studies described above and knowing that fluorine can be considered as a bioisostere
of both hydrogen and hydroxyl groups, we firstly decided to slightly modify this lead
structure by introducing a fluorine atom on the “east region” of the molecule (Figure 1,
see substituents for R). Herein we report, the chemical syntheses and first in vitro
biological evaluation of a series of new fluorinated PDE5 ligands and the selection of
the most promising ones for further labelling with fluorine-18 and evaluation in vivo.
2. Results and discussion
2.1.
To obtain compounds modified at the “east region” of the quinoline core, we firstly
decided to replace the hydroxyl group of the two key compounds 4a directly by a
fluorine atom (Scheme 1). The synthesis of derivative 4a was previously described by
Fiorito et al. [12] in three steps starting from intermediate quinoline (Scheme 1). This
Chemistry: introduction of fluorine at the “east region”
,
b
1
synthetic approach was used to produce the second reference alcohol 4b. Briefly, a
nucleophilic aromatic substitution of the chlorine atom at the C-4 position of the
quinoline core of
1
using (3-chloro-4-methoxyphenyl)methanamine hydrochloride [29]
. Then, the ethyl and cyclopropyl substituents were
in n-propanol yielded compound
2
introduced at the C-8 position via a classical Suzuki coupling in the presence of
tetrakis(triphenylphosphine)palladium[0] and caesium carbonate under thermal
heating. When using 1.5 equivalents of cyclopropylboronic acid, 3a was efficiently
produced in 70% yield. However, in the case of 3b, bearing an ethyl group, the
concomitant formation of the corresponding debrominated compound 3b1 was
observed. Interestingly, a large excess of ethylboronic acid (16 eq.) was mandatory to

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avoid the formation of the by-product and produce preferentially 3b in 63% yield. To
successfully afford 4a, b, reduction of the ester group at the C-3 position into the
corresponding hydroxymethyl group was performed using LiAlH(OtBu)3 as a mild
reducing agent to avoid the hydrolysis of the cyano group.
Scheme 1. Synthesis of key alcohols intermediates 4a, b.a
aReagents and conditions: (i) (3-Chloro-4-methoxyphenyl)methanamine hydrochloride,
DIPEA, n-propanol, reflux, 3 h; (ii) Cyclopropylboronic acid or ethylboronic acid, Pd(PPh3)4,
Cs2CO3, reflux, toluene, 24 h; (iii) LiAlH(tOBu)3, THF, 60 °C, 16 h for 4a or 50 °C, 27 h for 4b.
To introduce the fluorine atom at the C-3 position, we investigated the fluorination of
compound 4a using diethylaminosulfur trifluoride (DAST) which is a classical reagent to
convert straightforward aliphatic alcohols into their corresponding fluorinated derivatives.
Under these conditions, the desired fluorinated compound 5a could not be isolated.
Unfortunately, this reaction led to the major imine by-product 6a bearing a methyl group at
the C-3 position (Scheme 2). This was confirmed by NMR analyses (1H, 13C, HMBC, HSQC)
and HRMS analysis (m/z 376.1214, calculated for C22H19ClN3O m/z 376.1217) (See
supplementary material, Figure 1S).
Scheme 2. Formation of 6a.a
aReagents and conditions: (i) DAST, dichloromethane, -60 °C.

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We assume that compound 6a was formed during the purification step of 5a via column
1
chromatography. Indeed, the analysis of the H NMR spectrum of the crude product
confirmed the presence of 5a with a doublet at 5.46 ppm, integrating for two protons and
presenting a spin-spin coupling between H and F with a coupling constant of 49.2 Hz, typical
of a fluoromethyl group (See supplementary material, Figure 2S). To avoid the formation of
6a, different stationary phases (silica, alumina, RP C18, filtration through a pad of Celite® 545)
were tested with various combinations of solvents for elution but remained unsuccessful.
Moreover, we observed after one day that the crude compound 5a was completely converted
into 6a (1H NMR kinetic studies; data not shown). These results clearly demonstrated that the
desired compound 5a is highly unstable. As possible mechanism we propose the elimination
of HF as depicted in Figure 2.
Figure 2. Possible mechanism proposed for the formation of 6a.
We then tested an alternative synthetic route using a tosylate leaving group. Compound 4a
was treated with 4-toluenesulfonyl chloride in the presence of Et3N and DMAP in dry
dichloromethane at room temperature. As expected, the total conversion of the tosylate into
6a was observed (See supplementary material, Figure 2S). These findings support our
assumption of the instability of these derivatives bearing either a fluoromethyl group (for the
reference compounds) or a tosylate leaving group (corresponding precursors for fluorine-18
radiolabelling) at the C-3 position. Consequently, we focused our efforts on the development
of quinoline structures bearing a longer alkyl chain such as a fluoroethyl moiety at the C-3
position (Figure 1).
We designed a synthetic pathway based on a Gould-Jacobs reaction as previously
developed for the synthesis of starting material 1 (Scheme 3). Firstly, quinolone 10 was
obtained in three steps via formylation of diethyl succinate 7 according to the procedure
described by Holmes et al. [34] followed by a condensation with 2-bromo-4-cyanoaniline and
a thermal cyclisation in Dowtherm A at 250 °C. Trea tment of compound 10 with POCl3 gave
the corresponding 4-chloroquinoline 11 in 90% yield. The next step was the aromatic

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nucleophilic substitution of the chlorine atom to graft the benzylamino moiety at the C-4
position.
Scheme 3. First synthetic approach tested to introduce a fluoroethyl side chain at the C-3
position of the quinoline scaffold.a
aReagents and conditions: (i) Ethyl formate, sodium, Et2O, 0 °C to rt, 20 h; (ii) 2-bromo-4-
cyanoaniline, toluene, reflux, 8 h; (iii) Dowtherm A, 250 °C, 2 h; (iv) POCl 3, reflux, 7 h; (v)
nucleophilic aromatic substitution under thermal conditions; (vi) Cyclopropylboronic acid,
Pd(PPh3)4, Cs2CO3, reflux, toluene, 22 h; (vii) buchwald reaction under thermal or microwave
conditions.
The experimental conditions previously used to obtain compound 2 (Scheme 1) failed to
produce 12 even with further modifications of reaction parameters (solvents, temperatures,
nature of the base). Obviously, due to the addition of a methylene group between the ester
function and the carbon C-3 of the quinoline core, the electron withdrawing effect necessary
for the nucleophilic substitution at the C-4 position was suppressed. Therefore, we focused
our effort to insert the benzylamino moiety via a Buchwald-Hartwig coupling. Given that
compound 11 possesses both a chlorine and a bromine atom at C-4 and C-8 positions
respectively, we firstly performed the regioselective Suzuki reaction to obtain compound 13
from 11 with a good yield (62%). Then, the Buchwald-Hartwig reaction was performed with
compound 13 to introduce the benzylamine derivative at the C-4 position. Despite using
several combinations of catalyst, ligand, base and activation via thermal or microwave
heating, only the degradation of 13 was observed.
Therefore, we were forced to reconsider our synthetic approach and designed a new strategy
depicted in Scheme 4. Compound 4a previously synthesised, was oxidised into its

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corresponding aldehyde 15a. Then, we performed a Wittig reaction in the presence of
(methoxymethyl)triphenylphosphonium chloride to obtain the mixture of (E, Z)-16a. The
cleavage of the methoxy group of 16a using hydrochloric acid (4 M) unfortunately afforded
the cyclic compound 17a rather than the expected aldehyde 18a. The formation of the five-
membered cycle of 17a is probably due to the nucleophilic character of the secondary amine
at the C-4 position of the quinoline scaffold. Keeping in mind that this amine is an important
feature for the binding to the PDE5 enzyme [28] and cannot be blocked as a tertiary amine,
we decided to protect this function to avoid the cyclisation. We chose the allyloxycarbonyl
protecting group, because of its stability under acidic conditions needed to convert
intermediates 19a, b to the corresponding aldehydes 20a, b. Afterwards, the aldehyde
function was reduced using LiAlH(OtBu)3 to give the alcohols 21a, b which were finally
treated with DAST affording the desired fluorinated compounds 22a, b in 46% and 15% yield
respectively. However, during this reaction we observed the formation of major by-products,
identified as the cyclic compounds 23a, b.
Scheme 4. Modified synthetic approach starting from 4a and efficiently producing derivatives
22a, b bearing a fluoroethyl side chain at the C-3 position of the quinoline scaffold.a
aReagents and conditions: (i) MnO2, CH2Cl2, rt, 5 h for 15a or 20 h for 15b; (ii)
(Methoxymethyl)triphenylphosphonium chloride, n-Buli, THF, -15 °C to rt, 2 h; (iii) 4 M HCl in
H2O/THF (1/3, v/v), 50 °C, 4 h; (iv) AllocCl, NaH, DM F, rt, 1 h for 19a and 2 h for 19b; (v)
HCO2H, 50 °C, 20 h; (vi) LiAlH(O tBu)3, THF, 60 °C, 20 h; (vii) DAST, CH 2Cl2, -78 °C to rt, 3 h
for 22a and 1.5 h for 22b.

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To further exemplify this series of candidates modified at the “east region”, we intended to
develop fluorinated derivatives (24a, b and 29a, b) via O-alkylation of the hydroxymethyl
substituent by a 2-fluoroethyl group (Schemes 5 and 6). We firstly intended the direct
reaction of 4a and 2-fluoroethyl-4-methylbenzenesulfonate [35] to obtain 24a (Scheme 5).
However, we observed that the hydroxymethyl group was not reactive enough to substitute
the tosylate leaving group, despite modifying experimental conditions (temperature, solvents,
bases). Therefore, as we already proved that a halogen atom on the methylene moiety at the
C-3 position of the quinoline core is a good leaving group (See Scheme 1 and Figure 2), we
decided to convert the alcohol function of 4a into the corresponding chloromethyl function
using thionyl chloride. This chlorinated intermediate was used without purification for the
nucleophilic substitution with 2-fluoroethanol to obtain the desired compound 24a in
moderate yield over two steps (37%). These conditions were then applied to compound 4b to
synthesise the fluorinated derivative 24b.
Scheme 5. Syntheses of compounds 24a, b starting from 4a, b.a
aReagents and conditions: (i) 2-fluoroethyl-4-methylbenzenesulfonate, basic conditions, THF,
rt or reflux; (ii) SOCl2, rt, 30 min; (iii) 2-fluoroethanol, DMF, 80 °C, 27 h for 24a and 30 h for
24b.
After the first in vitro evaluation of these new fluorinated derivatives (see section “biology:
inhibitory effects on selected PDEs”) and identification of 24a, b as most potent analogues,
we explored the effect of replacing the cyano function of 24a, b by a trifluoromethyl group at
the “west region” (32a, b in Scheme 6).

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Scheme 6. Syntheses of analogues 32a, b bearing a trifluoromethyl group at the C-6
position of the quinoline ring.a
aReagents and conditions: (i) EMME, toluene, reflux, 11 h; (ii) diphenyl ether, 240 °C, 3 h; (iii)
POCl3, 120 °C, 6 h; (iv) (3-chloro-4-methoxyphenyl)meth anamine hydrochloride, DIPEA, n-
propanol, reflux, 16 h; (v) cyclopropylboronic acid or ethylboronic acid, Pd(PPh3)4, Cs2CO3,
reflux, toluene, 20 h; (vi) DIBALH, CH2Cl2, -70 °C to -30 °C, 3 h for 31a and DIBALH, THF, -
60 °C to rt, 16 h for 31b; (vii) (a) SOCl2, rt, 40 min for 32a or 30 min for 32b; (b) 2-
fluoroethanol, DMF, 80 °C, 30 h for 32a and 22 h for 32b.
The corresponding alcohols 31a, b were synthesised according to the procedure previously
optimised for alcohols 4a, b (Scheme 6). Quinoline 28 was produced in three steps from
commercially available 2-bromo-4-trifluoromethylaniline (26) [30]. Then, the aromatic
nucleophilic substitution of the chlorine atom at the C-4 position of 28 using (3-chloro-4-
methoxyphenyl)methanamine hydrochloride [29] efficiently produced compound 29. The
Suzuki coupling was performed to introduce either ethyl or cyclopropyl group at the C-8
position to obtain 30a, b. The ester function at the C-3 position was classically reduced using
DIBALH to afford alcohols 31a, b which were converted into their corresponding chlorinated
derivatives. The latter were not isolated and reacted immediately with 2-fluoroethanol to yield
the desired fluorinated derivatives 32a, b in 22-29% yields.

