Rigid: Versus flexible anilines or anilides confirm the bicyclic ring as the hydrophobic portion for optimal σ2 receptor binding and provide novel tools for the development of future σ2 receptor PET radiotracers

Article abstract of DOI:10.1039/c6ra15783a

Despite their uncertain identification, σ₂ receptors are promising targets for the development of diagnostics and therapeutics for tumor diseases. Among the σ₂ ligands developed, the class of the flexible benzamides furnished an opti

Full text of DOI:10.1039/c6ra15783a

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Rigid versus flexible anilines or anilides confirm the bicyclic ring as
the hydrophobic portion for optimal σ2 receptor binding and provide
novel tools for the development of future σ2 receptor PET radiotracer
Niso Mauro‡, Maria Laura Pati‡, Francesco Berardi‡, Carmen Abate*‡
‡Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ALDO MORO, Via
Orabona 4, I-70125 Bari, Italy
*To whom correspondence should be addressed. Tel.: +39ꢀ080ꢀ5442750. Fax.: +39ꢀ080ꢀ5442231.
Eꢀmail: carmen.abate@uniba.it

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Abstract
Despite their uncertain identification, σ2 receptors are promising targets for the development of
diagnostics and therapeutics for tumor diseases. Among the σ2 ligands developed, the class of the
flexible benzamides furnished an optimal pharmacophore for σ2 receptor high affinity ligands. A
recent investigation suggested that flexible benzamides bind σ2 receptor in a bicyclicꢀlike
conformation due to an intramolecular Hꢀbond, with 3,4ꢀdihydroisoquinolinone derivatives
reaching excellent σ2 affinity and selectivity. Herein, the bicyclicꢀpreferred conformation for σ2
binding was confirmed through the development of 3,4ꢀdihydroquinolinꢀ(1H)2ꢀone isomeric
derivatives, 1,2,3,4ꢀtetrahydroquinolines and the corresponding flexible anilides and anilines, all
linked to the 6,7ꢀdimethoxytetrahydroisoquinoline as basic moiety. 3,4ꢀDihydroisoquinolinꢀ(1H)2ꢀ
one (10a) and 1,2,3,4ꢀtetrahydroisoquinoline (11b) emerged for the high σ2 affinity combined to an
excellent σ2 versus σ1 selectivity. In particular, compound 11b with its low nanomolar σ2 affinity
and impressive 2807ꢀfold σ2 versus σ1 selectivity largely exceeded the biological profile of the best
3,4ꢀdihydroisoquinolinꢀ(2H)1ꢀone reference compounds (1). Because of the absence of a cytotoxic
effect, the modest interaction with the Pꢀgp, an appropriate lipophilicity and the presence of easily
radiolabeling functions, 11b deserves further investigation for the imaging of σ2 receptors via PET
in tumors.

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1. Introduction
Sigma (σ) receptors are intriguing targets for the therapy and diagnosis of cancer and
neurodegenerative diseases. They were first discovered in the 1976, when they were misidentified
as opioid receptors. First designated as ‘σꢀopioid’ receptors,1 they were later recognized as a
distinct class of proteins, and in the early 1990s two distinct sigma receptorꢀtypes were identified,
namely σ1 and σ2. The σ1 subtype has been cloned and recently crystallized and several agents
acting at the σ1 receptors have been developed.2 Its implication in pathologies such as anxiety,
depression, Alzheimer’s disease, juvenile lateral sclerosis has been shown, with role in
neuroprotection and microglia modulation.3ꢀ7 While considerable progress has been made over four
decades, the mechanisms activated by σ1 receptor are complex and deserve further characterization
because of the importance that it is gaining as a target for the treatment of human diseases. Less is
known about the σ2 receptor which has never been cloned. Attempts of identification led to propose
it as a histone protein first, and as a progesteroneꢀreceptor membrane component 1 (PGRMC1)
complex later.8ꢀ10 Recent literatures seem to agree about the identification of the σ2 as the PGRMC1
as they refer to the protein as to PGRMC1/σ2 protein.11ꢀ13 However, a number of controversies are
emerging pointing out a possible misidentification of the σ2 as the PGRMC1.14ꢀ16 Nevertheless,
despite the absence of clear information about the σ2 receptor structure, interest by the scientific
research is increasing because of the important therapeutic or diagnostic potentials that range from
the treatment and diagnosis of different cancers to the recently proposed involvement in
neurodegenerative pathologies such as the Alzheimer’s disease.17,12,13 Development of novel σ2
ligands is a crucial step to further clarify the binding site of the σ2 and finally push towards the
therapeutic and diagnostic exploitation of these receptors. Conformationally flexible benzamides
have provided a successful pharmacophore for the development of high affinity σ2 ligands.18 While
extending the investigation about these structures, we proposed the presence of an intramolecular
Hꢀbond in the binding mode of these molecules to the σ2 binding site, mimicking a bicycle

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structure, which is also common to other σ2 ligands such as the tetraline and the indoleꢀbased
derivatives.19ꢀ21 Support to this hypothesis came from freezing the Hꢀbond into the corresponding
isoquinolinone structures (Figure 1).22,23 Therefore, if the binding of this moleculeꢀtypes to the σ2
receptors prefers the bicyclicꢀlike conformation, then, the inverted amide, i.e. the corresponding
3,4ꢀdihydroquinolinone derivatives would bind σ2 receptors as well. In order to confirm this
hypothesis and produce novel σ2 high affinity and selectivity ligands, we produced 3,4ꢀ
dihydroquinolinꢀ(1H)2ꢀone derivatives and the corresponding 1,2,3,4ꢀtetrahydroquinoline that
according to our hypothesis would bind σ2 receptors equally well. Besides the 3,4ꢀdihydroquinolinꢀ
(1H)2ꢀone and 1,2,3,4ꢀtetrahydroquinoline rings, also the corresponding 5ꢀmethoxy or 6ꢀfluoro
functionalized rings were investigated because the same groups led to the most promising σ2
receptor ligands in the isoquinolinones series, providing convenient functions for Positron Emission
Tomography (PET) tracers development (Compounds 1 and 2, Figure 1). The corresponding
flexible anilides and anilines were also produced to investigate the σ2 receptor binding site and test
how the absence of the Hꢀbondꢀdriven bicyclic structures influences the interaction with the σ2
receptor. In view of a possible application of these agents for diagnostic purposes, we evaluated
their cytotoxicity as well as their interaction with Pꢀglycoprotein (Pꢀgp), since we frequently found
σ2 receptor ligands bearing cyclic basic moieties such as 6,7ꢀdimethoxytetrahydroisoquinoline, to
modulate the Pꢀgp efflux pump.22,24,25
2. Results and discussion
2.1. Chemistry
The synthesis of final compounds is described in Scheme 1 and 2. The already known 3ꢀ
chloropropanamides 3aꢀc26 which were obtained by alkylation of the appropriate anilines were used
to alkylate 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline to afford final compounds 4aꢀc. Reduction
of these amides with LiAlH4 afforded final amines 5aꢀc, which were then acetylated by acetyl
chloride to provide final acetamides 6aꢀc. Reduction of these latter with LiAlH4 provided final

