Novel Sigma Receptor Ligand-Nitric Oxide Photodonors: Molecular Hybrids for Double-Targeted Antiproliferative Effect

Article abstract of DOI:10.1021/acs.jmedchem.7b00791

This contribution reports the synthesis and evaluation of novel hybrid compounds that conjugate a sigma (σ) receptor pharmacophore and a nitric oxide (NO) photodonor. All compounds preserve their capability to generate NO under visible light and possess overall σ receptor nanomolar affinity, with one of them (8b) exhibiting remarkable σ₂ receptor selectivity. Compounds 8b, 11a, and 11b were tested on tumorigenic MCF-7 and A2058 cells expressing high levels of σ₂ and σ₁ receptor, respectively. Considerable loss of cell viability was detected under light excitation, while negligible effects in the dark were detected. Moreover, they did not show any significant cytotoxicity in the dark or under irradiation on nontumorigenic NCTC-2544 keratinocytes. NO-induced reduction of cellular viability was demonstrated by in-cell NO detection and total nitrite estimation. For the first time, a combination of σ receptor moieties and a NO photodonor is reported, providing distinctive ligands potentially useful for cancer management.

Full text of DOI:10.1021/acs.jmedchem.7b00791

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Article
Novel Sigma Receptors Ligands-Nitric Oxide Photodonor:
Molecular Hybrids for Double-Targeted Antiproliferative Effect
Emanuele Amata, Maria Dichiara, Emanuela Arena, Valeria Pittalà,
Venerando Pistarà, Venera Cardile, Adriana Carol Eleonora Graziano,
Aurore Fraix, Agostino Marrazzo, Salvatore Sortino, and Orazio Prezzavento
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00791 • Publication Date (Web): 27 Nov 2017
Downloaded from http://pubs.acs.org on November 29, 2017
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Novel Sigma Receptors LigandsꢀNitric Oxide
Photodonor: Molecular Hybrids for DoubleꢀTargeted
Antiproliferative Effect
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Emanuele Amata, Maria Dichiara, Emanuela Arena, Valeria Pittalà, Venerando Pistarà,
‡
‡
§
†
Venera Cardile, Adriana Carol Eleonora Graziano, Aurore Fraix, Agostino Marrazzo
§
,†
Salvatore Sortino* and Orazio Prezzavento*
†
Department of Drug Sciences, Medicinal Chemistry Section, University of Catania, Viale A.
Doria 6, 95125 Catania, Italy.
§
Department of Drug Sciences, Laboratory of Photochemistry, University of Catania, Viale A.
Doria 6, 95125 Catania, Italy.
#
Department of Drug Sciences, Laboratory of Organic Chemistry, University of Catania, Viale
A. Doria 6, 95125 Catania, Italy.
‡
Department of Biomedical and Biotechnological Sciences, Physiology Section, University of
Catania, Via Santa Sofia 97, 95123 Catania, Italy.
ABSTRACT
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This contribution reports the synthesis and evaluation of novel hybrid compounds that
conjugate a sigma (σ) receptors pharmacophore and a nitric oxide (NO) photodonor. All
compounds preserve their capability to generate NO under visible light and possess overall σ
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receptors nanomolar affinity, with one of them (8b) exhibiting remarkable σ receptor
2
selectivity. Compounds 8b, 11a, and 11b were tested on tumorigenic MCFꢀ7 and A2058 cells
expressing high levels of σ and σ receptor, respectively. Considerable loss of cell viability was
2
1
detected under light excitation while negligible effects in the dark were detected. Moreover, they
did not show any significant cytotoxicity in the dark or under irradiation on nonꢀtumorigenic
NCTCꢀ2544 keratinocytes. NOꢀinduced reduction of cellular viability was demonstrated by in
cell NO detection and total nitrite estimation. For the first time, a combination of σ receptors
moieties and a NO photodonor is reported, providing distinctive ligands potentially useful for
cancer management.
INTRODUCTION
One of the main drawbacks of many conventional anticancer drugs is the induction of
1
,2
significant toxicity to healthy cells. To minimize this toxicity issue, a commonly applied
strategy is the selective cellular targeting. This is achieved by combining the therapeutic agent
with components that specifically bind to biological targets significantly overexpressed in cancer
3
cells. Most of the current research on cellular targeting, is based on the use of antibodies,
although, this approach does not lead often to effective intracellular delivery. In contrast, the use
of small molecular units as targeting ligands significantly enhances the intracellular drug
4ꢀ6
delivery. Another and still poorly investigated form of cellular targeting is based on the
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production of cytotoxic species upon light activation. This strategy exploits suitable
photochemical precursors that generate cytotoxic agents under the exclusive excitation with
biocompatible visible light. This approach does not require, in principle, the selective
accumulation of the photoprecursor at the tumor site but only the lack of toxic effects in the dark.
Indeed, high selectivity can be achieved by restricting the application of the incident light to the
tumor area, and therein confining the release of the cytotoxic species therein. In this context,
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photodynamic therapy (PDT) is the most used treatment that has found a clinical application, and
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singlet oxygen ( O ) is accepted to be the foremost mediator of cytotoxic reactions in cells under
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,9
PDT treatments. Another type of photoactivated therapy is based on the photocontrolled
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release of nitric oxide (NO) by using suitable NO photodonors (NOPDs).
Although still
confined to the research environment, NOꢀbased therapy holds very promising features helpful in
1
3,14
cancer treatment.
Besides playing multiple roles in the bioregulation of a broad array of
1
5
physiological processes, NO has also proven to be an effective anticancer agent acting through
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different mechanisms. However, the role of NO in tumour biology is quite intricate, as this
inorganic free radical can act as a tumour progressor or suppressor depending on concentration,
1
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duration of exposure, and cells sensitivity to NO. For example, ꢁM range concentrations of NO
1
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result in antitumor effects, while pM–nM promote cytoprotective effects.
The development of NOPDs has become of interest due to the superb spatiotemporal control
that lightꢀtriggering offers if compared to thermal, pH, and metabolic stimuli. Analogously to
1
O , NO presents many advantages over conventional chemotherapeutics, i.e. multitarget
2
activity, absence of multidrug resistance problems. Moreover, due to its short lifetime, NO
region of action is confined to a short distance from the production site inside the cells (< 200
µm) reducing systemic toxicity effects common to many conventional anticancer drugs.