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2.2.
Biology: inhibitory effects on selected PDEs
In order to study the influence of the introduction of a fluorine atom into the lead structures
4a, b and select the most potent derivative for further development as new radiotracer for
PET imaging of PDE5, we evaluated the inhibitory effects of the synthesised fluorinated
ligands on human PDE5A1 and a panel of other human PDEs (SB drug discovery, Glasgow,
UK). Table 1 summarises the percentage of inhibition of compounds 22a, b, 24a, b and 32a,
b compared to lead alcohols 4a, b. At the beginning, all experiments were performed at
ligand concentrations of 100 nM. However, only inhibitory effects on the PDE5 enzyme could
be observed under these conditions. Therefore, for all other PDEs the ligand concentration
had to be increased to 1 µM in order to achieve analyzable signals. This behaviour already
indicated the high specificity of the new derivatives for PDE5. Compounds 22a, b, and 24a, b
showed a high and selective inhibitory activity on PDE5A1 (>88% at 100 nM vs. 0%-77% at 1
µM for other PDEs). Similar values were obtained with reference alcohols 4a, b highlighting
that the introduction of a fluorine atom at the “east region”, i.e. at the C-3 position of the
quinoline scaffold, did not significantly affect the affinity of the ligands for the PDE5 enzyme.
Only a slightly lower inhibitory activity was observed for products 32a, b obviously caused by
the substitution of a trifluoromethyl group instead of the cyano group at the C-6 position of
the quinoline scaffold.
Table 1: Percentage inhibition of selected PDEs by the synthesised compounds; the
compounds were studied using a concentration of 100 nM for inhibition of human
PDE5A1 and a concentration of 1 µM for inhibition of PDE2A3, PDE3A, PDE4A1,
PDE4C2, PDE6AB, PDE9A1, PDE10A1 and PDE11A1; NI: no inhibition observed.
% Inhibition of PDEs
PDE2
A3
PDE3
A
PDE4
A1
PDE4
C2
PDE5
A1
PDE6
AB
PDE9
A1
PDE10
A1
PDE11
A1
4a
4b
15.3
22.4
65.5
68.4
16.0
7.49
12.1
9.74
NI
NI
NI
NI
6.12
18.2
NI
36.9
45.4
44.5
74.7
13.2
9.03
6.5
22.4
40.9
27.3
40.3
35.9
32.4
17.3
21.4
84.2
90.3
96.1
100
89.9
88.4
57.1
74.6
32.4
49.8
26.0
26.3
54.8
60.4
2.36
26.4
NI
NI
18.0
22.5
14.8
NI
22.9
40.0
43.0
77.1
18.8
14.1
13.0
8.5
24.1
27.2
17.6
42.1
15.1
5.21
3.51
8.84
22a
22b
24a
24b
32a
32b
NI
40.9
NI
4.74
The compounds bearing a fluoroethyl chain at the C-3 position of the quinoline appeared to
be less selective than derivatives with a fluoroethoxymethyl moiety. For example, 24a
presented a 4-fold lower inhibitory activity on PDE2A3 compared to 22a. Therefore we
focused our attention on compounds 24a, b and 32a, b and determined their IC50 values for
inhibition of PDE5A1. The values obtained were 7.39, 1.86, 358 and 192 nM for 24a, b and
32a, b respectively. These results clearly highlight that the substituent at the C-6 position of
the quinoline heterocycle influences the binding to the enzyme. As already indicated by the
percentage of inhibition values in Table 1, when bearing a trifluoromethyl substituent, the IC50

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values reflect a significantly lower affinity for the PDE5A1 enzyme compared to the
compounds with a cyano group (358 and 192 nM for 32a, b vs. 7.39 and 1.86 nM for 24a, b
respectively). Under identical conditions, Sildenafil presented an IC50 value of 6.23 nM.
3. Conclusion
A new series of fluorinated inhibitors of the PDE5 enzyme was synthesised and evaluated in
vitro. The organic synthesis of six new derivatives was successfully performed. In vitro
inhibition experiments revealed that the introduction of small fluoro-containing alkyl side
chains on the “east region” of the selected lead structures 4a, b did not alter the inhibitory
potency of these compounds towards PDE5. According to the data obtained, the fluorinated
derivatives 24a, b appeared to be most promising regarding affinity and selectivity and were
selected for radiolabelling with fluorine-18 and biological evaluations in vitro and in vivo for
PET imaging of PDE5 in the brain [36]. The data collected for [18F]24a revealed that this
compound was fastly metabolized in vivo and formed brain penetrable radiometabolites
making it unsuitable for PET imaging of the PDE5 enzyme in the brain [36]. However, all
these findings revealed useful for the design of metabolically more stable fluorinated
quinoline analogs for further development of PET probes for imaging of PDE5 in brain. This
work is currently undertaken in our laboratory.
4. Experimental section
4.1. Materials for Chemical Syntheses
All commercially available reagents and solvents were purchased from the following
commercial suppliers: Sigma Aldrich, Acros Organics, Carlo Erba, and Alfa Aesar and
were used without further purification. Room temperature (rt) refers to 20-25 °C. All
solvents were dried using common techniques. Air and moisture sensitive reactions
were carried out under anhydrous argon atmosphere. Magnesium sulfate (MgSO4)
and sodium sulfate (Na2SO4) were used as drying agents. Monitoring of the reaction
progresses was performed using thin layer chromatography (TLC) on silica (60 F254)
or alumina (gel 60A + F254) plates and visualised with UV light (UV lamp Fisher
Bioblock Scientific, 365 nm or 254 nm) or with an appropriate staining agent. Column
chromatography was performed on silica gel (Chromagel 60 ACC, 40-63 µm, Carlo
Erba Reagents) or on alumina gel (Merck, column chromatographic adsorption
analysis ACC. to Brockmann, neutral aluminium oxide 90, standardise, 63-200 µm).
Uncorrected melting points (Mp) were measured on an IA9100 Digital Melting Point
Apparatus. Infrared spectra (IR) were recorded in the range of 4000-600 cm-1 on a

ACCEPTED MANUSCRIPT
Nicolet IS10 with attenuated total reflectance (ATR) accessory (ν: stretch, as:
asymmetric, s: symmetric, δ: deformation; op: out of plane). Nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker Avance 500 instrument (500
MHz for 1H, 125 MHz for 13C), a Bruker Avance 400 instrument (400 MHz for 1H, 100
MHz for 13C) or a Bruker Avance 200 instrument (200 MHz for 1H, 50 MHz for 13C). All
chemical shifts (δ) are reported in parts per million (ppm). Coupling constants (J) are
given in Hertz (Hz). Spectral coupling patterns are indicated as follow: s: singlet; d:
doublet; t: triplet; q: quartet; m: multiplet, brt: broad triplet and brs: broad singlet. All
new compounds were analysed by HRMS (High-Resolution Mass Spectrometry,
Waters® Micromass® Q-Tof micro™ Mass Spectrometer, UBP- START, Blaise
Pascal University, Clermont-Ferrand, France). The isotope peaks for chlorine and
bromine atoms are given with their relative intensities.
4.1.1.
Ethyl
4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-ethylquinoline
(3b). Ethylboronic acid (1.25 g, 16.8 mmol, 16.0
-3-
carboxylate
eq.),
tetrakis(triphenylphosphine)palladium (0) (122 mg, 0.11 mmol, 0.1 eq.), caesium carbonate
(858 mg, 2.63 mmol, 2.5 eq.) were added, under argon and stirring, to a solution of 212 (0.5
g, 1.05 mmol, 1.0 eq.) in anhydrous toluene (20 mL). The resulting mixture was
refluxed for 24 h. After cooling to rt, the mixture was filtered on celite® 545, washed
with ethyl acetate (100 mL). The filtrate was concentrated under reduced pressure.
The crude product was purified by column chromatography (SiO2, gradient: ethyl
acetate/cyclohexane, 1/9 to 5/5, v/v) to give, by order of elution: the desired product
3b (270 mg, 0.64 mmol, 60%) as a yellow solid and ethyl 4-[(3-chloro-4-
methoxybenzyl)amino]-6-cyanoquinoline-3-carboxylate (3b1) (150 mg, 0.38 mmol,
36%) as
a
yellow solid. Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyano-8-
ethylquinoline-3-carboxylate (3b) [30]. Rf (SiO2, cyclohexane/ethyl acetate, 7/3, v/v): 0.38;
Mp: 159-161 ºC; IR (cm-1): 2230 (νC≡N), 1681 (νC=O), 1577 (δN-H), 1507 (νC=C), 1289 (νC-O),
1190 (νC-O), 1114 (νC-O), 1029 (νC-O); 1H NMR (CDCl3, 400 MHz) δ 9.60 (brs, 1H, NH), 9.26 (s,
1H, H2), 8.39 (d, 1H, 4JH -H = 1.4 Hz, H5), 7.67 (d, 1H, 4J H -H = 1.4 Hz, H7), 7.40 (d, 1H, 4JH
-
2’
5
7
7
5
= 2.2 Hz, H2’), 7.26 (dd, 1H, 3JH
= 8.5 Hz, 4JH
= 2.2 Hz, H6’), 6.97 (d, 1H, 3JH
=
-H
5’ 6’
H
-H
6’ 5’
-H
6’ 2’
6’
8.5 Hz, H5’), 4.87 (d, 2H, 3JH -NH = 5.5 Hz, Hd), 4.38 (q, 2H, 3JH -H = 7.1 Hz, Hb), 3.92 (s, 3H,
d
b
c
He), 3.23 (q, 2H, 3JH -H = 7.5 Hz, Hf), 1.40 (t, 3H, 3JH -H = 7.1 Hz, Hc), 1.35(t, 3H, 3JH -Hf = 7.5
f
g
c
b
g
Hz, Hg). Ethyl 4-[(3-chloro-4-methoxybenzyl)amino]-6-cyanoquinoline-3-carboxylate (3b1). Rf
(SiO2, cyclohexane/ethyl acetate, 3/7, v/v): 0.20; Mp: 155-157 °C; IR (cm -1): 2226 (νC≡N),
1
1671 (νC=O), 1572 (δN-H), 1504 (νC=C), 1280 (νC-O), 1185 (νC-O), 1064 (νC-O); H NMR (CDCl3,

