Stereoselective synthesis of Z-acrylonitrile derivatives: Catalytic and acetylcholinesterase inhibition studies

Article abstract of DOI:10.1039/c3nj01384g

In the present study, a focused library of (Z)-acrylonitrile analogues (library A & B) were synthesized, which were typically accessed via a facile Knoevenagel condensation between p-nitrophenylacetonitrile and appropriately substituted aromatic aldehydes

Full text of DOI:10.1039/c3nj01384g

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Stereoselective synthesis of Z-acrylonitrile
derivatives: catalytic and acetylcholinesterase
inhibition studies†
Cite this: New J. Chem., 2014,
38, 1655
Mehtab Parveen,*a Ali Mohammed Malla,a Mahboob Alam,a Musheer Ahmadb and
Shahnawaz Rafiqb
In the present study, a focused library of (Z)-acrylonitrile analogues (library A & B) were synthesized,
which were typically accessed via a facile Knoevenagel condensation between p-nitrophenylacetonitrile
and appropriately substituted aromatic aldehydes (1a–i) and 3-formyl chromones (3a–c). This new synthetic
eco-friendly approach resulted in a remarkable improvement in the synthetic efficiency (83–92% yield), high
purity, minimizing the production of chemical wastes without using highly toxic reagents for the synthesis
and, more notably, it improved the selectivity for (Z)-acrylonitrile derivatives. By performing DFT calculations,
it was found that the (Z)-isomer of compound 2b is stabilized by 2.61 kcal molꢀ1 more than the (E)-isomer.
All of the compounds were tested for acetylcholinesterase (AChE) inhibition. Compounds 2a and 4c,
displayed the strongest inhibition, with IC50 values of 0.20 mM and 0.22 mM respectively. The methoxy
group at the para-position of phenyl ring A was found to be essential for AChE inhibition.
Received (in Montpellier, France)
8th November 2013,
Accepted 13th January 2014
DOI: 10.1039/c3nj01384g
www.rsc.org/njc
anti-proliferative activity and is a potent inducer of pro-
grammed cell death in human leukemia cells.25 Acrylonitrile
1. Introduction
Carbon–carbon bond formation reactions are the most important derivatives have been recently documented as selective acetyl-
reactions in organic synthesis.1–3 The Knoevenagel condensation cholinesterase inhibitors.26 The immense biological impor-
between aldehydes or ketones with activated methylene com- tance inspired researchers, therefore, to synthesize them and
pounds is one of the reactions which facilitates C–C bond study their potential biological activities.
formation4,5 and has been exploited in the synthesis of some
The synthesis of acrylonitrile compounds has previously been
vital drugs such as entacapone,6 pioglitazone7 and lumefantrine8 achieved by the use of Wittig reactions,27 McMurry coupling
(Fig. 1). Over the past few decades there has been an increasing reactions28 and the Heck reaction.29 The recognition of diethyl
prevalence in the number of biologically active compounds that amine as a catalyst, first by Knoevenagel,1 paved the way for the
contain an acrylonitrile moiety.9,10 The reactivity of heteroaryl- development of a large number of catalysts30–33 and reaction
acetonitriles has been exploited for the synthesis of some conditions.34–36 Despite the various methods, the Knoevenagel
nitrogen-bridged heterocycles.11 Acetonitrile derivatives are condensation is a simple and straightforward approach for the
useful intermediates and scaffolds which possess a wide range synthesis of acrylonitrile derivatives.37,38 This method involves
of promising biological properties such as antiproliferative,12 the acid or base-catalyzed condensation of active methylene
antifungal,13 antitumor,14 antibacterial,15 antitubercular,16 moieties with carbonyl compounds. In the quest to achieve
antioxidative,17 insecticidal,18 spasmolytic,19 estrogenic,20 higher efficiency, several catalysts and reaction conditions were
hypotensive,21 tuberculostatic,22 antitrichomonal23 and anti- tried, including the use of microwaves,39 Brønsted acid cata-
parasitic24 properties.
lysts,40 Lewis acids such as MgBr2ꢁOEt2,41 SnCl2,42 silica-
It was recently reported that 2-acetyl-3-(6-methoxybenzo- supported catalysts,43,44 bases such as NaOEt,45 piperidine46
thiazo)-2-yl-amino-acrylonitrile (AMBAN) possesses significant and potassium sorbate.47 Earlier investigations required long
reaction times, the use of hazardous reagents and tedious
workup procedures. In view of the numerous biological appli-
cations of acrylonitrile derivatives, the development of a simple
and convenient protocol is of considerable interest.
a Department of Chemistry, Aligarh Muslim University, Aligarh, 202002, India.
E-mail: mehtab.organic2009@gmail.com; Tel: +91 09897179498
b Indian Institute of Technology (IIT), Kanpur, 208016, India
In recent years, the use of reagents and catalysts supported
† Electronic supplementary information (ESI) available. CCDC 963259. For ESI
on solid supports48–50 has received considerable attention due
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c3nj01384g
to growing environmental concerns. Such reagents not only
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Fig. 1 Knoevenagel assisted synthesis of some vital drugs.
simplify the purification processes but also help in preventing the for (Z)-acrylonitrile derivatives, and minimized the production
discharge of toxic reaction residues into the environment. In this of chemical waste without using highly toxic reagents for the
respect, silica chloride (SiO2–Cl) is an attractive candidate. It is a synthesis. In addition to efficacy, a major requirement of novel
well known catalyst in organic synthesis and has been docu- supported reagents concerns their reusability, a factor that has
mented in several organic transformations.51–53 Silica chloride significant environmental and economic impact since the most
was prepared by employing standard procedures depicted in the costly components in a chemical reaction are often not the
literature.54 To a well stirred oven-dried silica gel in dry dichloro- starting materials but the catalyst.56,57 The Si–Cl bond is labile
methane, thionyl chloride was added dropwise at room tempera- and can give rise to Lewis acid centers on silica. The Cl is
ture, and the evolution of profuse amounts of HCl and SO2 displaced selectively by the carbonyl oxygen of the aldehyde by a
occurred instantaneously. After stirring for 2 hours, the solvent nucleophilic substitution reaction, generating a cationic centre on
was removed to dryness under reduced pressure. The silica the carbonyl carbon, which is believed to be easily attacked by the
chloride thus synthesized was used in the following experiments. nucleophile, i.e. the imide form of p-nitrophenylacetonitrile,58 to
The mechanistic pathway for the preparation of silica chloride is give the corresponding acrylonitrile derivatives.
shown in Scheme 1.55 It possesses environmentally benign prop-
In view of the comprehensive advantages of silica-supported
erties such as non-toxicity, biocompatibility, recyclability, physio- catalysts, herein we report the stereoselective synthesis of bio-
logical inertness, inexpensiveness and thermal stability. This new logically active (Z)-acrylonitrile derivatives of substituted
synthetic strategy resulted in a remarkable improvement in the chromones and aromatic aldehydes using silica chloride as
synthetic efficiency and more notably, it enhanced the selectivity the heterogeneous catalyst. The efficacy of the catalyst was
examined. The catalyst was reused five times and the results show
that the catalyst can be reused without a significant reduction of
the yield. The compounds were also screened for AChE inhibitory
activity against the standard drug tacrine.