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Differently to O photogeneration, NO release is independent of O availability. Therefore, NOꢀ
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based anticancer therapy can successfully complement PDT mainly where this latter may fail, as
under the hypoxic conditions typical of some tumours.
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Based on the above considerations, the development of molecular conjugates in which a
phototherapeutic agent is covalently joined to specific targeting ligands can give intriguing
candidates for “doubleꢀtargeted” cancer therapy. While this approach has already been proposed
for PDT agents, only a limited number of examples is known to date regarding targeted
18ꢀ24
NOPDs.
Sigma (σ) receptors have been shown to be overexpressed in cancer cells and can be useful for
2
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cancer cells targeted drug delivery.
The σ receptors, firstly introduced as subtypes of the
opioid receptors, are now considered a unique class of proteins classified into two subtypes,
sigmaꢀ1 (σ ) and sigmaꢀ2 (σ ) with peculiar structure, biological functions, and ligands
1
2
sensitivity. Indeed, σ binding sites display high affinity for dextro benzomorphan enantiomers
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like (+)ꢀpentazocine, while levo isomers bind to opioid receptors.
The σ receptor has been purified, cloned and crystallized and is well characterized as a
1
chaperone protein at the endoplasmic reticulum (ER)ꢀmitochondria interface where regulates
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+
30ꢀ33
Ca signaling and cell survival.
The σ
1
receptor agonists show neuroprotective, antiꢀ
amnestic, and antidepressant effects, while σ receptor antagonists possess modulatory effects on
1
3
4ꢀ40
opioid analgesia, as well as antiproliferative, and antiangiogenic activities.
The σ receptor is
1
highly expressed in lung, breast, and prostate cancer cell lines whereas low levels are found in
4
1,42
normal counterpart cells.
The σ receptor was purified, revealing its identity as the transmembrane protein 97
2
(
TMEM97), an endoplasmic reticulumꢀresident transmembrane protein that regulates the sterol
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transporter Niemann–Pick disease protein (NPC1). Some quantitative structureꢀactivity
relationship (QSAR) models for the determination of the σ receptor binding affinity were
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setup.
The σ receptor is found in lungs, liver, and kidney, and it is highly expressed in
2
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several malignant tumors including melanoma, neuroblastoma, glioblastoma, breast, and lung
48ꢀ50
cancer.
Generally, the density of the σ receptor in tumor cell lines is higher than that of the
2
σ1 receptor, having a tenꢀfold higher density in proliferating tumor cells than in quiescent tumor
4
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cells.
This evidence indicates that the σ receptor is also an important clinical biomarker for
2
determining the proliferative status of solid tumors. The σ receptors overexpression in tumor
cells has led to the development of fluorescent and radiolabeled σ receptor ligands as diagnostic
imaging tools using positron emission tomography (PET) or single photon emission computed
18
tomography (SPECT). [ F]ISOꢀ1, a promising PET ligand targeting σ receptor, has been
2
evaluated in clinical trial for the assessment of cellular proliferation in tumors by PET and
5
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additional phase I clinical trials on this compound are ongoing.
Moreover, this
overexpression boosted the development of σ receptor ligands derivatized nanocarriers for
41,49,50,55ꢀ59
targeted anticancer drug delivery.
On these grounds and motivated by our ongoing interest in developing multifunctional
1
0,60,61
molecular conjugates and nanomaterials photodelivering NO,
in this paper we report the
design, synthesis, characterization, and biological evaluation of a new class of molecular hybrids.
These latter compounds combine the capability to bind to σ receptors, allowing a preferential
accumulation within cancer cells, and the photoregulated release of NO upon visible light
excitation.
RESULTS AND DISCUSSION
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Design and synthesis. A crucial step when photoresponsive molecular units, such as NOPDs,
need to be combined with other functional components, i.e. targeting ligands, is the preservation
of the photochemical properties of the photoactive center. This is not a trivial result since the
response to light of the photoactive unit can be considerably influenced in nature, efficiency or
both, by the occurrence of competitive photoprocesses (i.e., photoinduced energy and/or electron
transfer, nonꢀradiative deactivation, etc.), occurring upon light absorption. With this in mind, we
have designed a series of molecular hybrids in which a nitroaniline derivative NOPD developed
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in our group, and extensively studied
possess the structural determinants for σ receptors recognition.
nitroꢀ3ꢀtrifluoromethyl aniline has been combined with an appropriate σ amino moieties, namely
ꢀbenzylpiperidine or 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline, separated by two to five
has been covalently joined to molecules that overall
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In particular, the NOPD 4ꢀ
4
methylene unit spacers (compounds 8–11, Figure 1).
Figure 1. General structure of compounds 8‒11
NO Photodonor
Cpd
N
n
Amino moiety
hv
Spacer
8
11a
11b
1 4
1 4
NO2
N
N
NO
O
O
N
Tumor cells
death
8
NH
CF3
n
Sigma receptors recognition
structural determinants
Compounds 8−11 were synthesized according to the steps illustrated in Scheme 1. Starting
from commercially available 4ꢀfluoroꢀ1ꢀnitroꢀ2ꢀ(trifluoromethyl)benzene, intermediates 1 and 2
were obtained with 2ꢀaminoethanol or 5ꢀaminopentanꢀ1ꢀol and then converted into the
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corresponding mesyl derivatives by reaction with mesyl chloride. The mesylated intermediates
underwent
benzylpiperidine to give the final compounds 8b and 11a‒b. Compound 9b was prepared from
ꢀnitroꢀ3ꢀ(trifluoromethyl)aniline via acylation with 3ꢀchloropropionylꢀchloride. The resulting
condensation
with
6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline
or
4ꢀ
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chlorine intermediate 3 was coupled with 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline to obtain
the amide 4b, which in turn was reduced to the resulting amine 9b using LiAlH4.
a
Scheme 1. Synthesis of compounds 8‒11.
a
Reagents and conditions: (i) Y=NH , ClCH CH COCl, Et N, CH Cl , 0 °C to rt, overnight;
2
2
2
3
2
2
(
ii) 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline, NaHCO , DMF, 70 °C, overnight; (iii)
3
LiAlH , THF, N , rt, overnight; (iv) Y=F, appropriate amino alcohol, Et N, EtOH, 100 °C, 30
4
2
3
min; (v) 1) mesyl chloride, Et N, CH Cl , 0 °C to rt, N , 2) appropriate amine, Et N, CH Cl , 0
3
2
2
2
3
2
2
°
C to rt, N ; (vi) BrCH CN or BrCH CH CN or BrCH CH CH CN, NaI, K CO , DMF, 60 °C,
2 2 2 2 2 2 2 2 3
overnight; (vii) 1) LiAlH , THF, rt, N , 2) 4ꢀfluoroꢀ1ꢀnitroꢀ2ꢀ(trifluoromethyl)benzene, Et N,
4
2
3
ACN, 60 °C, overnight.