ACCEPTED MANUSCRIPT
400 MHz) δ 9.85 (t, 1H, 3JNH-H = 5.1 Hz, NH), 9.19 (s, 1H, H2), 8.54 (d, 1H, 4JH -H7 = 1.5 Hz,
d
5
3
3
4
H5), 8.02 (d, 1H, JH8-H = 8.7 Hz, H8), 7.79 (dd, 1H, JH7-H = 8.7 Hz, JH7-H = 1.5 Hz, H7),
7
8
5
7.41 (d, 1H, 4JH
(d, 1H, 3JH
= 2.2 Hz, H2’), 7.27 (dd, 1H, 3JH
= 8.5 Hz, 4JH6’ 2’ = 2.2 Hz, H6’), 6.98
-H -H
6’ 5’
-H
2’ 6’
= 8.5 Hz, H5’), 4.90 (d, 2H, 3JH -NH = 5.5 Hz, Hd), 4.38 (q, 2H, 3JH -H = 7.1 Hz,
-H
5’ 6’
d
b
c
Hb), 3.92 (s, 3H, He), 1.41 (t, 3H, 3JH -H = 7.1 Hz, Hc).
c
b
4.1.2.
4-[(3-Chloro-4-methoxybenzyl)amino]-8-ethyl-3-(hydroxymethyl)quinoline-6-
carbonitrile (4b). An anhydrous 1.1 M solution of lithium tri-tert-butoxyaluminum hydride in
tetrahydrofuran (10.5 mL, 11.55 mmol, 7.0 eq.) was added, under stirring and argon, to a
solution of 3b (700 mg, 1.65 mmol, 1.0 eq.) in anhydrous tetrahydrofuran (30 mL). The
o
resulting mixture was heated at 50 C for 27 hours. After cooling to rt, methanol (2 mL) was
added slowly. This mixture was then diluted with dichloromethane (100 mL) before addition
of a 1.0 M aqueous sodium hydroxide solution (100 mL). After decantation, the aqueous
layer was then extracted with dichloromethane (2 × 100 mL). The combined organic layers
were washed successively with a 1.0 M aqueous sodium hydroxide solution (100 mL) and
brine (100 mL), dried over anhydrous magnesium sulfate, filtered and then concentrated
under reduced pressure. The crude product was purified by column chromatography (SiO2,
ethyl acetate/cyclohexane, 4/6, v/v) to give the desired product 4b30 (500 mg, 1.31 mmol,
79%) as a yellow solid. Rf (SiO2, cyclohexane/ethyl acetate, 1/9, v/v): 0.32; Mp: 177-179 ºC;
IR (cm-1): 3413 (νN-H), 2922 (νCH ), 2230 (νC≡N), 1548 (δN-H), 1504 (νC=C), 1260 (νC-O), 1068 (νC-
3
1
4
O); H NMR (DMSO-d6, 400 MHz) δ 8.79 (d, 1H, JH5-H = 1.5 Hz, H5), 8.49 (s, 1H, H2), 7.75
7
4
3
4
(d, 1H, JH7-H = 1.4 Hz, H7), 7.44 (t, 1H, JNH-H = 6.7 Hz, NH), 7.39 (d, 1H, JH2’ 6’ = 2.0 Hz,
-H
5
b
H2’), 7.24 (dd, 1H, 3JH
= 8.5 Hz, 4JH
= 2.0 Hz, H6’), 7.11 (d, 1H, 3JH
= 8.5 Hz, H5’),
-H
6’ 5’
-H
6’ 2’
-H
5’ 6’
3
3
5.38 (t, 1H, JOH-H = 5.2 Hz, OH), 4.81 (d, 2H, 3JH -NH = 6.7 Hz, Hb), 4.44 (d, 2H, JH -OH = 5.2
a
b
a
Hz, Ha), 3.81 (s, 3H, Hc), 3.13 (q, 2H, 3JH -H = 7.5 Hz, Hd), 1.25 (t, 3H, 3JH -H = 7.5 Hz, He).
d
e
e
d
4.1.3. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-cyclopropyl-3-(fluoromethyl)quinoline-6-
carbonitrile (5a). Diethylaminosulfur trifluoride (58 mg, 0.36 mmol, 2.0 eq.) was added
dropwise, at -60 ºC and under stirring to a solution of 4a12 (70 mg, 0.18 mmol, 1.0 eq.) in
anhydrous dichloromethane (20 mL). The temperature increased slowly to rt over 2 hours
and the mixture was stirred at this temperature for additional 2 hours. Then, a saturated
aqueous sodium hydrogen carbonate solution (30 mL) was added to the mixture cooled at -
20 ºC (pH = 7-8). After return back to rt and decantation, the aqueous layer was extracted
with dichloromethane (2 × 30 mL). The combined organic layers were washed with water (50
mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered and evaporated under
reduced pressure to afford a highly unstable white solid 5a (60 mg, 0.15 mmol, 83%). Mp:

ACCEPTED MANUSCRIPT
121-123 ºC; IR (cm-1): 3405 (νN-H), 2230 (νC≡N), 1504 (νC=C), 1260 (νC-O), 1068 (νC-O); 1H NMR
(CDCl3, 400 MHz) δ 8.65 (d, 1H, 4JH -F = 1.7 Hz, H2), 8.23 (m, 1H, H5), 7.36 (d, 1H, 4JH
=
-H
2’ 6’
2
3
4
2.1 Hz, H2’), 7.27 (m, 1H, H7), 7.20 (dd, 1H, JH6’ 5’ = 8.4 Hz, JH6’ 2’ = 2.1 Hz, H6’), 6.94 (d,
-H
-H
1H, 3JH
= 8.4 Hz, H5’), 5.46 (d, 2H, 3JH -F = 49.2 Hz, Ha), 4.78 (d, 2H, 3JH -NH = 5.7 Hz, Hb),
a b
-H
5’ 6’
13
3.91 (s, 3H, Hc), 3.08 (m, 1H, Hd), 1.23 (m, 2H, He or He’), 0.83 (m, 2H, He or He’); C NMR
(CDCl3, 100 MHz) δ 155.0 (C4’), 154.6 (C2), 152.2 (C4), 150.2 (C8a), 145.4 (C8), 131.6 (C1’),
129.4 (C2’), 126.8 (C6’), 125.8 (C5), 124.7 (C7), 123.2 (C3’), 119.7 (C4a), 119.4 (CN), 112.7
2
1
(C5’), 111.5 (d, JC3-F = 18 Hz, C3), 108.4 (C6), 81.4 (d, JCa-F = 164 Hz, Ca), 56.4 (Cc), 50.9
(Cb), 11.2 (Cd), 10.1 (2C, Ce, Ce’); ESI-MS calculated for C22H1935ClFN3O [M+H]+ m/z 396.12,
found C22H1935ClFN3O [M+H]+ m/z 396.12 (100%); C22H1937ClFN3O [M+H]+ m/z 398.09 (33%).
If the crude product was purified over silica gel, alumina or C-18, (Z,E)-4-[((3-chloro-4-
methoxyphenyl)methylene)amino]-8-cyclopropyl-3-methylquinoline-6-carbonitrile (6a) was
obtained. Rf (SiO2, ethyl acetate/cyclohexane, 3/7, v/v): 0.24; Mp: 216-218 ºC; IR (cm-1):
2227 (νC≡N), 1502 (νC=C), 1276 (νas C-O-C), 1187 (νs C-O-C); 1H NMR (CDCl3, 400 MHz) δ 8.91 (s,
1H, H2), 8.20 (s, 1H, Hb) 8.09 (d, 1H, 4JH
= 2.0 Hz, H2’), 7.95 (d, 1H, 4JH -H = 1.7 Hz, H5),
-H
2’ 6’
5 7
3
4
4
7.82 (dd, 1H, JH6’ 5’ = 8.5 Hz) , JH6’ 2’= 2.0 Hz, H6’), 7.23 (m, 1H, H7), 7.09 (d, 1H, JH5’ 6’
=
-H
-H
-H
8.5 Hz, H5’), 4.03 (s, 3H, Hc), 3.17 (m, 1H, Hd), 2.34 (s, 3H, Ha), 1.25 (m, 2H, He or He’), 0.86
(m, 2H, He or He’); 13C NMR (CDCl3, 100 MHz) δ 162.6 (Cb), 158.8 (C4’), 155.6 (C4), 154.6
(C2), 147.4 (C8a), 144.8 (C8), 130.6 (C2’), 129.9 (C6’), 128.5 (C1’), 127.0 (C5), 124.0 (C7), 123.4
(C3’), 121.4 (C4a), 119.4 (CN), 118.7 (C3), 112.1 (C5’), 109.6 (C6), 56.7 (Cc), 15.2 (Ca), 11.0
(Cd), 10.1 (2C, Ce, Ce’); HRMS calculated for C22H1835ClN3O [M+H]+ m / z 376.1217, found
C22H1835ClN3O [M+H]+ m/z 376.1214 (100%); C22H1837ClN3O [M+H]+ m/z 378.1169 (33%).
4.1.4. (E)-Diethyl 2-[((2-bromo-4-cyanophenyl)amino)methylene]succinate (9). 4-Amino-
3-bromobenzonitrile (2.0 g, 10.2 mmol, 1.0 eq.) was added to a mixture of 834 (5.16 g, 25.5
mmol, 2.0 eq.) in anhydrous toluene (24 mL) under stirring. The resulting mixture was
refluxed for 8 hours. After cooling to rt, the solvent was evaporated under reduced pressure.
The crude product was purified by column chromatography (Al2O3, ethyl
acetate/cyclohexane, 4/6, v/v) to give the desired product 9 (3.0 g, 7.87 mmol, 77%) as a
white solid. Rf (SiO2, cyclohexane/ethyl acetate, 6/4, v/v): 0.62; Mp: 138-140 ºC; IR (cm-1):
1
2223 (νC≡N), 1677 (νC=O), 1625 (νC=C), 1596 (δN-H), 1191 (νas C-O-C), 1023 (νs C-O-C); H NMR
3
4
(CDCl3, 400 MHz) δ 10.68 (d, 1H, JNH-H = 11.6 Hz, NH), 7.82 (d, 1H, JH3-H = 1.8 Hz, H3),
a
5
3
4
3
7.54 (dd, 1H, JH5-H = 8.6 Hz, JH5-H = 1.8 Hz, H5), 7.22 (d, 1H, JHa-NH = 11.6 Hz, Ha), 7.10
6
3
(d, 1H, 3JH -H = 8.6 Hz, H6), 4.27 (q, 2H, 3JH -H
= 7.1 Hz, He or He’), 4.17 (q, 2H, 3JH -H
or H -H
e’ f’
6
5
e
f
e
d
3
= 7.1 Hz, He or He’), 3.24 (s, 2H, Hc), 1.30 (t, 3H, JHf-H
= 7.1 Hz, He or He’),
or H -H
e’ d’
or H -H
f’ e’
e
1.25 (t, 3H, 3JH -H
= 7.1 Hz, Hf or Hf’); 13C NMR (CDCl3, 100 MHz) δ 171.8 (Cd), 168.5
or H -H
e f’ e’
f

