Synthesis and pharmacological evaluation of benzannulated derivatives as potent and selective sigma-1 protein ligands

Article abstract of DOI:10.1016/j.ejmech.2014.01.013

The σ₁ proteins are considered to be a new class of target structures for several central nervous system disorders, including depression, anxiety, psychosis, and Parkinson's and Alzheimer's diseases. Recently, the involvement of these receptors

Full text of DOI:10.1016/j.ejmech.2014.01.013

Accepted Manuscript
Synthesis And Pharmacological Evaluation Of Benzannulated Derivatives As Potent
And Selective Sigma-1 Protein Ligands
Marion Donnier-Maréchal, Pascal Carato, Delphine Le Broc, Christophe Furman,
Patricia Melnyk
PII:
S0223-5234(14)00044-0
10.1016/j.ejmech.2014.01.013
EJMECH 6666
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 June 2013
Revised Date: 8 January 2014
Accepted Date: 9 January 2014
Please cite this article as: M. Donnier-Maréchal, P. Carato, D. Le Broc, C. Furman, P. Melnyk, Synthesis
And Pharmacological Evaluation Of Benzannulated Derivatives As Potent And Selective Sigma-1
Protein Ligands, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.01.013.
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ACCEPTED MANUSCRIPT
Synthesis and pharmacological evaluation of benzannulated derivatives as potent and selective
sigma-1 protein ligands
Marion Donnier-Maréchala,b, Pascal Caratoa,b, Delphine Le Broca,c, Christophe Furmana,c and Patricia
Melnyka,b,*
aUniv Lille Nord de France, F-59000 Lille, France
bUDSL, EA 4481, UFR Pharmacie, F-59000 Lille, France
cUDSL, EA 4483, UFR Pharmacie, F-59000 Lille, France
O
N
n
Y
X
N
R1
N
R2
n = 0 or 1
Y = O or S
Linker
N
X
O
N
X = H, Br, Cl
N
N
N
N
A series of benzannulated derivatives were synthesised as sigma 1 ligands. Some of them showed
excellent affinity for sigma 1 receptor and good selectivity for sigma 2 receptor. The cytotoxic effects
were also evaluated.