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ethylamines 7aꢀc (Scheme 1). 5ꢀMethoxyꢀ3,4ꢀdihydroquinolinꢀ2(1H)ꢀone (8b) and 6ꢀfluoroꢀ3,4ꢀ
dihydroquinolinꢀ2(1H)ꢀone (8c) were obtained by chromatographic separation from the
corresponding 3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀone derivatives according to the synthesis previously
reported for the latter compounds,23 whereas 3,4ꢀdihydroquinolinꢀ2(1H)ꢀone (8a) was commercially
available. Alkylation of 3,4ꢀdihydroquinolinꢀ2(1H)ꢀone derivatives (8aꢀc) with 1ꢀbromoꢀ3ꢀ
chloropropane using NaH as a base afforded 3ꢀchloropropyl derivatives (9aꢀc) whose reaction with
6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline led to final compounds 10aꢀc (Scheme 2). Reduction
of 10aꢀc with BH3·DMS afforded final 1,2,3,4ꢀtetrahydroquinoline 11aꢀc. All of the final amine
compounds were converted into their hydrochloride salts with gaseous HCl, in anhydrous diethyl
ether except for compound 6b which was crystallized as oxalate salt. Physical properties of the salts
are listed in the Table of Physical Properties of Novel Compounds in the Supporting Information.
2.2. Biology
2.2.1. σ Receptors radioligand binding. Results from binding assays are expressed as inhibition
constants (Ki values) in Table 1 and 2. As expected for most of the compounds carrying the 6,7ꢀ
dimethoxytetrahydrisoquinoline as the basic moiety, the σ1 receptor affinity was low (Ki values
ranging from 144 nM to 4627 nM). Among the different series, the propanamides (4aꢀc) and the
propanamines (5aꢀc) displayed higher σ1 affinities in comparison with the other series such as the
corresponding acetamides (6aꢀc), ethylamines (7aꢀc), 3,4ꢀdihydroquinolinꢀ2(1H)ꢀones (10aꢀc) and
tetrahydroquinolines (11aꢀc) series. The presence of the Fluoro substituent on the benzene ring
consistently determined in all the anilines ꢀ flexible and rigid ꢀ an increase in the σ1 receptor
affinity, with 5c, 7c, and 11c displaying the highest affinities within their series (Ki values = 144
nM, 320 nM, 876 nM, respectively). As for the σ2 affinity, the flexible anilides displayed worse σ2
binding than corresponding anilines (compare 4aꢀc with 5aꢀc and 6aꢀc with 7aꢀc). This could reflect
the partial conjugation of the Nꢀatom lone pair with the benzene ring in the anilines, that determines
a partial rigidification driving a bicyclicꢀlike conformation like in the dihydroquinolines. This is not
the case in the anilide derivatives (4aꢀc and 6aꢀc) in which the delocalization of the Nꢀatom lone

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pair is shared with the amide function. On the other hand and in agreement with these observations,
when the amide is inserted in a rigid bicyclic structure, such as the 3,4ꢀdihydroisoquinolinꢀ(1H)2ꢀ
one derivatives (10aꢀc), excellent σ2 receptor affinity are displayed (Ki values from 7.11 nM to 34.2
nM). The functionalization on the benzene ring did not seem to drive any particular effect in the σ2
binding, except for the propanamides series where the insertion of a 3ꢀmethoxy or 4ꢀfluorine group
determined a 5ꢀfold increase in the σ2 receptor affinity. The highest σ2 receptor affinities were
recorded for anilines 5a (Ki = 3.58 nM) and 5c (Ki = 2.70 nM), for 3,4ꢀdihydroquinolinone 10a (Ki
= 7.11 nM) and tetrahydroquinoline 11b (Ki = 1.42 nM). However, excellent selectivity values
versus the σ1 subtype were reached by the bicyclic derivatives 10a (650ꢀfold) and particularly for
11b (2807ꢀfold), whose affinity and selectivity values were superior to those displayed by reference
3,4ꢀdihydroisoquinolinꢀ(2H)1ꢀone compounds 1 and 2, and in line with more recently developed
and most promising flexible benzamides.18 Moreover, 10a and 11b showed an improved σ2 receptor
biological profile compared to reference flexible benzamide RHMꢀ1 (σ1 receptor Ki = 3078 nM; σ2
receptor Ki = 10.3 nM)27,28 and its fluoroethoxy analogue ISOꢀ1 (σ1 receptor Ki = 330 nM; σ2
receptor Ki = 7.0 nM) which is currently recruiting for three different clinical trials for tumor
imaging.28ꢀ32 For compounds such as 7a, 7c, 11a and 11c, the Ki could not be determined because of
a nonꢀcompetitive binding with the σ2 reference ligand diꢀ1,2ꢀbis(oꢀtolyl)guanidine (DTG) that led
to high percentages of displacement of the radioligand (from 67 % to 80%) at very low
concentrations (10ꢀ11 M). The same binding assay was repeated with cell membranes from MCF7
cells, where σ2 receptors are overexpressed. However, the same results were recorded, indicating a
likely allosteric binding of these compounds with the σ2 receptors.
2.2.2. Calcein-AM assay. Pꢀgp inhibiting activity of the newly synthesized compounds 4aꢀc–7aꢀc,
10aꢀc, 11aꢀc and the reference compound 6,7ꢀdimethoxyꢀ2ꢀ{3ꢀ[4ꢀmethoxyꢀ3,4ꢀdihydroꢀ2Hꢀ
naphthalenꢀ(1E)ꢀylidene]propyl}ꢀ1,2,3,4ꢀtetrahydroisoquinoline (MC18)33 was determined by
fluorescence measurement using CalceinꢀAM probe in MDCKꢀMDR1 cell line as previously