ACCEPTED MANUSCRIPT
(Cd’), 142.5 (C1), 139.1 (Ca), 136.7 (C3), 132.5 (C5), 117.8 (CN), 112.7 (C6), 111.2 (C2 or C4),
104.9 (C2 or C4), 99.6 (Cb), 60.9 (Ce or Ce’), 60.6 (Ce or Ce’), 36.0 (Cc), 14.2 (2C, Cf, Cf’);
HRMS calculated for C16H1779BrN2O4 [M+H]+ m/z 381.0450, found C16H1779BrN2O4 [M+H]+
m/z 381.0476 (100%); C16H1781BrN2O4 [M+H]+ m/z 383.0457 (98%).
4.1.5. Ethyl 2-(8-bromo-6-cyano-4-oxo-1,4-dihydroquinolin-3-yl)acetate (10).
A
suspension of 9 (510 mg, 1.34 mmol, 1.0 eq.) in DowTherm A (10 mL) was placed into a
o
bath beforehand heated at 240-250 C. The mixture was stirred at this temperature for 2
hours. After cooling to rt and decantation overnight, the formed precipitate was collected by
filtration, washed with diethyl ether (30 mL) and dried to give the desired product 10 (230 mg,
0.69 mmol, 51%) as a brown solid. Mp: 134-136 ºC; IR (cm-1): 2232 (νC≡N), 1740 (νC=O), 1548
1
3
(δN-H), 1505 (νC=C), 1166 (νC-O); H NMR (DMSO-d6, 400 MHz) δ 11.55 (d, 1H, JNH-H = 5.9
2
Hz, NH), 8.48 (d, 1H, 3JH -H
= 1.8 Hz, H5 or H7), 8.46 (d, 1H, 3JH -H
5 = 1.8 Hz, H5
or H -H
7
or H -H
5
7
7
5
5
7
3
3
or H7), 8.02 (d, 1H, JH2-NH = 5.9 Hz, H2), 4.06 (q, 2H, JHc-H = 7.1 Hz, Hc), 3.51 (s, 2H, Ha),
d
3
1.18 (t, 3H, JH -H = 7.1 Hz, Hd); HRMS calculated for C14H1179BrN2O3 [M+H]+ m/z 335.0031,
d
c
found C14H1179BrN2O3 [M+H]+ m/z 335.0034 (100%); C14H1181BrN2O3 [M+H]+ m/z 337.0004
(98%).
4.1.6. Ethyl 2-(8-bromo-4-chloro-6-cyanoquinolin-3-yl)acetate (11). A mixture of 10 (0.53
g, 1.58 mmol, 1.0 eq.) in phosphorous oxychloride (15 mL) was stirred at reflux for 5 hours.
After cooling to rt, the solvent was evaporated. Then, ice (10 mL) and a saturated aqueous
sodium carbonate solution (30 mL) were slowly added. The aqueous layer was extracted
with dichloromethane (2 × 50 mL). The combined organic layers were washed with brine (80
mL), dried over anhydrous magnesium sulfate, filtered and concentrated under reduced
pressure. The brown residue was purified by column chromatography (SiO2, ethyl
acetate/cyclohexane, 3/7, v/v) to give the desired product 11 as a brown solid (0.45 g, 1.27
mmol, 81%). Rf (SiO2, cyclohexane/ethyl acetate, 5/5, v/v): 0.71; Mp: 179-181 ºC; IR (cm-1):
2235 (νC≡N), 1721 (νC=O), 1213 (νas C-O-C), 1151 (νs C-O-C); 1H NMR (CDCl3, 400 MHz) δ 8.99 (s,
1H, H2), 8.60 (d, 1H, 4JH -H = 1.7 Hz, H5), 8.20 (d, 1H, 4JH -H = 1.7 Hz, H7), 4.20 (q, 2H, 3JH -
5
7
7
5
c
3
= 7.1 Hz, Hc), 4.02 (s, 2H, Ha), 1.26 (t, 3H, JH -H = 7.1 Hz, Hd); 13C NMR (CDCl3, 100
H
d
d
c
MHz) δ 168.7 (Cb), 155.2 (C2), 146.4 (C8a), 143.0 (C4), 133.9 (C7), 130.2 (C5), 128.6 (C3),
126.9 (C4a or C8), 126.8 (C4a or C8), 116.9 (CN), 112.1 (C6), 61.8 (Cc), 37.1 (Ca), 14.2 (Cd);
HRMS calculated for C14H1079Br35ClN2O2 [M+H]+ m / z 352.9692, found C14H1079Br35ClN2O2
[M+H]+ m/z 352.9671 (77%); C14H1081Br35ClN2O2 or C14H1079Br37ClN2O2 [M+H]+ m/z 354.9647
(100%); C14H1081Br37ClN2O2 [M+H]+ m/z 356.9632 (24%).

ACCEPTED MANUSCRIPT
4.1.7.
Ethyl
2-(4-chloro-6-cyano-8-cyclopropylquinolin-3-yl)acetate
(13).
Cyclopropylboronic
acid
(73
mg,
0.85
mmol,
1.5
eq.),
tetrakis(triphenylphosphine)palladium(0) (39 mg, 33.7 µmol, 0.06 eq.), caesium carbonate
(460 mg, 1.41 mmol, 2.5 eq.) were added, under stirring and argon, to a solution of 11 (200
mg, 0.57 mmol, 1.0 eq.) in anhydrous toluene (10 mL). The resulting mixture was refluxed for
22 h. After cooling to rt, the reaction mixture was filtered over Celite® 545, washed with ethyl
acetate (40 mL). The filtrate was concentrated under reduced pressure. The crude product
was purified by column chromatography (SiO2, ethyl acetate/cyclohexane, 3/7, v/v) to give
the desired product 13 (110 mg, 0.35 mmol, 62%) as a light yellow solid. Rf (Al2O3,
cyclohexane/ethyl acetate, 8/2, v/v): 0.40; Mp: 126-128 ºC; IR (cm-1): 2232 (νC≡N), 1742
1
(νC=O), 1482 (νC=C), 1151 (νas C-O-C), 1043 (νs C-O-C); H NMR (DMSO-d6, 400 MHz) δ 8.93 (s,
1H, H2), 8.45 (d, 1H, 4JH -H = 1.6 Hz, H5), 7.31 (d, 1H, 4J H -H = 1.6 Hz, H7), 4.21 (q, 2H, 3JH -
5
7
7
5
c
d = 7.1 Hz, Hc), 4.01 (s, 2H, Ha), 3.19 (m, 1H, He), 1.26 (m, 5H, Hd, Hf or Hf’), 0.89 (m, 2H, Hf
H
or Hf’); 13C NMR (DMSO-d6, 100 MHz) δ 169.0 (Cb), 153.1 (C2), 148.0 (C8a), 145.8 (C8), 142.4
(C4), 127.2 (C3), 127.0 (C5), 125.5 (C4a), 123.9 (C7), 118.4 (CN), 111.2 (C6), 61.4 (Cc), 36.8
(Ca), 14.0 (Cd), 10.6 (Ce), 10.5 (2C, Cf, Cf’); HRMS calculated for C17H1535ClN2O2 [M+H]+ m/z
315.0900, found C17H1535ClN2O2 [M+H]+ m/z 315.0915 (100%); C17H1537ClN2O2 [M+H]+ m/z
317.0922 (33%).
4.1.8.
4-[(3-Chloro-4-methoxybenzyl)amino]-8-cyclopropyl-3-formylquinoline-6-
carbonitrile (15a). Manganese oxide (331 mg, 3.81 mmol, 10 eq.) was added to a solution
of 4a [12] (150 mg, 0.38 mmol, 1.0 eq.) in dichloromethane (20 mL). The resulting mixture
was stirred at rt for 5 hours, filtered on Celite® 545 and washed with dichloromethane (40
mL). The filtrate was evaporated under reduced pressure. The residue was purified by
column chromatography (SiO2, ethanol/dichloromethane, 3/97, v/v) to give the desired
product 15a (140 mg, 0.36 mmol, 95%) as a light yellow solid. Rf (SiO2,
ethanol/dichloromethane, 1/9, v/v): 0.49; Mp: 209-211 ºC; IR (cm-1): 2228 (νC≡N), 1642 (νC=O),
1
1571 (δN-H), 1501 (νC=C), 1181 (νC-O), 1022 (νC-O); H NMR (CDCl3, 400 MHz) δ 10.56 (t, 1H,
3JNH-H = 5.5 Hz, NH), 9.91 (s, 1H, Ha), 8.74 (s, 1H, H2), 8.34 (d, 1H, 4JH -H = 1.7 Hz, H5), 7.38
b
5
7
(d, 1H, 4JH
= 2.2 Hz, H2’), 7.26 (m, 2H, H7, H6’), 6.95 (d, 1H, 3JH5’ 6’ = 8.5 Hz, H5’), 4.92 (d,
-H
2’ 6’
-H
2H, 3JH -NH = 5.5 Hz, Hb), 3.89 (s, 3H, Hc), 3.08 (m, 1H, Hd), 1.19 (m, 2H, He or He’), 0.78 (m,
b
13
2H, He or He’); C NMR (CDCl3, 100 MHz) δ 192.7 (Ca), 157.0 (C2), 155.5 (C4), 155.3 (C4’),
151.8 (C8a), 145.5 (C8), 129.5 (C1’), 129.5 (C5), 129.4 (C2’), 127.0 (C6’), 126.9 (C7), 123.4
(C3’), 119.0 (CN), 118.0 (C3), 112.7 (C5’), 111.6 (C4a), 107.7 (C6), 56.3 (Cc), 51.1 (Cb), 11.4
(Cd), 10.1 (2C, Ce, Ce’); HRMS calculated for C22H1835ClN3O2 [M+H]+ m/z 392.1166, found
C22H1835ClN3O2 [M+H]+ m/z 392.1151 (100%); C22H1837ClN3O2 [M+H]+ m/z 394.1126 (33%).

ACCEPTED MANUSCRIPT
4.1.9.
4-[(3-Chloro-4-methoxybenzyl)amino]-8-ethyl-3-formylquinoline-6-carbonitrile
(15b). Manganese oxide (1.4 g, 16.1 mmol, 10.0 eq.) was added to a solution of 4b (0.6 g,
1.57 mmol, 1.0 eq.) in dichloromethane (40 mL). The resulting mixture was stirred at rt for 20
hours before filtration on celite® 545 and washing with dichloromethane (2 × 40 mL). The
filtrate was concentrated under reduced pressure. The residue was purified by column
chromatography (SiO2, cyclohexane/ethyl acetate, 7/3, v/v) to give the desired product 15b
(0.27 g, 0.71 mmol, 45%) as a yellow solid. Rf (SiO2, cyclohexane/ethyl acetate, 7/3, v/v):
0.25; Mp: 168-170 ºC; IR (cm-1): 2226 (νC≡N), 1651 (νC=O), 1576 (δNH), 1505 (νC=C), 1266 (νC-
O), 1069 (νC-O); 1H NMR (CDCl3, 400 MHz) δ 10.57 (t, 1H, 3JNH-Hb = 5.5 Hz, NH), 9.91 (s, 1H,
Ha), 8.72 (s, 1H, H2), 8.43 (d, 1H, 4JH -H = 1.7 Hz, H5), 7.70 (m, 1H, H7), 7.40 (d, 1H, 4JH
=
-H
2’ 6’
5
7
4
3
2.3 Hz, H2’), 7.27 (dd, 1H, JH6’ 2’ = 2.3 Hz, 3JH
= 8.6 Hz, H6’), 6.97 (d, 1H, JH5’ 6’ = 8.6
-H
-H
-H
6’ 5’
Hz, H5’), 4.94 (d, 2H, 3JH -NH = 5.5 Hz, Hb), 3.90 (s, 3H, Hc), 3.20 (q, 2H, 3JH -H = 7.5 Hz, Hd),
b
d
e
1.33 (t, 3H, 3JH -H = 7.5 Hz, He); 13C NMR (CDCl3, 100 MHz) δ 192.7 (Ca), 157.0 (C2), 155.4
e
d
(C4’), 155.3 (C4), 151.2 (C8a), 145.4 (C8), 131.5 (C7), 130.5 (C5), 129.5 (C1’), 129.4 (C2’), 126.9
(C6’), 123.4 (C3’), 119.0 (CN), 118.0 (C4a), 112.7 (C5’), 111.5 (C3), 107.6 (C6), 56.3 (Cc), 51.1
(Cb), 25.3 (Cd), 14.5 (Ce); HRMS calculated for C21H1835ClN3O2 [M+H]+ m/z 380.1166, found
C21H1835ClN3O2 [M+H]+ m/z 380.1159 (100%); C21H1837ClN3O2 [M+H]+ m/z 382.1113 (33%).
4.1.10.
(Z,
E)-4-[(3-Chloro-4-methoxybenzyl)amino]-8-cyclopropyl-3-(2-
methoxyvinyl)quinoline-6-carbonitrile (16a). A 1.6 M solution of n-butyllithium (4.1 mL,
o
6.56 mmol, 9.9 eq.) in hexane was slowly added at -7 C, under argon and stirring, to a
suspension of (methoxymethyl)triphenylphosphonium chloride (2.28 g, 6.65 mmol, 10.0 eq.)
in anhydrous tetrahydrofuran (20 mL). The resulting mixture was then stirred at rt for 20
minutes. After cooling to -15 oC, a solution of 15a (260 mg, 0.66 mmol, 1.0 eq.) in anhydrous
tetrahydrofuran (20 mL) was slowly added. The resulting mixture was stirred at rt for 2 hours
o
before addition of a saturated aqueous ammonium chloride solution (40 mL) at -5 C. This
aqueous layer was extracted with ethyl acetate (3 × 30 mL). The combined organic layers
were washed with brine (50 mL), dried over anhydrous magnesium sulfate, filtered, and
concentrated under reduced pressure. The residue obtained was purified by column
chromatography (SiO2, cyclohexane/ethyl acetate, 5/5, v/v) to give the desired product (Z, E)-
16a (0.19 g, 0.45 mmol, 68%) as a yellow solid (ratio Z/E = 1/0.4). Rf (SiO2,
cyclohexane/ethyl acetate, 5/5, v/v): 0.32, 0.23; Mp: 140-142 ºC; IR (cm-1): 3405 (νN-H), 2226
1
(νC≡N), 1651 (νC=C), 1498 (νC=C), 1255 (νC-O), 1099 (νC-O), 1061 (νC-O); H NMR (CDCl3, 400
MHz) δ 8.96 (s, 1H, H2(Z)), 8.69 (s, 1H, H2(E)), 8.15 (d, 1H, 4JH5(Z) 7(Z) = 1.6 Hz, H5(Z)), 8.12 (d,
-H
4
4
1H, JH5(E) 7(E) = 1.6 Hz, H5(E)), 7.32 (d, 1H, JH2’(Z/E) 6’(Z/E) = 2.2 Hz, H2’(E/Z)), 7.16 (m, 1H,
-H
-H
4
3
4
H7(Z/E)), 7.12 (dd, 1H, JH6’(Z) 2’(Z) = 2.2 Hz, JH6’(Z) 5’(Z) = 8.4 Hz, H6’(Z)), 7.11 (dd, 1H, JH6’(E)
-
-H
-H