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SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF BENZANNULATED
DERIVATIVES AS POTENT AND SELECTIVE SIGMA-1 PROTEIN LIGANDS
Marion Donnier-Maréchala,b, Pascal Caratoa,b,*, Delphine Le Broca,c, Christophe Furmana,c and
Patricia Melnyka,b
aUniv Lille Nord de France, F-59000 Lille, France
bUDSL, EA 4481, UFR Pharmacie, F-59000 Lille, France
cUDSL, EA 4483, UFR Pharmacie, F-59000 Lille, France
Mail adress : UFR Pharmacie, 3 rue du Pr Laguesse, BP83, 59006 Lille
List of e-mail adresses :
marion.donnier-marechal@univ-lille2.fr, pascal.carato@univ-lille2.fr, delphine.lebroc@univ-
lille2.fr, christophe.furman@univ-lille2.fr, patricia.melnyk@univ-lille2.fr
Corresponding author :
Pascal Carato, EA4481, UFR Pharmacie, 3 rue du Pr Laguesse, BP83, 59006 Lille
tel : 33 (0)3 20 96 49 66 – fax : 33 (0)3 20 96 49 13
mail : pascal.carato@univ-lille2.fr
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Abstract
The σ1 proteins are considered to be a new class of target structures for several central
nervous system disorders, including depression, anxiety, psychosis, and Parkinson's and
Alzheimer's diseases. Recently, the involvement of these receptors in neuropathic pain and
cancer has also been observed. So far, only a few ligands are in clinical trials. In a
continuation of our previous studies on the development of σ1 ligands, a new series of
benzannulated heterocycles was designed and synthesised. In vitro competition binding assays
showed that many of them possessed high σ1 receptor affinity (Ki = 0.6 to 10.3 nM), and good
σ2/σ1 subtype selectivity, without cytotoxic effects on SY5Y cells (human neuroblastoma cell
line).
Abbreviations:
DCM, Dichloromethane; DMEM, Dulbecco’s Modified Eagle Medium; DMSO,
Dimethylsulphoxide; PHPLC, Purity determined by HPLC; TLC, thin layer chromatography;
TFA, Trifluoroacetic acid; tR, HPLC retention time
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1. Introduction
Originally proposed as a subtype of opioid receptors [1], σ receptors are now recognised as
a unique protein family. They have a characteristic distribution in the central nervous system,
but they are also widely present in the peripheral organs and tissues such as lung, liver, kidney
and heart [2-4]. Based on ligand selectivity assays, two subtypes have been identified and
designated σ1 and σ2 [5-6]. These subtypes differ in size, anatomical distribution and ligand
selectivity. While the human σ1 receptor has been cloned from various tissues, and is well
characterised at functional and structural level, the σ2 receptor has not been yet cloned from
any species, and is less well known [7].
The σ1 receptors are integral membrane proteins consisting of 223 amino acids with two
transmembrane domains [8]. They primarily reside in the specialised endoplasmic reticulum
(ER) membrane directly apposing mitochondria, the so-called MAM (mitochondrial-
associated endoplasmic reticulum membrane), and modulate Ca2+ efflux from ER by acting as
molecular chaperones of inositol (1,4,5)-triphosphate receptors [9]. However, the localisation
of σ1 receptors is dynamic in nature. Indeed, they are also able to translocate from the MAM
to the plasma membrane [10], where they regulate a variety of functional proteins, including
ion channels, receptors and kinases. It has been shown that the σ1 receptors are involved in the
regulation of numerous neurotransmitter systems such as the cholinergic, dopaminergic and
glutamatergic neurotransmission [11-12]. Although the signal transduction pathway after
activation of σ1 receptors is not completely understood, there is more and more evidence to
suggest that they represent a potential therapeutic target in many diseases. Indeed, since their
discovery, the σ1 receptors have been implicated in various pathologies, including
neurological and psychiatric disorders. Thus, ligands that bind to these receptors have been
proposed to exhibit effects in several therapeutic areas such as mnesic disorders (Alzheimer’s
disease and amnesia) [13], drug addiction [14], depression, anxiety [15], epilepsy, multiple
sclerosis [16], Parkinson’s disease [17], stroke and pain. Recently, σ1 receptors have been
described as inter-organelle signalling modulators, thus being potentially involved in
misfolded protein diseases [18]. Furthermore, the σ1 receptors are overexpressed in tumour
cells, making them a possible target for cancer treatment [19]. Several compounds have
undergone clinical trials, but no selective σ1 receptor ligands have so far been marketed [20].
A phase 2 study to evaluate Anavex 2-73 in patients with Alzheimer’s disease is ongoing
[21]. Moreover, the effects of Igmesine on depressive patients have been studied in phase 3
clinical trials [22].
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Biological and pharmacological investigations have pointed out several classes of
structurally different compounds with high affinity for the σ1 receptors. Based on optimisation
studies, the pharmacophoric model of these ligands has been reported to consist of an amine
binding site flanked on either side by hydrophobic pockets that display bulk tolerance [23]. A
large diversity of hydrophobic moieties have already been described, including essentially
heterocycles such as benzomorphanes, benzofurans [24], benzothiazolinones [25] and
benzoxazolinones [26].
Work in our laboratory has focused on development of selective σ1 receptor ligands with
diverse therapeutic applications. In previous papers we presented the affinity of more
constrained series containing tetrahydroisoquinoline-hydantoin (tic-hydantoin) structure [27-
30]. Compounds 1 and 2 (Figure 1) were identified as efficient σ1 ligands [27-29] with
nanomolar affinity (1(S): Ki = 4.5 nM, 1(R): Ki = 7.1 nM, 2: Ki = 5.3 nM), low affinity for σ2
receptor ligands (1(S): Ki = 496 nM, 1(R): Ki = 1000 nM, 2: Ki = 545 nM), good σ2/σ1
selectivity (1(S): σ2/σ1 = 110, 1(R): σ2/σ1 = 141, 2: σ2/σ1 = 103) and very low cytotoxicity
(CC50 > 100 µM), providing a high selectivity index (CC50/IC50 > 14,000).
Figure 1
These σ1 ligands were evaluated in different pharmacological models. Compound 1(R) was
identified as a potent anti-cocaine agent which is able to increase cocaine-induced locomotor
stimulation and sensitisation [31]. The S-enantiomer of compound 1 brought about a 57%
decrease of infarct volume in an ischaemia model [32]. Finally, when evaluated as
neuroprotective agents, these compounds showed strong anti-inflammatory and
neuroprotective effects in an experimental autoimmune encephalomyelitis model [submitted
for publication]. Although high potency and efficacy in in vivo experiments is a prerequisite
for a candidate drug, the ADME profile is also essential in the perspective of drug
development. Tic-hydantoin derivative 1 showed all properties compatible with development
except metabolic stability. Indeed, despite chemical stability in neutral and acidic media, they
demonstrated a low metabolic stability [31]. The major part of metabolites resulted from tic-
hydantoin instability. However, among all the metabolites of compound 1, demethylated and
debenzylated compounds have been identified (respectively 5% and 9% of all metabolites,
unpublished data). An isoindoline moiety was thus introduced to avoid this metabolic
instability.
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In this paper, we report our efforts to replace the tic-hydantoin core with different
heterocycles. Many benzannulated moieties have already proved their interest in the design of
novel σ1 ligands. Based on the results obtained with benzoxazolinone derivatives [26], we
decided to focus our work on the evaluation of related heterocycles. In the light of previous
results with tic-hydantoin series, both side chains of lead compounds 1 and 2 (i.e.
propylbenzylmethylamine and propylisoindoline) were selected for the study. Moreover, the
presence of chlorine in many known σ1 ligands, such as haloperidol, guided our work on the
introduction of halogens on the heterocyclic moieties.
2. Chemistry
A series of benzannulated derivatives were synthesised and investigated for their affinity
towards σ1 receptors. The heterocycles used were either commercially available, such as 2-
methyl-1H-benzimidazole and 1-methyl-1,3-dihydro-2H-benzimidazol-2-one, or synthesised.
2-benzoxazolinone and 5-bromo-2-benzoxazolinone were synthesised from the corresponding
2-aminophenol derivatives according to the method previously reported by Nachman et al.
[33]. 2-Benzothiazolinone and 5-bromo-2-benzothiazolinone were prepared from the
corresponding 2-nitrobenzenethiol derivatives and triphosgene [34]. An aromatic bromination
reaction of 2-benzoxazolinone or 2-benzothiazolinone was performed in chloroform with
bromine to obtain 6-bromo derivatives [35,36]. Ring closure of 4-bromo-2-aminophenol with
ethyl bromoacetate in the presence of potassium ethoxide gave rise to 6-bromo-1,4-
benzoxazinone [37]. Finally, 6-bromo-1,4-benzothiazinone was synthesised from 1,4-
dibromo-2-nitrobenzene according to a multi-step reaction previously described by Badger et
al. [38].
According to the general procedure shown in scheme 1, a library of 18 novel
benzannulated derivatives was synthesised. Synthesis started from 3-bromo-1-chloropropane,
and reaction with the appropriate amines yielded amino side chains 4-5. The preparation of
compounds 6-7 involved direct nucleophilic substitution using various heterocycles and
potassium carbonate in DMF to obtain our desired derivatives. Heterocycles a-l were all
introduced on N-benzyl-3-chloro-N-methylpropan-1-amine 4. For derivatives 7, only
heterocycles a, c, e, g, i and k were used. The structures of all final compounds were
1
13
determined by H NMR, C NMR and LC-MS analyses. Purity was evaluated under HPLC
conditions and exceeded 96%. Results are presented in the experimental section. For the
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pharmacological evaluation, all final products were converted into their water soluble
hydrochloride salts.
Schema 1
3. Results and Discussion
3.1 Biological evaluations and SAR
Receptor affinities were investigated in competition experiments with radioligands
according to the methods of Ganapathy et al. [39]. In the σ1 assay, the selective ligand [3H]
(+)-pentazocine was employed as the radioligand. Since a σ2 selective radioligand was not
commercially available, the non selective ligand [3H]-DTG was employed for σ2 assay, in the
presence of an excess of non-tritiated (+)-pentazocine, which selectively occupies σ1
receptors. In both assays, Jurkat cell membranes were used as a source of receptors.
The Ki values for σ1 receptors were determined from the corresponding IC50 values for
each compound 6-7. For compounds showing high σ1 affinity, the Ki values for σ2 receptors
and the σ2/σ1 selectivity ratios were also calculated (Table 1).
The tic-hydantoin moiety was replaced with various benzannulated heterocycles such as
benzoxazolinone, benzothiazolinone, benzoxazinone, benzothiazinone, [40] 2-methyl-1H-
benzimidazole or 1-methyl-1,3-dihydro-2H-benzimidazol-2-one, substituted with halogen
atoms (Cl, Br) or unsubstituted. From the results obtained, it appears that the replacement of
the tic-hydantoin core with different heterocycles can strongly affect the σ1 affinity. Indeed,
although a variety of modulations leads to a dramatic loss of affinity compared to the lead
compounds 1 and 2, high affinity for σ1 receptors is maintained, or significantly improved for
some derivatives. The whole structure-activity relationship was described and analysed
afterwards.
With respect to the nature of the heterocycle, the presence of oxygen, nitrogen or sulphur
atoms appeared to influence σ1 affinity, except for the halogenated derivatives. The
benzothiazolinone derivative 6e (Ki = 10.3 nM) displayed an affinity 10-fold higher than the
benzoxazolinone derivative 6a (Ki > 100 nM), the 1-methyl-1,3-dihydro-2H-benzimidazol-2-
one derivative 6k (Ki > 100 nM) or even the 2-methyl-1H-benzimidazole derivative 6l (Ki >
100 nM). Moreover, if the size of the heterocycle was increased from 5 to 6 atoms, the
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presence of an oxygen atom conferred a complete loss of affinity (6i, Ki > 100 nM), whereas
the sulphur analogue 6j maintained good affinity (Ki = 2.2 nM).
Since many known σ1 ligands contain one or two halogens, the introduction of this kind of
atom on our structures should be of interest. For this study, bromine and chlorine were
inserted on the heterocyclic moiety. For the majority of halogenated compounds, a significant
increase in affinity was noticed compared to the non-halogenated analogues. In the case of
halogenated benzothiazolinonic compounds 6e and 6g, the affinity towards σ1 receptors was
increased from 2 to 10-fold. For benzoxazolinone derivatives 6a and 6c, the improvement was
even greater. For these two families of derivatives, whatever the nature of the heterocycle, the
halogenated compounds (6b-d and 6f-g) showed useful σ1 affinity, with a Ki value less than 7
nM. The nature of the halogen appeared to modulate the affinity for σ1 receptors. The
brominated compounds 6b and 6f showed σ1 affinity at least 2-fold better than their
chlorinated analogues 6d and 6h. Conversely, the position of the halogen atom seemed to
have no influence on affinity. Indeed, the 5-bromo derivatives 6b, 6f and the 6-bromo
derivatives 6c, 6g all showed a Ki value around 1-3 nM.
Modification of the amino side chain on the tic-hydantoin family had a beneficial effect on
metabolic stability without any significant decrease in the affinity of the ligands. However, for
the new benzannulated derivatives, replacement of the methylbenzylamine moiety with the
isoindoline moiety caused a dramatic loss of affinity. Indeed, compared to the
methylbenzylamine derivative 6c, which showed a Ki value of 0.9 nM, the isoindoline
analogue 7c displayed a poor affinity for σ1 receptors (Ki > 100 nM). Except for compounds
7e and 7g, which showed good affinity (Ki = 30.7 nM and 5.7 nM, respectively), all
derivatives 7 showed a Ki value above 100 nM. For these two compounds, the presence of the
bromo substituent should be noted.
In addition to the σ1 affinity, the selectivity of this new class of potent σ1 ligands for σ2
receptors was also investigated. The results clearly demonstrate a lower σ2 affinity of the
benzannulated derivatives 6-7, and thus indicate some selectivity for the σ1 over the σ2
subtype. From the results obtained, it seems that the benzothiazoline derivatives 6f, 6g and 6h
show better selectivity, with σ2/σ1 ratios between 35 and 48, than the oxygenated analogues
6b, 6c and 6d. In the case of sulphur heterocycles, the modification of the ring size seems to
have no influence on σ2/σ1 selectivity (compare 6b and 6c with 6j).
Table 1
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3.2 Evaluation of cytotoxic effects
To determine the potential cytotoxic effects of our synthetic derivatives, the human
neuroblastoma cell line SY5Y was treated with the whole compounds at different
concentrations up to 100 µM. The cell viability was calculated using MTT colorimetry. As
shown in Table 1, all benzannulated derivatives exhibited moderate to low cytotoxicity. In
general, benzothiazole derivatives showed higher cytotoxicity than benzoxazole and 2-
methyl-1H-benzimidazole derivatives. Moreover, the isoindoline side chain conferred lower
cytotoxicity than the methylbenzylamine moiety. Seven compounds (6a, 6d, 7a, 7c, 7e, 7i and
7k) resulted in an IC50 value above 100 µM. The other compounds displayed more moderate,
though reasonable, cytotoxicity, with IC50 values between 10 µM and 50 µM. Among these
18 novel benzannulated σ1 ligands, compounds 6b, 6c, 6d and 6g (IC50 = 14.2 µM, 58.8 µM,
100 µM and 13.2 µM, respectively) showed interesting results, with selectivity indices
(IC50(SY5Y)/Ki
ratio) above 10,000. The best compounds, 6c and 6g, respectively showed
(σ1)
selectivity indices of 63,291 and 23,250.
4. Conclusion
Sigma-1 receptors are associated with various neurological and psychiatric disorders, and
the identification of potent ligands for them is of great interest. We synthesised a series of
halogenated and non-halogenated benzannulated derivatives, and evaluated their binding
affinity for σ receptors and their cytotoxic effect on neuronal cells. Several high affinity σ1
ligands with significant selectivity for σ1 versus σ2, and with low toxicity were identified. The
presence of bromine or chlorine in the benzannulated part of our derivatives significantly
improved the affinity for σ1 receptors in most cases. Taking into account that the presence of
an halogen is high importance in this structure, the best result was obtained with 3-[3-(N-
benzyl-N-methylamino)propyl]-6-bromobenzothiazolin-2-one (6g), which displayed a high
affinity for σ1 receptor (Ki = 0.6 nM), a σ2/σ1 ratio of 48, and an SY5Y/σ1 ratio of 23,250.
This work underlines the importance of halogen atoms on the heterocyclic part of the ligands,
which is in agreement with the literature. Further work is in progress to characterise this
compound in several in vivo models of pathologies related to σ1 receptors.
5. Experimental section
5.1 Chemistry
5.1.1 General
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Chemicals and solvents were obtained from commercial sources, and used without further
purification unless otherwise noted. Reactions were monitored by TLC performed on
Macherey-Nagel Alugram® Sil 60/UV254 sheets (thickness 0.2mm). Purification of products
was carried out by either column chromatography or thick layer chromatography. Column
chromatography was carried out on using Macherey-Nagel silica gel (230-400 mesh). Thick
layer chromatography was performed on glass plates coated with Macherey-Nagel Sil/UV254
(thickness 2 mm), from which the pure compounds were extracted with the following solvent
system: DCM/MeOH(NH3), 90:10. NMR spectra were recorded on a Bruker DRX 300
1
spectrometer (operating at 300 MHz for Hand 75 MHz for 13C). Chemical shifts are
expressed in ppm relative to either tetramethylsilane (TMS) or to residual proton signal in
deuterated solvents. Chemical shifts are reported as position (δ in ppm), multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, p = pentet, dd = double doublet, br = broad and m
= multiplet), coupling constant (J in Hz), relative integral and assignment. The attributions of
protons and carbons were achieved by analysis of 2D experiments (COSY, HSQC and
HMBC). Mass spectra were recorded on a Varian triple quadrupole 1200W mass spectrometer
equipped with a non-polar C18 TSK-gel Super ODS (4.6 x 50 mm) column, using
electrospray ionisation and a UV detector (diode array). The purity of final compounds was
verified by two types of high pressure liquid chromatography (HPLC) columns: C18
Interchrom UPTISPHERE and C4 Interchrom UPTISPHERE. Analytical HPLC was
performed on a Shimadzu LC-2010AHT system equipped with a UV detector set at 254 nm
and 215 nm. Compounds were dissolved in 50 µL methanol and 950 µL buffer A, and
injected into the system. The following eluent systems were used: buffer A (H2O/TFA,
100:0.1) and buffer B (CH3CN/H2O/TFA, 80:20:0.1). HPLC retention times (HPLC tR) were
obtained at a flow rate of 0.2 mL/min for 35 min using the following conditions: a gradient
run from 100% of buffer A over1 min, then to 100% of buffer B over the next 30 min.
5.1.5 N-Benzyl-3-chloro-N-methylpropan-1-amine 4 [41]
A 2.0 g (16.9 mmol) amount of N-methylbenzylamine was dissolved in 50 mL DMF. After
addition of potassium carbonate (4.6 g, 33.0 mmol), the resulting mixture was heated at 70°C.
After 30 minutes, the solution was allowed to cool to room temperature. A 4.9 mL (49.5
mmol) amount of 1-bromo-3-chloropropane was added, and the reaction mixture stirred at
room temperature for 24 hours. Then the excess potassium carbonate was filtered off and the
solvent removed under reduced pressure. Purification by column chromatography (DCM) was
performed and enabled collection of the product as a colourless oil (3.2 g, 95%). TLC: Rf 0.4
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(DCM:MeOH(NH3), 99:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.40-7.22 (m, 5H, Haro); 3.65
(t, 3J = 7Hz, 2H, CH2); 3.52 (s, 2H, CH2); 2.55 (t, 3J = 7Hz, 2H, CH2); 2.21 (s, 3H, CH3); 1.98
3
(p, J = 7 Hz, 2H, CH2). LCMS (ESI+): Calc. for [M+H]: 198.09; 200.09. Found: 198.01;
199.96. HPLC (C4, 35 min): tR 8.01 min, PHPLC 96%; HPLC (C18, 35 min): tR 14.69 min,
PHPLC 96%.
5.1.6 N-(3-Chloropropyl)isoindoline 5
A 1.0 g (8.4 mmol) amount of isoindoline was dissolved in 25 mL of acetonitrile. After
addition of potassium carbonate (2.3 g, 16.8 mmol), the resulting mixture was heated at 70°C.
After 30 minutes, the solution was allowed to cool to room temperature. A 4.2 mL (47.2
mmol) amount of 1-bromo-3-chloropropane was added and the reaction mixture stirred at
room temperature for 24 hours. The solvent was removed under reduced pressure and 60 mL
water added to the residue. The product was extracted with 3 x 30 mL dichloromethane. The
combined organic fractions were washed with water and dried over sodium sulphate. The
solvent was evaporated under reduced pressure. Purification by column chromatography
(DCM:MeOH(NH3), 99:1 (v/v)) was performed and enabled collection of the product as a
1
brown liquid (1.27 g, 77%). TLC: Rf 0.7 (DCM:MeOH(NH3), 9:1, v/v). H NMR (300 MHz,
3
3
CDCl3) δ: 7.21 (s, 4H, Haro); 3.95 (s, 4H, 2 CH2); 3.69 (t, J = 7Hz, 2H, CH2); 2.90 (t, J =
3
13
7Hz, 2H, CH2); 2.07 (p, J = 7 Hz, 2H, CH2). C NMR (75 MHz, CDCl3) δ: 140.0 (2 Caro);
126.7 (2 Caro); 122.3 (2 Caro); 59.1 (2 CH2); 53.0 (CH2); 43.1 (CH2); 31.9 (CH2). LCMS
(ESI+): Calc. for [M+H]: 196.05; 198.07. Found: 195.95; 197.91.
5.1.7 General procedure for final compounds
One equivalent of appropriate heterocyclic derivative was dissolved in DMF. Three
equivalents of potassium carbonate and 1.2 equivalent of the appropriate 3-chloropropan-1-
amine derivative were added. The resulting mixture was heated at 70°C until disappearance of
the starting material. The reaction was monitored by TLC. After 24 to 96 hours, the solvent
was removed under reduced pressure, and water added to the residue. The crude product was
extracted with dichloromethane. The combined organic fractions were washed with water and
dried over magnesium sulphate. Purification by thick layer chromatography or column
chromatography was performed.
5.1.7.1 3-[3-(N-Benzyl-N-methylamino)propyl]benzoxazolin-2-one 6a
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The compound was purified by column chromatography (CycloHex:EtOAc, 3:2 (v/v) + 0.1%
MeOH(NH3)), and obtained as a yellow oil (346 mg, 79%). TLC: Rf 0.4 (CycloHex:EtOAc,
1
1:1, v/v). H NMR (300 MHz, CDCl3) δ: 7.40-7.25 (m, 5H, Haro); 7.23-6.98 (m, 4H, Haro);
3
3
3.93 (t, J = 7 Hz, 2H, CH2); 3.51 (s, 2H, CH2); 2.50 (t, J = 7 Hz, 2H, CH2); 2.22 (s, 3H,
CH3); 2.00 (p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 154.6 (CO); 142.7 (Caro);
138.9 (Caro); 131.4 (Caro); 129.0 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 123.8 (Caro); 122.2 (Caro);
110.0 (Caro); 108.4 (Caro); 62.3 (CH2); 54.2 (CH2); 42.1 (CH3); 40.3 (CH2); 25.6 (CH2). LCMS
(ESI+): Calc. for [M+H]: 297.15; Found: 297.00. HPLC (C4, 35 min): tR 9.57 min, PHPLC 99%;
HPLC (C18, 35 min): tR 17.06 min, PHPLC 99%.
5.1.7.2 3-[3-(N-Benzyl-N-methylamino)propyl]-5-bromobenzoxazolin-2-one 6b
The compound was purified by thick layer chromatography (CycloHex:EtOAc, 1:1 (v/v) +
0.1% MeOH(NH3)), and obtained as a brown oil (150 mg, 57%). TLC: Rf 0.4
(CycloHex:EtOAc 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.35-7.19 (m, 6H, H4, Haro); 7.16
3
4
3
3
(dd, J6-7= 9 Hz, J6-4 = 2 Hz, 1H, H6); 6.76 (d, J7-6 = 9 Hz, 1H, H7); 4.08 (t, J = 7 Hz, 2H,
3
3
CH2); 3.55 (s, 2H, CH2); 2.57 (t, J = 7 Hz, 2H, CH2); 2.29 (s, 3H, CH3); 1.96 (p, J = 7 Hz,
2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 153.9 (CO); 146.3 (Caro); 138.9 (Caro); 131.7 (Caro);
129.2 (2 Caro); 128.2 (2 Caro); 127.4 (Caro); 126.7 (Caro); 114.2 (Caro); 113.6 (Caro); 112.2 (Caro);
62.4 (CH2); 53.6 (CH2); 42.4 (CH3); 42.0 (CH2); 26.8 (CH2). LCMS (ESI+): Calc. for [M+H]:
375.04; 377.06. Found: 375.20; 377.20. HPLC (C4, 35 min): tR 13.93 min, PHPLC 96%; HPLC
(C18, 35 min): tR 17.48 min, PHPLC 98%.
5.1.7.3 3-[3-(N-Benzyl-N-methylamino)propyl]-6-bromobenzoxazolin-2-one 6c
The compound was purified by column chromatography (CycloHex:EtOAc, 1:1 (v/v) + 0.1%
MeOH(NH3)), and obtained as a yellow oil (500 mg, 95%). TLC: Rf 0.5 (CycloHex:EtOAc
1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.37-7.17 (m, 7H, H5, H7, Haro); 6.88 (d, 3J = 8 Hz,
1H, H4); 3.89 (t, 3J = 7 Hz, 2H, CH2); 3.48 (s, 2H, CH2); 2.45 (t, 3J = 7 Hz, 2H, CH2); 2.19 (s,
3
3H, CH3); 1.96 (p, J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 154.0 (CO); 143.1
(Caro); 138.9 (Caro); 130.6 (Caro); 128.9 (Caro); 128.4 (2 Caro); 127.1 (2 Caro); 126.7 (Caro); 114.4
(Caro); 113.5 (Caro); 109.4 (Caro); 62.4 (CH2); 54.1 (CH2); 42.1 (CH3); 40.5 (CH2); 25.6 (CH2).
LCMS (ESI+): Calc. for [M+H]: 375.06; 377.06. Found: 375.10; 377.00. HPLC (C4, 35 min):
tR 20.55 min, PHPLC 96%; HPLC (C18, 35 min): tR 20.21 min, PHPLC 96%.
11