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reported.34 In comparison to recently developed Pꢀgp modulators whose EC50 are in the low
nanomolar range34ꢀ37 all the compounds showed a moderate interaction with the efflux pump, with
4a displaying the weakest interaction (EC50 = 9.51 ꢀM) and 7b the strongest (EC50 = 0.86 ꢀM).
Higher modulating Pꢀgp activity was shown by the ethylamines (7aꢀc) and the bicyclic compounds
10aꢀc and 11aꢀc (EC50 ranging from 1.81 ꢀM to 2.74 ꢀM), without differences conferred by the
substituent on the benzene ring or by the presence of the amide function.
2.2.3. Antiproliferative activity. The antiproliferative activity of all of the novel compounds was
evaluated through the MTT assay in human breast adenocarcinoma MCF7 cells. These cells were
previously deeply investigated and represent a model for the evaluation of the σ2 mediated activity
because of the constitutive overexpression of σ2 receptors.14,38 None of the compounds exerted
antiproliferative activity (EC50 > 100 ꢀM) in accordance with data from the isoquinolinone
derivatives.22,23 The absence of antiproliferative activity as an indication of a low toxicity of these
compounds suggests future investigation of the highest affinity of these novel compounds as
diagnostic tools for the imaging of σ2 receptors in cancer pathologies.
3.1. Chemistry
Both column chromatography and flash column chromatography were performed with 60 Å pore
size silica gel as the stationary phase (1:30 w/w, 63−200 ꢁm particle size, from ICN, and 1:15 w/w,
15−40 ꢁm particle size, from Merck, respectively). Melting points were determined in open
1
capillaries on a Gallenkamp electrothermal apparatus. H NMR (499.801 MHz) and 13C NMR
(125.686 MHz) spectra were recorded on a 500ꢀvnmrs500 Agilent spectrometer. Chemical shift (δ)
1
in parts per million (ppm), and for H NMR multiplicity (s = singlet, d = doublet, t = triplet, m =
multiplet), integration, and coupling constant(s) in hertz were reported. 13C NMR spectra were
performed on representative final compounds in their hydrochloride or oxalate forms. Recording of
mass spectra was done on an Agilent 6890ꢀ5973 MSD gas chromatograph/mass spectrometer, or on
an Agilent 1100 series LCꢀMSD trap system VL mass spectrometer; only significant m/z peaks,

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with their percentage of relative intensity in parentheses, are reported. Chemicals were from
Aldrich, TCI and Alpha Aesar and were used without any further purification.
3.2. General procedure for the synthesis of N-phenyl-3-(6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)-propanamides (4aꢀc).
A solution in CH3CN (25 mL) of one among the intermediate propanamides 3aꢀc (2.5 mmol) was
added with K2CO3 (2.5 mmol, 0.34 g) and 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline (2.5 mmol,
0.48 g). The resulting mixture was stirred under reflux overnight. The solvent was then removed
under reduced pressure and the residue was taken up with water and extracted with ethyl acetate (3
× 20 mL). The collected organic layers were dried (Na2SO4) and evaporated under reduced pressure
to afford a crude oil which was purified by column chromatography (CH2Cl2/CH3OH, 95:5) to
afford the title compounds.
3.2.1. N-phenyl-3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propanamide (4a). Was
1
obtained as as yellow solid (0.51 g, 60% yield), mp = 135ꢀ137 ºC; H NMR (500 MHz, CDCl3) δ
2.63 (t, J = 5.8 Hz, 2H, ArCH2), 2.78ꢀ2.98 (m, 6H, COCH2, N(CH2)2), 3.72 (s, 2H, NCH2Ar), 3.87
(s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.85ꢀ7.38 (m, 7H, aromatic), 11.05 (broad s, 1H, D2O
exchanged); GCꢀMS m/z 339 (M+ ‒ 1, 0.5), 205 (20), 192 (100), 164 (30). Anal.
(C20H24N2O3ꢂHClꢂ0.25H2O) C, H, N.
3.2.2. N-(3-methoxyphenyl)-3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propanamide
(4b). Was obtained as yellow oil (0.62 g, 67% yield); 1H NMR (500 MHz, CDCl3) δ 2.62 (t, J = 5.8
Hz, 2H, ArCH2), 2.78ꢀ2.98 (m, 6H, COCH2, N(CH2)2), 3.70 (s, 3H, OCH3), 3.75 (s, 2H, NCH2Ar),
3.85 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.58ꢀ7.18 (m, 6H, aromatic), 11.18 (broad s, 1H, D2O
exchanged); 13C NMR (125 MHz, CD3OD) δ 27.30, 33.00, 53.04, 55.54, 55.62, 55.80, 56.10,
56.30, 108.31, 109.64, 110.21, 111.41, 116.46, 126.51, 126.83, 129.90, 139.50, 146.70, 148.20,
160.61, 173.70; GCꢀMS m/z 369 (M+ ‒ 1, 0.5), 205 (20), 192 (100), 164 (30). Anal.
(C21H26N2O4ꢂHClꢂ0.75H2O) C, H, N.