ACCEPTED MANUSCRIPT
3
3
= 2.2 Hz, JH6’(E) 5’(E) = 8.4 Hz, H6’(E)), 6.88 (d, 1H, JH5’(Z/E) 6’(Z/E) = 8.4 Hz, H5’(Z/E)), 6.85
H
-H
-H
2’(E)
(d, 1H, 3JH
= 12.8 Hz, Hb(E)), 6.27 (d, 1H, 3JH
= 6.8 Hz, Hb(Z)), 5.72 (d, 1H, 3JH
-H
b(E) a(E)
-H
b(Z) a(Z)
-
a(E)
3
= 12.8 Hz, Ha(E)), 5.28 (d, 1H, JHa(Z) b(Z) = 6.8 Hz, Ha(Z)), 4.58 (s, 2H, Hd(E)), 4.55 (s, 2H,
H
-H
b(E)
Hd(Z)), 3.88 (s, 3H, He(Z/E)), 3.73 (s, 3H, Hc(Z)), 3.65 (s, 3H, Hc(E)), 3.10 (m, 1H, Hf(Z/E)), 1.19 (m,
2H, Hg(Z/E) or Hg’(Z/E)), 0.81 (m, 2H, Hg(Z/E) or Hg’(Z/E)); 13C NMR (CDCl3, 100 MHz) δ 154.8
(C4’(E)), 154.7 (C4’(Z)), 154.0 (C2(Z)), 152.0 (C2(E)), 151.3 (Cb(E)), 149.1 (2C, Cb(Z), C4(Z/E)), 148.6
(C8a(E)), 148.4 (C8a(Z)), 145.3 (C8(E)), 144.9 (C8(Z)), 132.4 (C1’(Z)), 132.3 (C1’(E)), 129.5 (C2’(Z)),
129.4 (C2’(E)), 127.0 (C6’(Z)), 126.9 (C6’(E)), 126.6 (C5(Z)), 125.8 (C5(E)), 123.3 (C7(Z)), 123.1
(C7(E)), 123.0 (C3’(E)), 122.9 (C3’(E)), 121.0 (C4a(Z)), 120.7 (C4a(E)), 119.7 (CN(Z)), 119.6 (CN(E)),
117.4 (C3(E)), 116.7 (C3(Z)), 112.4 (C5’(E)), 112.4 (C5’(Z)), 108.4 (C6(E)), 108.0 (C6(Z)), 99.4 (Ca(Z)),
98.6 (Ca(E)), 60.8 (Cc(Z)), 56.9 (Cc(E)), 56.3 (Ce(Z/E)), 52.5 (Cd(Z)), 52.0 (Cd(E)), 11.2 (Cf(Z)), 11.1
(Cf(E)), 10.0 (2C, Cg(E)/Cg’(E)), 9.9 (2C, Cg(Z)/Cg’(Z)); HRMS calculated for C24H2235ClN3O2 [M+H]+
m/z 420.1479, found C24H2235ClN3O2 [M+H]+ m/z 420.1505 (100%); C24H2237ClN3O2 [M+H]+
m/z 422.1455 (33%).
4.1.11. (Z)-4-[(3-Chloro-4-methoxybenzyl)amino]-8-ethyl-3-(2-methoxyvinyl)quinoline-6-
carbonitrile (16b). A solution of 1.6 M n-butyllithium (4.1 mL, 6.56 mmol, 9.2 eq) in hexane
was added slowly to a suspension of (methoxymethyl)triphenylphosphonium chloride (2.4 g,
7.00 mmol, 9.9 eq) in anhydrous tetrahydrofuran (20 mL) under argon and stirring at -15 oC.
The resulting mixture was then stirred at rt for 20 minutes before addition of a solution of 15b
(270 mg, 0.71 mmol, 1.0 eq) in anhydrous tetrahydrofuran (15 mL) at -10 oC over 15 minutes.
The resulting mixture was stirred at rt for another 2 hours before addition of a saturated
o
o
aqueous solution of ammonium chloride (40 mL) at a temperature between -5 C and 0 C.
The resulting solution was extracted with ethyl acetate (3 × 30 mL) and the combined organic
layers were washed with brine (100 mL), dried over anhydrous magnesium sulfate, filtered,
and concentrated under reduced pressure. The residue was purified by column
chromatography (SiO2, cyclohexane/ethyl acetate, 5/5, v/v) to give the desired product (Z)-
16b (140 mg, 0.34 mmol, 48%) as a yellow solid. Rf (SiO2, cyclohexane/ethyl acetate, 5/5,
v/v): 0.24; Mp: 151-153 ºC; IR (cm-1): 3405 (νN-H), 2228 (νC≡N), 1499 (δN-H), 1390 (δCH3), 1255
1
(νC-O), 1099 (νC-O), 1060 (νC-O); H NMR (CDCl3, 400 MHz) δ 8.88 (s, 1H, H2), 8.14 (d, 1H,
4JH -H = 1.8 Hz, H5), 7.49 (m, 1H, H7), 7.27 (d, 1H, 4JH
= 2.2 Hz, H2’), 7.07 (dd, 1H, 3JH
-H
2’ 6’
-H
6’ 5’
5
7
4
3
3
= 8.4 Hz, JH6’ 2’ = 2.2 Hz, H6’), 6.82 (d, 1H, JH5’ 6’ = 8.4 Hz, H5’), 6.21 (d, 1H, JHb-H = 6.9
-H
-H
a
Hz, Hb), 5.22 (d, 1H, 3JH -H = 6.9 Hz, Ha), 4.64 (t, 1H, 3JNH-H = 6.2 Hz, NH), 4.47 (d, 2H, 3JH -
a
b
d
d
3
= 6.2 Hz, Hd), 3.82 (s, 3H, He), 3.67 (s, 3H, Hc), 3.17 (q, 2H, JHf-H = 7.5 Hz, Hf), 1.29 (t,
NH
g
3
3H, JH -H = 7.5 Hz, Hg); 13C NMR (CDCl3, 100 MHz) δ 154.7 (C4’), 154.2 (C2), 149.0 (Cb),
g
f

ACCEPTED MANUSCRIPT
148.9 (C4), 147.9 (C8a), 145.1 (C8), 132.5 (C1’), 129.5 (C2’), 127.5 (C7), 127.4 (C6’), 127.0 (C5),
122.9 (C3’), 121.1 (C4a), 119.8 (CN), 116.7 (C3), 112.4 (C5’), 107.9 (C6), 99.5 (Ca), 60.7 (Cc),
56.3 (Ce), 52.6 (Cd), 24.9 (Cf), 14.6 (Cg); HRMS calculated for C23H2235ClN3O2 [M+H]+ m/z
408.1479, found C23H2235ClN3O2 [M+H]+ m/z 408.1461 (100%); C23H2237ClN3O2 [M+H]+ m/z
410.1417 (33%).
4.1.12.
1-(3-Chloro-4-methoxybenzyl)-6-cyclopropyl-1H-pyrrolo[3,2-c]quinoline-8-
carbonitrile (17a). A 4 M aqueous hydrochloric acid solution (0.1 mL) was added to a
solution of 16a (20 mg, 47.6 µmol, 1.0 eq.) in tetrahydrofuran (0.3 mL). The resulting mixture
was stirred at 50 oC for 4 hours. After cooling to rt, an aqueous saturated sodium bicarbonate
solution (20 mL) was added. The resulting solution was extracted with ethyl acetate (2 × 15
mL) and the combined organic layers were washed with brine (30 mL), dried over anhydrous
magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography (SiO2, cyclohexane/ethyl acetate, 7/3, v/v) to give
product 17a as a white solid (10 mg, 25.8 µmol, 54%); Rf (SiO2, cyclohexane/ethyl acetate,
7/3, v/v): 0.53; Mp: 196-198 ºC; IR (cm-1): 2230 (νC≡N), 1502 (νC=C), 1258 (νas C-O), 1064 (νs C-
1
4
O); H NMR (CDCl3, 400 MHz) δ 9.32 (s, 1H, H4), 8.44 (d, 1H, JH9-H = 1.6 Hz, H9), 7.76 (d,
7
1H, 3JH -H = 3.1 Hz, H2), 7.31 (d, 1H, 4JH -H = 1.6 Hz, H7), 7.19 (d, 1H, 4JH2’ 6’ = 2.1 Hz, H2’),
-H
2
3
7
9
3
3
4
7.04 (d, 1H, JH5’ 6’ = 8.6 Hz, H5’), 6.98 (d, 1H, JH3-H = 3.1 Hz, H3), 6.88 (dd, 1H, JH6’ 2’
=
-H
-H
2
2.1 Hz, 3JH6’ 5’ = 8.6 Hz, H6’), 5.95 (s, 2H, Ha), 3.76 (s, 3H, Hb), 3.31 (m, 1H, Hc), 1.10 (m, 2H,
-H
Hd or Hd’), 0.88 (m, 2H, Hd or Hd’); 13C NMR (CDCl3, 100 MHz) δ 153.8 (C4’), 147.7 (C4), 144.9
(C6), 143.8 (C5a), 132.6 (C2), 132.3 (C9b), 130.3 (C1’), 127.7 (C2’), 126.0 (C6’), 124.2 (C9),
123.1 (C3a), 121.4 (C3’), 121.0 (C7), 119.3 (CN), 117.1 (C9a), 113.1 (C5’), 107.7 (C8), 102.7
(C3), 56.1 (Cb), 51.1 (Ca), 11.2 (Cc), 10.3 (2C, Cd, Cd’); HRMS calculated for C23H1835ClN3O
[M+H]+ m/z 388.1217, found C23H1835ClN3O [M+H]+ m/z 388.1197 (100%); C23H1837ClN3O
[M+H]+ m/z 390.1178 (33%).
4.1.13.
Allyl
(3-chloro-4-methoxybenzyl)-[(6-cyano-8-cyclopropyl-3-(2-
methoxyvinyl))quinolin-4-yl]carbamate (19a). Sodium hydride powder (moistened with oil,
55-65%, 91 mg, 2.26 mmol, 5.0 eq.) was added to a solution of 16a (190 mg, 0.45 mmol, 1.0
eq.) in anhydrous N,N-dimethylformamide (10 mL). The resulting mixture was stirred at rt for
20 minutes before careful addition of allyl chloroformate (192 µL, 1.81 mmol, 4.0 eq.). The
mixture was then stirred at rt for one hour before addition of water (20 mL). The resulting
solution was extracted with ethyl acetate (3 × 30 mL) and the combined organic layers were
washed with brine (3 × 50 mL), dried over anhydrous magnesium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified by column chromatography
(SiO2, cyclohexane/ethyl acetate, 6/4, v/v) to give the desired product 19a as yellow oil
corresponding to a mixture of Z/E isomers (ratio: Z/E = 1/0.7, 170 mg, 0.34 mmol, 76%). IR