ACCEPTED MANUSCRIPT
5.1.7.4 3-[3-(N-Benzyl-N-methylamino)propyl]-5-chlorobenzoxazolin-2-one 6d
The compound was purified by column chromatography (PE:EtOAc, 7:3 (v/v) + 0.1%
MeOH(NH3)), and obtained as a colourless oil (130 mg, 23%). TLC: Rf 0.3 (PE:EtOAc 7:3,
v/v). 1H NMR (300 MHz, CDCl3) δ: 7.37-7.20 (m, 5H, Haro); 7.17-6.98 (m, 3H, Haro); 3.88 (t,
3J = 7 Hz, 2H, CH2); 3.50 (s, 2H, CH2); 2.47 (t, 3J = 7 Hz, 2H, CH2); 2.20 (s, 3H, CH3); 1.97
(p, 3J = 7 Hz, 2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 154.4 (CO); 141.1 (Caro); 138.7 (Caro);
132.4 (Caro); 129.3 (Caro); 129.0 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 122.1 (Caro); 110.8 (Caro);
109.0 (Caro); 62.4 (CH2); 54.0 (CH2); 42.0 (CH3); 40.5 (CH2); 25.5 (CH2).LCMS (ESI+): Calc.
for [M+H]: 331.11; 333.11. Found: 330.94; 331.97. HPLC (C4, 35 min): tR 20.97 min, PHPLC
99%; HPLC (C18, 35 min): tR 19.50 min, PHPLC 99%.
5.1.7.5 3-[3-(N-Benzyl-N-methylamino)propyl]benzothiazolin-2-one 6e
The compound was purified by column chromatography (CycloHex:EtOAc, 3:2, (v/v) + 0.1%
MeOH(NH3)), and obtained as a pale yellow oil (223 mg, 54%). TLC: Rf 0.7
(CycloHex:EtOAc 1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.46-7.10 (m, 9H, H4, H5, H6, H7,
3
3
Haro); 4.03 (t, J = 7 Hz, 2H, CH2); 3.51 (s, 2H, CH2); 2.49 (t, J = 7 Hz, 2H, CH2); 2.32 (s,
3
3H, CH3); 1.95 (p, J = 7 Hz, 2H, CH2).13C NMR (75 MHz, CDCl3) δ: 169.9 (CO); 139.0
(Caro); 137.3 (Caro); 128.9 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 126.3 (Caro); 123.0 (Caro); 122.8
(Caro); 122.6 (Caro); 110.7 (Caro); 62.4 (CH2); 54.4 (CH2); 42.1 (CH3); 41.0 (CH2); 25.6 (CH2).
LCMS (ESI+): Calc. for [M+H]: 313.13. Found: 313.03. HPLC (C4, 35 min): tR 10.27 min,
PHPLC 98%; HPLC (C18, 35 min): tR 18.30 min, PHPLC 99%.
5.1.7.6 3-[3-(N-Benzyl-N-methylamino)propyl]-5-bromobenzothiazolin-2-one 6f
The pure compound was purified by column chromatography (PE:EtOAc, 8:2 (v/v) + 0.1%
MeOH(NH3)), and obtained as a yellowish oil (450 mg, 88%). TLC: Rf 0.6 (CycloHex:EtOAc
1
3
1:1, v/v). H NMR (300 MHz, CDCl3) δ: 7.41-7.20 (m, 8H, H4, H6, H7, Haro); 4.00 (t, J = 7
Hz, 2H, CH2); 3.53 (s, 2H, CH2); 2.48 (t, 3J = 7 Hz, 2H, CH2); 2.22 (s, 3H, CH3); 1.94 (p, 3J =
13
7 Hz, 2H, CH2). C NMR (75 MHz, CDCl3) δ: 169.5 (CO); 138.8 (Caro); 138.5 (Caro); 129.0
(2 Caro); 128.3 (2 Caro); 127.1 (Caro); 125.9 (Caro); 123.7 (Caro); 121.6 (Caro); 119.8 (Caro); 113.9
(Caro); 62.3 (CH2); 54.1 (CH2); 42.1 (CH3); 41.2 (CH2); 25.5 (CH2). LCMS (ESI+): Calc. for
[M+H]: 391.04; 393.04. Found: 391.10; 393.10. HPLC (C4, 35 min): tR 17.59 min, PHPLC
98%; HPLC (C18, 35 min): tR 20.67 min, PHPLC 98%.
12