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Conversely, amines 8a, 9a and 10a‒b were obtained via alkylation of 4ꢀbenzylpiperidine or
6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline with 2ꢀbromoacetonitrile or 3ꢀbromopropanenitrile
or 4ꢀbromobutanenitrile to give the corresponding nitrile derivatives 5a, 6a, and 7a‒b.The latter
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compounds were reduced with LiAlH to give the corresponding diamines and condensed with
4
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ꢀfluoroꢀ1ꢀnitroꢀ2ꢀ(trifluoromethyl)benzene through nucleophilic aromatic substitution to obtain
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the desired products 8a, 9a, and 10a‒b.
We then investigated how the chemical structure of the amino moieties and their distance from
the NO photodonor region influenced the σ receptors affinity and their ability in releasing NO.
Radioligand binding assay. The synthesized compounds bearing a 4ꢀbenzylpiperidine
(8a−11a) or a 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline moiety (8b−11b), were evaluated
for affinity at both σ and σ receptors through radioligand binding assay (Table 1).
1
2
Table 1. σ and σ , binding assays of compounds 8‒11
1
2
a
K (nM) ±SD
i
Compound
8a
n
1
2
3
4
1
2
σ1
σ2
σ1/σ2
0.53
1.87
2.38
1.68
40.7 ± 0.95
41.6 ± 0.84
20.0 ± 1.10
40.4 ± 0.81
3,916 ± 205
212.4 ± 17
76.1 ± 1.2
22.3 ± 1.0
8.4 ± 0.80
24 ± 0.85
23.8 ± 1.23
16.2 ± 0.76
9
1
1
8
9
a
0a
1a
b
164.5
13.11
b
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ꢀ
37.8 ± 0.91
39.4 ± 1.10
2.5 ± 0.40
23.0 ± 0.65
25.5 ± 0.78
16.0 ± 1.60
1.64
1.55
0.16
Haloperidol
a
Each value is the mean ± SD of three determinations.
Within the 4ꢀbenzylpiperidine series (8a−11a), all compounds have shown double digits
nanomolar affinity toward both receptor subtypes with K σ in the 20–40 nM range, and K σ in
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i 2
the 8–76 nM range. Compound 10a demonstrated particular high affinity for both receptor
subtypes (K σ = 20.0 nM and K σ = 8.4 nM). The spacer elongation did not alter significantly
i
1
i 2
the binding affinity or selectivity. Compounds bearing the 6,7ꢀdimethoxyꢀ1,2,3,4ꢀ
tetrahydroisoquinoline (8b−11b) moiety showed a preference for the σ receptor. Indeed,
2
compound 8b demonstrated high affinity and selectivity for this receptor subtype with K σ =
i
1
3,916 nM and K σ = 23.8 nM, having a σ /σ selectivity ratio of 164 fold. The spacer elongation
i
2
1
2
in this series brought to an improvement of the σ receptor affinity while keeping the σ receptor
1
2
affinity almost unchanged. Indeed, compounds 8b–9b with two or three methylene chain,
respectively, showed low affinity for the σ receptor (K σ = 3,916 and 212 nM, respectively),
1
i 1
and desirable σ receptor affinity (K σ = 23.8 and 16.2 nM, respectively), while compounds
2
i 2
1
0b–11b with longer spacer, reported a double digits nanomolar K against both receptor
i
subtypes (10b K σ = 37.8 nM and K σ = 23 nM; 11b K σ = 39.4 nM and K σ = 25.5 nM).
i
1
i
2
i
1
i 2
Affinity at σ receptor has been found in the 16.2–25.5 nM range, thus a clear spacer length
2
effect on σ receptor was not established for this series.
2
Overall within the designed compounds, the 4ꢀnitroꢀ3ꢀ(trifluoromethyl)aniline moiety is well
tolerated with regard to σ receptors binding affinity. The series bearing the 4ꢀbenzylpiperidine
moiety has demonstrated higher affinity against both receptor subtypes. While in the 6,7ꢀ
dimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline series a preference for the σ receptor was observed in
2
those compounds with a shorter spacer. Haloperidol, a typical σ receptors ligand used as
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1
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1
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2
2
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2
2
2
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5
6
reference compounds, has reported K values at both σ receptor subtypes, in accordance with
i
previously published data. With exception of compound 8a (K σ = 76.1 nM), all derivatives
i
2
have shown σ receptor affinity (8.4–25.5 nM range) comparable to that of haloperidol (K σ =
2
i 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
6.0 nM).
Spectroscopic and photochemical properties. All compounds synthesized are well soluble in
a water:methanol (1:1) mixture. As expected, the nitroaniline chromogenic unit strongly
dominates the absorption in the visible region. The molar absorptivity at the λmax for all
−1
−1
molecular hybrids is ca. 10.000 M cm . This value is similar to those already reported for the
NO photocage alone ruling out any significant interaction, in the ground state, between the
nitroaniline chromogenic unit and the targeting molecular components.
Figure 2 shows the absorption spectra for compounds 8b and 11a chosen as representative for
6
2,74
the two different targeting motifs having a short and a long spacer, respectively.
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1
2000
0000
1
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8
9
0
1
1a
8
000
8
b
6000
4000
2
000
0
250
300
350
400
450
500
λ
(nm)
Figure 2. Absorption spectra of 8b and 11a in water:methanol (1:1) solution.