ACCEPTED MANUSCRIPT
(cm-1): 2228 (νC≡N), 1703 (νC=O), 1635 (νC=C), 1501 (νC=C), 1257 (νC-O), 1220 (νC-O), 1118 (νC-O);
HRMS calculated for C28H2635ClN3O4 [M+H]+ m/z 504.1690, found C28H2635ClN3O4 [M+H]+ m/z
504.1676 (100%); C28H2637ClN3O4 [M+H]+ m/z 506.1830 (33%). During the purification by
column chromatography, a few fractions containing each pure isomer were obtained. The
quantities isolated allowed to characterise the isomers Z and E by NMR analyses separately.
Allyl (E)-(3-chloro-4-methoxybenzyl)-[(6-cyano-8-cyclopropyl-3-(2-methoxyvinyl))quinolin-4-
1
yl]carbamate ((E)-19a). Rf (SiO2, cyclohexane/ethyl acetate, 5/5, v/v): 0.62; H NMR (CDCl3,
400 MHz) δ 9.09 (s, 1H, H2), 7.61 (d, 1H, 4JH -H = 1.6 Hz, H5), 7.16 (d, 1H, 3JH -Ha = 13.1 Hz,
5
7
b
Hb), 7.15 (d, 1H, 4JH
= 2.2 Hz, H2’), 7.14 (d, 1H, 4JH -H = 1.6 Hz, H7), 7.01 (dd, 1H, 3JH
-H
7 5 6’ 5’
-H
2’ 6’
= 8.4 Hz, 4JH
= 2.2 Hz, H6’), 6.77 (d, 1H, 3JH
= 8.4 Hz, H5’), 5.66 (m, 1H, Hg), 5.40 (d,
-H
5’ 6’
-H
6’ 2’
1H, 3JH -H = 13.1 Hz, Ha), 5.02 (m, 2H, Hh), 4.90 (d, 1H, 2JH -H = 14.3 Hz, Hd), 4.58 (m, 1H,
a
b
d
d’
2
Hf), 4.53 (d, 1H, JHd’ = 14.3 Hz, Hd’), 4.46 (m, 1H, Hf’), 3.86 (s, 3H, Hi), 3.55 (s, 3H, Hc),
-H
d
3.17 (m, 1H, Hj), 1.23 (m, 2H, Hk or Hk’), 0.87 (m, 2H, Hk or Hk’); 13C NMR (CDCl3, 100 MHz)
δ 162.5 (Ce), 155.2 (C4’), 152.5 (Cb), 150.5 (C2), 147.9 (C8a), 145.8 (C8), 139.6 (C4), 132.1
(Cg), 131.6 (C2’), 129.4 (C6’), 129.3 (C1’), 125.9 (C5), 125.7 (C4a), 122.9 (C7), 122.5 (C3’),
119.5 (C3), 118.9 (CN), 118.2 (Ch), 112.0 (C5’), 111.1 (C6), 97.7 (Ca), 66.9 (Cf), 56.6 (Cc), 56.3
(Ci), 52.8 (Cd), 10.8 (Cj), 10.5 (2C, Ck, Ck’). Allyl (Z)-(3-chloro-4-methoxybenzyl)-[(6-cyano-8-
cyclopropyl-3-(2-methoxyvinyl))quinolin-4-yl]carbamate ((Z)-19a). Rf (SiO2, cyclohexane/ethyl
1
4
acetate, 5/5, v/v): 0.55; H NMR (CDCl3, 400 MHz) δ 9.77 (s, 1H, H2), 7.48 (d, 1H, JH5-H
=
7
1.6 Hz, H5), 7.11 (d, 1H, 4JH -H = 1.6 Hz, H7), 7.06 (dd, 1H, 3JH
= 8.4 Hz, 4JH 2’ = 2.1 Hz,
-H
6’
-H
6’ 5’
7
5
H6’), 7.02 (d, 1H, 4JH
= 2.1 Hz, H2’), 6.76 (d, 1H, 3JH
= 8.4 Hz, H5’), 6.26 (d, 1H, 3JH -H
-H
5’ 6’ b a
-H
2’ 6’
3
= 7.1 Hz, Hb), 5.64 (m, 1H, Hg), 5.04 (d, 1H, JHa-H = 7.1 Hz, Ha), 4.97 (m, 2H, Hh), 4.81 (d,
b
2
1H, JH -H = 14.2 Hz, Hd), 4.56 (d, 1H, 2JH
= 14.2 Hz, Hd’), 4.48 (m, 2H, Hf), 3.84 (s, 3H,
-H
d
d
d’
d’
Hi), 3.82 (s, 3H, Hc), 3.16 (m, 1H, Hj), 1.20 (m, 2H, Hk or Hk’), 0.84 (m, 2H, Hk or Hk’); 13C
NMR (CDCl3, 100 MHz) δ 155.1 (2C, Ce, C4’), 154.8 (C2), 151.5 (Cb), 147.4 (C8a), 145.4 (C8),
140.0 (C4), 132.1 (Cg), 131.6 (C2’), 129.4 (C6’), 129.3 (C1’), 128.7 (C3), 126.5 (C5), 125.7 (C4a),
122.9 (C7), 122.3 (C3’), 118.9 (CN), 117.8 (Ch), 112.0 (C5’), 110.4 (C6), 97.3 (Ca), 66.7 (Cf),
61.2 (Cc), 56.3 (Ci), 52.9 (Cd), 10.8 (Cj), 10.2 (2C, Ck, Ck’).
4.1.14.
(Z)-Allyl
3-chloro-4-methoxybenzyl[6-cyano-8-ethyl-3-(2-
methoxyvinyl)quinoline-4-yl]carbamate (19b). Sodium hydride powder (moistened with oil,
55-65%, 113 mg, 2.82 mmol, 5.0 eq.) was added to a solution of 16b (230 mg, 0.56 mmol,
1.0 eq.) in anhydrous N,N-dimethylformamide (10 mL). The resulting mixture was stirred at rt
for 20 minutes before careful addition of allyl chloroformate (240 µL, 2.26 mmol, 4.0 eq.). The
mixture was stirred for 2 hours before addition of water (20 mL). The resulting solution was
extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with

ACCEPTED MANUSCRIPT
brine (50 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatography (SiO2,
cyclohexane/ethyl acetate, 7/3, v/v) to give the desired product 19b as yellow oil (170 mg,
0.35 mmol, 63%). Rf (SiO2, cyclohexane/ethyl acetate, 7/3, v/v): 0.31; IR (cm-1): 2228 (νC≡N),
1
1707 (νC=O), 1645 (νC=C), 1502 (νC=C), 1281 (νC-O), 1258 (νC-O), 1102 (νC-O), 1065 (νC-O); H
NMR (CDCl3, 400 MHz) δ 9.75 (s, 1H, H2), 7.53 (m, 2H, H5 & H7), 7.08 (dd, 1H, 3JH 5’ = 8.4
-H
6’
4
4
3
Hz, JH6’ 2’ = 2.1 Hz, H6’), 7.01 (d, 1H, JH2’ 6’ = 2.1 Hz, H2’), 6.77 (d, 1H, JH5’ 6’ = 8.4 Hz,
-H
-H
-H
3
3
H5’), 6.26 (d, 1H, JHb-H = 7.1 Hz, Hb), 5.64 (m, 1H, Hg), 5.04 (d, 1H, JHa-H = 7.1 Hz, Ha),
a
b
4.98 (m, 2H, Hf), 4.82 (d, 1H, 2JH -H = 14.3 Hz, Hd), 4.54 (d, 1H, 2JH d = 14.3 Hz, Hd’), 4.47
-H
d’
d
d’
3
(m, 2H, Hh), 3.85 (s, 3H, Hi), 3.81 (s, 3H, Hc), 3.27 (q, 2H, JHj-H = 7.5 Hz, Hj), 1.36 (t, 3H,
k
3JH -H = 7.5 Hz, Hk); 13C NMR (CDCl3, 100 MHz) δ 155.1 (C4’ or Ce), 155.0 (C4’ or Ce), 154.6
k
j
(C2), 151.5 (Cb), 146.7 (C8a), 145.1 (C8), 139.9 (C4), 132.1 (Cg), 131.6 (C2’), 129.4 (C6’), 129.3
(C1’), 128.6 (C3), 127.5 (C5 or C7), 127.4 (C5 or C7), 125.7 (C4a), 122.2 (C3’), 118.9 (CN),
117.8 (Ch), 112.0 (C5’), 110.3 (C6), 97.2 (Ca), 66.7 (Cf), 61.3 (Cc), 56.3 (Ci), 53.0 (Cd), 24.6
(Cj), 14.7 (Ck); HRMS calculated for C27H2635ClN3O4 [M+H]+ m/z 492.1690, found
C27H2635ClN3O4 [M+H]+ m/z 492.1658 (100%); C27H2637ClN3O4 [M+H]+ m/z 494.1739 (33%).
4.1.15. Allyl 3-chloro-4-methoxybenzyl[6-cyano-8-cyclopropyl-3-(2-oxoethyl)quinoline-
4-yl]carbamate (20a). A mixture of 19a (170 mg, 0.34 mmol, 1.0 eq.) in formic acid (10 mL)
o
was stirred at 50 C for 20 hours. After cooling to rt, the solvent was evaporated under
reduced pressure. The residue was dissolved in ethyl acetate (20 mL) before addition of a
saturated aqueous sodium carbonate solution (25 mL). After decantation, the aqueous layer
was then extracted with ethyl acetate (2 × 20 mL). The combined organic layers were
washed with brine (60 mL), dried over anhydrous magnesium sulfate, filtered, and
concentrated under reduced pressure. The residue was purified by column chromatography
(SiO2, cyclohexane/ethyl acetate, 7/3, v/v) to give the desired product 20a (100 mg, 0.20
mmol, 60%) as yellow oil. Rf (SiO2, cyclohexane/ethyl acetate, 5/5, v/v): 0.33; IR (cm-1): 2228
(νC≡N), 1702 (νC=O), 1501 (νC=C), 1387 (δCH ), 1257 (νC-O), 1064 (νC-O), 1020 (νC-O); Analyses of
3
NMR spectra revealed that this product was a mixture of two rotamers, named A and B with
a ratio A/B = 1/0.3; 1H NMR (CDCl3, 400 MHz) δ 9.48 (s, 1H, Hb(A/B)), 8.96 (s, 1H, H2(B)), 8.93
(s, 1H, H2(A)), 7.80 (m, 1H, H5(B)), 7.72 (d, 1H, 4JH5(A) 7(A) = 1.6 Hz, H5(A)), 7.26 (m, 1H, H7(A/B)),
-H
4
7.09 (d, 1H, 4JH
= 2.1 Hz, H2’(A/B)), 7.03 (dd, 1H, 3JH
= 8.4 Hz, JH6’(A) 2’(A)
=
-H
2’(A/B) 6’(A/B)
-H
6’(A) 5’(A)
-H
2.1 Hz, H6’(A)), 6.89 (m, 1H, H6’(B)), 6.80 (d, 1H, 3JH
= 8.4 Hz, H5’(A)), 6.78 (d, 1H, 3JH
5’(B)
-H
5’(A) 6’(A)
-
= 8.4 Hz, H5’(B)), 6.11 (m, 1H, Hf(B)), 5.65 (m, 1H, Hf(A)), 5.46 (m, 2H, Hg(B)), 5.03 (m, 3H,
H
6’(B)
Hg(A), Hc(A/B)), 4.80 (m, 2H, He(B)), 4.49 (m, 2H, He(A)), 4.39 (d, 1H, 2JHc’(B) c(B) = 14.7 Hz, Hc’(B)),
-H
2
4.38 (d, 1H, JHc’(A) c(A) = 14.7 Hz, Hc’(A)), 3.87 (s, 3H, Hh(A/B)), 3.48 (m, 2H, Ha(A/B)), 3.23 (m,
-H