2. Results and discussion
2.1. Chemistry
The synthetic pathways of a series of new acrylonitrile deriva-
tives (2a–i) and (4a–c) are shown in Scheme 2 and 3 respectively.
Scheme 1 Synthetic scheme for the preparation of silica chloride.
Herein each library (A & B) was typically accessed via a facile
Scheme 2 Synthesis of compounds 2a–i.
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Scheme 3 Synthesis of compounds 4a–c.
Knoevenagel condensation between p-nitrophenylacetonitrile
The structural elucidation of the synthesized compounds
and appropriately substituted aromatic aldehydes (1a–i) and (2a–i) and (4a–c) was established on the basis of elemental
3-formyl chromones (3a–c). All of the compounds were obtained analysis, IR, 1H NMR, 13C NMR and mass spectral studies. The
in excellent yields (83–92%) with high purity. Among the two analytical results for C, H and N were within ꢂ0.3% of the
possible geometrical isomers (E/Z), (Z)-isomers were obtained theoretical values. All of the synthesized compounds displayed
as the sole product (Scheme 4). This Z-selectivity can be inter- no peak for the aldehydic carbonyl in the IR spectrum, which
preted as a way to minimize steric interactions between the confirmed the reaction at the carbonyl moiety. Moreover, all of
approaching nucleophile (imide form of p-nitrophenyl- the compounds displayed a characteristic peak for the cyano
acetonitrile) and the bulky substituted aryl substituent and group resonating at around 2210–2226 cmꢀ1; similarly, peaks
bound catalyst at the crowded, electron deficient carbonyl for the nitro group, aromatics (CQC) and other functional
carbon, favouring back side attack of the nucleophile in such moieties are discussed in the experimental section. In the
a way that the phenyl ring of the approaching nucleophile is 1H NMR spectra, each compound displayed a sharp singlet at
pushed away, thus favouring the formation of the Z-isomer. around d 6.98–8.85 ascribed to the olefinic proton (H-3), and a
Steric interactions seem to have an influence on the control of sharp double doublet appearing at a slightly downfield value
the Z-configurational isomers. This is further supported by d 8.02–8.34, due to an adjacent electron withdrawing nitro
single crystal X-ray crystallographic analysis of compound 2b group, corresponds to the H-300 and H-500 protons. Similarly, a
and also verified by DFT calculations.
double doublet at around d 7.35–7.99 representing two protons
Scheme 4 Plausible mechanism for the synthesis of compounds 2a–i and 4a–c.
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is attributed to the H-200 and H-600 protons and two independent the activation energy. However, there was a significant improve-
sharp singlets representing one proton each at d 7.20 and 7.23 ment in the yield when methanol was used as a solvent (Table 3,
are attributed to the H-20 protons (g-pyrone ring) of compounds entry 3). When the reaction was carried out in EtOH (Table 3,
4b and 4a respectively. In the 13C NMR spectra, a series of entry 2), the yield of the product improved significantly in a
signals emerging at around d 109.7–162.3 are ascribed to shorter time. A comparative study of a variety of catalysts was
aromatic carbons, peaks resonating at around d 115.9–118.4 conducted to show the superiority of silica chloride as a hetero-
correspond to the cyanide group and the peaks at d 177.4, 178.9 geneous acid catalyst. When the model reaction was investigated
and 180.1 correspond the to g-pyrone ring carbonyl group in with heterogeneous catalysts such as HClO4–SiO2, NaHSO4–SiO2
the case of compounds 4b, 4a and 4c respectively. The mass and NH4OAc–SiO2,the reaction required a prolonged time period,
spectral analysis was also in agreement with the proposed and the yields were not satisfactory (Table 4, entries 2–4), whereas
structure of the compounds.
using basic catalysts such as piperidine and NaOMe (Table 4,
During the present study, a series of acrylonitrile derivatives entries 5 and 6), the reaction again took an extended time period
of substituted aldehydes (2a–i) and substituted 3-formyl for completion, with lower yields, and impure products were
chromones (4a–c) were synthesized by the Knoevenagel con- obtained. When silica chloride was used as a catalyst (Table 4,
densation of differently substituted aromatic aldehydes and entry 1), the yield of the products increased significantly, suggest-
3-formyl chromones with p-nitrophenylacetonitrile under stirring ing silica chloride is the catalyst of choice for the Knoevenagel
using piperidine (0.5 mL) as a basic catalyst in ethanol. The condensation. The reusability of the silica chloride catalyst was
reaction took a longer time (4–5 hours) for completion with a also examined in a model reaction. The catalyst was reused five
moderate yield (67–76%) of the products (Table 1).
times and the results show that the catalyst can be reused without
In order to develop an eco-friendly approach, we explored a significant reduction of the yield (Table 5). The reusability of
the efficacy of silica chloride (SiO2–Cl) by carrying out the the catalyst reduces the cost of the reaction. After the first fresh
reaction of p-nitrophenylacetonitrile with a variety of substituted run with 91% yield, the catalyst was removed by filtration. The
aromatic aldehydes and 3-formyl chromones in equimolar ratios recovered catalyst was dried under vacuum at 120 1C for 10 h and
(1: 1), in the absence of piperidine at room temperature. The tested for five more reaction cycles. Recycling and reuse of the
reaction proceeded smoothly and resulted in the formation of catalyst showed minimal decreases in the yields. The product 2b
the corresponding products (2a–i) and (4a–c) in good yields was obtained in 90%, 88%, 87%, 85% and 85% yields after
(83–92%) within 1.5–2 hours, as monitored by TLC (Table 1). successive cycles (Table 5, entries 2–6), thus proving the catalyst’s
Although the intermediates were not separated and characteri- reusability.