ACCEPTED MANUSCRIPT
5.1.7.7 3-[3-(N-Benzyl-N-methylamino)propyl]-6-bromobenzothiazolin-2-one 6g
The compound was purified by column chromatography (PE:EtOAc, 8:2 (v/v) + 0.1%
MeOH(NH3)), and obtained as a yellow oil (440 mg, 86%). TLC: Rf 0.7 (CycloHex:EtOAc
1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.54 (d, 4J = 2Hz, 1H, H7); 7.39 (dd, 3J = 8 Hz, 4J =
3
3
2Hz, 1H, H5); 7.37-7.24 (m, 5H, Haro); 7.00 (d, J = 8 Hz, 1H, H4); 4.00 (t, J = 7 Hz, 2H,
3
3
CH2); 3.50 (s, 2H, CH2); 2.47 (t, J = 7 Hz, 2H, CH2); 2.21 (s, 3H, CH3); 1.94 (p, J = 7 Hz,
2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 169.1 (CO); 139.0 (Caro); 136.3 (Caro); 129.3 (Caro);
128.9 (2 Caro); 128.3 (2 Caro); 127.0 (Caro); 125.1 (C7); 124.6 (Caro); 115.3 (Caro); 111.9 (C4);
62.4 (CH2); 54.3 (CH2); 42.1 (CH3); 41.1 (CH2); 25.5 (CH2). LCMS (ESI+): Calc. for [M+H]:
391.04; 393.04. Found: 391.20; 393.20. HPLC (C4, 35 min): tR 21.56 min, PHPLC 97%; HPLC
(C18, 35 min): tR 20.99 min, PHPLC 97%.
5.1.7.8 3-[3-(N-Benzyl-N-methylamino)propyl]-5-chlorobenzothiazolin-2-one 6h
The compound was purified by thick layer chromatography (CycloHex:EtOAc, 1:1 (v/v) +
0.1% MeOH(NH3)), and obtained as a yellow oil (510 mg, 91%). TLC: Rf 0.6
1
(CycloHex:EtOAc 1:1, v/v). H NMR (300 MHz, CDCl3) δ: 7.38-7.22 (m, 6H, H7 and Haro);
4
3
4
3
7.19 (d, J = 2 Hz, 1H, H4); 7.12 (dd, J = 8 Hz, J = 2Hz, 1H, H5); 3.99 (t, J = 7 Hz, 2H,
3
3
CH2); 3.52 (s, 2H, CH2); 2.48 (t, J = 7 Hz, 2H, CH2); 2.22 (s, 3H, CH3); 1.93 (p, J = 7 Hz,
2H, CH2). 13C NMR (75 MHz, CDCl3) δ: 169.8 (CO); 138.7 (Caro); 138.3 (Caro); 132.4 (Caro);
129.0 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 123.4 (Caro); 123.1 (Caro); 121.0 (Caro); 111.2 (Caro);
62.2 (CH2); 54.1 (CH2); 42.1 (CH3); 41.2 (CH2); 25.5 (CH2). LCMS (ESI+): Calc. for [M+H]:
347.09; 349.09. Found: 347.20; 349.20. HPLC (C4, 35 min): tR 20.99 min, PHPLC 96%; HPLC
(C18, 35 min): tR 20.71 min, PHPLC 97%.
5.1.7.9
4-[3-(N-Benzyl-N-methylamino)propyl]-6-bromo-3-oxo-3,4-dihydro-[2H]-1,4-
benzoxazine 6i
The compound was purified by thick layer chromatography (CycloHex:EtOAc, 1:1 (v/v) +
0.1% MeOH(NH3)), and obtained as a colourless oil (65 mg, 62%). TLC: Rf 0.5
1
(CycloHex:EtOAc 1:1, v/v). H NMR (300 MHz, CDCl3) δ: 7.39-7.23 (m, 6H, H5 and Haro);
3
3
3
7.13 (dd, J7-8 = 8 Hz, J7-5 = 2 Hz, 1H, H7); 6.86 (d, J8-7= 8 Hz, 1H, H8); 4.58 (s, 2H, H2);
3.95(t, 3J= 7 Hz, 2H, CH2); 3.59 (s, 2H, CH2); 2.52 (t, 3J= 7 Hz, 2H, CH2); 2.28 (s, 3H, CH3);
3
1.89 (p, J= 7 Hz, 2H, CH2).13C NMR (75 MHz, CDCl3) δ: 163.9 (CO); 144.4 (Caro); 137.9
(Caro); 130.0 (Caro); 129.2 (2 Caro); 128.4 (2 Caro); 127.3 (Caro); 126.4 (C7); 118.5 (Caro); 118.0
13