ACCEPTED MANUSCRIPT
1H, Hi(A/B)), 1.26 (m, 2H, Hj(A/B) or Hj’(A/B)), 0.91 (m, 2H, Hj(A/B) or Hj’(A/B)); 13C NMR (CDCl3, 100
MHz) δ 197.0 (Cb(B)), 196.3 (Cb(A)), 155.3 (C4’(A/B)), 154.7 (Cd(A/B)), 154.6 (C2(A/B)), 149.3
(C8a(A/B)), 146.5 (C8(A/B)), 144.5 (C4(A/B)), 131.9 (Cf(B)), 131.7 (Cf(A)), 131.4 (C2’(A)), 131.3 (C2’(B)),
129.3 (C6’(A/B)), 129.0 (C1’(A)), 128.9 (C1’(B)), 126.0 (C3(A/B)), 125.9 (C5(A/B)), 125.1 (C4a(A/B)),
124.0 (C7(A/B)), 122.7 (C3’(A/B)), 119.9 (Cg(B)), 119.0 (Cg(A)), 118.7 (CN(A/B)), 112.2 (C5’(A/B)), 111.4
(C6(A/B)), 67.8 (Ce(B)), 67.3 (Ce(A)), 56.3 (Ch(A/B)), 53.5 (Cc(B)), 53.3 (Cc(A)), 43.8 (Ca(B)), 43.5
(Ca(A)), 11.0 (Ci(B)), 10.9 (Ci(A)), 10.7 (Cj(A) or Cj’(A)), 10.5 (Cj(B) or Cj’(B)); ESI-MS calculated for
C27H2435ClN3O4 [M+H]+ m/z 490.15, found C27H2435ClN3O4 [M+H]+ m/z 490.14 (100%);
C27H2437ClN3O4 [M+H]+ m/z 492.10 (33%).
4.1.16.
Allyl
3-chloro-4-methoxybenzyl[6-cyano-8-ethyl-3-(2-oxoethyl)quinoline-4-
yl]carbamate (20b). A mixture of 19b (170 mg, 0.35 mmol, 1.0 eq.) in formic acid (10 mL)
o
was stirred at 50 C for 20 hours. After cooling to rt, the solvent was evaporated under
reduced pressure. The residue was dissolved in ethyl acetate (30 mL) before addition of a
saturated aqueous sodium carbonate solution (30 mL). After decantation, the aqueous layer
was extracted ethyl acetate (2 × 20 mL). The combined organic layers were washed with
brine (100 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatography (SiO2,
cyclohexane/ethyl acetate, 6/4, v/v) to give the product 20b (0.11 g, 0.23 mmol, 67%) as
yellow oil. Rf (SiO2, cyclohexane/ethyl acetate, 6/4, v / v): 0.24; IR (cm-1): 2224 (νC≡N), 1705
(νC=O), 1260 (νC-O), 1065 (νC-O); Analyses of NMR spectra revealed that this product was a
mixture of two rotamers, named A and B with a ratio A/B = 1/0.3. 1H NMR (CDCl3, 400 MHz)
δ 9.48 (s, 1H, Hb(A/B)), 8.92 (s, 1H, H2(B)), 8.89 (s, 1H, H2(A)), 7.85 (m, 1H, H5(B)), 7.78 (d, 1H,
4
4JH
= 1.7 Hz, H5(A)), 7.68 (m, 1H, H7(A/B)), 7.08 (d, 1H, JH2’(A/B) 6’(A/B) = 2.1 Hz, H2’(A/B)),
-H
5(A) 7(A)
-H
3
4
7.03 (dd, 1H, JH6’(A) 5’(A) = 8.4 Hz, JH6’(A) 2’(A) = 2.1 Hz, H6’(A)), 6.89 (m, 1H, H6’(B)), 6.80 (d,
-H
-H
3
3
1H, JH5’(A) 6’(A) = 8.4 Hz, H5’(A)), 6.78 (d, 1H, JH5’(B) 6’(B) = 8.4 Hz, H5’(B)), 6.10 (m, 1H, Hf(B)),
-H
-H
5.65 (m, 1H, Hf(A)), 5.50 (d, 1H, 3JH (B) (B) = 17.2 Hz, Hg(B)), 5.42 (d, 1H, 3JH
(B) = 10.3 Hz,
-H
f
-H
g
f
g’
(B)
2
Hg’(B)), 5.02 (m, 3H, Hg(A), Hc(A/B)), 4.81 (m, 2H, He(B)), 4.48 (m, 2H, He(A)), 4.40 (d, 1H, JHc’(B)
-
= 14.7 Hz, Hc’(B)), 4.38 (d, 1H, 2JH
= 14.7 Hz, Hc’(A)), 3.86 (s, 3H, Hh(A/B)), 3.48 (m,
H
-H
c’(A) c(A)
c(B)
3
3
2H, Ha(A/B)), 3.32 (q, 2H, JHi(A/B)-Hj(A/B) = 7.5 Hz, Hi(A/B)), 1.39 (t, 3H, JHj(A/B)-Hi(A/B) = 7.5 Hz,
Hj(A/B)); 13C NMR (CDCl3, 100 MHz) δ 197.0 (Cb(B)), 196.4 (Cb(A)), 155.3 (C4’(A/B)), 154.8
(Cd(A/B)), 154.6 (C2(A/B)), 148.6 (C8a(A/B)), 146.1 (C8(A/B)), 144.5 (C4(A/B)), 131.7 (Cf(A/B)), 131.4
(C2’(A/B)), 129.3 (C6’(A/B)), 129.0 (C1’(A/B)), 128.8 (C5(A/B)), 127.1 (C7(A/B)), 125.9 (C3(A/B)), 125.2
(C4a(A/B)), 122.7 (C3’(A/B)), 119.9 (Cg(B)), 119.0 (Cg(A)), 118.7 (CN(A/B)), 112.2 (C5’(A/B)), 111.3
(C6(A/B)), 67.8 (Ce(B)), 67.3 (Ce(A)), 56.3 (Ch(A/B)), 53.5 (Cc(B)), 53.4 (Cc(A)), 43.8 (Ca(B)), 43.5
(Ca(A)), 24.7 (Ci(B)), 24.6 (Ci(A)), 14.8 (Cj(B)), 14.6 (Cj(A)); ESI-MS calculated for C26H2435ClN3O4

ACCEPTED MANUSCRIPT
[M+H]+ m/z 478.15, found C26H2435ClN3O4 [M+H]+ m/z 478.05 (100%); C26H2437ClN3O4 [M+H]+
m/z 480.11 (33%).
4.1.17.
4-[(3-Chloro-4-methoxybenzyl)amino]-8-cyclopropyl-3-(2-
hydroxyethyl)quinoline-6-carbonitrile (21a). A 1.1 M solution of lithium tri-tert-
butoxyaluminum hydride in tetrahydrofuran (1.7 mL, 1.87 mmol, 3.0 eq.) was added, under
argon and stirring, to a solution of 20a (300 mg, 0.61 mmol, 1.0 eq.) in anhydrous
tetrahydrofuran (30 mL). The resulting mixture was stirred at 60 oC for 20 hours. After cooling
to rt, water (50 mL) was slowly added under stirring. This aqueous layer was then extracted
with dichloromethane (100 mL) using a Soxhlet extractor for 20 hours. The organic layer was
concentrated under reduced pressure. The residue was purified by column chromatography
(SiO2, ethanol/dichloromethane, 1/9, v/v) to give the desired product 21a (130 mg, 0.32
mmol, 52%) as a yellow oil. Rf (SiO2, dichloromethane/ethanol, 9/1, v/v): 0.20; IR (cm-1): 2226
(νC≡N), 1500 (νC=C), 1257 (νC-O), 1062 (νC-O), 1020 (νC-O); 1H NMR (CDCl3, 400 MHz) δ 8.56 (s,
1H, H2), 8.20 (d, 1H, 4JH -H = 1.7 Hz, H5), 7.29 (d, 1H, 4JH
= 2.2 Hz, H2’), 7.17 (d, 1H, 4JH -
-H
2’ 6’
5
7
7
3
4
3
= 1.7 Hz, H7), 7.09 (dd, 1H, JH6’ 5’ = 8.4 Hz, JH6’ 2’ = 2.2 Hz, H6’), 6.86 (d, 1H, JH5’ 6’
=
H
-H
-H
-H
5
8.4 Hz, H5’), 4.48 (s, 2H, Hc), 3.86 (s, 3H, Hd), 3.83 (t, 2H, 3JH -H = 5.8 Hz, Hb), 2.92 (m, 1H,
b
a
He), 2.80 (t, 2H, 3JH -H = 5.8 Hz, Ha), 1.15 (m, 2H, Hf or Hf’), 0.77 (m, 2H, Hf or Hf’); 13C NMR
a
b
(CDCl3, 100 MHz) δ 154.7 (C4’), 153.4 (C2), 153.0 (C4), 149.0 (C8a), 144.4 (C8), 132.1 (C1’),
129.4 (C2’), 127.8 (C5), 127.0 (C6’), 123.8 (C7), 122.8 (C3’), 121.0 (C4a), 120.8 (C3), 119.4
(CN), 112.4 (C5’), 107.8 (C6), 63.2 (Cb), 56.3 (Cd), 53.0 (Cc), 32.8 (Ca), 11.3 (Ce), 9.6 (2C, Cf,
Cf’); HRMS calculated for C23H2235ClN3O2 [M+H]+ m/z 408.1479, found C23H2235ClN3O2 [M+H]+
m/z 408.1478 (100%); C23H2237ClN3O2 [M+H]+ m/z 410.1430 (33%).
4.1.18.
4-[(3-Chloro-4-methoxybenzyl)amino]-8-ethyl-3-(2-hydroxyethyl)quinoline-6-
carbonitrile (21b). A 1.1 M solution of lithium tri-tert-butoxyaluminum hydride in
tetrahydrofuran (0.63 mL, 0.69 mmol, 3.0 eq.) was added, under argon and stirring, to a
solution of 20b (110 mg, 0.23 mmol, 1.0 eq.) in anhydrous tetrahydrofuran (15 mL). The
o
resulting mixture was stirred at 60 C for 20 hours. After cooling to rt, water (3 mL) was
added slowly and the mixture was stirred at this temperature for 4 hours. The solid formed
was filtered and washed with ethyl acetate (20 mL). The filtrate was concentrated under
reduced pressure. The residue obtained was purified by column chromatography (SiO2,
ethanol/dichloromethane, 1/9, v/v) to give the desired product 21b (40 mg, 0.10 mmol, 44%)
as a yellow solid. Rf (SiO2, ethanol/dichloromethane, 1/9, v/v): 0.20; IR (cm-1): 2226 (νC≡N),
1
1504 (δN-H), 1279 (νC-O), 1261 (νC-O), 1048 (νC-O), 1024 (νC-O); H NMR (CDCl3, 400 MHz) δ
8.55 (s, 1H, H2), 8.28 (d, 1H, 4JH -H = 1.7 Hz, H5), 7.54 (m, 1H, H7), 7.30 (d, 1H, 4JH 6’ = 2.1
-H
2’
5
7
3
4
3
Hz, H2’), 7.12 (dd, 1H, JH6’ 5’ = 8.4 Hz, JH6’ 2’ = 2.1 Hz, H6’), 6.88 (d, 1H, JH5’ 6’ = 8.4 Hz,
-H
-H
-H