As outlined in the design section (vide supra), the NOPD photoreleasing capability need to be
preserved after its covalent conjugation in order to exploit the NOꢀinduced cytotoxic effects. The
most convenient methodology to demonstrate the NO release from the molecular conjugates
under visible light irradiation is the direct and realꢀtime monitoring of released NO amount. To
this end, we have employed an ultrasensitive NO electrode, which directly detects NO with nM
concentration sensitivity by an amperometric technique. In Figure 3 a representative amperogram
obtained for compound 11b is reported. The amperometric measurement gives an unambiguous
evidence that the compound is stable in the dark but supplies NO upon illumination with visible
light. It should be noted that the release process is strictly regulated by the external light inputs as
confirmed by the linear NO generation, which promptly stops as the light turns off and restarts as
the illumination turns on again.
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Dark
Dark
1200
Light
1000
Dark
0
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9
0
Light
800
600
400
200
0
Light
0
200
400
600
800
1000
1200
1400
1600
Time (s)
Figure 3. NO release profiles observed upon visible light irradiation (λexc = 405 nm) of a
water:methanol (1:1) solution of 11b (50 µM).
Of interest is that the absorption spectral changes of 11b as function of the irradiation time show
a photobleaching of the visible band without any significant shift in the absorption maximum
(
Figure 4). This spectral behavior is in excellent agreement with the photochemical pathway
leading to the NO release previously proposed for the single NO photodonor unit and rules out
the occurrence of any competitive reaction.
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.7
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9
0
0
.5
0 min
0.4
0
0
0
0
.3
.2
.1
.0
2
50
300
350
400
450
500
λ
(nm)
Figure 4. Absorption spectral changes observed upon visible light irradiation (λexc = 405 nm) of
a water:methanol (1:1) solution of 11b (50 µM) from 0 to 85 min.
All compounds showed a good capability to release NO under photoexcitation, with quantum
yields (Φ ) ranging from ca. 0.01 to 0.03 in the solvent mixture used (Table 2). In particular,
NO
while no significant differences were noted in the 4ꢀbenzylpiperidine series (8a−11a), the NO
photorelease quantum yields was dependent on the length of the spacer in the 6,7ꢀdimethoxyꢀ
1
,2,3,4ꢀtetrahydroisoquinoline series (8b−11b). This behavior can be tentatively attributed to
potential quenching effect by photoinduced electron transfer involving the
tetrahydroisoquinoline (as an electron donor) and the NO photoreleasing moiety (as an electron
acceptor) competitive with the photorelease of NO and taking place more effectively in the
compounds with shorter spacers.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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5
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5
5
5
5
5
6
Table 2. NO photorelease quantum yields (Φ ) for compounds 8−11
NO
Compound
8a
ΦNO
0.031
9
1
1
8
9
a
0.025
0.033
0.035
0.010
0.017
0.027
0.034
0
1
2
3
4
5
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8
9
0
1
2
3
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8
9
0
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8
9
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8
9
0
0a
1a
b
b
10b
1b
1
Cell viability assay
Considering that targeting therapeutic agents should be useful in reducing cytotoxicity in
healthy cells, a selected group of molecular hybrids was tested in in vitro studies. Tumorigenic
and nonꢀtumorigenic cell lines were used to evaluate the compounds putative function as cell
death inducers through the photorelease of the cytotoxic concentration of NO. Specifically, given
the overexpression of the σ receptor in rapidly proliferating cells compared to quiescent cells,
2
the different concentrations of the representative compounds (8b, 11a, and 11b) were evaluated
in MCFꢀ7 breast adenocarcinoma cell line expressing high levels of σ receptor and very low or
2
7
5
even no σ receptor expression. They were also tested in A2058 melanoma cell line, which, in
1
contrast, expresses the σ receptor. Finally, biological tests were also performed on nonꢀ
1
7
6
tumorigenic skin keratinocytes NCTCꢀ2544 cells. Compound 8b was selected because of its
remarkable affinity and selectivity towards the σ receptor, whereas compounds 11a and 11b
2
were selected because of their good affinities for both σ and σ receptors combined with their
1
2
good NO photoreleasing capabilities.
The first step was the determination of the toxic concentrations of tested compounds against
selected cell lines. For this purpose, all cell lines were incubated in the dark, in the presence of
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doxorubicin (12), 8b, 11a, or 11b for 24 hours (Table 3). The antineoplastic drug 12 was
evaluated in order to define cells sensitivity toward death induction. In all cell lines, and in a
concentrationꢀdependent manner, compound 12 demonstrated a significant reduction of viability
confirming that it does not well discriminate between normal and tumor cells. Compound 12
exhibited EC values of 15.3, 6.5, and 32.5 µM in MCFꢀ7, A2058, and NCTCꢀ2544 cell lines,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
respectively.
Table 3. MTT viability test on MCFꢀ7, A2058, and NCTCꢀ2544: EC in the dark at 24 hours
5
0
for compounds 12, 8b, 11a, and 11b.
a
b
EC (µM) (pEC ±SE)
50
50
Cell line
MCFꢀ7
A2058
12
8b
11a
11b
c
c
15.3 (4.81 ± 0.15)
6.5 (5.19 ± 0.34)
32.5 (4.49 ± 0.11)
>100 (<4)
14.9 (4.83 ± 0.05)
14.2 (4.85 ± 0.05)
15.0 (4.82 ± 0.03)
>100 (<4)
c
c
>100 (<4)
>100 (<4)
c
c
NCTCꢀ2544
>100 (<4)
>100 (<4)
a
EC50 values have been calculated with GraphPad Prism 5 for Windows using a nonlinear fit transform sigmoidal
doseꢀresponse (variable slope). Each value represents the mean of quadruplicate samplings from 96ꢀwell microplates
±
SEM. Cytotoxicity was measured by the MTT conversion into formazan crystals and expressed as the percentage
of viable cell respect to each of untreated controls. Cells were incubated for 24 h in the dark, in the presence of
b
c
various concentrations of tested compounds; pEC is defined as the –log(EC ); cell viability reduction lower than
50
50
5
0% at 100 µM.
