An efficient synthesis of pyridoxal oxime derivatives under microwave irradiation

Article abstract of DOI:10.3390/molecules19067610

Quaternary salts of pyridoxal oxime have been synthesized by the quaternization of pyridoxal oxime with substituted phenacyl bromides using microwave heating. Microwave-assisted rapid synthesis was done both in solvent (acetone) and under solvent-free con

Full text of DOI:10.3390/molecules19067610

Molecules 2014, 19, 7610-7620; doi:10.3390/molecules19067610
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
An Efficient Synthesis of Pyridoxal Oxime Derivatives under
Microwave Irradiation
Dajana Gašo-Sokač 1,*, Valentina Bušić 1, Mario Cetina 2,* and Marijana Jukić 3
1
Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Kuhačeva 20,
31000 Osijek, Croatia; E-Mail: valentina.busic@ptfos.hr
2
University of Zagreb, Faculty of Textile Technology, Department of Applied Chemistry,
Prilaz baruna Filipovića 28a, 10000 Zagreb, Croatia
3
Faculty of Food Technology and Biotechnology, Department of Chemistry and Biochemistry,
Pierottijeva 6, 10000 Zagreb, Croatia; E-Mail: mjukic@pbf.hr
* Authors to whom correspondence should be addressed; E-Mails: dajana.gaso@ptfos.hr (D.G.-S.);
mario.cetina@ttf.hr (M.C.); Tel.: +385-31-224-327 (D.G.-S.); +385-1-3712-590 (M.C.);
Fax: +385-31-207-115 (D.G.-S.); +385-1-3712-599 (M.C.).
Received: 7 May 2014 / in revised form: 28 May 2014 / Accepted: 3 June 2014 /
Published: 6 June 2014
Abstract: Quaternary salts of pyridoxal oxime have been synthesized by the quaternization
of pyridoxal oxime with substituted phenacyl bromides using microwave heating.
Microwave-assisted rapid synthesis was done both in solvent (acetone) and under
solvent-free conditions. Good to excellent yields (58%–94%) were obtained in acetone in
very short reaction times (3–5 min) as well as in the solvent-free procedure (42%–78%) in
very short reaction times (7–10 min) too. Effective metodologies for the preparation of
pyridoxal oxime quaternary salts, having the advantagies of being eco-friendly, easy to
handle, and performed in shorter reactions time are presented. The structure of compound
7, in which a 4-fluorophenacyl moiety is bonded to the pyridinium ring nitrogen atom, was
unequivocally confirmed by the single-crystal X-ray diffraction method.
Keywords: eco-friendly quaternization; microwave synthesis; phenacyl bromides;
pyridoxal oxime

Molecules 2014, 19
7611
1. Introduction
The development of cleaner technologies is a major emphasis in green chemistry. Among the
several aspects of green chemistry, avoiding the use of volatile organic solvents in the reaction
medium is recommended. The use of the large excesses of conventional volatile solvents required to
conduct a chemical reaction creates ecological and economical concerns. Therefore the search for a
nonvolatile and recyclable alternative, as well as performing reactions without solvents has a key role
in this field of research [1].
During recent years, microwaves (MW) have been extensively used for carrying out chemical
reactions and have become a useful non-conventional energy source for performing organic synthesis.
The application of microwaves in chemistry is extremely attractive and, from the very beginning it was
realized that a number of chemical processes can be carried out with substantial reduction of the
reaction time in comparison to conventional methods [2].
Several papers have been reported on the alkylation of N-containing heterocycles [3–6]. The first
solventless quaternization via microwave heating has been described for preparation of variety of ionic
liquids [1]. Perez and coworkers have described the N-alkylation of azoles with 4-bromophenacyl
bromide under MW irradiation under solvent-free conditions. The results obtained showed high yields
and selectivity [7].
Organophosphorus compounds are widely used in agriculture as insecticides, in industry and
technology, as well as in military technology as chemical warfare agents (sarin, soman, tabun). They
are extremely potent inhibitors of the enzyme acetylcholinesterase (AChE) that is responsible for the
termination of the action of acetylcholine at cholinergic synapses [8,9]. There are many commonly
used reactivators of inhibited AChE, such as 2-pralidoxime, trimedoxime, and toxogonin [10–12].
Unfortunately, none of the currently used oximes is sufficiently effective against all inhibitors and
there is no single reactivator having the ability to reactivate inhibited enzyme, regardless of the
inhibitor chemical structure [13,14].
Previously, we synthesized a series of novel pyridinium oximes and tested them as reactivators of
AChE inhibited by organophosphosphorus compounds tabun and paraoxon [15]. In view of the
emerging importance of the quartenary salts as antidotes, antibacterial and anticancerogenic agents and
our general interest in microwave-assisted chemical processes, we decided to explore the synthesis of
those compounds using MW irradiation in the presence of solvents and under solvent-free conditions.