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3.2.3.
N-(4-fluorophenyl)-3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propanamide
1
(4c). Was obtained as a white waxy solid (0.61 g, 68% yield), mp = 136ꢀ138 ºC; H NMR (500
MHz, CDCl3) δ 2.55ꢀ2.65 (m, 2H, ArCH2), 2.85ꢀ2.98 (m, 6H, COCH2, N(CH2)2), 3.65 (s, 3H,
OCH3), 3.83 (s, 2H, NCH2Ar), 3.88 (s, 3H, OCH3), 6.58 (s, 1H, aromatic), 6.65 (s, 1H, aromatic),
6.85ꢀ7.38 (m, 4H, aromatic), 11.05 (broad s, 1H, D2O exchanged). Anal.
(C20H23N2O3ꢂHClꢂ0.25H2O) C, H, N.
3.3. General procedure for the synthesis of N-phenyl-3-(6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)propanamines (5aꢀc). A solution of one of the appropriate amides
4aꢀc (2.0 mmol) in the same solvent (10 mL) was added in a dropwise manner to a suspension of
LiAlH4 (4.35 mmol, 0.16 g) in anhydrous THF (20 mL) kept under a stream of N2 and cooled in an
ice bath. After the addition, the mixture was refluxed for 5 h, cooled and the excess of LiAlH4 was
quenched with water. The aqueous layer was extracted with Et2O (2 × 10 mL) and ethyl acetate (20
mL). The collected organic layers were dried (Na2SO4) and evaporated under reduced pressure to
afford a crude oil as a dense brown oil which was either transformed into the corresponding
hydrochloride salt or used for the next step without any further purification.
3.3.1. N-phenyl-3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propanamine (5a). Was
1
obtained as orange oil (0.38 g, 58% yield); H NMR (500 MHz, CDCl3) δ 1.88ꢀ1.95 (m, 2H,
CH2CH2CH2), 2.64ꢀ2.68 (m, 2H, ArCH2), 2.74 (t, 2H, J = 5.8 Hz, NCH2), 2.86 (t, 2H, J = 5.8 Hz,
NCH2), 3.25 (t, 2H, J = 6.3 Hz, NHCH2), 3.58 (s, 2H, NCH2Ar), 3.82 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 6.52ꢀ7.18 (m, 7H, aromatic); GCꢀMS m/z 326 (M+, 8), 192 (100). Anal.
(C20H26N2O2ꢂ2HClꢂ0.75H2O) C, H, N.
3.3.2. N-(3-methoxyphenyl)-3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propanamine
1
(5b). Was obtained as brown oil (0.48 g, 68% yield); H NMR (500 MHz, CDCl3) δ 1.7 (br s, 1H,
NH), 1.70ꢀ1.95 (m, 2H, CH2CH2CH2), 2.60ꢀ2.78 (m, 6H, 2 NCH2 and ArCH2), 3.22 (t, 2H, J = 10.7
Hz, NHCH2), 3.58 (s, 2H, NCH2Ar), 3.73 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.85 (s, 3H, OCH3),
6.18ꢀ7.10 (m, 6H, aromatic); GCꢀMS m/z 356 (M+, 8), 192 (100). Anal.

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(C21H29N2O3·2HCl·0.25H2O) C, H, N.
3.3.3. N-(4-fluorophenyl)-3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propanamine
(5c). Was obtained as brown oil (0.521 g, 75% yield); 1H NMR (500 MHz, CDCl3) δ 1.87ꢀ1.94 (m,
2H, CH2CH2CH2), 2.64ꢀ2.76 (m, 4H, NCH2 and ArCH2), 2.85 (t, 2H, J = 5.8 Hz, NCH2), 3.20 (t,
2H, J = 6.3 Hz, NHCH2), 3.57 (s, 2H, NCH2Ar), 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.47ꢀ6.88
(m, 6H, aromatic); 13C NMR (125 MHz, CD3OD) δ 27.10, 27.35, 41.13, 56.11, 56.20, 56.31,
59.70, 63.40, 108.34, 111.41, 116.30, 118.90, 126.51, 126.90, 143.19, 146.72, 148.21, 155.70; GCꢀ
MS m/z 344 (M+, 8), 192 (100); Anal. (C20H25N2O2F·2HCl·0.25H2O) C, H, N.
3.4. General procedure for the synthesis of N-phenyl-N-[(6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)-propan-3-yl]acetamides (6aꢀc). A solution of one of the
appropriate amines 5aꢀc (2.0 mmol) in CH2Cl2 (20 mL) and Et3N (4.0 mmol, 0.5 mL) was added to
a solution of CH3COCl (2.0 mmol, 0.14 mL) in the same solvent (5 mL) cooled in an ice bath. After
the addition, the mixture was stirred to room temperature overnight. H2O was added to the mixture
and extracted with CH2Cl2 (3 × 10 mL). The collected organic layers, were dried (Na2SO4) and
concentrated under reduced pressure to provide a crude residue which was purified by column
chromatography with CH2Cl2/MeOH (95:5) as eluent.
3.4.1.
N-phenyl-N-[(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-propan-3-yl]acetamide
1
(6a). Was obtained as brown oil (0.23 g, 32% yield); H NMR (500 MHz, CDCl3) δ 1.80ꢀ1.88 (m,
5H, CH2CH2CH2, COCH3), 2.50ꢀ2.54 (m, 2H, ArCH2), 2.64ꢀ2.68 (m, 2H, NCH2), 2.78 (t, 2H, J =
5.87 Hz, NCH2), 3.50 (s, 2H, NCH2Ar), 3.76ꢀ3.80 (m, 2H, CONCH2), 3.82 (s, 3H, OCH3), 3.84 (s,
3H, OCH3), 6.49 (s, 1H, aromatic), 6.58 (s, 1H, aromatic), 7.18ꢀ7.45 (m, 5H, aromatic). GCꢀMS
m/z: 368 (M+, 0.5), 192 (100). Anal. (C22H28N2O3ꢂHClꢂ1.25H2O) C, H, N.
3.4.2.
N-(3-methoxyphenyl)-N-[(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-propan-3-
1
yl]acetamide (6b). Was obtained as brown oil (0.25 g, 31% yield); H NMR (500 MHz, CDCl3) δ
1.80ꢀ1.90 (m, 5H, CH2CH2CH2, COCH3), 2.50ꢀ2.54 (m, 2H, ArCH2), 2.67 (t, 2H, J = 5.87 Hz,
NCH2), 2.78 (t, 2H, J = 5.87 Hz, NCH2), 3.51 (s, 2H, NCH2Ar), 3.77 (t, 2H, J = 7.34 Hz,