zed, an attempt was made to establish the mechanism for
the formation of (Z)-acrylonitrile derivatives 2a–i and 4a–c in
2.2. AChE inhibition results
conformity with the previously reported analogous results In library A, we analyzed nine aromatic aldehydes, substituted
(Scheme 4).58,59 In order to optimize the reaction conditions, with different groups for AChE inhibition activity. Of the library
a model reaction was conducted using p-nitrophenylaceto- A analogues (2a–i) shown in Table 6, compound 2a, with a 3,4,5-
nitrile and 2,3-dimethoxy benzaldehyde (1b) under various trimethoxy-substituted benzene moiety (ring A), exhibited the
reaction conditions (including varying the catalyst loading strongest inhibition to AChE with an IC50 value of 0.20 mM,
and the effect of the solvent in terms of yields and time). In followed by compound 2d (IC50 = 0.23 mM), 2f (IC50 = 0.25 mM),
order to establish the best reaction conditions, we performed 2c (IC50 = 0.27 mM), 2b (IC50 = 0.29 mM), 2h (IC50 = 0.30 mM), 2g
an optimization study using a model substrate in the presence (IC50 = 0.35 mM), 2i (IC50 = 0.38 mM) and 2e (IC50 = 0.43 mM). The
of varying amounts of silica chloride. It is clear from Table 2 results indicate that all the tested compounds showed signifi-
(entry 5) that 2.5 mol% of the catalyst is sufficient to gain the cant AChE inhibitory activity. Among the methoxy-substituted
optimum yield in the shortest reaction time. When using less analogues 2a–d, compound 2a showed promising activity against
than 2.5 mol% catalyst (0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%), AChE inhibition. Compound 2d, with a 3,4-dimethoxy-substituted
moderate yields of the product (78–88%) were obtained with benzene ring (ring A), also exhibited encouraging activity but
higher reaction times, while with an excess mol% of catalyst 0.03 lower than 2a. Similarly, compound 2c, with methoxy
(42.5 mol%) there was no increase in the yield of the product, groups at the 3 and 5 positions of the benzene moiety (ring A),
probably due to the saturation of the catalyst. In order to study showed lower activity than 2d, while compound 2b, with methoxy
the solvent effect, the model reaction was carried out in groups at the 2 and 3 positions (ring A), exhibited lower activity
different solvent systems. The reaction was primarily tested than all the 4-methoxy-substituted derivatives. It can be inferred
under solvent-free conditions by the grinding method, however from the data that the position and number of methoxy groups
only traces of product were obtained (Table 3, entry 1). When attached to the benzene moiety inflicts a prominent effect on the
the reaction was performed in CH2Cl2 and DMF, moderate AChE inhibition. Compound 2a, with three methoxy groups
yields of the products were obtained after a prolonged reaction attached to the benzene moiety (ring A), is the most potent
period (Table 3, entries 4 and 6), whereas in acetic acid, the inhibitor. Furthermore, when the methoxy group at the 4 position
reaction again took longer (Table 3, entry 5) but there was an of the benzene moiety (ring A) in compound 2a is removed
improvement in the yield, probably due to the electromeric effect (compound 2c), the activity decreased significantly by 0.07. Thus
activating the carbonyl group of the reactants, thus decreasing the methoxy group at the 4 position in the benzene moiety
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Table 1 Silica chloride-catalyzed synthesis of acrylonitrile derivatives 2a–i and 4a–c
Reaction in presence of piperidine
Reaction in presence of silica chloride
Time (hours) Yield (%)
Time (hours)
Yield (%)
Derivative Structure
M.p. (1C)
2a
5
69
1.5
87
91–92 (90)60
2b
2c
2d
2e
4.5
67
71
73
67
2
91
93
89
88
190–191
5
1.7
179–180 (180)60
4
2
152–153
5
2
178–179
2f
5
70
1.8
85
231–232 (230)60
2g
2h
2i
4.6
69
68
67
2
87
84
83
145–146
233–234
181–182
4
1.3
5
2
4a
4
76
2
90
194–195
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Table 1 (continued)
Reaction in presence of piperidine
Reaction in presence of silica chloride
Time (hours)
Yield (%)
Time (hours)
Yield (%)
Derivative Structure
M.p. (1C)
4b
4.3
70
1.5
92
203–204
4c
4
69
2
87
187–188
Table 2 Effect of catalyst loading on the synthesis of (2b)a
Table 5 Reusability of the silica chloride catalyst in the synthesis of
compound 2b
Entry
Catalyst (mol%)
Timeb (hours)
Yieldc (%)
Entry
Reaction cycle
Isolated yield (%)
1
2
3
4
5
6
0.5
1.0
1.5
2.0
2.5
3.0
7
6
4
3
2
2
78
81
85
88
91
91
1
2
3
4
5
6
1st (fresh run)
2nd cycle
3rd cycle
4th cycle
5th cycle
91
90
88
87
85
85
6th cycle
a
Reaction conditions: 2,3-dimethoxybenzaldehyde (1b,1 mmol),
p-nitrophenylacetonitrile (1 mmol), stirring, room temperature (25 1C),
b
c
EtOH solvent. Reaction progress monitored by TLC. Isolated yield of
products.
Table 6 Quantitative estimation of the acetylcholinesterase inhibition
activity of compounds (2a–i) by a modified Ellaman’s coupled enzyme
assay method using tacrine as the reference (n = 3)
Table 3 Effect of various solvents on a model reactiona
Entry
Solvent
Timeb (hours)
Yieldc (%)
1
2
3
4
5
6
Solvent-freed
EtOH
MeOH
CH2Cl2
CH3COOH
DMF
—
2
3.5
6
5
8
Traces
91
83
60
69
Library A
IC50
54
(mM)a ꢂ SEMb
a
Reaction conditions: 2,3-dimethoxybenzaldehyde (1b,
1 mmol),
for hAChEc
p-nitrophenylacetonitrile (1 mmol), catalyst (2.5 mol%), different solvents Entry Product
Nature of substituents
inhibition
b
c
(15 mL), stirring at RT. Reaction progress monitored by TLC. Isolated
yield of products. d Solvent-free by grinding method.
1
2
2a
2b
R1 = H; R2 = R3 = R4 = OCH3
R1 = R2 = OCH3; R3 = R4 = H
R1 = R3 = H; R2 = R4 = OCH3
R1 = R4 = H; R2 = R3 = OCH3
R1 = R3 = R4 = H; R2 = NO2
R1 = R2 = R4 = H; R3 = N(CH3)2
0.20 ꢂ 0.03
0.29 ꢂ 0.02
0.27 ꢂ 0.05
0.23 ꢂ 0.03
0.43 ꢂ 0.05
0.25 ꢂ 0.01
3
4
2c
2d
Table 4 Comparison of the efficiency of silica chloride for the synthesis
of (2b)a
5
6
2e
2f
7
8
2g
2h
R1 = R4 = H; R2 = OCH3; R3 = OH 0.35 ꢂ 0.03
Entry
Catalyst
Timeb (hours)
Yieldc (%)
R1 = H; R2 = R3 = R4 = OH
R1 = R3 = H; R3 = R4 = OH
—
0.30 ꢂ 0.07
0.38 ꢂ 0.033
0.19 ꢂ 0.02
9
10
2i
1
2
3
4
5
6
Cl–SiO2
2
5
7
6
4.5
8
91
70
68
65
Standard
(tacrine)
HClO4–SiO2
NaHSO4–SiO2
NH4OAc–SiO2
Piperidine
NaOMe
a
IC50 values represent the concentration of inhibitor required to decrease
the enzyme activity by 50%. b SEM = standard error of the mean. hAChE =
human recombinant AChE from human serum was used.
c
67
58 (impure)
a
Reaction conditions: 2,3-dimethoxybenzaldehyde (1b,
1 mmol),
p-nitrophenylacetonitrile (1 mmol), different catalysts (2.5 mol%), EtOH displayed better AChE inhibition in comparison to 2b.
b
c
(15 mL), stirring at RT. Reaction progress monitored by TLC. Isolated
yield of products.