In our earlier paper [15] we prepared a series of novel pyridinium oximes under classical conditions.
These reactions have some major disadvantages, including long reaction times (1–3 weeks), the usage
of large amounts of solvents, etc. For these reasons we decided to use MW technology, as a
nonconventional method for their synthesis. Herein, we report an efficient method for the preparation
of quaternary salts that simply involves exposing neat reactants to MW using a Milestone single-mode
microwave reactor.
2. Results and Discussion
In this article two MW techniques are presented: MW irradiation in the presence of solvent
(acetone) and MW irradiation under solvent free-conditions. The work up was easy and the products

Molecules 2014, 19
7612
were obtained in excellent to moderate yields (as shown in Table 1). This method is efficient, versatile
and generates little waste. The results clearly demonstrate quaternization by the classical method is
possible as well as via the MW irradiation method, but the synthetic efficiency was significantly
different, as evidenced by the data of Table 1. Scheme 1 outlines the synthesis of 2–10. Optimum
conditions for carrying out the MW-assisted quaternizations were ascertained by carrying out a series
of reactions of pyridoxal oxime with substituted phenacyl bromides. The results showed that
maximum yield of 94% was obtained by microwave-assisted procedure in acetone for 5, while
maximum yield of 79% was obtained in MW solvent-free procedure for 6. All the products were
crystalline crude products.
Scheme 1. A general schematic representation for the preparation of quaternary salts of
pyridoxal oxime.
Table 1. The optimized conditions in the synthesis of quaternary salts under MW heating
in acetone A and solvent-free procedure B as well as under classical heating.
MW (A)
MW (B)
Conventional [15]
Compound
R
t/min Yield (%) t/min Yield (%) t/weeks Yield (%)
2
3
4'-Cl
4'-Br
5
4.5
5
75
90
74
94
80
70
90
79
58
10
10
7
63
74
53
76
79
48
71
46
42
3
3
66
44
38
62
60
36
67
46
12
4
4'-H
1
5
4'-CH3
4'-NO2
4'-F
4
8
1
6
4.5
3
10
10
10
10
10
1
7
3
8
4'-OCH3
4'-Ph
5
3
9
5
1
10
2'-OCH3
5
12
Interestingly, the same reactions carried out under conventional conditions using acetone, methanol and DMF
as a solvents gave 2–10 in lower yields, and required significantly longer reaction time [15].
The syntheses were done in a Milestone controllable single-mode microwave reactor. The reactor is
equipped with a magnetic stirrer as well as temperature and power controls. The effect of microwave
irradiation on a set of reactions using pyridoxal oxime and substituted phenacyl bromides as reactants
was examined. Under these conditions a very efficient, fast, and practical method for the preparation of
quaternary salts was developed. The time required to synthesize the salts compared to conventional
method is strongly reduced.

Molecules 2014, 19
7613
In case of the microwave-assisted reactions using organic volatile solvents, the reactants are usually
dissolved in the solvent, which often couples effectively with microwaves and thus acts as the energy
transfer medium. Acetone was selected as MW solvent because the highest yield has been obtained
with it compared to those obtained in DMF and methanol via the conventional technique [15].
The reaction carried out in acetone via MW irradiation required a maximum of 5 min of irradiation.
The white and light yellow color of the reactants turned yellow brownish as the mixing and
irradiation progressed.
In the microwave-assisted quaternizations in acetone the products required no rigorous purification
and pure products were obtained by simple recrystallization from an appropriate solvent. Therefore,
this method offers an easy practical access for the production of a series of quaternary salts.
Solvent-free methods are especially adapted to organic synthesis under green chemistry conditions.
When coupled to MW irradiation, these methods result in very efficient and noticeable improvements
over classical methods. The absence of solvent reduces the risk of explosions, moreover, aprotic
solvents with high boiling points are expensive and difficult to remove from the reaction mixtures.
This solvent-free approach requires only a few min of reaction time in contrast to few weeks under
conventional heating conditions. Although this synthesis offers several advantages over traditional
methods, there is still need for improving the postsynthetic treatment where solvents are also used.
Quaternization of pyridoxal oxime with substituted phenacyl bromides was accelerated under MW
irradiation. Adsorption of reactant molecules on the surface of silica gel was promoter force for this
reaction. It was interesting that without silica gel added the reaction was not successful. The silica gel
surface was an active catalyst for reaction of quaternization in this solventless procedure. In spite of
the fact that “dry” microwave procedure demand easy work up it is obvious that purification in
procedure done in acetone is increasingly shorter and less rigorous. The solventless quaternization
MW procedure requires only a few min of reaction time but purification was quite longer as compared
with MW procedure in acetone due to the presence of starting materials and some byproducts.