Compounds 8b and 11b have been found considerably safe in all three cell lines evaluated,
having shown EC values >100 µM (inhibition lower than 50% at 100 µM). This trend proves
5
0
that these compounds are not toxic for both normal and tumor cells at the concentrations
evaluated. Conversely, compound 11a induced a comparable loss of viability in all three cell
lines with EC50 of 14.9, 14.2, and 15.0 µM in MCFꢀ7, A2058, and NCTCꢀ2544, respectively.
According to these results, compound 11a seems to act as cytotoxic in both normal and tumor
cells. This result could be explained by alternative targets involved in the accumulation and/or
activity of compound 11a in the nonꢀtumorigenic NCTCꢀ2544 cell line.
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In a second set of experiments, these compounds were evaluated under light irradiation to test
their efficacy as NO donors. Different concentrations of tested compounds were applied to cell
lines for 24 hours. A potentiation of cytotoxicity was observed when subtoxic concentrations of
compound 8b were combined with light exposure in MCFꢀ7 cell line (Figure 5). At the three
tested concentration (10, 25, and 50 µM) compound 8b exerted a significant reduction of
viability under irradiation, in contrast with the negligible mortality observed in the dark (Figure
0
1
2
3
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0
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9
0
5
, Panel A). Interestingly, when the same experiment was conducted with either A2058 cell line
or nonꢀtumorigenic NCTCꢀ2544 cell line, compound 8b did not induce any significant reduction
neither in the dark nor under irradiation (Figure 5, Panels B and C). It is worthy of note that in
the selected cell lines the slight reduction of cellular viability observed in the dark was in line
with previous observations (Figure 5 and Table 3).
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8
9
0
Figure 5. Photodynamic activation of compound 8b. Various concentrations of compound 8b
were added to MCFꢀ7 (Panel A), A2058 (Panel B), and NCTCꢀ2544 (Panel C) cell lines for 24
hours. Cytotoxicity was determined by MTT assay both in dark condition and after 15 min
irradiation. Results are expressed as percentage of cell viability. Each data point represents the
mean of quadruplicate samplings from 96ꢀwell microplates ± SD.
The decrease of cell viability was associated to lightꢀinduced intracellular NO release that was
appreciable only in MCFꢀ7 cells treated with compound 8b. This result was also confirmed by
fluorescent method and nitrite quantification (Figure 6, Panel A and B).
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9
0
Figure 6. Intracellular NO detection and total nitrite estimation for compound 8b. (Panel A)
Intracellular NO evaluation in cell lines exposed to 50 ꢁM of 8b determined by DCFꢀDA and
visualized by fluorescence microscopy. Line 1: dark (10x); line 2: lightꢀtreated cells (10x); line
3
: lightꢀtreated cells (40x). (Panel B) Total nitrite content measured using Griess reagent in cells
treated with compound 8b (50 ꢁM) under dark (control) or light conditions.
A similar biological profile was found also for compound 11b. This conjugate induced a
marked and significant loss of viability in MCFꢀ7 cell line (Figure 7, Panel A) at the employed
concentrations. Negligible cell mortality was observed both in the dark and under irradiation in
A2058 and NCTCꢀ2544 cell lines at the same concentrations (Figure 7, Panels B and C). A
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significant lightꢀinduced reduction of viability for compound 11b in A2058 cells was observed
only at higher concentrations (data not shown).
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Photodynamic activation of compound 11b. Various concentrations of compound 11b
were added to MCFꢀ7 (Panel A), A2058 (Panel B), and NCTCꢀ2544 (Panel C) cell lines for 24
hours. Cytotoxicity was determined by MTT assay both in dark condition and after 15 min
irradiation. Results are expressed as the percentage of cell viability. Each data point represents
the mean of quadruplicate samplings from 96ꢀwell microplates ± SD.
An enhanced intracellular NO generation (Figure 8, Panel A) and accumulation of nitrite
content (Figure 8, Panel B) were found in lightꢀexposed and 11bꢀtreated MCFꢀ7 cells.
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Figure 8. Intracellular NO detection and total nitrite estimation for compound 11b. (Panel A)
Intracellular NO evaluation in cell lines exposed to 50 ꢁM of 11b determined by DCFꢀDA and
visualized by fluorescence microscopy. Line 1: dark (10x); line 2: lightꢀtreated cells (10x); line
3
: lightꢀtreated cells (40x). (Panel B) Total nitrite content measured using Griess reagent in cells
treated with compound 11b (50 ꢁM) under dark (control) or light conditions.
Regarding compound 11a, we have to take into account its considerable reduction of the
viability in the selected cell lines in the dark (Table 3). Thus, this conjugate was evaluated at
lower concentrations with respect to 8b and 11b (Figure 9). In MCFꢀ7 cell line, compound 11a
produced a significant photoinduced loss of cellular viability at the four nonꢀtoxic concentrations
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employed, in contrast to the negligible effect observed in the dark (Figure 9, Panel A). The
experiment performed in the A2058 cell line showed an appreciable lightꢀinduced mortality at
1.0 and 10 µM (Figure 9, Panel B). No relevant dark or photoinduced mortality was observed
when 11a was incubated with the nonꢀtumorigenic NCTCꢀ2544 cell line at the selected
concentrations (Figure 9, Panel C).
1
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Figure 9. Photodynamic activation of compound 11a. Various concentrations of compound 11a
were added to MCFꢀ7 (Panel A), A2058 (Panel B), and NCTCꢀ2544 (Panel C) cell lines for 24
hours. Cytotoxicity was determined by MTT assay both in dark condition and after 15 min
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irradiation. Results are expressed as the percentage of cell viability. Each data point represents
the mean of quadruplicate samplings from 96ꢀwell microplates ± SD.
The NO production and the nitrite accumulation were enhanced in MCFꢀ7 and A2058 cells
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after incubation with 10 µM of 11a and light irradiation (Figure 10, Panel A and B).
Figure 10. Intracellular NO detection and total nitrite estimation for compound 11a. (Panel A)
Intracellular NO evaluation in cell lines exposed to 10 ꢁM of 11a determined by DCFꢀDA and
visualized by fluorescence microscopy. Line 1: dark (10x); line 2: lightꢀtreated cells (10x); line
3
: lightꢀtreated cells (40x). (Panel B) Total nitrite content measured using Griess reagent in cells
treated with compound 11a (10 ꢁM) under dark (control) or light conditions.