Compound 2f, with
a N,N-dimethyl (CH3)2N substituent
attached at the 4 position of the benzene moiety, also showed
noteworthy activity, with an IC50 value of 0.25 mM. Compound 2e,
(ring A) is essential for activity. It can also be seen with with a nitro group at the 3 position (ring A), showed moderate
compounds 2b and 2d that although they have two methoxy activity among the tested compounds. Compounds 2g–i, with
groups each, compound 2d with a methoxy group at the 4 position hydroxy substituents attached to ring A, also showed good
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AChE inhibition. Compound 2h, bearing three hydroxyl groups to their bulky geometry which restricts them to fit better inside
at the 3, 4 and 5 positions on the benzene moiety (ring A), the cavity of AChE.
showed an IC50 value of 0.30 mM, with the removal of a hydroxyl
2.3. X-ray crystallographic study
group at the 4 position (compound 2i) significantly lowering its
activity by 0.08 in comparison to 2h. Similarly, compound 2g, Compound 2b, once isolated, is found to be air-stable and
with one of the hydroxyl groups at the 4 position, showed better soluble in all common organic solvents but insoluble in water.
activity than 2i, suggesting the vital role of the 4-hydroxyl group X-ray crystallographic analysis reveals that compound 2b crys-
for AChE inhibition.
tallizes in the monoclinic structure with space group P21/c. The
Of the library B analogues (4a–c) shown in Table 7, com- asymmetric unit of compound 2b is shown in Fig. 2, while the
pound 4c exhibited potent inhibition for AChE with an IC50 selected bond lengths and bond angles are listed in Table S1,
value of 0.22 mM, followed by compound 4b (IC50 = 0.31 mM) ESI.† It can be seen that the 3D supramolecular framework of
and compound 4a (IC50 = 0.57 mM). The moderate profile of compound 2b is stabilized via an intricate array of H-bonding
compounds 4a and 4b for AChE inhibition is believed to be due and weak pꢁ ꢁ ꢁp interactions which are depicted in Fig. 3.
Moreover, the packing diagram of compound 2b is shown in
Fig. 4a and b.
Table 7 Quantitative estimation of the acetylcholinesterase inhibition
activity of compounds (4a–c) by a modified Ellaman’s coupled enzyme
assay method using tacrine as the reference (n = 3)
2.4. Results of DFT calculations
In order to gain some insight into the influence of the inter-
molecular interactions on the molecular geometry, we have
performed quantum mechanical calculations of the equilibrium
geometry of the free molecule. The DFT calculations closely
reproduce the solid-state geometry of the molecule. The ground
Library B
state optimized structure of compound 2b is shown in Fig. 5a,
Nature of
substituents
IC50 (mM)a ꢂ SEMb for
hAChEc inhibition
wherein the dimethoxyphenyl part of the molecule is planar with
the acrylic moiety, while the nitrophenyl group is pushed out of
the plane, with a dihedral angle of B271 (C10–C12–C17–C18),
probably by the steric interactions between the two hydrogen
atoms (H11–H27). The optimized geometry exhibits the
(Z)-configuration of the dimethoxyphenyl and nitrophenyl groups
about the acrylic double bond (Fig. 5a). The HOMO depicts more
electronic density over the dimethoxyphenyl rings, with a ground
Entry Product
1
2
3
4
4a
4b
4c
R1 = R2 = H
R1 = H; R2 = Br
R1 = NH2; R2 = H 0.22 ꢂ 0.04
0.57 ꢂ 0.03
0.31 ꢂ 0.05
Standard (tacrine)
0.19 ꢂ 0.02
a
IC50 values represent the concentration of inhibitor required to decrease
the enzyme activity by 50%. b SEM = standard error of the mean. hAChE =
c
Human recombinant AChE from human serum was used.
Fig. 2 Asymmetric unit showing (a) thermal ellipsoids (50% probability level). Hydrogen atoms have been omitted for clarity. (b) Ball and stick model
of compound 2b.
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Fig. 3 Representation of (a) H-bonding interactions and (b) pꢁ ꢁ ꢁp interactions in compound 2b.
Fig. 4 Packing diagrams of compound 2b.
state dipole moment vector pointing towards the dimethoxy difference is the reason that during the crystallization process,
substituents with a magnitude of 9.49 D suggesting an inhomo- the (Z)-isomer exclusively crystallized out.
geneous distribution of charge in the HOMO (Fig. 6). Single
Theoretical calculations were also employed to predict the
point time-dependent density functional theory calculations infrared (IR) spectrum of the (Z)-form of the molecule (Fig. 7).
of the Franck–Condon state estimated maximum oscillator Based on the vibrational motion corresponding to different
strength for the HOMO to LUMO transition, with an energy spectral lines, various assignments were made. The dominant
difference of 378 nm. The electronic density is redistributed in line at 1363 cmꢀ1 corresponds to the stretching frequency of
the Franck–Condon state as predicted by its distribution in the the nitro group. The 1000–1500 cmꢀ1 region represents the
LUMO, wherein the density is delocalized more towards the combined skeletal vibrations of the molecule, and the lines at
nitrophenyl moiety (Fig. 6). Ground state optimization was also 1604 cmꢀ1 and 1615 cmꢀ1 are dominated by the CQC stretch-
carried out for the (E)-isomer (Fig. 5b) of this molecule, using ing vibration. The CN stretching vibrational mode is observed
the same level of theory, to confirm the relative stability of the at 2312 cmꢀ1. These results are nearly in concordance with
two isomers. It was found that the (Z)-isomer is stabilized by the experimentally obtained IR spectrum of the molecule
2.61 kcal molꢀ1 more than the (E)-isomer, and this small energy (Fig. S1, ESI†).
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Fig. 5 Ground state optimized structures of the (a) (Z)-isomer and (b) (E)-isomer of compound 2b.
Fig. 6 Electron density distribution in the (a) HOMO and (b) LUMO of the (Z)-isomer.