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CONCH2), 3.82 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.50 (s, 1H, aromatic),
13
6.58 (s, 1H, aromatic), 6.71ꢀ6.92 (m, 3H, aromatic), 7.32 (t, 1H, J = 8.32 Hz, aromatic). C NMR
(125 MHz, CD3OD) δ 23.10, 24.41, 27.32, 40.18, 55.81, 56.13, 56.22, 56.31, 59.71, 63.45, 108.34,
111.43, 116.45, 119.80, 124.05, 125.42, 126.53, 126.92, 142.72, 146.70, 148.21, 160.83, 168.92;
GCꢀMS m/z: 398 (M+, 0.5), 192 (100). Anal. (C25H30N2O4ꢂ(COOH)2ꢂ0.75H2O) C, H, N.
3.4.3.
N-(4-fluorophenyl)-N-[(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-propan-3-
1
yl]acetamide (6c). Was obtained as brown oil (0.62 g, 80% yield); H NMR (500 MHz, CDCl3) δ
1.92ꢀ2.00 (m, 5H, CH2CH2CH2, COCH3), 2.77ꢀ2.83 (m, 2H, ArCH2), 2.90ꢀ2.94 (m, 2H, NCH2),
2.98ꢀ3.02 (m, 2H, NCH2), 3.74ꢀ3.77 (m, 2H, CONCH2), 3.81 (s, 2H, NCH2Ar), 3.83 (s, 3H, OCH3),
3.85 (s, 3H, OCH3), 6.50 (s, 1H, aromatic), 6.60 (s, 1H, aromatic), 7.10ꢀ7.24 (m, 4H, aromatic).
GCꢀMS m/z: 386 (M+, 0.5), 192 (100). Anal. (C22H27N2O3FꢂHClꢂ0.25H2O) C, H, N.
3.5. General procedure for the synthesis of N-phenyl-N-ethyl-3-(6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl)propanamines (7aꢀc). A solution of one of the appropriate
acetamides 6aꢀc (2 mmol) in anhydrous THF (10 mL) was added in a dropwise manner to a
suspension of LiAlH4 (4.2 mmol, 0.16 g) in the same solvent (10 mL) kept under a stream of N2 and
cooled in an ice bath. After the addition, the mixture was refluxed for 2 h, cooled and the excess of
LiAlH4 was quenched with water. The aqueous layer was extracted with Et2O (2 × 10 mL) and
ethyl acetate (20 mL). The collected organic layers were dried (Na2SO4) and evaporated under
reduced pressure to afford a crude oil which was purified by column chromatography with
CH2Cl2/MeOH (98:2) as eluent.
3.5.1. N-phenyl-N-ethyl-3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propanamine (7a).
1
Was obtained as yellow oil (0.21 g, 30 %); H NMR (500 MHz, CDCl3) δ 1.16 (t, 3H, J = 6.85 Hz,
CH3CH2), 1.90ꢀ1.95 (m, 2H, CH2CH2CH2), 2.50ꢀ2.54 (m, 2H, ArCH2), 2.72 (t, 2H, J = 5.87 Hz,
NCH2), 2.84ꢀ2.88 (m, 2H, NCH2), 3.35ꢀ3.40 (m, 4H, ArN(CH2)2), 3.57 (s, 2H, NCH2Ar), 3.84 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 6.53 (s, 1H, aromatic), 6.62 (s, 1H, aromatic), 6.65 (t, 1H, J = 6.85

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Hz, aromatic), 6.72 (d, 2H, J = 8.80 Hz, aromatic), 7.20ꢀ7.23 (m, 2H, aromatic). GCꢀMS m/z: 354
(M+, 20), 232 (90), 192 (100). Anal. (C22H30N2O2ꢂ2HClꢂ0.75H2O) C, H, N.
3.5.2.
N-(3-methoxyphenyl)-N-ethyl-3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
1
yl)propanamine (7b). Was obtained as orange oil (0.38 g, 50%); H NMR (500 MHz, CDCl3) δ
1.16 (t, 3H, J = 6.85 Hz, CH3CH2), 1.88ꢀ1.95 (m, 2H, CH2CH2CH2), 2.54ꢀ2.60 (m, 2H, NCH2), 2.72
(t, 2H, J = 5.87 Hz, ArCH2), 2.84 (t, 2H, J = 5.87 Hz, NCH2), 3.34ꢀ3.40 (m, 4H, ArN(CH2)2) 3.56
(s, 2H, NCH2Ar), 3.78 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.23 (d, 1H, J =
8.31 Hz, aromatic), 6.27 (s, 1H, aromatic), 6.35 (d, 1H, J = 8.31 Hz, aromatic), 6.53 (s, 1H,
aromatic), 6.62 (s, 1H, aromatic), 7.12 (t, 1H, J = 8.31 Hz, aromatic). 13C NMR (125 MHz,
CD3OD) δ 9.32, 20.39, 24.42, 47.42,48.22, 50.17, 52.36, 52.46, 53.88, 55.05, 55.13, 109.39,
111.21, 113.64, 118.91, 122.76, 131.25, 131.36, 148.49, 149.33, 161.40, 161.45; GCꢀMS m/z: 384
(M+, 15), 232 (90), 192 (100). Anal. (C23H32N2O3ꢂ2HClꢂ1.25H2O) C, H, N.
3.5.3.
N-(4-fluorophenyl)-N-ethyl-3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
1
yl)propanamine (7c). Was obtained as orange oil (0.22 g, 30 %); H NMR (500 MHz, CDCl3) δ
1.13 (t, 3H, J = 6.85 Hz, CH3CH2), 1.84 (m, 2H, CH2CH2CH2), 2.52ꢀ2.55 (m, 2H, NCH2), 2.71 (t,
2H, J = 5.87 Hz, ArN), 2.84 (t, 2H, J = 5.87 Hz, NCH2), 3.30ꢀ3.36 (m, 4H, ArN(CH2)2), 3.55 (s,
2H, NCH2Ar), 3.84 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 6.53 (s, 1H, aromatic), 6.61 (s, 1H,
aromatic), 6.64ꢀ6.68 (m, 2H, aromatic), 6.89ꢀ6.94 (m, 2H, aromatic). GCꢀMS m/z: 372 (M+, 10),
232 (80), 192 (100). Anal. (C22H29N2O2Fꢂ2HClꢂ0.25H2O) C, H, N.
3.6. General procedure for the synthesis of final 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)propyl)-3,4-dihydroquinolin-2(1H)-one compounds (10a-c). To a solution of the
appropriate alkylchloride derivative (2.0 mmol) in CH3CN (20 mL), 6,7ꢀdimethoxyꢀ1,2,3,4ꢀ
tetrahydroisoquinoline hydrochloride (2.0 mmol, 0.46 g) and K2CO3 (3.0 mmol, 0.42 g) were added
and the mixture was refluxed overnight. After cooling to room temperature, the solvent was
evaporated under vacuum and the residue was treated with H2O and extracted with CH2Cl2 (3 × 10
mL). The collected organic layers, were dried (Na2SO4) and concentrated under reduced pressure to