ACCEPTED MANUSCRIPT
H5’), 4.49 (s, 2H, Hc), 3.88 (s, 3H, Hd), 3.86 (t, 2H, 3JH -H = 5.8 Hz, Hb), 3.19 (q, 2H, 3JH -H
=
b
a
e
f
3
3
7.5 Hz, He), 2.82 (t, 2H, JH -H = 5.8 Hz, Ha), 1.33 (t, 3H, JH -H = 7.5 Hz, Hf); 13C NMR
a
b
f
e
(CDCl3, 100 MHz) δ 154.7 (C4’), 153.8 (C2), 152.7 (C4), 148.6 (C8a), 144.8 (C8), 132.3 (C1’),
129.5 (C2’), 128.5 (C5), 127.6 (C7), 127.0 (C6’), 122.8 (C3’), 121.4 (C4a), 120.9 (C3), 119.6
(CN), 112.4 (C5’), 107.8 (C6), 63.4 (Cb), 56.3 (Cd), 53.2 (Cc), 32.8 (Ca), 24.8 (Ce), 14.5 (Cf);
HRMS calculated for C22H2235ClN3O2 [M+H]+ m/z 396.1479, found C22H2235ClN3O2 [M+H]+ m/z
396.1512 (100%); C22H2237ClN3O2 [M+H]+ m/z 398.1501 (33%).
4.1.19. 4-[(3-Chloro-4-methoxybenzyl)amino]-8-cyclopropyl-3-(2-fluoroethyl)quinoline-
6-carbonitrile (22a). Diethylaminosulfur trifluoride (42 µL, 0.32 mmol, 2.0 eq.) was added at
o
-78 C, under argon and stirring, to a solution of 21a (65 mg, 0.16 mmol, 1.0 eq.) in
anhydrous dichloromethane (10 mL). The temperature was slowly increased to rt over 3
hours. The mixture was then cooled to -10 °C before addition of a saturated sodium
bicarbonate solution (20 mL). After decantation, the aqueous layer was extracted with
dichloromethane (4 × 15 mL). The combined organic layers were washed with brine (50 mL),
dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure. The
mixture was purified by column chromatography (SiO2, dichloromethane/ethanol, 9/1, v/v) to
give by order of elution: the desired product 22a (30 mg, 73.2 µmol, 46%) as yellow oil and
the
by-product
1-(3-chloro-4-methoxybenzyl)-6-cyclopropyl-2,3-dihydro-1H-pyrrolo[3,2-
c]quinoline-8-carbonitrile 23a (10 mg, 25.6 µmol, 15%) as yellow oil. 4-[(3-chloro-4-
methoxybenzyl)amino]-8-cyclopropyl-3-(2-fluoroethyl)quinoline-6-carbonitrile (22a). Rf (SiO2,
dichloromethane/ethanol, 9/1, v/v): 0.56; 1H NMR (CDCl3, 400 MHz) δ 8.68 (s, 1H, H2), 8.25
4
(d, 1H, 4JH -H = 1.6 Hz, H5), 7.31 (d, 1H, 4JH
= 2.2 Hz, H2’), 7.23 (d, 1H, JH7-H = 1.6 Hz,
-H
2’ 6’
5
7
5
3
4
3
H7), 7.13 (dd, 1H, JH6’ 5’ = 8.4 Hz, JH6’ 2’ = 2.2 Hz, H6’), 6.91 (d, 1H, JH5’ 6’ = 8.4 Hz, H5’),
-H
-H
-H
4.64 (dt, 2H, 2JH -F = 47.0 Hz, 3JH -H = 5.8 Hz, Hb), 4.52 (d, 2H, 3JH -NH = 5.9 Hz, Hc), 3.91 (s,
b
b
a
c
3H, Hd), 3.10 (m, 1H, He), 3.02 (dt, 2H, 3JH -F = 26.8 Hz, 3JH -H = 5.8 Hz, Ha), 1.22 (m, 2H, Hf
a
a
b
or Hf’), 0.84 (m, 2H, Hf or Hf’); 13C NMR (CDCl3, 100 MHz) δ 155.0 (C4’), 154.1 (C2), 152.2
(C4), 149.3 (C8a), 145.2 (C8), 131.9 (C1’), 129.6 (C2’), 127.3 (C5), 127.1 (C6’), 123.7 (C7), 123.1
(C3’), 121.1 (C3), 119.5 (CN), 118.4 (C4a), 112.5 (C5’), 108.4 (C6), 84.1 (d, 2JC -F = 168 Hz, Cb),
b
3
56.4 (Cd), 53.4 (Cc), 30.9 (d, JCa-F = 20 Hz, Ca), 11.2 (Ce), 10.0 (2C, Cf, Cf’); HRMS
calculated for C23H2135ClFN3O [M+H]+ m/z 410.1435, found C23H2135ClFN3O [M+H]+ m/z
410.1420 (100%); C23H2137ClFN3O [M+H]+ m/z 412.1215 (33%). 1-(3-chloro-4-
methoxybenzyl)-6-cyclopropyl-2,3-dihydro-1H-pyrrolo[3,2-c]quinoline-8-carbonitrile (23a). Rf
(SiO2, dichloromethane/ethanol, 9/1, v/v): 0.12; IR (cm-1): 2222 (νC≡N), 1501 (νC=C), 1294 (νas
C-O-C), 1252 (νs C-O-C); 1H NMR (CDCl3, 400 MHz) δ 8.54 (s, 1H, H2), 7.94 (d, 1H, 4JH -H7 = 1.7
5
4
3
4
Hz, H5), 7.31 (d, 1H, JH2’ 6’ = 2.2 Hz, H2’), 7.15 (dd, 1H, JH6’ 5’ = 8.5 Hz, JH6’ 2’ = 2.2 Hz,
-H
-H
-H

ACCEPTED MANUSCRIPT
4
3
H6’), 7.09 (d, 1H, JH7-H = 1.7 Hz, H7), 6.91 (d, 1H, JH5’ 6’ = 8.5 Hz, H5’), 4.78 (s, 2H, Hc),
-H
5
3
3.85 (m, 5H, Hd, Hb), 3.24 (t, 2H, JHa-H = 9.4 Hz, Ha), 2.92 (m, 1H, He), 1.14 (m, 2H, Hf or
b
Hf’), 0.73 (m, 2H, Hf or Hf’); 13C NMR (CDCl3, 100 MHz) δ 155.3 (C4’), 154.7 (C4), 149.3 (C8a),
145.8 (C2), 144.3 (C8), 129.3 (C1’), 128.2 (C2’), 126.5 (C5 or C6’), 125.8 (C5 or C6’), 123.8 (C7),
123.3 (C3’), 121.2 (C3), 119.4 (CN), 115.0 (C4a), 112.7 (C5’), 106.6 (C6), 56.2 (Cd), 55.8 (Cb),
53.7 (Cc), 25.5 (Ca), 11.8 (Ce), 9.3 (2C, Cf, Cf’); HRMS calculated for C23H2035ClFN3O [M+H]+
m/z 390.1373, found C23H2035ClFN3O [M+H]+ m/z 390.1374 (100%); C23H2037ClFN3O [M+H]+
m/z 392.1176 (33%).
4.1.20.
4-[(3-Chloro-4-methoxybenzyl)amino]-8-ethyl-3-(2-fluoroethyl)quinoline-6-
carbonitrile (22b). Diethylaminosulfur trifluoride (27 µL, 0.20 mmol, 2.0 eq.) was added at -
o
78 C, under argon and stirring, to a solution of 21b (40 mg, 0.10 mmol, 1.0 eq.) in
anhydrous dichloromethane (10 mL). After return back to rt over 1.5 hours, a saturated
sodium hydrogen carbonate solution (15 mL) was added slowly at -10 oC. After decantation,
the aqueous layer was extracted with dichloromethane (4 × 15 mL). The combined organic
layers were washed with brine (50 mL), dried over anhydrous magnesium sulfate, filtered,
and concentrated under reduced pressure. The residue was purified by column
chromatography (SiO2, dichloromethane/ethanol, 9/1, v/v) to give, by order of elution: the
desired product 22b (6.0 mg, 15.1 µmol, 15%) as a yellow oil and the by-product 1-(3-chloro-
4-methoxybenzyl)-6-ethyl-2,3-dihydro-1H-pyrrolo[3,2-c]quinoline-8-carbonitrile 23b (25 mg,
66.2 µmol, 68%) as yellow oil. 4-[(3-chloro-4-methoxybenzyl)amino]-8-ethyl-3-(2-
fluoroethyl)quinoline-6-carbonitrile (22b); Rf (SiO2, dichloromethane/ethanol, 93/7, v/v): 0.51;
1H NMR (CDCl3, 400 MHz) δ 8.64 (s, 1H, H2), 8.31 (d, 1H, 4JH -H = 1.7 Hz, H5), 7.63 (m, 1H,
5
7
4
3
4
H7), 7.32 (d, 1H, JH2’ 6’ = 2.2 Hz, H2’), 7.15 (dd, 1H, JH6’ 5’ = 8.4 Hz, JH6’ 2’ = 2.2 Hz, H6’),
-H
-H
-H
6.93 (d, 1H, 3JH
= 8.4 Hz, H5’), 4.65 (dt, 2H, 2JH -F = 47.0 Hz, 3JH -H = 5.8 Hz, Hb), 4.52 (d,
-H
5’ 6’
b b a
2H, 3JH -NH = 5.7 Hz, Hc), 3.92 (s, 3H, Hd), 3.27 (q, 2H, 3JH -H = 7.4 Hz, He), 3.02 (dt, 2H, 3JH -F
c
e
f
a
3
3
= 27.0 Hz, JHa-H = 5.8 Hz, Ha), 1.38 (t, 3H, JHf-H = 7.4 Hz, Hf); HRMS calculated for
b
e
C22H2135ClFN3O [M+H]+ m/z 398.1435, found C22H2135ClFN3O [M+H]+ m/z 398.1451 (100%);
C22H2137ClFN3O [M+H]+ m/z 400.1242 (33%). 1-(3-chloro-4-methoxybenzyl)-6-ethyl-2,3-
dihydro-1H-pyrrolo[3,2-c]quinoline-8-carbonitrile (23b). Rf (SiO2, dichloromethane/ethanol,
93/7, v/v): 0.20; IR (cm-1): 2219 (νC≡N), 1501 (νC=C), 1254 (νas C-O), 1061 (νs C-O); Analyses of
NMR spectra revealed that this product was a mixture of two rotamers, named A and B with
1
a ratio A/B = 1/0.2; H NMR (CDCl3, 400 MHz) δ 8.62 (s, 1H, H2(B)), 8.52 (s, 1H, H2(A)), 8.31
(d, 1H, 4JH
= 1.8 Hz, H5(B)), 8.04 (d, 1H, 4JH5(A) 7(A) = 1.8 Hz, H5(A)), 7.58 (m, 1H, H7(B)),
-H
5(B) 7(B)
-H
7.47 (m, 1H, H7(A)), 7.36 (d, 1H, 4JH
= 2.2 Hz, H2’(A)), 7.32 (d, 1H, 4JH
6’(B) = 2.2 Hz,
-H
2’(B)
-H
2’(A) 6’(A)
H2’(B)), 7.19 (dd, 1H, 3JH
= 8.4 Hz, 4JH
= 2.2 Hz, H6’(A)), 7.13 (dd, 1H, 3JH
-H -H
6’(A) 2’(A) 6’(B) 5’(B)
-H
6’(A) 5’(A)