ACCEPTED MANUSCRIPT
(C8); 115.1 (Caro); 67.5 (CH2); 62.1 (CH2); 54.1 (CH2); 42.0 (CH3); 39.6 (CH2); 24.6 (CH2).
LCMS (ESI+): Calc. for [M+H]: 389.08; 391.08. Found: 388.80; 390.80. HPLC (C4, 35 min):
tR 11.30 min, PHPLC 98%; HPLC (C18, 35 min): tR 20.16 min, PHPLC 97%.
5.1.7.10
4-[3-(N-Benzyl-N-methylamino)propyl]-6-bromo-3-oxo-3,4-dihydro-[2H]-1,4-
benzothiazine 6j
The compound was purified by column chromatography (PE:EtOAc, 8:2 (v/v) + 0.1%
MeOH(NH3)), and obtained as a yellow oil (220 mg, 47%). TLC: Rf 0.5 (PE:EtOAc 7:3, v/v).
4
1H NMR (300 MHz, CDCl3) δ: 7.44 (d, J5-7 = 2 Hz, 1H, H5); 7.36-7.10 (m, 7H, H7, H8 and
Haro); 4.03 (t, 3J = 7Hz, 2H, CH2); 3.50 (s, 2H, CH2); 3.36 (s, 2H, H2); 2.44 (t, 3J = 7 Hz, 2H,
3
13
CH2); 2.21 (s, 3H, CH2); 1.86 (p, J = 7 Hz, 2H, CH2). C NMR (75 MHz, CDCl3) δ: 164.7
(CO); 140.7 (Caro); 139.0 (Caro); 129.5 (Caro); 129.0 (2 Caro); 128.3 (2 Caro); 127.0 (Caro); 126.2
(Caro); 122.9 (Caro); 121.0 (Caro); 120.6 (Caro); 62.3 (CH2); 54.2 (CH2); 43.4 (CH3); 42.2 (CH2);
31.4 (CH2); 25.2 (CH2). LCMS (ESI+): Calc. for [M+H]: 405.05; 407.05. Found: 404.92;
406.92. HPLC (C4, 35 min): tR 21.96 min, PHPLC 98%; HPLC (C18, 35 min): tR 12.75 min,
PHPLC 98%.
5.1.7.11 3-[3-(N-Benzyl-N-methylamino)propyl]-1-methyl-1,3-dihydro-2H-benzimidazol-2-
one 6k
The compound was purified by thick layer chromatography (DCM: MeOH(NH3), 98:2 (v/v)),
and obtained as a pale green oil (54 mg, 22%). TLC: Rf 0.2 DCM:MeOH(NH3), 98:2, v/v). 1H
3
NMR (300 MHz, CDCl3) δ: 7.36-6.95 (m, 9H, H4, H5, H6, H7, Haro); 3.97 (t, J= 7 Hz, 2H,
3
CH2); 3.43 (s, 2H, CH2); 3.53 (s, 3H, CH3); 2.51 (t, J= 7 Hz, 2H, CH2); 2.22 (s, 3H, CH3);
3
13
1.99 (p, J= 7 Hz, 2H, CH2). C NMR (75 MHz, CDCl3) δ: 154.4 (CO); 130.1 (Caro); 129.5
(Caro); 129.1 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 121.2 (Caro); 121.0 (2 Caro); 107.6 (Caro);
107.4 (Caro); 62.2 (CH2); 54.5 (CH2); 42.0 (CH3); 39.2 (CH2); 27.1 (CH3); 26.2 (CH2). LCMS
(ESI+): Calc. for [M+H]: 310.19. Found: 310.09. HPLC (C4, 35 min): tR 9.74 min, PHPLC 99%;
HPLC (C18, 35 min): tR 16.79 min, PHPLC 99%.
5.1.7.12 1-[3-(N-Benzyl-N-methylamino)propyl]-2-methyl-1H-benzimidazole 6l
The compound was purified by column chromatography (DCM:MeOH(NH3), 97:3 (v/v)), and
1
obtained as a colourless oil (283 mg, 64%). TLC: Rf 0.5 (DCM:MeOH(NH3), 9:1, v/v). H
NMR (300 MHz, CDCl3) δ: 7.70 (m, 1H, Haro); 7.38-7.18 (m, 8H, Haro); 4.19 (t, 3J = 7 Hz,
14