The lowest yields in both the solvent-free method (42%) and in acetone (58%) was obtained for the
quaternization of pyridoxal oxime with 2-bromo-1-(4-metoxyphenyl)ethanone. This low yield can be
explained by the existence of an ortho-methoxy substituent in the electrophile which prevents attack
on the pyridoxal oxime at the nitrogen atom due to steric hindrance.
The structure of compound 7 was unequivocally confirmed by single-crystal X-ray diffraction
method. In 7 (Figure 1), the 4-fluorophenacyl moiety is bonded to the pyridinium ring N1 atom. The
C2-N1-C6 bond angle in the pyridinium ring is widened [123.2(2)°]. However, sum of the endocyclic
bond angles is 720°, as expected for aromatic six-membered ring. The bond lengths and angles agree
quite closely with equivalent ones in structures of 3-hydroxy-4-hydroxyiminomethyl-5-
hydroxymethylpyridinium derivatives [16,17]. The C2 ring atom is in synclinal position with respect
to the C11 atom of the phenacyl moiety [C2−N1−C10−C11 = 77.2(3)°]. This indicates that phenyl ring
is rotated with respect to the pyridinium ring, as also shown by the dihedral angle between mean ring
planes of 55.38(13)°.

Molecules 2014, 19
7614
Figure 1. A molecular structure of 7, with the atom-numbering scheme. Displacement
ellipsoids for non-hydrogen atoms are drawn at the 50% probability level.
In the cation, one strong O−H···N intramolecular hydrogen bond [O1···N2 = 2.579(3) Å] forms
six-membered ring, and one C−H···O intramolecular hydrogen bond [C6···O3 = 2.648(3) Å] generates
five-membered ring (Figure 1).
The pyridinium cation and bromide are linked by various intermolecular interactions. Two
pyridinium cations and two bromides are assembled by one strong O−H···Br− hydrogen bond
[O3···Br1 = 3.451(2) Å] and two C−H···Br− hydrogen bonds [C10···Br1 = 3.667(3) Å; C6···Br1 =
3.817(3) Å], so forming ring consists of two cations and two anions (Figure 2).
Figure 2. Crystal packing diagram along the a axis, showing three-dimensional network
formed by O−H···Br−, C−H···Br−, C−H···O and C−H···F intermolecular hydrogen bonds.
Bromides are presented in ball and stick style.

Molecules 2014, 19
7615
This ring is further linked by O−H···Br−, C−H···O i C−H···F hydrogen bonds [O2···Br1 = 3.131(2) Å;
C8···O2 = 3.479(3) Å; C16···O4 = 3.486(4) Å; C7···F1 = 3.136(4) Å] into three-dimensional network.
One π...π interaction participates also in supramolecular assembling. The phenyl rings of the
neighbouring molecules are mutually parallel [α = 0°], a centroid separation is 3.6374(17) Å, an
interplanar spacing ca 3.33 Å and offset ca. 1.46 Å.
3. Experimental Section
3.1. General Information
All reactions were performed using a, Microwave Synthesis Labstation Start S controllable
single-mode microwave reactor (Milestone, Shelton, CT, USA). The reactor is equipped with a
magnetic stirrer as well as a temperature and power controls (220 V/50–60 Hz, 2.4 kW). Solvents and
reagents were purchased from Fluka (Milwaukee, WI, US) and Aldrich (St. Luis, MO, US) and used
without further purification. TLC was done by using chloroform–methanol (6:1.5, v/v) as the
developing solvent. The silica gel (Aldrich, 0.063–0.200 nm; 70–230 mesh) was used in solvent-free
method. IR spectra were measured on a FTIR-8400S spectrophotometer (Shimadzu, Kyoto, Japan) in
1
13
KBr pellets. H-NMR and C-NMR spectra were measured on a XL-GEM 300 spectrophotometer
(Varian, Rheinstetten, Germany) in DMSO-d6 solutions and chemical shifts are reported in δ values
downfield from TMS as an internal standard. The compounds are also characterized by elemental analyses.
Melting points were determined with a SMP3 melting point apparatus (Stuart, Staffordshire, UK).
3.2. General Procedure for the Synthesis of Compounds 2–10 with MW Procedure A
Pyridoxal oxime (0.18 g; 1 mmol) was dissolved in acetone (50 mL) at 50 °C. The reaction mixture
was cooled to room temperature, and substituted phenacyl bromide was added (1 mmol). The mixture
was placed in the Milestone single-mode microwave reactor and irradiated at with starting power at
440 W and reaction temperature 56 °C until TLC analysis has shown the presence of the product.
When the irradiation was stopped the mixture was left in the dark to cool and the crystalline crude
product was collected by filtration under reduce pressure and recrystallized from appropriate solvent.