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CONCLUSION
The here reported compounds combine a high σ receptors binding affinity with the capability
to release NO upon visible light excitation. Compounds 8b and 11b induced a marked loss of
viability in tumorigenic MCFꢀ7 cell line under light irradiation, whereas compound 11a
exhibited the same effect on both MCFꢀ7 and A2058 cell lines. The same group of compounds
showed negligible cell mortality of nonꢀtumorigenic NCTCꢀ2544 cells at chosen concentrations,
both in the dark and under irradiation. The NOꢀinduced reduction of cellular viability was also
quantitatively and qualitatively demonstrated by in cell intracellular NO detection and total
nitrite estimation. To the best of our knowledge, this is the first time that a successful
combination of σ receptors moieties and a NO photodonor is reported, providing distinctive
ligands with potential beneficial effect in the treatment of neoplastic disorders
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EXPERIMENTAL SECTION
General remarks. Reagent grade chemicals were purchased from SigmaꢀAldrich (St. Louis,
MO, USA) or Merck (Darmstadt, Germany) and were used without further purification. All
reactions involving airꢀsensitive reagents were performed under nitrogen in ovenꢀdried glassware
using the syringeꢀseptum cap technique. Flash chromatography purification was performed on a
Merck silica gel 60 0.040‒0.063 mm (230‒400 mesh) stationary phase. Nuclear magnetic
1
resonance spectra ( H NMR recorded at 200 and 500 MHz) were obtained on VARIAN INOVA
spectrometers using CDCl , DMSOꢀd6 or CD OD. TMS was used as an internal standard.
3
3
Chemical shifts (δ) are given in parts per million (ppm), and coupling constants (J) in Hertz
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(
Hz). The following abbreviations are used to designate the multiplicities: s = singlet, d =
doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br = broad. The purity of all tested
compounds, whether synthesized or purchased, reached at least 95% as determined by
microanalysis (C, H, N) that was performed on a Carlo Erba instrument model E1110; all the
results agreed within ± 0.4% of the theoretical values. Reactions were monitored by thinꢀlayer
chromatography (TLC) performed on 0.25 mm silica gel Merck 60 F254 coated aluminum plates;
the spots were visualized by UV light. UVꢀVis spectra absorption spectra were recorded with a
JascoVꢀ560 spectrophotometer. LCMS analysis was performed using a Perkin Elmer Flexar FXꢀ
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1
0 UHPLC System (software Analyst 1.6.1, columns Waters XꢀTerra RP18 3.0x150mm, 3.5ꢁm
or Thermo Fisher Scientific Hypersil WPꢀ300 C8 4.6x100mm, 5.0ꢁm), with singleꢀwavelength
UV–visible detector and API 2000 LC/MS/MS System AB Sciex (electrospray ionization).
Compounds nomenclature were generated with ChemBioDraw Ultra version 16.0.0.82.
General procedure for the nitrile formation (Procedure A). In a DMF (24 mL) solution of
the appropriate amine (8.71 mmol), K CO (13.06 mmol), NaI (0.043 mmol), and the appropriate
2
3
bromo nitrile (8.71 mmol) were added and the reaction mixture was refluxed for 3 h at 60 °C.
After the reaction was complete, the solvent was evaporated to dryness, the residue was
dissolved in CH Cl (30 mL), washed with a saturated solution of NaHCO (2 x 20 mL), and
2
2
3
brine (1 x 20 mL). The organic phase was dried with anhydrous Na SO , concentrated, and the
2
4
crude product was purified via silica gel chromatography to give the desired product. Products
synthesized according to Procedure A: 5a, 6a, and 7a–b.
General procedure for the amine preparation (Procedure B). To a solution of the appropriate
nitrile (1.4 mmol) in dry THF (6 mL), LiAlH (1M in THF, 4.20 mmol) was added dropwise at 0
4
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°
C under N atmosphere and the resulting mixture was allowed to warm to room temperature.
2
After 2 h stirring, the reaction was quenched with 1M NaOH (3 mL) at 0 °C, filtered through
Celite, and washed with MeOH. The solution was concentrated under reduced pressure and
dissolved in CH Cl . The organic phase was dried over anhydrous Na SO and concentrated to
1
1
1
1
1
1
1
1
1
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give the crude amino product that was used as it without any further purification. To the crude
product, dissolved in ACN (3 mL), 4ꢀnitroꢀ3ꢀ(trifluoromethyl)aniline (1.40 mmol) in ACN (3
mL) and Et N (1.54 mmol) were added. The resulting mixture was placed in an oil bath at 60 °C
3
and left to react overnight. After the reaction was complete, the mixture was washed with
hexane. The ACN solution was then evaporated to dryness and the residue purified via flash
chromatography. Products synthesized according to Procedure B: 8a, 9a, and 10a–b.
General procedure for amine preparation (Procedure C). A solution of the appropriate amino
alcohol (1.07 mmol) and Et N (2.14 mmol) in CH Cl (5 mL) was cooled to 0 °C under N
2
3
2
2
atmosphere. Methanesulfonyl chloride (1.07 mmol) dissolved in CH Cl (4 mL) was added and
2
2
the reaction mixture was allowed to warm to room temperature for 30 min while stirring. The
mixture was cooled to 0 °C and a solution of the appropriate amine (2.14 mmol) and Et N (4.28
3
mmol) in CH Cl (2 mL) was added. After 70 h, the mixture was washed with a saturated
2
2
solution of NaHCO (1 x 10 mL) and brine (1 x 10 mL). The organic phase was dried over
3
anhydrous Na SO and concentrated. The crude product was dissolved in EtOAc, washed with a
2
4
0
.2 N water solution of H SO and the mixture was filtered. The residue was washed with
2 4
EtOAc/CH Cl and left to dry. Then, it was dissolved in EtOAc and washed with NaHCO (1 x
2
2
3
1
0 mL) and brine (1 x 10 mL). The organic phase was dried over anhydrous Na SO ,
2 4
concentrated and the crude product purified via flash chromatography to give the desired
product. Products synthesized according to Procedure C: 8b, and 11a–b.