ACCEPTED MANUSCRIPT
2H, CH2); 3.51 (s, 2H, CH2); 2.62 (s, 3H, CH3); 2.42 (t, 3J = 7 Hz, 2H, CH2); 2.21 (s, 3H,
13
CH3); 1.98 (p, 3J = 7 Hz, 2H, CH2). C NMR (75 MHz, CDCl3) δ: 151.6 (C2); 142.7 (Caro);
138.8 (Caro); 135.1 (Caro); 128.9 (2 Caro); 128.3 (2 Caro); 127.1 (Caro); 121.9 (Caro); 121.7 (Caro);
119.0 (Caro); 109.2 (Caro); 62.3 (CH2); 54.0 (CH2); 42.1 (CH3); 41.5 (CH2); 27.4 (CH2); 13.9
(CH3). LCMS (ESI+): Calc. for [M+H]: 294.19. Found: 294.10. HPLC (C4, 35 min): tR 15.35
min, PHPLC 99%; HPLC (C18, 35 min): tR 12.34 min, PHPLC 98%.
5.1.7.13 3-[3-(Isoindolin-2-yl)propyl]benzoxazolin-2-one 7a
The compound was purified by column chromatography (CycloHex:EtOAc, 3:2 (v/v) + 0.1%
MeOH(NH3)), and obtained as a beige solid (152 mg, 70%). TLC: Rf 0.5 (CycloHex:EtOAc,
1:1, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.19 (s, 4H, Haro); 7.18-7.08 (m, 4H, Haro); 4.00 (t, 3J
3
3
= 7 Hz, 2H, CH2); 3.96 (s, 4H, 2CH2); 2.82 (t, J = 7 Hz, 2H, CH2); 2.09 (p, J = 7 Hz, 2H,
CH2).13C NMR (75 MHz, CDCl3) δ: 154.6 (CO); 142.7 (Caro); 139.5 (2 Caro); 131.5 (Caro);
126.9 (2 Caro); 123.8 (Caro); 122.3 (3 Caro); 110.0 (Caro); 108.5 (Caro); 58.9 (2 CH2); 52.5
(CH2); 40.0 (CH2); 27.0 (CH2). LCMS (ESI+): Calc. for [M+H]: 295.14. Found: 295.20.
HPLC (C4, 35 min): tR 9.57 min, PHPLC 99%; HPLC (C18, 35 min): tR 16.47 min, PHPLC 99%.
5.1.7.14 3-[3-(Isoindolin-2-yl)propyl]-6-bromobenzoxazolin-2-one 7c
The compound was purified by thick layer chromatography (CycloHex:EtOAc, 3:2 (v/v) +
0.1% MeOH(NH3)), and obtained as a brown oil (340 mg, 65%). TLC: Rf 0.4
1
(CycloHex:EtOAc, 3:2, v/v). H NMR (300 MHz, CDCl3) δ: 7.36-7.26 (m, 2H, H5 and H7);
3
3
7.22 (s, 4H, Haro); 7.00 (d, J = 9 Hz, 1H, H4); 3.98 (t, J = 7 Hz, 2H, CH2); 3.91 (s, 4H,
2CH2); 2.77 (t, 3J = 7 Hz, 2H, CH2); 2.05 (p, 3J = 7 Hz, 2H, CH2).13C NMR (75 MHz, CDCl3)
δ: 154.1 (CO); 143.0 (Caro); 139.8 (2 Caro); 130.8 (Caro); 126.9 (2 Caro); 126.7 (Caro); 123.7
(Caro); 122.3 (2 Caro); 114.5 (Caro); 113.5 (Caro); 109.6 (Caro); 58.8 (2 CH2); 52.2 (CH2); 40.1
(CH2); 26.9 (CH2). LCMS (ESI+): Calc. for [M+H]: 373.04; 375.04. Found: 372.90; 374.90.
HPLC (C4, 35 min): tR 12.24 min, PHPLC 96%; HPLC (C18, 35 min): tR 19.40 min, PHPLC 96%.
5.1.7.15 3-[3-(Isoindolin-2-yl)propyl]benzothiazolin-2-one 7e
The pure compound was purified by thick layer chromatography (CycloHex:EtOAc, 3:2 (v/v)
+ 0.1% MeOH(NH3)), and obtained as a brown oil (162 mg, 52%). TLC: Rf 0.3
(DCM:MeOH(NH3), 95:5, v/v). 1H NMR (300 MHz, CDCl3) δ: 7.36-7.14 (m, 8H, H4, H5, H6,
H7, Haro); 4.13 (t, 3J = 7 Hz, 2H, CH2); 3.97 (s, 4H, 2CH2); 2.82 (t, 3J = 7 Hz, 2H, CH2); 2.05
15