The products were obtained in a substantial yield (83–92%) with
high purity. The notable features of this procedure are mild
reaction conditions, operational simplicity, enhanced reaction
rates, cleaner reaction profiles and simple experimental and
product separation procedures, which make this method attrac-
tive. SAR studies reveal that the position and number of methoxy
groups attached to the benzene moiety inflict prominent effects
on AChE inhibition. The methoxy group at the 4 position in the
benzene moiety (ring A) was found to be obligatory for activity.
4. Experimental section
4.1. Materials and general methods
Fig. 7 Theoretically obtained infrared (IR) spectrum of the (Z)-isomer by
Chemicals were purchased from Merck and Sigma-Aldrich as
‘synthesis grade’ and used without further purification. The IR
spectra were recorded with a Shimadzu IR-408 Perkin-Elmer
DFT calculations.
1800 instrument (FTIR), and the values are given in cmꢀ1
.
3. Conclusions
1H NMR and 13C NMR spectra were run in CDCl3 on a Bruker
The present work reports the facile, convenient and eco-friendly, Avance-II 400 MHz instrument with TMS as the internal standard
silica chloride-assisted synthesis of (Z)-acrylonitrile derivatives and J values measured in Hertz. Chemical shifts are reported in
(library A & B) of substituted aromatic aldehydes and chromones. ppm (d) relative to TMS. Mass spectra were recorded on a JEOL
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D-300 mass spectrometer. Melting points were determined on a 13C NMR (100 MHz, DMSO, d, ppm): 162.3 (C-30 & C-50), 146.3
Kofler apparatus and are uncorrected. Elemental analysis (C, H, N) (C-400), 139.5 (C-100), 138.3 (C-3), 127.3 (C-10), 125.5 (C-200 & C-600),
was conducted using Carlo Erba analyzer model 1108. Thin layer 124.2 (C-300 & C-500), 120.6 (C-20 & C-60), 117.3 (CRN), 116.3 (C-40),
+
ꢄ
chromatography (TLC) glass plates (20 ꢃ 5 cm) were coated with 112.6 (C-2), 55.9 (OCH3). MS (ESI) m/z: 311 [M+H] .
silica gel G (Merck) and exposed to iodine vapor to check the
homogeneity as well as the progress of the reaction.
4.2.4. (Z)-3-(30,40-Dimethoxyphenyl)-2-(400-nitrophenyl)-acrylo-
nitrile (2d). Compound 2d recrystallized from CHCl3–MeOH as a
white creamy solid. Yield: 89%, m.p. 152–153 1C; anal. calc. for
C17H14N2O4: C, 65.80; H, 4.55; N, 9.03; found: C, 65.80; H, 4.56; N,
9.02. IR nKmBarx cmꢀ1: 2224 (CRN), 1580, 1455 (CQCaromatic), 1510,
4.2. General method for the synthesis of (Z)-2-(4-nitrophenyl)-
acrylonitrile derivatives of the substituted aromatic aldehydes
To a mixture of substituted aromatic aldehydes (1a–i) and 1335 (NO2), 1225 (C–O). 1H NMR (400 MHz, DMSO, d, ppm):
p-nitrophenylacetonitrile (1 mmol) in ethanol (20 mL), silica 8.02 (dd, 2H, H-300 & H-500), 7.71 (s, 1Holefinic, H-3), 7.62 (dd, 2H,
chloride was added (2.5 mol%). The reaction mixture was H-200 & H-600), 6.97 (dd, 2H, H-50 & H-60), 6.85 (s, 1H, H-20), 3.02
allowed to stir at room temperature for 1.5–2 hours. During (s, 6H, 2 ꢃ OCH3). 13C NMR (100 MHz, DMSO, d, ppm): 158.6
stirring, the clear solution of the reaction mixture began to turn (C-30 & C-40), 147.1 (C-400), 141.4 (C-100), 139.7 (C-3), 128.6 (C-10),
thick, and a solid product precipitated. After completion of the 124.9 (C-200 & C-600), 124.6 (C-300 & C-500), 123.8 (C-20 & C-50), 120.1
reaction, as evident from TLC, the formed solid was filtered and (C-60), 118.2 (CRN), 113.0 (C-2), 57.3 (OCH3). MS (ESI) m/z:
washed with hot methanol to recover the catalyst. The filtrate 311 [M+H]+
containing a soluble product was evaporated under reduced
4.2.5. (Z)-3-(30-Nitrophenyl)-2-(400-nitrophenyl)-acrylonitrile (2e).
ꢄ
.
pressure to obtain a crude product. The crude product obtained Compound 2e recrystallized from CHCl3–acetone as a yellowish
was washed with appropriate solvents, filtered, dried and solid. Yield: 88%, m.p. 178–179 1C; anal. calc. for C15H9N3O4: C,
crystallized from appropriate solvents. The catalyst was reused 61.02; H, 3.07; N, 14.23; found: C, 61.03; H, 3.07; N, 14.20.
KBr
without a significant reduction of the yield.
IR n
cmꢀ1: 2217 (CRN), 1585, 1445 (CQCaromatic), 1520,
max
1
4.2.1. (Z)-3-(30,40,50-Trimethoxyphenyl)-2-(400-nitrophenyl)-acrylo- 1325 (NO2). H NMR (400 MHz, DMSO, d, ppm): 8.21 (dd, 2H,
nitrile (2a). Compound 2a recrystallized from CHCl3–MeOH as a H-300 & H-500), 8.15 (s, 1H, H-20), 8.04 (dd, 1H, H-40), 7.69
brickish red solid. Yield: 87%, m.p. 91–92 1C [lit.,60 m.p. 90 1C]; anal. (s, 1Holefinic, H-3), 7.35 (dd, 2H, H-200 & H-600), 7.21 (m, 2H,
calc. for C18H16N2O5: C, 63.52; H, 4.74; N, 8.23; found: C, 63.54; H, H-50 & H-60). 13C NMR (100 MHz, DMSO, d, ppm): 148.3 (C-30 &
4.74; N, 8.22. IR nKmBarx cmꢀ1: 2214 (CRN), 1575, 1456 (CQCaromatic), C-400), 141.1 (C-100), 138.4 (C-3), 135.3 (C-10), 129.5 (C-50), 127.4
1508, 1329 (NO2), 1254 (C–O). 1H NMR (400 MHz, DMSO, d, ppm): (C-200 & C-600), 126.8 (C-300 & C-500), 125.7 (C-20), 125.4 (C-40), 121.5
8.32 (dd, 2H, H-300 & H-500), 7.95 (dd, 2H, H-200 & H-600), 7.85 (C-60), 116.9 (CRN), 109.2 (C-2). MS (ESI) m/z: 296 [M+H]+
.