3.3. General Procedure for the Solvent-Free Synthesis of Compounds 2–10 with MW Procedure B
Pyridoxal oxime (0.18 g; 1 mmol) and substituted phenacyl bromide (1 mmol) was added into the
mortar and the mixture was grinded with pestle in the presence of silica gel (500 mg) for 10 min. The
mixture was placed in the microwave reactor and irradiated with a starting power at 440 W and
reaction temperature 70 °C until TLC has shown the presence of the product. Reaction times are
reported in the Table 1. The mixture was dissolved in hot acetone. Silica gel was removed from the
mixture by filtration under reduced pressure and residual crude was collected by filtration under
1
reduced pressure and recrystallized from appropriate solvent. All products were analyzed by H- and
13C-NMR, IR spectroscopy and elemental analyses.

Molecules 2014, 19
7616
3.4. Characterization Data
1-(4-Chlorophenacyl)-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methylpyridinium bromide
(2). Yellow solid; Rf value: 0.18 (chloroform‒methanol, 6:1.5, v/v); mp after crystallization from
methanol 230–233 °C; FT-IR (KBr) νmax: 3385, 3073–2858, 1688, 1637–1589, 1261, 1051–1003 cm−1;
1H-NMR: δ 12.78 (1H, bs, NOH), 8.67 (1H, bs, OH), 8.59 (1H, s, H-6), 8.14–8.11 (2H, d,
J = 8.62 Hz, H-2', H-6'), 7.77–7.74 (2H, d, J = 8.58 Hz, H-3', H-5'), 6.89 (2H, s, CH2CO), 5.84 (1H,
bs, CH2OH), 4.81 (2H, s, CH2OH), 2.51 (3H, s, CH3); 13C-NMR: δ 189.82 (C, C=O), 152.55 (C, C-3),
145.46 (C, C-2), 139.65 (C, C-6), 137.28 (C, C-4), 135.10 (C, C-4'), 132.25 (C, C-1'), 130.43 (C-2',
C-6'), 129.76 (C-3', C-5'), 128.98 (C, C-5), 64.48 (CH2CO), 58.47 (CH2OH), 13.32 (CH3); MS (m/z):
415.0 [M]+ (54.16), 335.2 (43.05, Br), 333.0 (100), 302.8 (39.58), 164.1 (36.11); Anal. Calcd. for
C16H16N2O4BrCl: C 46.23, H 3.88, N 6.74; Found: C 46.34, H 3.86, N 6.72%.
1-(4'-Bromophenacyl)-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methylpyridinium bromide
(3) Yellow solid; Rf value: 0.27 (chloroform‒methanol, 6:1.5, v/v); mp after crystallization from
ethyl acetate 195–198 °C; FT-IR (KBr) νmax: 3385, 3073-2849, 1686, 1636–1456, 1261, 1049–980 cm−1;
1H-NMR: δ 12.99 (1H, bs, NOH), 8.66 (1H, bs, OH), 8.63 (1H, s, H-6), 8.04–8.01 (2H, d, J = 8,61 Hz,
H-2', H-6'), 7.92–7.89 (2H, d, J = 8.55 Hz, H-3', H-5'), 6.61 (2H, s, CH2CO), 5.88 (2H, s, CH2OH),
13
4.81 (1H, s, CH2OH), 2.3 (3H, s, CH3); C-NMR: δ 190.02 (C, C=O), 152.47 (C, C-3), 145.44 (C,
C-2), 137.28 (C, C-6), 135.11 (C, C-4), 132.56 (C, C-4'), 132.06 (C, C-1'), 130.43 (C-2', C-6'), 128.93
(C-3', C-5'), 128.16 (C, C-5), 64.37 (CH2CO), 58.46 (CH2OH), 13.28 (CH3); MS (m/z): 460.8 [M]+
(17.48), 379.1 (100), 361.1 (39.16), 349.1 (39,86), 199.0 (34.96), 160.7 (39.16); Anal. Calcd. for
C16H16N2O4Br2 : C 41.77; H 3.50; N 6.09; Found: C 41.96, H 3.61, N 5.97%.
3-Hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-phenacylpyridinium bromide (4). Yellow
solid; Rf value: 0.26 (chloroform‒methanol, 6:1.5, v/v); mp after crystallization from methanol
250–251 °C; FT-IR (KBr) νmax: 3275, 3107–2727, 1701, 1636–1449, 1229, 1084–980 cm−1; 1H-NMR:
δ 12.47 (1H, bs, NOH), 11.52 (1H, bs, OH), 8.62 (1H, s, H-6), 7.72–7.70 (2H, d, J = 7.98 Hz, H-2',
H-6'), 7.39–7.36 (2H, d, J = 8.16 Hz, H-3', H-5'), 6.00 (2H, s, CH2CO), 4.67 (2H, s, CH2OH), 2.51
13
(1H, s, CH2OH), 2.50 (3H, s, CH3). C-NMR: δ 190.70 (C, C=O), 152.50 (C, C-3), 137.21 (C, C-2),
135.11 (C, C-6), 135.10 (C, C-4), 130.44 (C, C-4'), 128.30 (C-2', C-6'), 128.26 (C, C-1'), 128.10, (C-3',
C-5'), 127.82 (C, C-5), 59.67 (CH2CO), 58.44, (CH2OH), 13.25 (CH3); MS (m/z): 381.0 [M]+ (83),
299.3 (100), 281.4 (52.45), 269.2 (37.06), 161.0 (47.55), 106.0 (31.46); Anal. Calcd. for C16H17N2O4Br:
C 50.41, H 4.49, N 7.35, Br 20.96; Found: C 50.58, H 4.33, N 7.48, Br 20.93%.