ACCEPTED MANUSCRIPT
3
(p, J = 7 Hz, 2H, CH2).13C NMR (75 MHz, CDCl3) δ: 170.0 (CO); 139.9 (2 Caro); 137.4
(Caro); 126.8 (2 Caro); 126.3 (Caro); 123.0 (Caro); 122.8 (Caro); 122.6 (Caro); 122.3 (2 Caro); 110.8
(Caro); 59.0 (2 CH2); 52.9 (CH2); 40.8 (CH2); 27.1 (CH2). LCMS (ESI+): Calc. for [M+H]:
311.12. Found: 311.10. HPLC (C4, 35 min): tR 10.50 min, PHPLC 97%; HPLC (C18, 35 min): tR
17.98 min, PHPLC 97%.
5.1.7.16 3-[3-(Isoindolin-2-yl)propyl]-6-bromobenzothiazolin-2-one 7g
The compound was purified by thick layer chromatography (CycloHex:EtOAc, 3:2 (v/v) +
0.1% MeOH(NH3)), and obtained as a colourless oil (324 mg, 64%). TLC: Rf 0.5
1
4
(CycloHex:EtOAc 3:2, v/v). H NMR (300 MHz, CDCl3) δ: 7.57 (d, J7-5= 2 Hz, 1H, H7);
3
4
3
7.43 (dd, J5-4= 8 Hz, J5-7 = 2 Hz, 1H, H5); 7.24 (s, 4H, Haro); 7.14 (d, J = 8 Hz, 1H, H4);
4.10 (t, 3J = 7 Hz, 2H, CH2); 3.97 (s, 4H, 2CH2); 2.80 (t, 3J = 7 Hz, 2H, CH2); 2.04 (p, 3J = 7
Hz, CH2).13C NMR (75 MHz, CDCl3) δ: 169.4 (CO); 139.6 (2 Caro); 136.5 (Caro); 129.3 (C5);
126.9 (2 Caro); 125.1 (C7); 124.5 (Caro); 122.3 (2 Caro); 115.4 (Caro); 112.1 (C4); 58.9 (2 CH2);
52.5 (CH2); 40.9 (CH2); 26.9 (CH2). LCMS (ESI+): Calc. for [M+H]: 389.03; 391.03. Found:
389.00; 391.02. HPLC (C4, 35 min): tR 13.36 min, PHPLC 99%; HPLC (C18, 35 min): tR 20.49
min, PHPLC 99%.
5.1.7.17 3-[3-(Isoindolin-2-yl)propyl]-6-bromo-3-oxo-3,4-dihydro-[2H]-1,4-benzoxazine 7i
The compound was purified by column chromatography (CycloHex:EtOAc, 9:1 (v/v) + 0.1%
MeOH(NH3)), and obtained as a yellowish oil (55 mg, 66%). TLC: Rf 0.5 (CycloHex:EtOAc
1
3
3:1, v/v). H NMR (300 MHz, CDCl3) δ: 7.39 (d, J 5-7 = 2 Hz, 1H, H5); 7.22 (s, 4H, Haro);
3
7.10 (dd, J7-8 = 8 Hz, 3J7-5 = 2 Hz, 1H, H7); 6.86 (d, 3J8-7= 8 Hz, 1H, H8); 4.60 (s, 2H, H2);
3
3
3
4.06 (t, J= 7 Hz, 2H, CH2); 3.97 (s, 4H, 2CH2); 2.81 (t, J= 7 Hz, 2H, CH2); 1.96 (p, J= 7
13
Hz, 2H, CH2). C NMR (75 MHz, CDCl3) δ: 163.9 (CO); 144.3 (Caro); 139.7 (2 Caro); 130.2
(Caro); 126.8 (2 Caro); 126.4 (C7); 122.3 (2 Caro); 118.4 (C8); 118.2 (C5); 115.1 (Caro); 65.5
(CH2); 59.0 (2 CH2); 52.8 (CH2); 39.5 (CH2); 26.3 (CH2). LCMS (ESI+): Calc. for [M+H]:
387.07; 389.07. Found: 386.90; 388.90. HPLC (C4, 35 min): tR 10.99 min, PHPLC 97%; HPLC
(C18, 35 min): tR 19.76 min, PHPLC 97%.
5.1.7.18 3-[3-(Isoindolin-2-yl)propyl]-1-methyl-1,3-dihydro-2H-benzimidazol-2-one 7k
The compound was purified by column chromatography (DCM:MeOH(NH3), 97:3 (v/v)), and
obtained as a brown oil (145 mg, 62%). TLC: Rf 0.4 DCM:MeOH(NH3), 95:5, v/v). 1H NMR
16