ꢄ
(s, 1Holefinic, H-3), 7.21 (s, 2H, H-20 & H-60), 3.73 (s, 9H, 3 ꢃ
4.2.6. (Z)-3-(40-Dimethylaminophenyl)-2-(400-nitrophenyl)-acrylo-
OCH3). 13C NMR (100 MHz, DMSO,0d0 , ppm): 150.7 (C-30 & C-50), nitrile (2f). Compound 2f recrystallized from CHCl3–MeOH as a
145.4 (C-400), 143.9 (C-40), 138.8 (C-1 ), 137.3 (C-3), 129.4 (C-200 & reddish solid. Yield: 85%, m.p. 231–232 1C [lit.,60 m.p. 230 1C];
C-600), 127.4 (C-10), 124.3 (C-300 & C-500), 121.4 (C-20 & C-60), 11ꢄ7.6 anal. calc. for C17H15N3O2: C, 69.61; H, 5.15; N, 14.33; found: C,
(CRN), 106.4 (C-2), 55.7 (OCH3). MS (ESI) m/z: 341 [M+H]+
.
69.60; H, 5.14; N, 14.33. IR nKmBarx cmꢀ1: 2212 (CRN), 1575, 1447
4.2.2. (Z)-3-(20,30-Dimethoxyphenyl)-2-(400-nitrophenyl)-acrylo- (CQCaromatic), 1513, 1310 (NO2), 1277 (C–N). 1H NMR (400 MHz,
nitrile (2b). Compound 2b recrystallized from CHCl3–MeOH as DMSO, d, ppm): 8.17 (dd, 2H, H-300 & H-500), 7.65 (dd, 2H, H-200 &
bluish crystals. Yield: 91%, m.p. 190–191 1C; anal. calc. for H-600), 7.53 (s, 1Holefinic, H-3), 7.25 (dd, 2H, H-20 & H-60), 6.58
C
17H14N2O4: C, 65.80; H, 4.55; N, 9.03; found: C, 65.80; H, 4.54; (dd, 2H, H-30 & H-50), 2.78 (s, 6H, 2 ꢃ CH3). 13C NMR (100 MHz,
KBr
max
N, 9.02. IR n
cmꢀ1: 2210 (CRN), 1571, 1476 (CQCaromatic), DMSO, d, ppm): 147.3 (C-400), 142.5 (C-40), 141.1 (C-100), 138.3
1511, 1345 (NO2), 1273 (C–O). 1H NMR (400 MHz, DMSO, (C-3), 127.3 (C-200 & C-600), 125.2 (C-300 & C-500), 124.8 (C-20 & C-60),
d, ppm): 8.34 (dd, 2H, H-300 & H-500), 8.17 (s, 1Holefinic, H-3), 121.5 (C-10), 117.3 (CRN), 113.4 (C-30 & C-50), 108.3 (C-2), 45.3
7.97 (dd, 2H, H-200 & H-600), 7.18–7.21 (m, 3H, H-40, H-50 & H-60), (CH3). MS (ESI) m/z: 294 [M+H]+
.
ꢄ
3.90 (s, 6H, 2 ꢃ OCH3). 13C NMR (100 MHz, DMSO, d, ppm):
4.2.7. (Z)-3-(30-Methoxy-40-hydroxyphenyl)-2-(400-nitrophenyl)-
152.2 (C-30), 148.3 (C-20), 147.3 (C-400), 140.6 (C-100), 140.1 (C-3), acrylonitrile (2g). Compound 2g recrystallized from CHCl3–MeOH
126.8 (C-200 & C-600), 126.7 (C-10), 123.9 (C-300 & C-500), 122.6 (C-50), as a white solid. Yield: 87%, m.p. 145–146 1C; anal. calc. for
119.2 (C-60), 116.5 (CRN), 115.5 (C-40), 109.9 (C-2), 55.6
C16H12N2O4: C, 64.86; H, 4.08; N, 9.46; found: C, 64.87; H, 4.08;
N, 9.44. IR n
(OCH3). MS (ESI) m/z: 311 [M+H]+
.
cmꢀ1: 3285 (OH), 2214 (CRN), 1576, 1440
KBr
max
ꢄ
4.2.3. (Z)-3-(30,50-Dimethoxyphenyl)-2-(400-nitrophenyl)-acrylo- (CQCaromatic), 1504, 1298 (NO2), 1227 (C–O). 1H NMR
nitrile (2c). Compound 2c recrystallized from CHCl3–ethyl acetate (400 MHz, DMSO, d, ppm): 9.82 (s, 1H, OH), 8.13 (dd, 2H,
as yellowish crystals. Yield: 93%, m.p. 179–180 1C [lit.,60 m.p. H-300 & H-500), 7.83 (s, 1Holefinic, H-3), 7.67 (dd, 2H, H-200 & H-600),
180 1C]; anal. calc. for C17H14N2O4: C, 65.80; H, 4.55; N, 9.03; 6.81–7.15 (m, 3H, H-20, H-50 & H-60), 3.14 (s, 3H, OCH3).
KBr
max
found: C, 65.79; H, 4.54; N, 9.04. IR n
cmꢀ1: 2215 (CRN), 13C NMR (100 MHz, DMSO, d, ppm): 151.4 (C-30), 145.3
1582, 1462 (CQCaromatic), 1506, 1338 (NO2), 1206 (C–O). 1H NMR (C-400), 143.1 (C-40), 137.6 (C-100), 135.6 (C-3), 127.6 (C-300
&
(400 MHz, DMSO, d, ppm): 8.19 (dd, 2H, H-300 & H-500), 7.81 C-500), 125.2 (C-200 & C-600), 123.6 (C-10), 119.4 (C-60), 118.2
(s, 1Holefinic, H-3), 7.65 (dd, 2H, H-200 & H-600), 7.15 (s, 2H, (CRN), 116.0 (C-50), 113.2 (C-20), 106.1 (C-1), 56.8 (OCH3).
H-20 & H-60), 7.01 (s, 1H, H-40), 3.71 (s, 6H, 2 ꢃ OCH3). MS (ESI) m/z: 297 [M+H]+
.