3-Hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(4-methylphenacyl)pyridinium bromide
(5). Yellow–light brown solid; Rf value: 0.22 (chloroform‒methanol, 6:1.5, v/v); mp after
crystallization from methanol 240–241.5 °C; FT-IR (KBr) νmax: 3385, 3064–2853, 1684, 1636–1541,
1
1234, 1088–980 cm−1; H-NMR: δ 12.87 (1H, bs, NOH), 8.67 (1H, bs, OH), 8.62 (1H, s, H-6),
8.03–8.00 (2H, d, J = 8.22 Hz; H-3', H-5'), 7.48–7.46 (2H, d, J = 8.07 Hz, H-2', H-6'), 6.42 (2H, s,
CH2CO), 4.81 (2H, s, CH2OH), 2.51, (1H, s, CH2OH), 2.49 (3H, s, CH3), 2,23 (3H, s, CH3). 13C-NMR:
δ 190.06 (C, C=O), 152.49 (C, C-3), 145.50 (C, C-2), 145.31 (C, C-6), 137.24 (C, C-4), 135.19 (C,
C-4'), 129.34 (C-2', C-6'), 128.59 (C, C-1'), 128.10 (C-3', C-5'), 127.80 (C, C-5), 64.42 (CH2CO),

Molecules 2014, 19
7617
58.43 (CH2OH), 13.25 (CH3); MS (m/z): 395.2 [M]+ (100), 313.0 (44.37), 295.5 (21.83), 264.8
(22.54), 162.1 (33.09), 132.9 (22.54); Anal. Calcd. for C17H19N2O4Br: C 51.66, H 4.85, N 7.09, Br
20.22; Found: C 51.65, H 4.83, N 7.27%.
3-Hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(4'-nitrophenacyl)pyridinium bromide
(6). Reddish brown solid; Rf value: 0.16 (chloroform‒methanol, 6:1.5, v/v); mp after crystallization
from ethyl acetate 235–237 °C; FT-IR (KBr) νmax: 3347, 3069–2862, 1715, 1604–1528, 1217,
1
1032–1001 cm−1; H-NMR: δ 12.54 (1H, bs, NOH), 8.66 (1H, bs, OH), 8.62 (1H, s, H-6), 8.59–8.53
(2H, d, J = 8.10 Hz, H-3', H-5'), 8.47–8.39 (2H, d, J = 8.12 Hz, H-2', H-6'), 6.51 (2H, s, CH2CO), 4.82
(2H, s, CH2OH), 4.67 (1H, bs, CH2OH), 2.29 (3H, s, CH3); 13C-NMR: δ 190.12 (C, C=O), 152.55 (C,
C-3), 150.68 (C, C-2), 146.90 (C, C-6), 145.60 (C, C-4), 145.51 (C, C-4'), 138.59 (C, C-1'), 138.23 (C,
C-2', C-6'), 137.36 (C-3', C-5'), 130.05 (C, C-5), 64.68 (CH2CO), 58.54 (CH2OH), 13.34 (CH3); MS (m/z):
425.1 [M]+ (5.63), 344.0 (100), 326.1 (31.69), 314.3 (21.13), 164.0 (52.81), 161.0 (36.62), 106,7 (26.76);
Anal. Calcd. for C16H16Br N3O6: C 45.09, H 3.78, N 9.86, Br 18.75; Found: C 44.70, H 4.00; N 9.67%.