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provide a crude residue which was purified by column chromatography with CH2Cl2/MeOH (95:5)
as eluent.
3.6.1.
2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-3,4-dihydroquinolin-
1
2(1H)-one (10a). Was obtained as waxy oil (0.55 g, 72%); H NMR (300 MHz) δ 1.85ꢀ2.00 (m,
2H, CH2CH2CH2), 2.55ꢀ2.90 [m, 10H, Ar(CH2)2, COCH2, N(CH2)2], 3.55 (s, 2H, NCH2Ar), 3.80 (s,
3H, OCH3), 3.82 (s, 3H, OCH3), 4.02 (t, 2H, J = 7.44 Hz, CONCH2), 6.50 (s, 1H, aromatic), 6.58 (s,
1H, aromatic), 6.62ꢀ7.20 (m, 4H, aromatic); 13C NMR (125 MHz, CD3OD) δ 24.40, 26.62, 27.16,
27.33, 40.41, 56.11, 56.33, 56.41, 59.73,63.40, 108.34, 111.42, 116.90, 124.83, 126.14, 126.56,
126.90, 127.83, 130.94, 138.92, 146.72, 148.22, 169.83; LCꢀMS (ESI+) m/z: 403 [M+Na]+. Anal.
(C23H28N2O3ꢂHCl) C, H, N.
3.6.2.
2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-5-methoxy-3,4-
dihydroquinolin-2(1H)-one (10b). Was obtained as yellow oil (0.57 g, 70%); 1H NMR (300 MHz)
δ 1.90ꢀ2.00 (m, 2H, CH2CH2CH2), 2.55ꢀ2.95 [m, 10H, Ar(CH2)2, COCH2, N(CH2)2], 3.58 (s, 2H,
NCH2Ar), 3.80 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.02 (t, J = 7.42 Hz,
CONCH2), 6.51 (s, 1H, aromatic), 6.59 (s, 1H, aromatic), 6.61ꢀ6.62 (m, 1H, aromatic), 6.74 (d, 1H,
J = 8.25 Hz, aromatic), 7.15 (t, 1H, J = 8.25 Hz, aromatic); GCꢀMS m/z: 410 (M+, 10), 192 (100).
Anal. (C24H30N2O4ꢂHClꢂ1.6H2O) C, H, N.
3.6.3.
2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-6-fluoro-3,4-
dihydroquinolin-2(1H)-one (10c). Was obtained as yellow oil (0.56 g, 70%); 1H NMR (300 MHz)
δ 1.95ꢀ2.05 (m, 2H, CH2CH2CH2), 2.58ꢀ2.95 [m, 10H, Ar(CH2)2, COCH2, N(CH2)2], 3.60 (s, 2H,
NCH2Ar), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.01 (t, J = 7.42 Hz, CONCH2), 6.49 (s, 1H,
aromatic), 6.59 (s, 1H, aromatic), 6.85ꢀ7.05 (m, 3H, aromatic); GCꢀMS m/z: 398 (M+, 0.5), 192
(100). Anal. (C23H27N2O3FꢂHClꢂ0.75H2O) C, H, N.
3.7. General procedure for the synthesis of final 1-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-
2(1H)-yl)propyl))-1,2,3,4-tetrahydroquinolines compounds (11a-c). A solution of one of the
appropriate amides 10aꢀc (2.0 mmol) in anhydrous THF (20 mL) was added with a solution of

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BH3·DMS (10 mmol, 1 mL) kept under a stream of N2 and cooled in an ice bath. After the addition,
the mixture was refluxed for 3 h, cooled and added with MeOH (20 mL). HCl 3N was added and
the mixture was refluxed for 1 h. After that the mixture was made basic with NaOH 3N and the
aqueous layer was extracted with CH2Cl2 (3 × 10 mL). The collected organic layers were dried
(Na2SO4) and evaporated under reduced pressure to afford a crude oil as a dense brown oil which
was purified by column chromatography (CH2Cl2/CH3OH, 95:5) to afford the title compound.
3.7.1.
1-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl))-1,2,3,4-
tetrahydroquinoline (11a). Was obtained as yellow oil (0.68 g, 90%); 1H NMR (500 MHz) δ 1.90ꢀ
2.00 [m, 4H, (CH2CH2CH2)2], 2.55ꢀ2.60 (m, 2H, ArCH2), 2.70ꢀ2.90 [m, 6H, ArCH2, N(CH2)2],
3.28ꢀ3.40 (m, 4H, ArN(CH2)2), 3.55 (s, 2H, NCH2Ar), 3.82 (s, 3H, OCH3), 3.86 (s, 3H, OCH3),
13
6.50ꢀ6.64 (m, 4H, aromatic), 6.92ꢀ7.06 (m, 2H, aromatic); C NMR (125 MHz, CD3OD) δ 16.20,
20.20, 24.31, 24.56, 48.59, 50.20, 52.48, 52.59, 54.30, 55.08, 55.17, 109.45, 111.27, 119.08,
122.08, 122.88, 127.45, 128.66, 130.85, 130.94, 136.31, 148.51, 149.35; GCꢀMS m/z: 366 (M+, 20),
192 (100). Anal. (C23H30N2O2ꢂ2HClꢂ0.25H2O) C, H, N.
3.7.2.
1-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-5-methoxy-1,2,3,4-
tetrahydroquinoline (11b). Was obtained as yellow oil (0.45 g, 55%); 1H NMR (500 MHz) δ 1.86ꢀ
1.98 [m, 4H, (CH2CH2CH2)2], 2.60ꢀ2.70 [m, 4H, N(CH2)2], 2.75ꢀ2.95 [m, 4H, 2 ArCH2], 3.20ꢀ3.25
(m, 2H, ArNCH2), 3.33ꢀ3.38 (m, 2H, ArNCH2), 3.60 (s, 2H, NCH2Ar), 3.78 (s, 3H, OCH3), 3.84 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 6.20ꢀ6.22 (m, 1H, aromatic), 6.33 (d, 1H, J = 8.25 Hz, aromatic),
6.51 (s, 1H, aromatic), 6.60 (s, 1H, aromatic), 6.99 (t, 1H, J = 8.32 Hz, aromatic); 13C NMR (125
MHz, CD3OD) δ 15.60, 18.94, 20.32, 24.55, 48.18, 50.21, 52.53, 52.62, 53.98, 55.05, 55.07, 55.16,
109.44, 111.24, 119.05, 119.24, 122.85, 127.56, 137.19, 148.53, 149.36, 158.23; GCꢀMS m/z: 396
(M+, 22), 192 (100). Anal. (C24H32N2O3ꢂ2HClꢂ0.25H2O) C, H, N.
3.7.3.
1-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-6-fluoro-1,2,3,4-
tetrahydroquinoline (11c). Was obtained as yellow oil (0.53 g, 67%); 1H NMR (500 MHz) δ 1.80ꢀ
1.89 (m, 2H, CH2CH2CH2), 1.90ꢀ1.96 (m, 2H, CH2CH2CH2), 2.52ꢀ2.58 (m, 2H, ArCH2), 2.68ꢀ2.76