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2
-((4-Nitro-3-(trifluoromethyl)phenyl)amino)ethanol (1). To a solution of 4ꢀnitroꢀ3ꢀ
(
trifluoromethyl)aniline (1.43 mmol, 0.30 g) in EtOH (5 mL), the appropriate 2ꢀaminoethanol
(7.17 mmol, 0.43 mL) and Et N (1.43 mmol, 0.20 mL) were added. After the reaction was
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complete, the mixture was concentrated and the residue was triturated with water and filtered
1
under vacuum to give the compound 1 as yellow solid (yield: 72%). H NMR (500 MHz,
CD OD) δ 7.92 (d, J = 9.2 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.71 (dd, J = 2.4, 9.2 Hz, 1H), 3.64
3
+
(
t, J = 5.8 Hz, 2H), 3.25 (t, J = 5.8 Hz, 2H). LCMS found 250.3, [M + H] .
5
-((4-Nitro-3-(trifluoromethyl)phenyl)amino)pentan-1-ol (2). To a solution of 4ꢀnitroꢀ3ꢀ
(trifluoromethyl)aniline (1.43 mmol, 0.30 g) in EtOH (5 mL), the appropriate 5ꢀaminopentanꢀ1ꢀ
ol (4.29 mmol, 0.44 g) and Et N (1.43 mmol, 0.20 mL) were added. After the reaction was
3
complete, the mixture was concentrated and the residue was dissolved in EtOAc, washed with a
saturated solution of NaHCO (1 x 10 mL) and brine (1 x 10 mL). The combined organic extracts
3
were dried over anhydrous Na SO and concentrated affording compound 2 as a yellow oil
2
4
1
(
yield: 98%). H NMR (200 MHz, CDCl ) δ 8.02 (d, J = 9.2 Hz, 1H), 6.87 (d, J = 2.6 Hz, 1H),
3
6
2
.64 (dd, J = 2.6, 9.2 Hz, 1H), 4.81 (br. s., 1H), 3.70 (t, J = 6.0 Hz, 2H), 3.24 (q, J = 6.70 Hz,
+
H), 1.42 ‒ 1.82 (m, 6H). LCMS found 292.6, [M + H] .
3
-Chloro-N-(4-nitro-3-(trifluoromethyl)phenyl)propanamide (3). To a solution of 4ꢀnitroꢀ3ꢀ
trifluoromethyl)aniline (4.85 mmol, 1 g) in dry CH Cl (15 mL), 3ꢀchloropropionylꢀchloride
(
2
2
(5.82 mmol, 0.55 mL) and Et N (5.82 mmol, 0.81 mL) were added dropwise at 0 °C under N2
3
atmosphere. The reaction mixture was stirred at room temperature overnight. After the reaction
was complete, it was cooled to 0 °C and quenched with water and a saturated solution of
NaHCO (3 mL) dropwise. The CH Cl solution was washed with a saturated solution of
3
2
2
NaHCO (2 x 10 mL), brine (1 x 10 mL) and dried over anhydrous Na SO . The solvent was
3
2
4
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concentrated under vacuum and the crude product was purified via silica gel chromatography,
1
eluting with 25% EtOAc in Hexane to give the desired product as white solid (yield: 60%). H
NMR (200 MHz, 47 DMSOꢀd6) δ 8.01 ‒ 8.17 (m, 2H), 7.82 ‒ 7.96 (m, 1H), 3.75 (t, J = 6.3 Hz,
+
1
1
1
1
1
1
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H), 2.77 (t, J = 6.3 Hz, 2H). MS found 296.5, [M + H] .
3
-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-N-(4-nitro-3-
(trifluoromethyl)phenyl)propanamide (4b). To a solution of compound 3 (2.15 mmol, 0.64 g) in
DMF (16 mL) were added 6,7ꢀdimethoxyꢀ1,2,3,4ꢀtetrahydroisoquinoline (2.15 mmol, 0.5 g) and
NaHCO (5.37 mmol, 0.45 g). The mixture was heated to 70 °C and stirred overnight. After the
3
reaction was complete, DMF was evaporated under vacuum and the residue was dissolved in
CH Cl (40 mL), washed with a saturated solution of NaHCO (2 x 15 mL) and brine (1x15 mL).
2
2
3
The solvent was dried over anhydrous Na SO , concentrated under vacuum and the crude was
2
4
purified by flash chromatography, eluting with 5 to 10% Hexane in EtOAc and finally with
1
100% EtOAc to give the desired product as yellow solid (yield: 47%). H NMR (200 MHz,
CDCl ) δ 7.65 ‒ 7.93 (m, 3H), 6.69 (s, 1H), 6.57 (s, 1H), 3.81 ‒ 3.96 (m, 6H), 3.76 (s, 2H), 2.90
3
+
‒
3.03 (m, 6H), 2.58 ‒ 2.73 (m, 2H). LCMS found 453.0, [M + H] .
2
-(4-Benzylpiperidin-1-yl)acetonitrile (5a). The compound has been prepared following
procedure A and using 2ꢀbromoacetonitrile (11.41 mmol, 0.76 mL). The crude product was
purified via silica gel chromatography, eluting with 10 to 40% EtOAc in Hexane to give
1
compound 5a as a white solid (yield: 75%). H NMR (500 MHz, CDCl ) δ 7.25 ‒ 7.30 (m, 2H),
3
7
2
2
.17 ‒ 7.22 (m, 1H), 7.11 ‒ 7.15 (m, 2H), 3.50 (s, 2H), 2.73 ‒ 2.80 (m, 2H), 2.54 (d, J = 7.34 Hz,
H), 2.28 (dt, J = 2.7, 11.6 Hz, 2H), 1.67 ‒ 1.73 (m, 2H), 1.50 ‒ 1.61 (m, 1H), 1.25 ‒ 1.36 (m,
+
H). LCMS found 214.2, [M + H] .