1-(4-Fluorophenacyl)-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methylpyridinium bromide
(7). Brown solid; Rf value: 0.31 (chloroform‒methanol, 6:1.5, v/v); mp after crystallization
from ethyl acetate 225–225,5 °C; FT-IR (KBr) νmax: 3447, 3064–2359, 1699, 1599–1456, 1233,
1
1063–1001 cm−1; H-NMR: δ 12.95 (1H, bs, NOH), 8.66 (1H, bs, OH), 8.56 (1H, s, H-6), 8.21–8.18
(2H, d, J = 8.82 Hz, H-2', H-6'), 7.52–7.49 (2H, d, J = 8.82 Hz, H-3', H-5'), 6.26 (2H, s, CH2CO), 5.38
13
(2H, s, CH2OH), 4.67 (1H, s, CH2OH), 2.27 (3H, s, CH3). C-NMR δ 189.31 (C, C=O), 164.17 (C,
C-3), 145.50 (C, C-2), 145.44 (C, C-6), 137.29 (C, C-4), 131.73 (C, C-4'), 131.62 (C-2', C-6'), 130.30 (C,
C-1'), 128.13 (C-3', C-5'), 128.10 (C, C-5), 64.32 (CH2CO), 58.49 (CH2OH), 13.25 (CH3); MS (m/z): 399.2
[M]+ (53.52), 319.1 (9.86), 317.25 (100), 299.1 (30.28), 287.3 (36.62), 137.0 (32.39), 133.0 (36.62); Anal.
Calcd. for C16H16BrFN2O4: C 48.14, H 4.04, N 7.02, Br 20.02; Found: C 47.53, H 4.22, N 6.95%.
3-Hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(4-methoxyphenacyl)pyridinium bromide
(8). Light brown solid; Rf value: 0.21 (chloroform‒methanol, 6:1.5, v/v); mp after crystallization from
ethyl acetate 267–268 °C; FT-IR (KBr) νmax: 3383, 3042–2843, 1676, 1638–1516, 1244, 1045–980
1
cm−1; H-NMR: δ 13.00 (1H, bs, NOH), 8.66 (1H, bs, OH), 8.56 (1H, s, H-6), 8.13 (2H, s, CH2CO),
8.08- 8.05 (2H, d, J = 8.34 Hz; H-3', H-5'), 7.20 – 7.17 (2H, d, J = 8.97 Hz; H-2', H-6'), 6.55 (2H, s,
13
CH2OH), 4.83 (1H, bs, CH2OH), 3.91 (3H, s, OCH3), 2.51 (3H, s, CH3). C-NMR: δ 188.73 (C,
C=O), 164.42 (C, C-3), 145.52 (C, C-2), 145.33 (C, C-6), 137.25 (C, C-4), 135.11 (C, C-4'), 130,97
(C-2', C-6'), 128.05 (C, C-1'), 128.32 (C-3', C-5'), 127.80 (C, C-5), 64.10 (CH2CO), 58.47 (CH2OH),
13.21 (CH3); MS (m/z): 411.2 [M]+ (96.5), 331.0 (3.49), 329.35 (100), 311.0 (17.48), 299.1 (42.65),
134.0 (57.34), 106.0 (60.84); Anal. Calcd. for C17H19N2O5Br: C 49.65, H 4.66, N 6.81, Br 19.43;
Found: C 49.79, H 4.46, N 6.83, Br 19.35%.
3-Hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(4'-phenylphenacyl)pyridinium bromide
(9). Yellow solid; Rf value: 0.39 (chloroform‒methanol, 6:1.5, v/v); mp after crystallization from
methanol 227–228 °C; FT-IR (KBr) νmax: 3397, 3075–2789, 1691, 1604–1451, 1237, 1088–995 cm−1;
1H-NMR: δ 13.02 (1H, bs, NOH), 12.78 (1H, bs, OH), 8.64 (1H, s, H-6), 8.19–8.166 (2H, d, J = 8.43
Hz, H-3', H-5'), 8.00–7.97 (2H, d, J = 8.43 Hz, H-2', H-6'); 7.58–7.45 (5H, m, Ph), 6.64 (2H, s,

Molecules 2014, 19
7618
13
CH2CO), 4.81 (2H, s, CH2OH); 4.75 (1H, bs, CH2OH), 2.50 (3H, s, CH3); C-NMR: δ 190.13 (C,
C=O), 152.46 (C, C-3), 146.02 (C, C-2), 145.49 (C, C-4), 145.38 (C, C-6), 142.86 (C, C-4'), 138.45
(C-2', C-6'), 138.05 (C, C-1'), 137.30 (C-3', C-5'), 136.83 (C, C-5), 64.40 (CH2CO), 58.48 (CH2OH),
13,22 (CH3); MS (m/z): 457.1 [M]+ (100), 375.3 (87.41), 357.3 (15.38), 345.0 (31.12), 164.3 (62,23);
Anal. Calcd. for C22H21N2O4Br: C 57.78, H 4.63, N 6.13, Br 17.47; Found: C 57.63, H 4.46, N 6.03%.