ꢄ
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4.2.8. (Z)-3-(30,40,50-Trihydroxyphenyl)-2-(400-nitrophenyl)- anal. calc. for C18H9BrN2O4: C, 54.43; H, 2.28; N, 7.05; found: C,
KBr
max
acrylonitrile (2h). Compound 2h recrystallized from CHCl3– 54.43; H, 2.27; N, 7.04. IR n
cmꢀ1: 2226 (CRN), 1650
MeOH as a brownish solid. Yield: 84%, m.p. 233–234 1C; anal. (CQOg-pyrone), 1599 (CQCg-pyrone), 1464 (CQCaromatic), 1519,
calc. for C15H10N2O5: C, 60.41; H, 3.38; N, 9.39; found: C, 60.40; 1341 (NO2), 599 (C–Br). 1H NMR (400 MHz, DMSO, d, ppm):
KBr
H, 3.37; N, 9.40. IR n
cmꢀ1: 3345, 3383 (OH), 2223 (CRN), 8.01 (dd, 2H, H-300 & H-500), 7.91 (dd, 2H, H-200& H-600), 7.82
max
1
1590, 1445 (CQCaromatic), 1494, 1305 (NO2), 1214 (C–O). H NMR (s, 1H, H-50), 7.61 (s, 1Holefinic, H-3), 7.32 (dd, 2H, H-70 & H-80),
(400 MHz, DMSO, d, ppm): 10.31 (s, OH), 9.34 (s, OH), 8.02 (dd, 7.20 (s, 1H, H-20). 13C NMR (100 MHz, DMSO, d, ppm): 177.4
2H, H-300 & H-500), 7.74 (s, 1Holefinic, H-3), 7.61 (dd, 2H, H-200 & H-600), (C-40), 161.2 (C-20), 155.8 (C-8a), 145.2 (C-400), 140.1 (C-100), 137.8
6.97 (s, 2H, H-20 & H-60). 13C NMR (100 MHz, DMSO, d, ppm): (C-2), 133.7 (C-70), 130.8 (C-200 & C-600), 128.3 (C-60), 124.1 (C-50),
146.6 (C-400), 145.2 (C-30 & C-50), 137.2 (C-100), 134.1 (C-3), 132.3 123.9 (C-4a), 120.7 (C-300 & C-500), 119.0 (C-80), 117.8 (CRN),
(C-40), 129.7 (C-10), 127.2 (C-300 & C-500), 126.4 (C-200 & C-600), 115.9 112.4 (C-3), 110.1 (C-30). MS (ESI) m/z: 397 [M+H]+
(CRN), 112.3 (C-20 & C-60), 107.4 (C-2). MS (ESI) m/z: 299 [M+H] .
.
ꢄ
+
ꢄ
4.3.3. (Z)-3-(20-Amino-4-oxo-4H-chromen-3-yl)-2-(400-nitro-
4.2.9. (Z)-3-(30,50-Dihydroxyphenyl)-2-(400-nitrophenyl)-acrylo- phenyl)-acrylonitrile (4c). Compound 4c recrystallized from
nitrile (2i). Compound 2i recrystallized from CHCl3–ethylacetate CHCl3–MeOH as a brownish solid. Yield: 87%, m.p. 187–188 1C;
as an orange solid. Yield: 83%, m.p. 181–182 1C; anal. calc. for anal. calc. for C18H11N3O4: C, 64.86; H, 3.33; N, 12.61; found: C,
C15H10N2O4; C, 63.83; H, 3.57; N, 9.92; found: C, 63.84; H, 3.56; 64.86; H, 3.32; N, 12.60. IR nKmBarx cmꢀ1: 3492 (NH2), 2223 (CRN),
N, 9.95. IR nKmBarx cmꢀ1: 3387, 3312 (OH), 2216 (CRN), 1583, 1460 1631 (CQOg-pyrone), 1603 (CQCg-pyrone), 1555, 1464 (CQCaromatic),
1
(CQCaromatic), 1485, 1315 (NO2), 1217 (C–O). 1H NMR (400 MHz, 1515, 1345 (NO2). H NMR (400 MHz, DMSO, d, ppm): 8.34 (dd,
DMSO, d, ppm): 9.87 (s, OH), 8.14 (dd, 2H, H-300 & H-500), 7.75 2H, H-300 & H-500), 7.97 (dd, 2H, H-200 & H-600), 7.89 (s, 2H, NH2, D2O
(s, 1Holefinic, H-3), 7.63 (dd, 2H, H-200 & H-600), 6.97 (s, 2H, H-20 & exchangeable), 7.23–7.85 (m, 4H, H-50, H-60, H-70 & H-80), 6.98
H-60), 6.75 (s, 1H, H-40). 13C NMR (100 MHz, DMSO, d, ppm): (s, 1Holefinic, H-3). 13C NMR (100 MHz, DMSO, d, ppm): 180.1
154.7 (C-30 & C-50), 144.8 (C-400), 141.2 (C-100), 138.5 (C-3), 127.3 (C-40), 166.8 (C-20), 154.7 (C-8a), 144.2 (C-400), 142.3 (C-100), 138.1
(C-300 & C-500), 125.9 (C-200 & C-600), 124.8 (C-20 & C-60), 121.6 (C-10), (C-2), 131.5 (C-70), 130.4 (C-200 & C-600), 129.1 (C-60), 125.6 (C-50),
+
ꢄ
118.4 (CRN), 109.7 (C-40), 107.5 (C-2). MS (ESI) m/z: 283 [M+H] . 123.4 (C-4a), 121.2 (C-300 & C-500), 118.3 (C-80), 116.4 (CRN), 114.2
+
(C-3), 112.7 (C-30). MS (ESI) m/z: 334 [M+H] .
ꢄ
4.3. General method for the synthesis of (Z)-2-(4-nitrophenyl)-
acrylonitrile derivatives of substituted 3-formyl chromones
4.4. Single crystal X-ray crystallographic studies of compound 2b
To a mixture of substituted 3-formyl chromones (3a–c) and Single crystal X-ray data of compound 2b was collected at 100 K
p-nitrophenylacetonitrile (1 mmol) in ethanol (20 mL), silica on a Bruker SMART APEX CCD diffractometer using graphite
chloride was added (2.5 mol%). The reaction mixture was monochromated MoKa radiation (l = 0.71073 Å). The linear
allowed to stir at room temperature for 1.5–2 hours. The clear absorption coefficients, scattering factors for the atoms and the
solution of the reaction mixture began to turn thick and the solid anomalous dispersion corrections were taken from the Inter-
product began to settle out. After completion of the reaction, as national Tables for X-ray Crystallography.61 The data integration
evident from TLC, the formed solid was filtered and washed with and reduction were carried out with SAINT software.62 An empirical
hot methanol to recover the catalyst. The filtrate containing the absorption correction was applied to the collected reflections with
soluble product was evaporated under reduced pressure to SADABS, and the space group was determined using XPREP.63 The
obtain the crude product. The crude product was washed with structure was solved by direct methods using SHELXTL-97 and
appropriate solvents, filtered, dried and crystallized from appro- refined on F2 by full-matrix least-squares using the SHELXL-9764
priate solvents. The catalyst was reused without significant program package. All non-hydrogen atoms were refined aniso-
reduction of the yield.
tropically. Pertinent crystallographic data for compound 2b is
4.3.1. (Z)-3-(40-Oxo-4H-chromen-3-yl)-2-(400-nitrophenyl)-acrylo- summarized in Table 8.
nitrile (4a). Compound 4a recrystallized from CHCl3–MeOH as
4.5. Density functional theory (DFT) calculations
a yellowish solid. Yield: 90%, m.p. 194–195 1C; anal. calc. for
18H10N2O4: C, 67.92; H, 3.17; N, 8.80; found: C, 67.93; H, 3.19; Quantum mechanical calculations were pursued for the mole-
C
KBr
max
N, 8.81. IR n
cmꢀ1: 2218 (CRN), 1650 (CQOg-pyrone), 1610 cule 2b, the crystal structure of which has been discussed.