ACCEPTED MANUSCRIPT
3
(300 MHz, CDCl3) δ: 7.14-6.96 (m, 8H, H4, H5, H6, H7, Haro); 4.05 (t, J= 7 Hz, 2H, CH2);
3
3
3.96 (s, 4H, 2CH2); 3.43 (s, 3H, CH3); 2.81 (t, J= 7 Hz, 2H, CH2); 2.06 (p, J= 7 Hz, 2H,
13
CH2). C NMR (75 MHz, CDCl3) δ: 154.5 (CO); 139.8 (2 Caro); 130.0 (Caro); 129.6 (Caro);
126.8 (2 Caro); 122.3 (2 Caro); 121.2 (Caro); 121.0 (Caro); 107.7 (Caro); 107.4 (Caro); 59.0 (2
CH2); 52.9 (CH2); 39.0 (CH2); 27.7 (CH2); 27.1 (CH3). LCMS (ESI+): Calc. for [M+H]:
308.17. Found: 308.20. HPLC (C4, 35 min): tR 10.03 min, PHPLC 97%; HPLC (C18, 35 min): tR
16.47 min, PHPLC 97%.
5.2 In vitro testing
5.2.1 Assay for binding to σ receptors
The σ binding assays were performed by CEREP (Poitiers, France), according to
Ganapathy et al.[36]. The σ1 binding assay was carried out by incubating Jurkat cell
membranes (10-20 mg protein per tube) with [3H](+)-pentazocine (15 nM) and a range of
concentrations of test compounds, at 37°C for 2 hours, in 5 mM Tris/HCl buffer (pH = 7.4).
The σ2 binding assay was performed by incubating Jurkat cell membranes (10-20 mg protein
per tube) with [3H]-DTG (25 nM) in the presence of (+)-pentazocine (1 µM) to saturate σ1
receptors, and a range of concentrations of test compounds, at room temperature for 1 hour in
5 mM TrisHCl buffer (pH = 7.4). The final assay volume was 0.5 mL. Binding was
terminated by rapid filtration through Whatman GF/B filters, which were then washed with 5
x 1 mL ice-cold NaCl solution and allowed to dry before bound radioactivity was measured
using liquid scintillation counting. Nonspecific binding was determined, in both assays, under
similar conditions, but in the presence of 10 µM unlabelled haloperidol. Inhibition constants
(Ki) were calculated from the IC50 values according to the method of Cheng and Prusoff
(1973).
5.2.2 Cell culture and cytotoxicity assay
The human neuroblastoma cell line (SY5Y) was cultured in DMEM (Dulbecco’s Modified
Eagle Medium) (Gibco) supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100
IU/mL penicillin, 1 mM non-essential amino acids and 10% (v/v) heat-inactivated foetal
bovine serum (Sigma Aldrich), and grown at 37°C in a humidified incubator with 5% CO2.
Cells were seeded at 2,000 cells per well onto 96-well plates in DMEM medium. Cells
were starved for 24 hours to obtain synchronous cultures, and were then incubated in culture
17

ACCEPTED MANUSCRIPT
medium that contained various concentrations of test compounds, each dissolved in less than
0.1% DMSO. After 72 hours of incubation, cell growth was estimated by the colorimetric
MTT (thiazolyl blue tetrazolium bromide) assay.
Acknowledgements
This work was supported by Lille 2 University, FRI “J’innove” PRES Univ Lille Nord de
France Grants. MDM is the recipient of a fellowship from Lille 2 University. The 300 MHz
NMR facilities were funded by the Région Nord-Pas de Calais (France), the Ministère de la
Jeunesse, de l’Education Nationale et de la Recherche (MJENR) and the Fonds Européens de
Développement Régional (FEDER). We express our thanks to Jiaqui He for her contribution
in organic synthesis.
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20

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Table 1: σ1 and σ2 affinity and cytotoxicity evaluation of target compounds 6 and 7
Compound
Ki (nM)
Ki (σ2)/
Ki (σ1)
IC50 (µM)
SY5Y
IC50/
Ki (σ1)
σ1
11.9
4.5
σ2
175
15
110
108
78
-
-
-
Haloperidol
496*
764*
416*
nd
> 100
19.5
-
> 20,000
2,746
1(S)
1(R)
2
7.1
5.3
> 100
1.2
> 100
14.2
58.8
100.0
10.0
26.5
13.2
9.9
-
6a
6b
6c
6d
6e
6f
15.9
34.9
100.5
13
37
16
11,463
0.9
63,291
6.4
15,500
10.3
3.1
973
110.5
27.3
165.4
nd
35
48
35
-
8,417
0.6
23,250
6g
6h
6i
4.7
2,124
> 100
2.2
13.4
18.7
73.8
50.2
> 100
> 100
> 100
31.4
> 100
> 100
-
47.7
nd
22
-
8,631
6j
> 100
> 100
>100
>100
30.3
5.7
-
6k
6l
nd
-
-
nd
-
-
7a
7c
7e
7g
7i
nd
-
-
nd
-
> 3,000
nd
5,471
> 100
> 100
nd
-
-
-
-
nd
7k
Mean IC50 values for 2-3 independent experiments are shown with less than 10% deviation. * Rat cerebral cortex
membranes were used as a source of σ2 receptors. nd: Not determined

ACCEPTED MANUSCRIPT
iii
N
Cl
N
N
i
4
X
6
Cl
Br
X = H, Br, Cl
3
N
N
Cl
N
ii
iii
X
5
7
Heterocycles: 6-7
O
O
O
O
e
f
g
h
X = H
a X = H
X = 5-Br
c X = 6-Br
X = 5-Cl
N
N
N
N
N
i
j
Y = O
Y = S
S
O
N
X = 5-Br
X = 6-Br
X = 5-Cl
N
b
l
k
Y
d
Br
X
X
Scheme 1: Reagents and conditions: i: N-methylbenzylamine (0.34 eq), K2CO3 (0.66 eq), DMF, 70°C
to rt, 24h (95%); ii: isoindoline (0.18 eq), K2CO3 (0.36 eq), CH3CN, 70°C to rt, 24 h (77%); iii: 3-
chloropropan-1-amine derivative 4 or 5 (1.2 eq), various heterocycles (1 eq), K2CO3 (3 eq), DMF,
70°C (23-95%).

ACCEPTED MANUSCRIPT
Figure 1. Structure of compounds 1and 2.
O
O
N
N
N
N
N
N
O
O
2
1(R)
1(S)

ACCEPTED MANUSCRIPT
Highlights:
> A series of sigma-1 ligands was synthesised.
> The affinity for sigma-1 and sigma-2 receptors was determined.
> Most ligands showed nanomolar affinity for sigma-1 receptor.
> A good selectivity towards sigma-2 was obtained.
> A very low cytotoxicity was measured on SY5Y cells.

ACCEPTED MANUSCRIPT
SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF BENZANNULATED
DERIVATIVES AS POTENT AND SELECTIVE SIGMA-1 PROTEIN LIGANDS
Marion Donnier-Maréchala,b, Pascal Caratoa,b,*, Delphine Le Broca,c, Christophe Furmana,c and Patricia
Melnyka,b
aUniv Lille Nord de France, F-59000 Lille, France
bUDSL, EA 4481, UFR Pharmacie, F-59000 Lille, France
cUDSL, EA 4483, UFR Pharmacie, F-59000 Lille, France
Mail adress : UFR Pharmacie, 3 rue du Pr Laguesse, BP83, 59006 Lille
List of e-mail adresses :
marion.donnier-marechal@univ-lille2.fr, pascal.carato@univ-lille2.fr, delphine.lebroc@univ-lille2.fr,
christophe.furman@univ-lille2.fr, patricia.melnyk@univ-lille2.fr
Corresponding author :
Pascal Carato, EA4481, UFR Pharmacie, 3 rue du Pr Laguesse, BP83, 59006 Lille
tel : 33 (0)3 20 96 49 66 – fax : 33 (0)3 20 96 49 13 Mail : pascal.carato@univ-lille2.fr

ACCEPTED MANUSCRIPT
N-Benzyl-3-chloro-N-methylpropan-1-amine 4
N-(3-Chloropropyl)isoindoline 5
3-[3-(N-Benzyl-N-methylamino)propyl]benzoxazolin-2-one 6a

ACCEPTED MANUSCRIPT
3-[3-(N-Benzyl-N-methylamino)propyl]-5-bromobenzoxazolin-2-one 6b

ACCEPTED MANUSCRIPT
3-[3-(N-Benzyl-N-methylamino)propyl]-6-bromobenzoxazolin-2-one 6c
3-[3-(N-Benzyl-N-methylamino)propyl]-5-chlorobenzoxazolin-2-one 6d