3-Hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(2-methoxyphenacyl)pyridinium bromide
(10). Brown solid; Rf value: 0.36 (chloroform‒methanol, 6:1.5, v/v); mp after crystallization from
methanol 217–219 °C; FT-IR (KBr) νmax: 3309, 3102–2949, 1674, 1597–1437, 1252, 1055–980 cm−1;
1H-NMR: δ 12.95 (1H, bs, NOH), 8.67 (1H, bs, OH), 8.61 (1H, s, H-6), 7.86–7.84 (2H, d, J = 6.03 Hz
H-3', H-5'), 7.36–7.33 (2H, d, J = 8.40 Hz H-2', H-6'), 7.66 (2H, s, CH2CO), 6.53 (1H, bs, CH2OH),
13
4.80 (3H, s, OCH3), 4.04 (2H, s, CH2OH), 2,51 (3H, s, CH3); C-NMR: δ 190.26 (C, C=O), 159.98
(C, C-3), 145.58 (C, C-2), 145.51 (C, C-6), 145.19 (C, C-4), 137.09 (C, C-4'), 136.97 (C, C-1'), 136.27
(C-2', C-6'), 130.25 (C-3', C-5'), 128.00 (C, C-5), 67.86 (CH2CO), 58.45 (CH2OH), 13.19 (CH3); anal.
C 49.65, H 4.66, N 6.81, Br 19.43% calcd for C17H19N2O5Br C 49.60, H 4.65, N 6.58%.
3.5. Crystal Structure Determination
Single crystal of 7 suitable for X-ray single crystal analysis was obtained at room temperature
by partial evaporation from ethyl-acetate solution. The intensities were collected on a Oxford
Diffraction Xcalibur2 diffractometer (Zagreb, Croatia) with a Sapphire 3 CCD detector using
graphite-monochromated MoKα radiation (λ = 0.71073 Å) at 150 K. The CrysAlisPro [18] program
was used for the data collection and processing. The intensities were corrected for absorption using the
multi-scan absorption correction method [18]. The structure was solved by direct methods with
SIR2004 [19] and refined by full-matrix least-squares calculations based on F2 using SHELXL-97 [20]
integrated in WinGX [21] program package. Hydrogen atoms of oxygen O1, O2 and O3 atoms have
been found in Fourier map and their coordinates and thermal isotropic parameters have been refined
freely. All other hydrogen atoms were treated using appropriate riding models, with SHELXL-97
defaults [20]. PLATON [22] and Mercury [23] programs were used for structure analysis and
molecular and crystal structure drawings preparation. The CCDC 990857 contains the supplementary
crystallographic data for this paper. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Crystal and refinement data
ത
for 7: C16H16BrFN2O4, Mr = 399.22; triclinic space group P1 (No. 2); a = 6.7128(3), b = 8.1316(4),
c = 15.9969(8) Å; α = 94.964(4), β = 92.224(4), γ = 111.877(5)°; V = 804.89(7) Å3; Z = 2;
dx = 1.647 g cm−3; θmax = 27.0°; RInt = 0.0290; S = 1.004; R[I ≥ 2σ(I)] = 0.0357, wR[all data] = 0.0991;
0.698 < ∆ρ < −0.760 eA−3.
4. Conclusions
In conclusion, a comparative study of the quaternization reaction of pyridoxal oxime with a series
of substituted phenacyl bromides under microwave irradiation is reported in this paper. The
preparation of quaternary pyridininium salts under microwave irradiation proved to be a fast,
environmentally friendly, and facile method. The microwave irradiation provided a remarkable rate of

Molecules 2014, 19
7619
acceleration for N-alkylation, reaction times decreased dramatically, the consumed energy decreased
considerably, the yields are higher and this method could be considered as eco-friendly.
The MW method performed in acetone offered easier work up, cleaner products and higher yields.
The solvent-free method was more eco-friendly because there was no solvent used in the synthesis, but
the disadvantage is longer work up, use of solvents in the post-synthetic stage, and less cleaner
products. One of the primary goals of the future experiment will be elimination of solvents from the
post-synthetic stage after solventless quaternization. The X-ray structure analysis of 7 shows that the
bond lengths and angles present no unexpected features, and that phenyl ring is rotated with respect to
the pyridinium ring. Several types of intermolecular hydrogen bonds and one π...π interaction link
cation and bromides into three-dimensional network. The continuation of this work to determine
antibacterial, antifungal and antidotal activity of the novel synthesized compounds is in progress.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/6/7610/s1.
Acknowledgments
Dajana Gašo-Sokač and Mario Cetina (Projects No 119-1193079-3069) would like to thank the
Ministry of Science, Education and Sport of the Republic of Croatia for financial support.
Author Contributions
Dajana Gašo-Sokač designed research and performed synthesis. Valentina Bušić prepared
compounds, Marijana Jukić was co-responsible for microwave synthesis and Mario Cetina was
responsible for X-Ray crystallography.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Varma, R.S.; Namboodiri, V.V. Solvent-free preparation of ionic liquids using a household
microwave oven. Pure. Appl. Chem. 2001, 73, 1309–1313.