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3
-(4-Benzylpiperidin-1-yl)propanenitrile (6a). The compound has been prepared following
procedure A and using 3ꢀbromopropanenitrile (11.41 mmol, 0.95 mL). The mixture was stirred
at 60 °C overnight. The crude product was purified via silica gel chromatography, eluting with
1
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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1
0 to 50% EtOAc in Hexane to give compound 6a as a clear oil (yield: 49%). H NMR (500
MHz, CDCl ) δ 7.25 ‒ 7.30 (m, 2H), 7.16 ‒ 7.21 (m, 1H), 7.10 ‒ 7.15 (m, 2H), 2.86 (br. d, J =
3
11.2 Hz, 2H), 2.67 (t, J = 7.3 Hz, 2H), 2.53 (d, J = 7.1 Hz, 2H), 2.48 (t, J = 7.1 Hz, 2H), 1.99 (dt,
J = 2.5, 11.6 Hz, 2H), 1.64 (br. d, J = 13.2 Hz, 2H), 1.46 ‒ 1.57 (m, 1H), 1.23 ‒ 1.35 (m, 2H).
+
LCMS found 228.5, [M + H] .
4-(4-Benzylpiperidin-1-yl)butanenitrile (7a). The compound has been prepared following
procedure A and using 4ꢀbromobutanenitrile (11.41 mmol, 1.13 mL). The mixture was stirred at
6
1
7
0 °C overnight. The crude product was purified via silica gel chromatography, eluting with
1
00% EtOAc to give compound 7a as a yellow oil (yield: 18%). H NMR (500 MHz, CDCl ) δ
3
.25 ‒ 7.30 (m, 2H), 7.16 ‒ 7.21 (m, 1H), 7.11 ‒ 7.15 (m, 2H), 2.83 (br. d, J = 11.5 Hz, 2H), 2.53
(
d, J = 7.1 Hz, 2H), 2.35 ‒ 2.42 (m, 4H), 1.88 (dt, J = 2.2, 11.7 Hz, 2H), 1.80 (quint, J = 7.0 Hz,
2
H), 1.62 (br. d, J = 13.7 Hz, 2H), 1.46 ‒ 1.56 (m, 1H), 1.22 ‒ 1.32 (m, 2H). LCMS found 242.0,
+
[
M + H] .
4
-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butanenitrile (7b). The compound has been
prepared following procedure A and using 4ꢀbromobutanenitrile (8.71 mmol, 0.86 mL). The
crude product was purified via silica gel chromatography, eluting with 30% EtOAc in Hexane, to
1
give compound 7b as a white solid (yield: 78 %). H NMR (200 MHz, CDCl ) δ 6.60 (s, 1H),
3
6
.52 (s, 1H), 3.79 ‒ 3.90 (m, 6H), 3.55 (br. s, 2H), 2.77 ‒ 2.87 (m, 2H), 2.68 ‒ 2.76 (m, 2H), 2.63
(t, J = 6.7 Hz, 2H), 2.48 (t, J = 7.0 Hz, 2H), 1.94 (quint, J = 7.0 Hz, 2H). LCMS found 260.4, [M
+
+
H] .
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N-(2-(4-Benzylpiperidin-1-yl)ethyl)-4-nitro-3-(trifluoromethyl)aniline (8a). The compound has
been prepared following procedure B and using 2ꢀ(4ꢀbenzylpiperidinꢀ1ꢀyl)acetonitrile (5a) (1.37
mmol, 0.29 g). The crude product was purified by flash chromatography in EtOAc to give the
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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final compound as yellow oil (yield: 54%). H NMR (200 MHz, CDCl ) δ 8.02 (d, J = 9.0 Hz,
3
1
H), 7.05 ‒ 7.37 (m, 5H), 6.89 (d, J = 2.4 Hz, 1H), 6.63 (dd, J = 2.4, 9.0 Hz, 1H), 5.52 (br. s.,
H), 3.22 (q, J = 5.0 Hz, 2H), 2.86 (d, J = 10.5 Hz, 2H), 2.46 ‒ 2.67 (m, 4H), 1.96 (t, J = 11.4
1
Hz, 2H), 1.46 ‒ 1.75 (m, 3H), 1.14 ‒ 1.41 (m, 2H). Anal. calcd for: C H F N O : C, 61.91; H,
2
1
24
3
3
2
+
5
.94; N, 10.31. Found: C, 61.78; H, 5.96; N, 10.40. LCMS found 407.4, [M + H] .
N-(2-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-4-nitro-3-(trifluoromethyl)aniline
(8b). The compound has been prepared following procedure C and using 2ꢀ((4ꢀnitroꢀ3ꢀ
trifluoromethyl)phenyl)amino)ethanol (1) (1.07 mmol, 0.26 g) and 6,7ꢀdimethoxyꢀ1,2,3,4ꢀ
(
tetrahydroisoquinoline (2.14 mmol, 0.41 g). The crude product was purified by flash
chromatography eluting 5% MeOH in EtOAc to give compound 8b as a yellow solid (yield:
1
3
6
5%). H NMR (200 MHz, CDCl ) δ 8.01 (d, J = 9.2 Hz, 1H), 6.89 (d, J = 2.2 Hz, 1H), 6.60 ‒
3
.69 (m, 2H), 6.54 (s, 1H), 5.58 (br. s., 1H), 3.78 ‒ 3.93 (m, 6H), 3.62 (s, 2H), 3.28 ‒ 3.42 (m,
2H), 2.73 ‒ 2.93 (m, 6H). Anal. calcd for: C H F N O : C, 56.47; H, 5.21; N, 9.88. Found: C,
2
0
22
3
3
4
+
5
6.29; H, 5.22; N, 9.78. LCMS found 425.0, [M + H] .
N-(3-(4-Benzylpiperidin-1-yl)propyl)-4-nitro-3-(trifluoromethyl)aniline (9a). The compound
has been prepared following procedure B and using 3ꢀ(4ꢀbenzylpiperidinꢀ1ꢀyl)propanenitrile (6a)
(1.37 mmol, 0.31 g). The crude product was purified by flash chromatography, eluting with 5 to
1
1
0% MeOH in EtOAc to give the final compound as yellow oil (yield: 20%). H NMR (200
MHz, CDCl ) δ 8.02 (d, J = 9.2 Hz, 1H), 7.06 ‒ 7.42 (m, 5H), 6.80 (d, J = 2.4 Hz, 1H), 6.51 (dd,
3
J = 2.4, 9.2 Hz, 1H), 3.21 ‒ 3.35 (m, 2H), 2.98 (br. d, J = 11.4 Hz, 2H), 2.45 ‒ 2.63 (m, 4H), 1.49
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