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[m, 4H, N(CH2)2], 2.82ꢀ2.86 (m, 2H, ArCH2), 3.22ꢀ3.26 (m, 2H, ArNCH2), 3.28ꢀ3.34 (m, 2H,
ArNCH2), 3.56 (s, 2H, NCH2Ar), 3.83 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 6.51 (s, 1H, aromatic),
6.52ꢀ6.56 (m, 1H, aromatic), 6.60 (s, 1H, aromatic), 6.65ꢀ6.74 (m, 2H, aromatic); 13C NMR (125
MHz, CD3OD) δ 16.43, 20.28, 24.55, 24.84,48.67, 50.21, 52.57, 52.63, 53.78, 55.04, 55.13, 109.40,
111.24, 116.80, 119.03, 122.82, 123.00, 132.95, 133.27, 148.54, 149.39, 160.24, 162.22; GCꢀMS
m/z: 384 (M+, 20), 192 (100). Anal. (C23H29N2O2Fꢂ2HClꢂ0.25H2O) C, H, N.
3.8. Biology
3.8.1. Materials. [3H]ꢀDTG (29 Ci/mmol) and (+)ꢀ[3H]ꢀpentazocine (32 Ci/mmol) were purchased
from PerkinElmer Life Sciences (Zavantem, Belgium). DTG was purchased from Tocris Cookson
Ltd., U.K. (+)ꢀPentazocine was obtained from SigmaꢀAldrichꢀRBI s.r.l. (Milan, Italy). Male Dunkin
guineaꢀpigs and Wistar Hannover rats (250ꢀ300 g) were from Harlan, Italy. Cell culture reagents
were purchased from EuroClone (Milan, Italy). CalceinꢀAM, MTT (3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]ꢀ
2,5ꢀdiphenyltetrazoliumbromide) were obtained from SigmaꢀAldrich (Milan, Italy).
3.8.2. Competition Binding Assays. All the procedures for the binding assays were previously
described. σ1 And σ2 receptor binding were carried out according to Berardi et al.21 The specific
radioligands and tissue sources were respectively: (a) σ1 receptor, (+)ꢀ[3H]ꢀpentazocine, guineaꢀpig
brain membranes without cerebellum; (b) σ2 receptor, [3H]ꢀDTG in the presence of 1 ꢁM (+)ꢀ
pentazocine to mask σ1 receptors, rat liver membranes. The following compounds were used to
define the specific binding reported in parentheses: (a) (+)ꢀpentazocine (73ꢀ87%), (b) DTG (85ꢀ
96%). Concentrations required to inhibit 50% of radioligand specific binding (IC50) were
determined by using six to nine different concentrations of the drug studied in two or three
experiments with samples in duplicate. Scatchard parameters (Kd and Bmax) and apparent inhibition
constants (Ki) values were determined by nonlinear curve fitting, using the Prism, version 3.0,
GraphPad software (1998).39
3.8.3. Cell Culture. MDCKꢀMDR1 cells was a gift of Prof. P. Borst, NKIꢀAVL Institute,
Amsterdam, Nederland. Human MCF7 breast adenocarcinoma cells was purchased from ICLC

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(Genoa, Italy). MCF7 and MDCKꢀMDR1 cells were grown in DMEM high glucose supplemented
with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 ꢀg/mL streptomycin, in a
humidified incubator at 37 °C with a 5 % CO2 atmosphere.
3.8.4. Cell Viability. Determination of cell growth was performed using the MTT assay at 48 h.40
On day 1, 25,000 cells/well were seeded into 96ꢀwell plates in a volume of 100 ꢀl. On day 2, the
various drugs concentration (0.1 ꢀMꢀ100 ꢀM) were added. In all the experiments, the various drugꢀ
solvents (EtOH, DMSO) were added in each control to evaluate a possible solvent cytotoxicity.
After the established incubation time with drugs (48 h), MTT (0.5 mg/ml) was added to each well,
and after 3ꢀ4 h incubation at 37 °C, the supernatant was removed. The formazan crystals were
solubilized using 100 ꢀl of DMSO/EtOH (1:1) and the absorbance values at 570 and 630 nm were
determined on the microplate reader Victor 3 from PerkinElmer Life Sciences.
3.8.5. Calcein-AM experiment. These experiments were carried out as described by Pati et al. with
minor modifications.34 CalceinꢀAM is a profluorescent probe and is a Pꢀgp substrate. In cells
overexpressing Pꢀgp, CalceinꢀAM is not able to permeate cell membrane whereas when the efflux
pump is not present or is inhibited, the probe enters living cells and is converted to fluorescent
Calcein by intracellular esterases. Calcein is not able to diffuse through the membrane since it is
hydrophilic and is not a Pꢀgp substrate; thus, Calcein accumulates in cells when the pump is
blocked. Therefore, the fluorescent signal is directly correlated to the amount of Pꢀgp inhibition.
MDCKꢀMDR1 cell line (50,000 cells per well) was seeded into black CulturePlate 96/wells plate
with 100 ꢁL medium and allowed to become confluent overnight. 100 ꢁL of test compounds were
solubilized in culture medium and added to monolayers. 96/Wells plate was incubated at 37 °C for
30 min. CalceinꢀAM was added in 100 ꢁL of Phosphate Buffered Saline (PBS) to yield a final
concentration of 2.5 ꢁM and plate was incubated for 30 min. Each well was washed 3 times with ice
cold PBS. Saline buffer was added to each well and the plate was read to Victor3 (PerkinElmer) at
excitation and emission wavelengths of 485 nm and 535 nm, respectively. In these experimental
conditions Calcein cell accumulation in the absence and in the presence of tested compounds was