2. Surati, M.A.; Jauhari, S.; Desai, K.R. A brief review: Microwave assisted organic reaction.
Arch. Appl. Sci. Res. 2012, 4, 645–661.
3. Bogdal, D.; Pielichowski, J.; Jaskott, K. Fast N-Alkylation of azaheterocycles under microwave
irradiation in dry media. Heterocycles 1997, 45, 715–722.
4. Bogdal, D.; Pielichowski, J.; Jaskott, K. New method of N-alkylation of carbazole under
microwave irradiation in dry media. Synth. Commun. 1997, 27, 1553–1560.
5. Bogdal, D. Fast solvent-free alkylation of amides and lactams under microwave irradiation.
Molecules 1999, 4, 333–337.
6. Deshayes, S.; Liagre, M.; Loupy, A.; Luche, J.L.; Petit, A. Microwave activation in phase transfer
catalysis. Tetrahedron 1999, 55, 10851–10870.

Molecules 2014, 19
7620
7. Perez, E.; Sotelo, E.; Loupy, A.; Mocelo, R.; Suarez, M.; Perez, R.; Autie, M. An easy and
efficient microwave-assisted method to obtain 1-(4-bromophenacyl)azoles in dry media. Heterocycles
1996, 43, 539–543.
8. Somani, S.M.; Romano, J.A. Chemical Warfare Agents: Toxicity at Low Levels; CRS Press: LLC
Boca Raton, FL, USA, 2001.
9. Bajgar J. Organophosphates/nerve agent poisoning: Mechanism of action, diagnosis, prophylaxis,
and treatment. J. Adv. Clin. Chem. 2004, 38, 151–216.
10. Kassa, J.; Cabal, J.; Kuča, K. A comparison of the efficacy of currently available oximes against
tabun in rats. Biologia 2005, 60, 77–79.
11. Kuča, K.; Bartošovál, D.; Patočka, J.; Cabal, J.; Kassa, J.; Kunešová, G. New quaternary pyridine
aldoximes as causal antidotes against nerve agents intoxications. Biomed. Papers 2005, 149, 75–82.
12. Kassa J. Review of oximes in the antidotal treatment of poisoning by organophosphorus nerve
agents. J. Clin. Toxicol. 2002, 40, 803–816.
13. Jokanović, M.; Stojiljković, M.P. Current understanding of the application of pyridinium oximes
as cholinesterase reactivators in treatment of organophosphate poisoning. Eur. J. Pharmacol.
2006, 553, 10–17.
14. Kovarik, Z.; Čalić, M.; Šinko, G.; Bosak, A.; Berend, S.; Lucić Vrdoljak, A.; Radić, B. Oximes:
Reactivators of phosphorylated acetylcholinesterase and antidotes in therapy against tabun
poisoning. Chem. Biol. Interact. 2008, 175, 173–179.
15. Gašo-Sokač, D.; Katalinić, M.; Kovarik, Z.; Bušić, V.; Kovač, S. Synthesis and evaluation of
novel analogues of vitamin B6 as reactivators of tabun and paraoxon inhibited
acetylcholinesterase. Chem. Biol. Interact. 2010, 187, 234–237.
16. Cetina, M.; Nagl, A.; Gašo-Sokač, D.; Kovač, S.; Bušić, V.; Saftić, D. Extensive intramolecular
and intermolecular interactions in two quaternary salts of the pyridoxal oxime. J Chem.
Christallogr. 2012, 42, 752–758.
17. Jukić, M.; Hergold-Brundić, A.; Cetina, M.; Nagl, A.; Vorkapić-Furač, J. The synthesis and
structures of the novel pyridoxal oxime derivatives. Struct. Chem. 2003, 14, 597–604.
18. Oxford Diffraction. Xcalibur CCD System, CrysAlisPro; Oxford Diffraction Ltd: Abingdon,
England, 2010.
19. Burla, M.C.; Caliandro, R.; Camalli, M.; Carrozzini, B.; Cascarano, G.L.; de Caro, L.;
Giacovazzo, C.; Polidori, G.; Spagna, R. SIR2004: An improved tool for crystal structure
determination and refinement. J. Appl. Crystallogr. 2005, 38, 381–388.
20. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. 2008, A64, 112–122.
21. Farrugia, L.J. WinGX and Ortep for Windows: An update. J. Appl. Crystallogr. 2012, 45, 849–854.
22. Spek, A.L. Structure validation in chemical crystallography. Acta Crystallogr. 2009, D65, 148–155.
23. Macrae, C.F.; Edgington, P.R.; McCabe, P.; Pidcock, E.; Shields, G.P.; Taylor, R.; Towler, M.;
van de Streek, J. Mercury: Visualization and analysis of crystal structures. J. Appl. Crystallogr.
2006, 39, 453–457.
Sample Availability: Samples of the compounds are not available from the authors.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).