(CQCg-pyrone), 1563, 1464 (CQCaromatic), 1515, 1341 (NO2). The B3LYP functional and 6-311+g(d,p) basis set were used to
1H NMR (400 MHz, DMSO, d, ppm): 8.32 (dd, 2H, H-300 & H-500), carry out the ground state optimization of the molecule in the
7.99 (dd, 2H, H-200& H-600), 7.85 (dd, 2H, H-50 & H-80), 7.65 (s, 1Holefinic
,
Gaussian 09 software package.65 A vibrational analysis was
H-3), 7.37 (dd, 2H, H-60 & H-70), 7.23 (s, 1H, H-20). 13C NMR performed for compound 2b to calculate the IR spectrum.
(100 MHz, DMSO, d, ppm): 178.9 (C-40), 162.5 (C-20), 155.2 (C-8a), We also calculated the single point energy for the optimized
147.3 (C-400), 139.2 (C-100), 136.8 (C-2), 134.3 (C-70), 129.7 (C-200 & C-600), structures of the (Z)- and (E)-conformations of compound 2b
125.5 (C-60), 124.8 (C-50), 123.6 (C-4a), 122.4 (C-300 & C-500), 118.6 (C-80), using the B3LYP/6-311+g(d,p) level of theory.
+
ꢄ
117.2 (CRN), 116.3 (C-3), 110.5 (C-30). MS (ESI) m/z: 318 [M+H] .
4.6. In vitro acetylcholinesterase inhibition activity
4.3.2. (Z)-3-(60-Bromo-40-oxo-4H-chromen-3-yl)-2-(400-nitro-
phenyl)-acrylonitrile (4b). Compound 4b recrystallized from CHCl3– The ability of compounds (2a–i) and (4a–c) to inhibit AChE
acetone as a grayish white solid. Yield: 92%, m.p. 203–204 1C; activity was assessed by Ellman’s method.66 AChE stock solution
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Table 8 Crystallographic data and structure refinement of compound 2b
References
Compound
Empirical formula
Formula wt
Crystal system
Space group
a (Å)
b (Å)
c (Å)
a (1)
b (1)
2b
1 E. Knoevenagel, Ber. Dtsch. Chem. Ges., 1898, 31, 2585–2596.
2 F. Bigi, L. Chesini, R. R. Maggi and G. Sartori, J. Org. Chem.,
1999, 64, 1033–1035.
3 N. Yu, J. M. Aramini, M. W. Germann and Z. Huang,
Tetrahedron Lett., 2000, 41, 6993–6996.
4 G. Jones, The Knoevenagel condensation reaction, Organic
Reactions, Wiley, New York, 1967, vol. 15, p. 204.
5 L. F. Tietze and U. Beifuss, Comprehensive Organic Synthesis,
Pergamon, New York, 1991, vol. 2, pp. 341–394.
6 S. M. J. Mukarram, R. A. R. Khan and R. P. Yadav, US Pat.,
0004935, 2007.
7 L. R. Madivada, R. R Anumala, G. Gilla, S. Alla, K. Charagondla,
M. Kagga, A. Bhattacharya and R. Bandichhor, Org. Process Res.
Dev., 2009, 13, 1190–1194.
C17H14N2O4
310.30
Monoclinic
P21/c
15.634(5)
4.720(2)
19.585(5)
90
91.229(5)
90
1444.9(16)
4
1.426
0.103
648
g (1)
U (Å3)
Z
rcalc (Mg mꢀ3
)
m (mmꢀ1
F(000)
)
Refl. collected
Independent refl.
GOF
Final R indices
[I 4 2s(I)]
R indices
(all data)
7121
2178
1.129
R1 = 0.0494a
wR2 = 0.1345b
R1 = 0.0633
wR2 = 0.1712
8 U. Beutler, P. C. Fuenfschilling and A. Steinkemper, Org.
Process Res. Dev., 2007, 11, 470–476.
9 A. Carta, P. Sanna, M. Palomba, L. Vargiub, M. La Colla and
R. Loddo, Eur. J. Med. Chem., 2002, 37, 891–900.
10 A. Carta, I. Briguglio, S. Piras, G. Boatto, P. L. Colla, R. Loddo,
M. Tolomeo, S. Grimaudo, A. D. Cristina, R. M. Pipitone,
E. Laurini, M. S. Paneni, P. Posocco, M. Fermeglia and S. Pricl,
Eur. J. Med. Chem., 2011, 46, 4151–4167.
P
P
P
a
b
R1 = ||Fo| ꢀ |Fc||/ |Fo| with Fo2 4 2s(Fo2). wR2 = [ w(|Fo2| ꢀ |Fc2|)2/
P
|Fo2|2]1/2
.
was prepared by dissolving human recombinant AChE (E.C.3.1.1.7)
lyophilized powder (Sigma Aldrich) in 0.1 M phosphate buffer
(pH = 8.0) containing Triton X-100 (0.1%). Five increasing
concentrations of test compounds were assayed to obtain the
% inhibition of the enzymatic activity in the range of 20–80. The
assay solution consisted of a 0.1 M phosphate buffer (pH 8.0),
with the addition of 340 mM 5,50-dithio-bis(2-nitrobenzoic acid),
0.02 units per mL of human recombinant AChE from human
serum and 550 mM of substrate (acetylthiocholine iodide, ATCh).
Increasing concentrations of the tested inhibitor were added to
the assay solution and pre-incubated for 20 min at 37 1C with the
enzyme, followed by the addition of the substrate. Initial rate
assays were performed at 37 1C with a Jasco V-530 double beam
spectrophotometer. The absorbance value at 412 nm was
recorded for 5 min and the enzyme activity was calculated from
the slope of the obtained linear trend. Assays were carried out
with a blank containing all of the components except AChE to
account for the non-enzymatic reaction. The reaction rates were
compared and the percent inhibition due to the presence of the
tested inhibitors was calculated. Each concentration was analyzed in
triplicate, and the IC50 values were determined graphically from log
concentration–inhibition curves (GraphPad Prism 4.03 software,
GraphPad Software Inc.). Tacrine was used as a standard inhibitor.
The AChE inhibition activity of the synthesized compounds is
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