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evaluated and fluorescence basal level was estimated by untreated cells. In treated wells the
increase of fluorescence with respect to basal level was measured. EC50 values were determined by
fitting the fluorescence increase percentage versus log[dose].
4. Conclusions
Herein we produced different series of rigid and flexible anilides and anilines linked to the 6,7ꢀ
dimethoxytetrahydroisoquinoline as basic moiety and we confirmed the preferential binding to σ2
receptors of ligands that contain a bicyclic or a bicyclicꢀlike moiety in their hydrophobic portion.
Except for the flexible amides, all of the novel compounds displayed notable σ2 affinity and
selectivity, with absence of antiproliferative activity and a modest interaction with the Pꢀgp. Two of
these compounds, belonging to the bicyclic series ꢀ the 3,4ꢀdihydroisoquinolinꢀ(1H)2ꢀone (10a) and
1,2,3,4ꢀtetrahydroisoquinoline (11b) ꢀ emerged for the high σ2 affinity combined to an excellent σ2
versus σ1 selectivity. Compound 11b with its low nanomolar σ2 affinity and a 2807ꢀfold σ2 versus
σ1 selectivity, exceeded the results obtained with the best 3,4ꢀdihydroisoquinolinꢀ(2H)1ꢀone
compounds developed so far (1 and 2). σ Binding profile of 11b represents also an improvement
compared to flexible benzamides RHMꢀ1 and ISOꢀ1, the former being successfully radiolabeled
and used to image tumors in preclinical studies and the latter currently under three different clinical
trials. Therefore, 10a and 11b in particular represent powerful tools for the study of these still
enigmatic receptors. In addition, the absence of a cytotoxic effect and the modest interaction with
the Pꢀgp, together with an appropriate lipophilicity (cLogP = 3.94 and cLogD7.4 = 3.23)41 suggest
that 11b should be further investigated to furnish a novel possible radiotracer for the imaging of σ2
receptors via PET in tumors.
Supplemental Information: Synthesis of intermediate compounds 3aꢀc, 9aꢀc; table of elemental
analyses, table of physical properties of novel compounds.

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Scheme 1a
R
H
NH
NH2
N
O
a
b
O
R
R
N
Cl
3a-c
c
a
b
c
: R = H
: R = 3ꢀOCH3
: R = 4ꢀF
4a-c
O
O
O
H
N
N
O
R
5a-c
d
O
O
N
N
O
R
6a-c
c
O
N
N
O
R
7a-c
aReagents: (a) 3ꢀChloropropionyl chloride, Et3N, CH2Cl2;
(b) 6,7ꢀdimethoxyꢀ1,2,3,4ꢀ
tetrahydroisoquinoline, K2CO3, CH3CN; (c) LiAlH4, anhydrous THF; d) CH3COCl, Et3N, CH2Cl2.

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Scheme 2a
Cl
H
N
O
N
O
a
R
R
9a-c
8a-c
a
b
c
: R = H
: R = 3ꢀOCH3
: R = 4ꢀF
b
O
O
N
N
O
R
10a-c
c
O
O
N
N
R
11a-c
aReagents: (a) 1ꢀBromoꢀ3ꢀchloropropane, NaH, DMF;
(b) 6,7ꢀdimethoxyꢀ1,2,3,4ꢀ
tetrahydroisoquinoline, K2CO3, CH3CN; (c) BH3·DMS, anhydrous THF.

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Figure 1.
O
N
Basic Moiety
H
O
R
CH3
Hydrogen bond driving the bicyclicꢀlike conformation
O
O
OCH3
OCH3
OCH3
N
N
N
N
OCH3
F
OCH3
1
2

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Table 1. Biological Data of Novel Anilide and Aniline Derivatives.
Ki ± SEMa (nM)
EC50 ± SEMa (ꢀM)
Pꢀgp MCF7
Comp
R
R’
X
σ1
σ2
278±17 9.51±1.1
H
3ꢀOCH3
4ꢀF
H
H
H
H
CO 397±24
> 100
> 100
> 100
> 100
4a
4b
4c
5a
CO 612±31 56.0±7.7 4.04±0.6
CO 1350±57 50.3±8.3 5.88±0.7
CH2 255±22 3.58±1.5 5.83±0.4
H
3ꢀOCH3
4ꢀF
H
H
CH2 604±31 9.80±2.7 2.75±0.2
CH2 144±17 2.70±1.1 3.70±0.2
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
5b
5c
6a
6b
6c
7a
7b
7c
H
COCH3 CH2
>5000
92.8±2.2 5.52±0.3
3ꢀOCH3 COCH3 CH2 3593±134 40.2±1.9 4.51±0.2
4ꢀF
H
COCH3 CH2 4033±219 83.7±6.9 4.64±0.7
70%b
CH2CH3 CH2 1166±67
1.77±0.3
3ꢀOCH3 CH2CH3 CH2 1070±69 11.3±0.2 0.86±0.1
4ꢀF
CH2CH3 CH2 320±42
3.40±0.7
67%b
1.41±0.2
(+)-pentazocine
DTG
28.5±4.2
1.1±0.3
MC18
b
aValues represent the mean of n ≥ 2 separate experiments in triplicate ± SEM. Percent
displacement at a concentration of 10ꢀ11 M was reported as a complete displacement curve was not
obtained.

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Table 2. Biological Data of Novel 1,2,3,4-Tetrahydroquinoline Derivatives.
Ki ± SEMa (nM)
EC50 ± SEMa (ꢀM)
Pꢀgp MCF7
Comp
10a
10b
10c
R
H
X
σ1
σ2
CO
4627±100
302±29
7.11±1.4 2.74±0.4
18.6±2.6 1.97±0.4
34.2±1.4 2.70±0.1
> 100
> 100
> 100
> 100
> 100
> 100
3ꢀOCH3 CO
4ꢀF
H
CO
3024±156
1462±81
CH2
80%b
1.97±0.2
11a
11b
11c
3ꢀOCH3 CH2
4ꢀF CH2
3987±134 1.42 ± 0.33 2.13±0.1
876±54
73%b
1.81±0.3
3.40±0.7
(+)-pentazocine
DTG
28.5±4.2
1.12±0.3
MC18
b
aValues represent the mean of n ≥ 2 separate experiments in triplicate ± SEM. Percent
displacement at a concentration of 10ꢀ11 M was reported as a complete displacement curve was not
obtained.

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Legend of Figure 1
Figure 1. Hydrogen bond driving the bicyclicꢀlike conformation in flexible benzamides, and 3,4ꢀ
dihydroisoquinolin(2H)1ꢀone reference σ2 ligands.